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Aduro Biotech Phase II Pancreatic Cancer Trial CRS-207 plus cancer vaccine GVAX Fails

Posted in Cancer - General, Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Immunology, Immunotherapy, tagged , , , , , , , , , on May 16, 2016| Leave a Comment »

Aduro Biotech Phase II Pancreatic Cancer Trial CRS-207 plus cancer vaccine GVAX Fails

Reporter: Stephen J. Williams, Ph.D

From Biospace News

May 16, 2016
By Alex Keown, BioSpace.com Breaking News Staff

BERKELEY, Calif. – Shares of Aduro Biotech (ADRO) have fallen more than 25 percent this morning following news that the company’s Phase II trial for its combination pancreatic cancer drug, CRS-207 did not meet its primary endpoint of survivability.

Aduro said its Eclipse trial of CRS-207 failed to show an improvement in overall survival for patients with pancreatic cancer who had failed at least two prior therapies in the metastatic setting. Median overall survival was 3.8 months for patients treated with the immunotherapy regimen of CRS-207 and the cancer vaccine GVAX pancreas, 5.4 months for patients treated with CRS-207 alone and 4.6 months for patients administered chemotherapy. Aduro said there were no reported safety concerns during the trial and full study findings will be presented at a later date.

Stephen T. Isaacs, chairman, president and chief executive officer of Aduro, called the findings a disappointing and “unexpected outcome.’

“While we are well aware of the very difficult-to-treat nature of late-stage metastatic pancreatic cancer, we are surprised by the divergence of these data from the results of our Phase IIa study. At the same time, we continue to look forward to the interim results later this year from our ongoing Stellar trial, which is evaluating CRS-207 and GVAX Pancreas with and without the anti-PD1 checkpoint inhibitor nivolumab as a second-line therapy for patients with metastatic pancreatic cancer,” Isaacs said in a statement.

For full story please see http://www.biospace.com/News/aduro-biotechs-stock-craters-after-pancreatic/419628/source=TopBreaking

Also from FierceBiotech

UPDATED: Aduro combo fails in a key pancreatic cancer study

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Cyclic Dinucleotides and Histone deacetylase inhibitors

Posted in Anaerobic Glycolysis, Apoptosis, Biological Networks, Cancer and Current Therapeutics, Cell Biology, Signaling & Cell Circuits, Cell death pathways, Chemical Biology and its relations to Metabolic Disease, Clinical & Translational, Enzyme Induction, Gene Regulation, Genetics & Pharmaceutical, Genomic Expression, Oxidative phosphorylation, Proteolysis, tagged , , , , , , on May 12, 2016| Leave a Comment »

Cyclic Dinucleotides and Histone deacetylase inhibitors

Curators: Larry H. Bernsten, MD, FCAP and Aviva Lev-Ari, PhD, RN

LPBI

 

New Class of Immune System Stimulants: Cyclic Di-Nucleotides (CDN): Shrink Tumors and bolster Vaccines, re-arm the Immune System’s Natural Killer Cells, which attack Cancer Cells and Virus-infected Cells

Reporter: Aviva Lev-Ari, PhD, RN

The Immunotherapeutics and Vaccine Research Initiative (IVRI), a UC Berkeley effort funded by Aduro Biotech, Inc.

https://pharmaceuticalintelligence.com/2016/04/24/new-class-of-immune-system-stimulants-cyclic-di-nucleotides-cdn-shrink-tumors-and-bolster-vaccines-re-arm-the-immune-systems-natural-killer-cells-which-attack-cancer-cells-and-virus-inf/

A new class of immune system stimulants called cyclic di-nucleotides have shown promise in shrinking tumors and bolstering vaccines against tuberculosis, and research that could help re-arm the immune system’s natural killer cells, which normally attack cancer cells and virus-infected cells, to better fight tumors.

Much of the excitement around combining these two areas — the immunology of cancer and the immunology of infectious disease — comes from the amazing success of immunotherapy against cancer, which started with the work of James Allison when he was a professor of immunology at UC Berkeley and director of the Cancer Research Laboratory from 1985 to 2004. Allison, now at the University of Texas MD Anderson Cancer Center, discovered a way to release a brake on the body’s immune response to cancer that has proved highly successful at unleashing the immune system to attack melanoma and is being tried against other types of cancer. Allison’s technique uses an antibody that blocks an immune suppressor called CTLA4, antibodies that block another immune suppressor, PD1. This has been successful in treating melanoma, renal cancer and a type of lung cancer. Both CTLA4 and PD1 antibodies are now FDA-approved as cancer therapies.

Another promising avenue involves a protein in cells that responds to foreign DNA to launch an innate immune response — the first response of the body’s immune system. The protein, dubbed STING, is triggered by small molecules called cyclic di-nucleotides (CDN), and makes immune cells release interferon and other cytokines that activate disease-fighting T cells and stimulate the production of antibodies that together kill invading pathogens and destroy cancer cells. Listeria bacteria, for example, secrete a CDN directly into infected cells that activates STING.

Russell Vance, a UC Berkeley professor of molecular and cell biology and current head of the Cancer Research Laboratory, discovered several years ago that the chemical structure of these di-nucleotides is critical to their ability to work in humans. Aduro has since developed a CDN designed to work in humans and found that injecting it directly into a tumor in mice caused the tumor to shrink.

Sarah Stanley, an assistant professor of public health, has found evidence that CDNs can help improve the imperfect vaccines we have today against tuberculosis.

 

Researchers at UC Berkeley will have access to Aduro’s novel technology platforms for research use, including its STING pathway activators, proprietary monoclonal antibodies and the engineered listeria bacteria, referred to as LADD (listeria attenuated double-deleted). David Raulet, professor of molecular and cell biology and director of IVRI has contributed to making these cells a new focus of cancer research. As tumors advance, NK cells inside the tumors appear to become desensitized, he said. Recent research shows that some cytokines and other immune mediators Raulet discovered are able to “wake them up” and get them to resume their elimination of cancer cells.

 

Histone deacetylase inhibitors: molecular mechanisms of action

W S Xu1,2, R B Parmigiani1,2 and P A Marks1

Oncogene (2007) 26, 5541–5552; http://dx.doi.org:/10.1038/sj.onc.1210620

This review focuses on the mechanisms of action of histone deacetylase (HDAC) inhibitors (HDACi), a group of recently discovered ‘targeted’ anticancer agents. There are 18 HDACs, which are generally divided into four classes, based on sequence homology to yeast counterparts. Classical HDACi such as the hydroxamic acid-based vorinostat (also known as SAHA and Zolinza) inhibits classes I, II and IV, but not the NAD+-dependent class III enzymes. In clinical trials, vorinostat has activity against hematologic and solid cancers at doses well tolerated by patients. In addition to histones, HDACs have many other protein substrates involved in regulation of gene expression, cell proliferation and cell death. Inhibition of HDACs causes accumulation of acetylated forms of these proteins, altering their function. Thus, HDACs are more properly called ‘lysine deacetylases.’ HDACi induces different phenotypes in various transformed cells, including growth arrest, activation of the extrinsic and/or intrinsic apoptotic pathways, autophagic cell death, reactive oxygen species (ROS)-induced cell death, mitotic cell death and senescence. In comparison, normal cells are relatively more resistant to HDACi-induced cell death. The plurality of mechanisms of HDACi-induced cell death reflects both the multiple substrates of HDACs and the heterogeneous patterns of molecular alterations present in different cancer cells.

histone deacetylase, histone deacetylase inhibitor, apoptosis, mitotic cell death, senescence, angiogenesis

Acetylation and deacetylation of histones play an important role in transcription regulation of eukaryotic cells (Lehrmann et al., 2002;Mai et al., 2005). The acetylation status of histones and non-histone proteins is determined by histone deacetylases (HDACs) and histone acetyl-transferases (HATs). HATs add acetyl groups to lysine residues, while HDACs remove the acetyl groups. In general, acetylation of histone promotes a more relaxed chromatin structure, allowing transcriptional activation. HDACs can act as transcription repressors, due to histone deacetylation, and consequently promote chromatin condensation. HDAC inhibitors (HDACi) selectively alter gene transcription, in part, by chromatin remodeling and by changes in the structure of proteins in transcription factor complexes (Gui et al., 2004). Further, the HDACs have many non-histone proteins substrates such as hormone receptors, chaperone proteins and cytoskeleton proteins, which regulate cell proliferation and cell death (Table 1). Thus, HDACi-induced transformed cell death involves transcription-dependent and transcription-independent mechanisms (Marks and Dokmanovic, 2005; Rosato and Grant, 2005; Bolden et al., 2006;Minucci and Pelicci, 2006).

Table 1 – Nonhistone protein substrates of HDACs (partial list).   Full table

http://www.nature.com/common/images/table_thumb.gif

In humans, 18 HDAC enzymes have been identified and classified, based on homology to yeast HDACs (Blander and Guarente, 2004;Bhalla, 2005; Marks and Dokmanovic, 2005). Class I HDACs include HDAC1, 2, 3 and 8, which are related to yeast RPD3 deacetylase and have high homology in their catalytic sites. Recent phylogenetic analyses suggest that this class can be divided into classes Ia (HDAC1 and -2), Ib (HDAC3) and Ic (HDAC8) (Gregoretti et al., 2004). Class II HDACs are related to yeast Hda1 (histone deacetylase 1) and include HDAC4, -5, -6, -7, -9 and -10 (Bhalla, 2005; Marks and Dokmanovic, 2005). This class is divided into class IIa, consisting of HDAC4, -5, -7 and -9, and class IIb, consisting of HDAC6 and -10, which contain two catalytic sites. All class I and II HDACs are zinc-dependent enzymes. Members of a third class, sirtuins, require NAD+ for their enzymatic activity (Blander and Guarente, 2004) (see review by E Verdin, in this issue). Among them, SIRT1 is orthologous to yeast silent information regulator 2. The enzymatic activity of class III HDACs is not inhibited by compounds such as vorinostat or trichostatin A (TSA), that inhibit class I and II HDACs. Class IV HDAC is represented by HDAC11, which, like yeast Hda 1 similar 3, has conserved residues in the catalytic core region shared by both class I and II enzymes (Gao et al., 2002).

HDACs are not redundant in function (Marks and Dokmanovic, 2005; Rosato and Grant, 2005; Bolden et al., 2006). Class I HDACs are primarily nuclear in localization and ubiquitously expressed, while class II HDACs can be primarily cytoplasmic and/or migrate between the cytoplasm and nucleus and are tissue-restricted in expression.

The structural details of the HDAC–HDACi interaction has been elucidated in studies of a histone deacetylase-like protein from an anerobic bacterium with TSA and vorinostat (Finnin et al., 1999). More recently, the crystal structure of HDAC8–hydroxamate interaction has been solved (Somoza et al., 2004; Vannini et al., 2004). These studies provide an insight into the mechanism of deacetylation of acetylated substrates. The hydroxamic acid moiety of the inhibitor directly interacts with the zinc ion at the base of the catalytic pocket.

This review focuses on the molecular mechanisms triggered by inhibitors of zinc-dependent HDACs in tumor cells that explain in part: (I) the effects of these compounds in inducing transformed cell death and (II) the relative resistance of normal and certain cancer cells to HDACi induced cell death. HDACi, for example, the hydroxamic acid-based vorinostat (SAHA, Zolinza), are promising drugs for cancer treatment (Richon et al., 1998; Marks and Breslow, 2007). Several HDACi are in phase I and II clinical trials, being tested in different tumor types, such as cutaneous T-cell lymphoma, acute myeloid leukemia, cervical cancer, etc (Bug et al., 2005; Chavez-Blanco et al., 2005; Kelly and Marks, 2005;Duvic and Zhang, 2006) (Table 2). Although considerable progress has been made in elucidating the role of HDACs and the effects of HDACi, these areas are still in early stages of discovery.

Table 2 – HDACi in clinical trials.  Full table

http://www.nature.com/common/images/table_thumb.gif

Recent phylogenetic analyses of bacterial HDACs suggest that all four HDAC classes preceded the evolution of histone proteins (Gregoretti et al., 2004). This suggests that the primary activity of HDACs may be directed against non-histone substrates. At least 50 non-histone proteins of known biological function have been identified, which may be acetylated and substrates of HDACs (Table 1) (Glozak et al., 2005; Marks and Dokmanovic, 2005;Rosato and Grant, 2005; Bolden et al., 2006; Minucci and Pelicci, 2006; Zhao et al., 2006). In addition, two recent proteomic studies identified many lysine-acetylated substrates (Iwabata et al., 2005; Kim et al., 2006). In view of all these findings, HDACs may be better called ‘N-epsilon-lysine deacetylase’. This designation would also distinguish them from the enzymes that catalyse other types of deacetylation in biological reactions, such as acylases that catalyse the deacetylation of a range of N-acetyl amino acids (Anders and Dekant, 1994).

Non-histone protein targets of HDACs include transcription factors, transcription regulators, signal transduction mediators, DNA repair enzymes, nuclear import regulators, chaperone proteins, structural proteins, inflammation mediators and viral proteins (Table 1). Acetylation can either increase or decrease the function or stability of the proteins, or protein–protein interaction (Glozak et al., 2005). These HDAC substrates are directly or indirectly involved in many biological processes, such as gene expression and regulation of pathways of proliferation, differentiation and cell death. These data suggest that HDACi could have multiple mechanisms of inducing cell growth arrest and cell death (Figure 1).

Figure 1.  Full figure and legend (90K)

Multiple HDACi-activated antitumor pathways. See text for detailed explanation of each pathway. HDAC6, histone deacetylase 6; HIF-1, hypoxia-induced factor-1; HSP90, heat-shock protein 90; PP1, protein phosphatase 1; ROS, reactive oxygen species; TBP2, thioredoxin binding protein 2; Trx, thioredoxin; VEGF, vascular endothelial growth factor.

http://www.nature.com/onc/journal/v26/n37/images/1210620f1.jpg

HDACi have been discovered with different structural characteristics, including hydroximates, cyclic peptides, aliphatic acids and benzamides (Table 2) (Miller et al., 2003; Yoshida et al., 2003; Marks and Breslow, 2007). Certain HDACi may selectively inhibit different HDACs. For example, MS-275 preferentially inhibits HDAC1 with IC50, at 0.3 m, compared to HDAC3 with an IC50 of about 8 m, and has little or no inhibitory effect against HDAC6 and HDAC8 (Hu et al., 2003). Two novel synthetic compounds, SK7041 and SK7068, preferentially target HDAC1 and 2 and exhibit growth inhibitory effects in human cancer cell lines and tumor xenograft models (Kim et al., 2003a). A small molecule, tubacin, selectively inhibits HDAC6 activity and causes an accumulation of acetylated -tubulin, but does not affect acetylation of histones, and does not inhibit cell cycle progression (Haggarty et al., 2003). No other HDACi for a specific HDAC has been reported.

HDACi selectively alters gene expression

HDACi-induced antitumor pathways

  • HDACi induces cell cycle arrest
  • HDACi activates the extrinsic apoptotic pathways
  • HDACi activates the intrinsic apoptotic pathways
  • HDACi induces mitotic cell death
  • HDACi induces autophagic cell death and senescence
  • ROS, thioredoxin and Trx binding protein 2 in HDACi-induced cell death
  • Antitumor effects of HDAC6 inhibition
  • Activation of protein phosphatase 1
  • Disruption of the function of chaperonin HSP90
  • Disruption of the aggresome pathway
  • HDACi inhibits angiogenesis

HDACi can block tumor angiogenesis by inhibition of hypoxia inducible factors (HIF) (Liang et al., 2006). HIF-1 and HIF-2 are transcription factors for angiogenic genes (Brown and Wilson, 2004). The oxygen level can control HIF activity through two mechanisms. First, under normoxic conditions, HIF-1 binds to von Hippel–Lindau protein (pVHL) and is degraded by the ubiquitination–proteasome system. Second, HIF activity depends on its transactivation potential (TAP), which is affected by the interaction with the coactivator p300/CBP among others. This complex can be disrupted by Factor Inhibiting HIF (FIH). Hypoxic conditions activate HIF through repression of the hydroxylases responsible for HIF degradation and loss of function.

 

Combination of HDACi with other antitumor agents

The HDACi have shown synergistic or additive antitumor effects with a wide range of antitumor reagents, including chemotherapeutic drugs, new targeted therapeutic reagents and radiation, by various mechanisms, some unique for particular combinations (Rosato and Grant, 2004; Bhalla, 2005; Marks and Dokmanovic, 2005; Bolden et al., 2006).

Clinical development of HDACi

At least 14 different HDACi are in some phase of clinical trials as monotherapy or in combination with retinoids, taxols, gemcitabine, radiation, etc, in patients with hematologic and solid tumors, including cancer of lung, breast, pancreas, renal and bladder, melanoma, glioblastoma, leukemias, lymphomas, multiple myeloma (see National Cancer Institute website for CTEP clinical trials, ctep.cancer.gov or clinicaltrials.gov, and website of companies developing HDACi; Table 2).

The resistance to HDACi

Conclusions and perspectives

HDACs have multiple substrates involved in many biological processes, including proliferation, differentiation, apoptosis and other forms of cell death. Indeed, the fact that HDACs have histone and multiple nonhistone protein substrates suggests these enzymes should be referred to as ‘lysine deacetylases’. HDACi can cause transformed cells to undergo growth arrest, differentiation and/or cell death. Normal cells are relatively resistant to HDACi. HDACi are selective in altering gene expression, which may reflect, in part, the proteins composing the transcription factor complex to which HDACs are recruited. Both altered gene expression and changes in non-histone proteins caused by HDACi-induced acetylation play a role in the antitumor activity of HDACi. This is reflected in the different inducer-activated antitumor pathways in transformed cells (Figure 1). The functions of HDACs are not redundant. Thus, a pan-HDAC inhibitor such as vorinostat may activate more antitumor pathways and have therapeutic advantages compared to HDAC isotype-specific inhibitors.

Almost all cancers have multiple defects in the expression and/or structure of proteins that regulate cell proliferation and death. Compared to other antitumor reagents, the plurality of action of HDACi potentially confers efficacy in a wide spectrum of cancers, which have heterogeneity and multiple defects, both among different types of cancer and within different individual tumors of the same type. The multiple defects in a cancer cell may be the reason for transformed cells being more sensitive than normal cells to HDACi. Thus, given the relatively rapid reversibility of vorinostat inhibition of HDACs, normal cells may be able to compensate for HDACi-induced changes more effectively than cancer cells.

HDACi have synergistic or additive antitumor effects with many other antitumor reagents – suggesting that combination of HDACi and other anticancer agents may be very attractive therapeutic strategies for using these agents. Complete understanding of the mechanisms underlying the resistance and sensitivity to HDACi has obvious therapeutic importance. Targeting resistant factors will enhance the antitumor efficacy of HDACi. Identifying markers that can predict response to HDACi is a high priority for expanding the efficacy of these novel anticancer agents.

References  ….

NEWS AND VIEWS   Blocking HDACs boosts regulatory T cells

Nature Medicine News and Views (01 Nov 2007)

RESEARCH   

Dimethyl sulfoxide to vorinostat: development of this histone deacetylase inhibitor as an anticancer drug

Nature Biotechnology Research (01 Jan 2007)

Comments of reviewer:

 

The complexity of cancer has been known for almost a century, in large part from the seminal work of Otto Warburg in the 1920s using manometry, and following the work of Louis Pasteur 60 years earlier with fungi.

 

The volume of work and our unlocking of mitotic activity, apoptosis, mitochondria, and the cytoskeleton has taken us further into the cell interior, cell function, metabolic regulation, and pathophysiology.  Despite the enormous contributions to our knowledge of genomics, there is a large body of work in pathways of cell function that resides in no small part in activity of protein catalysts and enzymes.

 

The work that has been described covers only cyclic dinucleotides and HDACi’s.  Some of the activities described have relevance to microorganisms as well as cancer.  As we have seen, blocking HDACs boosts the activity of regulatory T-cells. There are many specific functional alterations elucidated above.

 

The first presentation is of an antibody that blocks an immune suppressor called CTLA4, antibodies that block another immune suppressor, PD1. This also involves a protein in cells that responds to foreign DNA to launch an innate immune response — the first response of the body’s immune system. The protein, dubbed STING, is triggered by small molecules called cyclic di-nucleotides (CDN), and makes immune cells release interferon and other cytokines that activate disease-fighting T cells and stimulate the production of antibodies that together kill invading pathogens and destroy cancer cells. Listeria bacteria, for example, secrete a CDN directly into infected cells that activates STING.

 

The second is resident in acetylation status of histones and non-histone proteins is determined by histone deacetylases (HDACs) and histone acetyl-transferases (HATs). HATs add acetyl groups to lysine residues, while HDACs remove the acetyl groups. In general, acetylation of histone promotes a more relaxed chromatin structure, allowing transcriptional activation. HDACs can act as transcription repressors, due to histone deacetylation, and consequently promote chromatin condensation. HDAC inhibitors (HDACi) selectively alter gene transcription, in part, by chromatin remodeling and by changes in the structure of proteins in transcription factor complexes (Gui et al., 2004).  The description focuses on the molecular mechanisms triggered by inhibitors of zinc-dependent HDACs in tumor cells that explain in part: (I) the effects of these compounds in inducing transformed cell death and (II) the relative resistance of normal and certain cancer cells to HDACi induced cell death.

 

HDACs have multiple substrates involved in many biological processes, including proliferation, differentiation, apoptosis and other forms of cell death. Indeed, the fact that HDACs have histone and multiple nonhistone protein substrates suggests these enzymes should be referred to as ‘lysine deacetylases’. HDACi can cause transformed cells to undergo growth arrest, differentiation and/or cell death. Normal cells are relatively resistant to HDACi. HDACi are selective in altering gene expression, which may reflect, in part, the proteins composing the transcription factor complex to which HDACs are recruited. Both altered gene expression and changes in non-histone proteins caused by HDACi-induced acetylation play a role in the antitumor activity of HDACi.

 

 

 

 

 

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Bispecific and Trispecific Engagers: NK-T Cells and Cancer Therapy

Posted in Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Immuno-Oncology & Genomics, Immunology, Immunotherapy, tagged , , , , , , , , , on May 8, 2016| Leave a Comment »

Bispecific and Trispecific Engagers: NK-T Cells and Cancer Therapy

Curator: Larry H. Bernstein, MD, FCAP

 

 

Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer

Jeffrey S. Miller, Yvette Soignier, Angela Panoskaltsis-Mortari, …, Todd E. Defor, Linda J. Burns, Paul J. Orchard, Bruce R. Blazar, John E. Wagner, Arne Slungaard, Daniel J. Weisdorf, Ian J. Okazaki, and Philip B. McGlave
Blood. 2005;105:3051-3057   http://www.fortressbiotech.com/pdfs/Miller_NK%20adoptive%20immunotherapy.Blood.2005.pdf

We previously demonstrated that autologous natural killer (NK)–cell therapy after hematopoietic cell transplantation (HCT) is safe but does not provide an antitumor effect. We hypothesize that this is due to a lack of NK-cell inhibitory receptor mismatching with autologous tumor cells, which may be overcome by allogeneic NK-cell infusions. Here, we test haploidentical, related-donor NK-cell infusions in a nontransplantation setting to determine safety and in vivo NK-cell expansion. Two lower intensity outpatient immune suppressive regimens were tested: (1) lowdose cyclophosphamide and methylprednisolone and (2) fludarabine. A higher intensity inpatient regimen of high-dose cyclophosphamide and fludarabine (HiCy/Flu) was tested in patients with poorprognosis acute myeloid leukemia (AML). All patients received subcutaneous interleukin 2 (IL-2) after infusions. Patients who received lower intensity regimens showed transient persistence but no in vivo expansion of donor cells. In contrast, infusions after the more intense Hi-Cy/Flu resulted in a marked rise in endogenous IL-15, expansion of donor NK cells, and induction of complete hematologic remission in 5 of 19 poor-prognosis patients with AML. These findings suggest that haploidentical NK cells can persist and expand in vivo and may have a role in the treatment of selected malignancies used alone or as an adjunct to HCT.

Human natural killer (NK) cells are a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T-cell receptor (CD3).1 They recognize and kill transformed cell lines in a major histocompatibility complex (MHC)–unrestricted fashion and produce cytokines critical to the innate immune response. NK-cell function, distinct from the MHC-restricted cytolytic activity of T cells, may play a role in antitumor surveillance.2 The effects of NK-cell infusions have been studied in adoptive immunotherapy clinical trials. In these studies, autologous lymphokine-activated killer cells obtained from peripheral blood mononuclear cells (PBMCs) were administered to patients along with exogenous high-dose interleukin-2 (IL-2). Up to 20% of patients responded to these infusions of NK-cell– containing populations.3

In contrast to NK cells, T cells recognize targets through an antigen-specific T-cell receptor (TCR) and interact with targets only if human leukocyte antigen (HLA) MHC antigens are also recognized. Although NK-cell killing is MHC-unrestricted, NK cells display a number of activating and inhibitory receptors that ligate MHC molecules to modulate the immune response.4,5 NK-cell receptors that recognize antigens at the HLA-A, -B, or -C loci are members of the immunoglobulin superfamily and are termed killer immunoglobulin receptors (KIRs).6,7 Other receptor families (natural killer group 2 [NKG2]/CD94) that recognize antigens of the nonclassical HLA-E, -F, or -G loci and other ligand specificities have also been described.8-10 Engagement of these NK-cell receptors results in stimulation or inhibition of NK-cell effector function depending on intracellular signaling mediated through the cytoplasmic tail or adaptor molecules associated with each receptor.11-13 The NK-cell response to a target thus depends on the net effect of activating and inhibitory receptors.

Clinical trials have assessed the effects of low-dose IL-2 administration on activation of NK cells in patients with cancer. We have demonstrated the safety and feasibility of daily subcutaneous IL-2 injections following high-dose chemotherapy and autologous hematopoietic cell transplantation (HCT). Whereas IL-2 signifi- cantly expanded the number of circulating NK cells in vivo, these NK cells were not maximally cytotoxic as determined by in vitro assays.14 Subsequent studies tested infusion of IL-2–activated NK-cell–enriched populations or intravenous IL-2 infusions combined with subcutaneous IL-2. Although these approaches augmented in vivo NK-cell function, no consistent efficacy of autologous NK-cell therapy could be detected in cancer patients when compared with cohorts of matched controls.15

We then hypothesized that autologous NK cells may be suppressed by the physiologic response resulting from NK-cell recognition of “self” MHC molecules. This notion is supported by recent data from haploidentical T-cell–depleted transplantation studies. KIR mismatch with tumor MHC (ie, KIR ligand) may lead to greater tumor kill. In these studies, Ruggeri et al16 showed that stratifying patients by their KIR ligand mismatch would select for patients with alloreactive NK cells that protect against acute myeloid leukemia (AML) relapse. Although virtually untested in solid tumors, these clinical data strongly support a therapeutic role for allogeneic NK cells in myeloid leukemia.17 We present data on the biologic effects of haploidentical NK-cell infusions administered to cancer patients as cell-based immunotherapy with the goal of demonstrating a feasible and safe method that permits in vivo donor NK-cell expansion.

………

 

In this study, we demonstrate that adoptively transferred human NK cells derived from haploidentical related donors can be expanded in vivo. Of interest, in vivo NK-cell expansion occurs after preparation with a high dose (Hi-Cy/Flu) but not lower doses of immunosuppression (Lo-Cy/mPred or Flu). Successful lymphocyte adoptive transfer following intensive immunosuppresion is not surprising. Lymphopenia may change the competitive balance between transferred lymphocytes and endogenous lymphocytes. Alternatively, lymphopenia may induce survival factors or deplete cellular or soluble inhibitory factors.25,26 In murine studies, preparative regimens sufficient to induce lymphopenia allowed homeostatic T-cell expansion in vivo that potentiated effective antitumor immunity.27 This concept has been tested in human T-cell clinical trials by Rosenberg’s group.28 T-cell lymphopenia was induced by Hi-Cy/Flu, similar to what was used here. Successful adoptive transfer and expansion of NK cells may also require intense immunosuppression. Prlic et al20 showed that mature NK cells proliferated only in an NK-cell–deficient host where the endogenous NK-cell pool was absent.

We also demonstrate that NK-cell adoptive therapy is associated with a striking rise in endogenous IL-15 levels, reminiscent of the role IL-7 plays in CD4 T-cell homeostasis.29 IL-15 is required for the final steps of in vitro NK-cell differentiation from CD34 progenitors.22-24 Cooper et al21 was the first to show that IL-15 was absolutely required for in vivo expansion and survival of NK cells, in mice, in part through bcl-2 expression. Transfer of NK cells into IL-15/ hosts resulted in loss of NK cells by 4 days after transfer. IL-15 receptor alpha knockout mice generate IL-15 but do not have NK cells and are unable to undergo successful adoptive transfer. This implies that IL-15 responsiveness by cells other than NK cells may be important in driving this response. IL-15 transgenic mice markedly expand their NK cells and CD8 T cells, ultimately resulting in an NK/T-lymphocytic leukemia.30 The endogenous origin of IL-15 in our patients was unclear. Our data support the notion that IL-15 levels increased only after an intensive lymphocyte-depleting preparative regimen as demonstrated by the inverse correlation between IL-15 concentrations and the absolute lymphocyte count. This does not exclude the possibility that IL-15 may be produced following chemotherapy-induced damage to gastrointestinal mucosa or other cells of epithelial origin.31-34 The effects of exogenous IL-2 administration in these patients needs to be explored as it does add toxicity to the regimen. Further clinical testing may demonstrate that expansion will occur in the presence of IL-15 alone.

Donor NK-cell infusions were feasible and tolerated without unexpected toxicity except for the umbilical cord blood transplantation patient who developed EBV reactivation after treatment. The risk of posttransplantation lymphoproliferative disease approached 10% when HCT is performed using a T-cell–depleted and mismatched graft.35 Although a single event, this finding is important to understand the possible consequences of allogeneic NK-cell therapy in heavily pretreated immunosuppressed patients. It also emphasizes that the CD3- depleted final product, enriched for NK cells but containing B cells, may need further purification to lessen the possibility of this complication. Clinical ex vivo selection methods to address this issue using CD3 depletion followed by CD56 selection are now in place36 and will be tested. We have previously shown that monocytes serve as accessory cells for NK-cell expansion in vitro18 but the role of accessory cells in vivo, if any, is unknown. We need to verify that removal of monocytes and B cells does not change the in vivo expansion potential of NK cells seen here before recommending a purified NK-cell product in all future studies.

In summary, this is the first study to demonstrate that adoptively transferred human NK cells can be expanded in vivo. Expansion was dependent on the more intense Hi-Cy/Flu preparative regimen, which induced lymphopenia, and the more potent immunosuppression that was associated with high endogenous concentrations of IL-15, none of which was observed following Lo-Cy/mPred and Flu alone. It is intriguing that this same regimen is the basis for many transplantation regimens and may help explain the robust NK-cell reconstitution seen in that setting. In this study, NKenriched cells were obtained from related haploidentical donors by efficient depletion of CD3 from PBMCs, although contaminating B cells and monocytes remained in the final product. A maximum tolerated dose was not reached and the largest cell dose administered was that obtained during a single lymphapheresis collection. Although tumor response was not a primary goal of this study, 5 of 19 poor-prognosis patients with AML achieved complete remission after haploidentical NK-cell therapy, with a significantly higher complete remission rate when KIR ligand mismatched donors were used, a strategy that predicts NK-cell alloreactivity.16,37 The precise role of the cells versus the high-intensity chemotherapy regimen in responding patients cannot be definitively determined in this current study. However, the benefit of alloreactivity and the preferential expansion of functional NK cells in responding patients is consistent with at least a partial effect from the NK cells. Our data suggest that prospective selection of KIR ligand– mismatched donors is warranted when possible, which will be assessed in subsequent larger clinical trails.

 

The biology of natural killer cells in cancer, infection, and pregnancy.

Miller JS1      Exp Hematol. 2001 Oct;29(10):1157-68.    http://www.ncbi.nlm.nih.gov/pubmed/11602317
OBJECTIVE: NK cells are important cells of the immune system. They are ultimately derived from pluripotent hematopoietic stem cells. NK cell cytotoxicity and other functions are tightly regulated by numerous activating and inhibitory receptors including newly discovered receptors that selectively recognize major histocompatibility complex class I alleles. Based on their defining function of spontaneous cytotoxicity without prior immunization, NK cells have been thought to play a critical role in immune surveillance and cancer therapy. However, new insights into NK cell biology have suggested major roles for NK cells in infection control and uterine function. The purpose of this review is to provide an update on NK cell function, ontogeny, and biology in order to better understand the role of NK cells in health and disease.
DATA SOURCES: In the Medline database, the major subject heading “Natural Killer Cells” was introduced in 1983, identifying 16,848 citations as of December 31, 2000. Since 1986, there have been approximately 1000 citations per year under this subject heading. In this database, 68% of manuscripts are limited to human NK cells; 40% of citations cross with the major sub-heading of cytotoxicity, 40% with cytokines, 36% with neoplasm, 5% with antibody-dependent cellular cytotoxicity, 2.8% with pregnancy, and 1.3% with infection. Of references from the year 2000-2001, 46 were selected to combine with contributions from earlier literature.
CONCLUSIONS: NK cells should no longer be thought of as direct cytotoxic killers alone as they clearly serve a critical role in cytokine production which may be important to control cancer, infection, and fetal implantation. Understanding mechanisms of NK cell functions may lead to novel therapeutic strategies for the treatment of human disease.

NK cell-based immunotherapy for malignant diseases

Min Cheng, Yongyan Chen, Weihua Xiao, Rui Sun and Zhigang Tian
Cellular & Molecular Immunology (2013) 10, 230–252;   published online 22 April 2013     http://dx. doi.org:/10.1038/cmi.2013.10

Natural killer (NK) cells play critical roles in host immunity against cancer. In response, cancers develop mechanisms to escape NK cell attack or induce defective NK cells. Current NK cell-based cancer immunotherapy aims to overcome NK cell paralysis using several approaches. One approach uses expanded allogeneic NK cells, which are not inhibited by self histocompatibility antigens like autologous NK cells, for adoptive cellular immunotherapy. Another adoptive transfer approach uses stable allogeneic NK cell lines, which is more practical for quality control and large-scale production. A third approach is genetic modification of fresh NK cells or NK cell lines to highly express cytokines, Fc receptors and/or chimeric tumor-antigen receptors. Therapeutic NK cells can be derived from various sources, including peripheral or cord blood cells, stem cells or even induced pluripotent stem cells (iPSCs), and a variety of stimulators can be used for large-scale production in laboratories or good manufacturing practice (GMP) facilities, including soluble growth factors, immobilized molecules or antibodies, and other cellular activators. A list of NK cell therapies to treat several types of cancer in clinical trials is reviewed here. Several different approaches to NK-based immunotherapy, such as tissue-specific NK cells, killer receptor-oriented NK cells and chemically treated NK cells, are discussed. A few new techniques or strategies to monitor NK cell therapy by non-invasive imaging, predetermine the efficiency of NK cell therapy byin vivo experiments and evaluate NK cell therapy approaches in clinical trials are also introduced.

Surgery, chemotherapeutic agents and ionizing radiation have been used for decades as primary strategies to eliminate the tumors in patients; however, the development of resistance to drugs or radiation led to a significant incidence of tumor relapse. Therefore, investigating effective strategies to eliminate these resistant tumor cells is urgently needed. The importance of immune system in malignant diseases has been demonstrated by recent major scientific advances.

Both innate and adaptive immune cells actively prevent neoplastic development in a process called ‘cancer immunosurveillance’. Innate immune cells, including monocytes, macrophages, dendritic cells (DCs) and natural killer (NK) cells, mediate immediate, short-lived responses by releasing cytokines that directly lyse tumor cells or capture debris from dead tumor cells. Adaptive immune cells, including T and B cells, mediate long-lived, antigen-specific responses and effective memory.1 Despite these immune responses, malignant cells can develop mechanisms to evade immunosurveillance. Some tumors protect themselves by establishing an immune-privileged environment. For example, they can produce immunosuppressive cytokines IL-10 and transforming growth factor-β (TGF-β) to suppress the adaptive antitumor immune response, or skew the immune response toward a Th2 response with significantly less antitumor capacity.2,3,4 Some tumors alter their expressions of IL-6, IL-10, vascular epithelial growth factor or granulocyte monocyte-colony stimulating factor (GM-CSF), impairing DC functions via inactivation or suppressing maturation.5 In some cases, induced regulatory T cells suppress tumor-specific CD4+ and CD8+ T-cell responses.6 Tumor cells also minimally express or shed tumor-associated antigens, shed the ligands of NK cell-activating receptor such as the NKG2D ligands UL16-binding protein 2, major histocompatibility complex (MHC) class I chain-related molecules A and B molecules (MICA/MICB) or alter MHC-I and costimulatory molecule expression to evade the immune responses.7,8,9 Malignant cells may also actively eliminate immune cells by activation-induced cell death or Fas ligand (FasL) expression.10,11 In addition, primary cancer treatments like chemotherapy and ionizing radiation can compromise antitumor immune responses by their immunosuppressive side effects.

Tumor cells can be eliminated when immune responses are adequate; when they are not, tumor growth and immunourveillance enter into a dynamic balance until tumor cells evade immunosurveillance, at which point neoplasms appear clinically as a consequence. Therapies designed to induce either a potent passive or active antitumor response against malignancies by harnessing the power of the immune system, known as tumor immunotherapy, is an appealing alternative strategy to control tumor growth. Until now, the cancer immunotherapy field has covered a vast array of therapeutic agents, including cytokines, monoclonal antibodies, vaccines, adoptive cell transfers (T, NK and NKT) and Toll-like receptor (TLR) agonists.1,12,13 Adoptive NK cell transfer in particular has held great promise for over three decades. With progress in the NK cell biology field and in understanding NK function, developing NK cells to be a powerful cancer immunotherapy tool has been achieved in recent years. In this article, we will review recent advances in NK cell-based cancer immunotherapy, focusing on potential approaches and large-scale NK cell expansion for clinical practice, as well as on the clinical trials and future perspectives to enhance the efficacy of NK cells.

NK cells were first identified in 1975 as a unique lymphocyte subset that are larger in size than T and B lymphocytes and contain distinctive cytoplasmic granules.14,15 After more than 30 years, our understanding of NK cell biology and function lends important insights into their role in immunosurveillance. It has been known that NK cells develop in bone marrow (BM) from common lymphoid progenitor cells;16 however, NK cell precursors have still not been clearly characterized in humans.17 After development, NK cells distribute widely throughout lymphoid and non-lymphoid tissues, including BM, lymph nodes (LN), spleen, peripheral blood, lung and liver.18

NK cells, defined as CD3−CD56+ lymphocytes, are distinguished as CD56bright and CD56dim subsets. Approximately 90% of peripheral blood and spleen NK cells belong to the CD56dimCD16+ subset with marked cytotoxic function upon interacting with target cells.19,20In contrast, most NK cells in lymph nodes and tonsils belong to the CD56brightCD16− subset and exhibit predominantly immune regulation properties by producing cytokines such as interferon (IFN)-γ in response to IL-12, IL-15 and IL-18 stimulation.19,21

NK cells rapidly kill certain target cells without prior immunization or MHC restriction, whose activation is dependent on the balance between inhibitory and activating signals from invariant receptors.22,23,24 The activating receptors include the cytotoxicity receptors (NCRs) (NKp46, NKp30 and NKp44), C-type lectin receptors (CD94/NKG2C, NKG2D, NKG2E/H and NKG2F) and killer cell immunoglobulin-like receptors (KIRs) (KIR-2DS and KIR-3DS), while the inhibitory receptors include C-type lectin receptors (CD94/NKG2A/B) and KIRs (KIR-2DL and KIR-3DL). Since some structural families contain both activating and inhibitory receptors, trying to understand how NK cell activity is regulated is often complicated.25 At steady state, the inhibitory receptors (KIRs and CD94/NKG2A/B), which bind to various MHC-I molecules present on almost all cell types, inhibit NK cell activation and prevent NK cell-mediated killing. Under stress conditions, cells downregulate MHC-I expression, causing NK cells to lose inhibitory signaling and be activated in a process called ‘missing-self recognition’. Additionally, the non-MHC self molecules Clr-b (mouse), LLT-1 (human) and CD48 (mouse) recognized by the inhibitory receptors NKR-P1B, NKR-P1A and 2B4, respectively, also perform this function.26,27 In contrast to the self-expressed inhibitory receptor ligands, NK cell-activating receptors can recognize either pathogen-encoded molecules that are not expressed by the host, called ‘non-self recognition’, or self-expressed proteins that are upregulated by transformed or infected cells, called ‘stress-induced self recognition’. For example, mouse Ly49H recognizes cytomegalovirus-encoded m157, and NKG2D recognizes the self proteins human UL16-binding proteins and MICA/MICB.28,29 NK cells identify their targets by recognizing a set of receptors on target cells in an NK-target cell zipper formation; this results in the integration of multiple activating and inhibitory signals, the outcome of which depends on the nature of the interacting cells.26IFNs or DC/macrophage-derived cytokines, such as type I IFN, IL-12, IL-18 and IL-15, enhance the activation or promote the maturation of NK cells, which can also augment NK cell cytolytic activity against tumor cells.30,31,32 Cytotoxic activity of NK cells can increase approximately 20–200 fold after exposure to IFN-α/β or IL-12. Despite these known innate immune cell functions, accumulating evidence in both mice and humans demonstrates that NK cells are educated and selected during development, possess receptors with antigen specificity, undergo clonal expansion during infection and can generate long-lived memory cells.33,34

After over 30 years of researching NK cells, evidence supports that they play critical roles in the early control of viral infection, in hematopoietic stem cell (HSC) transplantation (improved grafting, graft-vs.-host disease and graft-vs.-tumor), in tumor immunosurveillance and in reproduction (uterine spiral artery remodeling). The roles of NK cells in controlling organ transplantation, parasitic and HIV infections, autoimmunity and asthma have also been suggested, but remain to be explored further.26 In particular, therapeutic strategies harnessing the power of NK cells to target multiple malignancies have been designed.

NK cells originally described as large granular lymphocytes, exhibited natural cytotoxicity against certain tumor cells in the absence of preimmunization or stimulation.35,36,37 CD56dim NK cells, which make up the majority of circulating cells, are the most potent cytotoxic NK cells against tumor cells. Evidence gathered from a mouse xenograft tumor model testing functionally deficient NK cells or antibody-mediated NK cell depletion supports that NK cells can eradicate tumor cells.38,39,40,41 An 11-year follow-up study in patients indicated that low NK-like cytotoxicity was associated with increased cancer risk.42 High levels of tumor infiltrating NK cells (TINKs) are associated with a favorable tumor outcome in patients with colorectal carcinoma, gastric carcinoma and squamous cell lung cancer, suggesting that NK-cell infiltration into tumor tissues represents a positive prognostic marker.43,44,45 As described above, NK-cell recognition of tumor cells by inhibitory and activating receptors is complex, and the three recognition models—‘missing-self’, ‘non-self’ and ‘stress-induced self’—might be used to sense missing- or altered-self cells. Activated NK cells are thus in a position to directly or indirectly exert their antitumor activity to control tumor growth and prevent the rapid dissemination of metastatic tumors by ‘immunosurveillance’ mechanisms (Figure 1).

Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Figure 1.

NK cells in tumor immunosurveillance. The diagram shows the potential roles of NK cells in tumor immunosurveillance. NK cells initially recognize the tumor cells via stress or danger signals. Activated NK cells directly kill target tumor cells through at least four mechanisms: cytoplasmic granule release, death receptor-induced apoptosis, effector molecule production or ADCC. Additionally, NK cells act as regulatory cells when reciprocally interact with DCs to improve their antigen uptake and presentation, facilitating the generation of antigen-specific CTL responses. Also, by producing cytokines such as IFN-γ, activated NK cells induce CD8+ T cells to become CTLs. Activated NK cells can also promote differentiation of CD4+ T cells toward a Th1 response and promote CTL differentiation. Cytokines produced by NK cells might also regulate antitumor Ab production by B cells. Ab, antibody; ADCC, antibody-dependent cellular cytotoxicity; CTL, cytotoxic T lymphocyte; DC, dendritic cell; IFN, interferon; NK, natural killer.

Full figure and legend (96K)

Direct tumor clearance by NK-mediated cytotoxicity

Upon cellular transformation, surface MHC-I expression on tumor cells is often reduced or lost to evade recognition by antitumor T cells. In parallel, cellular stress and DNA damage lead to upregulated expression of ligands on tumor cells for NK cell-activating receptors. Human tumor cells that have lost self MHC-I expression or bear ‘altered-self’ stress-inducible proteins are ideal NK cell targets, as NK cells are activated by initially recognizing certain ‘stress’ or ‘danger’ signals.46 The ‘missing-self’ model of tumor cell recognition by NK cells was first demonstrated by observing that MHC-I-deficient syngeneic tumor cells were selectively rejected by NK cells; additionally, NK cell inhibitory receptors were shown to detect this absence of MHC-I expression.47,48,49 NK cells can also kill certain MHC-I-sufficient tumor cells by detecting stress-induced self ligands through their activating receptors. Broad MICA/B expression has been detected on epithelial tumors, melanoma, hepatic carcinoma and some hematopoetic malignancies, representing a counter-measure by the immune system to combat tumor development.31 NK cell-mediated cytotoxicity is also important against tumor initiation and metastasis in vivo.50,51,52

NK cells directly kill target tumor cells through several mechanisms: (i) by releasing cytoplasmic granules containing perforin and granzymes that leads to tumor-cell apoptosis by caspase-dependent and -independent pathways.53,54 Cytotoxic granules reorient towards the tumor cell soon after NK–tumor cell interaction and are released into the intercellular space in a calcium-dependent manner; granzymes are allowed entry into tumor cells by perforin-induced membrane perforations, leading to apoptosis; (ii) by death receptor-mediated apoptosis. Some NK cells express tumor-necrosis factor (TNF) family members, such as FasL or TNF-related apoptosis-inducing ligand (TRAIL), which can induce tumor-cell apoptosis by interacting with their respective receptors, Fas and TRAIL receptor (TRAILR), on tumor cells.55,56,57,58,59 TNF-α produced by activated NK cells can also induce tumor-cell apoptosis;60 (iii) by secreting various effector molecules, such as IFN-γ, that exert antitumor functions in various ways, including restricting tumor angiogenesis and stimulating adaptive immunity.61,62 Cytokine activation or exposure to tumor cells is also associated with nitric oxide (NO) production, where NK cells kill target tumor cells by NO signaling;63,64 (iv) through antibody-dependent cellular cytotoxicity (ADCC) by expressing CD16 to destroy tumor cells.40 The antitumor activity of NK cells can be further enhanced by cytokine stimulation, such as by IL-2, IL-12, IL-18, IL-15 or those that induce IFN production.40,65,66,67,68,69,70

Indirect NK-mediated antitumor immunity

NK cells act as regulatory cells when reciprocally interact with DCs, macrophages, T cells and endothelial cells by producing various cytokines (IFN-γ, TNF-α and IL-10), as well as chemokines and growth factors.26,71 By producing IFN-γ, activated NK cells induce CD8+ T cells to become cytotoxic T lymphocytes (CTLs), and also help to differentiate CD4+ T cells toward a Th1 response to promote CTL differentiation.72,73 NK cell-derived cytokines might also regulate antitumor antibody (Ab) production by B cells.40 In addition, cancer cells killed by NK cells could provide tumor antigens for DCs, inducing them to mature and present antigen.74By lysing surrounding DCs that have phagocytosed and processed foreign antigens, activated NK cells also could provide additional antigenic cellular debris for other DCs. Thus, activated NK cells promote antitumor immunity by regulating DC activation and maturation,75 as these DCs can facilitate the generation of antigen-specific CTL responses through their ability to cross-present tumor-specific antigens (derived from NK cell-mediated tumor lysis) to CD8+ T cells.76,77

During tumor progression, tumor cells develop several mechanisms to either escape from NK-cell recognition and attack or to induce defective NK cells. These include losing expression of adhesion molecules, costimulatory ligands or ligands for activating receptors, upregulating MHC class I, soluble MIC, FasL or NO expression, secreting immunosuppressive factors such as IL-10, TGF-β and indoleam ine 2,3-d ioxygense (IDO) and resisting Fas- or perforin-mediated apoptosis.31,78,79 In cancer patients, NK cell abnormalities have been observed, including decreased cytotoxicity, defective expression of activating receptors or intracellular signaling molecules, overexpression of inhibitory receptors, defective proliferation, decreased numbers in peripheral blood and in tumor infiltrate, and defective cytokine production.60Given that NK cells play critical roles in the first-line of defense against malignancies by direct and indirect mechanisms, the therapeutic use of NK cells in human cancer immunotherapy has been proposed and followed in a clinical context (Table 1).

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For NK cell immunotherapy, obtaining a sufficient number of functional NK cells is critical in clinical protocols. Therefore, the number, purity and state of NK cell proliferation and activation are considered as the key factors.151 In Table 2, the purification/expansion of clinical-grade NK cells developed in recent years is summarized. They can be produced from cord blood, bone marrow, peripheral blood and embryonic stem cells. Overall, the summarized methods suggest that long-term ex vivoexpansion of NK cells may present a clinical benefit, but not the short-term activation which is not sufficient for augmenting the functions of NK cells.152

Table 2 – Expansion of NK cells in vitro for clinical practice*.Full table

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Results from treating hematological malignancies demonstrated a critical role for NK cells in clinical immunotherapy, as alloreactive NK cells highlighted the graft-vs.-leukemia effect in AML patients.172 The graft-vs.-tumor effect of alloreactive NK cells was also strengthened by mismatched IL-2-activated lymphocytes in patients with solid tumors or hematological malignancies.173 As discussed above, autologous NK cells, allogeneic NK cells, NK cell lines and genetically modified NK cells were investigated for effectiveness as tumor immunotherapies. The clinical study designs evaluating the efficacy of these various NK cell-mediated tumor therapies are summarized in Table 3.

Table 3 – Clinical trials of tumor immunotherapy by using NK cells.Full table

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NK cell-based immunotherapy holds great promise for cancer treatment. However, only modest clinical success has been achieved thus far using NK cell-based therapies in cancer patients. Progress in the field of understanding NK cell biology and function is therefore needed to assist in developing novel approaches to effectively manipulate NK cells for the ultimate benefit of treating cancer patients.

 

Present and Future of Allogeneic Natural Killer Cell Therapy

Okjae Lim,1 Mi Young Jung,1 Yu Kyeong Hwang,2 and Eui-Cheol Shin3,*
Front Immunol. 2015; 6: 286.  Published online 2015 Jun 3.    doi:  10.3389/fimmu.2015.00286

Natural killer (NK) cells are innate lymphocytes that are capable of eliminating tumor cells and are therefore used for cancer therapy. Although many early investigators used autologous NK cells, including lymphokine-activated killer cells, the clinical efficacies were not satisfactory. Meanwhile, human leukocyte antigen (HLA)-haploidentical hematopoietic stem cell transplantation revealed the antitumor effect of allogeneic NK cells, and HLA-haploidentical, killer cell immunoglobulin-like receptor ligand-mismatched allogeneic NK cells are currently used for many protocols requiring NK cells. Moreover, allogeneic NK cells from non-HLA-related healthy donors have been recently used in cancer therapy. The use of allogeneic NK cells from non-HLA-related healthy donors allows the selection of donor NK cells with higher flexibility and to prepare expanded, cryopreserved NK cells for instant administration without delay for ex vivo expansion. In cancer therapy with allogeneic NK cells, optimal matching of donors and recipients is important to maximize the efficacy of the therapy. In this review, we summarize the present state of allogeneic NK cell therapy and its future directions.

Cancer is a major threat for humans worldwide, with approximately 14 million new cases and 8.2 million cancer-related deaths in 2012 (1). Although most common cancer treatments include surgery, chemotherapy, and radiotherapy, unsatisfactory cure rates require new therapeutic approaches, especially for refractory cancers. For this purpose, cancer immunotherapies with various cytokines, antibodies, and immune cells have been clinically applied to patients to encourage their own immune system to help fight the cancer (2).

Adoptive cellular immunotherapies have employed several types of immune cells, including dendritic cells (DCs), cytotoxic T lymphocytes (CTLs), lymphokine-activated killer (LAK) cells, cytokine-induced killer (CIK) cells, and natural killer (NK) cells. Although there has been recent progress in DC therapy and CTL therapy, clinical applications are somewhat limited because cancer antigens must first be characterized and autologous cells must be used. By contrast, LAK cells, CIK cells, and NK cells have antigen-independent cytolytic activity against tumor cells. In particular, NK cells can be used from not only autologous sources but also allogeneic sources and, recently, allogeneic NK cells have been employed more often in cancer treatment. Whereas autologous NK cells from cancer patients may have functional defects (3), allogeneic NK cells from healthy donors have normal function and can be safely administered to cancer patients (4). Allogeneic NK cell therapy is particularly beneficial because it can enhance the anti-cancer efficacy of NK cells via donor–recipient incompatibility in terms of killer cell immunoglobulin-like receptors (KIRs) on donor NK cells and major histocompatibility complex (MHC) class I on recipient tissues.

Natural killer cells are innate lymphocytes that provide a first line of defense against viral infections and cancer (5). Human NK cells are recognized as CD3−CD56+ lymphocytes. They can be further subdivided into two subsets based on the surface expression level of CD56. The CD56dim population with low-density expression of CD56 comprises approximately 90% of human blood NK cells and has a potent cytotoxic function, whereas the CD56bright population (approximately 10% of blood NK cells) with high-density expression of CD56 displays a potent cytokine producing capacity and has immunoregulatory functions (6). The CD56dim NK cell subset also expresses high levels of the Fc receptor for IgG (FcγRIII, CD16), which allows them to mediate antibody-dependent cellular cytotoxicity (ADCC) (7). NK cells comprise 5–15% of circulating lymphocytes and are also found in peripheral tissues, including the liver, peritoneal cavity, and placenta. Activated NK cells are capable of extravasation and infiltration into tissues that contain pathogens or malignant cells while resting NK cells circulate in the blood (8).

The NK cell activity is regulated by signals from activating and inhibitory receptors (9, 10). The activating signal is mediated by several NK receptors including NKG2D and natural cytotoxicity receptors (NCRs) (9–11). By contrast, NK cell activity is suppressed by inhibitory receptors, including KIRs, which bind to human leukocyte antigen (HLA) class I molecules on target cells (9, 10, 12). NKG2A is also an important inhibitory receptor binding to non-classical HLA molecule, HLA-E (13). If target cells lose or downregulate HLA expression (14), the NK inhibitory signal is abrogated, allowing NK cells to become activated and kill malignant targets. However, NK cell function is impaired in cancer patients by various mechanisms, particularly in tumor microenvironment (15).

Although NK cell activity is determined by the summation of signals from activating and inhibitory receptors, the inhibitory signal through KIRs is a main regulator of NK cell function particularly in allogeneic settings. Inhibitory KIRs have long cytoplasmic tails containing two immunoreceptor tyrosine-based inhibition motifs (ITIMs). Each KIR has its cognate ligand and consists of two (KIR2DL) or three (KIR3DL) extracellular Ig-domains. KIR2DL1 and KIR2DL2/3 recognize group 2 HLA-C (called C2, Lys80) and group 1 HLA-C (called C1, Asn80), respectively. KIR3DL1 recognizes HLA-Bw4 (16). The KIR repertoire on human NK cells is randomly determined and independent of the number and allotype of HLA class I ligands (17).

The antitumor activity of allogeneic NK cells has been demonstrated in the setting of hematopoietic stem cell transplantation (HSCT). Allogeneic HSCT is an established curative treatment for hematologic malignancies. In allogeneic HSCT, donor T cells contribute to graft-versus-host disease (GVHD) and graft-versus-tumor (GVT) effects (18). In T cell-depleted HSCT, however, donor NK cells are the major effector cells responsible for controlling residual cancer cells before T cell reconstitution (19, 20).

Natural killer cells are the first lymphoid population to recover after allogeneic HSCT. In the first month of transplantation, reconstituted NK cells represent the predominant lymphoid cells and play a crucial role in controlling the host immune system. Allogeneic NK cells prevent viral infections and restrain residual cancer cells in the early phase of transplantation (21). Of note, the GVT activity of donor NK cells is significantly improved when KIRs of donor and HLA class I of the recipient are incompatible, and consequently when inhibitory signals are absent, as observed in HLA-haploidentical HSCT (22). Therefore, increased GVT activity of NK cells with KIR-HLA incompatibility is the underlying rationale for the development of allogeneic NK cell therapy.

Following the discovery of inhibitory KIRs and the understanding that they play a role in preventing NK cell killing of self MHC class I-expressing tumor cells, investigators began to research the possibility of using allogeneic donor NK cells instead of autologous NK cells for cancer therapy. Several groups have infused activated, expanded donor NK cells to patients early after allogeneic HSCT to provide antitumor effects (23). In Table Table1,1, clinical trials with allogeneic NK cells as therapeutics are summarized.

Table 1   Selected clinical trials with expanded allogeneic NK cells
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As summarized in Table Table2,2, two clinical trials are investigating the use of CAR-expressing allogeneic NK cells. The aim of both studies is to assess the safety, feasibility, and efficacy of expanded, activated, and CD19-redirected haploidentical NK cells in ALL patients who have persistent disease after intensive chemotherapy or HSCT (NCT00995137, NCT01974479). Further, other tumor antigens, such as CS1, CEA, CD138, and CD33, are targeted by CARs expressed by NK cells, although NK-92, YT, or NKL cell lines were used (48–51).
Table 2  Genetically modified, expanded allogeneic NK cells.
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Therapeutic regimens

In allogeneic NK cell therapy, optimal therapeutic regimens for clinical applications should be considered because adoptively transferred NK cells not only target tumor cells but also interact with the immunological environment. To potentiate the therapeutic efficacy of allogeneic NK cells, proper strategies, including pre-conditioning or combination therapy, could be applied (34).

Upregulation of NKG2D ligands by spironolactone (63) or histone deacetylase inhibitors (64, 65) and upregulation of TRAIL-R2 by doxorubicin (66) result in enhanced antitumor efficacy of NK cells. Proteasome inhibitors also sensitize tumor cells to NK cell-mediated killing via TRAIL and FasL pathways. In addition, c-kit tyrosine kinase inhibitor (67) and JAK inhibitors (68) increase the susceptibility of tumor cells to NK cytotoxicity and enhance antitumor responses by increased IFN-γ production from NK cells. However, protein kinase inhibitors should be used cautiously because some protein kinase inhibitors, such as sorafenib, inhibit the effector function of NK cells (69).

Immunomodulatory drugs can augment NK cell function. Lenalidomide enhances rituximab-induced killing of non-Hodgkin’s lymphoma and B-cell chronic lymphocytic leukemia through NK cell and monocyte-mediated ADCC mechanisms (70). Combination therapy using IL-2 and anti-CD25 shows anti-leukemic effects by depletion of regulatory T cells in addition to activation and expansion of NK cells (71). Alloferon, an immunomodulatory peptide, enhances the expression of NK-activating receptor 2B4 and granule exocytosis from NK cells against cancer cells (72).

Therapeutic antibodies can be combined with allogeneic NK cell therapy (73). Antibodies against tumor antigens (e.g., CD20 and CS1) can induce ADCC of NK cells (74, 75). Antibodies to activating NK receptors (e.g., 4-1BB, GITR, NKG2D, DNAM-1, and NCRs) can enhance NK activation (74, 76–79). In addition, inhibitory receptors (e.g., KIR2DL, PD-1, PD-L1, and NKG2A) can be blocked by antibodies (80–85). Bispecific and trispecific killer cell engagers directly activate NK cells through CD16 signaling and thus, induce cytotoxicity and cytokine production against tumor targets (86, 87).

Conclusion

Antitumor activity of allogeneic NK cells was first observed in a setting of HLA-haploidentical HSCT. Allogeneic NK cell therapy was tried mostly using HLA-haploidentical NK cells with or without allogeneic HSCT and, recently, allogeneic NK cells from unrelated, random donors have been used in a non-HSCT setting. The efficacy of allogeneic NK cell therapy can be enhanced by optimal donor selection in terms of the KIR genotype of donors and donor KIR-recipient MHC incompatibility. Furthermore, efficacy can be increased by genetic modification of NK cells and optimized therapeutic regimens. In the future, allogeneic NK cell therapy can be an effective therapeutic modality for cancer.

δγ T cells for immune therapy of patients with lymphoid malignancies

http://dx.doi.org:/10.1182/blood-2002-12-3665                      Prepublished online  Blood March 6, 2003; 2003 102: 200-206
Martin Wilhelm, Volker Kunzmann, Susanne Eckstein, Peter Reimer, Florian Weissinger, Thomas Ruediger and Hans-Peter Tony

There is increasing evidence that gammadelta T cells have potent innate antitumor activity. We described previously that synthetic aminobisphosphonates are potent gammadelta T cell stimulatory compounds that induce cytokine secretion (ie, interferon gamma [IFN-gamma]) and cell-mediated cytotoxicity against lymphoma and myeloma cell lines in vitro. To evaluate the antitumor activity of gammadelta T cells in vivo, we initiated a pilot study of low-dose interleukin 2 (IL-2) in combination with pamidronate in 19 patients with relapsed/refractory low-grade non-Hodgkin lymphoma (NHL) or multiple myeloma (MM). The objectives of this trial were to determine toxicity, the most effective dose for in vivo activation/proliferation of gammadelta T cells, and antilymphoma efficacy of the combination of pamidronate and IL-2. The first 10 patients (cohort A) who entered the study received 90 mg pamidronate intravenously on day 1 followed by increasing dose levels of continuous 24-hour intravenous (IV) infusions of IL-2 (0.25 to 3 x 106 IU/m2) from day 3 to day 8. Even at the highest IL-2 dose level in vivo, gammadelta T-cell activation/proliferation and response to treatment were disappointing with only 1 patient achieving stable disease. Therefore, the next 9 patients were selected by positive in vitro proliferation of gammadelta T cells in response to pamidronate/IL-2 and received a modified treatment schedule (6-hour bolus IV IL-2 infusions from day 1-6). In this patient group (cohort B), significant in vivo activation/proliferation of gammadelta T cells was observed in 5 patients (55%), and objective responses (PR) were achieved in 3 patients (33%). Only patients with significant in vivo proliferation of gammadelta T cells responded to treatment, indicating that gammadelta T cells might contribute to this antilymphoma effect. Overall, administration of pamidronate and low-dose IL-2 was well tolerated. In conclusion, this clinical trial demonstrates, for the first time, that gammadelta T-cell-mediated immunotherapy is feasible and can induce objective tumor responses.

Despite significant improvement in the treatment of low-grade non-Hodgkin lymphoma (NHL) and multiple myeloma (MM), most patients relapse or become resistant to conventional treatment strategies such as chemotherapy or radiation. Therefore, there is need for alternative tumor therapies. One possibility is manipulating the immune system to target and eliminate neoplastic cells. Most current immunotherapeutic approaches aim at inducing antitumor response via stimulation of the adaptive immune system, which is dependent on major histocompatibility complex (MHC)– restricted T cells. Despite major advances in our understanding of the adaptive immunity toward tumors and the introduction of vaccine-based strategies, durable responses are rare, and active immunotherapy is still not an established treatment modality. Adaptive immunotherapeutic approaches have several disadvantages: T cells need specific tumor-associated antigens (TAAs) and appropriate costimulatory molecules for activation. Failure or loss of TAAs, MHC molecules, and/or costimulatory molecules renders tumor cells resistant to T-cell–mediated cytotoxicity or induces anergy of specific T cells.1

Mice deficient in innate effector cells such as natural killer (NK) cells, NK T cells, or T cells show a significantly increased incidence of tumors and provide clear evidence for an immune surveillance function of the innate immune system.2-4 Recognition of transformed cells by the innate immune system seems to be dependent on expression of stress-induced ligands and/or loss of MHC class I molecules on tumor cells.5 Several studies have demonstrated a role for human T cells in recognition of transformed cells.6,7 T cells exhibit a potent MHC-unrestricted lytic activity against different tumor cells in vitro.8-10 In addition, T cells have been found with increased frequency in disease-free survivors of acute leukemia following allogeneic bone marrow transplantation.11 Adoptive transfer of ex vivo–expanded human T cells in a mouse tumor model further supports the in vivo antitumor effects of T cells.12 V9V2 T cells, which represent most of the human circulating T cells, recognize small nonpeptide compounds with an essential phosphate residue (ie, microbial metabolites) or alkylamines.13-17 As we have shown previously, also synthetic aminobisphosphonates such as pamidronate are potent T-cell– stimulatory compounds.18 In addition, we could demonstrate that pamidronate-activated T cells produce cytokines (ie, interferon [IFN-]), exhibit specific cytotoxicity against lymphoma or myeloma cell lines, and lead to reduced survival of autologous myeloma cells.8

The aim of this pilot study is to evaluate the feasibility of activation and/or expansion of T cells in vivo using the combination of pamidronate and interleukin 2 (IL-2) in patients with refractory/relapsed lymphoma or myeloma, to determine the most effective IL-2 dose, to assess the toxicity of this regimen, and to evaluate its ability to exert antitumor effects.   …..

There has been no study published so far on in vivo stimulation of T cells in humans, and the consequences of a selective activation of T cells in vivo were not known. Therefore, evaluation of toxicity was one major end point of this study. We started with a low IL-2 dose of 0.25 106 IU IL-2/m2 and subsequently increased the IL-2 dose to 3 106 IU IL-2/m2 in cohort A and to 2 106 IU IL-2/m2 in cohort B. Overall, the combination of pamidronate and IL-2 was well tolerated, and no dose-limiting toxicity was observed. Most of the patients developed self-limiting fever and thrombophlebitis at the infusion site. Local thrombophlebitis has been described as a rare side effect in
patients receiving pamidronate alone.20,21 The high frequency of local thrombophlebitis in patients receiving pamidronate in combination with IL-2 might reflect immune-mediated effects on endothelial cells. It has also been recently shown that aminobisphosphonates have dose-dependent effects on proliferation-inhibition and apoptosis-induction of human endothelial cells in vitro.22

Next we asked whether the combination of pamidronate and IL-2 induces activation and proliferation of T cells in vivo. None of the first 10 patients included in this pilot study (cohort A, Table 1) developed a measurable T-cell response in vivo. The inability to induce T-cell proliferative response in vivo correlated with the negative in vitro proliferation of T cells in response to pamidronate/IL-2 in 4 of 5 analyzable patients. Therefore, extensive prior in vitro testing was initiated for all further eligible patients. Using this strategy, we found that a much lower proportion of patients with hematologic malignancies showed positive in vitro proliferation of T cells in response to pamidronate/IL-2 compared with a control group of healthy donors (49% versus 88%). Although the exact mechanisms of this defect are currently under investigation, a severe immunodeficiency caused by extensive prior chemotherapy in these relapsed/ refractory patients and/or the underlying disease itself may account for this observation. Indeed, the type of underlying disease seems to influence the in vitro proliferative response to pamidronate/IL-2 (Table 2). The failure of patients with B-CLL to develop a measurable T-cell proliferative response may be a result of the very small number of T cells in peripheral blood, which were often below the detection limit in our series. However, a larger number of patients with distinct disease entities and at different disease stages (eg, untreated versus treated) need to be evaluated to support this observation and to identify additional clinical parameters influencing T-cell reactivity. Furthermore, extensive prior in vitro testing in eligible patients revealed that T-cell proliferation in response to pamidronate can be significantly enhanced by concomitant addition of IL-2 to PBMC cultures on day 1 instead of day 3 (as previously done).

Thus, for all further patients the treatment schedule was changed (concomitant administration of IL-2 on day 1), and only patients with significant in vitro proliferation of T cells in the presence of pamidronate and IL-2 were included (cohort B, Table 1). After these modifications, significant in vivo expansion of T cells could be observed in 5 of 9 patients (55%) (Table 1). In vivo proliferation of T cells was associated with a robust up-regulation of early (CD69) and late (HLA-DR) activation markers, whereas pamidronate and IL-2 failed to induce comparable effects on T cells and NK cells (Table 3). These data support in vitro findings that the action of pamidronate is highly specific and, except for V9V2 T cells, it does not activate other immune effector cells.8,23,24 However, at higher IL-2 doses unspecific stimulation effects of IL-2 became more evident because a proportion of patients showed a moderate up-regulation of activation markers on T cells and NK cells at the highest dose level of IL-2 tested in this study. On the basis of the analysis of activation marker expression and proliferation we conclude that 1 106 IU IL-2/m2 IL-2 per day seems to be the most effective dose with respect to specific and effective T-cell stimulation in vivo.

Another aim of our study was to assess the clinical response. None of the 9 analyzable patients of cohortA(Table 1) achieved an objective tumor response. After change of protocol and inclusion criteria (cohort B, Table 1) 3 of 9 patients (33%) achieved an objective tumor response (3 PR). Clinical response could be associated with T-cell proliferation in vivo, because all 4 patients from cohort B without T-cell proliferation in vivo did not experience an objective tumor response, and 4 of 5 patients with T-cell proliferation in vivo responded (3 PR, 1 stable disease [SD]). These results suggest that the observed tumor regression in our patients is dependent on T-cell activation and proliferation. The relevance of this correlation is underlined by the fact that pamidronate-stimulated T cells possess an increased capacity for killing tumor cells in vitro.8,10 It is still open which mechanisms may have been responsible for the clinical responses. Several other antitumor effects have been attributed to aminobisphosphonates. However, at pharmacologically achievable concentrations in vivo, only the specific stimulation of V9V2T cells can be observed.8 Alternatively, the occurrence of clinical remissions may be attributed to an IL-2–mediated effect on other immune effector cells. However, our immunologic monitoring indicates that the combination of pamidronate and low-dose IL-2 does not induce specific activation and expansion of T cells or NK cells compared with the effect on T cells. In addition, the concentrations of IL-2 used here are much lower than the doses required in other immunotherapeutic approaches for these malignancies.25-27

The important question of what precise mechanisms are involved in tumor recognition and eradication by T cells is out of the scope of this study and will require further in vitro and in vivo studies. However, tumor cell recognition by T cells seems to be modulated by a balance of positive and negative signals.28 Although killer inhibitory receptors (KIRs) are obviously involved in the mediation of negative signals, the positive signals are only incompletely understood. One example of such a positive signal is the NKG2D-DAP10 receptor complex, which is known to interact with stress-induced ligands on tumor cells such as MICA and Rae-1.29 The very slow response profiles of most of the patients in our series strongly argue for an indirect influence on lymphoma cells rather than a sole cytotoxic effect. One possible mechanism may be secretion of cytokines, which influence tumor cells or their microenvironment.30 We have already shown that IFN- is the major cytokine secreted by pamidronate-activated T cells.8,31 IFN- has multiple antitumor effects such as direct inhibition of tumor growth, blocking angiogenesis, or stimulation of macrophages.32 Recently, a significant negative correlation between angiogenetic factors (ie, VEGF) and IFN- serum levels was described in patients treated with pamidronate.33 Therefore, IFN- might be one of the key cytokines involved in the T-cell– mediated antitumor response.

In conclusion, this study indicates for the first time that in vivo T-cell stimulation by pamidronate and low-dose IL-2 is a safe and promising immunotherapy approach in the treatment of
patients with low-grade B-NHL and MM. Further studies are necessary to confirm the clinical efficacy of this novel strategy. Our immunologic and clinical monitoring data provide further insight into the capacity of T cells to induce an antitumor immune response. However, this study also reveals that the function of T cells can be impaired in some patients with lymphoid malignancies. Therefore, the results of this study provide principles relevant to the design of future trials, including appropriate prior in vitro testing.

EXPANSION OF HIGHLY CYTOTOXIC HUMAN NATURAL KILLER CELLS FOR CANCER CELL THERAPY

Cancer Res. 2009 May 1; 69(9): 4010–4017.       Published online 2009 Apr 21.    doi:  10.1158/0008-5472.CAN-08-3712

Infusions of natural killer (NK) cells are an emerging tool for cancer immunotherapy. The development of clinically applicable methods to produce large numbers of fully functional NK cells is a critical step to maximize the potential of this approach. We determined the capacity of the leukemia cell line K562 modified to express a membrane-bound form of interleukin-15 and 4-1BB ligand (K562-mb15-41BBL) to generate human NK cells with enhanced cytotoxicity. Seven-day coculture with irradiated K562-mb15-41BBL induced a median 21.6-fold expansion of CD56+CD3– NK cells from peripheral blood (range, 5.1-86.6-fold; n = 50), which was considerably superior to that produced by stimulation with interleukin (IL)-2, IL-12, IL-15 and/or IL-21 and caused no proliferation of CD3+ lymphocytes. Similar expansions could also be obtained from the peripheral blood of patients with acute leukemia undergoing therapy (n = 11). Comparisons of the gene expression profiles of the expanded NK cells and of their unstimulated or IL-2-stimulated counterparts demonstrated marked differences. The expanded NK cells were significantly more potent than unstimulated or IL-2-stimulated NK cells against acute myeloid leukemia (AML) cells in vitro. They could be detected for more than one month when injected into immunodeficient mice and could eradicate leukemia in murine models of AML. We therefore adapted the K562-mb15-41BBL stimulation method to large-scale clinical-grade conditions, generating large numbers of highly cytotoxic NK cells. The results that we report here provide rationale and practical platform for clinical testing of expanded and activated NK cells for cell therapy of cancer.

Natural killer (NK) cells can kill cancer cells in the absence of prior stimulation and hold considerable potential for cell-based therapies targeting human malignancies (1–4). This notion is corroborated by the observation that, among patients with leukemia undergoing hematopoietic stem cell transplantation, the antileukemic effect of the transplant was significantly greater when the donor NK cells exhibited a killer inhibitory receptor (KIR) profile that predicted a higher cytotoxicity against the leukemic cells of the recipient (3;5–7). Moreover, allogeneic NK cells might be beneficial when directly infused into patients, a procedure that was shown to induce clinical remission in patients with high-risk acute myeloid leukemia (AML) (8). Infusions of NK cells have also been proposed as a means to improve the treatment of other cancers (9).

Because NK cells represent a small fraction of peripheral blood mononuclear cells, generating them in numbers sufficient to meet clinical requirements, especially if multiple infusions are planned, is problematic. Hence, NK cell-based therapies would greatly benefit from reliable methods to produce large numbers of fully functional NK cells ex vivo. Unlike T and B lymphocytes, which readily respond to a variety of stimuli, NK cells typically do not undergo sustained proliferation. Indeed, their reported proliferative responses to cytokines with or without coculture with other cells have generally been modest and of short duration in most studies (10–16).

We previously found that the K562 leukemia cell line genetically modified to express membrane-bound interleukin (IL)-15 and 41BB ligand specifically activates NK cells, drives them into the cell cycle and allows their genetic modification (17). In this study, we determined the capacity of NK cells stimulated by contact with K562-mb15-41BBL cells to exert anti-AML cytotoxicity.

…….

We found that K562-mb15-41BBL cells induce sustained and specific proliferation of human NK cells. NK cell expansion was observed in all donors tested, including patients with acute leukemia undergoing therapy, with no apparent proliferative advantage of any particular NK cell subset. Gene expression of NKAES-NK cells was markedly different than that of unstimulated and IL-2-stimulated cells, not only in regards to their expression of cell proliferation-associated genes but also in that of molecules that might regulate NK-cell function and their interaction with other cell types. NKAES-NK cells had powerful cytotoxicity against AML cell lines and AML cells from patients, and were more potent than unstimulated or IL-2-activated NK cells from the same donors. Based on these findings, and on the effectiveness of NKAES-NK cells in murine models of AML, we developed a Master Cell Bank of K562-mb15-41BBL cells under cGMP guidelines, and demonstrated that large-scale expansion and activation of human NK cells for clinical studies was feasible, producing expansions of CD56+CD3– cells that were even higher than those observed in the initial small-scale experiments while maintaining high anti-AML cytotoxicity.

IL-2 can induce proliferative responses in human NK cells but only a minor fraction sustains continued growth (10;26;27). Conceivably, some NK-cell subsets might be more responsive, as suggested by early reports of up to 50-fold expansion after culture with IL-2 for 2 weeks of an NK subset that adheres to plastic (28–31). It is unclear, however, whether some CD3+ cells might have had, at least in part, contributed to the increased cell numbers (29;30). More recently, anti-CD3 and IL-2 reportedly induced 190-fold NK expansions after 21 days from the blood of healthy individuals (32) and, surprisingly, 1600-fold expansions after 20 days from that of patients with myeloma (25). However, these cells’ cytotoxicity against K562 cells was <10% at 1 : 1 E : T (25), a ratio at which NKAES-NK cells from healthy donors or leukemia patients had a median cytotoxicity of 69% cells. Our results with IL-2 alone or in combination with other cytokines are in line with those of earlier reports (10;26;27;33). Indeed, most investigators have indicated that sustained expansions of CD56+CD3– cells require additional signals (14;16), such as the presence of B-lymphoblastoid cells (26;34;35). B-lymphoblastoid cells, however, also induce vigorous expansions of T lymphocytes, whereas NKAES cultures do not stimulate T-cell proliferation. In the setting of allogeneic NK-cell therapy, this could be an important practical advantage as it would facilitate the complete removal of residual T cells at the end of the cultures (to avoid the risk of graft-versus-host disease). Because K562-mb15-41BBL cells are lethally-irradiated before culture and they are lysed by the expanding NK cells, the risk of infusing viable K562-mb15-41BBL is negligible. Nevertheless, we have incorporated safeguards in our clinical protocol. We prepare cultures of irradiated K562-mb15-41BBL cells, and monitor their growth and DNA-synthesis rate. We also test for the presence of viable K562-mb15-41BBL cells at the end of the culture by flow cytometry, using GFP as a marker. The clinical product is released only if there is no cell growth and no viable of K562-mb15-41BBL cell at the end of the cultures.

Most patients with AML respond to initial treatment and achieve remission, but occult resistant leukemia persists in approximately half of the patients, leading to overt (and usually fatal) relapse (36;37). NK cell infusions have shown to be clinically effective in patients with high-risk AML (8); they are being considered for the therapy of other hematological malignancies (9;38). Conceivably, NK-cell therapy will be most powerful when the number of NK cells infused is sufficiently high to produce a high E : T ratio. In our murine models of AML, multiple injections of NKAES-derived cells were required to eradicate leukemia and achieve long-term remissions. The number of NK cells that can be generated with the method that we describe should meet the requirement for a high E : T ratio, particularly in the setting of minimal residual disease, and allow multiple NK cell infusions. We found that administration of IL-2 significantly prolonged the survival of NKAES-NK cells in immunodeficient mice. It is possible that other cytokines not yet available for clinical studies, such as IL-15, might prove to be superior for this purpose. Of note, it was shown in clinical studies that lymphodepletion of the recipients, a procedure essential to ensure prolonged engraftment of the infused cells (39), resulted in high levels of serum IL-15 (8).

Although infusion of allogeneic unstimulated or IL-2-stimulated NK cells has proven to be safe, with no significant graft-versus-host disease detected, the safety of NKAES-NK cell infusions must be established. To this end, we have begun a Phase I dose-escalation clinical study of haploidentical NKAES-NK cells in patients with refractory leukemia. In addition to AML and other hematologic malignancies, some solid tumors should also be susceptible to NK cell cytotoxicity (9). Therefore, patients with these malignancies could also be eligible for clinical studies of NK cell therapy.

ADOPTIVE T CELL THERAPY: HARNESSING THE IMMUNE SYSTEM TO FIGHT CANCER

August 15, 2014 | by Hiu Chung So    http://www.cityofhope.org/blog/adoptive-t-cell-fight-cancer

Immunotherapy — using one’s immune system to treat a disease — has been long lauded as the “magic bullet” of cancer treatments, one that can be more effective than the conventional therapies of surgery, radiation or chemotherapy. One specific type of immunotherapy, called adoptive T cell therapy, is demonstrating promising results for blood cancers and may have potential against other types of cancers, too.

In adoptive T cell therapy, T cells (in blue, above) are extracted from the patient and re-engineered to recognize and attack cancer cells. They are then re-infused back into the patient, where it can then target and kill cancer cells throughout the body. (Photo credit: Lawrence Berkeley Laboratory)

In adoptive T cell therapy, T cells (in blue, above) are extracted from the patient and modified to recognize unique cancer markers and attack the cells carrying those markers. They are then reinfused back into the patient, where they can kill cancer cells throughout the body. (Photo credit: Lawrence Berkeley Laboratory)

What is adoptive T cell therapy and how does it work to treat cancer?

Every day, our immune system works to recognize and destroy abnormal, mutated cells. But the abnormal cells that eventually become cancer are the ones that slip past this defense system. The idea behind this therapy is to make immune cells (specifically, T lymphocytes) sensitive to cancer-specific abnormalities so that malignant cells can be targeted and attacked throughout the body.

Who would be good candidates for this type of therapy?

Currently, adoptive T cell therapy is mostly used to treat lymphoma and lymphoid leukemia, because these cancer cells have unique surface markers that we can reprogram T cells to recognize and attack. However, we also studying how to adapt this approach to treat other cancers as well, including myeloid leukemia, multiple myeloma and solid tumors.

What happens to the patient during this therapy?

First, we collect the patient’s own T cells from the bloodstream, which takes about four hours. The cells are then modified to recognize the patient’s cancer; a two- to three-week process in our laboratories. They are then frozen for later use as needed.

While the T cells are being modified, the patient undergoes an autologous stem cell transplant. Afterward, the re-engineered T cells are infused back into the patient so that they can kill any residual cancer cells that remained after the transplant. Depending on the type of cancer, its stage, the patient’s health and other factors, some patients may receive the modified T cell infusions shortly after their transplant; others may get their infusions later on, when tests showed that the cancer has relapsed.

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The Application of Natural Killer Cell Immunotherapy for the Treatment of Cancer

Katayoun Rezvani1* and Rayne H. Rouce2,3
THIS ARTICLE IS PART OF THE RESEARCH TOPIC   NK cell-based cancer immunotherapy
Front. Immunol., 17 November 2015 | http://dx.doi.org/10.3389/fimmu.2015.00578

 

Natural killer (NK) cells are essential components of the innate immune system and play a critical role in host immunity against cancer. Recent progress in our understanding of NK cell immunobiology has paved the way for novel NK cell-based therapeutic strategies for the treatment of cancer. In this review, we will focus on recent advances in the field of NK cell immunotherapy, including augmentation of antibody-dependent cellular cytotoxicity, manipulation of receptor-mediated activation, and adoptive immunotherapy with ex vivo-expanded, chimeric antigen receptor (CAR)-engineered, or engager-modified NK cells. In contrast to T lymphocytes, donor NK cells do not attack non-hematopoietic tissues, suggesting that an NK-mediated antitumor effect can be achieved in the absence of graft-vs.-host disease. Despite reports of clinical efficacy, a number of factors limit the application of NK cell immunotherapy for the treatment of cancer, such as the failure of infused NK cells to expand and persist in vivo. Therefore, efforts to enhance the therapeutic benefit of NK cell-based immunotherapy by developing strategies to manipulate the NK cell product, host factors, and tumor targets are the subject of intense research. In the preclinical setting, genetic engineering of NK cells to express CARs to redirect their antitumor specificity has shown significant promise. Given the short lifespan and potent cytolytic function of mature NK cells, they are attractive candidate effector cells to express CARs for adoptive immunotherapies. Another innovative approach to redirect NK cytotoxicity towards tumor cells is to create either bispecific or trispecific antibodies, thus augmenting cytotoxicity against tumor-associated antigens. These are exciting times for the study of NK cells; with recent advances in the field of NK cell biology and translational research, it is likely that NK cell immunotherapy will move to the forefront of cancer immunotherapy over the next few years.

Natural killer (NK) cell-mediated cytotoxicity contributes to the innate immune response against various malignancies, including leukemia (1, 2). The antitumor effect of NK cells is a subject of intense investigation in the field of cancer immunotherapy. In this review, we will focus on recent advances in NK cell immunotherapy, including

  • augmentation of antibody-dependent cytotoxicity,
  • manipulation of receptor-mediated activation, and
  • adoptive immunotherapy with ex vivo-expanded,
  • chimeric antigen receptor (CAR)-engineered, or
  • engager-modified NK cells.

 

Biology of NK Cells Relevant to Adoptive Immunotherapy

Natural killer cells are characterized by the lack of CD3/TCR molecules and by the expression of CD16 and CD56 surface antigens. Around 90% of circulating NK cells are CD56dim, characterized by their distinct ability to mediate cytotoxicity in response to target cell stimulation (3, 4). This subset includes the alloreactive NK cells that play a central role in targeting leukemia cells in the setting of allogeneic hematopoietic stem cell transplant (HSCT) (5). The remaining NK cells, predominantly housed in lymphoid organs, are CD56bright, and although less mature (“unlicensed”) (3, 6, 7), they have a greater capability to secrete and respond to cytokines (8, 9). CD56bright and CD56dim NK cells are also distinguished by their differential expression of FcγRIII (CD16), an integral determinant of NK-mediated antibody-dependent cellular cytotoxicity (ADCC), with CD56dim NK cells expressing high levels of the receptor, while CD56bright NK cells are CD16 dim or negative (6). In contrast to T and B lymphocytes, NK cells do not express rearranged, antigen-specific receptors; rather, NK effector function is dictated by the integration of signals received through germ-line-encoded receptors that can recognize ligands on their cellular targets. Functionally, NK cell receptors are classified as activating or inhibitory. NK cell function, including cytotoxicity and cytokine release, is governed by a balance between signals received from inhibitory receptors, notably the killer Ig-like receptors (KIRs) and the heterodimeric C-type lectin receptor (NKG2A), and activating receptors, in particular the natural cytotoxicity receptors (NCRs) NKp46, NKp30, NKp44, and the C-type lectin-like activating immunoreceptor NKG2D (9).

The inhibitory KIRs (iKIRs) with known HLA ligands include KIR2DL2 and KIR2DL3, which recognize the HLA-C group 1-related alleles characterized by an asparagine residue at position 80 of the α-1 helix (HLA-CAsn80); KIR2DL1, which recognizes the HLA-C group 2-related alleles characterized by a lysine residue at position 80 (HLA-CLys80); and KIR3DL1, which recognizes the HLA-Bw4 alleles (9, 10). NK cells also express several activating receptors that are potentially specific for self-molecules. KIR2DS1 has been shown to interact with group 2 HLA-C molecules (HLA-C2), while KIR2DS2 was recently shown to recognize HLA-A*11 (10, 11). Hence, these receptors require mechanisms to prevent inadvertent activation against normal tissues, processes referred to as “tolerance to self.” Engagement of iKIR receptors by HLA class I leads to signals that block NK-cell triggering during effector responses. These receptors explain the “missing self” hypothesis, which postulates that NK cells survey tissues for normal levels of the ubiquitously expressed MHC class I molecules (12, 13). Upon cellular transformation or viral infection, surface MHC class I expression on the cell surface is often reduced or lost to evade recognition by antitumor T cells. When a mature NK cell encounters transformed cells lacking MHC class I, their inhibitory receptors are not engaged, and the unsuppressed activating signals, in turn, can trigger cytokine secretion and targeted attack of the virus-infected or transformed cell (13, 14). In parallel, cellular stress and DNA damage (occurring in cells during viral or malignant transformation) results in upregulation of “stress ligands” that can be recognized by activating NK receptors. Thus, human tumor cells that have lost self-MHC class I expression or bear “altered-self” stress-inducible proteins are ideal targets for NK recognition and killing (14–16). NK cells directly kill tumor cells through several mechanisms, including release of cytoplasmic granules containing perforin and granzyme (16–18), expression of tumor necrosis factor (TNF) family members, such as FasL or TNF-related apoptosis-inducing ligand (TRAIL), which induce tumor cell apoptosis by interacting with their respective receptors Fas and TRAIL receptor (TRAILR) (16–19) as well as ADCC (9).

 

Interaction Between Natural Killer Cells and Other Immune Subsets

Increasing understanding of NK cell biology and their interaction with other cells of the immune system has led to several novel immunotherapeutic approaches as discussed in this review. NK cells produce cytokines that can exert regulatory control of downstream adaptive immune responses by influencing the magnitude of T cell responses, specifically T helper-1 (TH1) function (20). NK cell function, in turn, is regulated by cytokines, such as IL-2, IL-15, IL-12, and IL-18 (21), as well as by interactions with other cell types, such as dendritic cells, macrophages, and mesenchymal stromal cells (10, 22, 23). IL-15 has emerged as a pivotal cytokine required for NK cell development and maintenance. Whereas mice deficient in IL-2 (historically the cytokine of choice to expand and activate NK cells) have normal NK cells, IL-15-deficient mice lack NK cells (24).

Several cytokines are also known to inhibit NK cell activation and function, thus playing a crucial role in tumor escape from NK immune surveillance. Recently, considerable attention has been paid to the inhibitory effects of transforming growth factor-beta (TGF-β) and IL-10 on NK cell cytotoxicity (12, 25, 26). Several groups have shown that secretion of TGF-β by tumor cells results in downregulation of activating receptors, such as NKp30 and NKG2D, with resultant NK dysfunction (25,26). Similarly, IL-10 production by acute myeloid leukemia (AML) blasts induces upregulation of NKG2A with significant impairment in NK function (3).

 

Modulation of Antibody-Dependent Cellular Cytotoxicity

The CD56dim subset of NK cells expresses the Fcγ receptor CD16, through which NK cells mount ADCC, providing opportunities for its modulation to augment NK effector function (27, 28). In fact, a number of clinically approved therapeutic antibodies targeting tumor-associated antigens (such as rituximab or cetuximab) function at least partially through triggering NK cell-mediated ADCC. Several studies using mouse tumor models have established that efficient antibody–Fc receptor (FcR) interactions are essential for the efficacy of monoclonal antibody (mAb) therapy, a mainstay of cancer therapy (28, 29). Based on this premise, Romain et al. successfully engineered the Fc region of the IgG mAb, HuM195 targeting the AML leukemia antigen CD33, by introducing the triple mutation S293D/A330L/I332E (DLE). Using timelapse imaging microscopy in nanowell grids (TIMING, a method of analyzing kinetics of thousands of NK cells and mAb-coated targets), they demonstrated that the DLE-HuM195 antibody increased both the quality and quantity of NK cell-mediated ADCC by recruiting NK cells to participate in cytotoxicity via CD16-mediated signaling. NK cells encountering DLE-HuM195-coated targets induced rapid target cell apoptosis by promoting conjugation to multiple target cells (leading to increased “serial killing” of targets), thus inducing apoptosis in twice the number of targets as the wild-type mAb (27).

Additional approaches under investigation to enhance NK cell-mediated ADCC include antibody engineering and therapeutic combination of antibodies predicted to have synergistic activity. For example, mogamulizumab (an anti-CCR4 mAb recently approved in Japan) is defucosylated to increase binding by FcγRIIIA and thereby enhances ADCC. Mogamulizumab successfully induced ADCC activity against CCR4-positive cell lines and inhibited the growth of EBV-positive NK-cell lymphomas in a murine xenograft model (30). These findings suggest that mogamulizumab may be a therapeutic option against EBV-associated T and NK-lymphoproliferative diseases (30). Obinutuzumab (GA101) is a novel type II glycoengineered mAb against CD20 with increased FcγRIII binding and ADCC activity. In contrast to rituximab, GA101 induces activation of NK cells irrespective of their inhibitory KIR expression, and its activity is not negatively affected by KIR/HLA interactions (31). These data show that modification of the Fc fragment to enhance NK-mediated ADCC can be an effective strategy to augment the efficacy of therapeutic mAbs (31).

Although enhanced NK-mediated ADCC occurs in the presence of certain mAbs, in the case of non-engineered mAbs (such as rituximab), this NK-mediated cytotoxicity is typically still under the jurisdiction of KIR-mediated inhibition. However, ADCC responses can be potentiated in vitro in the presence of antibodies that block NK cell inhibitory receptor interaction with MHC class I ligands (32). These include the use of anti-KIR Abs to block the interaction of iKIRs with their cognate HLA class I ligands. To exploit this pathway pharmacologically, a fully humanized anti-KIR mAb 1-7F9 (IPH2101) (33) with the ability to block KIR2DL1/L2/L3 and KIR2DS1/S2 was generated. In vitro, anti-KIR mAbs can augment NK cell-mediated lysis of HLA-C-expressing tumor cells, including autologous AML blasts and autologous CD138+ multiple myeloma (MM) cells (34). Additionally, in a dose-escalation phase 1 clinical trial in elderly patients with AML, 1-7F9 mAb was reported to be safe and could block KIRs for prolonged periods (35). A recombinant version of this mAb with a stabilized hinge (lirilumab) was recently developed. Lirilumab is a fully humanized IgG4 anti-KIR2DL1, -L2, -L3, -S1, and -S2 mAb. The iKIRs targeted by lirilumab collectively recognize virtually all HLA-C alleles, and the blockade of the three KIR2DLs allows targeting of every patient without the need for prior HLA or KIR typing (33, 34). Furthermore, the combination of an anti-KIR mAb with the immunomodulatory drug lenalidomide was shown to potentiate ADCC and is being tested in a phase 1 clinical trial in patients with MM [NCT01217203 (35)]. A potential concern is related to how inhibitory KIR blockade may impact on the ability of NK cells to discriminate self, healthy cells from abnormal virally infected or cancerous cells. Preliminary in vitro data suggest that Ab blockade of iKIRs will preferentially augment the ADCC response, without increasing cytotoxicity against self healthy cells (32). It is reassuring that in the IPH2101 phase 1 studies, no alterations in the expression of major inhibitory or activating NK receptors or frequencies of circulating peripheral lymphocytes were reported, indicating that the Ab does not induce clinically significant targeting of normal cells by NK cells (35). Lin et al. recently reported on the application of an agonistic NK cell-targeted mAb to augment ADCC (36). Following FcR triggering during ADCC, expression of the activation marker CD137 is increased. Agonistic antibodies targeting CD137 have been reported to augment NK-cell function, including degranulation, secretion of IFN-γ, and antitumor cytotoxicity in in vitro and in vivo preclinical models of tumor (36–39). The combination of the agonistic anti-CD137 antibody with rituximab is currently being evaluated in a phase 1 trial in patients with lymphoma [NCT01307267 (35–37)].

Other factors, such as specific CD16 polymorphisms and NKG2D engagement, can also influence ADCC, with certain polymorphisms (such as FcγRIIIa-V158F polymorphism) resulting in a stronger IgG binding (40). These findings are clinically relevant, as supported by the observation that patients with non-Hodgkin lymphoma (NHL) with the FcγRIIIa-V158F polymorphism experienced improved clinical response to rituximab (41, 42). In summary, several antibody combinations designed to boost ADCC have shown promising results in preclinical and early clinical trials, thus warranting further study of this strategy to enhance NK cell activity against tumor cells.

 

Adoptive Transfer of Autologous NK Cells

The early studies of adoptive NK cell therapy focused on enhancing the antitumor activity of endogenous NK cells (43). Initial trials of adoptive NK therapy in the autologous setting involved using CD56 beads to select NK cells from a leukapheresis product and subsequently infusing the bead-selected autologous NK cells into patients (43, 44). Infusions were followed by administration of systemic cytokines (most commonly IL-2) to provide additional in vivo stimulation and support their expansion. This strategy met with limited success due to a combination of factors (44). Although cytokine stimulation promoted NK cell activation and resulted in greater cytotoxicity against malignant targets in vitro, only limited in vivoantitumor activity was observed (43–45). Similar findings were observed when autologous NK cells and systemic IL-2 were given as consolidation treatment to patients with lymphoma who underwent autologous BMT (46). The poor clinical outcomes observed with adoptive transfer of ex vivo activated autologous NK cells followed by systemic IL-2 were attributed to three factors: (1) development of severe life-threatening side effects, such as vascular leak syndrome as a result of IL-2 therapy; (2) IL-2-induced expansion of regulatory T cells known to directly inhibit NK cell function and induce activation-induced cell death (47–49); and (3) lack of antitumor effect related to the inhibition of autologous NK cells by self-HLA molecules. Strategies to overcome this autologous “checkpoint,” thus redirecting autologous NK cells to target and kill leukemic blasts are the subject of intense investigation (33–35). These include the use of anti-KIR Abs (such as the aforementioned lirilumab) to block the interaction of inhibitory receptors on the surface of NK cells with their cognate HLA class I ligand.

 

Exploiting the Alloreactivity of Allogeneic NK Cells – Adoptive Immunotherapy and Beyond

An alternative strategy is to use allogeneic instead of autologous NK cells, thus taking advantage of the inherent alloreactivity afforded by the “missing self” concept (13). Over the past decade, adoptive transfer of ex vivo-activated or -expanded allogeneic NK cells has emerged as a promising immunotherapeutic strategy for cancer (24, 50–52). Allogeneic NK cells are less likely to be subject to the inhibitory response resulting from NK cell recognition of self-MHC molecules as seen with autologous NK cells. A number of studies have shown that infusion of haploidentical NK cells to exploit KIR/HLA alloreactivity is safe and can mediate impressive clinical activity in some patients with AML (50–52). In fact, algorithms have been developed to ensure selection of stem cell donors with the greatest potential for NK cell alloreactivity for allogeneic HSCT (50).

Promising results in the HSCT setting suggest that the application of this strategy in the non-transplant setting may be a plausible option. Miller et al. were among the first to show that adoptive transfer of ex vivo-expanded haploidentical NK cells after lymphodepleting chemotherapy is safe, and can result in expansion of NK cells in vivo without inducing graft-vs.-host disease (GVHD) (50). In a phase I dose-escalation trial, 43 patients with either hematologic malignancies (poor prognosis AML or Hodgkin lymphoma) or solid tumor (metastatic melanoma or renal cell carcinoma) received up to 2 × 107cells/kg of haploidentical NK cells following either low intensity [low-dose cyclophosphamide (Cy) and methylprednisolone or fludarabine (Flu)] or high intensity regimens (Hi-Cy/Flu). All patients received subcutaneous IL-2 after NK cell infusion. Whereas adoptively infused NK cells persisted only transiently following low intensity regimens, AML patients who received the more intense Hi-Cy/Flu regimen had a marked rise in endogenous IL-15 associated with expansion of donor NK cells and induction of complete remission (CR) in five of 19 very high-risk patients. The superior NK expansion observed after high-dose compared to low-dose chemotherapy was attributed to a combination of factors including prevention of host T cell-mediated rejection and higher levels of cytokines, such as IL-15. These findings provided the first evidence that haploidentical NK cells are safe and can persist and expand in vivo, supporting the proof of concept that NK cells may be applied for the treatment of selected malignancies either alone or as an adjunct to HSCT (50).

Another pivotal pilot study, the NKAML trial (Pilot Study of Haploidentical NK Transplantation for AML), reported that infusion of KIR-HLA-mismatched donor NK cells can reduce the risk of relapse in childhood AML (51). Ten pediatric patients with favorable or intermediate risk AML in first CR were enrolled following completion of 4–5 cycles of chemotherapy. All patients received a low-dose conditioning regimen consisting of Cy/Flu prior to infusion of NK cells (median, 29 × 106/kg NK cells) from a haploidentical donor, followed by six doses of IL-2. NK infusions were well tolerated with limited non-hematologic toxicity. All patients had transient engraftment of NK cells for a median of 10 days (range 2–189 days) with significant expansion of KIR-mismatched NK cells. With a median follow-up of 964 days, all patients remained in remission, suggesting that donor-recipient HLA-mismatched NK cells may reduce the risk of relapse in childhood AML (51).

Other strategies currently under investigation include the infusion of KIR-ligand-mismatched haploidentical NK cells as part of the pre-HSCT conditioning regimen (NCT00402558), and NK cell infusion to prevent relapse or as therapy for minimal residual disease in patients after haploidentical HSCT (NCT01386619).

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Human NK Cell Lines as a Source of NK Immunotherapy

The adoptive transfer of NK cell lines has several theoretical advantages over the use of patient- or donor-derived NK cells. These are primarily related to the lack of expression of iKIRs, presumed lack of immunogenicity, ease of expansion and availability as an “off-the-shelf” product (85). Several human NK cell lines, such as NK-92 and KHYG-1, have been documented to exert antitumor activity in both preclinical and clinical settings (86–88). NK-92, the most extensively characterized NK-cell line, was established in 1994 from the PB of a male Caucasian patient with NHL. NK-92 cells are IL-2-dependent, harbor a CD2+CD56+CD57+ phenotype and exert potent in vitro cytotoxicity (86). Infusion of up to 1010 cells/m2NK-92 cells into patients with advanced lung cancer and other advanced malignancies was well tolerated and the cells persisted for a minimum of 48 h with encouraging clinical responses (86, 88–91). However, potential limitations of using NK cell lines, such as NK-92 cells, include the requirement for irradiation to reduce the risk of engrafting cells with potential in vivo tumorigenicity, and the need for pre-infusion conditioning to avoid host rejection. Furthermore, infusion of allogeneic NK cell lines may induce T and B cell alloimmune responses, limiting their in vivo persistence and precluding multiple infusions. A number of studies are testing NK-92 cells (Neukoplast®) in patients with solid tumors, such as Merkel cell cancer and renal cell carcinoma, as well as in hematological malignancies (85).

While results from clinical studies of NK cell adoptive therapy are encouraging (48–52, 70), significant gaps remain in our understanding of the optimal conditions for NK cell infusion. Based on the pioneering work from Rosenberg et al. demonstrating the importance of lymphodepletion to support the expansion of tumor-infiltrating T cells (92) and given its emergence as a key determinant of efficacy with CAR therapy, several groups are actively investigating the ideal preparative regimen to promote the expansion and persistence of adoptively infused NK cells (53, 69, 70, 75). Available data support the use of high-dose Cy/Flu regimen as the frontrunner, considering it is reasonably well tolerated and shown to support the in vivo expansion of NK cells (51, 70). IL-15 is an ideal candidate cytokine for the expansion of NK cells in vivo, especially since it does not promote expansion of regulatory T cells (66), which have been shown to suppress NK cell effector function in IL-2-based trials (69, 70). In a recent phase 1 study in patients with metastatic melanoma or renal cell carcinoma, rhIL-15 was shown to activate NK cells, monocytes, γδ, and CD8 T cells (93). However, as an intravenous bolus dose, rhIL-15 proved too difficult to administer because of significant clinical toxicities (93). Based on these promising data, alternative dosing strategies are being investigated, including continuous intravenous infusions. To this effect, systemic IL-15 along with infusion of donor NK cells are currently being tested in a phase I clinical trial for AML (NCT01385423).

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Bispecific and Trispecific Engagers

An innovative immunoglobulin-based strategy to redirect NK cytotoxicity towards tumor cells is to create either bispecific or trispecific antibodies (BiKE, TriKE) (113). BiKEs are constructed by joining a single-chain Fv against CD16 and a single-chain Fv against a tumor-associated antigen (BiKE), or two tumor-associated antigens (TriKE). Gleason et al. showed that bispecific (bscFv) CD16/CD19 and trispecific (tscFv) CD16/CD19/CD22 engagers directly trigger NK cell activation through CD16, significantly increasing NK cell cytolytic activity and cytokine production against various CD19-expressing B cell lines. The same group also developed and tested a CD16 × 33 BiKE in refractory AML and demonstrated that the potent killing by NK cells could overcome the inhibitory effect of KIR signaling (113, 114).

Notably, activated NK cells lose CD16 (FcRγIII) and CD62L through a metalloprotease called ADAM17, which is expressed on NK cells, which may in turn impact on the efficacy of Fc-mediated cytotoxicity (115). Romee et al. recently showed that selective inhibition of ADAM17 enhances CD16-mediated NK cell function by preserving CD16 on the NK cell surface, thus enhancing ADCC (115). Additionally, Fc-induced production of cytokines by NK cells exposed to rituximab-coated B cell targets can be further enhanced by ADAM17 inhibition. These findings support a role for targeting ADAM17 to prevent CD16 shedding and to improve the efficacy of therapeutic mAbs. The same group subsequently discovered that ADAM17 inhibition enhances CD16 × 33 BiKE responses against primary AML targets (114).

 

NK Cells – What Does the Future Hold?

Recent advances in the understanding of NK cell immunobiology have paved the way for novel and innovative anti-cancer therapies. Here, we have discussed a representation of these novel immunotherapeutic strategies to potentiate NK cell function and enhance antitumor activity including ADCC-inducing mAbs, ex vivo activated or genetically modified NK cells and bi- or trispecific engagers (Figure 1).

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Microbe meets cancer

Posted in Bacterial Resistance, Biochemical pathways, Biological Networks, Gene Regulation and Evolution, Cancer - General, Cancer Informatics, Cell Biology, Signaling & Cell Circuits, Chemical Biology and its relations to Metabolic Disease, Clinical & Translational, Clinical Genomics, Curation, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Gastroenterology, Gene Regulation, Genome Biology, Inflammasome, Liver & Digestive Diseases Research, Metabolism, Methylation, Microbiologial genetics, Microbiology, Molecular Genetics & Pharmaceutical, Oxidative phosphorylation, Warburg effect, tagged , , , , , , on April 10, 2016| Leave a Comment »

Microbe meets cancer

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Microbes Meet Cancer

Understanding cancer’s relationship with the human microbiome could transform immune-modulating therapies.

By Kate Yandell | April 1, 2016  http://www.the-scientist.com/?articles.view/articleNo/45616/title/Microbes-Meet-Cancer

 © ISTOCK.COM/KATEJA_FN; © ISTOCK.COM/FRANK RAMSPOTT  http://www.the-scientist.com/images/April2016/feature1.jpg

In 2013, two independent teams of scientists, one in Maryland and one in France, made a surprising observation: both germ-free mice and mice treated with a heavy dose of antibiotics responded poorly to a variety of cancer therapies typically effective in rodents. The Maryland team, led by Romina Goldszmidand Giorgio Trinchieri of the National Cancer Institute, showed that both an investigational immunotherapy and an approved platinum chemotherapy shrank a variety of implanted tumor types and improved survival to a far greater extent in mice with intact microbiomes.1 The French group, led by INSERM’s Laurence Zitvogel, got similar results when testing the long-standing chemotherapeutic agent cyclophosphamide in cancer-implanted mice, as well as in mice genetically engineered to develop tumors of the lung.2

The findings incited a flurry of research and speculation about how gut microbes contribute to cancer cell death, even in tumors far from the gastrointestinal tract. The most logical link between the microbiome and cancer is the immune system. Resident microbes can either dial up inflammation or tamp it down, and can modulate immune cells’ vigilance for invaders. Not only does the immune system appear to be at the root of how the microbiome interacts with cancer therapies, it also appears to mediate how our bacteria, fungi, and viruses influence cancer development in the first place.

“We clearly see shifts in the [microbial] community that precede development of tumors,” says microbiologist and immunologist Patrick Schloss, who studies the influence of the microbiome on colon cancer at the University of Michigan.

But the relationship between the microbiome and cancer is complex: while some microbes promote cell proliferation, others appear to protect us against cancerous growth. And in some cases, the conditions that spur one cancer may have the opposite effect in another. “It’s become pretty obvious that the commensal microbiota affect inflammation and, through that or through other mechanisms, affect carcinogenesis,” says Trinchieri. “What we really need is to have a much better understanding of which species, which type of bug, is doing what and try to change the balance.”

Gut feeling

In the late 1970s, pathologist J. Robin Warren of Royal Perth Hospital in Western Australia began to notice that curved bacteria often appeared in stomach tissue biopsies taken from patients with chronic gastritis, an inflammation of the stomach lining that often precedes the development of stomach cancer. He and Barry J. Marshall, a trainee in internal medicine at the hospital, speculated that the bacterium, now called Helicobacter pylori, was somehow causing the gastritis.3 So committed was Marshall to demonstrating the microbe’s causal relationship to the inflammatory condition that he had his own stomach biopsied to show that it contained no H. pylori, then infected himself with the bacterium and documented his subsequent experience of gastritis.4 Scientists now accept that H. pylori, a common gut microbe that is present in about 50 percent of the world’s population, is responsible for many cases of gastritis and most stomach ulcers, and is a strong risk factor for stomach cancer.5 Marshall and Warren earned the 2005 Nobel Prize in Physiology or Medicine for their work.

H. pylori may be the most clear-cut example of a gut bacterium that influences cancer development, but it is likely not the only one. Researchers who study cancer in mice have long had anecdotal evidence that shifts in the microbiome influence the development of diverse tumor types. “You have a mouse model of carcinogenesis. It works beautifully,” says Trinchieri. “You move to another institution. It works completely differently,” likely because the animals’ microbiomes vary with environment.

IMMUNE INFLUENCE: In recent years, research has demonstrated that microbes living in and on the mammalian body can affect cancer risk, as well as responses to cancer treatment. Although the details of this microbe-cancer link remain unclear, investigators suspect that the microbiome’s ability to modulate inflammation and train immune cells to react to tumors is to blame.
See full infographic: WEB | PDF© AL GRANBERG

Around the turn of the 21st century, cancer researchers began to systematically experiment with the rodent microbiome, and soon had several lines of evidence linking certain gut microbes with a mouse’s risk of colon cancer. In 2001, for example, Shoichi Kado of the Yakult Central Institute for Microbiological Research in Japan and colleagues found that a strain of immunocompromised mice rapidly developed colon tumors, but that germ-free versions of these mice did not.6 That same year, an MIT-based group led by the late David Schauer demonstrated that infecting mice with the bacterium Citrobacter rodentium spurred colon tumor development.7 And in 2003, MIT’s Susan Erdman and her colleagues found that they could induce colon cancer in immunocompromised mice by infecting them with Helicobacter hepaticus, a relative of? H. pylori that commonly exists within the murine gut microbiome.8

More recent work has documented a similar link between colon cancer and the gut microbiome in humans. In 2014, a team led by Schloss sequenced 16S rRNA genes isolated from the stool of 90 people, some with colon cancer, some with precancerous adenomas, and still others with no disease.9 The researchers found that the feces of people with cancer tended to have an altered composition of bacteria, with an excess of the common mouth microbes Fusobacterium or Porphyromonas. A few months later, Peer Bork of the European Molecular Biology Laboratory performed metagenomic sequencing of stool samples from 156 people with or without colorectal cancer. Bork and his colleagues found they could predict the presence or absence of cancer using the relative abundance of 22 bacterial species, including Porphyromonas andFusobacterium.10 They could also use the method to predict colorectal cancer with about the same accuracy as a blood test, correctly identifying about 50 percent of cancers while yielding false positives less than 10 percent of the time. When the two tests were combined, they caught more than 70 percent of cancers.

Whether changes in the microbiota in colon cancer patients are harbingers of the disease or a consequence of tumor development remained unclear. “What comes first, the change in the microbiome or tumor development?” asks Schloss. To investigate this question, he and his colleagues treated mice with microbiome-altering antibiotics before administering a carcinogen and an inflammatory agent, then compared the outcomes in those animals and in mice that had received only the carcinogenic and inflammatory treatments, no antibiotics. The antibiotic-treated animals had significantly fewer and smaller colon tumors than the animals with an undisturbed microbiome, suggesting that resident bacteria were in some way promoting cancer development. And when the researchers transferred microbiota from healthy mice to antibiotic-treated or germ-free mice, the animals developed more tumors following carcinogen exposure. Sterile mice that received microbiota from mice already bearing malignancies developed the most tumors of all.11

Most recently, Schloss and his colleagues showed that treating mice with seven unique combinations of antibiotics prior to exposing them to carcinogens yielded variable but predictable levels of tumor formation. The researchers determined that the number of tumors corresponded to the unique ways that each antibiotic cocktail modulated the microbiome.12

“We’ve kind of proven to ourselves, at least, that the microbiome is involved in colon cancer,” says Schloss, who hypothesizes that gut bacteria–driven inflammation is to blame for creating an environment that is hospitable to tumor development and growth. Gain or loss of certain components of the resident bacterial community could lead to the release of reactive oxygen species, damaging cells and their genetic material. Inflammation also involves increased release of growth factors and blood vessel proliferation, potentially supporting the growth of tumors. (See illustration above.)

Recent research has also yielded evidence that the gut microbiota impact the development of cancer in sites far removed from the intestinal tract, likely through similar immune-modulating mechanisms.

Systemic effects

In the mid-2000s, MIT’s Erdman began infecting a strain of mice predisposed to intestinal tumors withH. hepaticus and observing the subsequent development of colon cancer in some of the animals. To her surprise, one of the mice developed a mammary tumor. Then, more of the mice went on to develop mammary tumors. “This told us that something really interesting was going on,” Erdman recalls. Sure enough, she and her colleagues found that mice infected with H. hepaticus were more likely to develop mammary tumors than mice not exposed to the bacterium.13The researchers showed that systemic immune activation and inflammation could contribute to mammary tumors in other, less cancer-prone mouse models, as well as to the development of prostate cancer.

MICROBIAL STOWAWAYS: Bacteria of the human gut microbiome are intimately involved in cancer development and progression, thanks to their interactions with the immune system. Some microbes, such as Helicobacter pylori, increase the risk of cancer in their immediate vicinity (stomach), while others, such as some Bacteroides species, help protect against tumors by boosting T-cell infiltration.© EYE OF SCIENCE/SCIENCE SOURCE
http://www.the-scientist.com/images/April2016/immune_2.jpg

 

 

© DR. GARY GAUGLER/SCIENCE SOURCE  http://www.the-scientist.com/images/April2016/immune3.jpg

At the University of Chicago, Thomas Gajewski and his colleagues have taken a slightly different approach to studying the role of the microbiome in cancer development. By comparing Black 6 mice coming from different vendors—Taconic Biosciences (formerly Taconic Farms) and the Jackson Laboratory—Gajewski takes advantage of the fact that the animals’ different origins result in different gut microbiomes. “We deliberately stayed away from antibiotics, because we had a desire to model how intersubject heterogeneity [in cancer development] might be impacted by the commensals they happen to be colonized with,” says Gajewski in an email to The Scientist.

Last year, the researchers published the results of a study comparing the progression of melanoma tumors implanted under the mice’s skin, finding that tumors in the Taconic mice grew more aggressively than those in the Jackson mice. When the researchers housed the different types of mice together before their tumors were implanted, however, these differences disappeared. And transferring fecal material from the Jackson mice into the Taconic mice altered the latter’s tumor progression.14

Instead of promoting cancer, in these experiments the gut microbiome appeared to slow tumor growth. Specifically, the reduced tumor growth in the Jackson mice correlated with the presence of Bifidobacterium, which led to the greater buildup of T?cells in the Jackson mice’s tumors. Bifidobacteriaactivate dendritic cells, which present antigens from bacteria or cancer cells to T?cells, training them to hunt down and kill these invaders. Feeding Taconic mice bifidobacteria improved their response to the implanted melanoma cells.

“One hypothesis going into the experiments was that we might identify immune-suppressive bacteria, or commensals that shift the immune response towards a character that was unfavorable for tumor control,” says Gajewski.  “But in fact, we found that even a single type of bacteria could boost the antitumor immune response.”

http://www.the-scientist.com/images/April2016/immune4.jpg

 

Drug interactions

Ideally, the immune system should recognize cancer as invasive and nip tumor growth in the bud. But cancer cells display “self” molecules that can inhibit immune attack. A new type of immunotherapy, dubbed checkpoint inhibition or blockade, spurs the immune system to attack cancer by blocking either the tumor cells’ surface molecules or the receptors on T?cells that bind to them.

CANCER THERAPY AND THE MICROBIOME

In addition to influencing the development and progression of cancer by regulating inflammation and other immune pathways, resident gut bacteria appear to influence the effectiveness of many cancer therapies that are intended to work in concert with host immunity to eliminate tumors.

  • Some cancer drugs, such as oxaliplatin chemotherapy and CpG-oligonucleotide immunotherapy, work by boosting inflammation. If the microbiome is altered in such a way that inflammation is reduced, these therapeutic agents are less effective.
  • Cancer-cell surface proteins bind to receptors on T cells to prevent them from killing cancer cells. Checkpoint inhibitors that block this binding of activated T cells to cancer cells are influenced by members of the microbiota that mediate these same cell interactions.
  • Cyclophosphamide chemotherapy disrupts the gut epithelial barrier, causing the gut to leak certain bacteria. Bacteria gather in lymphoid tissue just outside the gut and spur generation of T helper 1 and T helper 17 cells that migrate to the tumor and kill it.

As part of their comparison of Jackson and Taconic mice, Gajewski and his colleagues decided to test a type of investigational checkpoint inhibitor that targets PD-L1, a ligand found in high quantities on the surface of multiple types of cancer cells. Monoclonal antibodies that bind to PD-L1 block the PD-1 receptors on T?cells from doing so, allowing an immune response to proceed against the tumor cells. While treating Taconic mice with PD-L1–targeting antibodies did improve their tumor responses, they did even better when that treatment was combined with fecal transfers from Jackson mice, indicating that the microbiome and the immunotherapy can work together to take down cancer. And when the researchers combined the anti-PD-L1 therapy with a bifidobacteria-enriched diet, the mice’s tumors virtually disappeared.14

Gajewski’s group is now surveying the gut microbiota in humans undergoing therapy with checkpoint inhibitors to better understand which bacterial species are linked to positive outcomes. The researchers are also devising a clinical trial in which they will give Bifidobacterium supplements to cancer patients being treated with the approved anti-PD-1 therapy pembrolizumab (Keytruda), which targets the immune receptor PD-1 on T?cells, instead of the cancer-cell ligand PD-L1.

Meanwhile, Zitvogel’s group at INSERM is investigating interactions between the microbiome and another class of checkpoint inhibitors called CTLA-4 inhibitors, which includes the breakthrough melanoma treatment ipilimumab (Yervoy). The researchers found that tumors in antibiotic-treated and germ-free mice had poorer responses to a CTLA-4–targeting antibody compared with mice harboring unaltered microbiomes.15 Particular Bacteroides species were associated with T-cell infiltration of tumors, and feedingBacteroides fragilis to antibiotic-treated or germ-free mice improved the animals’ responses to the immunotherapy. As an added bonus, treatment with these “immunogenic” Bacteroides species decreased signs of colitis, an intestinal inflammatory condition that is a dangerous side effect in patients using checkpoint inhibitors. Moreover, Zitvogel and her colleagues showed that human metastatic melanoma patients treated with ipilimumab tended to have elevated levels of B. fragilis in their microbiomes. Mice transplanted with feces from patients who showed particularly strong B. fragilis gains did better on anti-CTLA-4 treatment than did mice transplanted with feces from patients with normal levels of B. fragilis.

“There are bugs that allow the therapy to work, and at the same time, they protect against colitis,” says Trinchieri. “That is very exciting, because not only [can] we do something to improve the therapy, but we can also, at the same time, try to reduce the side effect.”

And these checkpoint inhibitors aren’t the only cancer therapies whose effects are modulated by the microbiome. Trinchieri has also found that an immunotherapy that combines antibodies against interleukin-10 receptors with CpG oligonucleotides is more effective in mice with unaltered microbiomes.1He and his NCI colleague Goldszmid further found that the platinum chemotherapy oxaliplatin (Eloxatin) was more effective in mice with intact microbiomes, and Zitvogel’s group has shown that the chemotherapeutic agent cyclophosphamide is dependent on the microbiota for its proper function.

Although the mechanisms by which the microbiome influences the effectiveness of such therapies remains incompletely understood, researchers once again speculate that the immune system is the key link. Cyclophosphamide, for example, spurs the body to generate two types of T?helper cells, T?helper 1 cells and a subtype of T?helper 17 cells referred to as “pathogenic,” both of which destroy tumor cells. Zitvogel and her colleagues found that, in mice with unaltered microbiomes, treatment with cyclophosphamide works by disrupting the intestinal mucosa, allowing bacteria to escape into the lymphoid tissues just outside the gut. There, the bacteria spur the body to generate T?helper 1 and T?helper 17 cells, which translocate to the tumor. When the researchers transferred the “pathogenic” T?helper 17 cells into antibiotic-treated mice, the mice’s response to chemotherapy was partly restored.

Microbiome modification

As the link between the microbiome and cancer becomes clearer, researchers are thinking about how they can manipulate a patient’s resident microbial communities to improve their prognosis and treatment outcomes. “Once you figure out exactly what is happening at the molecular level, if there is something promising there, I would be shocked if people don’t then go in and try to modulate the microbiome, either by using pharmaceuticals or using probiotics,” says Michael Burns, a postdoc in the lab of University of Minnesota genomicist Ran Blekhman.

Even if researchers succeed in identifying specific, beneficial alterations to the microbiome, however, molding the microbiome is not simple. “It’s a messy, complicated system that we don’t understand,” says Schloss.

So far, studies of the gut microbiome and colon cancer have turned up few consistent differences between cancer patients and healthy controls. And the few bacterial groups that have repeatedly shown up are not present in every cancer patient. “We should move away from saying, ‘This is a causal species of bacteria,’” says Blekhman. “It’s more the function of a community instead of just a single bacterium.”

But the study of the microbiome in cancer is young. If simply adding one type of microbe into a person’s gut is not enough, researchers may learn how to dose people with patient-specific combinations of microbes or antibiotics. In February 2016, a team based in Finland and China showed that a probiotic mixture dubbed Prohep could reduce liver tumor size by 40 percent in mice, likely by promoting an anti-inflammatory environment in the gut.16

“If it is true that, in humans, we can alter the course of the disease by modulating the composition of the microbiota,” says José Conejo-Garcia of the Wistar Institute in Philadelphia, “that’s going to be very impactful.”

Kate Yandell has been a freelance writer living Philadelphia, Pennsylvania. In February she became an associate editor at Cancer Today.

GENETIC CONNECTION

The microbiome doesn’t act in isolation; a patient’s genetic background can also greatly influence response to therapy. Last year, for example, the Wistar Institute’s José Garcia-Conejo and Melanie Rutkowski, now an assistant professor at the University of Virginia, showed that a dominant polymorphism of the gene for the innate immune protein toll-like receptor 5 (TLR5) influences clinical outcomes in cancer patients by changing how the patients’ immune cells interact with their gut microbes (Cancer Cell, 27:27-40, 2015).

More than 7 percent of people carry a specific mutation in TLR5 that prevents them from mounting a full immune response when exposed to bacterial flagellin. Analyzing both genetic and survival data from the Cancer Genome Atlas, Conejo-Garcia, Rutkowski, and their colleagues found that estrogen receptor–positive breast cancer patients who carry the TLR5 mutation, called the R392X polymorphism, have worse outcomes than patients without the mutation. Among patients with ovarian cancer, on the other hand, those with the TLR5 mutation were more likely to live at least six years after diagnosis than patients who don’t carry the mutation.

Investigating the mutation’s contradictory effects, the researchers found that mice with normal TLR5produce higher levels of the cytokine interleukin 6 (IL-6) than those carrying the mutant version, which have higher levels of a different cytokine called interleukin 17 (IL-17). But when the researchers knocked out the animals’ microbiomes, these differences in cytokine production disappeared, as did the differences in cancer progression between mutant and wild-type animals.

“The effectiveness of depleting specific populations or modulating the composition of the microbiome is going to affect very differently people who are TLR5-positive or TLR5-negative,” says Conejo-Garcia. And Rutkowski speculates that many more polymorphisms linked to cancer prognosis may act via microbiome–immune system interactions. “I think that our paper is just the tip of the iceberg.”

References

  1. N. Iida et al., “Commensal bacteria control cancer response to therapy by modulating the tumor microenvironment,” Science, 342:967-70, 2013.
  2. S. Viaud et al., “The intestinal microbiota modulates the anticancer immune effects of cyclophosphamide,” Science, 342:971-76, 2013.
  3. J.R. Warren, B. Marshall, “Unidentified curved bacilli on gastric epithelium in active chronic gastritis,”Lancet, 321:1273-75, 1983.
  4. B.J. Marshall et al., “Attempt to fulfil Koch’s postulates for pyloric Campylobacter,” Med J Aust, 142:436-39, 1985.
  5. J. Parsonnet et al., “Helicobacter pylori infection and the risk of gastric carcinoma,” N Engl J Med, 325:1127-31, 1991.
  6. S. Kado et al., “Intestinal microflora are necessary for development of spontaneous adenocarcinoma of the large intestine in T-cell receptor β chain and p53 double-knockout mice,” Cancer Res, 61:2395-98, 2001.
  7. J.V. Newman et al., “Bacterial infection promotes colon tumorigenesis in ApcMin/+ mice,” J Infect Dis, 184:227-30, 2001.
  8. S.E. Erdman et al., “CD4+ CD25+ regulatory T lymphocytes inhibit microbially induced colon cancer in Rag2-deficient mice,” Am J Pathol, 162:691-702, 2003.
  9. J.P. Zackular et al., “The human gut microbiome as a screening tool for colorectal cancer,” Cancer Prev Res, 7:1112-21, 2014.
  10. G. Zeller et al., “Potential of fecal microbiota for early-stage detection of colorectal cancer,” Mol Syst Biol, 10:766, 2014.
  11. J.P. Zackular et al., “The gut microbiome modulates colon tumorigenesis,” mBio, 4:e00692-13, 2013.
  12. J.P. Zackular et al., “Manipulation of the gut microbiota reveals role in colon tumorigenesis,”mSphere, doi:10.1128/mSphere.00001-15, 2015.
  13. V.P. Rao et al., “Innate immune inflammatory response against enteric bacteria Helicobacter hepaticus induces mammary adenocarcinoma in mice,” Cancer Res, 66:7395, 2006.
  14. A. Sivan et al., “Commensal Bifidobacterium promotes antitumor immunity and facilitates anti-PD-L1 efficacy,” Science, 350:1084-89, 2015.
  15. M. Vétizou et al., “Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota,”Science, 350:1079-84, 2015.

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Koji Atarashi · Takeshi Tanoue · Minoru Ando ·Nobuhiko Kamada ·….. · Yoshinori Umesaki · Kenya Honda ·

Microbially Driven TLR5-Dependent Signaling Governs Distal Malignant Progression through Tumor-Promoting Inflammation

Melanie R. Rutkowski, Tom L. Stephen, Nikolaos Svoronos, …., Julia Tchou,  Gabriel A. Rabinovich, Jose R. Conejo-Garcia
Cancer cell    12 Jan 2015; Volume 27, Issue 1, p27–40  http://dx.doi.org/10.1016/j.ccell.2014.11.009
Figure thumbnail fx1
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  • •TLR5-dependent IL-6 mobilizes MDSCs that drive galectin-1 production by γδ T cells
  • •IL-17 drives malignant progression in IL-6-unresponsive tumors
  • •TLR5-dependent differences in tumor growth are abrogated upon microbiota depletion
  • •A common dominant TLR5 polymorphism influences the outcome of human cancers

The dominant TLR5R392X polymorphism abrogates flagellin responses in >7% of humans. We report that TLR5-dependent commensal bacteria drive malignant progression at extramucosal locations by increasing systemic IL-6, which drives mobilization of myeloid-derived suppressor cells (MDSCs). Mechanistically, expanded granulocytic MDSCs cause γδ lymphocytes in TLR5-responsive tumors to secrete galectin-1, dampening antitumor immunity and accelerating malignant progression. In contrast, IL-17 is consistently upregulated in TLR5-unresponsive tumor-bearing mice but only accelerates malignant progression in IL-6-unresponsive tumors. Importantly, depletion of commensal bacteria abrogates TLR5-dependent differences in tumor growth. Contrasting differences in inflammatory cytokines and malignant evolution are recapitulated in TLR5-responsive/unresponsive ovarian and breast cancer patients. Therefore, inflammation, antitumor immunity, and the clinical outcome of cancer patients are influenced by a common TLR5 polymorphism.

see also… Immune Influence

In recent years, research has demonstrated that microbes living in and on the mammalian body can affect cancer risk, as well as responses to cancer treatment.

By Kate Yandell | April 1, 2016

http://www.the-scientist.com/?articles.view/articleNo/45644/title/Immune-Influence

Although the details of this microbe-cancer link remain unclear, investigators suspect that the microbiome’s ability to modulate inflammation and train immune cells to react to tumors is to blame. Here are some of the hypotheses that have come out of recent research in rodents for how gut bacteria shape immunity and influence cancer.

HOW THE MICROBIOME PROMOTES CANCER

Gut bacteria can dial up inflammation locally in the colon, as well as in other parts of the body, leading to the release of reactive oxygen species, which damage cells and DNA, and of growth factors that spur tumor growth and blood vessel formation.

http://www.the-scientist.com/images/April2016/ImmuneInfluence1_640px.jpg

http://www.the-scientist.com/images/April2016/ImmuneInfluence2_310px1.jpg

Helicobacter pylori can cause inflammation and high cell turnover in the stomach wall, which may lead to cancerous growth.

HOW THE MICROBIOME STEMS CANCER

Gut bacteria can also produce factors that lower inflammation and slow tumor growth. Some gut bacteria (e.g., Bifidobacterium)
appear to activate dendritic cells,
which present cancer-cell antigens to T cells that in turn kill the cancer cells.

http://www.the-scientist.com/images/April2016/ImmuneInfluence3_310px1.jpg

http://www.the-scientist.com/images/April2016/ImmuneInfluence4_310px1.jpg

Read the full story.

 

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Meeting Announcement: Cancer Immunotherapy and Combinations June 15-16 2016

Posted in Cancer - General, Cancer and Current Therapeutics, FDA, FDA Regulatory Affairs, Immunodiagnostics, Immunology, Immunotherapy, tagged , , , , , , , on February 12, 2016| Leave a Comment »

Meeting Announcement: Cancer Immunotherapy and Combinations June 15-16 2016

Reporter: Stephen J. Williams, PhD

 

Cancer Immunotherapy & Combinations – June 15-16, 2016 in Boston, MA

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Final Brochure PDF | Learn More | Sponsorship & Exhibit Details | Register by March 4 & SAVE up to $400!

Cambridge Healthtech Institute’s inaugural Cancer Immunotherapy and Combinations meeting will convene immuno-oncology researchers, cancer immunotherapy developers, and technology providers to discuss next-generation approaches and combinations, including small molecule development, to enhance the efficacy of checkpoint inhibitors.

BISPECIFIC ANTIBODIES – DUAL TARGETING

FEATURED PRESENTATION: ANTI-PD1 OR CD137 ENHANCES NK-CELL CYTOTOXICITY TOWARDS CD30+ HODGKIN LYMPHOMA INDUCED BY CD30/CD16A TANDAB AFM13
Martin Treder, Ph.D., CSO, R&D, Affimed

In vivo Efficacy of Bispecific Antibodies Targeting Two Immune-Modulatory Receptors
Jacqueline Doody, Ph.D., Vice President, Immunology, F-star Biotechnology, Ltd

Dual-Targeting Bispecific Antibodies for Selective Neutralization of CD47 on Cancer Cells
Krzysztof Masternak, Ph.D., Head, Biology, Therapeutic Antibody Discovery, Novimmune

Update on MCLA-134: A Biclonics® Binding Two Immunomodulatory Targets Expressed by T Cells
Mark Throsby, Ph.D., CSO, Merus

The ImmTAC Technology: A Cutting-Edge Immunotherapy for Cancer Treatment
Samir Hassan, Ph.D., Director, Translational Research & Development, Immunocore Ltd.

RADIOTHERAPY AND CHEMOTHERAPY – PD-1 COMBINATIONS

Rational Development of Combinations of Antiangiogenic Therapy with Immune Checkpoint Blockers Using Mouse Models of HCC and Cirrhosis
Dan Duda, D.M.D., Ph.D., Associate Professor, Steele Laboratories for Tumor Biology, Department of Radiation Oncology, Massachusetts General Hospital and Harvard Medical School

Harnessing the Immune Microenvironment of Gastrointestinal Cancers Using Combined Modalities
Osama Rahma, M.D., Assistant Professor, Internal Medicine/Oncology, University of Virginia

AGONIST – PD-1 AND CTLA-4 COMBINATIONS

The Role of the Target in the Disposition and Immunogenicity of an Anti-GITR Agonist Antibody
Enrique Escandón, Ph.D., Senior Principal Scientist, DMPK and Disposition, Merck

Combination of 4-1BB Agonist and PD-1 Antagonist Promotes Antitumor Effector/Memory CD8 T Cells
Changyu Wang, Ph.D., Director, Cancer Immunology, Pfizer

Combination Immunotherapy with Checkpoint Blockade, Agonist Anti-OX40 mAb, and Vaccination Rescues Anergic CD8 T Cells
William Redmond, Ph.D., Associate Member, Laboratory of Cancer Immunotherapy, Earle A. Chiles Research Institute, Providence Portland Medical Center

Interactive Breakout Discussion Groups with Continental Breakfast

This session features various discussion groups that are led by a moderator/s who ensures focused conversations around the key issues listed. Attendees choose to join a specific group and the small, informal setting facilitates sharing of ideas and active networking. Continental breakfast is available for all participants.

Topic: Small Molecule Targeting of IDO1 and TDO for Cancer Immunotherapy

Moderator: Rogier Buijsman, Ph.D., Head, Chemistry, Netherlands Translational Research Center B.V. (NTRC)

  • Overcoming challenges of current IDO1 inhibitors lacking selectivity over TDO and having suboptimal drug-like properties
  • Advances in IDO1 and TDO inhibitor screening
  • Is selective IDO1 or TDO inhibitors is required, or a dual IDO1/TDO inhibitor is preferred to obtain optimal efficacy and safety in the clinic?

Topic: Strategies for Developing Bispecific Antibodies for Cancer Immunotherapy

Moderator: Krzysztof Masternak, Ph.D., Head, Biology, Therapeutic Antibody Discovery, Novimmune

  • Considerations for efficacy in vitro and in vivo: selectivity for tumor cells, ADCP, ADCC, in vivo efficacy (xenograft models)
  • Insights into mechanisms of action
  • Safety considerations: binding selectivity, PK and tox studies

Topic: Combining Standard Antiangiogenic Therapy with Immune Checkpoint Inhibitors

Moderator: Dan Duda, D.M.D., Ph.D., Associate Professor, Steele Laboratories for Tumor Biology, Department of Radiation Oncology, Massachusetts General Hospital and Harvard Medical School

  • Will checkpoint combination with chemotherapy or other targeted agents prove to have too many toxicity issues?
  • How do we minimize overlapping toxic effects of radiation and immunotherapy?
  • How to optimize the sequencing of these two treatment modalities?

SMALL MOLECULE INHIBITORS AS SINGLE AND CHECKPOINT COMBINATION AGENTS

Selective Small Molecule Inhibitors of IDO1 and TDO for Cancer Immunotherapy
Rogier Buijsman, Ph.D., Head, Chemistry, Netherlands Translational Research Center B.V. (NTRC)

Potent and Selective Small Molecule USP7 Inhibitors for Cancer Immunotherapy
Suresh Kumar, Ph.D., Director, R&D, Progenra, Inc.

Epigenetic Agents for Combination with Cancer Immunotherapy
Svetlana Hamm, Ph.D., Head, Biology, Translational Pharmacology, 4SC Group

VACCINES AND CHECKPOINT BLOCKADE IMMUNOTHERAPY

Immunotherapy for Mesothelioma with an in vivo DC Vaccine and PD-1/PD-L1 Blockade
Huabiao Chen, M.D., Ph.D., Principal Investigator, Vaccine and Immunotherapy Center, Massachusetts General Hospital

Bringing Together Checkpoint Inhibition with Vaccines Using Customizing Capsids
Willie Quinn, Ph.D., President & CEO, Bullet Bio

Recommended All Access Package:

June 14 SC1: Immunosequencing: Generating a New Class of Cancer Immunotherapy Diagnostics*

June 14 SC5: Convergence of Immunotherapy and Epigenetics for Cancer Treatment*

June 14 SC8: Rational Design of Cancer Combination Therapies*

June 15-16: Cancer Immunotherapy and Combinations

June 16-17: Tumor Models for Cancer Immunotherapy

* Separate registration required.

Exhibit booth space has sold out! The few remaining spaces are being sold via sponsorship only. To customize yoursponsorship package, please contact:
Joseph Vacca, M.Sc., Associate Director, Business Development, 781-972-5431, jvacca@healthtech.com

For more information visit

WorldPreclinicalCongress.com/Cancer-Immunotherapy-Combinations

Cambridge Healthtech Institute, 250 First Avenue, Suite 300, Needham, MA 02494 healthtech.com
Tel: 781-972-5400 | Fax: 781-972-5425

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Leaders in the CAR-T Field Are Proceeding With Cautious Hope

Posted in Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Clinical & Translational, Immuno-Oncology & Genomics, Immunology, Immunotherapy, tagged , , , , , , , , , , , , on December 8, 2015| Leave a Comment »

 

Leaders in the CAR-T Field Are Proceeding With Cautious Hope

Reporter: Stephen J. Williams, Ph.D.

It wasn’t a long time ago, in fact the May 26, 2014 Cover Story in Forbes entitled “Is This How We’ll Cure Cancer” with cover photo of Novartis CEO Joseph Jimenez and subtitle “Will This man Cure Cancer?” highlighted the promise of CAR-T therapy as the ‘magic bullet’ therapy which will eventually cure all cancer. However, over the years, the pioneers of such therapy, while offering impressive clinical results, caution not to get to eager in calling CAR-T as the end-all-be-all cure but insist there are many issues that need be resolved.

The Allogenic Approach

In an interview for LabBiotech.eu Phillip Hemme had a discussion (and wonderful writeup) with André Choulika, the CEO of the French CAR-T miracle Cellectis on the current state of CAR-T therapy for cancer. Below is the interview in full as ther ae multiple important point Dr. Choulika mentioned, including how much is needed to be done in the field.

Cellectis’ CEO: “I’m just trying to be realistic, CAR-T is not THE miracle cure for Cancer”

 

 

Cellectis

CAR-T is solidifying in everybody’s mind as the next revolution in Cancer treatment. But there is still a lot to do…That’s basically what came out from my discussion with André Choulika, the CEO of the French CAR-T miracle Cellectis.

Cellectis is probably the most successful Biotech in France. It was founded in 1999 by Choulika himself (not alone though), following the discovery of meganucleases ability to change gene editing. Today, Cellectis is a well-known Biotech company counting over 100 employees end of October and having a market cap north of 1 Billion euros.

The company is now focused on the development of allogenic CAR-T (from generic donors  – i.e. not from the patient themself). With these universal CAR-T candidates (UCARTs). Cellectis has signed a massive partnership with the French pharma Servier, as well as Pfizer (which owns 8% of Cellectis), and has just announced two big milestones for the company within the last few weeks.

It is now able to produce it’s allogenic CAR-T in a GMP settings and it releases results from the “miracle” treatment of a 11-month old girl from the UK with multi-resistant leukemia.

 

Let’s start directly with the latest news…People seemed over-enthusiastic about UCART19…even the New York Times wrote about it. What do you think?

It’s a great news for Cellectis even though it’s still a very early result, in a single patient only. What’s important for us is that the first human patient received our treatment without showing any adverse effects (such as no cytokinetic storm) and our CAR-T cells were still active in the body 3 months after the injections.

Now, we have to expand the clinical trials to several patients and showing data from a cohort of patients. We are now on track to file the clinical trial application by the end of the year.

Your approach in the CAR-T is pretty unique. You are using donor’s cells to treat many different patients, whereas most CAR-T approaches are autologous (i.e. engineered the patient’s own cells).  Is the future in CAR-T the allogenic approach alone?

When we started to move into the CAR-T field we were pretty reluctant because there are not many examples of commercial success in the field yet. But CAR-T has still attracted many big players such as Novartis, Celgene, Juno or Kite. These each have a strong involvement in making autologous therapies work commercially (Celgene especially, which makes most of its revenue from groundbreaking and pricey cancer drugs).

On our side, we want to make this therapy accessible to a larger population and have good market access at the end. We have already pretty good reason to think it could work out well for us. We’ll see though…

Comment: Reuters published a report a few weeks ago estimating the cost of autologous CAR-T could be above $450K per treatment, which would make it economically not realistic for the healthcare payers.

CAR-T seems to be extremely hype right now. At BIO-Europe 2015, I had the impression everybody was talking about CAR-T. Do you think it could have the same impact as monoclonal antibodies?

What’s interesting with CAR-T is that you can target cells which expresses less receptors (10k receptors instead of 100k for monoclonal antibodies). This increases the targets for CAR-T and the possibilities linked.

But there are also downsides. Tissues with low expressions can become targets too and CAR-T cells would start attacking healthy cells.

People should not overemphasise CAR-T. We are still at the beginning of the beginning of this technology. And it will probably have to be combined with surgery or checkpoint inhibitors.

 

You seem pessimistic about CAR-T…?

I am just trying to be more realistic, even though I am super positive about the technology. It will bring something really great to Haematology field, but is not a cure for Cancer. It’s more of a long-haul race in the right direction as opposed to fast results, and we expect great things perhaps 20 years down the line as opposed to 2016.

But yes, it will probably not be the miracle product some people are talking about.

As for every early technology, there are many challenges associated with its development. What are the main ones worth discussing?

I would say you have four main challenges…

The administration of the cells will be challenging. We have to find way of injecting repeated doses of the product (to ensure the therapy is fully effective seeing as CAR-T cells have a limited lifespan). This is difficult because of immunogenicty against the therapy.

Secondly, combination will play an essential role too and checkpoint inhibitors should be involved.

The last two are linked to the targets.

As I mentioned before, CAR-T can be too sensitive and one way to control that would be to induce “logic gates” where the cells would only act if a combination of receptors would be present. The last challenge is to find other antigens.

Most of the CAR-T cells today target the same antigen: CD19+. We should find new antigens and many companies are on the track, including us.

 

CD19 CART

An anti-CD19 CAR-expressing T cell recognizing a CD19+ (Source: Kochenderfer et al., Nature Reviews Clinical Oncology 10, 267-276, doi: 10.1038/nrclinonc.2013.46)

Autologous CART therapy

Dr. Carl June of University of Pennsylvania, who has helped pioneer the field of CAR-T therapy for leukemia, has also been cautiously hopeful on the progress of the therapy. In his 2015 AACR National Meeting address, he highlighted some achievements they had with CAR-T therapy in both hematologic as well as solid tumors however it was stressed that there is much work to do with regards to optimization of the system, characterization of new tumor antigens for diverse tumor types, as well as the need to develop optimal treatment strategies to mitigate toxicities. Indeed many of the pioneers in the field have been proactive in helping to develop pharmacovigilance, safety, and regulatory strategies (highlighted in a post found here: NIH Considers Guidelines for CAR-T therapy: Report from Recombinant DNA Advisory Committee and mitigating toxicities in a post Steroids, Inflammation, and CAR-T Therapy) and much credit should be given to these researchers.

https://youtu.be/1sA_oz_1P5E

Cancer Research Institute’s Breakthroughs in Cancer Immunotherapy Webinar Series are offered free to the public and feature informative updates from leaders in cancer immunotherapy, followed by a moderated Q&A. On June 10, 2013, Carl H. June, M.D., a specialist in T cell biology and lymphocyte activation at the Perelman School of Medicine, University of Pennsylvania, discussed his groundbreaking work that has led to remarkable remissions of advanced cancer. He focused on recent and ongoing successes in developing treatments with T cells that have been genetically engineered to target cancer. Called chimeric antigen receptor T cells (CAR T cells), these modified immune cells have proven effective at eliminating cancer in some patients, and offer great hope for this emerging strategy in cancer immunotherapy. For more information on this webinar, or to register for upcoming webinars, please visit www.cancerresearch.org/webinars.

Below are reports from the 2015 American Society of Hematology Conference by Novartis on results from CTL109 CART therapy trials. One trial is on response rate in B-cell lymphomas and follicular cell lymphomas while the second report is ongoing trial results in childhood refractory ALL, both conducted at University of Pennsylvania.

Novartis presents response rate data for CART therapy CTL019 in lymphoma

(Ref: Global Post, NASDAQ, PR Newswire)

posted on FirstWorldPharma.com December 6th, 2015

By: Matthew Dennis

Novartis announced Sunday data from an ongoing Phase IIa study demonstrating that the experimental chimaeric antigen receptor T-cell (CART) therapy CTL019 led to an overall response rate (ORR) at three months of 47 percent in adults with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) and an ORR of 73 percent in adults with follicular lymphoma. The results of the trial were presented at the American Society of Hematology annual meeting.

Findings from the study, which was conducted by the University of Pennsylvania’s Perelman School of Medicine, include 15 adults with DLBCL and 11 with follicular lymphoma who were evaluable for response. Results showed that three patients with DLBCL who achieved a partial response (PR) to treatment at three months converted to complete response (CR) by six months. In addition, three patients with follicular lymphoma who achieved a PR at three months converted to CR by six months.

Novartis added that one DLBCL patient with a PR at three months experienced disease progression at six months after treatment. Further, one follicular lymphoma patient with a PR at three months who remained in PR at nine months experienced disease progression at approximately 12 months after treatment. The company indicated that median progression-free survival was 11.9 months for patients with follicular lymphoma and 3 months for those with DLBCL.

In the study, four patients developed cytokine release syndrome (CRS) of grade 3 or higher. Novartis noted that during CRS, patients typically experience varying degrees of influenza-like symptoms with high fevers, nausea, muscle pain, and in some cases, low blood pressure and breathing difficulties. Meanwhile, neurologic toxicity occurred in two patients in the trial, including one grade three episode of delirium and one possibly related grade five encephalopathy.

“These data add to the growing body of clinical evidence on CTL019 and illustrate its potential benefit in the treatment of relapsed and refractory non-Hodgkin lymphoma,” commented lead investigator Stephen Schuster. Novartis indicated that the findings keep CTL019 on track for submission to the FDA in 2017. Usman Azam, global head of Novartis’ cell and gene therapies unit, said “we remain consistent again with the data set.”

“It’s an attractive population, it’s a population that continues to have a huge unmet need, it’s a cornerstone of our investments,” Azam remarked. Analysts expect CART therapies, once approved, to cost up to $450 000 per patient. Novartis acknowledged that prices will be high, but declined to give further details. “With any disruptive innovation that comes, initially, cost of goods is very challenging,” Azam said, adding “as time goes on, and more patients are treated, we will simplify that cost base.”

Source: http://www.firstwordpharma.com/node/1338217?tsid=28&region_id=2#axzz3tfDVaT1f

 

 

Novartis AG (NVS)’s Experimental Therapy Wipes Out Blood Cancer in 93 Percent of Patients

Reported in Biospace.com (for full article see here)

Novaritis and University of Pennsylvania reported results of the CTL019 CART trials for the treatment of children with relapsed/refractory acute lymphoblastic leukemia at the 2015 Annual Hemotologic Society Meeting. 55 of 59 patients, or 93 percent, experienced complete remissions with CTL019. The study did show that at the end of one year, 55 percent of patients had a remission-free survival rate and that 18 patients continued to show complete remission following one year

 

Other posts on the Open Access Journal on CAR-T therapy include

 

CAR-T therapy in leukemia

Steroids, Inflammation, and CAR-T Therapy

NIH Considers Guidelines for CAR-T therapy: Report from Recombinant DNA Advisory Committee

 

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Cancer Immunotherapy

Posted in Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cancer Informatics, Clinical & Translational, Clinical Diagnostics, Computational Biology/Systems and Bioinformatics, Curation, Disease Biology, Genetics & Innovations in Treatment, Genetics & Pharmaceutical, Genome Biology, Human Immune System in Health and in Disease, Immuno-Oncology & Genomics, Immunology, tagged , , on October 27, 2015| Leave a Comment »

Cancer Immunotherapy

Curator: Larry H. Bernstein, MD, FCAP, Curator

 

 

Research explains limits of cancer immunotherapy drugs

Finding offers possibility to improve response to therapies

http://www.uofmhealth.org/news/archive/201510/research-explains-limits-cancer-immunotherapy-drugs

 

A new study from the University of Michigan Comprehensive Cancer Center reveals molecular changes within the tumor that are preventing the immunotherapy drugs from killing off the cancer.

Clinical trials with PD-L1 and PD-1 blockade suggested that tumors with a high number of inflammation-causing T cells were more responsive to the immunotherapy-based PD-L1 and PD-1 inhibitors. Tumors with low inflammation, or low T cells, were less responsive. But what controls T cells in the tumor microenvironment is poorly understood.

graphical image that shows how turning off a mechanism leads to more inflammation which means better drug response

http://www.uofmhealth.org/sites/default/files/styles/large/public/Immunotherapy.png?itok=_oihl0jj

 

“We defined a molecular mechanism to explain why some tumors are inflamed and others are not – and consequently why some patients will be responsive to therapy and others not,” says senior author Weiping Zou, M.D., Ph.D.

“If we can reprogram this epigenetic mechanism, then the therapy might work for more patients,” says Zou, Charles B. de Nancrede Professor of Surgery, Immunology and Biology at the University of Michigan Medical School.

Zou’s group was the first to show PD-L1 expression, regulation and functional blockade in dendritic cells in the human cancer microenvironment.

In this study, published in Nature, researchers studied human and mouse models of ovarian cancer cells. They applied epigenetic drugs and found that the numbers of T cells in the tumor increased. They also saw that the epigenetic drugs synergized the anti-tumor effect of PD-L1 blockade in their models.

“We hope this could be developed into a clinical trial testing a combination of PD-L1 and PD-1 blockade with epigenetic therapy. We want to see if we can make the responders more responsive and turn the non-responders into responders,” Zou says.

Additional authors: From U-M: Dongjun Pen, Ilona Kryczek, Nisha Nagarsheth, Lili Zhao, Shuang Wei, Weimin Wang, Yuqing Sun, Ende Zhao, Linda Vatan, Wojciech Szeliga, Yali Dou, Kathleen Cho, Rebecca Liu; from Medical University in Lublin, Poland: Jan Kotarski, Rafal Tarkowski; from Henry Ford Health System: Sharon Hensley-Alford, Adnan Munkarah

Funding: National Institutes of Health grants CA190176, CA123088, CA099985, CA193136, CA152470, CA171306, 5P30 CA46592; Rivkin Ovarian Cancer Center, Ovarian Cancer Research Fund, Barbara and Don Leclair

Cancer immunotherapy research is evolving to more targeted strategies

Discoveries in immune pathway research have helped refine cancer immunotherapy strategies to become more targeted.1,2

Image of timeline showing cancer immunotherapy research has evolved from general to targeted approaches over the years

 

http://www.researchcancerimmunotherapy.com/overview/targeted-cancer-research

  • CAR=chimeric antigen receptor;
  • CSF-1R=colony-stimulating factor 1; CTLA4=cytotoxic
  • T-lymphocyte antigen-4; IDO=indoleamine
  • 2,3-dioxygenase; PD-1=programmed death-1; PD-L1=programmed death-ligand 1.

http://www.researchcancerimmunotherapy.com/images/overview/evolution-research/history-of-immunotherapy.png

 

 

Engaging the immune response: a unique approach to cancer management

Cancer immunotherapy strategies are designed to engage the immune system against tumors. This approach is unique in the oncology setting and introduces new considerations for cancer management.1,2

  • TARGETED

    to tumor-specific antigens

  • RAPID

    activation of the immune response

  • ADAPTABLE

    as the tumor mutates and evolves

  • SELF-PROPAGATING

    with each revolution of the cancer immunity cycle

  • DURABLE

    response over time

 

CONSIDERATIONS FOR CANCER IMMUNOLOGY

Duration of response

The immune response has the ability to adapt with cancer as it evolves, and can become self-propagating once the cancer immunity cycle is initiated. Immune-directed strategies aim to leverage these attributes, with the goal of inducing a durable antitumor effect.3-5

Pseudo-progression

T-cell infiltration to the tumor site may cause an apparent increase in tumor size or the appearance of new lesions. This inflammatory effect can be misinterpreted as progressive disease, as it can be difficult to differentiate the different cell types in radiographic imaging. New criteria have been developed to better capture immune-related response patterns, and may guide evaluation of immunotherapies in clinical trials, and potentially in clinical care.1,2,6

Image showing T-cell infiltration into the tumor site can cause pseudoprogression]

REFERENCES

  1. Chen DS, Mellman I. Oncology meets immunology: the cancer-immunity cycle. Immunity. 2013;39:1-10. PMID: 23890059
  2. Mellman I, Coukos G, Dranoff G. Cancer immunotherapy comes of age. Nature. 2011;480:480-489. PMID: 22193102
  3. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144:646-674. PMID: 21376230

Immune-related adverse events

While the goal of cancer immunotherapy research is to understand how to activate specific components of the immune response, the potential for off-target effects exists. Adverse event profiles may vary among different immune-directed strategies. As strategies grow more targeted, the recognition and management of immune-related adverse events will evolve.1,3

EVADING IMMUNE DESTRUCTION
EVOLUTION OF RESEARCH

REFERENCES

  1. Mellman I, Coukos G, Dranoff G. Cancer immunotherapy comes of age. Nature. 2011;480:480-489. PMID: 22193102
  2. Hoos A, Eggermont AM, Janetzki S, et al. Improved endpoints for cancer immunotherapy trials. J Natl Cancer Inst. 2010;102:1388-1397.PMID: 20826737
  3. Chen DS, Mellman I. Oncology meets immunology: the cancer-immunity cycle. Immunity. 2013;39:1-10. PMID: 23890059
  4. Topalian SL, Weiner GJ, Pardoll DM. Cancer immunotherapy comes of age. J Clin Oncol. 2011;29:4828-4836. PMID: 22042955
  5. Chen DS, Irving BA, Hodi FS. Molecular pathways: next-generation immunotherapy—inhibiting programmed death-ligand 1 and programmed death-1. Clin Cancer Res. 2012;18:6580-6587. PMID: 23087408
  6. Wolchok JD, Hoos A, O’Day S, et al. Guidelines for the evaluation of immune therapy activity in solid tumors: immune-related response criteria. Clin Cancer Res. 2009;15:7412-7420. PMID: 19934295

 

Tumors can evade immune destruction

By disrupting the processes of the cancer immunity cycle throughout the body, tumors can avoid detection by the immune system and limit the extent of immune destruction.1-3

Image of dendritic cell capturing tumor antigens, step 2 of cancer immunity cycle

Tumor microenvironment

Disrupting antigen detection

http://www.researchcancerimmunotherapy.com/images/overview/evading-immune-destruction/tumor-microenv.png

 

Lymph node

Inhibiting T-cell activation by dendritic cells

http://www.researchcancerimmunotherapy.com/images/overview/evading-immune-destruction/lymph-node.png

Image of dendritic cell activating T cell, step 3 of cancer immunity cycle

Blood vessel

Blocking T-cell infiltration into tumor

http://www.researchcancerimmunotherapy.com/images/overview/evading-immune-destruction/blood-vessel.png

Image of T cell infiltrating tumor, step 5 of cancer immunity cycle

Tumor microenvironment

Suppressing cytotoxic T-cell activity

http://www.researchcancerimmunotherapy.com/images/overview/evading-immune-destruction/suppressing-tcell.png

 

EVADING IMMUNE DESTRUCTION IS AN EMERGING HALLMARK OF CANCER

Alt text: Image of hallmarks of cancer, which includes evading immune destruction

http://www.researchcancerimmunotherapy.com/images/overview/evading-immune-destruction/hallmark-cancer.png

The growing body of research into the mechanisms of immune evasion has led to its addition as an emerging hallmark of cancer, a distinct biological capability that enables tumors to grow and metastasize.3 

 

MUTATION RATE BY CANCER TYPE4

Image of mutation chart; melanoma, lung, and bladder cancer have high mutation rates

http://www.researchcancerimmunotherapy.com/images/tumor-types/immunogenic-cancer/immunogenic-cancer.png

REFERENCES

  1. Chen DS, Mellman I. Oncology meets immunology: the cancer-immunity cycle. Immunity. 2013;39:1-10. PMID: 23890059
  2. Chen DS, Irving BA, Hodi FS. Molecular pathways: next-generation immunotherapy—inhibiting programmed death-ligand 1 and programmed death-1. Clin Cancer Res. 2012;18:6580-6587. PMID: 23087408
  3. Rajasagi M, Shokla SA, Fritsch EF, et al. Systematic identification of personal tumor-specific neoantigens in chronic lymphocytic leukemia.Blood. 2014;124:453-462. PMID: 24891321
  4. Lawrence MS, Stojanov P, Polak P, et al. Mutational heterogeneity in cancer and the search for new cancer-associated genes. Nature.2013;499:214-218. PMID: 23770567

 

Atezolizumab (Anti-PDL1) clinical trials   

Atezolizumab clinical trials are being conducted for the following types of cancers: lung, bladder, kidney, hematological malignancies, and breast cancer. Please check back for updates or search clinicaltrials.gov with the search term “Atezolizumab.”

 

EXPLORING A MORE PERSONALIZED APPROACH TO CANCER IMMUNOTHERAPY RESEARCH

With the evolution to more targeted strategies, research is focusing on identifying predictors of individual immune response through specific tumor characteristics and factors in the tumor microenvironment, such as

  • The presence of tumor-infiltrating immune cells8
    • The ability of immune cells to infiltrate the tumor microenvironment may be a key criterion for a variety of immune-directed strategies, and could indicate which tumors are more likely to respond
  • Gene expression patterns in tumors, particularly the genes involved in immune response9
  • Cell surface protein expression
    • PD-L1 expression on tumor cells and tumor-infiltrating immune cells10,11
    • MUC1 expression on tumor cells12     

REFERENCES

  1. Chen DS, Mellman I. Oncology meets immunology: the cancer-immunity cycle. Immunity. 2013;39:1-10. PMID: 23890059
  2. Mellman I, Coukos G, Dranoff G. Cancer immunotherapy comes of age. Nature. 2011;480:480-489. PMID: 22193102
  3. Lesterhuis WJ, Haanen JB, Punt CJ. Cancer immunotherapy—revisited. Nat Rev Drug Discov. 2011;10:591-600. PMID: 21804596
  4. National Institutes of Health ClinicalTrials.gov. https://clinicaltrials.gov/ct2/show/NCT01494688. Accessed March 4, 2015.
  5. National Institutes of Health ClinicalTrials.gov. https://clinicaltrials.gov/ct2/show/NCT00739609. Accessed March 4, 2015.
  6. Glienke W, Esser R, Priesner C, et al. Advantages and applications of CAR-expressing natural killer cells. Front Pharmacol. 2015;6:21. doi: 10.3389/fphar.2015.00021. PMID: 25729364
  7. National Institutes of Health ClinicalTrials.gov. https://clinicaltrials.gov/ct2/show/NCT01303705. Accessed March 4, 2015.
  8. Gajewski TF, Schreiber H, Fu YX. Innate and adaptive immune cells in the tumor microenvironment. Nat Immunol. 2013;14:1014-1022.PMID: 24048123
  9. Ji RR, Chasalow SD, Wang L, et al. An immune-active tumor microenvironment favors clinical response to ipilimumab. Cancer Immunol Immunother. 2012;61:1019-1031. PMID: 22146893
  10. Taube JM, Anders RA, Young GD, et al. Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape. Sci Transl Med. 2012;4:127ra37. PMID: 22461641
  11. Chen DS, Irving BA, Hodi FS. Molecular pathways: next-generation immunotherapy—inhibiting programmed death-ligand 1 and programmed death-1. Clin Cancer Res. 2012;18:6580-6587. PMID: 23087408
  12. Stojnev S, Ristic-Petrovic A, Velickovic LJ, et al. Prognostic significance of mucin expression in urothelial bladder cancer. Int J Clin Exp Pathol. 2014;7:4945-4958. PMID: 25197366
  13. Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer. 2012;12:252-264. PMID: 22437870
  14. Taylor RC, Patel A, Panageas KS, Busam KJ, Brady MS. Tumor-infiltrating lymphocytes predict sentinel lymph node positivity in patients with cutaneous melanoma. J Clin Oncol. 2007;25:869-875. PMID: 17327608

 

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Monoclonal Antibody Therapy and Market

Posted in CANCER BIOLOGY & Innovations in Cancer Therapy, Genome Biology, Personalized and Precision Medicine & Genomic Research, Translational Science, tagged , , , , on October 14, 2015| Leave a Comment »

Monoclonal Antibody Therapy and Market

Curator: Demet Sag, PhD, CRA, GCP 

 

Monoclonal Antibody treatment means a biological therapy where monoclonal antibodies is used to initiate development of specific antibodies (protein molecules produced by the B cells as a primary immune defense), so that they can fight against antigens (substances that are capable of inducing a specific immune response) specifically to kill extracellular/ cell surface target.  Thus, the application of this types of therapies are not limited to cancer but also rheumatoid arthritis, multiple sclerosis, Alzheimer’s disease, and some infectious diseases such as Ebola.

To eliminate or reduce the effects of chemotherapeutic agents. Thus chemotherapeutics agents attached to monoclonal antibodies.

Diagnostic process:

Monoclonal antibodies again used as a vehicle to locate the tumorigenic cancer cells in the body. There can be several methods but one of them is carrying radioactive substances to cancer cells so that they can be labelled in vivo.  However, there are less invasive ways to do as well. As a result, there are new combination of methods such as:

  • nuclear imaging,
  • surgical mapping, and
  • direct therapy in multiple settings either alone, or in conjunction with chemotherapeutic agents, adjuvant.

How do monoclonal antibody drugs work?

 525px-Monoclonal_antibodies.svg

  1. Naked monoclonal antibodies:
  • Make the cancer cell more visible to the immune system.

Action is to boost immune system.

Example: Alemtuzumab (Campath®), chronic lymphocytic leukemia (CLL) by binding to the CD52 antigen on lymphocytes.

 

T cell targets for immunoregulatory antibody therapy

  • Block immune checkpoint inhibitor proteins

        

 Treatments that target PD-1 or PD-L1.

 PD-1 is a checkpoint protein on T cells, called “off switch” of T cells since PD-1 prevents from attacking other cells in the body. Yet, when it is overexpressed on the cancer cells, tumors escape from immune system, because when PD-1 binds to PD-L1, T cells thinks these cells are body’s own normal cells.

http://www.nature.com/nature/journal/v515/n7528/images/515496a-f1.jpg

Checkpoint blockade activates antitumour immunity.

a, Tumour cells express both cancer-driving mutations and ‘passenger’ mutations that cause the expression of neoantigens — ‘new’ molecular structures that, when presented by MHC proteins on the cell surface, are recognized by T cells of the immune system as being foreign, leading to an immune response against the tumour. However, interactions between the receptor PD-1 and its ligand PD-L1, which are expressed on tumour cells, T cells and other immune cells such as macrophages, activate signalling pathways that inhibit T-cell activity and thus inhibit the antitumour immune response. b, Antibodies that block the PD-1 pathway by binding to PD-1 or PD-L1 can reactivate T-cell activity and proliferation, leading to enhanced antitumour immunity.

Examples are:

  • Pembrolizumab (Keytruda®)
  • Nivolumab (Opdivo®)

There is a possibility of developing an autoimmune reaction. The most common side effects include fatigue, cough, nausea, skin rash, and itching. Rarely more serious problems in the lungs, intestines, liver, kidneys, hormone-making glands, or other organs may occur.

 Treatments that target CTLA-4

 Another protein is CTLA-4 to control T cells, “off switch”.

Generation and regulation of anti-tumor immunity Biologic activities of CTLA-4 antibody blockade

Example: Ipilimumab (Yervoy®) is a monoclonal antibody that attaches to CTLA-4 and stops it from working. This can boost the body’s immune response against cancer cells.

  • Block antigens on cancer cells (or other nearby cells).

Example: Trastuzumab, when HER2 is activated, binds to these proteins and stops antigens from becoming active in breast and stomach cancer cells.

Example: Rituxan specifically attaches to CD20 that is found only on B cells so when these labelled B cells can be visible to immune system. There are certain types of lymphomas predisposed due to malfunctioning B cells.

  • Block growth signals. Prevent signal amplification for cell growth.

The cells like to amplify their message in danger or during certain metabolisms so they secrete or produce a type of chemicals called growth factors.  These factors then attaches to specific receptors on the surface of normal cells and cancer cells. Thus, signaling the cells to grow faster than the normal cells. The action is preventing the signals to be received by monoclonal.

 Example:

Cetuximab (Erbitux), targets epidermal growth factor. Thus its function utilized to cure colon cancer, head and neck cancers.

  • Stop new blood vessels from forming.

Tumors needs to grow so in the body they need blood vessel formation to feed the cell growth (angiogenesis)

Example; Bevacizumab (Avastin) targets vascular endothelial growth factor (VEGF) and blocks the angiogenesis.

  1. Conjugated monoclonal antibodies (tagged, labeled, or loaded antibodies).

 Deliver chemotherapy to cancer cells.

They are monoclonal antibodies (mAbs) joined to a chemotherapy drug or to a radioactive particle to locate cancer cells directly through targeting specific antigen after circulating in the bloodstream. They are used as a homing device.

Chemo-labeled antibodies: Also called as antibody-drug conjugates (ADCs) and provide powerful chemotherapy (or other) drugs attached to them.

  • Brentuximab vedotin (Adcetris®), an antibody that targets the CD30 antigen on lymphocytes, attached to MMAE (a chemo drug) against Hodgkin lymphoma and anaplastic large cell lymphoma.
  • Ado-trastuzumab emtansine (Kadcyla®, also called TDM-1), an antibody that targets the HER2 protein, attached to DM1 (a chemo drug) against cells overexpressing HER2 in breast cancer

 Toxin attached protein: Denileukin diftitox (Ontak®) is not an antibody but it is a protein, cytokine known as interleukin-2 (IL-2) and attached to diphtheria toxin that recognizes CD25 antigen to treat lymphoma of the skin (cutaneous T-cell lymphoma).

 Radiolabeled antibodies: Deliver radiation to cancer cells.

The other method, less preferred, is radiation-linked monoclonal antibodies.  This time low radiation in long term used to target the cancer cells but it is suggested that this method has elevated outcome to kill the cancer cells than conventional high-dose external beam radiation.

Example; Ibritumomab (Zevalin), is an approved treatment.  The targeted disease is for non-Hodgkin’s lymphoma.

Treatment with this type of antibody also referred as radioimmunotherapy (RIT).

  1. Bispecific monoclonal antibodies

 If the drug contains two parts of 2 different mAbs, meaning they can attach to 2 different proteins at the same time, they are called Bispecific monoclonal antibodies since they attack two proteins at the same time.

 

Example:  Blinatumomab (Blincyto), can attach CD 19 which is found on some leukemia and lymphoma cells and CD3 on T cells.  Thus, brings opponents, immune and malignant cancer cells, to defeat cancer.

  nature_graphic_immune-system_08.01.15

THE OTHER SIDE OF THE COIN: SAFETY

 Possible side effects of monoclonal antibodies

 Delivery is intravenously and since Mabs are themselves are proteins sometimes presents side effects like an allergic reaction yet compared to chemotherapy drugs these effects are much less. .

  • Fever
  • Chills
  • Weakness
  • Headache
  • Nausea
  • Vomiting
  • Diarrhea
  • Low blood pressure
  • Rashes

Examples:

  • Bevacizumab (Avastin®), high blood pressure, bleeding, poor wound healing, blood clots, and kidney damage.
  • Cetuximab (Erbitux®), serious rashes in some people.

Manufacturing of Monoclonal Antibodies and Market

“Since 2000, the therapeutic market for monoclonal antibodies has grown exponentially. The current “big 5” therapeutic antibodies on the market are bevacizumab, trastuzumab (both oncology), adalimumab, infliximab (both autoimmune and inflammatory disorders, ‘AIID’) and rituximab (oncology and AIID) accounted for 80% of revenues in 2006. In 2007, eight of the 20 best-selling biotechnology drugs in the U.S. are therapeutic monoclonal antibodies. Scolnik, Pablo A. (2009). “mAbs: A business perspective”. MAbs 1 (2): 179–184. doi:10.4161/mabs.1.2.7736. PMC 2725420. PMID 20061824.

This rapid growth in demand for monoclonal antibody production has been well accommodated by the industrialization of mAb manufacturing”. Kelley, Brian (2009). “Industrialization of mAb production technology”. MAbs 1 (5): 443–452. doi:10.4161/mabs.1.5.9448. PMC 2759494. PMID 20065641.

mabs0105_0443_fig001http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2759494/bin/mabs0105_0443_fig001.jpg

Model mAb production plant design and capabilities. A model large scale mAb production plant employs multiple bioreactors configured to supply a single purification train. A plant having six individual 15 kL bioreactors is potentially capable of supplying 10 tons of purified mAb per year using conventional technologies, or 4–5 products with 1 ton demands. This enormous capacity per plant would result in a marked decrease in drug substance production costs, and results in significant excess capacity throughout the biopharmaceutical industry.

Production:

Production capacity estimates for mammalian cell-derived mAbsa

Year CMO Product company Total Capacity at 2 g/L Capacity at 5 g/L
2007 500 kL 1,800 kL 2,300 kL 70 tons/yr 170 tons/yr
2010 700 kL 2,700 kL 3,400 kL 100 tons/yr 255 tons/yr
2013 1,000 kL 3,000 kL 4,000 kL 120 tons/yr 300 tons/yr

aCapacity estimates from ref. Ransohoff TC, Ecker DM, Levine HL, Miller J. Cell culture manufacturing capacity: trends and outlook through 2013. PharmSource. 2008

mabs0105_0443_fig002

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2759494/bin/mabs0105_0443_fig003.jpg

Estimated demand for therapeutic mAbs and Fc-fusion products in 2009. The total demand for the top 15 mAbs and Fc-fusions in 2009 is estimated to be approximately 7 tons, with the four largest volume products requiring approximately one ton per year. More than half of the products were estimated to require less than 200 kg per year.

mabs0105_0443_fig004 mabs0105_0443_fig003

Distribution of average wholesale prices for mAb and Fc-fusions in 2008. The average U.S. wholesale prices per gram for 15 commercial mAbs and Fc-fusions are shown. The minimum is approximately $2,000 per gram, and the median is approximately $8,000 per gram. Note that a significant price erosion (50% of the minimum shown here) for a product with modest demand (100 kg/yr) could result in an unprofitable market, as revenues for the therapeutic product ($100 million/yr) may never provide a positive return on investment.

Sensitivity analysis of mAb drug substance COGs for the model plant (six 15kL bioreactors)

Titer (g/L) Plant capacity (tons/yr) Raw materials ($/gm) Depreciation & labor ($/gm)b Fill/Finish costs per vial ($) Total Drug Product Cost ($/vial)
Cell culturea Purification 100 mg 1 gm
0.5 1 20 100 22 134
2 4 4 4 25 10 13 43
5 10 2 10 12 26

aAssumes medium cost of $8/L.

bBased on the model plant ($500 M capital investment + 250 staff = $100 M per year).

Estimated cost breakdown for three production scenarios

Model large-scale plant Small-scale plant using disposables CMO
Basis: 5 g/L 6 × 15 kL n × 2 kL 15 kL
Capital Investmenta $500 M $125 M – Difference in annual cost for two best alternatives ($M/yr)
Depreciationb($/yr) $50 M $12.5 M –
Raw Materialsc $10/gm $20/gm $10/gm
Labor ($/yr)d $50 M $20 M –
CMO – – $3 M/batche
COGs $/gm 10 ton/yr 20 23 60 $30 M
1 ton/yr 110 53 60 $7 M
0.1 ton/yr 1,010 345 60 $29 M

aThe new facility based on disposables is assumed to cost just one-quarter of model plant to build, and uses only the number of bioreactors (‘n’) needed to satisfy the demand.

bA 10-year straight line depreciation is used to estimate the depreciation costs.

cRaw material costs per gram are assumed to be slightly higher for the disposable facility.

dLabor costs for the new facility are assumed to be just 40% of the model plant (100 vs 250 staff, respectively).

eA constant cost per batch is assumed for the CMO, all-inclusive of production, testing and release.

Sales and Marketing

PMC full text: MAbs. 2009 Mar-Apr; 1(2): 179–184.

FDA-approved marketed mAbs

Name Structure Target Indication Path Approval (Y) Sales % Top 20
Generic Trade Landing Expansion
First Tier (U.S. $B)
infliximab Remicade® Ch TNF CD RA O, A, P, F 4.6 $5.0 9.84
AS
PA
UC
PP
rituximab Rituxan®, Ch CD20 NHL RA O, P 5.1 $4.9 9.62
MabThera® DLBC
1-NHL
trastuzumab Herceptin® Hm HER2 mBC BC F, P 7.5 $4.3 8.45
bevacizumab Avastin® Hm VEGF mCRC mCRC F, P 7.1 $3.6 7.15
NSCLC
HER2- BCa
adalimumab Humira® Hu TNF RA RA O 3.7 $3.1 6.04
JIA
PA
AS
CD
PP
cetuximab Erbitux® Ch EGFR mCRC SCCHN A, P 9.7 $1.4 2.73
ranibizumab Lucentis® Hm VEGF AMD P 6.8 $1.2 2.39
palivizumab Synagis® Hm RSV RSV P 3.6 $1.1 2.25
Second Tier (U.S. $M)
tositumomab Bexxar® Mu CD20 NHLb NHLc 13.7 $10.3 0.02
alemtuzumab Campath® Hm CD52 B-CLL B-CLLd A, P, F 10.4e $108.0 0.21
certolizumab pegol Cimzia® Hm TNF CD P n/a n/a n/a
gemtuzumab ozogamicin Mylotarg® Hm CD33 AML P, A, O 6.5 $60.0 0.12
muromonab-CD3 Orthoclone Okt3® Mu CD3 OR OR n/a $150.0 0.30
efalizumab Raptçiva® Hm CD11a PS 10e $163.0 0.32
abciximab ReoPro® Ch GP IIb/IIIa AC CI O n/a $380.0 0.75
basiliximab Simulect® Ch CD25 OR O, P n/a $300.0 0.59
eculizumab Soliris® Hm C5 PNH O, P n/a $230.0 0.45
natalizumab Tysabri® Hm a-4 integrin MS CD A 10.6e $100.0 0.20
panitumumab Vectibix® Hu EGFR mCRC A, P, F 7.4 $365.0 0.72
omalizumab Xolair® Hm IgE AA 9.7 $472.0 0.93
daclizumab Zenapax® Hm CD25 OR ORp O, P n/a $60.0 0.12
ibritumomab tiuxetan Zevalin® Mu CD20 NHL P, A, O, F 10.2 $17.0 0.03

Abbreviations: Structure: Ch, chimeric; Hm, humanized; Hu, human; Mu, murine. Regulatory Path: A, accelerated approval; F, fast-track; P, priority review; O, orphan indication. 1-, first-line therapy; a, conditional approval; b, rituximab refractory; c, refractory to chemotherapy; d, single-agent; e, estimate; m, metastatic; n/a, information not available; p, prophylaxis. Sources: 20 Compounds that defined biotech, Signals online magazine at www.signalsmag.com; ReCap database; Biopharmaceutical Products in the U.S. and European markets 6th edition, Ronald A. Rader, ed; Pharma Sales and BioPharmInsights databases; Reichert JM, Ph. D.; personal communications. Development times and sales estimates for some Second Tier mAbs are based on limited information.

References for Cancer Immunotherapy

Ault KA, Future II study group. Effect of prophylactic human papillomavirus L1 virus-like-particle vaccine on risk of cervical intraepithelial neoplasia grade 2, grade 3, and adenocarcinoma in situ: A combined analysis of four randomised clinical trials. Lancet. 2007;369:1861−1868.

Brahmer J, Reckamp KL, Baas P, et al. Nivolumab versus docetaxel in advanced squamous-cell non-small-cell lung cancer. N Engl J Med. 2015;373:123−135.

Chesney J, Rasku M, Clem A, Miller D. Denileukin diftitox depletes T regulatory cells and causes regression of melanoma metastases in humans. Eur J Cancer Suppl. 2006;4:12:84.

Cheung NV. Chapter 32: Therapeutic antibodies and immunologic conjugates. In: Niederhuber JE, Armitage JO, Doroshow JH, Kastan MB, Tepper JE, eds. Abeloff’s Clinical Oncology. 5th ed. Philadelphia, Pa: Elsevier; 2014.

Dudley ME, Wunderlich JR, Robbins PF, et al. Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes. Science. 2002;298:850−854.

Fry TJ, Mackall CL. T-cell adoptive immunotherapy for acute lymphoblastic leukemia. Hematology Am Soc Hematol Educ Program. 2013;2013:348−353.

Garon EB, Rizvi NA, Hui R, et al. Pembrolizumab for the treatment of non-small-cell lung cancer. N Engl J Med. 2015;372:2018−2028.

Grupp SA, Kalos M, Barrett D, et al. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med. 2013;368:1509−1518.

Hamid O, Robert C, Daud A, et al. Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma. N Engl J Med. 2013;369:134−144.

Higano CS, Schellhammer PF, Small EJ, et al. Integrated data from 2 randomized, double-blind, placebo-controlled, phase 3 trials of active cellular immunotherapy with sipuleucel-T in advanced prostate cancer. Cancer. 2009;115:3670−3679.

Hodi FS, O’Day SJ, McDermott DF, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med. 2010;363:711−723.

Larkin J, Chiarion-Sileni V, Gonzalez R, et al. Combined nivolumab and ipilimumab or monotherapy in untreated melanoma. N Engl J Med. 2015;373:23−34.

Le DT, Uram JN, Wang H, et al. PD-1 blockade in tumors with mismatch-repair deficiency. N Engl J Med. 2015;372:2509−2520.

Morgan RA, Dudley ME, Wunderlich JR, et al. Cancer regression in patients after transfer of genetically engineered lymphocytes. Science. 2006;314:126−129.

National Cancer Institute. CAR T-Cell Therapy: Engineering Patients’ Immune Cells to Treat Their Cancers. 2014. Accessed at http://www.cancer.gov/about-cancer/treatment/research/car-t-cells on July 20, 2015.

Pardoll D. Chapter 6: Cancer immunology. In: Niederhuber JE, Armitage JO, Doroshow JH, Kastan MB, Tepper JE, eds. Abeloff’s Clinical Oncology. 5th ed. Philadelphia, Pa: Elsevier; 2014.

Robbins PF, Morgan RA, Feldman SA, et al. Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1. J Clin Oncol. 2011;29:917−924.

Robert C, Schachter J, Long GV, et al. Pembrolizumab versus ipilimumab in advanced melanoma. N Engl J Med. 2015;372:2521−2532.

Borghaei H, Robinson MK, Adams GP, Weiner LM. Chapter 29: Monoclonal antibodies. In: DeVita VT, Lawrence TS, Rosenberg SA, eds. DeVita, Hellman, and Rosenberg’s Cancer: Principles & Practice of Oncology. 10th ed. Philadelphia, Pa: Lippincott Williams & Wilkins; 2015.

Rosenberg SA, Robbins PF, Phan GQ, Feldman SA, Kochenderfer JN. Chapter 14: Cancer immunotherapy. In: DeVita VT, Lawrence TS, Rosenberg SA, eds. DeVita, Hellman, and Rosenberg’s Cancer: Principles & Practice of Oncology. 10th ed. Philadelphia, Pa: Lippincott Williams & Wilkins; 2015.

Schlom J. Therapeutic cancer vaccines: Currrent status and moving forward. J Natl Cancer Inst. 2012;104:599−613.

Verma S, Miles D, Gianni L, et al. Trastuzumab emtansine for HER2-positive advanced breast cancer. N Engl J Med. 2012;367:1783−1791.

Bertotti A, Papp E, Jones S, Adleff V, Anagnostou V, Lupo B, Sausen M, Phallen J, Hruban CA, Tokheim C, Niknafs N, Nesselbush M, Lytle K, Sassi F, Cottino F, Migliardi G, Zanella ER, Ribero D, Russolillo N, Mellano A, Muratore A, Paraluppi G, Salizzoni M, Marsoni S, Kragh M, Lantto J, Cassingena A, Li QK, Karchin R, Scharpf R, Sartore-Bianchi A, Siena S, Diaz LA Jr, Trusolino L, Velculescu VE. The genomic landscape of response to EGFR blockade in colorectal cancer.Nature. 2015 Oct 8;526(7572):263-7. doi: 10.1038/nature14969. Epub 2015 Sep 30. PubMed PMID: 26416732.

Kondo A, Shahpasand K, Mannix R, Qiu J, Moncaster J, Chen CH, Yao Y, Lin YM, Driver JA, Sun Y, Wei S, Luo ML, Albayram O, Huang P, Rotenberg A, Ryo A,

Goldstein LE, Pascual-Leone A, McKee AC, Meehan W, Zhou XZ, Lu KP. Antibody against early driver of neurodegeneration cis P-tau blocks brain injury and tauopathy. Nature. 2015 Jul 23;523(7561):431-6. doi: 10.1038/nature14658. Epub 2015 Jul 15. PubMed PMID: 26176913.

Spranger S, Bao R, Gajewski TF Melanoma-intrinsic β-catenin signalling prevents anti-tumour immunity.  Nature. 2015 Jul 9;523(7559):231-5. doi:

10.1038/nature14404. Epub 2015 May 11. PubMed PMID: 25970248.

Caskey M, Klein F, Lorenzi JC, Seaman MS, West AP Jr, Buckley N, Kremer G,Nogueira L, Braunschweig M, Scheid JF, Horwitz JA, Shimeliovich I, Ben-Avraham S,Witmer-Pack M, Platten M, Lehmann C, Burke LA, Hawthorne T, Gorelick RJ, Walker BD, Keler T, Gulick RM, Fätkenheuer G, Schlesinger SJ, Nussenzweig MC. Viraemia suppressed in HIV-1-infected humans by broadly neutralizing antibody3BNC117. Nature. 2015 Jun 25;522(7557):487-91. doi: 10.1038/nature14411. Epub 2015 Apr 8. PubMed PMID: 25855300.

Ledford H. First biosimilar drug set to enter US market.Nature. 2015 Jan15;517(7534):253-4. doi: 10.1038/517253a. PubMed PMID: 25592512.

Gitlin AD, Nussenzweig MC. IImmunology: Fifty years of B lymphocytes. Nature.

2015 Jan 8;517(7533):139-41. doi: 10.1038/517139a. PubMed PMID: 25567266.

Gubin MM, Zhang X, Schuster H, Caron E, Ward JP, Noguchi T, Ivanova Y, Hundal J, Arthur CD, Krebber WJ, Mulder GE, Toebes M, Vesely MD, Lam SS, Korman AJ, Allison JP, Freeman GJ, Sharpe AH, Pearce EL, Schumacher TN, Aebersold R, Rammensee HG, Melief CJ, Mardis ER, Gillanders WE, Artyomov MN, Schreiber RD. Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens. Nature. 2014 Nov 27;515(7528):577-81. doi: 10.1038/nature13988. PubMed PMID: 25428507; PubMed Central PMCID: PMC4279952.

Herbst RS, Soria JC, Kowanetz M, Fine GD, Hamid O, Gordon MS, Sosman JA,McDermott DF, Powderly JD, Gettinger SN, Kohrt HE, Horn L, Lawrence DP, Rost S,Leabman M, Xiao Y, Mokatrin A, Koeppen H, Hegde PS, Mellman I, Chen DS, Hodi FS.Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancerpatients. Nature. 2014 Nov 27;515(7528):563-7. doi: 10.1038/nature14011. PubMed PMID: 25428504.

Powles T, Eder JP, Fine GD, Braiteh FS, Loriot Y, Cruz C, Bellmunt J, Burris HA, Petrylak DP, Teng SL, Shen X, Boyd Z, Hegde PS, Chen DS, Vogelzang NJ.

MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer. Nature. 2014 Nov 27;515(7528):558-62. doi: 10.1038/nature13904. PubMed PMID: 25428503.

Wolchok JD, Chan TA. Cancer: Antitumour immunity gets a boost. Nature. 2014 Nov 27;515(7528):496-8. doi: 10.1038/515496a. PubMed PMID: 25428495.

Hoag H. Drug development: a chance of survival. Nature. 2014 Nov 20;515(7527):S118-20. doi: 10.1038/515S118a. PubMed PMID: 25407709.

Ledford H. Cancer treatment: The killer within. Nature. 2014 Apr 3;508(7494):24-6. doi: 10.1038/508024a. PubMed PMID: 24695297.

Roederer M, Keele BF, Schmidt SD, Mason RD, Welles HC, Fischer W, Labranche C, Foulds KE, Louder MK, Yang ZY, Todd JP, Buzby AP, Mach LV, Shen L, Seaton KE, Ward BM, Bailer RT, Gottardo R, Gu W, Ferrari G, Alam SM, Denny TN, Montefiori DC, Tomaras GD, Korber BT, Nason MC, Seder RA, Koup RA, Letvin NL, Rao SS, Nabel GJ, Mascola JR. Immunological and virological mechanisms of vaccine-mediated protection against SIV and HIV. Nature. 2014 Jan 23;505(7484):502-8. doi: 10.1038/nature12893. Epub 2013 Dec 18. PubMed PMID: 24352234; PubMed Central PMCID: PMC3946913.

Weintraub K. Drug development: Releasing the brakes. Nature. 2013 Dec 19;504(7480):S6-8. doi: 10.1038/504S6a. PubMed PMID: 24352363.

Elert E. Calling cells to arms. Nature. 2013 Dec 19;504(7480):S2-3. doi: 10.1038/504S2a. PubMed PMID: 24352361.

Ledford H. Immunotherapy’s cancer remit widens. Nature. 2013 May 30;497(7451):544. doi: 10.1038/497544a. PubMed PMID: 23719439.

Ledford H. Sizing up a slow assault on cancer. Nature. 2013 Apr 4;496(7443):14-5. doi: 10.1038/496014a. PubMed PMID: 23552923.

Savage N. Modelling: Computing cancer. Nature. 2012 Nov 22;491(7425):S62-3. PubMed PMID: 23320290.

Diaz LA Jr, Williams RT, Wu J, Kinde I, Hecht JR, Berlin J, Allen B, Bozic I, Reiter JG, Nowak MA, Kinzler KW, Oliner KS, Vogelstein B. The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers. Nature. 2012 Jun 28;486(7404):537-40. doi: 10.1038/nature11219. PubMed PMID: 22722843; PubMed Central PMCID: PMC3436069.

Misale S, Yaeger R, Hobor S, Scala E, Janakiraman M, Liska D, Valtorta E, Schiavo R, Buscarino M, Siravegna G, Bencardino K, Cercek A, Chen CT, Veronese S,

Zanon C, Sartore-Bianchi A, Gambacorta M, Gallicchio M, Vakiani E, Boscaro V, Medico E, Weiser M, Siena S, Di Nicolantonio F, Solit D, Bardelli A. Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer. Nature. 2012 Jun 28;486(7404):532-6. doi: 10.1038/nature11156. PubMed PMID: 22722830; PubMed Central PMCID: PMC3927413.

Vilar E, Tabernero J. Cancer: Pinprick diagnostics. Nature. 2012 Jun 27;486(7404):482-3. doi: 10.1038/486482a. PubMed PMID: 22739312.

Hayden EC. Antibody alarm call rouses immune response to cancer. Nature. 2012 Jun 6;486(7401):16. doi: 10.1038/486016a. PubMed PMID: 22678259.

Steeg PS. Perspective: The right trials. Nature. 2012 May 30;485(7400):S58-9. doi: 10.1038/485S58a. PubMed PMID: 22648501.

Prahallad A, Sun C, Huang S, Di Nicolantonio F, Salazar R, Zecchin D,

Beijersbergen RL, Bardelli A, Bernards R. Unresponsiveness of colon cancer to BRAF(V600E) inhibition through feedback activation of EGFR. Nature. 2012 Jan 26;483(7387):100-3. doi: 10.1038/nature10868. PubMed PMID: 22281684.

Mellman I, Coukos G, Dranoff G. Cancer immunotherapy comes of age. Nature. 2011 Dec 21;480(7378):480-9. doi: 10.1038/nature10673. Review. PubMed PMID: 22193102; PubMed Central PMCID: PMC3967235.

Ledford H. Melanoma drug wins US approval. Nature. 2011 Mar 31;471(7340):561. doi: 10.1038/471561a. PubMed PMID: 21455150.

Dolgin E. FDA narrows drug label usage. Nature. 2009 Aug 27;460(7259):1069. doi: 10.1038/4601069a. PubMed PMID: 19713906.

Ellis LM, Reardon DA. Cancer: The nuances of therapy. Nature. 2009 Mar 19;458(7236):290-2. doi: 10.1038/458290a. PubMed PMID: 19295595.

Tammela T, Zarkada G, Wallgard E, Murtomäki A, Suchting S, Wirzenius M, Waltari M, Hellström M, Schomber T, Peltonen R, Freitas C, Duarte A, Isoniemi H, Laakkonen P, Christofori G, Ylä-Herttuala S, Shibuya M, Pytowski B, Eichmann A, Betsholtz C, Alitalo K. Blocking VEGFR-3 suppresses angiogenic sprouting and vascular network formation.  Nature. 2008 Jul 31;454(7204):656-60. doi: 10.1038/nature07083. Epub 2008 Jun 25. PubMed PMID: 18594512.

Schatton T, Murphy GF, Frank NY, Yamaura K, Waaga-Gasser AM, Gasser M, Zhan Q, Jordan S, Duncan LM, Weishaupt C, Fuhlbrigge RC, Kupper TS, Sayegh MH, Frank Identification of cells initiating human melanomas. . Nature. 2008 Jan 17;451(7176):345-9. doi: 10.1038/nature06489. PubMed PMID: 18202660; PubMed Central PMCID: PMC3660705.

Wadman M. London’s disastrous drug trial has serious side effects for research. Nature. 2006 Mar 23;440(7083):388-9. PubMed PMID: 16554763.

Check E. Mouse opens door for study of autoimmune diseases.Nature. 2004 Apr 22;428(6985):786. PubMed PMID: 15103338.

Gura T. Therapeutic antibodies: magic bullets hit the target. Nature. 2002 Jun 6;417(6889):584-6. PubMed PMID: 12050630.

Izumi Y, Xu L, di Tomaso E, Fukumura D, Jain RK. Tumour biology: herceptin acts as an anti-angiogenic cocktail.  Nature. 2002 Mar 21;416(6878):279-80. PubMed PMID: 11907566.

Hojo M, Morimoto T, Maluccio M, Asano T, Morimoto K, Lagman M, Shimbo T, Suthanthiran M. Cyclosporine induces cancer progression by a cell-autonomous mechanism. Nature. 1999 Feb 11;397(6719):530-4. PubMed PMID: 10028970.

Jameson BA, McDonnell JM, Marini JC, Korngold R. A rationally designed CD4 analogue inhibits experimental allergic encephalomyelitis. Nature. 1994 Apr 21;368(6473):744-6. PubMed PMID: 8152486.

Kim KJ, Li B, Winer J, Armanini M, Gillett N, Phillips HS, Ferrara N. Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo. Nature. 1993 Apr 29;362(6423):841-4. PubMed PMID: 7683111.

Basu TN, Gutmann DH, Fletcher JA, Glover TW, Collins FS, Downward J. Aberrant regulation of ras proteins in malignant tumour cells from type 1 neurofibromatosis patients. Nature. 1992 Apr 23;356(6371):713-5. PubMed PMID: 1570015.

Bix M, Liao NS, Zijlstra M, Loring J, Jaenisch R, Raulet D. Rejection of class I MHC-deficient haemopoietic cells by irradiated MHC-matched mice.Nature.

1991 Jan 24;349(6307):329-31. PubMed PMID: 1987491.

Pérarnau B, Siegrist CA, Gillet A, Vincent C, Kimura S, Lemonnier FA. Beta 2-microglobulin restriction of antigen presentation. Nature. 1990 Aug 23;346(6286):751-4. PubMed PMID: 1697039.

Sredni B, Caspi RR, Klein A, Kalechman Y, Danziger Y, Ben Ya’akov M, Tamari T, Shalit F, Albeck M. A new immunomodulating compound (AS-101) with potential therapeutic application. Nature. 1987 Nov 12-18;330(6144):173-6. PubMed PMID: 3118216.

Cobbold SP, Waldmann H. Therapeutic potential of monovalent monoclonal antibodies. Nature. 1984 Mar 29-Apr 4;308(5958):460-2. PubMed PMID: 6608694.

Adorini L, Pini C, De Santis R, Robbiati F, Doria G, Ricciardi-Castagnoli P. Monoclonal suppressor T-cell factor displaying V H restriction and fine antigenic specificity. Nature. 1983 Jun 23-29;303(5919):704-6. PubMed PMID: 6190092.

Shouval D, Shafritz DA, Zurawski VR Jr, Isselbacher KJ, Wands JR. Immunotherapy in nude mice of human hepatoma using monoclonal antibodies against hepatitis B virus. Nature. 1982 Aug 5;298(5874):567-9. PubMed PMID: 7099252.

Thorpe PE, Mason DW, Brown AN, Simmonds SJ, Ross WC, Cumber AJ, Forrester JA. Selective killing of malignant cells in a leukaemic rat bone marrow using an antibody-ricin conjugate. Nature. 1982 Jun 17;297(5867):594-6. PubMed PMID:7088145.

Beverley PC. Antibodies and cancer therapy. Nature. 1982 Jun 3;297(5865):358-9. PubMed PMID: 7078646.

Trowbridge IS. Cancer monoclonals. Nature. 1981 Nov 19;294(5838):204. PubMed PMID: 7300906.

Blythman HE, Casellas P, Gros O, Gros P, Jansen FK, Paolucci F, Pau B, Vidal Immunotoxins: hybrid molecules ofmonoclonal antibodiesand a toxin subunit specifically kill tumour cells.Nature. 1981 Mar 12;290 (5802):145-6. PubMed PMID: 7207595.

Wolchok JD, Chan TA. Cancer: Antitumour immunity gets a boost. Nature. 2014 Nov 27;515(7528):496-8. doi: 10.1038/515496a. PubMed PMID: 25428495.

Hoag H. Drug development: a chance of survival. Nature. 2014 Nov 20;515(7527):S118-20. doi: 10.1038/515S118a. PubMed PMID: 25407709.

Ledford H. Cancer treatment: The killer within. Nature. 2014 Apr 3;508(7494):24-6. doi: 10.1038/508024a. PubMed PMID: 24695297.

Weintraub K. Drug development: Releasing the brakes. Nature. 2013 Dec 19;504(7480):S6-8. doi: 10.1038/504S6a. PubMed PMID: 24352363.

Elert E. Calling cells to arms. Nature. 2013 Dec 19;504(7480):S2-3. doi: 10.1038/504S2a. PubMed PMID: 24352361.

Mellman I, Coukos G, Dranoff G. Cancer immunotherapy comes of age. Nature. 2011 Dec 21;480(7378):480-9. doi: 10.1038/nature10673. Review. PubMed PMID: 22193102; PubMed Central PMCID: PMC3967235.

Dolgin E. FDA narrows drug label usage. Nature. 2009 Aug 27;460(7259):1069. doi: 10.1038/4601069a. PubMed PMID: 19713906.

Ellis LM, Reardon DA. Cancer: The nuances of therapy. Nature. 2009 Mar 19;458(7236):290-2. doi: 10.1038/458290a. PubMed PMID: 19295595.

Izumi Y, Xu L, di Tomaso E, Fukumura D, Jain RK. Tumour biology: herceptin acts as an anti-angiogenic cocktail.  Nature. 2002 Mar 21;416(6878):279-80. PubMed PMID: 11907566.

Kim KJ, Li B, Winer J, Armanini M, Gillett N, Phillips HS, Ferrara N. Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo. Nature. 1993 Apr 29;362(6423):841-4. PubMed PMID: 7683111.

Sredni B, Caspi RR, Klein A, Kalechman Y, Danziger Y, Ben Ya’akov M, Tamari T, Shalit F, Albeck M. A new immunomodulating compound (AS-101) with potential therapeutic application. A new immunomodulating compound (AS-101) with potential therapeutic application. Nature. 1987 Nov 12-18;330(6144):173-6. PubMed PMID: 3118216.

Cobbold SP, Waldmann H. Therapeutic potential of monovalent monoclonal antibodies. Nature. 1984 Mar 29-Apr 4;308(5958):460-2. PubMed PMID: 6608694.

Shouval D, Shafritz DA, Zurawski VR Jr, Isselbacher KJ, Wands JR. Immunotherapy in nude mice of human hepatoma using monoclonal antibodies against hepatitis B virus. Nature. 1982 Aug 5;298(5874):567-9. PubMed PMID: 7099252.

Thorpe PE, Mason DW, Brown AN, Simmonds SJ, Ross WC, Cumber AJ, Forrester JA. Selective killing of malignant cells in a leukaemic rat bone marrow using an antibody-ricin conjugate. Nature. 1982 Jun 17;297(5867):594-6. PubMed PMID: 7088145.

Beverley PC. Antibodies and cancer therapy. Nature. 1982 Jun 3;297(5865):358-9. PubMed PMID: 7078646.

Trowbridge IS. Cancer monoclonals.  Nature. 1981 Nov 19;294(5838):204. PubMed PMID: 7300906.

Blythman HE, Casellas P, Gros O, Gros P, Jansen FK, Paolucci F, Pau B, Vidal Immunotoxins: hybrid molecules ofmonoclonal antibodiesand a toxin subunit specifically kill tumour cells. Nature. 1981 Mar 12;290(5802):145-6. PubMed PMID: 7207595.

Waldmann, Thomas A. (2003). “Immunotherapy: past, present and future”. Nature Medicine 9 (3): 269–277. doi:10.1038/nm0303-269. PMID 12612576.

Sharma, Pamanee; Allison, James P. (April 3, 2015). “The future of immune checkpoint therapy”. Science. doi:10.1126/science.aaa8172. Retrieved June 2015.

Gene Garrard Olinger, Jr., James Pettitt, Do Kim, Cara Working, Ognian Bohorov, Barry Bratcher, Ernie Hiatt, Steven D. Hume, Ashley K. Johnson, Josh Morton, Michael Pauly, Kevin J. Whaley, Calli M. Lear, Julia E. Biggins, Corinne Scully, Lisa Hensley, and Larry Zeitlin (2012). “Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques”. PNAS 109 (44): 18030–5.doi:10.1073/pnas.1213709109. PMC 3497800. PMID 23071322.

Janeway, Charles; Paul Travers; Mark Walport; Mark Shlomchik (2001).Immunobiology; Fifth Edition. New York and London: Garland Science. ISBN 0-8153-4101-6.

Janeway CA, Jr.; et al. (2005). Immunobiology. (6th ed.). Garland Science. ISBN 0-443-07310-4.

Modified from Carter P (November 2001). “Improving the efficacy of antibody-based cancer therapies”. Nat. Rev. Cancer 1 (2): 118–29. doi:10.1038/35101072.PMID 11905803.

Prof FC Breedveld (2000). “Therapeutic monoclonal antibodies”. Lancet.doi:10.1016/S0140-6736(00)01034-5.

Köhler G, Milstein C (August 1975). “Continuous cultures of fused cells secreting antibody of predefined specificity”. Nature 256 (5517): 495–7.Bibcode:1975Natur.256..495K. doi:10.1038/256495a0. PMID 1172191.

Nadler LM, Stashenko P, Hardy R, et al. (September 1980). “Serotherapy of a patient with a monoclonal antibody directed against a human lymphoma-associated antigen”.Cancer Res. 40 (9): 3147–54. PMID 7427932.

Ritz J, Schlossman SF (January 1982). “Utilization of monoclonal antibodies in the treatment of leukemia and lymphoma”. Blood 59 (1): 1–11. PMID 7032624.

Stern M, Herrmann R (April 2005). “Overview of monoclonal antibodies in cancer therapy: present and promise”. Crit. Rev. Oncol. Hematol. 54 (1): 11–29.doi:10.1016/j.critrevonc.2004.10.011. PMID 15780905.

(John, Martin et al. 2005, Robert, Ann et al. 2006, Albert, Edvardas et al. 2012, Claro, Karen et al. 2012, Gideon, Nancy et al. 2013, Michael, Ke et al. 2013, Thomas, Albert et al. 2013, Hyon-Zu, Barry et al. 2014, Larkins, Scepura et al. 2015, Sandra, Ibilola et al. 2015, Sean, Gideon et al. 2015)Hudson PJ, Souriau C (January 2003). “Engineered antibodies”. Nat. Med. 9 (1): 129–34. doi:10.1038/nm0103-129. PMID 12514726.

Carter P, Presta L, Gorman CM, et al. (May 1992). “Humanization of an anti-p185HER2 antibody for human cancer therapy”. Proc. Natl. Acad. Sci. U.S.A. 89 (10): 4285–9.Bibcode:1992PNAS…89.4285C. doi:10.1073/pnas.89.10.4285. PMC 49066.PMID 1350088.

Presta LG, Lahr SJ, Shields RL, et al. (September 1993). “Humanization of an antibody directed against IgE”. J. Immunol. 151 (5): 2623–32. PMID 8360482.

Chothia C, Lesk AM, Tramontano A, et al. (1989). “Conformations of immunoglobulin hypervariable regions”. Nature 342 (6252): 877–83. Bibcode:1989Natur.342..877C.doi:10.1038/342877a0. PMID 2687698.

Jefferis, Roy; Marie-Paule Lefranc (July–August 2009). “Human immunoglobulin allotypes”. MAbs 1 (4): 332–338. doi:10.4161/mabs.1.4.9122. PMC 2726606.PMID 20073133.

Chapman, Kathryn; Nick Pullen, Lee Coney, Maggie Dempster, Laura Andrews, Jeffrey Bajramovic, Paul Baldrick, Lorrene Buckley, Abby Jacobs, Geoff Hale, Colin Green, Ian Ragan and Vicky Robinson (2009). “Preclinical development of monoclonal antibodies”.MAbs 1 (5): 505–516. doi:10.4161/mabs.1.5.9676. PMC 2759500. PMID 20065651.

Francis RJ, Sharma SK, Springer C, et al. (2002). “A phase I trial of antibody directed enzyme prodrug therapy (ADEPT) in patients with advanced colorectal carcinoma or other CEA producing tumours”. Br J Cancer 87 (6): 600–7. doi:10.1038/sj.bjc.6600517.PMC 2364249. PMID 12237768.

Krauss WC, Park JW, Kirpotin DB, Hong K, Benz CC (2000). “Emerging antibody-based HER2 (ErbB-2/neu) therapeutics”. Breast Dis 11: 113–124. PMID 15687597.

Joyce1, Johanna A.; Fearon, Douglas T. (April 3, 2015). “T cell exclusion, immune privilege, and the tumor microenvironment”. Science 348 (6230 74-80).doi:10.1126/science.aaa6204.

Rang, H. P. (2003). Pharmacology. Edinburgh: Churchill Livingstone. p. 241. ISBN 0-443-07145-4.

Hooks MA, Wade CS, Millikan WJ (1991). “Muromonab CD-3: a review of its pharmacology, pharmacokinetics, and clinical use in transplantation”. Pharmacotherapy 11(1): 26–37. PMID 1902291.

Goel, Niti; Stephens, Sue (2010). “Certolizumab Pegol”. MAbs 2 (2): 137–147.doi:10.4161/mabs.2.2.11271. PMC 2840232. PMID 20190560.

Chames, Patrick; Baty, Daniel (2009). “Bispecific antibodies for cancer therapy: The light at the end of the tunnel?”. MAbs 1 (6): 539–547. doi:10.4161/mabs.1.6.10015.PMC 2791310. PMID 20073127.

Linke, Rolf; Klein, Anke; Seimetz, Diane (2010). “Catumaxomab: Clinical development and future directions”. MAbs 2 (2): 129–136. doi:10.4161/mabs.2.2.11221.

Scolnik, Pablo A. (2009). “mAbs: A business perspective”. MAbs 1 (2): 179–184.doi:10.4161/mabs.1.2.7736. PMC 2725420. PMID 20061824.

Kelley, Brian (2009). “Industrialization of mAb production technology”. MAbs 1 (5): 443–452. doi:10.4161/mabs.1.5.9448. PMC 2759494. PMID 20065641.

Selected FDA Approved Mab Drugs:

(John, Martin et al. 2005, Robert, Ann et al. 2006, Albert, Edvardas et al. 2012, Claro, Karen et al. 2012, Gideon, Nancy et al. 2013, Michael, Ke et al. 2013, Thomas, Albert et al. 2013, Hyon-Zu, Barry et al. 2014, Larkins, Scepura et al. 2015, Sandra, Ibilola et al. 2015, Sean, Gideon et al. 2015)

Albert, D., K. Edvardas, G. Joseph, C. Wei, S. Haleh, L. L. Hong, D. R. Mark, B. Satjit, W. Jian, G. Christine, B. Julie, B. B. Laurie, R. Atiqur, S. Rajeshwari, F. Ann and P. Richard (2012). “U.S. Food and Drug Administration Approval: Ruxolitinib for the Treatment of Patients with Intermediate and High-Risk Myelofibrosis.” Clinical Cancer Research: 3212-3217.

Claro, R. A. d., M. Karen, K. Virginia, B. Julie, K. Aakanksha, H. Bahru, O. Yanli, S. Haleh, L. Kyung, K. Kallappa, R. Mark, S. Marjorie, B. Francisco, C. Kathleen, C. Xiao Hong, B. Janice, A. Lara, K. Robert, K. Edvardas, F. Ann and P. Richard (2012). “U.S. Food and Drug Administration Approval Summary: Brentuximab Vedotin for the Treatment of Relapsed Hodgkin Lymphoma or Relapsed Systemic Anaplastic Large-Cell Lymphoma.” Clinical Cancer Research: 5845-5849.

Gideon, M. B., S. S. Nancy, C. Patricia, C. Somesh, T. Shenghui, S. Pengfei, L. Qi, R. Kimberly, M. P. Anne, T. Amy, E. K. Kathryn, G. Laurie, L. R. Barbara, C. W. Wendy, C. Bo, T. Colleen, H. Patricia, I. Amna, J. Robert and P. Richard (2013). “First FDA approval of dual anti-HER2 regimen: pertuzumab in combination with trastuzumab and docetaxel for HER2-positive metastatic breast cancer.” Clinical cancer research : an official journal of the American Association for Cancer Research: 4911-4916.

Hyon-Zu, L., W. M. Barry, E. K. Virginia, R. Stacey, D. Pedro, S. Haleh, G. Joseph, B. Julie, F. Jeffry, M. Nitin, K. Chia-Wen, N. Lei, S. Marjorie, T. Mate, C. K. Robert, K. Edvardas, J. Robert, T. F. Ann and P. Richard (2014). “U.S. Food and drug administration approval: obinutuzumab in combination with chlorambucil for the treatment of previously untreated chronic lymphocytic leukemia.” Clinical cancer research : an official journal of the American Association for Cancer Research: 3902-3907.

John, R. J., C. Martin, S. Rajeshwari, C. Yeh-Fong, M. W. Gene, D. John, G. Jogarao, B. Brian, B. Kimberly, L. John, H. Li Shan, C. Nallalerumal, Z. Paul and P. Richard (2005). “Approval Summary for Erlotinib for Treatment of Patients with Locally Advanced or Metastatic Non–Small Cell Lung Cancer after Failure of at Least One Prior Chemotherapy Regimen.” Clinical Cancer Research 11(18).

Larkins, E., B. Scepura, G. M. Blumenthal, E. Bloomquist, S. Tang, M. Biable, P. Kluetz, P. Keegan and R. Pazdur (2015). “U.S. Food and Drug Administration Approval Summary: Ramucirumab for the Treatment of Metastatic Non-Small Cell Lung Cancer Following Disease Progression On or After Platinum-Based Chemotherapy.” The oncologist.

Michael, A., L. Ke, J. Xiaoping, H. Kun, W. Jian, Z. Hong, K. Dubravka, P. Todd, D. Zedong, R. Anne Marie, M. Sarah, K. Patricia and P. Richard (2013). “U.S. Food and Drug Administration approval: vismodegib for recurrent, locally advanced, or metastatic basal cell carcinoma.” Clinical cancer research : an official journal of the American Association for Cancer Research: 2289-2293.

Robert, C. K., T. F. Ann, S. Rajeshwari and P. Richard (2006). “United States Food and Drug Administration approval summary: bortezomib for the treatment of progressive multiple myeloma after one prior therapy.” Clinical cancer research : an official journal of the American Association for Cancer Research: 2955-2960.

Sandra, J. C., F.-A. Ibilola, J. L. Steven, Z. Lillian, J. Runyan, L. Hongshan, Z. Liang, Z. Hong, Z. Hui, C. Huanyu, H. Kun, D. Michele, N. Rachel, K. Sarah, K. Sachia, H. Whitney, K. Patricia and P. Richard (2015). “FDA Approval Summary: Ramucirumab for Gastric Cancer.” Clinical cancer research : an official journal of the American Association for Cancer Research: 3372-3376.

Sean, K., M. B. Gideon, Z. Lijun, T. Shenghui, B. Margaret, F. Emily, H. Whitney, L. Ruby, S. Pengfei, P. Yuzhuo, L. Qi, Z. Ping, Z. Hong, L. Donghao, T. Zhe, H. Ali Al, B. Karen, K. Patricia, J. Robert and P. Richard (2015). “FDA approval: ceritinib for the treatment of metastatic anaplastic lymphoma kinase-positive non-small cell lung cancer.” Clinical cancer research : an official journal of the American Association for Cancer Research: 2436-2439.

Thomas, M. H., D. Albert, K. Edvardas, C. K. Robert, M. K. Kallappa, D. R. Mark, H. Bahru, B. Julie, D. B. Jeffrey, H. Jessica, R. P. Todd, J. Josephine, A. William, M. Houda, B. Janice, D. Angelica, S. Rajeshwari, T. F. Ann and P. Richard (2013). “U.S. Food and Drug Administration Approval: Carfilzomib for the Treatment of Multiple Myeloma.” Clinical Cancer Research: 4559-4563.

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Humanized Mice May Revolutionize Cancer Drug Discovery

Posted in BioTechnology - Venture Creation, BioTechnology - Venture Creation, Venture Capital, Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Monoclonal Immunotherapy, Patents, Pharmaceutical Drug Discovery, Pharmacodynamics and Pharmacokinetics, tagged , , , , , , , , , , , , , , on October 9, 2015| Leave a Comment »

Humanized Mice May Revolutionize Cancer Drug Discovery

Curator: Stephen J. Williams, Ph.D.

Humanized Mice May Revolutionize Cancer Drug Discovery

Word Cloud by Zach Day

Decades ago cancer research and the process of oncology drug discovery was revolutionized by the development of mice deficient in their immune system, allowing for the successful implantation of human-derived tumors. The ability to implant human tumors without rejection allowed researchers to study how the kinetics of human tumor growth in its three-dimensional environment, evaluate potential human oncogenes and drivers of oncogenesis, and evaluate potential chemotherapeutic therapies. Indeed, the standard preclinical test for antitumor activity has involved the subcutaneous xenograft model in immunocompromised (SCID or nude athymic) mice. More detail is given in the follow posts in which I describe some early pioneers in this work as well as the development of large animal SCID models:

Heroes in Medical Research: Developing Models for Cancer Research

The SCID Pig: How Pigs are becoming a Great Alternate Model for Cancer Research

The SCID Pig II: Researchers Develop Another SCID Pig, And Another Great Model For Cancer Research

This strategy (putting human tumor cells into immunocompromised mice and testing therapeutic genes and /or compounds) has worked extremely well for most cytotoxic chemotherapeutics (those chemotherapeutic drugs with mechanisms of action related to cell kill, vital cell functions, and cell cycle). For example the NCI 60 panel of human tumor cell lines has proved predictive for the chemosensitivity of a wide range of compounds.

Even though the immunocompromised model has contributed greatly to the chemotherapeutic drug discovery process. using these models to develop the new line of immuno-oncology products has been met with challenges three which I highlight below with curated database of references and examples.

From a practical standpoint development of a mouse which can act as a recipient for human tumors yet have a humanized immune system allows for the preclinical evaluation of antitumoral effect of therapeutic antibodies without the need to use neutralizing antibodies to the comparable mouse epitope,   thereby reducing the complexity of the study and preventing complications related to pharmacokinetics.

Champions Oncology Files Patents for Use of PDX Platform in Immune-Oncology

Hackensack, NJ – August 17, 2015 – Champions Oncology, Inc. (OTC: CSBR), engaged in the development of advanced technology solutions and services to personalize the development and use of oncology drugs, today announced that it has filed two patent applications with the United States Patent and Trademark Office (USPTO) relating to the development and use of mice with humanized immune systems to test immune-oncology drugs and therapeutic cancer vaccines.

Dr. David Sidransky, the founder and Chairman of Champions Oncology commented, “Drug development ‎in the immune-oncology space is fundamentally changing our approach to cancer treatment. These patents represent potentially invaluable tools for developing and personalizing immune therapy based on cutting edge sequence analysis, bioinformatics and our unique in vivo models.”

Joel Ackerman, Chief Executive Officer of Champions Oncology stated, “Developing intellectual property related to our Champions TumorGraft® platform has been an important component of strategy. The filing of these patents is an important milestone in leveraging our research and development investment to expand our platform and create proprietary tools for use by our pharmaceutical partners. We continue to look for additional revenue streams to supplement our fee-for-service business and we believe these patents will help us capture more of the value we create for our customers in the future.”

The first patent filing covers the methodology used by the Company to create a mouse model, containing a humanized immune system and a human tumor xenograft, which is capable of testing the efficacy of immune-oncology agents, both as single agents and in combination with anti-neoplastic drugs. The second patent filing relates to the detection of neoantigens and their role in the development of anti-cancer vaccines.

Keren Pez, Chief Scientific Officer, explained, “In the last few years, there has been a significant increase in cancer research that focuses on exploring the power of the human immune system to attack tumors. However, it’s challenging to test immune-oncology agents in traditional animal models due to the major differences between human and murine immune systems. The Champions ImmunoGraft™ platform has the unique ability of mimicking a human adaptive immune response in the mice, which allows us to specifically evaluate a variety of cancer therapeutics that modulate human immunity.

“Therapeutic vaccines that trigger the immune system to mount a response against a growing tumor are another area of intense interest. The development of an effective vaccine remains challenging but has an outstanding curative potential. Tumors harbor mutations in DNA that result in the translation of aberrant proteins. While these proteins have the potential to provoke an immune response that destructs early-stage cancer development, often the immune response becomes insufficient. Vaccines can trigger it by proactively challenging the system with these specific mutated peptides. Nevertheless, developing anti-cancer vaccines that effectively inhibit tumor growth has been complicated, partially due to challenges in finding the critical mutations, among others difficulties. With the more recent advances in genome sequencing, it’s now possible to identify tumor-specific antigens, or neoantigens, that naturally develop as an individual’s tumor grows and mutates,” she continued.

Traumatic spinal cord injury in mice with human immune systems.

Carpenter RS, Kigerl KA, Marbourg JM, Gaudet AD, Huey D, Niewiesk S, Popovich PG.

Exp Neurol. 2015 Jul 17;271:432-444. doi: 10.1016/j.expneurol.2015.07.011. [Epub ahead of print]

Inflamm Bowel Dis. 2015 Jul;21(7):1652-73. doi: 10.1097/MIB.0000000000000446.

Use of Humanized Mice to Study the Pathogenesis of Autoimmune and Inflammatory Diseases.

Koboziev I1, Jones-Hall Y, Valentine JF, Webb CR, Furr KL, Grisham MB.

Author information

Abstract

Animal models of disease have been used extensively by the research community for the past several decades to better understand the pathogenesis of different diseases and assess the efficacy and toxicity of different therapeutic agents. Retrospective analyses of numerous preclinical intervention studies using mouse models of acute and chronic inflammatory diseases reveal a generalized failure to translate promising interventions or therapeutics into clinically effective treatments in patients. Although several possible reasons have been suggested to account for this generalized failure to translate therapeutic efficacy from the laboratory bench to the patient’s bedside, it is becoming increasingly apparent that the mouse immune system is substantially different from the human. Indeed, it is well known that >80 major differences exist between mouse and human immunology; all of which contribute to significant differences in immune system development, activation, and responses to challenges in innate and adaptive immunity. This inconvenient reality has prompted investigators to attempt to humanize the mouse immune system to address important human-specific questions that are impossible to study in patients. The successful long-term engraftment of human hematolymphoid cells in mice would provide investigators with a relatively inexpensive small animal model to study clinically relevant mechanisms and facilitate the evaluation of human-specific therapies in vivo. The discovery that targeted mutation of the IL-2 receptor common gamma chain in lymphopenic mice allows for the long-term engraftment of functional human immune cells has advanced greatly our ability to humanize the mouse immune system. The objective of this review is to present a brief overview of the recent advances that have been made in the development and use of humanized mice with special emphasis on autoimmune and chronic inflammatory diseases. In addition, we discuss the use of these unique mouse models to define the human-specific immunopathological mechanisms responsible for the induction and perpetuation of chronic gut inflammation.

J Immunother Cancer. 2015 Apr 21;3:12. doi: 10.1186/s40425-015-0056-2. eCollection 2015.

Human tumor infiltrating lymphocytes cooperatively regulate prostate tumor growth in a humanized mouse model.

Roth MD1, Harui A1.

Author information

Abstract

BACKGROUND:

The complex interactions that occur between human tumors, tumor infiltrating lymphocytes (TIL) and the systemic immune system are likely to define critical factors in the host response to cancer. While conventional animal models have identified an array of potential anti-tumor therapies, mouse models often fail to translate into effective human treatments. Our goal is to establish a humanized tumor model as a more effective pre-clinical platform for understanding and manipulating TIL.

METHODS:

The immune system in NOD/SCID/IL-2Rγnull (NSG) mice was reconstituted by the co-administration of human peripheral blood lymphocytes (PBL) or subsets (CD4+ or CD8+) and autologous human dendritic cells (DC), and animals simultaneously challenged by implanting human prostate cancer cells (PC3 line). Tumor growth was evaluated over time and the phenotype of recovered splenocytes and TIL characterized by flow cytometry and immunohistochemistry (IHC). Serum levels of circulating cytokines and chemokines were also assessed.

RESULTS:

A tumor-bearing huPBL-NSG model was established in which human leukocytes reconstituted secondary lymphoid organs and promoted the accumulation of TIL. These TIL exhibited a unique phenotype when compared to splenocytes with a predominance of CD8+ T cells that exhibited increased expression of CD69, CD56, and an effector memory phenotype. TIL from huPBL-NSG animals closely matched the features of TIL recovered from primary human prostate cancers. Human cytokines were readily detectible in the serum and exhibited a different profile in animals implanted with PBL alone, tumor alone, and those reconstituted with both. Immune reconstitution slowed but could not eliminate tumor growth and this effect required the presence of CD4+ T cell help.

CONCLUSIONS:

Simultaneous implantation of human PBL, DC and tumor results in a huPBL-NSG model that recapitulates the development of human TIL and allows an assessment of tumor and immune system interaction that cannot be carried out in humans. Furthermore, the capacity to manipulate individual features and cell populations provides an opportunity for hypothesis testing and outcome monitoring in a humanized system that may be more relevant than conventional mouse models.

Methods Mol Biol. 2014;1213:379-88. doi: 10.1007/978-1-4939-1453-1_31.

A chimeric mouse model to study immunopathogenesis of HCV infection.

Bility MT1, Curtis A, Su L.

Author information

Abstract

Several human hepatotropic pathogens including chronic hepatitis C virus (HCV) have narrow species restriction, thus hindering research and therapeutics development against these pathogens. Developing a rodent model that accurately recapitulates hepatotropic pathogens infection, human immune response, chronic hepatitis, and associated immunopathogenesis is essential for research and therapeutics development. Here, we describe the recently developed AFC8 humanized liver- and immune system-mouse model for studying chronic hepatitis C virus and associated human immune response, chronic hepatitis, and liver fibrosis.

PMID:

25173399

[PubMed – indexed for MEDLINE]

PMCID:

PMC4329723

Free PMC Article

Immune humanization of immunodeficient mice using diagnostic bone marrow aspirates from carcinoma patients.

Werner-Klein M, Proske J, Werno C, Schneider K, Hofmann HS, Rack B, Buchholz S, Ganzer R, Blana A, Seelbach-Göbel B, Nitsche U, Männel DN, Klein CA.

PLoS One. 2014 May 15;9(5):e97860. doi: 10.1371/journal.pone.0097860. eCollection 2014.

From 2015 AACR National Meeting in Philadelphia

LB-050: Patient-derived tumor xenografts in humanized NSG mice: a model to study immune responses in cancer therapy
Sunday, Apr 19, 2015, 3:20 PM – 3:35 PM
Minan Wang1, James G. Keck1, Mingshan Cheng1, Danying Cai1, Leonard Shultz2, Karolina Palucka2, Jacques Banchereau2, Carol Bult2, Rick Huntress2. 1The Jackson Laboratory, Sacramento, CA; 2The Jackson Laboratory, Bar Harbor, ME

References

  1. Paull KD, Shoemaker RH, Hodes L, Monks A, Scudiero DA, Rubinstein L, Plowman J, Boyd MR. J Natl Cancer Inst. 1989;81:1088–1092. [PubMed]
  2. Shi LM, Fan Y, Lee JK, Waltham M, Andrews DT, Scherf U, Paull KD, Weinstein JN. J Chem Inf Comput Sci. 2000;40:367–379. [PubMed]
  3. Monks A, Scudiero D, Skehan P, Shoemaker R, Paull K, Vistica D, Hose C, Langley J, Cronise P, Vaigro-Wolff A, et al. J Natl Cancer Inst. 1991;83:757–766. [PubMed]
  4. Potti A, Dressman HK, Bild A, et al. Genomic signatures to guide the use of chemotherapeutics. Nat Med. 2006;12:1294–1300. [PubMed]
  5. Baggerly KA, Coombes KR. Deriving chemosensitivity from cell lines: forensic bioinformatics and reproducible research in high-throughput biology. Ann Appl Stat. 2009;3:1309–1334.
  6. Carlson, B. Putting Oncology Patients at Risk Biotechnol Healthc. 2012 Fall; 9(3): 17–21.
  7. Salter KH, Acharya CR, Walters KS, et al. An Integrated Approach to the Prediction of Chemotherapeutic Response in Patients with Breast Cancer. Ouchi T, ed. PLoS ONE. 2008;3(4):e1908. NOTE RETRACTED PAPER

Other posts on this site on Animal Models, Disease and Cancer Include:

Heroes in Medical Research: Developing Models for Cancer Research

Guidelines for the welfare and use of animals in cancer research

Model mimicking clinical profile of patients with ovarian cancer @ Yale School of Medicine

Vaccines, Small Peptides, aptamers and Immunotherapy [9]

Immunotherapy in Cancer: A Series of Twelve Articles in the Frontier of Oncology by Larry H Bernstein, MD, FCAP

Mouse With ‘Humanized Version’ Of Human Language Gene Provides Clues To Language Development

The SCID Pig: How Pigs are becoming a Great Alternate Model for Cancer Research

The SCID Pig II: Researchers Develop Another SCID Pig, And Another Great Model For Cancer Research

Read Full Post »

Steroids, Inflammation, and CAR-T Therapy

Posted in Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, FDA, FDA Regulatory Affairs, Immuno-Oncology & Genomics, Innovation in Immunology Diagnostics, Lymphoma, Personal Health Applications: Tech Innovations serves HealhCare, Personalized and Precision Medicine & Genomic Research, tagged , , , , , , , , , , , , , , on September 14, 2015| Leave a Comment »

Steroids, Inflammation, and CAR-T Therapy [6.3.8] (UPDATED)

Reporter: Stephen J. Williams, Ph.D.

UPDATED: 08/31/2020 (CRISPR edited CAR-T clinical trials)

Word Cloud by Daniel Menzin

Corticosteroids have been used as anticancer agents since the 1940s, with activity reported in a wide variety of solid tumors, including breast and prostate cancer, and the lymphoid hematologic malignancies. They are commonly found in regimens for acute lymphocytic leukemia, Hodgkin’s and non-Hodgkin’s lymphoma, myeloma, and chronic lymphocytic leukemia.

A great review on the mechanism of action of prednisone’s antitumoral activity is seen in

Corticosteroids in the Treatment of Neoplasms Lorraine I. McKay, PhD and John A. Cidlowski, PhD. in Holland-Frei Cancer Medicine. 6th edition.

It was first discovered that cortisone caused tumor regression in a transplantable mouse lymphosarcoma,81 a finding soon extended to a wide variety of murine lymphatic tumors. The effects of corticosteroids were also evaluated on many nonendocrine and nonlymphoid transplantable rodent tumors. Pharmacologic doses of steroid inhibited growth of various tumor systems.82 Tissue culture studies confirmed that lymphoid cells were the most sensitive to glucocorticoids, and responded to treatment with decreases in DNA, ribonucleic acid (RNA), and protein synthesis.83 Studies of proliferating human leukemic lymphoblasts supported the hypothesis that glucocorticoids have preferential lymphocytolytic effects. The mechanism of action was initially thought to be caused by impaired energy use via decreased glucose transport and/or phosphorylation; it was later discovered that glucocorticoids induce apoptosis, or programmed cell death, in certain lymphoid cell populations.84,85

–For review on corticosteroids in cancer therapy see more at: http://www.cancernetwork.com/review-article/corticosteroids-advanced-cancer#sthash.IwHbekuI.dpuf

However, as Dana Farber’s Dr. George Canellos, M.D. ponders in Can MOPP be replaced in the treatment of advanced Hodgkin’s disease? Semin Oncol. Canellos GP1. 1990 Feb;17(1 Suppl 2):2-6., many dose-limiting toxicities occur with MOPP (mechlorethamine, vincristine, procarbazine, prednisone) therapy used in advanced Hodgkin’s disease.  Although, at the time, he generally was looking to establish combination therapies with less side effect, the advent of more personalized therapies as well as immunotherapies may indeed replace the older regimens for B-cell malignancies and Hodgkin’s disease, and their panels of toxicities.

Short-term side effects of prednisone (Cancer.gov prednisone description with side effects) as with all glucocorticoids, include high blood glucose levels (especially in patients with diabetes mellitus or on other medications that increase blood glucose, such as tacrolimus) and mineralocorticoid effects such as fluid retention.[10] The mineralocorticoid effects of prednisone are minor, which is why it is not used in the management of adrenal insufficiency, unless a more potent mineralocorticoid is administered concomitantly.

Long-term side effects include Cushing’s syndrome, steroid dementia syndrome, truncal weight gain, osteoporosis, glaucoma and cataracts, type II diabetes mellitus, and depression upon dose reduction or cessation.

Therefore the oncology world has been moving toward therapies which are more selective with less dose-limiting toxicities (e.g. Rituximab), and are looking to CAR-T therapies as a possible replacement for standard chemotherapeutic regimens. However, as with prednisone, there have been serious adverse events in some CAR-T clinical trials. Luckily clinicians, as discussed below, have found supportive therapies to alleviate the most severe side effects to CAR-T.

This section will be refer to supportive therapies as those adjuvant therapy given to alleviate patient discomfort, reduce toxicities and adverse event, or support patient well-being during their course of chemotherapy, not adjuvant therapy to enhance antitumoral effect.

For more background information of CAR-T therapies and related issues please see my previous post

NIH Considers Guidelines for CAR-T therapy: Report from Recombinant DNA Advisory Committee

The following is a brief re-post of some of the important points for reference to this new posting.

1. Evolution of Chimeric Antigen Receptors

Early evidence had suggested that adoptive transfer of tumor-infiltrating lymphocytes, after depletion of circulating lymphocytes, could result in a clinical response in some tumor patients however developments showed autologous T-cells (obtained from same patient) could be engineered to express tumor-associated antigens (TAA) and replace the TILS in the clinical setting.

A brief history of construction of 2nd and 3rd generation CAR-T cells given by cancer.gov:

http://www.cancer.gov/cancertopics/research-updates/2013/CAR-T-Cells

cartdiagrampic

Differences between  second- and third-generation chimeric antigen receptor T cells. (Adapted by permission from the American Association for Cancer Research: Lee, DW et al. The Future Is Now: Chimeric Antigen Receptors as New Targeted Therapies for Childhood Cancer. Clin Cancer Res; 2012;18(10); 2780–90. doi:10.1158/1078-0432.CCR-11-1920)

Constructing a CAR T Cell (from cancer.gov)

The first efforts to engineer T cells to be used as a cancer treatment began in the early 1990s. Since then, researchers have learned how to produce T cells that express chimeric antigen receptors (CARs) that recognize specific targets on cancer cells.

The T cells are genetically modified to produce these receptors. To do this, researchers use viral vectors that are stripped of their ability to cause illness but that retain the capacity to integrate into cells’ DNA to deliver the genetic material needed to produce the T-cell receptors.

The second- and third-generation CARs typically consist of a piece of monoclonal antibody, called a single-chain variable fragment (scFv), that resides on the outside of the T-cell membrane and is linked to stimulatory molecules (Co-stim 1 and Co-stim 2) inside the T cell. The scFv portion guides the cell to its target antigen. Once the T cell binds to its target antigen, the stimulatory molecules provide the necessary signals for the T cell to become fully active. In this fully active state, the T cells can more effectively proliferate and attack cancer cells.

2. Consideration for Design of Trials and Mitigating Toxicities

  • Early Toxic effects– Cytokine Release Syndrome– The effectiveness of CART therapy has been manifested by release of high levels of cytokines resulting in fever and inflammatory sequelae. One such cytokine, interleukin 6, has been attributed to this side effect and investigators have successfully used an IL6 receptor antagonist, tocilizumab (Acterma™), to alleviate symptoms of cytokine release syndrome (see review Adoptive T-cell therapy: adverse events and safety switches by Siok-Keen Tey).
  • Early Toxic effects – Over-activation of CAR T-cells; mitigation by dose escalation strategy (as authors in reference [3] proposed). Most trials give billions of genetically modified cells to a patient.
  • Late Toxic Effects – long-term depletion of B-cells . For example CART directing against CD19 or CD20 on B cells may deplete the normal population of CD19 or CD20 B-cells over time; possibly managed by IgG supplementation

References

  1. Ertl HC, Zaia J, Rosenberg SA, June CH, Dotti G, Kahn J, Cooper LJ, Corrigan-Curay J, Strome SE: Considerations for the clinical application of chimeric antigen receptor T cells: observations from a recombinant DNA Advisory Committee Symposium held June 15, 2010. Cancer research 2011, 71(9):3175-3181.
  2. Morgan RA, Yang JC, Kitano M, Dudley ME, Laurencot CM, Rosenberg SA: Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Molecular therapy : the journal of the American Society of Gene Therapy 2010, 18(4):843-851.
  3. Kandalaft LE, Powell DJ, Jr., Coukos G: A phase I clinical trial of adoptive transfer of folate receptor-alpha redirected autologous T cells for recurrent ovarian cancer. Journal of translational medicine 2012, 10:157.

3. Case Reports of Adverse Events and Their Amelioration During CAR-T Therapy

CAR-T Therapy have Had reports of Serious Adverse Events

From FierceBiotech UPDATED: Two deaths force MSK to hit the brakes on engineered T cell cancer study

April 6, 2014 | By John Carroll

Safety concerns forced investigators at Memorial Sloan-Kettering Cancer Center to suspend patient recruitment for an early-stage study of a closely watched approach to reengineering the immune system to fight cancer. Several days ago MSK updated a site on clinicaltrials.gov to note that it was halting recruitment for a small study using T cells reengineered with chimeric antigen receptors (CARs) against CD19-positive B cells for aggressive non-Hodgkin lymphoma, triggering concerns about the potential fallout at Juno Therapeutics, the biotech formed to commercialize the effort. And Sunday evening representatives for MSK revealed at the meeting of the American Association for Cancer Research in San Diego that the deaths of two patients spurred investigators to rethink the trial protocol on recruitment, revamping the patient profile to account for the threat of comorbidities while adjusting the dose “based on the extent of disease at the time of treatment.”

For more on this story please see

Source: http://www.fiercebiotech.com/story/memorial-sloan-kettering-hits-brakes-engineered-t-cell-cancer-study/2014-04-06

Keynote presentation by Carl H. June, recipient of The Richard V. Smalley MD 2013 Award

As reported in 2013 in Highlights and summary of the 28th annual meeting of the Society for Immunotherapy of Cancer by Paolo A Ascierto1†, David H Munn2†, Anna K Palucka34† and Paul M Sondel in Journal of ImmunoTherapy of Cancer

“

Since 2005, SITC honors a luminary in the field who has significantly contributed to the advancement of cancer immunotherapy research by presenting the annual Richard V. Smalley MD Memorial Award, which is associated with the Smalley keynote lecture at the Annual SITC meeting. The awardee this year Carl H. June of the University of Pennsylvania, has led innovative translational research for over 25 years, with the most recent focus being the development of the Chimeric Antigen Receptor modified T-cell (CART) approach. Carl June summarized how the past 15 years of progress have expanded upon the original concept presented by Zelig Eshhar [4], in which variable regions of tumor-reactive monoclonal antibodies (mAbs) (VH and VL) are linked to transmembrane and signaling domains of T cell activating molecules to create membrane based receptors with specificity for the tumor antigen recognized by the original mAb [4]. These receptors can be transfected into T cells, for example with lentiviruses. Pre-clinical work demonstrated how CD3-ζ and 41BB signaling components enhanced proliferation and survival of T cells in hypoxic conditions. The initial clinical work has been done with CART reactive to CD-19 on malignant B cells, with progress particularly in chronic lymphocytic leukemia (CLL) in adults and acute lymphoblastic leukemia (ALL) in children [5,6]. As of the SITC meeting, CarlJune’s team had treated 35 patients with CLL and 20 with ALL. Of the 20 with ALL, ½ had relapsed after allogeneic BMT. Of these 20 children, 17 were in complete remission, and with persistent B cell aplasia; documenting the persistent effects of the CART cells. Toxicities included the persistent B cell aplasia and profound tumor lysis and cytokine storm, seen 1–2 weeks into the treatment for ALL. This cytokine storm has been ameliorated by using anti-IL6 mAb. The B cell aplasia, while undesired, is acceptable, as patients can receive passive replacement of IgG, thus making their B cells “expendable”. These CART cells can traffic into the CNS. In ALL patients, it appears that each individual CART cell (or its progeny) can destroy 1000 tumor cells. Ongoing efforts in CarlJune’s program, and at other centers, are now moving into analyses of CART reactive with other tumor targets, by using mAbs that recognize antigens expressed on other tumors. Among these are EGFR on glioblastoma, PSMA on prostate cancer, mesothelin on ovarian cancer, HER2 on breast (and other) cancers, and several other targets. Because some of these targets are also expressed on normal tissues that are “not expendable”, novel approaches are being developed to decrease the potency or longevity of the CART effect, to decrease potential toxicity. This includes generating “short lived” CART cells by inducing CAR expression with short-lived RNA, rather than transfecting with a DNA construct that remains permanently.

“

In T-Cell Immunotherapy: Looking Forward Molecular Therapy (2014); 22 9, 1564–1574. doi:10.1038/mt.2014.148 many of the leading CAR-T clinicians and investigators reported on some of the adverse events related o CAR-T therapy including

  • 40 severe adverse events (SAE) had been reported from 2010 to 2013.
  • B-cell aplasia
  • Systemic inflammatory release syndrome (CRS) {the most sever toxicity seen}
  • Tumor lysis syndrome
  • CNS toxicity
  • Macrophage activation syndrome

According to the investigators the systemic inflammatory release syndrome (CRS) is the most severe toxicity seen

“

The most commonly reported adverse event is CRS,49 with about three-quarters of the patients with CRS requiring admission to an intensive care unit. In the case of CAR therapy, the onset of CRS is related to the particular signaling domain in the CAR, with early-onset CRS in the first several days after infusion related to CARs that encode a CD28 signaling domain.4,16 By contrast, CARs encoding a 4-1BB signaling domain tend to have delayed-onset CRS (range, 7 to 50 days) after CAR T-cell infusion.6 CRS has also been reported after the infusion of TCR-modified T cells, with onset typically five to seven days after infusion. The development of CRS is often, but not invariably, associated with clinically beneficial tumor regression. Several cytokines have been reported to be elevated in the serum—most commonly, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-6. Management of CRS has included supportive care, corticosteroids, etanercept, tocilizumab, and alemtuzumab. The role of suicide genes in the management of CRS remains unknown.50

“

This supportive therapy have now been included in all protocols now and sites are engaged in developing pharmacovigilance protocol development for CAR-T therapy.

UPDATED 08/31/2020

The following articles discuss the use of the CRISPR Cas9 system to improve the efficacy and reduce immune tolerance and rejection of engineered CAR-T therapies and the evolution of the CAR-T therapy in clinical trials for lung cancer and leukemias.

CRISPR-engineered immune cells reach the bedside

UPDATED 10/04/2021 (Advances in CAR-T therapy for solid tumors)

The following contains a curation of the latest advances on CAR-T therapy for solid tumors, which has been a challenge for the field.

NK cells expressing a chimeric activating receptor eliminate MDSCs and rescue impaired CAR-T cell activity against solid tumors

From: Parihar, R., Rivas, C., Huynh, M., Omer, B., Lapteva, N., Metelitsa, L. S., Gottschalk, S. M., & Rooney, C. M. (2019). NK Cells Expressing a Chimeric Activating Receptor Eliminate MDSCs and Rescue Impaired CAR-T Cell Activity against Solid Tumors. Cancer immunology research, 7(3), 363–375. https://doi.org/10.1158/2326-6066.CIR-18-0572

Abstract

Solid tumors are refractory to cellular immunotherapies in part because they contain suppressive immune effectors such as myeloid-derived suppressor cells (MDSCs) that inhibit cytotoxic lymphocytes. Strategies to reverse the suppressive tumor microenvironment (TME) should also attract and activate immune effectors with antitumor activity. To address this need, we developed gene-modified natural killer (NK) cells bearing a chimeric receptor in which the activating receptor NKG2D is fused to the cytotoxic ζ-chain of the T-cell receptor (NKG2D.ζ). NKG2D.ζ–NK cells target MDSCs, which overexpress NKG2D ligands within the TME. We examined the ability of NKG2D.ζ–NK cells to eliminate MDSCs in a xenograft TME model and improve the antitumor function of tumor-directed chimeric antigen receptor (CAR)-modified T cells. We show that NKG2D.ζ–NK cells are cytotoxic against MDSCs, but spare NKG2D ligand-expressing normal tissues. NKG2D.ζ–NK cells, but not unmodified NK cells, secrete pro-inflammatory cytokines and chemokines in response to MDSCs at the tumor site and improve infiltration and antitumor activity of subsequently infused CAR-T cells, even in tumors for which an immunosuppressive TME is an impediment to treatment. Unlike endogenous NKG2D, NKG2D.ζ is not susceptible to TME-mediated down-modulation and thus maintains its function even within suppressive microenvironments. As clinical confirmation, NKG2D.ζ–NK cells generated from patients with neuroblastoma killed autologous intra-tumoral MDSCs capable of suppressing CAR-T function. A combination therapy for solid tumors that includes both NKG2D.ζ–NK cells and CAR-T cells may improve responses over therapies based on CAR-T cells alone.

INTRODUCTION

T lymphocytes can be engineered to target tumor-associated antigens by forced expression of CARs (). Although successful when directed against leukemia-associated antigens such as CD19 (, ), CAR-T cell therapy for solid tumors has been less effective, with best responses in patients with minimal disease (, ). Solid tumors recruit inhibitory cells such as myeloid-derived suppressor cells (MDSCs) (). These immature myeloid cells are a component of innate immunity and strengthen the suppressive TME (, ). The frequency of circulating or intra-tumoral MDSCs correlates with cancer stage, disease progression, and resistance to standard chemo- and radio-therapies (). Hence, MDSCs are worth targeting in the quest to enhance CAR-T cell efficacy against solid tumors.

Natural killer (NK) cells, a lymphoid component of the innate immune system, produce MHC-unrestricted cytotoxicity and secrete pro-inflammatory cytokines and chemokines (). NK cells also modulate the activity of antigen-presenting myeloid cells within lymphoid organs, and recruit and activate effector T cells at sites of inflammation (, ). NK cells express NKG2D, a cytotoxicity receptor that is activated by non-classical MHC molecules expressed on cells stressed by events such as DNA damage, hypoxia, or viral infection (). NKG2D ligands are overexpressed on several solid tumors and on tumor-infiltrating MDSCs (). NK cells, therefore, could alter the TME in favor of an antitumor response by eliminating suppressive elements such as MDSCs. However, the NKG2D cytotoxic adapter molecule, DAP10, is downregulated by suppressive molecules of the TME, such as TGFβ (), limiting the antitumor functions of NK cells.

To overcome the repressive effect of the solid TME on NKG2D function, we used a retroviral vector to modify NK cells with a chimeric NKG2D receptor (NKG2D.ζ) comprising the extracellular domain of the native NKG2D molecule fused to the intracellular cytotoxic ζ-chain of the T-cell receptor (). We hypothesized that primary human NK cells expressing NKG2D.ζ (NKG2D.ζ–NK cells) would maintain NKG2D.ζ expression within the suppressive TME, kill NKG2D ligand-expressing MDSCs, secrete pro-inflammatory cytokines and chemokines, and recruit and activate effector cells, including CAR-T cells, derived from the adaptive immune system. These benefits are not attainable from NK cells expressing the native NKG2D receptor as its functions are down-modulated in the TME. Here we show that when NK cells express NKG2D.ζ, immune suppression is sufficiently countered to enable tumor-specific CAR-T cells to persist within the TME and eradicate otherwise resistant tumors.

MATERIALS AND METHODS

Cytokines, cell lines, and antibodies.

Recombinant human interleukin (IL)2 was obtained from National Cancer Institute Biologic Resources Branch (Frederick, MD). Recombinant human IL6, GM-CSF, IL7, and IL15 were purchased from Peprotech (Rocky Hill, NJ, USA). The human neuroblastoma cell line LAN-1 was purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM culture medium supplemented with 2 mM L-glutamine (Gibco-BRL) and 10% FBS (Hyclone, Waltham, MA, USA). The human CML cell line K562 was purchased from American Type Culture Collection and cultured in complete-RPMI culture medium composed of RPMI-1640 medium (Hyclone) supplemented with 2 mM L-glutamine and 10% FBS. A modified version of parental K562 cells, genetically modified to express a membrane-bound version of IL15 and 41BB-ligand, K562-mb15–41BB-L, was kindly provided by Dr. Dario Campana (National University of Singapore). All cell lines were verified by either genetic or flow cytometry-based methods (LAN-1 and K562 authenticated by ATCC in 2009) and tested for mycoplasma contamination monthly via MycoAlert (Lonza) mycoplasma enzyme detection kit (last mycoplastma testing of LAN-1, K562 parental line, and K562-mb15–41BB-L on November 2, 2018; all negative). All cell lines were used within one month of thawing from early-passage (< 3 passages of original vial) lots.

CAR-encoding retroviral vectors.

The construction of the SFG-retroviral vector encoding GD2-CAR.41BB.ζ, as shown in Supplementary Fig. S1A, was previously described (). The SFG-retroviral vector encoding NKG2D.ζ, an internal ribosomal entry site (IRES), and truncated CD19 (tCD19), was generated by sub-cloning NKG2D.ζ from a retroviral vector () kindly provided by Dr. Charles L. Sentman (Dartmouth Geisel School of Medicine, Hanover, NH, USA) into pSFG.IRES.tCD19 (). RD114-speudotyped viral particles were produced by transient transfection in 293T cells, as previously described ().

Expansion and retroviral transduction of human NK and T cells.

Human NK cells were activated, transduced with retroviral constructs (Fig. 1A) and expanded as previously described by our laboratory (). Briefly, peripheral blood mononuclear cells (PBMCs) obtained from healthy donors under Baylor College of Medicine IRB-approved protocols, were cocultured with irradiated (100 Gy) K562-mb15–41BB-L at a 1:10 (NK cell:irradiated tumor cell) ratio in G-Rex® cell culture devices (Wilson Wolf, St. Paul, MN, USA) for 4 days in Stem Cell Growth Medium (CellGenix) supplemented with 10% FBS and 500 IU/mL IL2. Cell suspensions on day 4 (containing 50–70% expanded/activated NK cells) were transduced with SFG-based retroviral vectors, as previously described (). The transduced cell population was then subjected to secondary expansion to generate adequate cell numbers for experiments in G-Rex® devices at the same NK cell:irradiated tumor cell ratio with 100 IU/mL IL2. This 17-day human gene-modified NK cell protocol resulted in > 97% pure CD56+/CD3− NK cell population with avg. 77.4% ± 18.2% (n = 25) of NK cells transduced with the construct of interest. For most experiments, transduced NK cells were purified to > 95% by magnetic column selection of truncated CD19 selection marker-positive cells.

 
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NKG2D.ζ–NK cells expand and kill ligand-expressing targets.

(A) Schematic of SFG-based retroviral vector constructs for transduction of human NK cells. (B) Human NK cells were expanded as described in Methods and percentage of CD56+/CD3− NK cells at time of retroviral transduction (day 4) is shown. Expanded NK cells (red circle in A) purified via depletion of CD3+ cells were transduced with NKG2D.ζ retroviral vector or empty vector control (referred to as “unmodified”), and transduction efficiencies are shown inset. (C) NKG2D expression on NK cells (MFI, inset) was assessed with isotype antibody as control. Non-transduced NK cells exhibited similar NKG2D expression to empty vector-transduced NK cells. * p = 0.003 vs. unmodified condition. (D) Expression of NKG2D (absolute MFI on y-axis) on NK cells from each donor (n = 25) transduced with either empty vector or NKG2D.ζ construct was determined by flow cytometry. Each pair of data points connected by a line represent cells from a single donor, to confirm surface expression of our chimeric molecule after transduction. Black line with grey block next to each group are mean MFI ± SEM. (E) NKG2D.ζ–NK cell cytotoxicity against K562 and LAN-1 tumor targets in a 4-hour 51Cr-release assay. Given that K562 and LAN-1 are both NK-sensitive targets, low E:T ratios were utilized to observe differences. Experiment is representative of at least three separate determinations from n = 10 donors. * p < 0.01 vs. unmodified NK cells at same E:T ratio. (F) NKG2D.ζ–NK cells were expanded after transduction culture (as shown in schema), and fold-expansion and cytotoxicity both pre- (day 7) and post- (day 17) secondary expansion were determined.

For production of GD2.CAR-T cells (autologous to MDSCs and NK cells), PBMCs from healthy donors were suspended in T-cell medium (TCM) consisting of RPMI-1640 supplemented with 45% Click’s Medium (Gibco-BRL), 10% FBS, and 2 mM L-glutamine, and cultured in wells pre-coated with CD3 (OKT3, CRL-8001, American Type Culture Collection) and CD28 (Clone CD28.2, BD Biosciences) antibodies for activation. Human IL15 and IL7 were added on day +1, and cells underwent retroviral transduction on day +2, as previously described (). T-cells were used for experiments between days +9 to +14 post-transduction, with phenotype as shown in Supplementary Figs. S1B–C.

Induction and enrichment of human MDSCs.

Our method for ex vivo generation of human PBMC-derived MDSCs was derived from published reports (), with slight modifications. Briefly, PBMCs were sequentially depleted of CD25hi-expressing cells and CD3-expressing cells by magnetic column separation (Miltenyi Biotec). Resultant CD25lo/−, CD3− PBMCs were plated at 4×106 cells/mL in complete-RPMI medium with human IL6 and GM-CSF (both at 20 ng/mL) onto 12-well culture plates (Sigma Corning) at 1 mL/well. Plates were incubated for 7 days with medium and cytokines being replenished on days 3 and 5. Resultant cells were harvested by gentle scraping and MDSCs were purified by magnetic selection using CD33 magnetic microbeads (Miltenyi Biotec). Cells were analyzed by multi-color flow cytometry for CD33, CD14, CD15, HLA-DR, CD11b, CD83, and CD163 (BD Biosciences). MDSCs were defined as either monocytic (M-MDSCs; CD14+, HLA-DRlow/−), PMN-MDSCs (CD14−, CD15+, CD11b+), or early-stage MDSCs (lineage−, HLA-DRlow/−, CD33+), as per published guidelines (). In addition to the above markers, MDSCs were stained for PD-L1, PD-L2, and NKG2D ligands via an NKG2D-Fc chimera (BD Biosciences) followed by FITC-labeled anti-Fc. This pan-ligand staining approach was determined to be the most efficient way to assess NKG2D ligand expression on human MDSCs because (1.) NKG2D ligand expression had not previously been reported for human MDSCs and thus simultaneous evaluation of the eight different NKG2D ligands would have been required, and (2.) we found poor reproducibility in staining patterns using individual commercially-available ligand antibodies, even within the same donor.

In vitro T-cell suppression assay.

T-cell proliferation was assessed using Cell Trace Violet (Thermo Fisher) dye dilution analysis, as per manufacturer’s recommendations. Briefly, 1×105 Cell Trace Violet-labeled T cells (isolated at the time of MDSC generation) were plated onto 96-well plates in the presence of plate-bound 1 μg/mL CD3 and 1 μg/mL CD28 antibodies with 50 IU/mL IL2 in the absence or presence of autologous MDSCs or peripheral blood monocytes (as a myeloid cell control) at 1:1, 4:1 and 8:1 T cell:MDSC ratios. In some experiments, only the 4:1 ratio is shown as this was determined as optimal for assessment of suppression. After 4 days of coculture, T cells were labeled with CD3 antibody and assessed for cell division using Cell Trace Violet dye dilution by flow cytometry. Percent suppression was calculated as follows: [(% proliferating T cells in the absence of MDSCs – % proliferating T cells in presence of MDSCs)/% proliferating T cells in the absence of MDSCs] x 100. Proliferation was defined as % T cells undergoing active division as represented by Cell Trace Violet dilution peaks, as previously described ().

In vitro CAR-T chemotaxis assay.

Transwell 5-μm pore inserts (Corning, Somerset, NJ) for migration experiments were prepared by coating with 0.01% gelatin at 37 °C overnight, followed by 3 μg of human fibronectin (Life Technologies, Grand Island, NY) at 37 °C for 3 hours to mimic endothelial and extracellular matrix components, as previously described (). Briefly, 2×105 purified GD2.CAR-T cells were placed in 100 μL of TCM in the upper chambers of the pre-coated Transwell inserts that were then transferred into wells of a 24-well plate. Culture supernatants (400 μL) from NKG2D.ζ or unmodified NK cells cultured with autologous MDSCs or monocytes, were placed in the lower chambers of the wells. Plain medium or medium supplemented with 1 μg/mL of the T-cell recruiting chemokine, MIG, served as negative and positive controls, respectively. The plates were then incubated for 4 hours at 37 °C with 5% CO2, followed by a 10-minute incubation at 4 °C to loosen any cells adhering to the undersides of the insert membranes. The fluid in the lower chambers was collected separately and migrated cells were counted using trypan blue exclusion. The cells were analyzed for CAR expression by flow cytometry to confirm phenotype of migrated T cells.

In vivo tumor microenvironment model.

12–16 week old female NSG mice were implanted subcutaneously in the dorsal right flank with 1×106 Firefly luciferase(FfLuc)-expressing LAN-1 neuroblastoma cells admixed with 3×105 ex vivo-generated MDSCs, suspended in 100 μL of basement membrane Matrigel (Corning). Matrigel basement membrane was important in keeping tumor and MDSCs confined so as to establish a localized solid TME. 10–14 days later, when tumors measured at least 100 mm3 by caliper measurement, mice were injected intravenously with 5×106 GD2.CAR-T cells. Tumor growth was measured twice weekly by live bioluminescence imaging using the IVIS® system (IVIS, Xenogen Corporation) 10 minutes after 150 mg/kg D-luciferin (Xenogen)/mouse was injected intraperitoneally. In experiments examining the ability of NKG2D.ζ–NK cells to reduce intra-tumoral MDSCs, 1×107 unmodified or NKG2D.ζ–NK cells were injected intravenously when tumors measured at least 100 mm3. At end of experiment, tumors were harvested en bloc, digested ex vivo, and intra-tumoral human MDSCs (CD33+, HLA-DRlow cells) were enumerated by flow cytometry. The absolute number of human MDSCs within a tumor digest was enumerated per mouse (n = 5 mice/group), compared to pre-treatment MDSC numbers, and presented as mean % MDSCs remaining per treatment group. In experiments examining the effects of NKG2D.ζ–NK cells on GD2.CAR-T cell antitumor activity, 5×106 (cell dose chosen to mitigate direct antitumor effects of NK cells) unmodified or NKG2D.ζ–NK cells were injected intravenously 3 days prior to GD2.CAR-T injection. In GD2.CAR-T cell homing experiments, CAR-T were transduced with GFP-luciferase retroviral construct prior to injection into mice bearing unmodified tumor cells (). Mice received 5000 IU human IL2 intraperitoneally three times per week for 3 weeks following NK cell injection to promote NK cell survival in NSG mice (). Tumor size was measured twice weekly with calipers and the mice were imaged for bioluminescence signal from T cells at the same time. Mice were euthanized for excessive tumor burden, as per protocol guidelines. The animal studies protocol was approved by Baylor College of Medicine Institutional Animal Care and Use Committee and mice were treated in strict accordance with the institutional guidelines for animal care.

Immunohistochemistry of neuroblastoma xenografts.

On day 32 of in vivo experiments, animals were sacrificed, tumors were harvested and sectioned bluntly ex vivo to separate tumor periphery (outer 1/3 of tumor volume) vs. core (non-necrotic inner 2/3 of tumor volume), and n = 5 sections/tumor sample were analyzed for presence of GD2.CAR-T and NKG2D.ζ–NK cells by H&E and human CD3 and CD57 immunostaining performed by the Human Tissue Acquisition and Pathology Core of Baylor College of Medicine. Lack of CD57 expression on infused GD2.CAR-T was confirmed by flow cytometry prior to administration. CD57 was chosen as the marker for NK cells in tumor tissue in our study because LAN-1 tumors naturally express the prototypical NK marker CD56, truncated CD19 expression was inadequate for in situ staining, and CD57 had previously been used as a marker for tissue-localized activated NK cells (). The number of human CD3+ and CD57+ cells in representative sections of tumors from periphery vs. core of the treatment groups indicated were enumerated per high-powered field (HPF) at 40x magnification and percent of the total number of cells enumerated within tumors found in the periphery vs. core in each treatment group indicated from tumors with and without MDSCs is shown as mean ± SEM of n = 5 sections/periphery or core, n = 5 tumors/group.

Analysis of intra-tumoral MDSCs from patients with neuroblastoma.

Tumor tissue and matched peripheral blood of neuroblastoma patients obtained in the context of a specimen/laboratory study after patient identification had been removed were thawed and analyzed for MDSC subsets by flow cytometry or utilized in in vitro assays, as described in legends or Results. The tissue acquisition protocol was performed after review and approval by the Baylor College of Medicine Institutional Review Board. Briefly, subjects with a diagnosis of high-risk or intermediate-risk neuroblastoma were eligible to participate. Written informed consent, or appropriate assent for participation, in accordance with the Declaration of Helsinki was obtained from each subject or subject’s guardian for procurement of patient blood and tumor tissue and for subsequent analyses of stored patient materials.

Statistics.

Data are presented as mean ± SEM of either experimental replicates or number of donors, as indicated. Paired two-tailed t-test was used to determine significance of differences between means with p < 0.05 indicating a significant difference. For in vivo bioluminescence, changes in tumor radiance from baseline at each time point were calculated and compared between groups using two-sample t-test. Multiple group comparisons were conducted via ANOVA via GraphPad Prism v7 software. Survival determined from the time of tumor cell injection was analyzed by Kaplan-Meier and differences in survival between groups were compared by the log-rank test.

RESULTS

NKG2D.ζ NK cells expand and have cytotoxicity against target cells.

To increase killing of NKG2D ligand-expressing MDSCs, we generated primary human NK cells stably expressing NKG2D.ζ and a truncated CD19 (tCD19) marker from a retroviral vector (Fig. 1A). NK cells were expanded from PBMCs obtained from normal donors, transduced with retroviral construct expressing chimeric NKG2D, then cultured for 3 additional days. Transduction efficiency, as measured by the expression of tCD19 on CD56+CD3− NK cells after the additional 3 days, was 71.3 ± 16% (n = 25 normal donors) and produced a 5.4 ± 1.1-fold increase in NKG2D expression on the NK cell surface (Fig. 1B–D). NKG2D.ζ–NK cells showed greater cytotoxicity (79.2 ± 5.6%, n = 10 normal donors) against wild-type K562, a highly NK cell-sensitive tumor cell line that naturally expresses NKG2D ligands, than mock vector-transduced (hereafter referred to as, unmodified) NK cells (40.5 ± 2.1%) at 2:1 E:T ratio in a 4-hr cytotoxicity assay (Fig. 1E). In contrast, transgenic NKG2D.ζ expression did not increase NK cell killing of LAN-1 neuroblastoma cells that are marginally NK-sensitive, but lack NKG2D ligands. To determine if in vitro expansion affected the cytotoxic function of NKG2D.ζ–NK cells, we secondarily expanded NKG2D.ζ–NK cells for an additional 10-days (Fig. 1F schema). As seen in Fig. 1F, NKG2D.ζ–NK cells expanded (120 ± 7.3-fold by day 17 of culture; n = 10 donors) similarly to unmodified and non-transduced NK cells and maintained stable cytotoxic function between days 7 and 17 of expansion. Thus, we generated and expanded high numbers of primary human NKG2D.ζ-expressing NK cells capable of cytotoxicity against ligand-expressing targets, even after prolonged culture.

Transgenic NKG2D.ζ is unaffected by TGFβ or soluble NKG2D ligands.

Expression of the native NKG2D receptor on NK cells is down-modulated by tumor-derived TGFβ and soluble NKG2D ligands, both of which are abundant in the TME (, ) and likely impair NK cell function in solid tumors. To determine the effect of TGFβ and soluble NKG2D ligands on NKG2D.ζ receptor expression and function, we cultured NKG2D.ζ–NK cells in the presence of TGFβ or the soluble NKG2D ligands, MICA and MICB, and examined NKG2D expression and NK cytotoxicity after 24-, 48-, and 72-hours. After exposure to TGFβ or soluble MICA/B, unmodified NK cells significantly down-regulated NKG2D (MFI of 25 vs. 95 in non-exposed NK cells at 48 hours) and were less cytotoxic (20 ± 5.1% killing vs. 40 ± 3.7% killing by non-exposed NK cells at 48 hours) to NKG2D ligand-expressing K562 targets (Fig. 2A, ​,B).B). In contrast, NKG2D.ζ–NK cells maintained NKG2D expression and cytotoxicity after exposure to the same concentrations of TGFβ and soluble MICA/B (Fig. 2C, ​,D).D). This lack of sensitivity to down-regulation by these tumor-associated components should benefit the function of NKG2D.ζ–NK cells within the TME.

 
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Transgenic NKG2D.ζ is unaffected by TGFβ or soluble NKG2D ligands.

NKG2D.ζ or unmodified NK cells (n = 5 donors) were cultured in the presence of TGFβ (5 ng/mL) (A, B) or the soluble NKG2D ligands MICA and MICB (C, D) for 24-, 48-, and 72-hours. NKG2D receptor expression was determined by flow cytometry and NK cytotoxicity against K562 targets was assessed in a 4-hr Cr-release assay at an 5:1 E:T ratio using 48-hr exposed NK cells. Viability of transduced NK cells after exposure to TGFβ for 24, 48, and 72 hours, as assessed by 7-AAD vital staining, was > 90%. * p = 0.001 vs. non-TGFβ/MICA-treated NK groups at same time-points.

Human MDSCs express NKG2D ligands and are killed by NKG2D.ζ–NK cells.

To study the effects of human NK cells on autologous MDSCs, we generated human MDSCs by culture of CD3-/CD25lo PBMC with IL6 plus GM-CSF for 7 days, followed by CD33+ selection, as described in the Methods. The phenotypic characterization of these MDSCs and confirmation of their suppressive capacity is shown in Supplementary Fig. S2. Routinely, our ex vivo-generated MDSCs contained monocytic (M)-MDSC and early(e)-MDSC subsets, with few (avg. < 1%) polymorphonuclear (PMN)-MDSCs (Supplementary Fig. S2A), roughly reflecting the subset composition reported in patients with solid tumors (, ). The MDSCs expressed the suppressive factors TGFβ, IL6, IL10, and PDL-1 in amounts often greater than tumor cells (Supplementary Figs. S2B–C), and suppressed proliferation and cytokine secretion by autologous T cells stimulated with plate-bound CD3/CD28 antibodies (Supplementary Figs. S2D–E) and by 2nd generation GD2.CAR-T cells encoding 4–1BB and CD3-ζ endodomains stimulated with the GD2+ tumor line LAN-1 (Supplementary Figs. S2F–G). As seen in Fig. 3A, MDSCs expressed as much or more NKG2D ligand than the positive control tumor line, K562 (ligand MFI of 78.2 vs. 29.7, respectively). Freshly isolated peripheral blood T cells did not express NKG2D ligands, whereas immature and mature dendritic cells expressed little, consistent with previous data (). The neuroblastoma cell line, LAN-1, subsequently used in our in vivo TME model, did not express NKG2D ligands.

 
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Human MDSCs express ligands for NKG2D and are killed by NKG2D.ζ–NK cells.

(A) NKG2D ligand expression on human MDSCs by flow cytometry. Immature dendritic cells (iDC) and mature DCs (mDC) were used as myeloid controls. T cells activated with CD3 and CD28 mAbs plus 100 IU/mL IL2 for 24 hours were used as lymphocyte control. LAN-1 and K562 cells were used as negative and positive controls, respectively. MFI of NKG2D ligand expression in parenthesis. Representative data from single donor (of n = 25 normal donors). Isotype control for NKG2D staining routinely fell within the 1st log. (B) NKG2D.ζ–NK cell cytotoxicity against autologous MDSCs as targets in a 4-hour 51Cr-release assay. In some wells of the cytotoxicity assay, a blocking mAb to NKG2D was added. Representative data from triplicate samples per data point from a single donor (of n = 25 normal donors) is shown. * p < 0.01 vs. unmodified NK cells at same E:T ratio. (C) In the same experiment as (B), the same batch of NKG2D.ζ–NK cells were analyzed for cytotoxicity against autologous B cells, monocytes, monocyte-derived iDC and mDC, and activated T cells (n = 10 donors examined). (D) M-MDSC frequency by flow cytometry from neuroblastoma tumor samples obtained from high-risk patients, as described in Methods. (E) Cytotoxicity by NKG2D.ζ–NK cells derived from patient PBMC (harvested and frozen at time of tumor sampling) against autologous tumor-derived MDSCs in a 4-hour 51Cr-release assay. Data shown are from triplicate samples per data point at a 10:1 E:T ratio. * p < 0.001 vs. unmodified NK cells from same donor. (F) NKG2D.ζ–NK cells were cocultured with autologous MDSCs at 1:1 ratio plus low-dose 50 IU/mL IL2 to maintain NK survival, and fold change in the number of each cell type from the start of coculture was determined by flow cytometry at indicated time-points. * p < 0.001 vs. NK/MDSC fold-change in unmodified NK cell cocultures. (G) Cell-free supernatants were harvested from cocultures at day 3 and analyzed for IFNγ, TNFα, IL6, and IL10 by ELISA. # p < 0.01 vs. corresponding cytokine in cocultures with unmodified NK cells. (H) NKG2D ligand expression was determined for activated T cells (ATCs) expressing NKG2D.ζ and NKG2D.ζ–NK cells. Expression of NKG2D ligands on non-transduced ATCs as control for T-cell activation. (I) NKG2D.ζ–NK cells or NKG2D.ζ T cells were cocultured with autologous ATCs at 1:1 ratio and fold change in the number of each cell type from the start of coculture was determined by flow cytometry at indicated time-points. * p < 0.001 vs. ATC fold-change at days 0 and 3 cocultures.

To evaluate MDSC susceptibility to killing by NKG2D.ζ–NK cells, we performed both short- and long-term killing assays. Fig. 3B shows enhanced killing of MDSCs by autologous NKG2D.ζ–NK cells compared to unmodified NK cells (35 ± 5.5% vs. 8 ± 2.4% cytotoxicity, respectively, at an E:T ratio of 5:1) in a 4-hr chromium-release assay. MDSC killing was dependent on NKG2D, as pre-incubation with an NKG2D blocking Ab reduced the cytotoxicity to levels achieved by unmodified NK cells. NKG2D.ζ–NK cells mediated no cytotoxicity against other autologous immune cells such as freshly-isolated monocytes, monocyte-derived mature dendritic cells, T cells, or B cells (Fig. 3C). Only immature dendritic cells, which expressed little NKG2D ligand (approx. 7% of cells; MFI 11.4), were mildly susceptible to lysis by NKG2D.ζ–NK cells (4.2 ± 1.7 % lysis at an E:T ratio of 20:1). As confirmation of the clinical applicability of our approach, we assessed whether NKG2D.ζ–NK cells generated from patient PBMCs were able to kill highly suppressive MDSCs isolated from the patient’s tumor. Tumor samples obtained from two patients with high-risk neuroblastoma at time of first biopsy/resection contained M-MDSCs (Fig. 3D). NKG2D.ζ–NK cells generated from patient PBMCs (harvested and frozen at time of tumor sampling) mediated significant cytotoxicity in vitro against M-MDSCs purified from patient tumors, whereas unmodified patient NK cells did not (Fig. 3E). These results provide further clinical evidence for the capacity of NKG2D.ζ–NK cells to eliminate MDSCs in patients with suppressive TMEs.

To determine whether NKG2D.ζ–NK cells could control MDSC survival in long-term cultures, we cocultured NKG2D.ζ–NK cells with autologous MDSCs at a 1:1 ratio for 7 days in the presence of low-dose IL2 to maintain NK survival, and quantified each cell type by flow cytometry every two days. As shown in Fig. 3F, NKG2D.ζ–NK cells expanded in cocultures (mean 9.5 ± 0.7-fold increase) with a concomitant reduction in MDSCs (mean 81.3 ± 9.4-fold decrease), whereas unmodified NK cells failed to expand or eliminate MDSCs. NK cells cultured alone or with autologous monocyte controls did not expand (0.8 ± 0.1-fold change). As seen in Fig. 3G, NK cell expansion and MDSC reduction correlated with a shift in the culture cytokine milieu from one that is immune suppressive (more IL6 and IL10; less IFN-γ and TNF-α) in cocultures containing unmodified NK cells, to one that is immune stimulatory and enhances CAR-T antitumor function (less IL6 and IL10; more IFN-γ and TNF-α) in cocultures containing NKG2D.ζ–NK cells. Hence, NKG2D.ζ–NK cells mediate potent cytotoxicity against suppressive MDSCs via their highly expressed NKG2D ligands. In addition, through selective depletion of MDSCs in combination with immune stimulatory cytokine secretion, NKG2D.ζ–NK cells skew the cytokine microenvironment to one that can support CAR-T effector functions ().

Previous studies have reported that expression of chimeric NKG2D constructs in T lymphocytes can direct these cells to target NKG2D ligand-expressing tumors (, ). However, activated T cells (ATCs) themselves upregulate NKG2D ligands (), with variable ligand expression intensity dependent on the T-cell activation protocol employed, leading to fratricide when the chimeric NKG2D is expressed. To determine if this off-tumor side-effect occurred when the same NKG2D.ζ was expressed in NK cells, we compared the killing of ATCs by autologous NK cells or by autologous T cells expressing our NKG2D.ζ transgene. ATCs and NKG2Dζ.-T cells both upregulated NKG2D ligands during ex vivo expansion with CD3/CD28 antibodies plus IL7 and IL15, whereas NKG2D.ζ-transduced NK cells undergoing expansion in our K562-mb15–41BB-L culture system did not (Fig. 3H). Coculture without additional stimulation of NKG2D.ζ-T cells with autologous ATCs produced fratricide, of both the NKG2D.ζ effector T cells (35 ± 7.2% decrease in cell number) and the non-transduced ATC targets (98 ± 11.5% decrease in cell number) (n = 3). By contrast, ATC numbers were unaffected by coculture with autologous NKG2D.ζ–NK cells (Fig. 3I). These results show that NK cells expressing NKG2D.ζ can kill autologous MDSCs while sparing other NKG2D ligand expressing populations, thus avoiding the fratricide seen with NKG2D.ζ-expressing T cells.

NKG2D.ζ–NK cells eliminate intra-tumoral MDSCs and reduce tumor burden.

To determine if NKG2D.ζ–NK cells could eliminate MDSCs from tumor sites in vivo, we created an MDSC-containing TME in a xenograft model of neuroblastoma. We chose NKG2D ligand-negative LAN-1 tumor for this experiment so that the effects of NKG2D.ζ–NK cells on MDSCs were not confused with their effects on the tumor cells. LAN-1 tumor cells admixed with human MDSCs were inoculated subcutaneously in NSG mice. These animals had increases in the suppressive cytokines IL10 (10-fold vs. tumor alone) and TGFβ (2.6-fold vs. tumor alone) in circulation by day 16 as compared to animals bearing tumors initiated without MDSCs, and the resultant tumors grew more rapidly due to increased neovascularization and tumor-associated stroma (Supplementary Fig. S3A–D), consistent with clinical reports of MDSC-dense tumors (). As seen in Fig. 4A, in mice bearing NKG2D ligand-negative tumors without MDSCs, a single infusion of 1×107 NKG2D.ζ–NK cells resulted in a small delay in tumor growth but eventual progression, suggesting that the LAN-1 tumor itself (a marginally NK-sensitive target) can be killed at higher NK cell doses independent of NKG2D ligand expression. In mice bearing MDSC-containing tumors, 1×107 NKG2D.ζ–NK cells inhibited tumor growth (Fig. 4B), reduced NKG2D ligand-expressing intra-tumoral MDSCs with only 8.7 ± 3.5% of the input MDSCs remaining (Fig. 4C), and prolonged mouse survival (median survival of 73 days vs. 29 days after unmodified NK cells; Fig. 4D). Since LAN-1 tumor cells do not express NKG2D ligands and are only marginally sensitive to ligand-independent lysis, tumors subsequently regrew in these mice once the NKG2D.ζ–NK cells had disappeared (> day 40). Thus, NKG2D.ζ–NK cells can traffic to tumor sites and reduce intra-tumoral MDSCs but cannot themselves eradicate NKG2D ligand-negative malignant cells in our model.

 
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NKG2D.ζ–NK cells eliminate intra-tumoral MDSCs and reduce tumor burden.

LAN-1 tumor cells, either alone (A) or admixed with human MDSCs (B), were injected S.C. in the flanks of NSG mice. When tumors reached a volume of approx. 100 mm3 (day 14, gray block arrow inset), no NK cells (PBS control), 1×107 unmodified or NKG2D.ζ–NK cells were injected I.V. and tumor growth was measured over time via calipers. * p < 0.03 vs. other conditions shown at same time point. (C) On day 26, intra-tumoral human MDSCs (CD33+, HLA-DRlow) were enumerated by flow cytometry and are presented as mean % MDSCs remaining per treatment group. ** p < 0.005 vs. unmodified NK treatment. (D) Survival of groups by Kaplan-Meyer analysis. # p = 0.024. Representative experiment of three performed.

NKG2D.ζ–NK cells secrete chemokines that recruit GD2.CAR-T cells.

To determine if NKG2D.ζ–NK cells can recruit T cells modified with a tumor-specific CAR to tumor sites containing MDSCs, we cocultured NKG2D.ζ–NK cells with autologous MDSCs and analyzed culture supernatants for chemokines by multiplex ELISA. Compared to unmodified NK cells, NKG2D.ζ–NK cells produce significantly greater CCL5 (RANTES; 10-fold increase), CCL3 (MIP-1α; 2-fold increase), and CCL22 (MDC; 5-fold increase) in response to autologous MDSCs (Fig. 5A). Large amounts of CXCL8 (IL8) were also produced, but there was no significant difference from the production by unmodified NK cells. Analysis of chemokine receptor expression on 2nd generation GD2.CAR-T cells revealed CXCR1 (binds CXCL8), CCR2 (binds CCL2), CCR5 (binds CCL3), and CCR4 (binds CCL5) (see Supplementary Fig. S1C). These GD2.CAR-T cells were assayed for chemotaxis to supernatants derived from unmodified or NKG2D.ζ–NK cells cocultured with autologous MDSCs. Supernatants from NKG2D.ζ–NK cell-containing cocultures induced chemotaxis of 41.1 ± 5.5% of GD2.CAR-T cells (Fig. 5B), whereas supernatants from unmodified NK cells induced chemotaxis no greater than produced by medium (14.9 ± 6.4% vs. 17.3 ± 1.9%, respectively). Chemotaxis was not induced by supernatants from unmodified or NKG2D.ζ–NK cells cocultured with monocytes. Thus, following their encounter with MDSCs, NKG2D.ζ–NK cells secrete chemokines that recruit CAR-Ts in vitro.

 
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NKG2D.ζ–NK cells secrete chemokines that recruit GD2.CAR-T cells.

(A) NKG2D.ζ or unmodified NK cells were cocultured with autologous MDSCs and cell-free culture supernatants harvested at 48 hours were analyzed for chemokines CXCL8, CCL5, CCL3, and CCL22 by ELISA. Shown are mean chemokine concentration ± SEM for n = 5 cocultures/donor (data from one of five representative donors is shown). * p < 0.005 vs. unmodified NK cocultures. (B) GD2.CAR-T cells were assayed for chemotaxis in Transwells (described in Methods) in response to supernatants derived from unmodified or NKG2D.ζ–NK cells cocultured with autologous MDSCs. Supernatants derived from monocyte (non-suppressive myeloid cell control)-stimulated NK cells were also used. # p < 0.001 vs. Medium, ** p = 0.009 vs. “unmodified NK plus MDSCs” condition. (C) LAN-1 tumor cells, alone or admixed with human MDSCs, were injected S.C. into the flank of NSG mice. When tumors reached a volume ~100 mm3, 5×106 GD2.CAR-T cells were injected I.V. alone on day 13 (GD2.CAR-T), or preceded by 5×106 NKG2D.ζ–NK cells I.V. injected on day 10 (chNK + GD2.CAR-T). GD2.CAR-T signal at tumor site was measured over time via live-animal bioluminescence imaging. (D) Shown is mean ± SEM (n=5 mice/group) bioluminescent signal of GD2.CAR-T cells expressed as radiance. * p = 0.01 vs. all other groups.

NKG2D.ζ NK cells improve GD2.CAR-T cell trafficking to tumor sites.

To determine the effects of the MDSC-induced, NKG2D.ζ–NK cell chemokines on CAR-T cell recruitment in vivo, we used our MDSC-containing TME xenograft model (see Fig. 4). When tumors reached a volume of ~100 mm3 (day 10), 5×106 NKG2D.ζ–NK cells were infused, followed three days later (day 13) by infusion of 5×106 luciferase gene-transduced GD2.CAR-T cells. Tumor localization and expansion of GD2.CAR-T cells was measured over time via live-animal bioluminescence imaging. As seen in Fig. 5C, GD2.CAR-T cells injected alone on day 13 after tumor inoculation (without pre-administration of NKG2D.ζ–NK cells) into mice bearing tumors devoid of MDSCs localized effectively to subcutaneous tumors in the flank (4 of 5 mice showed bioluminescent signal on days 14 and 18; Fig. 5C). There was a 10.5 ± 0.8-fold increase in bioluminescent signal on day 18, with CAR-T cell bioluminescence remaining above baseline levels for the duration of the experiment (Fig. 5D). However, in tumors containing MDSCs, CAR-T cells localized poorly: only 1 of 5 mice exhibited bioluminescent signal (Fig. 5C), with only a 1.02 ± 0.1-fold increase in bioluminescent signal on day 18 and bioluminescence falling below pre-infusion levels within 10 days after injection (Fig. 5D). In contrast, pre-administration of NKG2D.ζ–NK cells on day 10 into mice bearing MDSC-containing tumors allowed subsequently infused GD2.CAR-T cells to localize effectively to tumor sites, with bioluminescence in 5 of 5 mice at the tumor site and a 10.9 ± 0.2-fold increase in bioluminescent signal on day 18, within 5 days of injection (Fig. 5D).

To determine if NKG2D.ζ–NK cells could promote GD2.CAR-T infiltration into the tumor bed, we compared the frequency of human GD2.CAR-T and human NK cells in the tumor periphery and the tumor core by immunohistochemistry (Supplementary Fig. S4A–B). In tumors without MDSCs, 89 ± 11% of the total T cells in the tumor had infiltrated into the tumor core. In contrast, a much smaller fraction (39 ± 16%) infiltrated into the core of tumors containing MDSCs, suggesting TME suppression of CAR-T infiltration. However, pre-treatment of tumors containing MDSCs with NKG2D.ζ–NK cells increased the fraction of intra-tumoral CAR-T cells (70 ± 13%) within the tumor core. Equal numbers of NKG2D.ζ–NK cells were observed within both peripheral and core samples from MDSC-positive and MDSC-negative tumors (Supplementary Fig. S5), suggesting the ability of NK cells to traffic well within tumors despite the presence of MDSCs.

Elimination of MDSCs increases antitumor activity of GD2.CAR-T cells.

To determine if the activities of NKG2D.ζ–NK cells described above enhance the antitumor function of CAR-T cells, we treated mice bearing subcutaneous, luciferase-labeled neuroblastoma containing MDSCs with GD2.CAR-T cells preceded by NKG2D.ζ–NK cells, in a similar set-up to experiments in Fig. 5C. As seen in Fig. 6A–B, a single injection of 5×106 NKG2D.ζ–NK cells (a dose that achieved intra-tumoral MDSC depletion with only 26.8 ± 5.8% of the input MDSCs remaining) resulted in no significant tumor regression or prolongation of survival in mice bearing xenografts containing human MDSCs. A single infusion of 5×106 GD2.CAR-T cells significantly reduced tumor in mice whose xenografts lacked human MDSCs with a median survival of 95 days (Fig. 6C–D). However, the same GD2.CAR-T cells were ineffective against xenografts containing human MDSCs, worsening overall median survival to 39 days (Fig. 6B). In contrast, when the same GD2.CAR-T cell injection was preceded 3 days earlier by a single injection of 5×106 NKG2D.ζ–NK cells (that had no direct antitumor effect by themselves within the other arm of the same experiment, see Fig. 6A–B), the antitumor activity of the GD2.CAR-T cells in mice bearing MDSC-containing tumors was restored to the level observed in mice whose tumors lacked MDSCs (Fig. 6C). NKG2D.ζ–NK cells pre-injection also improved the overall survival of the mice with MDSC-containing tumors to a median 120 days with durable cure in 2 of 5 mice (Fig. 6D). Taken together, our results suggest that NKG2D.ζ–NK cells not only eliminate MDSCs from the TME, but also recruit CAR-T cells to intra-tumoral sites which facilitates antitumor efficacy.

 
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Elimination of MDSCs by NKG2D.ζ–NK cells increases antitumor activity of GD2.CAR-T cells.

Luciferase gene-transduced LAN-1 tumor cells, alone or admixed with human MDSCs, were injected S.C. into NSG mice. (A) When tumors reached a volume ~100 mm3, no treatment (No Tx; PBS control) or 5×106 NKG2D.ζ–NK cells alone (chNK) were injected I.V. on day 10 and tumor growth was measured over time via live-animal bioluminescence imaging. Shown is mean ± SEM (n=5 mice/group) bioluminescent signal expressed as radiance. # ns, p = 0.18 vs. No treatment (+MDSC) group. (B) Survival of groups in A was determined by Kaplan-Meyer analysis. # ns, p = 0.059. (C) In other groups of mice within the same experiment, 5×106 GD2.CAR-T cells were injected I.V. alone on day 13 (GD2.CAR-T), or preceded by 5×106 NKG2D.ζ–NK cells injected on day 10 (chNK+GD2.CAR-T). * p = 0.001; # ns, p = 0.59 vs. each other. (D) Survival of groups in C by Kaplan-Meyer analysis. Representative experiment of 5 separate experiments. * p = 0.002; ** p = 0.001.

DISCUSSION

We have developed a TME-disrupting approach that eliminates MDSCs and rescues MDSC-mediated impairment of tumor-directed CAR-T cells. We show that when co-implanted with a neuroblastoma cell line, human MDSCs both enhance tumor growth and suppress the infiltration, expansion, and antitumor efficacy of tumor-specific CAR T-cells. In this model, NK cells bearing a chimeric version of the activating receptor NKG2D (NKG2D.ζ–NK cells) are directly cytotoxic to autologous MDSCs, thus eliminating MDSCs from tumors. In addition, NKG2D.ζ–NK cells secrete pro-inflammatory cytokines and chemokines in response to MDSCs at the tumor site, improving CAR-T cell infiltration and function, and resulting in tumor regression and prolonged survival compared to treatment with CAR-T cells alone. Our cell therapy approach utilizes an engineered innate immune effector that targets the TME, and shows potential to enhance efficacy of combination immune-based therapies for solid tumors.

NKG2D.ζ–NK cells directly killed highly suppressive MDSCs generated in vitro as well as those from patient tumors. NKG2D.ζ–NK cells also secreted cytokines that favored immune activation in response to MDSCs. Unmodified NK cells were unable to mediate these effects. The ability of NKG2D.ζ–NK cells to eliminate MDSCs from the TME should have several beneficial effects for antitumor immunity. First, as MDSCs express suppressive cytokines such as TGFβ and the checkpoint ligands PDL-1 and PDL-2, elimination of MDSCs should help relieve the suppression of endogenous T cell responses and potentiate the activity of adoptive T cell therapies. Given that high baseline numbers of MDSCs have been reported as a biomarker of poor response in the context of trials with the checkpoint inhibitors ipilimumab and pembrolizumab (, ), elimination of MDSCs by NKG2D.ζ–NK cells may also enhance checkpoint inhibition. Second, elimination of MDSCs should also decrease other MDSC-associated effects, including neovascularization via their expression of VEGF, production of immunosuppressive metabolic products such as PGE2 and adenosine, and establishment of tumor-supportive stroma via their expression of iNOS, FGF, and matrix metalloproteinases (). In short, the ability of NKG2D.ζ–NK cells to eliminate MDSCs alters the tumor microenvironment in multiple ways that should improve antitumor immunity.

Previous strategies for modulation of MDSCs within the TME have included use of agents that block single functions such as secretion of nitric oxide () or expression of checkpoint molecules (); induce MDSC differentiation such as with all-trans retinoic acid (); or eliminate MDSCs such as with the cytotoxic agents doxorubicin or cyclophosphamide (). The MDSC eliminating effects were dependent on continued administration of the agents, with a rapid rebound in MDSCs after discontinuation. Moreover, many of these agents have off-target toxicities that include damage to endogenous tumor-specific T cells. In contrast, NKG2D.ζ–NK cells produce prolonged and specific elimination of MDSCs with the potential to kill MDSCs that are recruited to the tumor from the bone marrow, while continually secreting cytokines and chemokines which respectively alter TME suppression and recruit and activate tumor-specific T cells. Thus, NKG2D.ζ–NK cells exert a prolonged combination of simultaneous immune modulatory effects that enhance antitumor immune function in ways that could not be achieved by previous methods that target MDSCs.

We observed no toxicity against normal hematopoietic cells when NKG2D.ζ was expressed in autologous human NK cells. Previous studies overexpressing an NKG2D.ζ receptor containing co-stimulatory endodomains (e.g., CD28 or 41BB) and DAP10, a signaling adaptor molecule for enhanced surface expression of NKG2D, in T cells showed activity against NKG2D ligand-overexpressing tumors, but at the cost of fratricide in vitro and lethal toxicity in mice (, , ). Using our standard T-cell activation/expansion protocol (), we also observed upregulation of NKG2D ligands, leading to fratricide in T cells expressing NKG2D.ζ. When NKG2D.ζ-T cells engage NKG2D ligands expressed on normal tissues, they will not receive the physiologic NK cell-directed inhibitory inputs that would safely regulate this potent and unopposed chimeric receptor activity. By contrast, when NKG2D.ζ is expressed on NK cells, they are able to recognize inhibitory NK cell ligands such as self-MHC expressed on healthy self-tissues, counteracting otherwise unopposed positive signals from NKG2D ligands. Thus, an NK cell platform for NKG2D enhancement may limit toxicity while taking advantage of the cytotoxic and immune modulatory potential of the receptor-ligand system.

Unlike wild-type NKG2D, transgenic NKG2D.ζ expression and activity were not sensitive to down-modulation by TGFβ or soluble NKG2D ligands, allowing improved function in the TME. Native NKG2D relies solely on the intra-cytoplasmic adaptor DAP10 for mediating its cytolytic activity in human NK cells (). TGFβ1 and soluble NKG2D ligands both decrease DAP10 gene transcription and protein activity, and thus reduce NKG2D function in endogenous NK cells (, ). In contrast, transgenic NKG2D.ζ does not rely on DAP10-based signaling for its activity, since signaling occurs through the ζ-chain. Thus, this construct provides a stable cytolytic pathway capable of circumventing TME-mediated down-modulation of native NKG2D activity. A previous study expressing a chimeric NKG2D.ζ molecule that incorporated DAP10 reported enhanced NK cytotoxicity compared to NKG2D.ζ alone in vitro against a variety of human cancer cell lines as well as in a xenograft model of osteosarcoma (). However, this report did not address the susceptibility of this complex to down-modulation by TGFβ or soluble NKG2D ligands, or whether these NK cells had activity against MDSCs.

NKG2D.ζ–NK cells countered immunosuppression mediated by MDSCs leading to enhanced CAR-T cell tumor infiltration and expansion at tumor sites, CAR-T functions that are impaired in patients with solid tumors (). Unlike the GD2.CAR-T cells in our model, NKG2D.ζ–NK cells homed effectively to MDSC-engrafted tumors and released an array of chemokines that increased T cell infiltration of tumor. Unlike pharmacologic strategies aimed at enhancing leukocyte trafficking, including administration of lymphotactin or TNFα (), our approach does not require continuous cytokine administration. In fact, the ability of chimeric NKG2D to augment NK immune function specifically within the immunosuppressive TME provides for the local release of chemotactic factors, reflecting a more homeostatic method by which to increase CAR-T infiltration. Once there, CAR-T cells should meet an environment favorably modified by NKG2D.ζ–NK cell mediated elimination of MDSCs and production of pro-inflammatory cytokines. Indeed, elimination of MDSCs from a GD2+ tumor xenograft enhanced the activity of GD2.CAR-T cells in our model, including T-cell survival and intratumoral expansion. Given the suppressive effects of MDSCs in neuroblastoma (, ), the model shows how reversal of an MDSC-mediated suppressive microenvironment can improve antitumor functions of effector T cells.

Clinical neuroblastoma contains intense infiltrates of MDSCs (), which are not included in tumor xenograft models currently used to study human cell therapeutics. Our data suggest that co-inoculation of tumors with suppressive components (such as MDSCs) can model TME-mediated suppression of CAR-T activity against solid tumors, and provides a method by which to understand and counter immunosuppression. Although NSG mice lack a complete immune system in which to examine the effects of multiple endogenous immune components, our ability to engraft exogenous components (e.g., human MDSCs) within our TME model provides the possibility of simulating different immunosuppressive aspects of the solid TME. In fact, further model development utilizing human inhibitory macrophages and regulatory T cells (Tregs) as additional suppressive components of the TME is currently underway in our laboratory.

In summary, we describe an approach to reverse the suppressive TME using engineered human NK cells. We have shown that generation and expansion of our NK cell product is feasible and that NKG2D.ζ–NK cells have antitumor activity within a suppressive solid tumor microenvironment without toxicity to normal NKG2D ligand-expressing tissues. Hence, the elimination of suppressive MDSCs by NKG2D.ζ–NK cells may safely enhance adoptive cellular immunotherapy for neuroblastoma and for many other tumors that are supported and protected by MDSCs.

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