Funding, Deals & Partnerships: BIOLOGICS & MEDICAL DEVICES; BioMed e-Series; Medicine and Life Sciences Scientific Journal – http://PharmaceuticalIntelligence.com
Wednesday, January 27, 2016 | By John Carroll
Building on years of work on developing new insulin-producing cells that could one day control glucose levels and cure diabetes, a group of investigators led by scientists at MIT and Boston Children’s Hospital say they’ve developed a promising new gel capsule that protected the cells from an immune system assault.
Dr. Jose Oberholzer, a professor of bioengineering at the University of Illinois at Chicago, tested a variety of chemically modified alginate hydrogel spheres to see which ones would be best at protecting the islet cells created from human stem cells.
The team concluded that 1.5-millimeter spheres of triazole-thiomorphine dioxide (TMTD) alginate were best at protecting the cells and allowing insulin to seep out without spurring an errant immune system attack or the development of scar tissue–two key threats to making this work in humans.
They maintained healthy glucose levels in the rodents for 174 days, the equivalent to decades for humans.
“While this is a very promising step towards an eventual cure for diabetes, a lot more testing is needed to ensure that the islet cells don’t de-differentiate back toward their stem-cell states or become cancerous,” said Oberholzer.
Millions of diabetics have effectively controlled the chronic disease with existing therapies, but there’s still a huge unmet medical need to consider. While diabetes companies like Novo ($NVO) like to cite the fact that a third of diabetics have the disease under control, a third are on meds but don’t control it well and a third haven’t been diagnosed. An actual cure for the disease, which has been growing by leaps and bounds all over the world, would be revolutionary.
The transplantation of glucose-responsive, insulin-producing cells offers the potential for restoring glycemic control in individuals with diabetes1. Pancreas transplantation and the infusion of cadaveric islets are currently implemented clinically2, but these approaches are limited by the adverse effects of immunosuppressive therapy over the lifetime of the recipient and the limited supply of donor tissue3. The latter concern may be addressed by recently described glucose-responsive mature beta cells that are derived from human embryonic stem cells (referred to as SC-β cells), which may represent an unlimited source of human cells for pancreas replacement therapy4. Strategies to address the immunosuppression concerns include immunoisolation of insulin-producing cells with porous biomaterials that function as an immune barrier5, 6. However, clinical implementation has been challenging because of host immune responses to the implant materials7. Here we report the first long-term glycemic correction of a diabetic, immunocompetent animal model using human SC-β cells. SC-β cells were encapsulated with alginate derivatives capable of mitigating foreign-body responses in vivo and implanted into the intraperitoneal space of C57BL/6J mice treated with streptozotocin, which is an animal model for chemically induced type 1 diabetes. These implants induced glycemic correction without any immunosuppression until their removal at 174 d after implantation. Human C-peptide concentrations and in vivo glucose responsiveness demonstrated therapeutically relevant glycemic control. Implants retrieved after 174 d contained viable insulin-producing cells.
Figure 1: SC-β cells encapsulated with TMTD alginate sustain normoglycemia in STZ-treated immune-competent C57BL/6J mice.close
(a) Top, schematic representation of the last three stages of differentiation of human embryonic stem cells to SC-β cells. Stage 4 cells (pancreatic progenitors 2) co-express pancreatic and duodenal homeobox 1 (PDX-1) and NK6 homeobox 1…
Two new scientific papers published on Monday demonstrated tools that could result in potential therapies for patients diagnosed with type 1 diabetes, a condition in which the immune system limits the production of insulin, typically in adolescents. See —
Bubble Technique Could Create Type 1 Diabetes Therapy
Two new scientific papers published on Monday demonstrated tools that could result in potential therapies for patients diagnosed with type 1 diabetes, a condition in which the immune system limits the production of insulin, typically in adolescents.
Previous treatments for this disease have involved injecting beta cells from dead donors into patients to help their pancreas generate healthy-insulin cells, writes STAT. However, this method has resulted in the immune system targeting these new cells as “foreign” so transplant recipients have had to take immune-suppressing medications for the rest of their lives.
The first paper published in the journal Nature Biotechnology explained how scientists analyzed a seaweed extract called alginate to gauge its effectiveness in supporting the flow of sugar and insulin between cells and the body. An estimated 774 variations were tested in mice and monkeys in which results indicated only a handful could reduce the body’s response to foreign invaders, explains STAT.
The other paper in the journal Nature Medicine detailed a process where scientists developed small capsules infused with alginate and embryonic stem cells. A six-month observation period revealed this “protective bubble” technique “began to produce insulin in response to blood glucose levels” after transplantation in mice subjects with a condition similar to type 1 diabetes, reports Gizmodo.
Essentially, this cured the mice of their diabetes, and the beta cells worked as well as the body’s own cells, according to the researchers. Human trials could still be a few years away, but this experiment could yield a safer alternative to insulin injections.
Combinatorial hydrogel library enables identification of materials that mitigate the foreign body response in primates
The foreign body response is an immune-mediated reaction that can lead to the failure of implanted medical devices and discomfort for the recipient1, 2, 3, 4, 5, 6. There is a critical need for biomaterials that overcome this key challenge in the development of medical devices. Here we use a combinatorial approach for covalent chemical modification to generate a large library of variants of one of the most widely used hydrogel biomaterials, alginate. We evaluated the materials in vivo and identified three triazole-containing analogs that substantially reduce foreign body reactions in both rodents and, for at least 6 months, in non-human primates. The distribution of the triazole modification creates a unique hydrogel surface that inhibits recognition by macrophages and fibrous deposition. In addition to the utility of the compounds reported here, our approach may enable the discovery of other materials that mitigate the foreign body response.
Video 1: Intravital imaging of 300 μm SLG20 microcapsules.
Video 2: Intravital imaging of 300 μm Z2-Y12 microcapsules.
Video 3: NHP Laparoscopic procedure for the retrieval of Z2-Y12 spheres.
Clinical Focus on Follicular Lymphoma: CAR T-Cells Active in Relapsed Blood Cancers
MedPage Today
CAR T-Cells Active in Relapsed Blood Cancers
Complete responses in half of patients
by Charles Bankhead
Patients with relapsed and refractory B-cell malignancies have responded to treatment with modified T-cells added to conventional chemotherapy, data from an ongoing Swedish study showed.
Six of the first 11 evaluable patients achieved complete responses with increasing doses of chimeric antigen receptor (CAR)-modified T-cells that target the CD19 antigen, although two subsequently relapsed.
Five of the six responding patients received preconditioning chemotherapy the day before CAR T-cell infusion, in addition to chemotherapy administered up to 90 days before T-cell infusion to reduce tumor-cell burden. The remaining five patients received only the earlier chemotherapy, according to a presentation at the inaugural International Cancer Immunotherapy Conference in New York City.
“The complete responses in lymphoma patients despite the fact that they received only low doses of preconditioning compared with other published data surprised us,” Angelica Loskog, PhD, of Uppsala University in Sweden, said in a statement. “The strategy of both providing tumor-reductive chemotherapy for weeks prior to CAR T-cell infusion combined with preconditioning just before CAR T-cell infusion seems to offer promise.
CAR T-cells have demonstrated activity in a variety of studies involving patients with B-cell malignancies. Much of the work has focused on patients with leukemia, including trials in the U.S. B-cell lymphomas have proven more difficult to treat with CAR T-cells because the diseases are associated with higher concentration of immunosuppressive cells that can inhibit CAR T-cell activity, said Loskog. Moreover, blood-vessel abnormalities and accumulation of fibrotic tissue can hinder tumor penetration by therapeutic T-cells.
Each laboratory has its own process for modifying T-cells. Loskog and colleagues in Sweden and at Baylor College of Medicine in Houston have developed third-generation CAR T-cells that contain signaling domains for CD28 and 4-1BB, which act as co-stimulatory molecules. In preclinical models, third-generation CAR T-cells have demonstrated increased activation and proliferation in response to antigen challenge. Additionally, they have chosen to experiment with tumor burden-reducing chemotherapy, a preconditioning chemotherapy to counter the higher immunosuppressive cell count in lymphoma patients.
Loskog reported details of an ongoing phase I/IIa clinical trial involving patients with relapsed or refractory CD19-positive B-cell malignancies. Altogether, investigators have treated 12 patients with increasing doses (2 x 107 to 2 x 108 cells/m2) of CAR T-cells. One patient (with mixed follicular/Burkitt lymphoma) has yet to be evaluated for response. The remaining 11 included three patients with diffuse large B-cell lymphoma (DLBCL), one with follicular lymphoma transformed to DLBCL, two with chronic lymphocytic leukemia, two with mantle cell lymphoma, and three with acute lymphoblastic leukemia.
All of the patients with lymphoma received standard tumor cell-reducing chemotherapy, beginning 3 to 90 days before administration of CAR T-cells. Beginning with the sixth patient in the cohort, patients also received preconditioning chemotherapy (cyclophosphamide/fludarabine) 1 to 2 days before T-cell infusion to reduce the number and activity of immunosuppressive cells.
Cytokine release syndrome is a common effect of CAR T-cell therapy and occurred in several patients treated. In general, the syndrome has been manageable and has not interfered with treatment or response to the modified T-cells.
On the basis of the data produced thus far, the investigators have proceeded with patient evaluation and enrollment. They have already begun cell production for the next patient that will be treated with autologous CAR T-cells.
Although laboratories have their own cell production techniques, the treatment strategy has broad applicability to the treatment of B-cell malignancies, said Loskog.
“The results using different CARs and different techniques for manufacturing them is very similar in the clinic, in terms of initial complete response,” she told MedPage Today. “By using 4-1BB as a co-stimulator in the CAR intracellular region, it seems possible to achieve long-term complete responses in some patients. However, preconditioning of the patients with chemotherapy to reduce the regulatory immune cells seems crucial for effect.”
In an effort to manage the effect of patients’ immunosuppressive cells, the investigators have begun studying each the immune profile before and after treatment. Preliminary results suggest that the population of immunosuppressive cells increases over time, which has the potential to interfere with CAR T-cell responses.
“Especially for lymphoma, it may be crucial to deplete such cells prior to CAR infusion,” said Loskog. “It may even be necessary with supportive treatment for some time after CAR T-cell infusion. A supportive treatment needs to specifically regulate the suppressive cells while sparing the effect of CARs.”
The immunotherapy conference is jointly sponsored by the American Association for Cancer Research, the Cancer Research Institute, the Association for Cancer Immunotherapy, and the European Academy of Tumor Immunology.
PET-CT Best for FL Response Assessment
PET-CT associated with better progression-free and overall survival rates in follicular lymphoma.
Kay Jackson
PET-CT (PET) rather than contrast-enhanced CT scanning should be considered the new gold standard for response assessment after first-line rituximab therapy for high-tumor burden follicular lymphoma (FL), a pooled analysis of a central review in three multicenter studies indicated.
Rhizen Pharmaceuticals is developing RP-6530, a PI3K delta and gamma dual inhibitor, for the potential oral treatment of cancer and inflammation In November 2013, a phase I trial in patients with hematologic malignancies was initiated in Italy ]\. In September 2015, a phase I/Ib study was initiated in the US, in patients with relapsed and refractory T-cell lymphoma. At that time, the study was expected to complete in December 2016
PI3K delta/gamma inhibitor RP6530 An orally active, highly selective, small molecule inhibitor of the delta and gamma isoforms of phosphoinositide-3 kinase (PI3K) with potential immunomodulating and antineoplastic activities. Upon administration, PI3K delta/gamma inhibitor RP6530 inhibits the PI3K delta and gamma isoforms and prevents the activation of the PI3K/AKT-mediated signaling pathway. This may lead to a reduction in cellular proliferation in PI3K delta/gamma-expressing tumor cells. In addition, this agent modulates inflammatory responses through various mechanisms, including the inhibition of both the release of reactive oxygen species (ROS) from neutrophils and tumor necrosis factor (TNF)-alpha activity. Unlike other isoforms of PI3K, the delta and gamma isoforms are overexpressed primarily in hematologic malignancies and in inflammatory and autoimmune diseases. By selectively targeting these isoforms, PI3K signaling in normal, non-neoplastic cells is minimally impacted or not affected at all, which minimizes the side effect profile for this agent. Check for active clinical trials using this agent. (NCI Thesaurus)
Company
Rhizen Pharmaceuticals S.A.
Description
Dual phosphoinositide 3-kinase (PI3K) delta and gamma inhibitor
Dual PI3Kδ/γ Inhibition By RP6530 Induces Apoptosis and Cytotoxicity In B-Lymphoma Cells
Swaroop Vakkalanka, PhD*,1, Srikant Viswanadha, Ph.D.*,2, Eugenio Gaudio, PhD*,3, Emanuele Zucca, MD4, Francesco Bertoni, MD5, Elena Bernasconi, B.Sc.*,3, Davide Rossi, MD, Ph.D.*,6, and Anastasios Stathis, MD*,7
1Rhizen Pharmaceuticals S A, La Chaux-de-Fonds, Switzerland, 2Incozen Therapeutics Pvt. Ltd., Hyderabad, India, 3Lymphoma & Genomics Research Program, IOR-Institute of Oncology Research, Bellinzona, Switzerland, 4IOSI Oncology Institute of Southern Switzerland, Bellinzona, Switzerland, 5Lymphoma Unit, IOSI-Oncology Institute of Southern Switzerland, Bellinzona, Switzerland, 6Italian Multiple Myeloma Network, GIMEMA, Italy, 7Oncology Institute of Southern Switzerland, Bellinzona, Switzerland
RP6530 is a potent and selective dual PI3Kδ/γ inhibitor that inhibited growth of B-cell lymphoma cell lines with a concomitant reduction in the downstream biomarker, pAKT. Additionally, the compound showed cytotoxicity in a panel of lymphoma primary cells. Findings provide a rationale for future clinical trials in B-cell malignancies.
Blood 2013 122:4411; published ahead of print December 6, 2013
Swaroop Vakkalanka, Srikant Viswanadha, Eugenio Gaudio, Emanuele Zucca, Francesco Bertoni, Elena Bernasconi, Davide Rossi, Anastasios Stathis
Dual PI3K delta/gamma Inhibition By RP6530 Induces Apoptosis and Cytotoxicity
RP6530, a novel, small molecule PI3K delta/gamma
Activity and selectivity of RP6530 for PI3K delta and gamma isoforms
Introduction Activation of the PI3K pathway triggers multiple events including cell growth, cell cycle entry, cell survival and motility. While α and β isoforms are ubiquitous in their distribution, expression of δ and γ is restricted to cells of the hematopoietic system. Because these isoforms contribute to the development, maintenance, transformation, and proliferation of immune cells, dual targeting of PI3Kδ and γ represents a promising approach in the treatment of lymphomas. The objective of the experiments was to explore the therapeutic potential of RP6530, a novel, small molecule PI3Kδ/γ inhibitor, in B-cell lymphomas.
Methods Activity and selectivity of RP6530 for PI3Kδ and γ isoforms and subsequent downstream activity was determined in enzyme and cell-based assays. Additionally, RP6530 was tested for potency in viability, apoptosis, and Akt phosphorylation assays using a range of immortalized B-cell lymphoma cell lines (Raji, TOLEDO, KG-1, JEKO, OCI-LY-1, OCI-LY-10, MAVER, and REC-1). Viability was assessed using the colorimetric MTT reagent after incubation of cells for 72 h. Inhibition of pAKT was estimated by Western Blotting and bands were quantified using ImageJ after normalization with Actin. Primary cells from lymphoid tumors [1 chronic lymphocytic leukemia (CLL), 2 diffuse large B-cell lymphomas (DLBCL), 2 mantle cell lymphoma (MCL), 1 splenic marginal zone lymphoma (SMZL), and 1 extranodal MZL (EMZL)] were isolated, incubated with 4 µM RP6530, and analyzed for apoptosis or cytotoxicity by Annexin V/PI staining.
Results RP6530 demonstrated high potency against PI3Kδ (IC50=24.5 nM) and γ (IC50=33.2 nM) enzymes with selectivity over α (>300-fold) and β (>100-fold) isoforms. Cellular potency was confirmed in target-specific assays, namely anti-FcεR1-(EC50=37.8 nM) or fMLP (EC50=39.0 nM) induced CD63 expression in human whole blood basophils, LPS induced CD19+ cell proliferation in human whole blood (EC50=250 nM), and LPS induced CD45R+ cell proliferation in mouse whole blood (EC50=101 nM). RP6530 caused a dose-dependent inhibition (>50% @ 2-7 μM) in growth of immortalized (Raji, TOLEDO, KG-1, JEKO, REC-1) B-cell lymphoma cells. Effect was more pronounced in the DLBCL cell lines, OCI-LY-1 and OCI-LY-10 (>50% inhibition @ 0.1-0.7 μM), and the reduction in viability was accompanied by corresponding inhibition of pAKT with EC50 of 6 & 70 nM respectively. Treatment of patient-derived primary cells with 4 µM RP6530 caused an increase in cell death. Fold-increase in cytotoxicity as evident from PI+ staining was 1.6 for CLL, 1.1 for DLBCL, 1.2 for MCL, 2.2 for SMZL, and 2.3 for EMZL. Cells in early apotosis (Annexin V+/PI-) were not different between the DMSO blank and RP6530 samples.
Conclusions RP6530 is a potent and selective dual PI3Kδ/γ inhibitor that inhibited growth of B-cell lymphoma cell lines with a concomitant reduction in the downstream biomarker, pAKT. Additionally, the compound showed cytotoxicity in a panel of lymphoma primary cells. Findings provide a rationale for future clinical trials in B-cell malignancies.
While alpha and beta isoforms are ubiquitous in their distribution, expression of delta and gamma is restricted to circulating hematogenous cells and endothelial cells. Unlike PI3K-alpha or beta, mice lacking expression of gamma or delta do not show any adverse phenotype indicating that targeting of these specific isoforms would not result in overt toxicity. Dual delta/gamma inhibition is strongly implicated as an intervention strategy in allergic and non-allergic inflammation of the airways and other autoimmune diseases. Scientific evidence for PI3K-delta and gamma involvement in various cellular processes underlying asthma and COPD stems from inhibitor studies and gene-targeting approaches. Also, resistance to conventional therapies such as corticosteroids in several COPD patients has been attributed to an up-regulation of the PI3K delta/gamma pathway. Disruption of PI3K-delta/gamma signalling therefore provides a novel strategy aimed at counteracting the immuno-inflammatory response. Due to the pivotal role played by PI3K-delta and gamma in mediating inflammatory cell functionality such as leukocyte migration and activation, and mast cell degranulation, blocking these isoforms may also be an effective strategy for the treatment of rheumatoid arthritis as well.
Given the established criticality of these isoforms in immune surveillance, inhibitors specifically targeting the delta and gamma isoforms would be expected to attenuate the progression of immune response encountered in airway inflammation and rheumatoid arthritis.
Rhizen has identified an orally active Lead Molecule, RP-6530, that has an excellent pre-clinical profile. RP-6530 is currently in non-GLP Tox studies and is expected to enter Clinical Development in H2 2013.
In December 2013, Rhizen announced the start of a Phase I clinical trial. The study entitled A Phase-I, Dose Escalation Study to Evaluate Safety and Efficacy of RP6530, a dual PI3K delta /gamma inhibitor, in patients with Relapsed or Refractory Hematologic Malignancies is designed primarily to establish the safety and tolerability of RP6530. Secondary objectives include clinical efficacy assessment and biomarker response to allow dose determination and potential patient stratification in subsequent expansion studies.
Partners by Region
Rhizen’s pipeline consists of internally discovered (with 100% IP ownership) novel small molecule programs aimed at high value markets of Oncology, Immuno-inflammtion and Metabolic Disorders. Rhizen has been successful in securing critical IP space in these areas and efforts are on for further expansion in to several indications. Rhizen seeks partnerships to unlock the potential of these valuable assets for further development from global pharmaceutical partners. At present global rights on all programs are available and Rhizen is flexible to consider suitable business models for licensing/collaboration.
In 2012, Rhizen announced a joint venture collaboration with TG Therapeutics for global development and commercialization of Rhizen’s Novel Selective PI3K Kinase Inhibitors. The selected lead RP5264 (hereafter, to be developed as TGR-1202) is an orally available, small molecule, PI3K specific inhibitor currently being positioned for the treatment of hematological malignancies.
PATENT
WO2014195888, DUAL SELECTIVE PI3 DELTA AND GAMMA KINASE INHIBITORS
This scheme provides a synthetic route for the preparation of compound of formula wherein all the variables are as described herein in above
15 14 10 12 12a
REFERENCES
April 2015, preclinical data were presented at the 106th AACR Meeting in Philadelphia, PA. RP-6530 had GI50 values of 17,028 and 22,014 nM, respectively
December 2014, data were presented at the 56th ASH Meeting in San Francisco, CA.
December 2013, preclinical data were presented at the 55th ASH Meeting in New Orleans, LA.
June 2013, preclinical data were presented at the 18th Annual EHA Congress in Stockholm, Sweden. RP-6530 inhibited PI3K delta and gamma isoforms with IC50 values of 24.5 and 33.2 nM, respectively.
01 Sep 2015 Phase-I clinical trials in Hematological malignancies (Second-line therapy or greater) in USA (PO) (NCT02567656)
18 Nov 2014 Preclinical trials in Multiple myeloma in Switzerland (PO) prior to November 2014
18 Nov 2014 Early research in Multiple myeloma in Switzerland (PO) prior to November 2014
Process for preparation of optically pure and optionally substituted 2- (1 -hydroxy- alkyl) – chromen – 4 – one derivatives and their use in preparing pharmaceuticals
Companion diagnostics (CDx), in vitro diagnostic devices or imaging tools that provide information essential to the safe and effective use of a corresponding therapeutic product, have become indispensable tools for oncologists. As a result, analysts expect the global CDx market to reach $8.73 billion by 2019, up from from $3.14 billion in 2014.
Use of CDx during a clinical trial to guide therapy can improve treatment responses and patient outcomes by identifying and predicting patient subpopulations most likely to respond to a given treatment.
These tests not only indicate the presence of a molecular target, but can also reveal the off-target effects of a therapeutic, predicting toxicities and adverse effects associated with a drug.
For pharma manufacturers, using CDx during drug development improves the success rate of drugs being tested in clinical trials. In a study estimating the risk of clinical trial failure during non-small cell lung cancer drug development in the period between 1998 and 2012 investigators analyzed trial data from 676 clinical trials with 199 unique drug compounds.
The data showed that Phase III trial failure proved the biggest obstacle to drug approval, with an overall success rate of only 28%. But in biomarker-guided trials, the success rate reached 62%. The investigators concluded from their data analysis that the use of a CDx assay during Phase III drug development substantially improves a drug’s chances of clinical success.
The Regulatory Perspective
According to Patricia Keegen, M.D., supervisory medical officer in the FDA’s Division of Oncology Products II, the agency requires a companion diagnostic test if a new drug works on a specific genetic or biological target that is present in some, but not all, patients with a certain cancer or disease. The test identifies individuals who would benefit from the treatment, and may identify patients who would not benefit but could also be harmed by use of a certain drug for treatment of their disease. The agency classifies companion diagnosis as Class III devices, a class of devices requiring the most stringent approval for medical devices by the FDA, a Premarket Approval Application (PMA).
On August 6, 2014, the FDA finalized its long-awaited “Guidance for Industry and FDA Staff: In Vitro Companion Diagnostic Devices,” originally issued in July 2011. The final guidance stipulates that FDA generally will not approve any therapeutic product that requires an IVD companion diagnostic device for its safe and effective use before the IVD companion diagnostic device is approved or cleared for that indication.
Close collaboration between drug developers and diagnostics companies has been a key driver in recent simultaneous pharmaceutical-CDx FDA approvals, and partnerships between in vitro diagnostics (IVD) companies have proliferated as a result. Major test developers include Roche Diagnostics, Abbott Laboratories, Agilent Technologies, QIAGEN), Thermo Fisher Scientific, and Myriad Genetics.
But an NGS-based test has yet to make it to market as a CDx for cancer. All approved tests include PCR–based tests, immunohistochemistry, and in situ hybridization technology. And despite the very recent decision by the FDA to grant marketing authorization for Illumina’s MiSeqDx instrument platform for screening and diagnosis of cystic fibrosis, “There still seems to be a number of challenges that must be overcome before we see NGS for targeted cancer drugs,” commented Jan Trøst Jørgensen, a consultant to DAKO, commenting on presentations at the European Symposium of Biopathology in June 2013.
Illumina received premarket clearance from the FDA for its MiSeqDx system, two cystic fibrosis assays, and a library prep kit that enables laboratories to develop their own diagnostic test. The designation marked the first time a next-generation sequencing system received FDA premarket clearance. The FDA reviewed the Illumina MiSeqDx instrument platform through its de novo classification process, a regulatory pathway for some novel low-to-moderate risk medical devices that are not substantially equivalent to an already legally marketed device.
Dr. Jørgensen further noted that “We are slowly moving away from the ‘one biomarker: one drug’ scenario, which has characterized the first decades of targeted cancer drug development, toward a more integrated approach with multiple biomarkers and drugs. This ‘new paradigm’ will likely pave the way for the introduction of multiplexing strategies in the clinic using gene expression arrays and next-generation sequencing.”
The future of CDxs therefore may be heading in the same direction as cancer therapy, aimed at staying ahead of the tumor drug resistance curve, and acknowledging the reality of the shifting genomic landscape of individual tumors. In some cases, NGS will be applied to diseases for which a non-sequencing CDx has already been approved.
Illumina believes that NGS presents an ideal solution to transforming the tumor profiling paradigm from a series of single gene tests to a multi-analyte approach to delivering precision oncology. Mya Thomae, Illumina’s vice president, regulatory affairs, said in a statement that Illumina has formed partnerships with several drug companies to develop a universal next-generation sequencing-based oncology test system. The collaborations with AstraZeneca, Janssen, Sanofi, and Merck-Serono, announced in 2014 and 2015 respectively, seek to “redefine companion diagnostics for oncology focused on developing a system for use in targeted therapy clinical trials with a goal of developing and commercializing a multigene panel for therapeutic selection.”
On January 16, 2014 Illumina and Amgen announced that they would collaborate on the development of a next-generation sequencing-based companion diagnostic for colorectal cancer antibody Vectibix (panitumumab). Illumina will develop the companion test on its MiSeqDx instrument.
In 2012, the agency approved Qiagen’s Therascreen KRAS RGQ PCR Kit to identify best responders to Erbitux (cetuximab), another antibody drug in the same class as Vectibix. The label for Vectibix, an EGFR-inhibiting monoclonal antibody, restricts the use of the drug for those metastatic colorectal cancer patients who harbor KRAS mutations or whose KRAS status is unknown.
The U.S. FDA, Illumina said, hasn’t yet approved a companion diagnostic that gauges KRAS mutation status specifically in those considering treatment with Vectibix. Illumina plans to gain regulatory approval in the U.S. and in Europe for an NGS-based companion test that can identify patients’ RAS mutation status. Illumina and Amgen will validate the test platform and Illumina will commercialize the test.
Treatment Options
Foundation Medicine says its approach to cancer genomic characterization will help physicians reveal the alterations driving the growth of a patient’s cancer and identify targeted treatment options that may not have been otherwise considered.
FoundationOne, the first clinical product from Foundation Medicine, interrogates the entire coding sequence of 315 cancer-related genes plus select introns from 28 genes often rearranged or altered in solid tumor cancers. Based on current scientific and clinical literature, these genes are known to be somatically altered in solid cancers.
These genes, the company says, are sequenced at great depth to identify the relevant, actionable somatic alterations, including single base pair change, insertions, deletions, copy number alterations, and selected fusions. The resultant fully informative genomic profile complements traditional cancer treatment decision tools and often expands treatment options by matching each patient with targeted therapies and clinical trials relevant to the molecular changes in their tumors.
As Foundation Medicine’ s NGS analyses are increasingly applied, recent clinical reports describe instances in which comprehensive genomic profiling with the FoundationOne NGS-based assay result in diagnostic reclassification that can lead to targeted drug therapy with a resulting dramatic clinical response. In several reported instances, NGS found, among the spectrum of aberrations that occur in tumors, changes unlikely to have been discovered by other means, and clearly outside the range of a conventional CDx that matches one drug to a specific genetic change.
TRK Fusion Cancer
In July 2015, the University of Colorado Cancer Center and Loxo Oncology published a research brief in the online edition of Cancer Discovery describing the first patient with a tropomyosin receptor kinase (TRK) fusion cancer enrolled in a LOXO-101 Phase I trial. LOXO-101 is an orally administered inhibitor of the TRK kinase and is highly selective only for the TRK family of receptors.
While the authors say TRK fusions occur rarely, they occur in a diverse spectrum of tumor histologies. The research brief described a patient with advanced soft tissue sarcoma widely metastatic to the lungs. The patient’s physician submitted a tumor specimen to Foundation Medicine for comprehensive genomic profiling with FoundationOne Heme, where her cancer was demonstrated to harbor a TRK gene fusion.
Following multiple unsuccessful courses of treatment, the patient was enrolled in the Phase I trial of LOXO-101 in March 2015. After four months of treatment, CT scans demonstrated almost complete tumor disappearance of the largest tumors.
The FDA’s Elizabeth Mansfield, Ph.D., director, personalized medicine staff, Office of In Vitro Diagnostics and Radiological Health, said in a recent article, “FDA Perspective on Companion Diagnostics: An Evolving Paradigm” that “even as it seems that many questions about co-development have been resolved, the rapid accumulation of new knowledge about tumor biology and the rapid evolution of diagnostic technology are challenging FDA to continually redefine its thinking on companion diagnostics.” It seems almost inevitable that a consolidation of diagnostic testing should take place, to enable a single test or a few tests to garner all the necessary information for therapeutic decision making.”
Whether this means CDx testing will begin to incorporate NGS sequencing remains to be seen.
Despite high rates of response to initial chemoimmunotherapy, patients with follicular lymphoma experience frequent relapses, and better treatment options are needed. Several novel biologic agents have been developed based on a greater understanding of the intrinsic factors driving the development of this heterogeneous disease. Such therapies target extracellular surface proteins and intracellular signaling pathways, as well as manipulate and engage the tumor microenvironment. Many of these agents have shown great promise in early-phase studies and are the focus of ongoing clinical investigations.
Introduction As the second most common form of non-Hodgkin lymphoma (NHL), follicular lymphoma affects thousands of new patients in the United States each year. Although follicular lymphoma is considered an indolent disease, its clinical course is highly variable. Asymptomatic patients with low tumor burden can be monitored closely with the “watch and wait” strategy, given that the early intervention of chemotherapy or immunotherapy has not demonstrated a survival benefit.[1,2] The most widely accepted indications for treatment are one or more of the following criteria from the Groupe d’Etude des Lymphomes Folliculaires (GELF): a single lesion > 7 cm, three nodal sites > 3 cm, splenomegaly, effusions, threat or evidence of organ compression, or constitutional symptoms.[3] Whereas patients with limited-stage disease have several treatment options—including single-agent rituximab, radiation, and chemoimmunotherapy, those with advanced-stage disease typically receive chemoimmunotherapy.[4,5] Both the German Study Group Indolent Lymphomas (StiL) NHL-2 study and the pharmaceutical company–sponsored Bendamustine Rituximab Investigational Non-Hodgkin’s Trial (BRIGHT) have established the front-line role of combination therapy with bendamustine and rituximab in the treatment of follicular lymphoma, based on comparable efficacy and better tolerability than standard regimens such as R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone).[6,7] Despite high response rates with initial therapy, follicular lymphoma is characterized by frequent relapses, and patients need improved treatment options.
Table: Targeted Therapies in Development and FDA-Approved for the Treatment of Follicular Lymphomas
Since the discovery of rituximab, there has been significant innovation in drug development, based on a greater understanding of the pathogenesis of the disease. The multistep process leading to follicular lymphoma is theorized to begin with an initial genetic insult, the hallmark t(14;18) translocation, which results in overexpression of the anti-apoptotic B-cell lymphoma protein, BCL-2.[8] This translocation is not the sole factor in malignant transformation, as it is a naturally occurring anomaly, often identified in healthy individuals. Furthermore, preclinical studies have indicated a positive correlation between increasing numbers of genetic alterations and the progression from follicular lymphoma in situ to grade 3A follicular lymphoma.[9] The B-cell receptor is a critical cellular factor in the development of the disease. Its active tonic signaling leads to recruitment of the spleen tyrosine kinase (SYK) and activation of multiple downstream pathways, including phosphatidylinositol 3-kinase (PI3K), nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), and mitogen-activated protein kinase (MAPK). Activation of these pathways ultimately results in the maturation, proliferation, and survival of malignant lymphocytes.[10] Other key components include immune cells such as T cells, dendritic cells, and reticular cells, which infiltrate among the centrocytes.[11] In addition to stimulating the B-cell receptor, these immune cells induce exhaustion of cytotoxic T cells and allow for T-cell evasion via disruption of synapses and secretion of interleukin-12.[12] Expression of the inhibitory receptor programmed cell death 1 (PD-1) is another contributing factor, as its presence is believed to affect the ability of T cells to mount appropriate antitumor responses.[13] Several novel therapies have been developed to target these various aspects of the disease in the hope of identifying more effective, yet tolerable, treatment options for patients with follicular lymphoma. (See the Table for a summary of therapies approved and in development for treatment of follicular lymphoma.)
Since the approval of rituximab, there has been significant investigation into the development of a superior anti-CD20 monoclonal antibody. Second- and third-generation versions vary in their structures and mechanisms of action. Two anti-CD20 antibodies, ofatumumab and obinutuzumab, have been approved by the US Food and Drug Administration (FDA) for indications in chronic lymphocytic leukemia (CLL). Ofatumumab, a type I human monoclonal antibody, was initially approved for patients whose disease is refractory to fludarabine and alemtuzumab.[14] Obinutuzumab was approved in combination with chlorambucil for patients with preexisting comorbidities that preclude conventional chemoimmunotherapy.[15] Although designed to induce stronger complement-dependent cytotoxicity than rituximab, ofatumumab demonstrated minimal activity in rituximab-refractory follicular lymphoma (overall response rate [ORR], 10% to 13%; median progression-free survival [PFS], 5.8 months).[16] In phase II trials of ofatumumab in combination with chemotherapy, including bendamustine and CHOP, results appeared comparable to those achieved with rituximab-based regimens, although no direct comparisons have been made.[17,18]
Obinutuzumab, a type II glycoengineered humanized antibody, is further in development for use in NHL. This agent, which works primarily by triggering antibody-dependent cellular cytotoxicity (ADCC) and apoptosis, has demonstrated superiority to rituximab in preclinical studies, including those employing whole blood B cell–depletion assays, human lymphoma xenograft mice models, and nonhuman primates.[19] When compared with rituximab in patients with indolent B-cell NHL, obinutuzumab produced a higher ORR by independent radiology review (45% vs 27%; P = .01); however, complete response (CR) and PFS rates were similar.[20] Like ofatumumab, treatment with obinutuzumab is associated with a higher rate of infusion-related reactions than rituximab (grade 1–4, 72% vs 49%; and grade 3/4, 11% vs 5%, respectively). In contrast to ofatumumab, it has efficacy in rituximab-refractory indolent NHL, producing an ORR of 50% and median PFS of 12 months among 10 patients.[21] The phase III GADOLIN study evaluated obinutuzumab in combination with bendamustine followed by maintenance obinutuzumab in the same disease setting (N = 413).[22] While the response rates by independent review were similar to those observed in the comparative arm of patients randomized to single-agent bendamustine (bendamustine-obinutuzumab ORR, 69% [CR, 11%] vs bendamustine alone ORR, 63% [CR, 12%]), the median PFS was significantly higher with the combination (not reached vs 14.9 months; P = .00011). Treatment with the combination of bendamustine and obinutuzumab was associated with a similar incidence of grade ≥ 3 adverse events compared with bendamustine monotherapy (68% vs 62%), which included neutropenia (33% vs 26.3%) and infusion-related reactions (9% vs 3.5%). While there was no difference in overall survival (OS) noted, the study did demonstrate the clinical benefit of obinutuzumab in rituximab-refractory disease. The role of the drug in this setting is becoming less clear, as more patients now receive bendamustine-rituximab as front-line therapy. In the phase Ib GAUDI study, bendamustine-obinutuzumab and obinutuzumab-CHOP produced similar response rates in patients with previously untreated follicular lymphoma, with ORRs of 93% (CR, 39%) and 95% (CR, 35%), respectively.[23] The incidences of grade 3/4 neutropenia and infection were similar to historical data on rituximab chemotherapy. These data prompted the front-line phase III GALLIUM study of chemotherapy (CHOP, CVP [cyclophosphamide, vincristine, and prednisone], or bendamustine) with obinutuzumab or rituximab followed by maintenance obinutuzumab or rituximab in advanced-stage indolent B-cell NHL (ClinicalTrials.gov identifier: NCT01332968). Newer monoclonal antibodies directed against CD20, such as ublituximab, and rituximab biosimilars are also in development.
Monoclonal antibodies to alternative targets
Monoclonal antibodies directed against other B-cell antigens have also been developed. Galiximab, a chimeric human-macaque anti-CD80 antibody, and epratuzumab, a humanized anti-CD22 antibody, were two of the first antibodies directed against these targets to be explored in follicular lymphoma. Both antibodies have shown activity as single agents and in combination with rituximab in follicular lymphoma. However, neither is being studied further due to the availability of newer, more promising therapies.[24-28] MEDI-551, an afucosylated humanized anti-CD19 antibody, induces cell death via ADCC and cytotoxic T-cell response. The lack of a fucose moiety on the Fc portion of the antibody is believed to enhance the activity of ADCC. Phase I studies of MEDI-551 in heavily pretreated follicular lymphoma have reported ORRs ranging from 31% to 82%.[29,30] The median PFS from the earlier study was nearly 10 months. MEDI-551 is currently being evaluated in aggressive lymphomas in combination with rituximab and salvage chemoimmunotherapy (ClinicalTrials.gov identifiers: NCT00983619 and NCT01453205). Also targeting CD19 is MOR208, a humanized monoclonal antibody that has been engineered to have a higher affinity to FcγRIIIa and FcγRIIa, resulting in stronger ADCC. In a phase II study of patients with relapsed and refractory B-cell NHL who had received a median of two prior therapies, the ORR was 26% among those with follicular lymphoma (n = 31).[31] The median duration of response (DOR) was 2.6 months; however, the longest DOR was 15.4 months. Upcoming trials with MOR208 include combination studies with lenalidomide in diffuse large B-cell lymphoma (DLBCL) and CLL (ClinicalTrials.gov identifiers: NCT02399085 and NCT02005289).
Radioimmunotherapy
One of the first attempts to improve upon the efficacy of the naked monoclonal antibody was radioimmunotherapy, which produced ORRs of 65% to 74% in patients with relapsed and refractory indolent B-cell lymphomas.[32,33] 90Y-ibritumomab tiuxetan, the first radioimmunotherapy to receive FDA approval, was approved in February 2002 for relapsed or refractory low-grade, follicular, or transformed B-cell NHL. In 2009, it was granted expanded approval as consolidation therapy in previously untreated follicular lymphoma patients with a partial or complete response to first-line chemotherapy. 131I-tositumomab was approved in June 2003 (along with tositumomab) for CD20-positive follicular NHL, with and without transformation, in relapsed rituximab-refractory patients with relapse following chemotherapy. The use of 131I-tositumomab and 90Y-ibritumomab tiuxetan has declined significantly over the past several years; the manufacture and sale of 131I-tositumomab (marketed in the United States and Canada as Bexxar) was stopped in February 2014. Radioimmunotherapy can be difficult; there are strict hematologic criteria (< 25% lymphomatous marrow involvement, platelet count > 100 × 109, leukocyte count > 1.5 × 109), and its administration requires a certified nuclear medicine physician. In addition, the patient must not have had prior radiation to > 25% of the bone marrow nor undergone stem cell transplantation.[34]
Antibody-drug conjugates (ADCs)
Recent efforts in augmenting antibody-based therapy include the use of ADCs. Once bound to its target antigen, the ADC is engulfed via endocytosis, trafficked to the lysosome for degradation, and ultimately released, whereupon it causes damage to tubulin and DNA. The calicheamicin-bound anti-CD22, inotuzumab ozogamicin, was one of the first to be studied in patients with follicular lymphoma who were refractory to CD20-targeted therapy, yielding an ORR of 66%.[35] The ORR increased to 87% when inotuzumab ozogamicin was combined with rituximab in patients with relapsed and refractory follicular lymphoma, prompting a trial in which it was compared with combination treatment with rituximab plus chemotherapy.[36] The trial was closed early due to poor accrual. Inotuzumab ozogamicin is currently being studied in combination with the mammalian target of rapamycin (mTOR) inhibitor temsirolimus in relapsed and refractory CD22-expressing NHL (ClinicalTrials.gov identifier: NCT01535989). Pinatuzumab vedotin and polatuzumab vedotin, which target CD22 and CD79b, respectively, are ADCs linked to the anti-tubulin molecule monomethyl auristatin E. While both agents have demonstrated activity in indolent NHL (with reported ORRs of 50% and 47%, respectively), polatuzumab vedotin is being taken further in development.[37,38] When polatuzumab vedotin was administered at a higher dose (2.4 mg/kg) with rituximab in patients with relapsed and refractory follicular lymphoma (n = 25), the ORR was 76% (CR, 44%) and median PFS was 15 months.[39] The cohort of 20 patients treated at the lower rituximab dose (1.8 mg/kg) had a similar response rate (ORR, 70%; CR, 40%), and median PFS and DOR were not reached. Peripheral neuropathy, a common toxicity with ADCs, occurred less frequently with the lower dose of polatuzumab vedotin and was ameliorated in some patients by dose delay and reduction. Ongoing studies with polatuzumab vedotin include phase I/II combinations with bendamustine-rituximab or obinutuzumab-bendamustine in relapsed and refractory follicular lymphoma (ClinicalTrials.gov identifier: NCT02257567) and R-CHOP in B-cell NHL patients who have received less than one prior therapy (ClinicalTrials.gov identifier: NCT01992653). Coltuximab ravtansine (formerly SAR3419) is an anti-CD19 ADC that has also been associated with neurologic complications, primarily dose-limiting ocular toxicity.[40] IMGN529, which targets the overexpressed CD37 protein, is another B-cell–directed ADC in development.[41]
Despite high rates of response to initial chemoimmunotherapy, patients with follicular lymphoma experience frequent relapses, and better treatment options are needed. Several novel biologic agents have been developed based on a greater understanding of the intrinsic factors driving the development of this heterogeneous disease. Such therapies target extracellular surface proteins and intracellular signaling pathways, as well as manipulate and engage the tumor microenvironment. Many of these agents have shown great promise in early-phase studies and are the focus of ongoing clinical investigations.
Small-Molecule Inhibitors – PI3K inhibitors
In contrast to the various antibody-based therapies under investigation for treatment of follicular lymphoma, several small molecules have been designed to inhibit key intracellular pathways of the malignant B cell. The majority of these agents are directed against kinases downstream of the B-cell receptor, and many have been combined with bendamustine-rituximab, given this combination’s efficacy and tolerability. Idelalisib, a potent PI3K-δ inhibitor, was the first PI3K inhibitor to be approved by the FDA for follicular lymphoma. It received an indication for patients who have received at least two prior systemic therapies, based on results of a phase II study in rituximab-refractory indolent NHL[42] As reported at the 2015 American Society of Clinical Oncology Annual Meeting, of the 72 patients in the study who had follicular lymphoma, 54% were considered high-risk by the Follicular Lymphoma International Prognostic Index.[43] The patients had received a median of four prior therapies, and 86% had disease that was refractory to the last regimen they received. The ORR was 56% and the median PFS was 11 months. The median PFS for the 10 patients who achieved a CR was 27 months. Notable grade 3/4 adverse events included neutropenia (27% of all patients), diarrhea/colitis (16%), elevations in hepatic transaminases (13%), and pneumonia (7%).
Idelalisib was subsequently administered with rituximab, bendamustine, and bendamustine-rituximab in a phase I study of patients with relapsed (n = 79) and refractory (n = 59) indolent NHL, the majority of whom had follicular lymphoma.[44] The ORRs were similar between the arms (75% to 88%); however, treatment with bendamustine-rituximab-idelalisib was associated with the highest CR (43%) and longest median PFS (37.1 months). A phase III trial of bendamustine-rituximab with or without idelalisib in relapsed and refractory indolent B-cell NHL is ongoing (ClinicalTrials.gov identifier: NCT01732926). In phase I investigations, combined treatment with idelalisib and lenalidomide plus the second-generation SYK inhibitor entospletinib has demonstrated considerable toxicity, including hepatic transaminitis, sepsis, and refractory pneumonitis.[45,46] Second-generation PI3K inhibitors, including duvelisib (IPI-145), TGR-1202, and INCB040093, are in development. Duvelisib, a dual inhibitor of the delta and gamma isoforms of PI3K, has demonstrated an ORR of 69% (CR, 38%) in a heavily pretreated follicular lymphoma cohort (n = 13).[47] Based on these encouraging data, duvelisib is being administered with rituximab or obinutuzumab in patients with previously untreated follicular lymphoma (ClinicalTrials.gov identifier: NCT02391545) and with bendamustine and/or rituximab in those with relapsed B-cell malignancies (ClinicalTrials.gov identifier: NCT01871675).
Bruton tyrosine kinase (BTK) inhibitors
Ibrutinib, a selective and irreversible inhibitor of BTK, may also have some impact on the tumor microenvironment via cytokine and chemokine inhibition.[48] Approved in CLL, mantle cell lymphoma, and Waldenström macroglobulinemia, it has demonstrated activity in a number of B-cell malignancies.[49-53] In a phase II study of relapsed and refractory follicular lymphoma, ibrutinib yielded an ORR of 30% (with one CR) and a median PFS of 9.9 months. The 40 enrolled patients had received a median of three prior therapies, and 36% were considered refractory to treatment. Common adverse events included mild diarrhea, rash, and fatigue; rare events included atrial fibrillation and bleeding.[54] Like idelalisib, ibrutinib has been combined with bendamustine-rituximab in the treatment of B-cell NHL.[55] This triplet produced an ORR of 90% (CR, 50%) in a cohort of 10 patients with previously treated follicular lymphoma. At the time these results were reported, the median PFS had not been reached. Notable grade 3/4 adverse events included neutropenia (33%), rash (25%), and thrombocytopenia (19%). Results from SELENE, a randomized phase III trial of ibrutinib with bendamustine-rituximab or R-CHOP in previously treated follicular lymphoma and marginal zone lymphoma, will provide more insight into the role of ibrutinib in the management of indolent NHL (ClinicalTrials.gov identifier: NCT01974440). Phase I trials of combinations with targeted agents include the Alliance for Clinical Trials in Oncology study of rituximab, lenalidomide, and ibrutinib in previously untreated follicular lymphoma[56] and the pharmaceutical-sponsored trial of combination therapy with ublituximab, TGR-1202, and ibrutinib in relapsed and refractory B-cell malignancies.[57] Second-generation BTK inhibitors, including ACP-196 and ONO-4059, are also in development.
B-cell lymphoma–2 (BCL-2) inhibitors
The chromosomal translocation t(14;18) allows for dysregulation of the BCL-2 oncogene and overexpression of the anti-apoptotic BCL-2 family of proteins, contributing to development of follicular lymphoma. Venetoclax, formerly known as ABT-199, is a second-generation selective BCL-2 inhibitor in the early stages of clinical investigation. When this agent was administered at a dose greater than 600 mg daily to six patients with relapsed and refractory follicular lymphoma, three patients achieved a response.[58] Common toxicities reported included mild nausea (34%) and diarrhea (25%), and grade 3/4 myelosuppression occurred in less than 15% of patients. Two patients (one with DLBCL and one with mantle cell lymphoma) in the entire NHL cohort (of 44 patients then enrolled in the study) developed laboratory evidence of grade 3 tumor lysis syndrome. When venetoclax was combined with bendamustine-rituximab in 21 patients with relapsed and refractory follicular lymphoma, the ORR was 71% (CR, 29%).[59] While a maximum tolerated dose was not reached, dose-limiting toxicities included thrombocytopenia, neutropenia, and Stevens-Johnson syndrome. Venetoclax is being evaluated in relapsed and refractory follicular lymphoma, in a three-arm phase II study of bendamustine-rituximab vs rituximab-venetoclax vs bendamustine-rituximab-venetoclax (ClinicalTrials.gov identifier: NCT02187861). It will be studied with ibrutinib in a phase I/II trial of relapsed follicular and marginal zone lymphoma (Ujjani C, principal investigator). Small-molecule inhibitors aimed at less well known targets are also under investigation, including selinexor (a selective inhibitor of nuclear export), MK-2206 (an AKT inhibitor), alisertib (an Aurora-A kinase inhibitor), and cerdulatinib (a dual SYK/Janus tyrosine kinase [JAK] inhibitor).
TO PUT THAT INTO CONTEXT
Loretta J. Nastoupil, MD
Department of Lymphoma/Myeloma, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center Houston, Texas
What Are the Challenges of Treating Follicular Lymphoma?
Follicular lymphoma, the most common indolent lymphoma, is characterized by high rates of initial response to chemoimmunotherapy, but it is not curable with standard therapy. The clinical course of the disease is highly variable and somewhat unpredictable. As a result, the optimal management of follicular lymphoma, including the most effective sequencing of therapy, is undefined. Identifying the subsets of patients at risk for early failure and those with indolent disease that remains quiescent would assist clinicians in tailoring therapy for individual patients. Given the heterogeneity of treatment options and possible clinical outcomes, improvement in risk stratification and personalization in follicular lymphoma is needed, particularly given the expanding treatment options outlined by Dr. Ujjani.
What Can We Expect in the Future?
Historically, prognostication for patients with follicular lymphoma has relied primarily on clinical characteristics. The Follicular Lymphoma International Prognostic Index (FLIPI) can distinguish patients with low or intermediate risk from those at high risk, but it is not routinely used to guide risk-adapted therapy. More recently, the development of m7-FLIPI, a multivariable risk model incorporating the mutation status of seven genes with established clinical relevance in follicular lymphoma, improved the ability to predict early treatment failure in patients receiving front-line chemoimmunotherapy. Identifying the follicular lymphoma patients at highest risk for early treatment failure with standard therapy allows for their prioritization to a clinical trial assessing some of the novel therapies outlined by Dr. Ujjani.
Given the number of therapeutic agents under investigation in follicular lymphoma, and the vast combinatorial possibilities, consideration of toxicity is as imperative as the need to conduct correlational studies to unravel the complexity of this disease.
Tumor Microenvironment Immunomodulatory agents
As stated previously, the tumor microenvironment plays a critical role in the pathogenesis of follicular lymphoma. Approaches to promoting a functional immune system have allowed for effective treatment of the disease. The second-generation immunomodulatory agent lenalidomide has been the most extensively studied of these therapies. Its mechanisms of action include activation of natural killer cells and T cells, stimulation of apoptosis, and inhibition of tumor necrosis factor (TNF)-α and vascular endothelial growth factor (VEGF).[60] In follicular lymphoma cell lines, lenalidomide has been shown to restore immunologic synapses between malignant lymphocytes and T cells and augment rituximab-mediated ADCC.[61] Lenalidomide has demonstrated modest activity as a single agent in relapsed or refractory follicular lymphoma (with ORRs ranging from 27% to 49%); however, when lenalidomide was added to rituximab the ORR improved to 76% and the median event-free survival time was 2 years.[62,63] The doublet (dubbed “R2,” for Revlimid [lenalidomide] plus rituximab) was evaluated by the Alliance study as a front-line regimen, producing an ORR of 93% (CR, 72%) and 2-year PFS of 89% (n = 65).[64] Minimal toxicity was noted with the regimen; common grade 3/4 adverse events included neutropenia (in 19% of patients), rash (8%), and infection (8%). In a similar study from the University of Texas MD Anderson Cancer Center, 35 of the 50 patients with follicular lymphoma achieved a CR (70%) and 5 had an unconfirmed CR.[65] The Swiss Group for Clinical Cancer Research and the Nordic Lymphoma Group reported an ORR of 78% (CR, 61%) with the R2 combination (n = 77).[66] The impressive activity noted in the Alliance and MD Anderson studies prompted the pharmaceutical-sponsored phase III RELEVANCE trial of R2 vs rituximab with chemotherapy (CVP, CHOP, or bendamustine) in previously untreated advanced-stage follicular lymphoma (ClinicalTrials.gov identifier: NCT01650701). R2 has been studied in combination with other regimens such as CHOP, producing an ORR of 94% (CR/unconfirmed CR, 74%) in patients with previously untreated follicular lymphoma.[67] These data are relatively comparable to previously reported results with R2 and call into question the need for CHOP. The Alliance has conducted subsequent studies of R2 with targeted agents including ibrutinib and idelalisib. Results with ibrutinib are pending; however, the trial of idelalisib was closed owing to considerable toxicity.[45,56] Lenalidomide has also been combined with obinutuzumab in the treatment of relapsed and refractory disease, yielding an ORR of 68% (CR, 35%) among the 20 patients enrolled in the phase I portion of a phase I/II study.[68]
Immune checkpoint modulators
In patients with follicular lymphoma, the overexpression of PD-1 in the intratumoral T cells results in an impairment in antitumor immune surveillance. Inhibition of PD-1 or its ligands, PD-L1 and PD-L2, has shown promise in the treatment of follicular lymphoma. Pidilizumab, a humanized PD-1 monoclonal antibody, was the first PD-1 inhibitor to be explored. Although minimally active as a single agent, when pidilizumab was administered in conjunction with rituximab in the setting of relapsed follicular lymphoma, the ORR was 66% and median PFS was 18.8 months (n = 29).[69,70] The majority of the responses were complete (52%), and the median PFS had not been reached for those who achieved a response. Nivolumab, a fully human monoclonal antibody approved for the treatment of melanoma and squamous non–small-cell lung cancer, has also demonstrated activity in relapsed and refractory follicular lymphoma. A phase I evaluation has reported an ORR of 40% (CR, 10%) in 10 patients.[71] Nivolumab is currently being studied in combination with ibrutinib in patients with relapsed B-cell malignancies (ClinicalTrials.gov identifier: NCT02329847). Pembrolizumab and MEDI-0680 are humanized PD-1 antibodies also under clinical investigation in CLL and other low-grade B-cell NHLs, as well as in relapsed and refractory aggressive B-cell lymphomas (ClinicalTrials.gov identifiers: NCT02332980 and NCT02271945, respectively). MEDI4736, a human anti–PD-L1 antibody, is also being studied with ibrutinib in patients with relapsed lymphoma (ClinicalTrials.gov identifier: NCT02401048). Similar to PD-1, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) is a negative regulator of T-cell function. Inhibition of CTLA-4 with ipilimumab, also approved in melanoma, has demonstrated some activity in lymphoid malignancies, including in one patient with follicular lymphoma.[72]
Bispecific T-cell engager (BiTE)
The BiTE is a unique form of immunotherapy that stimulates T-cell function via binding simultaneously to CD3 on the surface of the T cell and a specific marker on the malignant B cell, resulting in caspase-mediated apoptosis. Blinatumomab, a CD19-specific BiTE approved for treatment of relapsed and refractory B-cell acute lymphoblastic leukemia (ALL), has demonstrated activity in other CD19-positive lymphoid diseases.[73] It produced an ORR of 100% in a phase I study of 13 patients with relapsed indolent NHL, the majority of whom had a follicular or mantle cell histology.[74] Four patients achieved a CR, and eight remained in remission at 13 months. A single cycle of blinatumomab requires a 4-week continuous IV infusion and is associated with significant, yet reversible, neurologic toxicity. The current focus of clinical investigations with this agent is ALL, and its role in follicular lymphoma is unclear.
Chimeric antigen receptor (CAR)-modified T cells
CAR-modified T cells are one of the newest, most intriguing, forms of immunotherapy. These autologous T cells have been genetically transduced using lentiviral vectors to express tumor cell–specific antigen receptors. Having demonstrated activity in CLL and ALL, CAR-modified T cells are now being explored in CD19-positive NHL. A phase II study at the University of Pennsylvania demonstrated an ORR of 100% among seven patients with relapsed and refractory follicular lymphoma who lacked curative treatment options.[75] Six patients achieved a CR by 6 months, and responses appeared to be durable. In all seven patients there was evidence of severe cytokine release syndrome (cytokine storm); in two of the patients this was a grade 3/4 toxicity. One patient developed grade 5 encephalopathy 2 months after completing therapy. Although quite promising, further investigation is necessary to fully understand this new method.
Conclusion
The treatment of follicular lymphoma has changed dramatically over the past several years. The availability of newer, novel forms of therapy has enabled the field to continue to evolve. In addition to having tumor-specific activity, these newer agents provide the possibility of a more favorable toxicity profile than conventional chemotherapy. Although chemoimmunotherapy has been the traditional front-line induction for patients with advanced-stage disease, this concept is being challenged by the remarkable efficacy of the R2 regimen (with ORR > 90%; CR, 61% to 72%).[64,66] If the phase III RELEVANCE trial demonstrates results with R2 that are even equivalent to those achieved with standard regimens such as R-CHOP or bendamustine-rituximab, a major paradigm shift will occur; R2 would then be the first chemotherapy-free option for the initial treatment of follicular lymphoma. Given that the attainment of a CR has been associated with a survival benefit in this setting, there is still room for improvement.[76] Although approved as a single agent, idelalisib is being studied in combination with rituximab in previously untreated and relapsed patients (ClinicalTrials.gov identifiers: NCT02258529 and NCT01732913). Ongoing clinical investigations, such as the Alliance phase I trial of R2 plus ibrutinib in previously untreated patients, are exploring the benefit of multitargeted agents in this population. Studies such as the phase II trial of bendamustine-rituximab vs rituximab-venetoclax vs bendamustine-rituximab-venetoclax are exploring the utility of other targeted agents in comparison to standard chemoimmunotherapy.
While the concept of multitargeted therapy is quite appealing, these regimens must be explored with caution. Early-phase investigations of idelalisib with R2 and entospletinib produced significant adverse events, requiring study closures.[45,46] In addition to understanding how to combine treatment with these agents safely and efficaciously, research efforts must incorporate sound correlative science. Through whole-exome sequencing, Woyach et al have already discovered mutations associated with resistance to ibrutinib in CLL.[77] The identification of other predictive biomarkers is imperative to tailor therapy effectively and to develop superior regimens for individual patients. Furthermore, this information may enable us to provide appropriate treatment options that are also financially prudent. Given the lengthy follow-up period required to achieve the traditional objectives of clinical trials, it is important to explore earlier, yet meaningful, surrogate endpoints. Residual positron emission tomography activity on post-induction imaging, the presence of minimal residual disease, and relapse within 2 years of chemoimmunotherapy have been associated with an inferior PFS and OS outcome; in contrast, the presence of a CR at 30 months has been correlated with a significantly reduced risk of progression in patients with follicular lymphoma.[78-81] By incorporating novel therapies into innovative clinical investigations, we may achieve significantly better outcomes and improve the outlook for patients with this incurable disease.
Financial Disclosure: Dr. Ujjani has served on advisory boards for Genentech, Inc., and Pharmacyclics, Inc.
Using computational modeling, researchers at Carnegie Mellon University, the Colorado School of Mines and the University of California, Davis have come up with a design for a better liposome. Their findings, while theoretical, could provide the basis for efficiently constructing new vehicles for nanodrug delivery.
Liposomes are small containers with shells made of lipids, the same material that makes up the cell membrane. In recent years, liposomes have been used for targeted drug delivery. In this process, the membrane of a drug-containing liposome is engineered to contain proteins that will recognize and interact with complementary proteins on the membrane of a diseased or dysfunctional cell. After the drug-containing liposomes are administered, they travel through the body, ideally connecting with targeted cells where they release the drug.
This packaging technique is often used with highly toxic nanodrugs, like chemotherapy drugs, in an attempt to prevent the free drug from damaging non-cancerous cells. However, studies of this model of delivery have shown that in many cases less than 10 percent of the drugs transported by liposomes end up in tumor cells. Often, the liposome breaks open before it reaches a tumor cell and the drug is absorbed into the body’s organs, including the liver and spleen, resulting in toxic side effects.
“Even with current forms of targeted drug delivery, treatments like chemotherapy are still very brutal. We wanted to see how we could make targeted drug delivery better,” said Markus Deserno, professor of physics at Carnegie Mellon and a member of the university’s Center for Membrane Biology and Biophysics.
Deserno and colleagues propose that targeted drug delivery can be improved by making more stable liposomes. Using three different types of computer modeling, they have shown that liposomes can be made sturdier by incorporating a nanoparticle core made of a material like gold or iron and connecting that core to the liposome’s membrane using polymer tethers. The core and tethers act as a hub-and-spoke-like scaffold and shock-absorber system that help the liposome to weather the stresses and strains it encounters as it travels through the body to its target.
Francesca Stanzione and Amadeu K. Sum of the Colorado School of Mines conducted a fine-grained simulation that looked at how the polymer tethers anchor the liposome’s membrane at an atomistic level. Roland Faller of UC Davis did a meso-scale simulation that looked how a number of tethers held on to a small patch of membrane. Each of these simulations allowed researchers to look at smaller components of the liposome, nanoparticle core and tethers, but not the entire structure.
To see the entire structure, Carnegie Mellon’s Deserno and Mingyang Hu developed a coarse-grained model that represents groupings of components rather than individual atoms. For example, one lipid in the cell membrane might have 100 atoms. In a fine-grain simulation, each atom would be represented. In Deserno’s coarse grain simulation, those atoms might be represented by only three pieces instead of 100.
“Its unfeasible to look at the complete construct at an atomistic level. There are too many atoms to consider, and the timescale is too long. Even with the most advanced supercomputer, we wouldn’t have the power to run an atom-level simulation,” Deserno said. “But the physics that matters isn’t locally specific. It’s more like soft matter physics, which can be described at a much coarser resolution.”
Deserno’s simulation allowed the researchers to see how the entire reinforced liposome construct responded to stress and strain. They proposed that if a liposome was given the right-sized hub and tethers, its membrane would be much more resilient, bending to absorb impact and pressure.
Additionally, they were able to simulate how to best assemble the liposome, hub and tether system. They found that if the hub and tether are attached and placed in a solution of lipids, and solvent conditions are suitably chosen, a correctly sized liposome would self-assemble around the hub and tethers.
The researchers hope that chemists and drug developers will one day be able to use their simulations to determine what size core and polymer tethers they would need to effectively secure a liposome designed to deliver a specific drug or other nanoparticle. Using such simulations could narrow down the design parameters, speed up the development process and reduce costs.
NIHS to use the Lipotype Shotgun Lipidomics Technology for lipid analysis.
Lipotype GmbH and the Nestlé Institute of Health Sciences (NIHS) have collaborated to employ the innovative Lipotype Shotgun Lipidomics Technology to analyze lipids in blood for nutritional research. Recently, Lipotype and NIHS have jointly published results of the robustness of the Lipotype Technology. Lipotype envisions a future use of its technology in clinical diagnostics screens for establishing reliable lipid diagnostic biomarkers.
Innovative Lipotype Technology for lipid analysis
The purpose of this collaboration is to enable NIHS to use the Lipotype Shotgun Lipidomics Technology for lipid analysis. The mass spectrometry-based Lipotype technology covers a broad spectrum of lipid molecules and delivers quantitative results in high-throughput. The Nestlé Institute of Health Sciences uses this technology platform for nutritional research. NIHS is a specialized biomedical research institute and is part of Nestlé’s global Research & Development network.
Joint research project reveals robustness of Lipotype Technology
During the collaboration, Lipotype and NIHS conducted a joint research project and demonstrated that the Lipotype technology was robust enough to deliver data with high precision and negligible technical variation between different sites. In addition, important features are the high coverage and throughput, which were confirmed when applying the Lipotype technology.
Lipotype envisions these as important features, required for future use in clinical diagnostics screens, in order to establish and validate reliable lipid diagnostic biomarkers. The results have been published in October 2015, in the European Journal of Lipid Science and Technology (Surma et al. “An Automated Shotgun Lipidomics Platform for High Throughput, Comprehensive, and Quantitative Analysis of Blood Plasma Intact Lipids.”).
Lipids play an important role for health and disease
Lipotype is a spin-off company of the Max-Planck-Institute of Molecular Cell Biology and Genetics in Dresden, Germany. Prof. Kai Simons, CEO of Lipotype explains: “We developed a novel Shotgun-Lipidomics technology to analyze lipids in blood and other biological samples. Our analysis is quick and covers hundreds of lipid molecules at the same time. Our technology can be used to identify disease related lipid signatures.”
Researchers at the University of Liverpool, working with a global healthcare company, have helped develop a new treatment for obesity.
The treatment, which is a once-daily injectable derivative of a metabolic hormone called GLP-1 conventionally used in the treatment of type 2 diabetes, has proved successful in helping non-diabetic obese patients lose weight.
Professor John Wilding, who leads Obesity and Endocrinology research in the Institute of Ageing and Chronic Disease, investigates the pathophysiology and treatment of both obesity and type 2 diabetes and is applying his expertise in this area to work with, and often act as a consultant for, a number of large pharmaceutical companies looking to develop new treatments for obesity and diabetes.
Exciting development
Professor Wilding, said: “The biology of GLP-1 has been a focus of my research for 20 years; in particular when I was working at Hammersmith Hospital in London, I was part of the team that demonstrated that it was involved in appetite regulation; work on GLP-1 has continued during my time in Liverpool. Being involved in the development of a treatment, from the basic research right through to clinical trials in patients is very exciting”.
“It is likely that the treatment will be used initially in very specific situations, such as helping patients who are severely obese. It differs from current treatments used for diabetes, as it has stronger appetite regulating effects but no greater effect on glucose control.”
In 2014 more than 1.9 billion adults worldwide were classed as obese by the World Health Organisation; in the UK numbers have more than tripled since 1980. This Obesity can lead to other serious health-related illnesses including type 2 diabetes, hypertension and obstructive sleep apnoea as well as increasing the risk for many common cancers.
The drug has been approved in the European Union, but has not yet launched in the UK.
Professor Wilding added: “Consultancy like this can help relationship and reputation building and informs my research keeping it at the forefront of developments. It also brings many other benefits such as publications and income generation, which can help support other research, for example by such as funding for pilot projects that can lead to grant applications and investigator-initiated trials funded by the company”.
Researchers in the West Midlands have made a breakthrough in explaining how an incurable type of blood cancer develops from an often symptomless prior blood disorder.
The findings could lead to more effective treatments and ways to identify those most at risk of developing the cancer.
All patients diagnosed with myeloma, a cancer of the blood-producing bone marrow, first develop a relatively benign condition called ‘monoclonal gammopathy of undetermined significance’ or ‘MGUS’.
MGUS is fairly common in the older population and only progresses to cancer in approximately one in 100 cases. However, currently there is no way of accurately predicting which patients with MGUS are likely to go on to get myeloma.
Myeloma is diagnosed in around 4,000 people each year in the UK. It specifically affects antibody-producing white blood cells found in the bone marrow, called plasma cells. The researcher team from the University of Birmingham, New Cross and Heartlands Hospitals compared the cellular chemistry of bone marrow and blood samples taken from patients with myeloma, patients with MGUS and healthy volunteers.
Surprisingly, the researchers found that the metabolic activity of the bone marrow of patients with MGUS was significantly different to plasma from healthy volunteers, but there were very few differences at all between the MGUS and myeloma samples. The research was funded by the blood cancer charity Bloodwise, which changed its name from Leukaemia & Lymphoma in September.
The findings suggest that the biggest metabolic changes occur with the development of the symptomless condition MGUS and not with the later progression to myeloma.
Dr Daniel Tennant, who led the research at the University of Birmingham, said, “Our findings show that very few changes are required for a MGUS patient to progress to myeloma as we now know virtually all patients with myeloma evolve from MGUS. A drug that interferes with these specific initial metabolic changes could make a very effective treatment for myeloma, so this is a very exciting discovery.”
The research team found over 200 products of metabolism differed between the healthy volunteers and patients with MGUS or myeloma, compared to just 26 differences between MGUS patients and myeloma patients. The researchers believe that these small changes could drive the key shifts in the bone marrow required to support myeloma growth.
Non-Hodgkin’s lymphoma (NHL) is the most common hematological malignancy in adults, with B-cell lymphomas accounting for 85% of all NHLs. The most substantial advancement in the treatment of B-cell malignancies, since the advent of combination chemotherapy, has been the addition of the monoclonal anti-CD20 antibody rituximab (Rituxan). Since its initially reported single-agent activity in indolent lymphomas in 1997, the role of rituximab has expanded to cover both indolent and aggressive lymphomas.
This article focuses on the impact of rituximab on the treatment, survival, and long-term outcomes of patients with indolent and aggressive lymphomas over the past two decades.
Keywords: rituximab, non-Hodgkin’s lymphoma
Non-Hodgkin’s lymphoma (NHL) is the most common adult hematological cancer. In 2009, almost 66,000 cases were anticipated in the U.S. alone.1The incidence of NHL in the U.S. over the previous 15 years has increased by approximately 4% annually, despite the decline in age-adjusted incidence rates for all cancers combined.
NHL encompasses a heterogeneous group of lymphomas that have been classified in various ways. In 1995, the World Health Organization developed a classification that included a combination of morphology, immunotyping, genetic features, and clinical syndromes. The goal was to define disease entities of B cells, T cells, and natural killer (NK) cells that pathologists could recognize and that had clinical relevance. The lymphomas were further subdivided into categories based on their clinical behavior (indolent, aggressive, or highly aggressive).2 More recent updates of this classification have clarified some less common entities but have left the overall schema intact.3
B-cell lymphomas account for about 85% of all NHL diagnoses.4 Although many subtypes of NHL exist clinically, most are grouped as either indolent (characterized by a prolonged median survival but generally considered incurable) or aggressive (characterized by rapid growth but with the potential for cure). Because patients with indolent lymphoma eventually die with this disease if they do not die of intercurrent illness, new treatments are needed to prolong survival, with the ultimate goal to provide cure. For patients with aggressive lymphoma, unmet needs include higher initial cure rates, improved salvage chemotherapy options, and less toxic therapies for old and frail patients.
Conventional methods of treatment, including chemotherapy and radiation, are associated with toxicity and lack specific antitumor-targeted activity. Cell–surface proteins, such as CD19, CD20, and CD22, are highly expressed on B-cell lymphomas and represent key potential targets for treatment.
Antibody therapy directed against CD20 has had the most important clinical impact to date. CD20 is thought to be involved in the regulation of intracellular calcium, cell cycle, and apoptosis. CD20 is not shed, modulated, or internalized significantly upon antibody binding, thus making it an ideal target for passive immunotherapy.5
Over the past two decades, significant progress has been made in the development of new therapies for B-cell lymphoma. Perhaps the most important advance is the addition of rituximab (Rituxan, Genentech/Biogen Idec), which the FDA approved for use in the U.S. in 1997. Rituximab is a chimeric (mouse and human) monoclonal antibody directed against the B-cell antigen CD20. It depletes B cells by several mechanisms, including direct antibody-dependent cellular cytotoxicity (ADCC), complement-mediated cell death, and signaling apoptosis.6–11
Phase 1 trials of two doses of rituximab (500 mg/m2 and 375 mg/m2 for four weeks) showed clinical responses with no dose-limiting toxicity.12 The weekly 375-mg/m2 dose, given for four weeks, was selected for further phase 2 evaluation and is currently the standard single-agent dose and schedule. Since this first reported activity, the role of rituximab has expanded to include both indolent and aggressive lymphomas.
This article addresses the effect of rituximab on survival and long-term outcomes in patients with NHL.
Indolent, Low-Grade Lymphomas
Unlike aggressive lymphomas, indolent B-cell lymphomas are not considered curable with conventional therapies. Many patients are observed for prolonged periods without requiring treatment.13 In one study, more than 50% of the patients remained untreated for a median period of almost six years after diagnosis.14 Treatment goals focus on maintaining good quality of life with minimal symptoms. The indications for treatment include the presence of B symptoms (fevers, night sweats, and weight loss), compromise of normal organ function, bulky disease, or the presence of cytopenias resulting from marrow involvement. Transformation to an aggressive histological pattern warrants treatment for the aggressive component.
Although many active therapies are available for indolent NHL, patients ultimately die of this disease, which is incurable. Additional therapeutic options with improved efficacy and reduced toxicity are still needed for patients with indolent NHL. In light of this unmet need, the FDA’s approval of rituximab for the treatment of relapsed or refractory CD20-positive (CD20+) NHL in 1997 was an important clinical advance. The approval was based on the pivotal trial reported by McLaughlin et al., in which single-agent rituximab brought about significant response rates in heavily pretreated patients with indolent lymphoma.15
Initial Therapy for Indolent (Follicular) Lymphoma
Rituximab as first-line therapy has been widely studied in patients with indolent lymphomas, both as a single agent and in combination with conventional chemotherapy (Table 1). Witzig et al. evaluated the use of single-agent rituximab, 375 mg/m2 weekly for four doses, as an initial therapy for patients with stage III or IV grade 1 follicular lymphoma (FL). In this small phase 2 trial of only 37 patients, the reported objective response rate (ORR) was 72% and the complete remission rate (CRR) was 36%.16 Similarly, a phase 2 study by Hainsworth et al., which evaluated initial therapy in patients with indolent lymphomas, showed response rates in the range of 50%.17
Randomized Trials Using Rituximab in First-Line and Relapsed Settings in Indolent Lymphomas
Using rituximab as a first-line therapy in patients with low-tumor-burden, indolent NHL, Colombat et al. reported an ORR of 73%.18 Long-term follow-up results of the completed randomized phase 3 Rituximab ExtendedSchedule Or Re-treatment Trial (RESORT, ECOG 4402 [Eastern Cooperative Oncology Group]) are still pending. In this randomized study, patients received four weekly rituximab treatments. Retreatment is then given as a single dose of rituximab every three months or upon disease progression with four weekly doses. The aim of the study is to define the benefit of maintenance therapy or re-treatment with rituximab (in terms of time to requiring a therapy other than rituximab) when progressive disease is documented.
The benefit of adding rituximab to combination chemotherapy during the initial treatment of FL has been documented in multiple clinical trials over the past decade. The phase 3 trial by Marcus et al. compared cyclophosphamide, vincristine, and prednisone (CVP), with and without rituximab, in 318 previously untreated patients with stage III and IV CD20+ FL.19 The addition of rituximab to CVP (R-CVP) significantly improved time to disease progression (34 months with R-CVP vs. 15 months with CVP, respectively; P < 0.0001) and duration of response (38 months vs. 14 months, respectively; P < 0.0001). Disease-free survival was 21 months with CVP but has not yet been determined in the group receiving R-CVP.
The East German Study Group evaluated the combination of rituximab with mitoxantrone, chlorambucil, and prednisone (MCP), followed by maintenance interferon in treatment-naive patients with stage III/IV CD20+ FL.20 The ORR was 92% with rituximab and 75% with chemotherapy alone (P = 0.0009).
Rituximab was also tested in combination with cyclophosphamide, hydroxydaunorubicin (doxorubicin), Oncovin (vincristine), and prednisone (R-CHOP) as first-line therapy in 428 patients with FL in a randomized phase 3 study with three years of follow-up.21 This combination showed a significant prolongation of time to treatment failure (P < 0.001) and prolonged duration of remission (P = 0.001) with the addition of rituximab. A higher ORR was observed in the group receiving R-CHOP (96%), compared with CHOP alone (90%) (P = 0.011). Even with a short follow-up, overall survival rates improved in the group receiving chemotherapy and rituximab (P = 0.016).
Similar results were seen in the GELA–GOELAMS FL 2000 trial (Groupe d’Etude des Lymphomes de l’Adulte/Groupe Ouest Est des Leucémies etAutres Maladies du Sang). This study was designed to examine the combination of rituximab with cyclophosphamide, hydroxydaunorubicin (doxorubicin), etoposide (VP-16), and prednisolone (CHVP) plus interferon-2 .22 A significant improvement in event-free survival at five years was noted for the rituximab patients (37% vs. 53%, respectively; P = 0.0004).
A meta-analysis of seven randomized controlled trials assessed the value of adding rituximab to conventional chemotherapy for 1,943 patients with FL, mantle-cell lymphoma, and other indolent lymphomas.23 This analysis demonstrated improved overall survival with the combination, as follows:
hazard ratio (HR) for mortality, 0.65
95% confidence interval (CI), 0.51–0.79
disease control (HR for the disease event, 0.62; 95% CI, 0.55–0.71)
response rates (relative risk for response 1.21; 91% CI, 1.16–1.27)
Specifically in FL, overall survival was better with rituximab plus chemotherapy (HR for mortality, 0.60; 95% CI, 0.37–0.98).23 The study authors concluded that the combination of rituximab and chemotherapy for patients with indolent lymphomas was superior to chemotherapy alone with respect to overall survival, disease-free survival, and response rates.23
The pivotal trial upon which the initial approval of rituximab was based showed the drug’s efficacy as a single agent in relapsed/refractory indolent NHL.15 Re-treatment with rituximab alone in 57 patients with low-grade FL who had previously responded to single-agent rituximab yielded a response rate of 40% and a similar duration of response, indicating sensitivity to re-treatment with the same agent.24
Davis et al. studied the use of single-agent rituximab in patients with bulky lesions (larger than 10 cm) and relapsed NHL.25 Patients receiving rituximab 375 mg/m2 weekly for four doses had an ORR of 43%. Among patients with a partial response, lesion size decreased by 76%.
The addition of rituximab to standard chemotherapy was found to be beneficial in the treatment of FL patients with relapsed/refractory NHL (see Table 1). An international trial by van Oers et al. evaluated the combination of six cycles of CHOP with rituximab 375 mg/m2 given intravenously on day 1 of each cycle, compared with chemotherapy alone in 465 patients with advanced disease.26 The ORR was higher with the addition of rituximab (85% with R-CHOP vs. 72% with CHOP alone; P < 0.001), and the median progression-free survival rate was also significantly improved in the rituximab group (33.1 vs. 20 months; P < 0.001). The addition of rituximab to the combination of fludarabine, cyclophosphamide, and mitoxantrone (FCM) in a similar group of patients also showed superior responses.27
Rituximab with bendamustine (Treanda, Cephalon) was studied in a phase 2 trial in patients with relapsed disease. This combination was found to be very effective, with an ORR of 92%.28
Maintenance Therapy for Follicular Lymphoma
Some authors consider rituximab to be an ideal medication to use as maintenance therapy for an incurable disease such as FL because of its minimal toxicity and long half-life, which obviates the need for frequent administration.29 The use of rituximab as maintenance therapy after induction treatment has been the subject of several studies (Table 2) and is being evaluated by two large phase 3 trials: Primary Rituximab and Maintenance (PRIMA) and RESORT.30,31
Trials Showing Benefit for Maintenance Rituximab after Induction Therapy for Indolent Lymphomas
In the PRIMA trial, patients with previously untreated FL requiring therapy received a rituximab–chemotherapy regimen designated by the participating center as R-CHOP, R-CVP, or R-FCM. Responding patients were then randomly assigned to receive observation or scheduled re-treatment with rituximab as a single dose every eight weeks for two years. The RESORT trial was designed for asymptomatic patients with low-tumor-burden, indolent NHL (as discussed in detail on page 149).
The Swiss Group for Clinical Cancer Research (SAKK) evaluated maintenance rituximab following induction with rituximab monotherapy in a phase 3 trial in patients with newly diagnosed NHL and in previously treated patients with FL.32 In this study, the maintenance schedule consisted of four infusions at two-month intervals. Event-free survival was significantly longer among patients who received this maintenance schedule.
A phase 2 trial by the Minnie Pearl Cancer Research Network compared maintenance rituximab (four weekly doses repeated every six months for two years) with re-treatment using rituximab upon disease progression.33The study showed significant prolongation of progression-free survival in the maintenance therapy group (31.3 vs. 7.4 months, respectively; P = 0.007), although no difference in overall survival or duration benefit from rituximab was observed between the two cohorts.
ECOG 1496 was a study that compared the use of maintenance rituximab with observation after induction with a non-rituximab chemotherapy regimen (CVP) in 282 patients with newly diagnosed FL.34 Improvement in progression-free survival at three years (68% with rituximab vs. 33% with CVP; P < 0.001) and overall survival at three years (91% vs. 86%, respectively; P = 0.08) were noted in the maintenance arm. In the relapsed setting, the prolonged use of rituximab was found to be beneficial with improved progression-free survival when it was used after CHOP or R-CHOP and after treatment with R-FCM.26,35
Although significant evidence exists for the improved progression-free survival with the use of maintenance therapy for FL, the benefit in terms of overall survival is still controversial. Moreover, because different dosing schedules were used in these studies, no data are available for the optimal dosing schedule of maintenance therapy and the recommended duration of this treatment.
Rituximab plus Chemotherapy: Effect on Survival In Follicular Lymphoma
There is no doubt that the clinical development of rituximab has been a significant breakthrough in the field of indolent lymphomas. However, its effect on overall survival in this group of patients is still open to debate.
An analysis of survival in patients 15 years of age and older with NHL diagnosed between 1990 and 2004, using data from the Surveillance, Epidemiology and End Results (SEER) program, revealed a markedly improved outcome for patients with NHL in recent years. This finding may be related, in part, to the addition of rituximab.36
A large retrospective analysis by Swenson et al. was conducted to examine survival rates of 14,564 patients with FL diagnosed between 1978 and 1999 in the U.S.37 Improvement in survival was noted over the past 25 years, and a reduction in the relative risk of death by 1.8% per year was observed from 1983 to1999.
In a second analysis, the Southwest Oncology Group (SWOG) looked at the survival of patients with FL on three large randomized clinical trials between 1974 and 2000.38 Overall survival rates improved over this period of time. The greatest improvement was observed with the most recent treatment approach consisting of CHOP with an anti-CD20 monoclonal antibody.
The study by Marcus et al., published in 2008, showed improved survival rates among untreated patients receiving CVP plus rituximab when compared with CVP alone (four-year survival, 83% vs. 77%, respectively; P= 0.029).19 Other studies, including a Cochrane meta-analysis, have shown similar trends toward improved survival.23,26,34
These observations can be attributed to multiple factors, including improved supportive care measures, enhancements in education of physicians and patients, and better treatments of relapsed and transformed cases.39 Despite these uncertainties regarding its effect on overall survival, it is clear that rituximab has substantially advanced the treatment of indolent lymphomas in the last decade.
Diffuse Large B-Cell Lymphoma
As the most common high-grade form of NHL, diffuse large B-cell lymphoma (DLBCL) accounts for more than 30% of new diagnoses. The median age of presentation is 60 years. Unlike indolent lymphomas, DLBCL is an aggressive lymphoma; if it is untreated, survival can be measured in months. More than 70% of patients with DLBCL present at an advanced stage, and systemic chemotherapy is the foundation of treatment. Since its development in the 1970s, CHOP has been the mainstay of treatment for this group of patients.
A milestone phase 3 trial found that complex regimens that included the addition of other chemotherapy agents to CHOP did not demonstrate any significant difference in overall survival, disease-free survival, or remission rates over CHOP.40–43 Moreover, CHOP was associated with significantly less toxicity and cost.
Based on these results, CHOP remained the gold standard of therapy for DLBCL. Nonetheless, long-term remission occurred in only about 45% of patients, so that more than half of patients relapsed with the best therapy possible in the early 1990s. A relatively small percentage of relapsed DLBCL patients (25%–50%) might have been “salvaged” with high-dose chemotherapy and stem-cell support, yet many patients were not even eligible for such therapy.
Thus, in the early 1990s, the addition of more chemotherapy drugs into complex regimens had not improved results with CHOP, and there was a sense that future improvements in therapy would not come from additional “standard” drugs. While rituximab was approved for treatment of low-grade lymphoma in 1997, several trials combining rituximab with CHOP (R-CHOP) for aggressive lymphomas began prior to that time. Because rituximab-related toxicities were not overlapping with those of CHOP, both CHOP and rituximab could be administered at full doses. Results from large international, randomized trials have demonstrated the significant benefits of the addition of rituximab to standard chemotherapy for DLBCL. These trials are summarized next.
Previously Untreated Diffuse Large B-Cell Lymphoma
Based on the efficacy of rituximab in low-grade lymphomas, Vose et al. conducted a phase 2 study of rituximab with CHOP chemotherapy in 33 previously untreated patients with advanced-stage, aggressive B-cell lymphoma.44 Rituximab at a dose of 375 mg/m2 was administered on day 1 of each of six cycles of CHOP. The ORR was 94%; 61% of patients had complete responses (CRs), and 33% had partial responses (PRs). This was the first report that demonstrated an improved efficacy of the combination without worsening toxicity.
GELA investigators randomized previously untreated elderly patients (60–80 years of age) to eight cycles of CHOP alone (197 patients) or eight cycles of R-CHOP given on day 1 of each cycle (202 patients).45 The rate of CRs was significantly higher in the rituximab group (76% vs. 63% receiving CHOP alone, P = 0.005). Sixty percent of patients exhibited features of poor risk, with age-adjusted International Prognostic Index (aaIPI) scores of 2 to 3. With a median follow-up of two years, event-free survival rates (57% vs. 38%; P < 0.001) and overall survival rates (70% vs. 57%; P = 0.007) were significantly higher with rituximab (Table 3). Furthermore, toxicity was not greater with the addition of rituximab.
Trials Using Rituximab for Diffuse Large B-Cell Lymphomas in the First-Line Setting
A long-term analysis at seven years has confirmed the benefit of the addition of rituximab.46 Event-free survival (42% with R-CHOP vs. 25%; P < 0.0001), progression-free survival (52% vs. 29%, respectively; P < 0.0001) and disease-free survival (66% vs. 42% respectively, P = 0.0001) were all statistically better for patients treated with combination therapy.
A retrospective analysis of the GELA trial suggested that R-CHOP increased overall survival preferentially in bcl-2–positive patients compared with CHOP alone.47 These data suggested that rituximab may overcome chemotherapy resistance associated with bcl-2 in patients with DLBCL. However, other retrospective analyses have led to conflicting results on whether the benefit of R-CHOP is primarily or only observed in bcl-2expressing DLBCL.
Habermann et al. randomly assigned patients older than 60 years of age to receive CHOP or R-CHOP, with a second random assignment to maintenance rituximab therapy or observation in responders (see Table 3).48This study demonstrated the benefit of the addition of rituximab to CHOP using a modified schedule of rituximab administration. Three-year failure-free survival rates were 53% and 46% (P = 0.04). Failure-free survival was higher for patients who received maintenance therapy with rituximab after CHOP but not for patients who received R-CHOP initially.
The trials described above established R-CHOP as standard first-line therapy for elderly patients with DLBCL. With respect to younger patients, the MabThera (rituximab) International Trial (MInT) confirmed the benefit of adding rituximab to standard chemotherapy in 824 patients (18 to 60 years of age) with only zero (0) to one risk factor, as assessed by the IPI (seeTable 3).49 Patients with stage II to IV or stage I disease with bulky lymphadenopathy were randomly assigned to six cycles of CHOP-like chemotherapy with or without the addition of rituximab. Radiation therapy was subsequently administered to initial sites of bulky disease. Three-year event-free survival rates (79% vs. 59%; P < 0.0001) and overall survival rates (93% vs. 84%; P = 0.00001) were both significantly higher for patients treated with the addition of rituximab. There were no additional major adverse effects.
Sehn et al. compared outcomes during a three-year period; 18 months pre- and post-inclusion of rituximab in standard treatment protocols guided care for patients with newly diagnosed advanced-stage DLBCL in British Columbia.50 All age and risk factor groups were included. Adding rituximab resulted in dramatic improvement in both progression-free survival and overall survival (Figure 1). These studies have indicated significant benefit for the addition of rituximab to chemotherapy for the treatment of DLBCL in a wide range of patient ages and risk categories. Although adding other cytotoxic chemotherapy agents to CHOP failed to improve outcomes, R-CHOP is now the gold standard for treating DLBCL in all subgroups.43
Overall survival according to treatment regimen in diffuse large B-cell lymphoma. Results of a randomized trial of non-rituximab containing regimens as historical controls for British Columbia outcome data with R-CHOP. MACOP-B = methotrexate, Adriamycin, …
Relapsed/Refractory Diffuse Large B-Cell Lymphoma
Coiffier et al. conducted a randomized phase 2 trial to evaluate the efficacy and tolerability of rituximab in patients with relapsed/refractory DLBCL, mantle-cell lymphoma, or other intermediate-grade or high-grade B-cell lymphomas and previously untreated patients older than 60 years of age.51 Fifty-four patients received eight weekly infusions of rituximab 375 mg/m2 in arm A or one infusion of 375 mg/m2, followed by seven weekly infusions of 500 mg/m2in arm B. A total of five complete responses and 12 partial responses were observed among the 54 enrolled patients, with no difference between the two doses. The ORR was 31%. An analysis of prognostic factors showed that response rates were lower in patients with refractory disease, in patients with lymphoma not classified as DLBCL, and patients with a tumor larger than 5 cm in diameter. Single-agent rituximab is active in aggressive NHL but not as active as in indolent NHL. This finding led to the use of combinations of rituximab plus chemotherapy in such patients.
The combination of rituximab, ifosfamide, carboplatin, and etoposide (R-ICE) was evaluated in relapsed DLBCL for cytoreduction prior to autologous hematopoietic stem cell transplantation (HSCT).52 Thirty-six eligible patients received rituximab plus ICE (RICE), and 34 patients received all three planned cycles. The CR rate was 53%, significantly better than the 27% CR rate (P = 0.01) achieved among 147 similar consecutive historical control patients with DLBCL treated with ICE; the PR rate was 25%. Progression-free survival in patients who underwent transplantation after RICE was marginally better than for 95 consecutive historical controls who underwent transplantation after ICE alone, but the results did not reach statistical significance (54% with RICE vs. 43% with ICE alone at two years, respectively; P = 0.25). Preliminary results of the CORAL study (Collaborative Trial in Relapsed Aggressive Lymphoma) demonstrated a decreased response to rituximab in the salvage setting of patients previously treated with rituximab-containing regimens.53
In a phase 2 study, rituximab was evaluated in addition to etoposide, prednisone, Oncovin (vincristine), doxorubicin, and cyclophosphamide (EPOCH) in patients with relapsed or refractory aggressive NHL.54 The ORR of 68% included 28% of patients in complete remission. At three years, event-free survival and overall survival rates were 28% and 38%, respectively.
Few studies have explored the use of rituximab as an adjunct to autologous HSCT after high-dose chemotherapy in patients with relapsed DLBCL.55–57 These studies have reported positive results with rituximab in this setting. Larger, randomized trials are needed to establish a definitive role for rituximab in these patients. While overall the data on rituximab efficacy in aggressive NHL is not as strong in relapsed patients as for initial R-CHOP, rituximab is active and additional confirmatory studies are needed in various relapsed settings.
Maintenance Therapy for Diffuse Large B-Cell Lymphoma
Unlike the situation with indolent lymphomas, there is no apparent benefit to maintenance rituximab in DLBCL. In an ECOG trial, responding patients were randomly assigned to receive maintenance rituximab or to observation alone.48 Two-year failure-free survival was 76% for maintenance therapy and 61% for observation alone, but these figures were confounded depending on whether rituximab was used initially. No significant differences in survival were seen when rituximab was included either as maintenance or as induction therapy. Failure-free survival was prolonged with maintenance therapy after CHOP but not after R-CHOP. This study confirmed the role of R-CHOP as standard first-line therapy in older DLBCL patients, with maintenance therapy to be used only for patients not previously treated with rituximab.
ADVERSE EFFECTS
Rituximab is usually well tolerated, and toxicities are generally mild.12,24,58Common side effects include pruritus, nausea, vomiting, dizziness, headaches, fevers, and rigors. A major concern is the potential for an infusion-related reaction, such as rigors, chills, anaphylactic reactions potentially leading to myocardial infarction and cardiogenic shock. These reactions occur most commonly during the first administration of rituximab. Although infusion reactions are rarely fatal, predisposing cardiac conditions can increase the risk of death. Pre medication with acetaminophen and antihistamines is recommended prior to infusion. Reactions usually abate if the infusion is discontinued and can then be restarted at a slower rate. The benefit of premedication with glucocorticoids is not entirely clear, but they are useful if a reaction occurs. Mucocutaneous reactions, including Stevens–Johnson syndrome, have also been reported within one to 13 weeks following rituximab exposure.
Tumor lysis syndrome has also occurred in patients with bulky lymphoma. Hepatitis B reactivation with fulminant hepatitis, hepatic failure, and death have been reported in patients with previous hepatitis B infection who have been treated with rituximab. Consultation with a hepatologist and administration of antiviral therapy should be considered if hepatitis B antigen is detectable. The risk of reactivation of hepatitis C is not well defined. The use of live vaccines, including those against herpes zoster, is not recommended during rituximab therapy secondary to the risk of causing an active infection. Rituximab-treated patients are also at risk for other viral infections, including cytomegalovirus, herpes simplex, parvovirus B19, and West Nile virus.
Late-onset neutropenia has been described as a possible complication of adding rituximab to chemotherapy.59 In a retrospective review, patients who received chemotherapy plus rituximab for CD20+, B-cell NHL had a higher rate of late-onset neutropenia compared with historical controls receiving chemotherapy alone.
A study published in 2009 reported 57 cases of progressive multifocal leukoencephalopathy (PML) following the administration of rituximab, usually with additional therapy, in HIV-negative patients.60 PML, a viral infection that affects the white matter of the brain, is usually fatal. This cohort of patients was treated with a median of six doses of rituximab. The median time from last rituximab dose to PML diagnosis was 5.5 months, and median survival after the diagnosis of PML was two months. In accordance with these data, the FDA issued a boxed (black-box) warning.
Reversible posterior leukoencephalopathy (RPLE), a subacute neurological syndrome manifested as headaches, cortical blindness, and seizures with a characteristic appearance on magnetic resonance imaging (MRI), has also been described in rare cases.61,62 It is not clear whether these events are directly related to rituximab, because most of these patients have received multiple therapies, but RPLE has also been reported after other antibody and small-molecule therapeutics. Cardiac arrhythmias, renal toxicity, and bowel obstruction with perforation have also been reported.57
Rituximab induces B-cell depletion, which may compromise the immune system; however, recovery of the normal B-cell population usually occurs six to nine months after discontinuation of therapy.15 Despite this depletion, rituximab has not been definitively shown to cause a significant decrease in circulating immunoglobulin levels, although this may occur with more prolonged maintenance strategies. Stable immunoglobulin levels are likely to reflect that plasma cells are long-lived and do not express CD20.
In a prospective study, van der Kolk et al. investigated the effect of rituximab on the humoral immune response to two primary antigens and two recall antigens.63 After rituximab treatment, the humoral immune response to the recall antigens was significantly decreased when compared with the response before treatment.
FUTURE DIRECTIONS
Attempts to improve upon rituximab have focused on antibody engineering, including humanized instead of chimeric antibodies, stronger binding affinity for CD20, or enhancing effector functions such as antibody-dependent, cell-mediated cytotoxicity (ADCC) or complement activation. Ofatumumab (Arzerra, GlaxoSmithKline) is a humanized monoclonal anti-CD20 antibody that targets a small loop epitope of CD20. Compared with rituximab, in the laboratory it delivers stronger complement-dependent cytotoxicity, even in lymphoma cells with low expression of CD20. Approved by the FDA in October 2009 for the treatment of fludarabine and alemtuzumab–refractory chronic lymphocytic leukemia (CLL), the drug also showed activity in relapsed/refractory FL.64,65
Additional humanized antibodies under development include some with enhanced ADCC, stronger binding to low-affinity polymorphisms of FcgRIII, or targeting other epitopes on the CD20 molecule. Whether these agents are more effective, less immunogenic, or faster to infuse with fewer infusion reactions resulting may be difficult to determine.
Other proteins on the surface of B cells are also potential antibody targets. CD22 has a pattern of expression similar to that of CD20 on normal and malignant B lymphocytes, and it is targeted by epratuzumab (UCB/Immunomedics).66,67 Because CD22 is internalized upon antibody binding, it might be better suited for delivering toxins inside CD22+ cells. Examples of this approach include inotuzumab ozogamicin (CMC-544, Wyeth), an anti-CD22 immunoconjugate with the antitumor antibiotic calicheamicin, and CAT-3888 (Cambridge Antibody Technology), formerly called BL22, which uses a Pseudomonas exotoxin fragment.68,69
CONCLUSION
Rituximab (Rituxan) has changed the treatment paradigms and outcomes for all CD20+ NHL and represents arguably the most noteworthy advance in lymphoma treatment over the past decade. In patients with NHL, the addition of rituximab to standard treatment significantly enhanced response to therapy and overall outcomes. Rituximab is currently approved for treatment of relapsed and refractory indolent lymphomas as single-agent therapy and as initial therapy in combination with standard chemotherapy regimens. In patients with DLBCL, it is approved for use as initial therapy with CHOP or other anthracycline-based chemotherapy. The drug was also recently approved for use with chemotherapy in previously treated and untreated patients with CLL.
Benefits have been sustained among all age groups, and the drug has been safe and well tolerated in elderly patients as well. Overall survival of patients with NHL has improved over the last two decades. While some of this improvement may stem from earlier or more precise diagnosis and better supportive care, the results of many trials reviewed in this article indicate significant improvement in outcomes with the addition of rituximab to the therapeutic armamentarium.
Despite these advances, questions remain, mainly in the field of indolent lymphomas. More research is under way to establish the optimal schedule, timing, and duration for maintenance rituximab. Reports of clinical trials demonstrating longer follow-up of indolent lymphoma are eagerly awaited in an attempt to clarify the effect of rituximab on overall survival.
Rituximab represents a paradigm shift in treatment of B-cell NHL; it marks the beginning of a new age of targeted therapies in oncology, being the first approved therapeutic monoclonal antibody for cancer. In the years to come, we anticipate more clinical trials combining rituximab with targeted treatments that might further improve outcomes while minimizing toxicity.
REFERENCES
1. Jemal A, Siegel R, Ward E. Cancer statistics, 2008. CA Cancer J Clin.2008;58(2):71–96. [PubMed]
2. Rudiger T, Muller-Hermelink HK. WHO classification of malignant lymphomas. Radiologe. 2002;42(12):936–942. [PubMed]
3. Harris NL, Jaffe ES, Diebold J, et al. The World Health Organization Classification of Neoplastic Diseases of the Hematopoietic and Lymphoid Tissues. Report of the Clinical Advisory Committee meeting. Airlie House, Virginia, November 1997; Ann Oncol; 1999. pp. 1419–1432. [PubMed]
4. Armitage JO, Weisenburger DD. New approach to classifying non-Hodgkin’s lymphomas: Clinical features of the major histologic subtypes. Non-Hodgkin’s Lymphoma Classification Project. J Clin Oncol.1998;16(8):2780–2795. [PubMed]
5. Tedder TF, Engel P. CD20: A regulator of cell–cycle progression of B lymphocytes. Immunol Today. 1994;15(9):450–454. [PubMed]
6. Silverman GJ, Weisman S. Rituximab therapy and autoimmune disorders: Prospects for anti-B cell therapy. Arthritis Rheum. 2003;48(6):1484–1492.[PubMed]
7. Shan D, Ledbetter JA, Press OW. Apoptosis of malignant human B cells by ligation of CD20 with monoclonal antibodies. Blood. 1998;91(5):1644–1652. [PubMed]
8. Reff ME, Carner K, Chambers KS, et al. Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20. Blood.1994;83(2):435–445. [PubMed]
9. Golay J, Zaffaroni L, Vaccari T, et al. Biologic response of B lymphoma cells to anti-CD20 monoclonal antibody rituximab in vitro: CD55 and CD59 regulate complement-mediated cell lysis. Blood. 2000;95(12):3900–3908.[PubMed]
10. Clynes RA, Towers TL, Presta LG, Ravetch JV. Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets. Nat Med.2000;6(4):443–446. [PubMed]
11. Alas S, Bonavida B. Rituximab inactivates signal transducer and activation of transcription 3 (STAT3) activity in B–non-Hodgkin’s lymphoma through inhibition of the interleukin 10 autocrine/paracrine loop and results in down-regulation of Bcl-2 and sensitization to cytotoxic drugs.Cancer Res. 2001;61(13):5137–5144. [PubMed]
12. Maloney DG, Liles TM, Czerwinski DK, et al. Phase I clinical trial using escalating single-dose infusion of chimeric anti-CD20 monoclonal antibody (Idec-C2B8) in patients with recurrent B-cell lymphoma. Blood.1994;84(8):2457–2466. [PubMed]
13. Horning SJ, Rosenberg SA. The natural history of initially untreated low-grade non-Hodgkin’s lymphomas. N Engl J Med. 1984;311(23):1471–1475. [PubMed]
14. Advani R, Rosenberg SA, Horning SJ. Stage I and II follicular non-Hodgkin’s lymphoma: Long-term follow-up of no initial therapy. J Clin Oncol. 2004;22(8):1454–1459. [PubMed]
15. McLaughlin P, Grillo-Lopez AJ, Link BK, et al. Rituximab chimeric anti-CD20 monoclonal antibody therapy for relapsed indolent lymphoma: Half of patients respond to a four-dose treatment program. J Clin Oncol.1998;16(8):2825–2833. [PubMed]
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
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
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.
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
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:
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)
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
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.
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
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.”
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.
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.
In a clinical trial, CRISPR editing of T cells to fight lung cancer was feasible and safe.
Lung cancer is the most prevalent and fatal malignancy worldwide. Whereas the overall five-year cancer survival rates in the United States increased by 16.7% over the past five decades, lung cancer five-year survival rates only increased by 5.9%. Immune checkpoint inhibitors, such as antibodies against programmed cell death protein 1 (PD-1), are front-line anticancer treatments because PD-1 ligand on tumor cells efficiently inhibits immune responses.
Lu et al. published the results of a clinical trial, which aimed at knocking out PD-1 on T cells to confer antitumor activity against metastatic non–small cell lung cancer. The T cells were expanded ex vivo, then reinfused into 12 patients. The primary aim of the trial was to test feasibility and safety, and the secondary aims included efficiency and tracking of edited T cells. Overall, the treatment was well tolerated, with only mild to moderate adverse effects and no dose-limiting toxicities. Considerable care was taken to analyze clustered regularly interspaced short palindromic repeats (CRISPR)–induced off-target effects, and the study showed that median mutation frequency was 0.05% in off-target sites. Edited T cells were detected in the peripheral blood of 11 out of 12 patients and in tumor biopsy specimens from one patient up to 52 weeks after infusion. After infusion, two patients had stable disease, and it lasted up to 76 weeks in one patient.
This trial was performed cautiously to avoid major issues with off-target effects, and the researchers used T cells with relatively low editing rates (median of 5.8%). Nevertheless, the authors observed signs of biological activity in patients with metastatic disease who had failed several other lines of treatment. To improve biological efficiency, it will be critical to increase editing rates without increasing off-target effects. Although the off-target effects were close to background level in this study, genome-wide unbiased screens will be necessary, particularly when editing rates are boosted. A separate problem to address before broader clinical translation is T cell expansion, which failed in five patients in this study.
These early data suggest that CRISPR editing of T cells in humans is feasible and safe.Several other trials are ongoing and should help assess the potential benefit of ex vivo edited immune cells. With CRISPR-edited immune cells, it may be possible to get a better grip on metastatic lung cancer and other malignancies.
Lu, J. Xue, T. Deng, X. Zhou, K. Yu, L. Deng, M. Huang, X. Yi, M. Liang, Y. Wang, H. Shen, R. Tong, W. Wang, L. Li, J. Song, J. Li, X. Su, Z. Ding, Y. Gong, J. Zhu, Y. Wang, B. Zou, Y. Zhang, Y. Li, L. Zhou, Y. Liu, M. Yu, Y. Wang, X. Zhang, L. Yin, X. Xia, Y. Zeng, Q. Zhou, B. Ying, C. Chen, Y. Wei, W. Li, T. Mok, Safety and feasibility of CRISPR-edited T cells in patients with refractory non-small-cell lung cancer. Nat. Med. 26, 732–740 (2020).
Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 editing of immune checkpoint genes could improve the efficacy of T cell therapy, but the first necessary undertaking is to understand the safety and feasibility. Here, we report results from a first-in-human phase I clinical trial of CRISPR–Cas9 PD-1-edited T cells in patients with advanced non-small-cell lung cancer (ClinicalTrials.gov NCT02793856). Primary endpoints were safety and feasibility, and the secondary endpoint was efficacy. The exploratory objectives included tracking of edited T cells. All prespecified endpoints were met. PD-1-edited T cells were manufactured ex vivo by cotransfection using electroporation of Cas9 and single guide RNA plasmids. A total of 22 patients were enrolled; 17 had sufficient edited T cells for infusion, and 12 were able to receive treatment. All treatment-related adverse events were grade 1/2. Edited T cells were detectable in peripheral blood after infusion. The median progression-free survival was 7.7 weeks (95% confidence interval, 6.9 to 8.5 weeks) and median overall survival was 42.6 weeks (95% confidence interval, 10.3–74.9 weeks). The median mutation frequency of off-target events was 0.05% (range, 0–0.25%) at 18 candidate sites by next generation sequencing. We conclude that clinical application of CRISPR–Cas9 gene-edited T cells is generally safe and feasible. Future trials should use superior gene editing approaches to improve therapeutic efficacy.
BY EDWARD A. STADTMAUER, JOSEPH A. FRAIETTA, MEGAN M. DAVIS, ADAM D. COHEN, KRISTY L. WEBER, ERIC LANCASTER, PATRICIA A. MANGAN, IRINA KULIKOVSKAYA, MINNAL GUPTA, FANG CHEN, LIFENG TIAN, VANESSA E. GONZALEZ, JUN XU, IN-YOUNG JUNG, J. JOSEPH MELENHORST, GABRIELA PLESA, JOANNE SHEA, TINA MATLAWSKI, AMANDA CERVINI, AVERY L. GAYMON, STEPHANIE DESJARDINS, ANNE LAMONTAGNE, JANUARY SALAS-MCKEE, ANDREW FESNAK, DONALD L. SIEGEL, BRUCE L. LEVINE, JULIE K. JADLOWSKY, REGINA M. YOUNG, ANNE CHEW, WEI-TING HWANG, ELIZABETH O. HEXNER, BEATRIZ M. CARRENO, CHRISTOPHER L. NOBLES, FREDERIC D. BUSHMAN, KEVIN R. PARKER, YANYAN QI, ANSUMAN T. SATPATHY, HOWARD Y. CHANG, YANGBING ZHAO, SIMON F. LACEY, CARL H. JUNE
CRISPR-Cas9 is a revolutionary gene-editing technology that offers the potential to treat diseases such as cancer, but the effects of CRISPR in patients are currently unknown. Stadtmauer et al. report a phase 1 clinical trial to assess the safety and feasibility of CRISPR-Cas9 gene editing in three patients with advanced cancer (see the Perspective by Hamilton and Doudna). They removed immune cells called T lymphocytes from patients and used CRISPR-Cas9 to disrupt three genes (TRAC, TRBC, and PDCD1) with the goal of improving antitumor immunity. A cancer-targeting transgene, NY-ESO-1, was also introduced to recognize tumors. The engineered cells were administered to patients and were well tolerated, with durable engraftment observed for the study duration. These encouraging observations pave the way for future trials to study CRISPR-engineered cancer immunotherapies.
Structured Abstract
INTRODUCTION
Most cancers are recognized and attacked by the immune system but can progress owing to tumor-mediated immunosuppression and immune evasion mechanisms. The infusion of ex vivo engineered T cells, termed adoptive T cell therapy, can increase the natural antitumor immune response of the patient. Gene therapy to redirect immune specificity combined with genome editing has the potential to improve the efficacy and increase the safety of engineered T cells. CRISPR coupled with CRISPR-associated protein 9 (Cas9) endonuclease is a powerful gene-editing technology that potentially allows the ability to target multiple genes in T cells to improve cancer immunotherapy.
RATIONALE
Our first-in-human, phase 1 clinical trial (clinicaltrials.gov; trial NCT03399448) was designed to test the safety and feasibility of multiplex CRISPR-Cas9 gene editing of T cells from patients with advanced, refractory cancer. A limitation of adoptively transferred T cell efficacy has been the induction of T cell dysfunction or exhaustion. We hypothesized that removing the endogenous T cell receptor (TCR) and the immune checkpoint molecule programmed cell death protein 1 (PD-1) would improve the function and persistence of engineered T cells. In addition, the removal of PD-1 has the potential to improve safety and reduce toxicity that can be caused by autoimmunity. A synthetic, cancer-specific TCR transgene (NY-ESO-1) was also introduced to recognize tumor cells. In vivo tracking and persistence of the engineered T cells were monitored to determine if the cells could persist after CRISPR-Cas9 modifications.
RESULTS
Four cell products were manufactured at clinical scale, and three patients (two with advanced refractory myeloma and one with metastatic sarcoma) were infused. The editing efficiency was consistent in all four products and varied as a function of the single guide RNA (sgRNA), with highest efficiency observed for the TCR α chain gene (TRAC) and lowest efficiency for the TCR β chain gene (TRBC). The mutations induced by CRISPR-Cas9 were highly specific for the targeted loci; however, rare off-target edits were observed. Single-cell RNA sequencing of the infused CRISPR-engineered T cells revealed that ~30% of cells had no detectable mutations, whereas ~40% had a single mutation and ~20 and ~10% of the engineered T cells were double mutated and triple mutated, respectively, at the target sequences. The edited T cells engrafted in all three patients at stable levels for at least 9 months. The persistence of the T cells expressing the engineered TCR was much more durable than in three previous clinical trials during which T cells were infused that retained expression of the endogenous TCR and endogenous PD-1. There were no clinical toxicities associated with the engineered T cells. Chromosomal translocations were observed in vitro during cell manufacturing, and these decreased over time after infusion into patients. Biopsies of bone marrow and tumor showed trafficking of T cells to the sites of tumor in all three patients. Although tumor biopsies revealed residual tumor, in both patients with myeloma, there was a reduction in the target antigens NY-ESO-1 and/or LAGE-1. This result is consistent with an on-target effect of the engineered T cells, resulting in tumor evasion.
CONCLUSION
Preliminary results from this pilot trial demonstrate that multiplex human genome engineering is safe and feasible using CRISPR-Cas9. The extended persistence of the engineered T cells indicates that preexisting immune responses to Cas9 do not appear to present a barrier to the implementation of this promising technology.
CRISPR-Cas9 engineering of T cells in cancer patients. T cells were isolated from the blood of a patient with cancer. CRISPR-Cas9 rebonuclear protein complexes loaded with three sgRNAs were electroporated into the normal T cells, resulting in gene editing of the TRAC, TRBC1, TRBC2, and PDCD1 (encoding PD-1) loci. The cells were then transduced with a lentiviral vector to express a TCR specific for the cancer-testis antigens NY-ESO-1 and LAGE-1 (right). The engineered T cells were then returned to the patient by intravenous infusion, and patients monitored for safety and efficacy. Taken from Science 28 Feb 2020:Vol. 367, Issue 6481, eaba7365 DOI: 10.1126/science.aba7365
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 (1). Although successful when directed against leukemia-associated antigens such as CD19 (2, 3), CAR-T cell therapy for solid tumors has been less effective, with best responses in patients with minimal disease (4, 5). Solid tumors recruit inhibitory cells such as myeloid-derived suppressor cells (MDSCs) (6). These immature myeloid cells are a component of innate immunity and strengthen the suppressive TME (7, 8). The frequency of circulating or intra-tumoral MDSCs correlates with cancer stage, disease progression, and resistance to standard chemo- and radio-therapies (9). 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 (10). 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 (11, 12). 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 (13). NKG2D ligands are overexpressed on several solid tumors and on tumor-infiltrating MDSCs (14). 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 (15), 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 (16). 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.
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 (17). 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 (18) kindly provided by Dr. Charles L. Sentman (Dartmouth Geisel School of Medicine, Hanover, NH, USA) into pSFG.IRES.tCD19 (19). RD114-speudotyped viral particles were produced by transient transfection in 293T cells, as previously described (20).
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 (21). 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 (22). 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.
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 (22). 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 (23), 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 (24). 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 (25).
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 (26). 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 3×105ex 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 (27). 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 (28). 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 (29). 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.
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 (15, 30) 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.
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 (9, 31). 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 (13). The neuroblastoma cell line, LAN-1, subsequently used in our in vivo TME model, did not express NKG2D ligands.
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 (32).
Previous studies have reported that expression of chimeric NKG2D constructs in T lymphocytes can direct these cells to target NKG2D ligand-expressing tumors (16, 33). However, activated T cells (ATCs) themselves upregulate NKG2D ligands (34), 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 (35). 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.
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.
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.
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.
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 (36, 37), 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 (8). 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 (38) or expression of checkpoint molecules (39); induce MDSC differentiation such as with all-trans retinoic acid (40); or eliminate MDSCs such as with the cytotoxic agents doxorubicin or cyclophosphamide (41). 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 (16, 33, 34). Using our standard T-cell activation/expansion protocol (22), 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 (42). TGFβ1 and soluble NKG2D ligands both decrease DAP10 gene transcription and protein activity, and thus reduce NKG2D function in endogenous NK cells (43, 44). 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 (45). 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 (46). 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 (47), 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 (48, 49), 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 (50), 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.
Other posts on this site on Immunotherapy and Cancer include
The most widely used therapies are combinations of chemotherapyand radiation therapy.
Biological therapy, which targets key features of the lymphoma cells, is used in many cases nowadays.
The goal of medical therapy in lymphoma is complete remission. This means that all signs of the disease have disappeared after treatment. Remission is not the same as cure. In remission, one may still have lymphoma cells in the body, but they are undetectable and cause no symptoms.
When in remission, the lymphoma may come back. This is called recurrence.
The duration of remission depends on the type, stage, and grade of the lymphoma. A remission may last a few months, a few years, or may continue throughout one’s life.
Remission that lasts a long time is called durable remission, and this is the goal of therapy.
The duration of remission is a good indicator of the aggressiveness of the lymphoma and of the prognosis. A longer remission generally indicates a better prognosis.
Remission can also be partial. This means that the tumor shrinks after treatment to less than half its size before treatment.
The following terms are used to describe the lymphoma’s response to treatment:
Improvement: The lymphoma shrinks but is still greater than half its original size.
Stable disease: The lymphoma stays the same.
Progression: The lymphoma worsens during treatment.
Refractory disease: The lymphoma is resistant to treatment.
The following terms to refer to therapy:
Induction therapy is designed to induce a remission.
If this treatment does not induce a complete remission, new or different therapy will be initiated. This is usually referred to as salvage therapy.
Once in remission, one may be given yet another treatment to prevent recurrence. This is called maintenance therapy.
Chemotherapy
Many different types of chemotherapy may be used for Hodgkin lymphoma. The most commonly used combination of drugs in the United States is called ABVD. Another combination of drugs, known as BEACOPP, is now widely used in Europe and is being used more often in the United States. There are other combinations that are less commonly used and not listed here. The drugs that make up these two more common combinations of chemotherapy are listed below.
ABVD: Doxorubicin (Adriamycin), bleomycin (Blenoxane), vinblastine (Velban, Velsar), and dacarbazine (DTIC-Dome). ABVD chemotherapy is usually given every two weeks for two to eight months.
BEACOPP: Bleomycin, etoposide (Toposar, VePesid), doxorubicin, cyclophosphamide (Cytoxan, Neosar), vincristine (Vincasar PFS, Oncovin), procarbazine (Matulane), and prednisone (multiple brand names). There are several different treatment schedules, but different drugs are usually given every two weeks.
The type of chemotherapy, number of cycles of chemotherapy, and the additional use of radiation therapy are based on the stage of the Hodgkin lymphoma and the type and number of prognostic factors.
Adult non-Hodgkin lymphoma is a disease in which malignant (cancer) cells form in the lymph system.
Because lymph tissue is found throughout the body, adult non-Hodgkin lymphoma can begin in almost any part of the body. Cancer can spread to the liver and many other organs and tissues.
Non-Hodgkin lymphoma in pregnant women is the same as the disease in nonpregnant women of childbearing age. However, treatment is different for pregnant women. This summary includes information on the treatment of non-Hodgkin lymphoma during pregnancy
Non-Hodgkin lymphoma can occur in both adults and children. Treatment for children, however, is different than treatment for adults. (See the PDQ summary on Childhood Non-Hodgkin Lymphoma Treatment for more information.)
There are many different types of lymphoma.
Lymphomas are divided into two general types: Hodgkin lymphoma and non-Hodgkin lymphoma. This summary is about the treatment of adult non-Hodgkin lymphoma. For information about other types of lymphoma, see the following PDQ summaries:
Age, gender, and a weakened immune system can affect the risk of adult non-Hodgkin lymphoma.
If cancer is found, the following tests may be done to study the cancer cells:
Immunohistochemistry: A test that uses antibodies to check for certain antigens in a sample of tissue. The antibody is usually linked to a radioactive substance or a dye that causes the tissue to light up under a microscope. This type of test may be used to tell the difference between different types of cancer.
Cytogenetic analysis: A laboratory test in which cells in a sample of tissue are viewed under a microscope to look for certain changes in the chromosomes.
Immunophenotyping: A process used to identify cells, based on the types of antigens ormarkers on the surface of the cell. This process is used to diagnose specific types of leukemia and lymphoma by comparing the cancer cells to normal cells of the immune system.
Certain factors affect prognosis (chance of recovery) and treatment options.
The prognosis (chance of recovery) and treatment options depend on the following:
The stage of the cancer.
The type of non-Hodgkin lymphoma.
The amount of lactate dehydrogenase (LDH) in the blood.
The amount of beta-2-microglobulin in the blood (for Waldenström macroglobulinemia).
The patient’s age and general health.
Whether the lymphoma has just been diagnosed or has recurred (come back).
Stages of adult non-Hodgkin lymphoma may include E and S.
Adult non-Hodgkin lymphoma may be described as follows:
E: “E” stands for extranodal and means the cancer is found in an area or organ other than the lymph nodes or has spread to tissues beyond, but near, the major lymphatic areas.
S: “S” stands for spleen and means the cancer is found in the spleen.
Stage I adult non-Hodgkin lymphoma is divided into stage I and stage IE.
Stage I: Cancer is found in one lymphatic area (lymph node group, tonsils and nearby tissue, thymus, or spleen).
Stage IE: Cancer is found in one organ or area outside the lymph nodes.
Stage II adult non-Hodgkin lymphoma is divided into stage II and stage IIE.
Stage II: Cancer is found in two or more lymph node groups either above or below the diaphragm (the thin muscle below the lungs that helps breathing and separates the chest from the abdomen).
Stage IIE: Cancer is found in one or more lymph node groups either above or below the diaphragm. Cancer is also found outside the lymph nodes in one organ or area on the same side of the diaphragm as the affected lymph nodes.
Stage III adult non-Hodgkin lymphoma is divided into stage III, stage IIIE, stage IIIS, and stage IIIE+S.
Stage III: Cancer is found in lymph node groups above and below the diaphragm (the thin muscle below the lungs that helps breathing and separates the chest from the abdomen).
Stage IIIE: Cancer is found in lymph node groups above and below the diaphragm and outside the lymph nodes in a nearby organ or area.
Stage IIIS: Cancer is found in lymph node groups above and below the diaphragm, and in the spleen.
Stage IIIE+S: Cancer is found in lymph node groups above and below the diaphragm, outside the lymph nodes in a nearby organ or area, and in the spleen.
In stage IV adult non-Hodgkin lymphoma, the cancer:
is found throughout one or more organs that are not part of a lymphatic area (lymph node group, tonsils and nearby tissue, thymus, or spleen), and may be in lymph nodes near those organs; or
is found in one organ that is not part of a lymphatic area and has spread to organs or lymph nodes far away from that organ; or
is found in the liver, bone marrow, cerebrospinal fluid (CSF), or lungs (other than cancer that has spread to the lungs from nearby areas).
Adult non-Hodgkin lymphomas are also described based on how fast they grow and where the affected lymph nodes are in the body. Indolent & aggressive.
The treatment plan depends mainly on the following:
The type of non-Hodgkin’s lymphoma
Its stage (where the lymphoma is found)
How quickly the cancer is growing
The patient’s age
Whether the patient has other health problems
If there are symptoms present such as fever and night sweats (see above)
Radiation therapy, also called radiotherapy or irradiation, can be used to treat leukemia, lymphoma, myeloma and myelodysplastic syndromes. The type of radiation used for radiotherapy (ionizing radiation) is the same that’s used for diagnostic x-rays. Radiotherapy, however, is given in higher doses.
Radiotherapy works by damaging the genetic material (DNA) within cells, which prevents them from growing and reproducing. Although the radiotherapy is directed at cancer cells, it can also damage nearby healthy cells. However, current methods of radiotherapy have been improved upon, minimizing “scatter” to nearby tissues. Therefore its benefit (destroying the cancer cells) outweighs its risk (harming healthy cells).
When radiotherapy is used for blood cancer treatment, it’s usually part of a treatment plan that includes drug therapy. Radiotherapy can also be used to relieve pain or discomfort caused by an enlarged liver, lymph node(s) or spleen.
Radiotherapy, either alone or with chemotherapy, is sometimes given as conditioning treatment to prepare a patient for a blood or marrow stem cell transplant. The most common types used to treat blood cancer are external beam radiation (see below) and radioimmunotherapy.
External Beam Radiation
External beam radiation is the type of radiotherapy used most often for people with blood cancers. A focused radiation beam is delivered outside the body by a machine called a linear accelerator, or linac for short. The linear accelerator moves around the body to deliver radiation from various angles. Linear accelerators make it possible to decrease or avoid skin reactions and deliver targeted radiation to lessen “scatter” of radiation to nearby tissues.
The dose (total amount) of radiation used during treatment depends on various factors regarding the patient, disease and reason for treatment, and is established by a radiation oncologist. You may receive radiotherapy during a series of visits, spread over several weeks (from two to 10 weeks, on average). This approach, called dose fractionation, lessens side effects. External beam radiation does not make you radioactive.
Autologous bone marrow transplant: The term auto means self. Stem cells are removed from you before you receive high-dose chemotherapy or radiation treatment. The stem cells are stored in a freezer (cryopreservation). After high-dose chemotherapy or radiation treatments, your stems cells are put back in your body to make (regenerate) normal blood cells. This is called a rescue transplant.
Allogeneic bone marrow transplant: The term allo means other. Stem cells are removed from another person, called a donor. Most times, the donor’s genes must at least partly match your genes. Special blood tests are done to see if a donor is a good match for you. A brother or sister is most likely to be a good match. Sometimes parents, children, and other relatives are good matches. Donors who are not related to you may be found through national bone marrow registries.
Umbilical cord blood transplant: This is a type of allogeneic transplant. Stem cells are removed from a newborn baby’s umbilical cord right after birth. The stem cells are frozen and stored until they are needed for a transplant. Umbilical cord blood cells are very immature so there is less of a need for matching. But blood counts take much longer to recover.
Before the transplant, chemotherapy, radiation, or both may be given. This may be done in two ways:
Ablative (myeloablative) treatment: High-dose chemotherapy, radiation, or both are given to kill any cancer cells. This also kills all healthy bone marrow that remains, and allows new stem cells to grow in the bone marrow.
Reduced intensity treatment, also called a mini transplant: Patients receive lower doses of chemotherapy and radiation before a transplant. This allows older patients, and those with other health problems to have a transplant.
A stem cell transplant is usually done after chemotherapy and radiation is complete. The stem cells are delivered into your bloodstream usually through a tube called a central venous catheter. The process is similar to getting a blood transfusion. The stem cells travel through the blood into the bone marrow. Most times, no surgery is needed.
Donor stem cells can be collected in two ways:
Bone marrow harvest. This minor surgery is done under general anesthesia. This means the donor will be asleep and pain-free during the procedure. The bone marrow is removed from the back of both hip bones. The amount of marrow removed depends on the weight of the person who is receiving it.
Leukapheresis. First, the donor is given 5 days of shots to help stem cells move from the bone marrow into the blood. During leukapheresis, blood is removed from the donor through an IV line in a vein. The part of white blood cells that contains stem cells is then separated in a machine and removed to be later given to the recipient. The red blood cells are returned to the donor.
Why the Procedure is Performed
A bone marrow transplant replaces bone marrow that either is not working properly or has been destroyed (ablated) by chemotherapy or radiation. Doctors believe that for many cancers, the donor’s white blood cells can attach to any remaining cancer cells, similar to when white cells attach to bacteria or viruses when fighting an infection.
Your doctor may recommend a bone marrow transplant if you have:
Certain cancers, such as leukemia, lymphoma, and multiple myeloma
A disease that affects the production of bone marrow cells, such as aplastic anemia, congenital neutropenia, severe immunodeficiency syndromes, sickle cell anemia, thalassemia
Had chemotherapy that destroyed your bone
2.5.3 Autologous stem cell transplantation
Phase II trial of 131I-B1 (anti-CD20) antibody therapy with autologous stem cell transplantation for relapsed B cell lymphomas
25 patients with relapsed B-cell lymphomas were evaluated with trace-labelled doses (2·5 mg/kg, 185-370 MBq [5-10 mCi]) of 131I-labelled anti-CD20 (B1) antibody in a phase II trial. 22 patients achieved 131I-B1 biodistributions delivering higher doses of radiation to tumor sites than to normal organs and 21 of these were treated with therapeutic infusions of 131I-B1 (12·765-29·045 GBq) followed by autologous hemopoietic stem cell reinfusion. 18 of the 21 treated patients had objective responses, including 16 complete remissions. One patient died of progressive lymphoma and one died of sepsis. Analysis of our phase I and II trials with 131I-labelled B1 reveal a progression-free survival of 62% and an overall survival of 93% with a median follow-up of 2 years. 131I-anti-CD20 (B1) antibody therapy produces complete responses of long duration in most patients with relapsed B-cell lymphomas when given at maximally tolerated doses with autologous stem cell rescue.
An autologous transplant (or rescue) is a type of transplant that uses the person’s own stem cells. These cells are collected in advance and returned at a later stage. They are used to replace stem cells that have been damaged by high doses of chemotherapy, used to treat the person’s underlying disease.
In most cases, stem cells are collected directly from the bloodstream. While stem cells normally live in your marrow, a combination of chemotherapy and a growth factor (a drug that stimulates stem cells) called Granulocyte Colony Stimulating Factor (G-CSF) is used to expand the number of stem cells in the marrow and cause them to spill out into the circulating blood. From here they can be collected from a vein by passing the blood through a special machine called a cell separator, in a process similar to dialysis.
Most of the side effects of an autologous transplant are caused by the conditioning therapy used. Although they can be very unpleasant at times it is important to remember that most of them are temporary and reversible.
Procedure of Hematopoietic Stem Cell Transplantation
Hematopoietic stem cell transplantation (HSCT) is the transplantation of multipotent hematopoietic stem cells, usually derived from bone marrow, peripheral blood, or umbilical cord blood. It may be autologous (the patient’s own stem cells are used) or allogeneic (the stem cells come from a donor).
Hematopoietic stem cell transplantation (HSCT) involves the intravenous (IV) infusion of autologous or allogeneic stem cells to reestablish hematopoietic function in patients whose bone marrow or immune system is damaged or defective.
The image below illustrates an algorithm for typically preferred hematopoietic stem cell transplantation cell source for treatment of malignancy.
An algorithm for typically preferred hematopoietic stem cell transplantation cell source for treatment of malignancy: If a matched sibling donor is not available, then a MUD is selected; if a MUD is not available, then choices include a mismatched unrelated donor, umbilical cord donor(s), and a haploidentical donor.
Supportive Therapies
2.5.4 Blood transfusions – risks and complications of a blood transfusion
Allogeneic transfusion reaction (acute or delayed hemolytic reaction)
Allergic reaction
Viruses Infectious Diseases
The risk of catching a virus from a blood transfusion is very low.
HIV. Your risk of getting HIV from a blood transfusion is lower than your risk of getting killed by lightning. Only about 1 in 2 million donations might carry HIV and transmit HIV if given to a patient.
Hepatitis B and C. The risk of having a donation that carries hepatitis B is about 1 in 205,000. The risk for hepatitis C is 1 in 2 million. If you receive blood during a transfusion that contains hepatitis, you’ll likely develop the virus.
Variant Creutzfeldt-Jakob disease (vCJD). This disease is the human version of Mad Cow Disease. It’s a very rare, yet fatal brain disorder. There is a possible risk of getting vCJD from a blood transfusion, although the risk is very low. Because of this, people who may have been exposed to vCJD aren’t eligible blood donors.
Fever
Iron Overload
Lung Injury
Graft-Versus-Host Disease
Graft-versus-host disease (GVHD) is a condition in which white blood cells in the new blood attack your tissues.
Also called hematopoietin or hemopoietin, it is produced by interstitial fibroblasts in the kidney in close association with peritubular capillary and proximal convoluted tubule. It is also produced in perisinusoidal cells in the liver. While liver production predominates in the fetal and perinatal period, renal production is predominant during adulthood. In addition to erythropoiesis, erythropoietin also has other known biological functions. For example, it plays an important role in the brain’s response to neuronal injury.[1] EPO is also involved in the wound healing process.[2]
Granulocyte-colony stimulating factor (G-CSF or GCSF), also known as colony-stimulating factor 3 (CSF 3), is a glycoprotein that stimulates the bone marrow to produce granulocytes and stem cells and release them into the bloodstream.
Plasmapheresis is a term used to refer to a broad range of procedures in which extracorporeal separation of blood components results in a filtered plasma product.[1, 2] The filtering of plasma from whole blood can be accomplished via centrifugation or semipermeable membranes.[3] Centrifugation takes advantage of the different specific gravities inherent to various blood products such as red cells, white cells, platelets, and plasma.[4] Membrane plasma separation uses differences in particle size to filter plasma from the cellular components of blood.[3]
Traditionally, in the United States, most plasmapheresis takes place using automated centrifuge-based technology.[5] In certain instances, in particular in patients already undergoing hemodialysis, plasmapheresis can be carried out using semipermeable membranes to filter plasma.[4]
In therapeutic plasma exchange, using an automated centrifuge, filtered plasma is discarded and red blood cells along with replacement colloid such as donor plasma or albumin is returned to the patient. In membrane plasma filtration, secondary membrane plasma fractionation can selectively remove undesired macromolecules, which then allows for return of the processed plasma to the patient instead of donor plasma or albumin. Examples of secondary membrane plasma fractionation include cascade filtration,[6] thermofiltration, cryofiltration,[7] and low-density lipoprotein pheresis.
The Apheresis Applications Committee of the American Society for Apheresis periodically evaluates potential indications for apheresis and categorizes them from I to IV based on the available medical literature. The following are some of the indications, and their categorization, from the society’s 2010 guidelines.[2]
The only Category I indication for hemopoietic malignancy is Hyperviscosity in monoclonal gammopathies
2.5.8 Platelet Transfusions
Indications for platelet transfusion in children with acute leukemia
In an attempt to determine the indications for platelet transfusion in thrombocytopenic patients, we randomized 56 children with acute leukemia to one of two regimens of platelet transfusion. The prophylactic group received platelets when the platelet count fell below 20,000 per mm3 irrespective of clinical events. The therapeutic group was transfused only when significant bleeding occurred and not for thrombocytopenia alone. The time to first bleeding episode was significantly longer and the number of bleeding episodes were significantly reduced in the prophylactic group. The survival curves of the two groups could not be distinguished from each other. Prior to the last month of life, the total number of days on which bleeding was present was significantly reduced by prophylactic therapy. However, in the terminal phase (last month of life), the duration of bleeding episodes was significantly longer in the prophylactic group. This may have been due to a higher incidence of immunologic refractoriness to platelet transfusion. Because of this terminal bleeding, comparison of the two groups for total number of days on which bleeding was present did not show a significant difference over the entire study period.
INTRODUCTION — Hemostasis depends on an adequate number of functional platelets, together with an intact coagulation (clotting factor) system. This topic covers the logistics of platelet use and the indications for platelet transfusion in adults. The approach to the bleeding patient, refractoriness to platelet transfusion, and platelet transfusion in neonates are discussed elsewhere.
Pooled Platelets – A single unit of platelets can be isolated from every unit of donated blood, by centrifuging the blood within the closed collection system to separate the platelets from the red blood cells (RBC). The number of platelets per unit varies according to the platelet count of the donor; a yield of 7 x 1010 platelets is typical [1]. Since this number is inadequate to raise the platelet count in an adult recipient, four to six units are pooled to allow transfusion of 3 to 4 x 1011 platelets per transfusion [2]. These are called whole blood-derived or random donor pooled platelets.
Advantages of pooled platelets include lower cost and ease of collection and processing (a separate donation procedure and pheresis equipment are not required). The major disadvantage is recipient exposure to multiple donors in a single transfusion and logistic issues related to bacterial testing.
Apheresis (single donor) Platelets – Platelets can also be collected from volunteer donors in the blood bank, in a one- to two-hour pheresis procedure. Platelets and some white blood cells are removed, and red blood cells and plasma are returned to the donor. A typical apheresis platelet unit provides the equivalent of six or more units of platelets from whole blood (ie, 3 to 6 x 1011 platelets) [2]. In larger donors with high platelet counts, up to three units can be collected in one session. These are called apheresis or single donor platelets.
Advantages of single donor platelets are exposure of the recipient to a single donor rather than multiple donors, and the ability to match donor and recipient characteristics such as HLA type, cytomegalovirus (CMV) status, and blood type for certain recipients.
Both pooled and apheresis platelets contain some white blood cells (WBC) that were collected along with the platelets. These WBC can cause febrile non-hemolytic transfusion reactions (FNHTR), alloimmunization, and transfusion-associated graft-versus-host disease (ta-GVHD) in some patients.
Platelet products also contain plasma, which can be implicated in adverse reactions including transfusion-related acute lung injury (TRALI) and anaphylaxis. (See ‘Complications of platelet transfusion’ .)
2012pharmaceutical
Amandeep Kaur
Aashir Awan, Phd
Abhisar Anand
Adina Hazan
Alan F. Kaul, PharmD., MS, MBA, FCCP
alexcrystal6
anamikasarkar
apreconasia
aviralvatsa
David Orchard-Webb, PhD
danutdaagmailcom
Demet Sag, Ph.D., CRA, GCP
Dror Nir
dsmolyar
Ethan Coomber
evelinacohn
Gail S Thornton
Irina Robu
jayzmit48
jdpmd
jshertok
kellyperlman
Ed Kislauskis
larryhbern
Madison Davis
marzankhan
megbaker58
ofermar2020
Dr. Pati
pkandala
ritusaxena
Rick Mandahl
sjwilliamspa
Srinivas Sriram
stuartlpbi
Dr. Sudipta Saha
tildabarliya
vaishnavee24
zraviv06
zs22