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Recently, researchers at Mount Sinai were able to develop a therapeutic agent that shows high levels of effectiveness in Vitro disrupting a biological pathway that allow cancer to survive. This finding is according to a paper which was published in Cancer Discovery, which is a Journal of the American Association of cancer research in July 2021.
The therapy in which they focus on is a molecule named MS21, which causes the degradation of AKT which is an enzyme that is very active and present in cancers. In this study there was much evidence that pharmacological degradation of AKT is a feasible treatment for cancer’s which have a mutation in certain genes.
AKT is a cancer gene that encodes an enzyme that is abnormally activated in cancer cells to stimulate tumor growth. The degradation of AKT reverses all these processes which ultimately inhibits further tumor growth.
“Our study lays a solid foundation for the clinical development of an AKT degrader for the treatment of human cancers with certain gene mutations,” said Ramon Parsons, MD, Ph.D., Director of The Tisch Cancer Institute and Ward-Coleman Chair in Cancer Research and Chair of Oncological Sciences at the Icahn School of Medicine at Mount Sinai. “Examination of 44,000 human cancers identified that 19 percent of tumors have at least one of these mutations, suggesting that a large population of cancer patients could benefit from therapy with an AKT degrader such as MS21.”
MS21 was tested and human cancer derived cell lines, is used in Laboratories as a model to study the efficacy of different cancer therapies.
At Mount Sinai they were looking to develop MS21 with an industry partner in order to open clinical trials for patients.
“Translating these findings into effective cancer therapies for patients is a high priority because the mutations and the resulting cancer-driving pathways that we lay out in this study are arguably the most commonly activated pathways in human cancer, but this effort has proven to be particularly challenging,” said Jian Jin, Ph.D., Mount Sinai Professor in Therapeutics Discovery and Director of the Mount Sinai Center for Therapeutics Discovery at Icahn Mount Sinai. “We look forward to an opportunity to develop this molecule into a therapy that is ready to be studied in clinical trials.”
Advancing cancer precision medicine by creating a better toolbox for cancer therapy
Jian Jin1,2,3,4,5*, Arvin C. Dar1,2,3,4, Deborah Doroshow1
A
mong approximately 20,000 proteins in the human proteome, 627 have been identified by cancer-dependency studies as priority cancer targets, which are functionally important for various cancers. Of these 600-plus priority targets, 232 are enzymes and 395 are nonenzyme proteins (1). Tremendous progress has been made over the past several decades in targeting enzymes, in particular kinas-es, which have suitable binding pockets that can be occupied by small-molecule inhibitors, leading to U.S. Food and Drug Administration (FDA) approvals of many small-molecule drugs as targeted anticancer thera-
1Tisch Cancer Institute; 2Department of Oncological Sciences; 3Department of Pharmacological Sciences; 4Mount Sinai Center for Therapeutics Discovery; 5Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY
pies. However, most of the 395 nonenzyme protein targets, including transcription factors (TFs), do not have suitable binding pockets that can be effectively targeted by small molecules. These targets have consequently been considered undruggable; however, new cutting-edge approaches and technologies have recently been developed to target some of these “un-druggable” proteins in order to advance precision oncology.
TPD, a promising approach to precision cancer therapeutics
Targeted protein degradation (TPD) refers to the process of chemically eliminating proteins of interest (POIs) by utilizing small molecules, which are broadly divided into two types of modalities: PROteolysis Targeting Chimeras (PROTACs) and molecular glues (2). PROTACs are het-erobifunctional small molecules that contain two moieties: one binding the POI, linked to another binding an ubiquitin E3 ligase. The induced proximity between the POI and ubiquitination machinery leads to selective polyubiquitylation of the POI and its subsequent degradation by the ubiquitin–proteasome system (UPS). Molecular glues are monovalent small molecules, which, when built for TPD, directly induce interactions between the POI and an E3 ligase, also resulting in polyubiquitylation and subsequent degradation of the POI by the UPS. One of the biggest potential advantages of these therapeutic modalities over traditional inhibitors is that PROTACs and molecular glues can target undruggable proteins. Explosive growth has been seen in the TPD field over recent years (2, 3). Here, we highlight several recent advancements.
TF-PROTAC, a novel platform for targeting undruggable
tumorigenic TFs
Many undruggable TFs are tumorigenic. To target them, TF-PROTAC was developed (4), which exploits the fact that TFs bind DNA in a sequence-specific manner. TF-PROTAC was created to selectively bind a TF and E3 ligase simultaneously, by conjugating a DNA oligonucleotide specific for the TF of interest to a selective E3 ligase ligand. As stated earlier, this simultaneous binding and induced proximity leads to selective polyubiquitination of the TF and its subsequent degradation by the UPS. TF-PROTAC is a cutting-edge technology that could potentially provide a universal strategy for targeting most undruggable tumorigenic TFs.
Development of novel PROTAC degraders
WDR5, an important scaffolding protein, not an enzyme, is essential for sustaining tumorigenesis in multiple cancers, including MLL-rearranged (MLL-r) leukemia. However, small-molecule inhibitors that block the pro-tein–protein interaction (PPI) between WDR5 and its binding partners exhibit very modest cancer cell–killing effects, likely due to the confounding fact that these PPI inhibitors target only some—but not all—of WDR5’s on-cogenic functions. To address this shortcoming, a novel WDR5 PROTAC, MS67, was recently created using a powerful approach that effectively eliminates the protein and thereby all WDR5 functions via ternary complex structure-based design (Figure 1) (5). MS67 is a highly effective WDR5 degrader that potently and selectively degrades WDR5 and effectively suppresses the proliferation of tumor cells both in vitro and in vivo. This study provides strong evidence that pharmacological degradation of WDR5 as a novel therapeutic strategy is superior to WDR5 PPI inhibition for treating WDR5-dependent cancers.
EZH2 is an oncogenic methyltransferase that catalyzes histone H3 lysine 27 trimethylation, mediating gene repression. In addition to this canonical function, EZH2 has numerous noncanonical tumorigenic functions. EZH2 enzymatic inhibitors, however, are generally ineffective in
suppressing tumor growth in triple-negative breast cancer (TNBC) and MLL-r leukemia models and fail to phenocopy antitumor effects induced by EZH2 knockdown strategies. To target both canonical and noncanon-ical oncogenic functions of EZH2, several novel EZH2 degraders were recently developed, including MS1943, a hydrophobic tag–based EZH2 degrader (6), and MS177, an EZH2 PROTAC (7). MS1943 and MS177 effectively degrade EZH2 and suppress in vitro and in vivo growth in TNBC and MLL-r leukemia, respectively, suggesting that EZH2 degraders could provide a novel and effective therapeutic strategy for EZH2-dependent tumors.
MS21, a novel AKT PROTAC degrader, was developed to target activated AKT, the central node of the PI3K–AKT–mTOR signaling pathway (8). MS21 effectively suppresses the proliferation of PI3K–PTEN pathway-mutant cancers with wild-type KRAS and BRAF, which represent a large percentage of all human cancers. Another recent technology that expands the bifunctional toolbox for TPD is the demonstration that the E3 ligase KEAP1 can be leveraged for PROTAC development using a selective KEAP1 ligand (9). Overall, tremendous progress has been made in discovering novel degraders, some of which have advanced to clinical development as targeted therapies (2, 3).
Novel approaches to selective TPD in cancer cells
To minimize uncontrolled protein degradation in normal tissues, which may cause potential toxicity, a new technology was developed that incorporates a light-inducible switch, termed “opto-PROTAC” (10). This switch serves as a caging group that renders opto-PROTAC inactive in all cells in the absence of ultraviolet (UV) light. Upon UV irradiation, however, the caging group is removed, resulting in the release of the active degrader and spatiotemporal control of TPD in cancer cells. Another strategy to achieve selective TPD in cancer over normal cells is to cage degraders with a folate group (11, 12). Folate-caged degraders are inert and selectively concentrated within cancer cells, which overexpress folate receptors compared to normal cells. The caging group is subsequently removed inside tumor cells, releasing active degraders and achieving selective TPD in these cells. These novel approaches potentially enable degraders to be precision cancer medicines.
11
Frontiers of Medical Research: Cancer
Trametiglue, a novel and atypical molecular glue
The RAS–RAF–MEK–ERK signaling pathway, one of the most frequently mutated pathways in cancer, has been intensively targeted. Several drugs, such as the KRAS G12C inhibitor sotorasib and the MEK inhibitor trametinib, have been approved by the FDA. A significant advancement in this area is the discovery that trametinib unexpectedly binds a pseudokinase scaffold termed “KSR” in addition to MEK through interfacial contacts (13). Based on this structural and mechanistic insight, tra-metiglue, an analog of trametinib, was created as a novel molecular glue to limit adaptive resistance to MEK inhibition by enhancing interfacial binding between MEK, KSR, and the related homolog RAF. This study provides a strong foundation for developing next-generation drugs that target the RAS pathway.
TF-DUBTAC, a novel technology to stabilize undruggable tumor-suppressive TFs
Complementary to degrading tumorigenic TFs, stabilizing tumor-suppressive TFs could provide another effective approach for treating cancer. While most tumor-suppressive TFs are undruggable, TF-DUBTAC was recently developed as a generalizable platform to stabilize tumor-suppressive TFs (14). Deubiquitinase-targeting chimeras (DUBTACs) are heterobifunctional small molecules with a deubiquitinase (DUB) ligand linked to a POI ligand, which stabilize POIs by harnessing the deubiq-uitination machinery (15). Similar to TF-PROTAC, TF-DUBTAC exploits the fact that most TFs bind specific DNA sequences. TF-DUBTAC links a DNA oligonucleotide specific to a tumor-suppressive TF with a selective DUB ligand, resulting in simultaneous binding of the TF and DUB. The induced proximity between the TF and DUB leads to selective deubiquiti-
Putting a bull’s-eye on cancer’s back
Scientists are aiming the immune systems’ “troops” directly at tumors to better treat cancer
Joshua D. Brody, Brian D. Brown
I
mmunotherapy has transformed the treatment of several types of cancers. In particular, immune checkpoint blockade (ICB), which reinvigorates killer T cells, has helped extend the lives of many patients with advanced-stage lung, bladder, kidney, or skin cancers. Unfortunately, ~80% of patients do not respond to current immunotherapies or even-tually relapse. Emerging data indicate that one of the most profound ways cancers resist immunotherapy is by keeping killer T cells out of the tumor and putting other immune cells in a suppressed state (1). This understanding is giving rise to a new frontier in immunotherapy that is using synthetic biology and other approaches to reprogram the tumor from immune “cold” to immune “hot,” so T cells can be recruited to the tumor, and enter, target, and destroy the cancer cells (2) (Figure 1).
Cancers protect themselves by keeping out immune cells
Cancers grow in tissues like foreign invaders. Though they start from healthy cells, mutations turn cells malignant and allow them to grow unchecked. T cells can kill malignant cells that express mutated proteins, but cancers employ strategies to fend off the T cells. One way they do this is
12
nation of the TF and its stabilization. As an exciting new technology, TF-DUBTAC provides a potential general strategy to stabilize most undrugga-ble tumor-suppressive TFs for treating cancer.
Future outlook
The breathtaking pace we are seeing in the development of innovative approaches and technologies for advancing cancer therapies is only expected to accelerate. The promising clinical results achieved by PROTACs with established targets are particularly encouraging and pave the way for development of PROTACs for newer and more innovative targets. These groundbreaking discoveries have now put opportunities to fully realize cancer precision medicine within our reach.
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Other related articles published on this Open Access Online Scientific Journal include the following:
Machine Learning (ML) in cancer prognosis prediction helps the researcher to identify multiple known as well as candidate cancer diver genes
New evidence has shown how coronavirus has caused much damage to the brain. There is a new evidence that shows that COVID-19 assault on the brain I has the power to be multipronged. What this means is that it can attack on certain Brain cells such as reduce the amount of blood flow that the brain needs to the brain tissue.
Along with brain damage COVID-19 has also caused strokes and memory loss. A neurologist at yell University Serena Spudich says, “Can we intervene early to address these abnormalities so that people don’t have long-term problems?”
We’re on 80% of the people who have been hospitalized due to COVID-19 have showed brain symptoms which seem to be correlated to coronavirus.
At the start of the pandemic a group of researchers speculated that coronavirus they can damage the brain by infecting the neurons in the cells which are important in the process of transmitting information. After further studies they found out that coronavirus has a harder time getting past the brains defense system and the brain barrier and that it does not affect the neurons in anyway.
An expert in this study indicated that a way in which SARS-CoV-2 may be able to get to the brain is by going through the olfactory mucosa which is the lining of the nasal cavity. It is found that this virus can be found in the nasal cavity which is why we swab the nose one getting tested for COVID-19.
Spudich quotes, “there’s not a tonne of virus in the brain”.
Recent studies indicate that SARS-CoV-2 have ability to infect astrocytes which is a type of cell found in the brain. Astrocytes do quite a lot that supports normal brain function,” including providing nutrients to neurons to keep them working, says Arnold Kriegstein, a neurologist at the University of California, San Francisco.
Astrocytes are star-shaped cells in the central nervous system that perform many functions, including providing nutrients to neurons.
Kriegstein and his fellow colleagues have found that SARS-CoV-2 I mostly infects the astrocytes over any of the other brain cells present. In this research they expose brain organoids which is a miniature brain that are grown from stem cells into the virus.
As quoted in the article” a group including Daniel Martins-de-Souza, head of proteomics at the University of Campinas in Brazil, reported6 in a February preprint that it had analysed brain samples from 26 people who died with COVID-19. In the five whose brain cells showed evidence of SARS-CoV-2 infection, 66% of the affected cells were astrocytes.”
The infected astrocytes could indicate the reasoning behind some of the neurological symptoms that come with COVID-19. Specifically, depression, brain fog and fatigue. Kreigstein quotes, “Those kinds of symptoms may not be reflective of neuronal damage but could be reflective of dysfunctions of some sort. That could be consistent with astrocyte vulnerability.”
A study that was published on June 21 they compared eight different brands of deceased people who did have COVID-19 along with 14 brains as the control. The results of this research were that they found that there was no trace of coronavirus Brain infected but they found that the gene expression was affected in some of the astrocytes.
As a result of doing all this research and the findings the researchers want to know more about this topic and how many brain cells need to be infected for there to be neurological symptoms says Ricardo Costa.
Further evidence has also been done on how SARS-CoV-2 can affect the brain by reducing its blood flow which impairs the neurons’ function which ends up killing them.
Pericytes can be found on the small blood vessels which are called capillaries and are found all throughout the body and in the brain. In a February pre-print there was a report about how SARS-CoV-2 can infect the pericyte in the brain organoids.
David Atwell, a neuroscientist at the University College London, along with his other colleagues had published a pre-print which has evidence to show that SARS-CoV-2 odes In fact pericytes behavior. I researchers saw that in the different part of the hamsters brain SARS-CoV-2 blocks the function of receptors on the pericytes which ultimately causes the capillaries found inside the tissues to constrict.
As stated in the article, It’s a “really cool” study, says Spudich. “It could be something that is determining some of the permanent injury we see — some of these small- vessel strokes.”
Attwell brought to the attention that the drugs that are used to treat high blood pressure may in fact be used in some cases of COVID-19. Currently there are two clinical trials that are being done to further investigate this idea.
There is further evidence showing that the neurological symptoms and damage could in fact be happening because of the bodies on immune system reacting or misfiring after having COVID-19.
Over the past 15 years it has become evident that people’s immune system’s make auto antibodies which attack their own tissues says Harald Prüss in the article who has a Neuroimmunologist at the German Center for neurogenerative Diseases in Berlin. This may cause neuromyelitis optica which is when you can experience loss of vision or weakness in limbs. Harald Prüss summarized that the autoantibodies can pass through the blood brain barrier and ultimately impact neurological disorders such as psychosis.
Prüss and his colleagues published a study last year that focused on them isolating antibodies against SARS-CoV-2 from people. They found that one was able to protect hamsters from lung damage and other infections. The purpose of this was to come up with and create new treatments. During this research they found that some of the antibodies from people. They found that one was able to protect hamsters from lung damage and other infections. The purpose of this was to come up with and create new treatments. During this research they found that some of the antibodies can bind to the brain tissue which can ultimately damage it. Prüss states, “We’re currently trying to prove that clinically and experimentally,” says Prüss.
Was published online in December including Prüss sorry the blood and cerebrospinal fluid of 11 people who were extremely sick with COVID-19. These 11 people had neurological symptoms as well. All these people were able to produce auto antibodies which combined to neurons. There is evidence that when the patients were given intravenous immunoglobin which is a type of antibody it was successful.
Astrocytes, pericytes and autoantibodies we’re not the only pathways. However it is likely that people with COVID-19 experience article symptoms for many reasons. As stated, In the article, Prüss says a key question is what proportion of cases is caused by each of the pathways. “That will determine treatment,” he says.
Comparing COVID-19 Vaccine Schedule Combinations, or “Com-COV” – First-of-its-Kind Study will explore the Impact of using eight different Combinations of Doses and Dosing Intervals for Different COVID-19 Vaccines
test has a specificity over 90% and intended to used along with guideline
The Circulating Cell-free Genome Atlas Study (clinical trial NCT02889978) (CCGA) study divided into three substudies: highest performing assay, refining assay, validation of assays
methylation based assays worked better than sequencing (bisulfite sequencing)
used a machine learning algorithm to help refine assay
prediction was >90%; subgroup for high clinical suspicion of cancer
HCS sensitivity was 100% and specificity very high; but sensitivity on training set was 40% and results may have been confounded by including kidney cancer
TOO tissue of origin was predicted in greater than 99% in both training and validation sets
A first-of-its-kind prospective study of a multi-cancer blood test to screen and manage 10,000 women with no history of cancer
DETECT-A study: prospective interventional study; can multi blood test be used prospectively and can lead to a personalized care; can the screen be used to complement current therapy?
10,000 women aged 65-75; these women could not have previous cancer and conducted through Geisinger Health Network; multi test detects DNA and protein and standard of care screening
the study focused on safety so a committee was consulted on each case, and used a diagnostic PET-CT
blood test alone not good but combined with protein and CT scans much higher (5 fold increase) detection for breast cancer
there are mutiple opportunities yet at same time there are still challenges to utilize these cell free tests in therapeutic monitoring, diagnostic, and screening however sensitivities for some cancers are still too low to use in large scale screening however can supplement current screening guidelines
we have to ask about false positive rate and need to concentrate on prospective studies
we must consider how tests will be used, population health studies will need to show improved survival
Phylogenetic tracking and minimal residual disease detection using ctDNA in early-stage NSCLC: A lung TRACERx study Chris Abbosh@ucl
TRACERx study in collaboration with Charles Swanton.
multiplex PCR to track 200 SNVs: correlate tumor tissue biopsy with ctDNA
spike in assay shows very good sensitivity and specificity for SNVs variants tracked, did over 400 TRACERx libraries
sensitivity increases when tracking more variants but specificity does go down a bit
tracking variants can show evidence of subclonal dynamics and evolution and copy number deletion events; they also show neoantigen editing or changing of their neoantigens
this assay can detect low variants in a reproducible manner
The TRACERx (TRAcking Cancer Evolution through therapy (Rx)) lung study is a multi-million pound research project taking place over nine years, which will transform our understanding of non-small cell lung cancer (NSCLC) and take a practical step towards an era of precision medicine. The study will uncover mechanisms of cancer evolution by analysing the intratumour heterogeneity in lung tumours from approximately 850 patients and tracking its evolutionary trajectory from diagnosis through to relapse. At £14 million, it’s the biggest single investment in lung cancer research by Cancer Research UK, and the start of a strategic UK-wide focus on the disease, aimed at making real progress for patients.
Led by Professor Charles Swanton at UCL, the study will bring together a network of experts from different disciplines to help integrate clinical and genomic data and identify patients who could benefit from trials of new, targeted treatments. In addition, it will use a whole suite of cutting edge analytical techniques on these patients’ tumour samples, giving unprecedented insight into the genomic landscape of primary and metastatic tumours and the impact of treatment upon this landscape.
In future, TRACERx will enable us to define how intratumour heterogeneity impacts upon cancer immunity throughout tumour evolution and therapy. Such studies will help define how the clinical evaluation of intratumour heterogeneity can inform patient stratification and the development of combinatorial therapies incorporating conventional, targeted and immune based therapeutics.
Intratumour heterogeneity is increasingly recognised as a major hurdle to achieve improvements in therapeutic outcome and biomarker validation. Intratumour genetic diversity provides a substrate for tumour adaptation and evolution. However, the evolutionary genomic landscape of non-small cell lung cancer (NSCLC) and how it changes through the disease course has not been studied in detail. TRACERx is a prospective observational study with the following objectives:
Primary Objectives
Define the relationship between intratumour heterogeneity and clinical outcome following surgery and adjuvant therapy (including relationships between intratumour heterogeneity and clinical disease stage and histological subtypes of NSCLC).
Establish the impact of adjuvant platinum-containing regimens upon intratumour heterogeneity in relapsed disease compared to primary resected tumour.
Key Secondary Objectives
Develop and validate an intratumour heterogeneity (ITH) ratio index as a prognostic and predictive biomarker in relation to disease-free survival and overall survival.
Infer a complete picture of NSCLC evolutionary dynamics – define drivers of genomic instability, metastatic progression and drug resistance by identifying and tracking the dynamics of somatic mutational heterogeneity, and chromosomal structural and numerical instability present in the primary tumour and at metastatic sites. Individual tumour phylogenetic tree analysis will:
Establish the order of somatic events in relation to genomic instability onset and metastatic progression
Decipher genetic “bottlenecking” events following metastasis and drug therapy
Establish dynamics of tumour evolution during the disease course from early to late stage NSCLC.
Initiate a longitudinal biobank of circulating tumour cells (CTCs) and circulating-free tumour DNA (cfDNA) to develop analytical methods for the early detection and monitoring of tumour evolution over time.
Develop a longitudinal tissue resource to serve as a platform to assess the relationship between genetic intratumour heterogeneity and the host immune response.
Define relationships between intratumour heterogeneity and targeted/cytotoxic therapeutic outcome.
Use a lung cancer specific gene panel in a certified Good Clinical Practice (GCP) laboratory environment to define clonally dominant disease drivers to address the role of clonal driver dominance in targeted therapeutic response and to guide stratification of lung cancer treatment and future clinical study inclusion (paired primary-metastatic site comparisons in at least 270 patients with relapsed disease).
Utility of longitudinal circulating tumor DNA (ctDNA) modeling to predict RECIST-defined progression in first-line patients with epidermal growth factor receptor mutation-positive (EGFRm) advanced non-small cell lung cancer (NSCLC)
Martin Johnson
Impact of the EML4-ALK fusion variant on the efficacy of lorlatinib in patients (pts) with ALK-positive advanced non-small cell lung cancer (NSCLC) Todd Bauer
Lorlatinib, a smallmolecule inhibitor of ALK and ROS1, was granted accelerated U.S. Food and Drug Administration approval in November 2018 for patients with ALK-positive metastatic NSCLC whose disease has progressed on crizotinib and at least one other ALK inhibitor or whose disease has progressed on alectinib or ceritinib as the first ALK inhibitor therapy for metastatic disease. Todd M. Bauer, MD, a medical oncologist and senior investigator at Sarah Cannon Research Institute/Tennessee Oncology, PLLC, in Nashville, has been very involved with the development of lorlatinib since the beginning. In the following interview, Dr. Bauer discusses some of lorlatinib’s unique toxicities, as well as his first-hand experiences with the drug.
BACKGROUND: Lorlatinib is a potent, brain-penetrant, third-generation inhibitor of ALK and ROS1 tyrosine kinases with broad coverage of ALK mutations. In a phase 1 study, activity was seen in patients with ALK-positive non-small-cell lung cancer, most of whom had CNS metastases and progression after ALK-directed therapy. We aimed to analyse the overall and intracranial antitumour activity of lorlatinib in patients with ALK-positive, advanced non-small-cell lung cancer.
METHODS: In this phase 2 study, patients with histologically or cytologically ALK-positive or ROS1-positive, advanced, non-small-cell lung cancer, with or without CNS metastases, with an Eastern Cooperative Oncology Group performance status of 0, 1, or 2, and adequate end-organ function were eligible. Patients were enrolled into six different expansion cohorts (EXP1-6) on the basis of ALK and ROS1 status and previous therapy, and were given lorlatinib 100 mg orally once daily continuously in 21-day cycles. The primary endpoint was overall and intracranial tumour response by independent central review, assessed in pooled subgroups of ALK-positive patients. Analyses of activity and safety were based on the safety analysis set (ie, all patients who received at least one dose of lorlatinib) as assessed by independent central review. Patients with measurable CNS metastases at baseline by independent central review were included in the intracranial activity analyses. In this report, we present lorlatinib activity data for the ALK-positive patients (EXP1-5 only), and safety data for all treated patients (EXP1-6). This study is ongoing and is registered with ClinicalTrials.gov, number NCT01970865.
FINDINGS: Between Sept 15, 2015, and Oct 3, 2016, 276 patients were enrolled: 30 who were ALK positive and treatment naive (EXP1); 59 who were ALK positive and received previous crizotinib without (n=27; EXP2) or with (n=32; EXP3A) previous chemotherapy; 28 who were ALK positive and received one previous non-crizotinib ALK tyrosine kinase inhibitor, with or without chemotherapy (EXP3B); 112 who were ALK positive with two (n=66; EXP4) or three (n=46; EXP5) previous ALK tyrosine kinase inhibitors with or without chemotherapy; and 47 who were ROS1 positive with any previous treatment (EXP6). One patient in EXP4 died before receiving lorlatinib and was excluded from the safety analysis set. In treatment-naive patients (EXP1), an objective response was achieved in 27 (90·0%; 95% CI 73·5-97·9) of 30 patients. Three patients in EXP1 had measurable baseline CNS lesions per independent central review, and objective intracranial responses were observed in two (66·7%; 95% CI 9·4-99·2). In ALK-positive patients with at least one previous ALK tyrosine kinase inhibitor (EXP2-5), objective responses were achieved in 93 (47·0%; 39·9-54·2) of 198 patients and objective intracranial response in those with measurable baseline CNS lesions in 51 (63·0%; 51·5-73·4) of 81 patients. Objective response was achieved in 41 (69·5%; 95% CI 56·1-80·8) of 59 patients who had only received previous crizotinib (EXP2-3A), nine (32·1%; 15·9-52·4) of 28 patients with one previous non-crizotinib ALK tyrosine kinase inhibitor (EXP3B), and 43 (38·7%; 29·6-48·5) of 111 patients with two or more previous ALK tyrosine kinase inhibitors (EXP4-5). Objective intracranial response was achieved in 20 (87·0%; 95% CI 66·4-97·2) of 23 patients with measurable baseline CNS lesions in EXP2-3A, five (55·6%; 21·2-86·3) of nine patients in EXP3B, and 26 (53·1%; 38·3-67·5) of 49 patients in EXP4-5. The most common treatment-related adverse events across all patients were hypercholesterolaemia (224 [81%] of 275 patients overall and 43 [16%] grade 3-4) and hypertriglyceridaemia (166 [60%] overall and 43 [16%] grade 3-4). Serious treatment-related adverse events occurred in 19 (7%) of 275 patients and seven patients (3%) permanently discontinued treatment because of treatment-related adverse events. No treatment-related deaths were reported.
INTERPRETATION: Consistent with its broad ALK mutational coverage and CNS penetration, lorlatinib showed substantial overall and intracranial activity both in treatment-naive patients with ALK-positive non-small-cell lung cancer, and in those who had progressed on crizotinib, second-generation ALK tyrosine kinase inhibitors, or after up to three previous ALK tyrosine kinase inhibitors. Thus, lorlatinib could represent an effective treatment option for patients with ALK-positive non-small-cell lung cancer in first-line or subsequent therapy.
loratinib could be used for crizotanib resistant tumors based on EML4-ALK variants present in ctDNA
Each sheet in the workbook is separated by current COVID-19 vaccine trials, currents COVID-19 trials with the IL6R (interleukin 6 receptor) antagonist tocilizumab, and all COVID related trials. The Excel spreadsheet also contains links to more information about the trials.
As of April 15, 2020 the number of listed trials are as follows:
clinicaltrials.gov search terms
Number of results
Number of completed trials
Number of trials currently recruiting
COVID-19 or SARS-CoV-2
410
5 completed
5 withdrawn
192
1st row terms + vaccine
28
0
15
1st row terms + tocilizumab
16
0
10
1st row terms + hydroxychloroquine
61
1
22
A few highlights of the COVID related trials on clinicaltrials.gov
This is an open label, randomized, controlled, pilot clinical study in patients with COVID-19, to obtain preliminary biologic, physiologic, and clinical data in patients with COVID-19 treated with rhACE2 or control patients, to help determine whether a subsequent Phase 2B trial is warranted.
Condition or disease
Intervention/treatment
Phase
COVID-19
Drug: Recombinant human angiotensin-converting enzyme 2 (rhACE2)
Not Applicable
Detailed Description:
This is a small pilot study investigating whether there is any efficacy signal that warrants a larger Phase 2B trial, or any harm that suggests that such a trial should not be done. It is not expected to produce statistically significant results in the major endpoints. The investigators will examine all of the biologic, physiological, and clinical data to determine whether a Phase 2B trial is warranted.
Primary efficacy analysis will be carried only on patients receiving at least 4 doses of active drug. Safety analysis will be carried out on all patients receiving at least one dose of active drug.
It is planned to enroll more than or equal to 24 subjects with COVID-19. It is expected to have at least 12 evaluable patients in each group.
Experimental group: 0.4 mg/kg rhACE2 IV BID and standard of care Control group: standard of care
Intervention duration: up to 7 days of therapy
No planned interim analysis.
Study was withdrawn before participants were enrolled.
Gut dysbiosis co-exists in patients with coronavirus pneumonia. Some of these patients would develop secondary bacterial infections and antibiotic-associated diarrhea (AAD). The recent study on using washed microbiota transplantation (WMT) as rescue therapy in critically ill patients with AAD demonstrated the important clinical benefits and safety of WMT. This clinical trial aims to evaluate the outcome of WMT combining with standard therapy for patients with 2019-novel coronavirus pneumonia, especially for those patients with dysbiosis-related conditions.
Detailed Description:
An ongoing outbreak of 2019 novel coronavirus was reported in Wuhan, China. 2019-nCoV has caused a cluster of pneumonia cases, and posed continuing epidemic threat to China and even global health. Unfortunately, there is currently no specific effective treatment for the viral infection and the related serious complications. It is in urgent need to find a new specific effective treatment for the 2019-nCoV infection. According to Declaration of Helsinki and International Ethical Guidelines for Health-related Research Involving Humans, the desperately ill patients with 2019-nCov infection during disease outbreaks have a moral right to try unvalidated medical interventions (UMIs) and that it is therefore unethical to restrict access to UMIs to the clinical trial context.
There is a vital link between the intestinal tract and respiratory tract, which was exemplified by intestinal complications during respiratory disease and vice versa. Some of these patients can develop secondary bacterial infections and antibiotic-associated diarrhea (AAD). The recent study on using washed microbiota transplantation (WMT) as rescue therapy in critically ill patients with AAD demonstrated the important clinical benefits and safety of WMT. Additionally, the recent animal study provided direct evidence supporting that antibiotics could decrease gut microbiota and the lung stromal interferon signature and facilitate early influenza virus replication in lung epithelia. Importantly, the above antibiotics caused negative effects can be reversed by fecal microbiota transplantation (FMT) which suggested that FMT might be able to induce a significant improvement in the respiratory virus infection. Another evidence is that the microbiota could confer protection against certain virus infection such as influenza virus and respiratory syncytial virus by priming the immune response to viral evasion. The above results suggested that FMT might be a new therapeutic option for the treatment of virus-related pneumonia. The methodology of FMT recently was coined as WMT, which is dependent on the automatic facilities and washing process in a laboratory room. Patients underwent WMT with the decreased rate of adverse events and unchanged clinical efficacy in ulcerative colitis and Crohn’s disease. This clinical trial aims to evaluate the outcome of WMT combining with standard therapy for patients with novel coronavirus pneumonia, especially for those patients with dysbiosis-related conditions.
Responsible Party:
Faming Zhang, Director of Medical Center for Digestive Diseases, The Second Hospital of Nanjing Medical University
The 2019 novel coronavirus pneumonia outbroken in Wuhan, China, which spread quickly to 26 countries worldwide and presented a serious threat to public health. It is mainly characterized by fever, dry cough, shortness of breath and breathing difficulties. Some patients may develop into rapid and deadly respiratory system injury with overwhelming inflammation in the lung. Currently, there is no effective treatment in clinical practice. The present clinical trial is to explore the safety and efficacy of Human Umbilical Cord Mesenchymal Stem Cells (UC-MSCs) therapy for novel coronavirus pneumonia patients.
Detailed Description:
Since late December 2019, human pneumonia cases infected by a novel coronavirus (2019-nCoV) were firstly identified in Wuhan, China. As the virus is contagious and of great epidemic, more and more cases have found in other areas of China and abroad. Up to February 24, a total of 77, 779 confirmed cases were reported in China. At present, there is no effective treatment for patients identified with novel coronavirus pneumonia. Therefore, it’s urgent to explore more active therapeutic methods to cure the patients.
Recently, some clinical researches about the 2019 novel coronavirus pneumonia published in The Lancet and The New England Journal of Medicine suggested that massive inflammatory cell infiltration and inflammatory cytokines secretion were found in patients’ lungs, alveolar epithelial cells and capillary endothelial cells were damaged, causing acute lung injury. It seems that the key to cure the pneumonia is to inhibit the inflammatory response, resulting to reduce the damage of alveolar epithelial cells and endothelial cells and repair the function of the lung.
Mesenchymal stem cells (MSCs) are widely used in basic research and clinical application. They are proved to migrate to damaged tissues, exert anti-inflammatory and immunoregulatory functions, promote the regeneration of damaged tissues and inhibit tissue fibrosis. Studies have shown that MSCs can significantly reduce acute lung injury in mice caused by H9N2 and H5N1 viruses by reducing the levels of proinflammatory cytokines and the recruitment of inflammatory cells into the lungs. Compared with MSCs from other sources, human umbilical cord-derived MSCs (UC-MSCs) have been widely applied to various diseases due to their convenient collection, no ethical controversy, low immunogenicity, and rapid proliferation rate. In our recent research, we confirmed that UC-MSCs can significantly reduce inflammatory cell infiltration and inflammatory factors expression in lung tissue, and significantly protect lung tissue from endotoxin (LPS) -induced acute lung injury in mice.
The purpose of this clinical study is to investigate safety and efficiency of UC-MSCs in treating pneumonia patients infected by 2019-nCoV. The investigators planned to recruit 48 patients aged from 18 to 75 years old and had no severe underlying diseases. In the cell treatment group, 24 patients received 0.5*10E6 UC-MSCs /kg body weight intravenously treatment 4 times every other day besides conventional treatment. In the control group, other 24 patients received conventional treatment plus 4 times of placebo intravenously. The lung CT, blood biochemical examination, lymphocyte subsets, inflammatory factors, 28-days mortality, etc will be evaluated within 24h and 1, 2, 4, 8 weeks after UC-MSCs treatment.
Sponsor:
Puren Hospital Affiliated to Wuhan University of Science and Technology
Collaborator:
Wuhan Hamilton Bio-technology Co., Ltd
Study was withdrawn before participants were enrolled.
There are currently no clinical studies reporting clinical characteristics difference between the hypertension patients with and without ACEI treatment when suffered with novel coronavirus infection in China
Detailed Description:
At present, the outbreak of the new coronavirus (2019-nCoV) infection in Wuhan and Hubei provinces has attracted great attention from the medical community across the country. Both 2019-nCoV and SARS viruses are coronaviruses, and they have a large homology.
Published laboratory studies have suggested that SARS virus infection and its lung injury are related to angiotensin-converting enzyme 2 (ACE2) in lung tissue. And ACE and ACE2 in the renin-angiotensin system (RAS) are vital central links to maintain hemodynamic stability and normal heart and kidney function in vivo.
A large amount of evidence-based medical evidence shows that ACE inhibitors are the basic therapeutic drugs for maintaining hypertension, reducing the risk of cardiovascular, cerebrovascular, and renal adverse events, improving quality of life, and prolonging life in patients with hypertension. Recent experimental studies suggest that treatment with ACE inhibitors can significantly reduce pulmonary inflammation and cytokine release caused by coronavirus infection.
ACEI treatment
hypertension patients with ACEI treatment when suffered with novel coronavirus infection in China
Control
hypertension patients without ACEI treatment when suffered with novel coronavirus infection in China
Locations
China
The First Affiliated Hospital of Chongqing Medical University Chongqing, China
Sponsors and Collaborators Chongqing Medical University
Responsible PI:
Dongying Zhang, Associate Professor, Chongqing Medical University
Withdrawn (Similar projects have been registered, and it needs to be withdrawn.)
3.3.21 Multiple Barriers Identified Which May Hamper Use of Artificial Intelligence in the Clinical Setting, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 2: CRISPR for Gene Editing and DNA Repair
In a commentary article from Jennifer Couzin-Frankel entitled “Medicine contends with how to use artificial intelligence” the barriers to the efficient and reliable adoption of artificial intelligence and machine learning in the hospital setting are discussed. In summary these barriers result from lack of reproducibility across hospitals. For instance, a major concern among radiologists is the AI software being developed to read images in order to magnify small changes, such as with cardiac images, is developed within one hospital and may not reflect the equipment or standard practices used in other hospital systems. To address this issue, lust recently, US scientists and government regulators issued guidance describing how to convert research-based AI into improved medical images and published these guidance in the Journal of the American College of Radiology. The group suggested greater collaboration among relevant parties in developing of AI practices, including software engineers, scientists, clinicians, radiologists etc.
As thousands of images are fed into AI algorithms, according to neurosurgeon Eric Oermann at Mount Sinai Hospital, the signals they recognize can have less to do with disease than with other patient characteristics, the brand of MRI machine, or even how a scanner is angled. For example Oermann and Mount Sinai developed an AI algorithm to detect spots on a lung scan indicative of pneumonia and when tested in a group of new patients the algorithm could detect pneumonia with 93% accuracy.
However when the group from Sinai tested their algorithm from tens of thousands of scans from other hospitals including NIH success rate fell to 73-80%, indicative of bias within the training set: in other words there was something unique about the way Mt. Sinai does their scans relative to other hospitals. Indeed, many of the patients Mt. Sinai sees are too sick to get out of bed and radiologists would use portable scanners, which generate different images than stand alone scanners.
The results were published in Plos Medicine as seen below:
PLoS Med. 2018 Nov 6;15(11):e1002683. doi: 10.1371/journal.pmed.1002683. eCollection 2018 Nov.
Variable generalization performance of a deep learning model to detect pneumonia in chest radiographs: A cross-sectional study.
There is interest in using convolutional neural networks (CNNs) to analyze medical imaging to provide computer-aided diagnosis (CAD). Recent work has suggested that image classification CNNs may not generalize to new data as well as previously believed. We assessed how well CNNs generalized across three hospital systems for a simulated pneumonia screening task.
METHODS AND FINDINGS:
A cross-sectional design with multiple model training cohorts was used to evaluate model generalizability to external sites using split-sample validation. A total of 158,323 chest radiographs were drawn from three institutions: National Institutes of Health Clinical Center (NIH; 112,120 from 30,805 patients), Mount Sinai Hospital (MSH; 42,396 from 12,904 patients), and Indiana University Network for Patient Care (IU; 3,807 from 3,683 patients). These patient populations had an age mean (SD) of 46.9 years (16.6), 63.2 years (16.5), and 49.6 years (17) with a female percentage of 43.5%, 44.8%, and 57.3%, respectively. We assessed individual models using the area under the receiver operating characteristic curve (AUC) for radiographic findings consistent with pneumonia and compared performance on different test sets with DeLong’s test. The prevalence of pneumonia was high enough at MSH (34.2%) relative to NIH and IU (1.2% and 1.0%) that merely sorting by hospital system achieved an AUC of 0.861 (95% CI 0.855-0.866) on the joint MSH-NIH dataset. Models trained on data from either NIH or MSH had equivalent performance on IU (P values 0.580 and 0.273, respectively) and inferior performance on data from each other relative to an internal test set (i.e., new data from within the hospital system used for training data; P values both <0.001). The highest internal performance was achieved by combining training and test data from MSH and NIH (AUC 0.931, 95% CI 0.927-0.936), but this model demonstrated significantly lower external performance at IU (AUC 0.815, 95% CI 0.745-0.885, P = 0.001). To test the effect of pooling data from sites with disparate pneumonia prevalence, we used stratified subsampling to generate MSH-NIH cohorts that only differed in disease prevalence between training data sites. When both training data sites had the same pneumonia prevalence, the model performed consistently on external IU data (P = 0.88). When a 10-fold difference in pneumonia rate was introduced between sites, internal test performance improved compared to the balanced model (10× MSH risk P < 0.001; 10× NIH P = 0.002), but this outperformance failed to generalize to IU (MSH 10× P < 0.001; NIH 10× P = 0.027). CNNs were able to directly detect hospital system of a radiograph for 99.95% NIH (22,050/22,062) and 99.98% MSH (8,386/8,388) radiographs. The primary limitation of our approach and the available public data is that we cannot fully assess what other factors might be contributing to hospital system-specific biases.
CONCLUSION:
Pneumonia-screening CNNs achieved better internal than external performance in 3 out of 5 natural comparisons. When models were trained on pooled data from sites with different pneumonia prevalence, they performed better on new pooled data from these sites but not on external data. CNNs robustly identified hospital system and department within a hospital, which can have large differences in disease burden and may confound predictions.
Surprisingly, not many researchers have begun to use data obtained from different hospitals. The FDA has issued some guidance in the matter but considers “locked” AI software or unchanging software as a medical device. However they just announced development of a framework for regulating more cutting edge software that continues to learn over time.
Still the key point is that collaboration over multiple health systems in various countries may be necessary for development of AI software which is used in multiple clinical settings. Otherwise each hospital will need to develop their own software only used on their own system and would provide a regulatory headache for the FDA.
Other articles on Artificial Intelligence in Clinical Medicine on this Open Access Journal include:
May 16, 2016
By Alex Keown, BioSpace.com Breaking News Staff
BERKELEY, Calif. – Shares of Aduro Biotech (ADRO) have fallen more than 25 percent this morning following news that the company’s Phase II trial for its combination pancreatic cancer drug, CRS-207 did not meet its primary endpoint of survivability.
Aduro said its Eclipse trial of CRS-207 failed to show an improvement in overall survival for patients with pancreatic cancer who had failed at least two prior therapies in the metastatic setting. Median overall survival was 3.8 months for patients treated with the immunotherapy regimen of CRS-207 and the cancer vaccine GVAX pancreas, 5.4 months for patients treated with CRS-207 alone and 4.6 months for patients administered chemotherapy. Aduro said there were no reported safety concerns during the trial and full study findings will be presented at a later date.
Stephen T. Isaacs, chairman, president and chief executive officer of Aduro, called the findings a disappointing and “unexpected outcome.’
“While we are well aware of the very difficult-to-treat nature of late-stage metastatic pancreatic cancer, we are surprised by the divergence of these data from the results of our Phase IIa study. At the same time, we continue to look forward to the interim results later this year from our ongoing Stellar trial, which is evaluating CRS-207 and GVAX Pancreas with and without the anti-PD1 checkpoint inhibitor nivolumab as a second-line therapy for patients with metastatic pancreatic cancer,” Isaacs said in a statement.
Aduro Biotech says its leading clinical trial for its cancer-fighting combination of GVAX and CRS-207 was a flop. The biotech reported this morning that its combo not only failed to hit the primary endpoint, it did worse than the two solo groups in the study.
“Median overall survival (MOS) in this third-line and greater setting was 3.8 months for patients treated with the immunotherapy regimen of CRS-207 and GVAX Pancreas, 5.4 months for patients treated with CRS-207 alone and 4.6 months for patients administered chemotherapy,” the company reported.
Aduro’s stock ($ADRO) plunged 30% on the news in premarket trading.
“While we are well aware of the very difficult-to-treat nature of late-stage metastatic pancreatic cancer, we are surprised by the divergence of these data from the results of our Phase 2a study,” said CEO Stephen Isaacs. But the company hasn’t given up hope on its lead program.
“In the overall survival analysis, we were intrigued by activity seen with CRS-207 as a single-agent. While the median duration of 5.4 months appears greater for CRS-207, the overall survival curve of CRS-207 alone was comparable to overall survival seen with chemotherapy. Of note, due to a disproportionately high dropout rate in the single-agent chemotherapy arm, most of these patients instead received a variety of combination treatments, including chemotherapies, immunotherapies and targeted therapies,” said Dirk Brockstedt, executive vice president, research and development for Aduro.
In a conference call with analysts Aduro execs noted that they are now shelving plans for a pivotal Phase III study in pancreatic cancer, focusing on an ongoing study in the pipeline that includes a checkpoint inhibitor.
The history of cancer vaccines not customized to individual patients has been a litany of failure, with programs for Stimuvax and others experiencing a string of defeats in the clinic. Celldex joined that circle of failure recently with a Phase III miss for Rintega. Aduro, though, doubled down on the cancer vaccine strategy and was able to strike some big-money pacts along the way on the basis of some promising midstage data.
GVAX Pancreas is based on a pancreatic cell line modified to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine known to stimulate the immune system. CRS-207 is made up of attenuated (deactivated) Listeria monocytogenes that express mesothelin, sparking another immune response to a protein concentrated on pancreatic cancer cells. Together, they’re supposed to provide a one-two punch against cancer, with an option to add on additional drugs to continue to build an attack on cancer cells.
That was the only workable strategy with GVAX, which, like other cancer vaccines, wasn’t able to spur a strong enough response to make a significant difference in the fight against cancer as a solo therapy, allowing Aduro to buy it for a song in early 2013. Monday’s conference call included several skeptical remarks from analysts about GVAX’s future, if any.
Aduro was on hand at the 2013 ASCO confab to review the data from a midstage study involving 93 patients with advanced pancreatic cancer. A combination of chemo plus GVAX pancreas and CRS-207 significantly outperformed GVAX and chemo, bumping up overall survival from 3.9 months to 6.1 months. That’s not a big gain, but it was a sign of progress in a very tough field. And it was good enough for the FDA to grant a breakthrough drug designation.
Now the company is forced into a classic defensive crouch, citing its significant cash reserves as it looks to regroup.
We previously demonstrated that autologous natural killer (NK)–cell therapy after hematopoietic cell transplantation (HCT) is safe but does not provide an antitumor effect. We hypothesize that this is due to a lack of NK-cell inhibitory receptor mismatching with autologous tumor cells, which may be overcome by allogeneic NK-cell infusions. Here, we test haploidentical, related-donor NK-cell infusions in a nontransplantation setting to determine safety and in vivo NK-cell expansion. Two lower intensity outpatient immune suppressive regimens were tested: (1) lowdose cyclophosphamide and methylprednisolone and (2) fludarabine. A higher intensity inpatient regimen of high-dose cyclophosphamide and fludarabine (HiCy/Flu) was tested in patients with poorprognosis acute myeloid leukemia (AML). All patients received subcutaneous interleukin 2 (IL-2) after infusions. Patients who received lower intensity regimens showed transient persistence but no in vivo expansion of donor cells. In contrast, infusions after the more intense Hi-Cy/Flu resulted in a marked rise in endogenous IL-15, expansion of donor NK cells, and induction of complete hematologic remission in 5 of 19 poor-prognosis patients with AML. These findings suggest that haploidentical NK cells can persist and expand in vivo and may have a role in the treatment of selected malignancies used alone or as an adjunct to HCT.
Human natural killer (NK) cells are a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T-cell receptor (CD3).1 They recognize and kill transformed cell lines in a major histocompatibility complex (MHC)–unrestricted fashion and produce cytokines critical to the innate immune response. NK-cell function, distinct from the MHC-restricted cytolytic activity of T cells, may play a role in antitumor surveillance.2 The effects of NK-cell infusions have been studied in adoptive immunotherapy clinical trials. In these studies, autologous lymphokine-activated killer cells obtained from peripheral blood mononuclear cells (PBMCs) were administered to patients along with exogenous high-dose interleukin-2 (IL-2). Up to 20% of patients responded to these infusions of NK-cell– containing populations.3
In contrast to NK cells, T cells recognize targets through an antigen-specific T-cell receptor (TCR) and interact with targets only if human leukocyte antigen (HLA) MHC antigens are also recognized. Although NK-cell killing is MHC-unrestricted, NK cells display a number of activating and inhibitory receptors that ligate MHC molecules to modulate the immune response.4,5 NK-cell receptors that recognize antigens at the HLA-A, -B, or -C loci are members of the immunoglobulin superfamily and are termed killer immunoglobulin receptors (KIRs).6,7 Other receptor families (natural killer group 2 [NKG2]/CD94) that recognize antigens of the nonclassical HLA-E, -F, or -G loci and other ligand specificities have also been described.8-10 Engagement of these NK-cell receptors results in stimulation or inhibition of NK-cell effector function depending on intracellular signaling mediated through the cytoplasmic tail or adaptor molecules associated with each receptor.11-13 The NK-cell response to a target thus depends on the net effect of activating and inhibitory receptors.
Clinical trials have assessed the effects of low-dose IL-2 administration on activation of NK cells in patients with cancer. We have demonstrated the safety and feasibility of daily subcutaneous IL-2 injections following high-dose chemotherapy and autologous hematopoietic cell transplantation (HCT). Whereas IL-2 signifi- cantly expanded the number of circulating NK cells in vivo, these NK cells were not maximally cytotoxic as determined by in vitro assays.14 Subsequent studies tested infusion of IL-2–activated NK-cell–enriched populations or intravenous IL-2 infusions combined with subcutaneous IL-2. Although these approaches augmented in vivo NK-cell function, no consistent efficacy of autologous NK-cell therapy could be detected in cancer patients when compared with cohorts of matched controls.15
We then hypothesized that autologous NK cells may be suppressed by the physiologic response resulting from NK-cell recognition of “self” MHC molecules. This notion is supported by recent data from haploidentical T-cell–depleted transplantation studies. KIR mismatch with tumor MHC (ie, KIR ligand) may lead to greater tumor kill. In these studies, Ruggeri et al16 showed that stratifying patients by their KIR ligand mismatch would select for patients with alloreactive NK cells that protect against acute myeloid leukemia (AML) relapse. Although virtually untested in solid tumors, these clinical data strongly support a therapeutic role for allogeneic NK cells in myeloid leukemia.17 We present data on the biologic effects of haploidentical NK-cell infusions administered to cancer patients as cell-based immunotherapy with the goal of demonstrating a feasible and safe method that permits in vivo donor NK-cell expansion.
………
In this study, we demonstrate that adoptively transferred human NK cells derived from haploidentical related donors can be expanded in vivo. Of interest, in vivo NK-cell expansion occurs after preparation with a high dose (Hi-Cy/Flu) but not lower doses of immunosuppression (Lo-Cy/mPred or Flu). Successful lymphocyte adoptive transfer following intensive immunosuppresion is not surprising. Lymphopenia may change the competitive balance between transferred lymphocytes and endogenous lymphocytes. Alternatively, lymphopenia may induce survival factors or deplete cellular or soluble inhibitory factors.25,26 In murine studies, preparative regimens sufficient to induce lymphopenia allowed homeostatic T-cell expansion in vivo that potentiated effective antitumor immunity.27 This concept has been tested in human T-cell clinical trials by Rosenberg’s group.28 T-cell lymphopenia was induced by Hi-Cy/Flu, similar to what was used here. Successful adoptive transfer and expansion of NK cells may also require intense immunosuppression. Prlic et al20 showed that mature NK cells proliferated only in an NK-cell–deficient host where the endogenous NK-cell pool was absent.
We also demonstrate that NK-cell adoptive therapy is associated with a striking rise in endogenous IL-15 levels, reminiscent of the role IL-7 plays in CD4 T-cell homeostasis.29 IL-15 is required for the final steps of in vitro NK-cell differentiation from CD34 progenitors.22-24 Cooper et al21 was the first to show that IL-15 was absolutely required for in vivo expansion and survival of NK cells, in mice, in part through bcl-2 expression. Transfer of NK cells into IL-15/ hosts resulted in loss of NK cells by 4 days after transfer. IL-15 receptor alpha knockout mice generate IL-15 but do not have NK cells and are unable to undergo successful adoptive transfer. This implies that IL-15 responsiveness by cells other than NK cells may be important in driving this response. IL-15 transgenic mice markedly expand their NK cells and CD8 T cells, ultimately resulting in an NK/T-lymphocytic leukemia.30 The endogenous origin of IL-15 in our patients was unclear. Our data support the notion that IL-15 levels increased only after an intensive lymphocyte-depleting preparative regimen as demonstrated by the inverse correlation between IL-15 concentrations and the absolute lymphocyte count. This does not exclude the possibility that IL-15 may be produced following chemotherapy-induced damage to gastrointestinal mucosa or other cells of epithelial origin.31-34 The effects of exogenous IL-2 administration in these patients needs to be explored as it does add toxicity to the regimen. Further clinical testing may demonstrate that expansion will occur in the presence of IL-15 alone.
Donor NK-cell infusions were feasible and tolerated without unexpected toxicity except for the umbilical cord blood transplantation patient who developed EBV reactivation after treatment. The risk of posttransplantation lymphoproliferative disease approached 10% when HCT is performed using a T-cell–depleted and mismatched graft.35 Although a single event, this finding is important to understand the possible consequences of allogeneic NK-cell therapy in heavily pretreated immunosuppressed patients. It also emphasizes that the CD3- depleted final product, enriched for NK cells but containing B cells, may need further purification to lessen the possibility of this complication. Clinical ex vivo selection methods to address this issue using CD3 depletion followed by CD56 selection are now in place36 and will be tested. We have previously shown that monocytes serve as accessory cells for NK-cell expansion in vitro18 but the role of accessory cells in vivo, if any, is unknown. We need to verify that removal of monocytes and B cells does not change the in vivo expansion potential of NK cells seen here before recommending a purified NK-cell product in all future studies.
In summary, this is the first study to demonstrate that adoptively transferred human NK cells can be expanded in vivo. Expansion was dependent on the more intense Hi-Cy/Flu preparative regimen, which induced lymphopenia, and the more potent immunosuppression that was associated with high endogenous concentrations of IL-15, none of which was observed following Lo-Cy/mPred and Flu alone. It is intriguing that this same regimen is the basis for many transplantation regimens and may help explain the robust NK-cell reconstitution seen in that setting. In this study, NKenriched cells were obtained from related haploidentical donors by efficient depletion of CD3 from PBMCs, although contaminating B cells and monocytes remained in the final product. A maximum tolerated dose was not reached and the largest cell dose administered was that obtained during a single lymphapheresis collection. Although tumor response was not a primary goal of this study, 5 of 19 poor-prognosis patients with AML achieved complete remission after haploidentical NK-cell therapy, with a significantly higher complete remission rate when KIR ligand mismatched donors were used, a strategy that predicts NK-cell alloreactivity.16,37 The precise role of the cells versus the high-intensity chemotherapy regimen in responding patients cannot be definitively determined in this current study. However, the benefit of alloreactivity and the preferential expansion of functional NK cells in responding patients is consistent with at least a partial effect from the NK cells. Our data suggest that prospective selection of KIR ligand– mismatched donors is warranted when possible, which will be assessed in subsequent larger clinical trails.
The biology of natural killer cells in cancer, infection, and pregnancy.
OBJECTIVE: NK cells are important cells of the immune system. They are ultimately derived from pluripotent hematopoietic stem cells. NK cell cytotoxicity and other functions are tightly regulated by numerous activating and inhibitory receptors including newly discovered receptors that selectively recognize major histocompatibility complex class I alleles. Based on their defining function of spontaneous cytotoxicity without prior immunization, NK cells have been thought to play a critical role in immune surveillance and cancer therapy. However, new insights into NK cell biology have suggested major roles for NK cells in infection control and uterine function. The purpose of this review is to provide an update on NK cell function, ontogeny, and biology in order to better understand the role of NK cells in health and disease.
DATA SOURCES: In the Medline database, the major subject heading “Natural Killer Cells” was introduced in 1983, identifying 16,848 citations as of December 31, 2000. Since 1986, there have been approximately 1000 citations per year under this subject heading. In this database, 68% of manuscripts are limited to human NK cells; 40% of citations cross with the major sub-heading of cytotoxicity, 40% with cytokines, 36% with neoplasm, 5% with antibody-dependent cellular cytotoxicity, 2.8% with pregnancy, and 1.3% with infection. Of references from the year 2000-2001, 46 were selected to combine with contributions from earlier literature.
CONCLUSIONS: NK cells should no longer be thought of as direct cytotoxic killers alone as they clearly serve a critical role in cytokine production which may be important to control cancer, infection, and fetal implantation. Understanding mechanisms of NK cell functions may lead to novel therapeutic strategies for the treatment of human disease.
NK cell-based immunotherapy for malignant diseases
Min Cheng, Yongyan Chen, Weihua Xiao, Rui Sun and Zhigang Tian Cellular & Molecular Immunology (2013) 10, 230–252; published online 22 April 2013 http://dx. doi.org:/10.1038/cmi.2013.10
Natural killer (NK) cells play critical roles in host immunity against cancer. In response, cancers develop mechanisms to escape NK cell attack or induce defective NK cells. Current NK cell-based cancer immunotherapy aims to overcome NK cell paralysis using several approaches. One approach uses expanded allogeneic NK cells, which are not inhibited by self histocompatibility antigens like autologous NK cells, for adoptive cellular immunotherapy. Another adoptive transfer approach uses stable allogeneic NK cell lines, which is more practical for quality control and large-scale production. A third approach is genetic modification of fresh NK cells or NK cell lines to highly express cytokines, Fc receptors and/or chimeric tumor-antigen receptors. Therapeutic NK cells can be derived from various sources, including peripheral or cord blood cells, stem cells or even induced pluripotent stem cells (iPSCs), and a variety of stimulators can be used for large-scale production in laboratories or good manufacturing practice (GMP) facilities, including soluble growth factors, immobilized molecules or antibodies, and other cellular activators. A list of NK cell therapies to treat several types of cancer in clinical trials is reviewed here. Several different approaches to NK-based immunotherapy, such as tissue-specific NK cells, killer receptor-oriented NK cells and chemically treated NK cells, are discussed. A few new techniques or strategies to monitor NK cell therapy by non-invasive imaging, predetermine the efficiency of NK cell therapy byin vivo experiments and evaluate NK cell therapy approaches in clinical trials are also introduced.
Surgery, chemotherapeutic agents and ionizing radiation have been used for decades as primary strategies to eliminate the tumors in patients; however, the development of resistance to drugs or radiation led to a significant incidence of tumor relapse. Therefore, investigating effective strategies to eliminate these resistant tumor cells is urgently needed. The importance of immune system in malignant diseases has been demonstrated by recent major scientific advances.
Both innate and adaptive immune cells actively prevent neoplastic development in a process called ‘cancer immunosurveillance’. Innate immune cells, including monocytes, macrophages, dendritic cells (DCs) and natural killer (NK) cells, mediate immediate, short-lived responses by releasing cytokines that directly lyse tumor cells or capture debris from dead tumor cells. Adaptive immune cells, including T and B cells, mediate long-lived, antigen-specific responses and effective memory.1 Despite these immune responses, malignant cells can develop mechanisms to evade immunosurveillance. Some tumors protect themselves by establishing an immune-privileged environment. For example, they can produce immunosuppressive cytokines IL-10 and transforming growth factor-β (TGF-β) to suppress the adaptive antitumor immune response, or skew the immune response toward a Th2 response with significantly less antitumor capacity.2,3,4 Some tumors alter their expressions of IL-6, IL-10, vascular epithelial growth factor or granulocyte monocyte-colony stimulating factor (GM-CSF), impairing DC functions via inactivation or suppressing maturation.5 In some cases, induced regulatory T cells suppress tumor-specific CD4+ and CD8+ T-cell responses.6 Tumor cells also minimally express or shed tumor-associated antigens, shed the ligands of NK cell-activating receptor such as the NKG2D ligands UL16-binding protein 2, major histocompatibility complex (MHC) class I chain-related molecules A and B molecules (MICA/MICB) or alter MHC-I and costimulatory molecule expression to evade the immune responses.7,8,9 Malignant cells may also actively eliminate immune cells by activation-induced cell death or Fas ligand (FasL) expression.10,11 In addition, primary cancer treatments like chemotherapy and ionizing radiation can compromise antitumor immune responses by their immunosuppressive side effects.
Tumor cells can be eliminated when immune responses are adequate; when they are not, tumor growth and immunourveillance enter into a dynamic balance until tumor cells evade immunosurveillance, at which point neoplasms appear clinically as a consequence. Therapies designed to induce either a potent passive or active antitumor response against malignancies by harnessing the power of the immune system, known as tumor immunotherapy, is an appealing alternative strategy to control tumor growth. Until now, the cancer immunotherapy field has covered a vast array of therapeutic agents, including cytokines, monoclonal antibodies, vaccines, adoptive cell transfers (T, NK and NKT) and Toll-like receptor (TLR) agonists.1,12,13 Adoptive NK cell transfer in particular has held great promise for over three decades. With progress in the NK cell biology field and in understanding NK function, developing NK cells to be a powerful cancer immunotherapy tool has been achieved in recent years. In this article, we will review recent advances in NK cell-based cancer immunotherapy, focusing on potential approaches and large-scale NK cell expansion for clinical practice, as well as on the clinical trials and future perspectives to enhance the efficacy of NK cells.
NK cells were first identified in 1975 as a unique lymphocyte subset that are larger in size than T and B lymphocytes and contain distinctive cytoplasmic granules.14,15 After more than 30 years, our understanding of NK cell biology and function lends important insights into their role in immunosurveillance. It has been known that NK cells develop in bone marrow (BM) from common lymphoid progenitor cells;16 however, NK cell precursors have still not been clearly characterized in humans.17 After development, NK cells distribute widely throughout lymphoid and non-lymphoid tissues, including BM, lymph nodes (LN), spleen, peripheral blood, lung and liver.18
NK cells, defined as CD3−CD56+ lymphocytes, are distinguished as CD56bright and CD56dim subsets. Approximately 90% of peripheral blood and spleen NK cells belong to the CD56dimCD16+ subset with marked cytotoxic function upon interacting with target cells.19,20In contrast, most NK cells in lymph nodes and tonsils belong to the CD56brightCD16− subset and exhibit predominantly immune regulation properties by producing cytokines such as interferon (IFN)-γ in response to IL-12, IL-15 and IL-18 stimulation.19,21
NK cells rapidly kill certain target cells without prior immunization or MHC restriction, whose activation is dependent on the balance between inhibitory and activating signals from invariant receptors.22,23,24 The activating receptors include the cytotoxicity receptors (NCRs) (NKp46, NKp30 and NKp44), C-type lectin receptors (CD94/NKG2C, NKG2D, NKG2E/H and NKG2F) and killer cell immunoglobulin-like receptors (KIRs) (KIR-2DS and KIR-3DS), while the inhibitory receptors include C-type lectin receptors (CD94/NKG2A/B) and KIRs (KIR-2DL and KIR-3DL). Since some structural families contain both activating and inhibitory receptors, trying to understand how NK cell activity is regulated is often complicated.25 At steady state, the inhibitory receptors (KIRs and CD94/NKG2A/B), which bind to various MHC-I molecules present on almost all cell types, inhibit NK cell activation and prevent NK cell-mediated killing. Under stress conditions, cells downregulate MHC-I expression, causing NK cells to lose inhibitory signaling and be activated in a process called ‘missing-self recognition’. Additionally, the non-MHC self molecules Clr-b (mouse), LLT-1 (human) and CD48 (mouse) recognized by the inhibitory receptors NKR-P1B, NKR-P1A and 2B4, respectively, also perform this function.26,27 In contrast to the self-expressed inhibitory receptor ligands, NK cell-activating receptors can recognize either pathogen-encoded molecules that are not expressed by the host, called ‘non-self recognition’, or self-expressed proteins that are upregulated by transformed or infected cells, called ‘stress-induced self recognition’. For example, mouse Ly49H recognizes cytomegalovirus-encoded m157, and NKG2D recognizes the self proteins human UL16-binding proteins and MICA/MICB.28,29 NK cells identify their targets by recognizing a set of receptors on target cells in an NK-target cell zipper formation; this results in the integration of multiple activating and inhibitory signals, the outcome of which depends on the nature of the interacting cells.26IFNs or DC/macrophage-derived cytokines, such as type I IFN, IL-12, IL-18 and IL-15, enhance the activation or promote the maturation of NK cells, which can also augment NK cell cytolytic activity against tumor cells.30,31,32 Cytotoxic activity of NK cells can increase approximately 20–200 fold after exposure to IFN-α/β or IL-12. Despite these known innate immune cell functions, accumulating evidence in both mice and humans demonstrates that NK cells are educated and selected during development, possess receptors with antigen specificity, undergo clonal expansion during infection and can generate long-lived memory cells.33,34
After over 30 years of researching NK cells, evidence supports that they play critical roles in the early control of viral infection, in hematopoietic stem cell (HSC) transplantation (improved grafting, graft-vs.-host disease and graft-vs.-tumor), in tumor immunosurveillance and in reproduction (uterine spiral artery remodeling). The roles of NK cells in controlling organ transplantation, parasitic and HIV infections, autoimmunity and asthma have also been suggested, but remain to be explored further.26 In particular, therapeutic strategies harnessing the power of NK cells to target multiple malignancies have been designed.
NK cells originally described as large granular lymphocytes, exhibited natural cytotoxicity against certain tumor cells in the absence of preimmunization or stimulation.35,36,37 CD56dim NK cells, which make up the majority of circulating cells, are the most potent cytotoxic NK cells against tumor cells. Evidence gathered from a mouse xenograft tumor model testing functionally deficient NK cells or antibody-mediated NK cell depletion supports that NK cells can eradicate tumor cells.38,39,40,41 An 11-year follow-up study in patients indicated that low NK-like cytotoxicity was associated with increased cancer risk.42 High levels of tumor infiltrating NK cells (TINKs) are associated with a favorable tumor outcome in patients with colorectal carcinoma, gastric carcinoma and squamous cell lung cancer, suggesting that NK-cell infiltration into tumor tissues represents a positive prognostic marker.43,44,45 As described above, NK-cell recognition of tumor cells by inhibitory and activating receptors is complex, and the three recognition models—‘missing-self’, ‘non-self’ and ‘stress-induced self’—might be used to sense missing- or altered-self cells. Activated NK cells are thus in a position to directly or indirectly exert their antitumor activity to control tumor growth and prevent the rapid dissemination of metastatic tumors by ‘immunosurveillance’ mechanisms (Figure 1).
NK cells in tumor immunosurveillance. The diagram shows the potential roles of NK cells in tumor immunosurveillance. NK cells initially recognize the tumor cells via stress or danger signals. Activated NK cells directly kill target tumor cells through at least four mechanisms: cytoplasmic granule release, death receptor-induced apoptosis, effector molecule production or ADCC. Additionally, NK cells act as regulatory cells when reciprocally interact with DCs to improve their antigen uptake and presentation, facilitating the generation of antigen-specific CTL responses. Also, by producing cytokines such as IFN-γ, activated NK cells induce CD8+ T cells to become CTLs. Activated NK cells can also promote differentiation of CD4+ T cells toward a Th1 response and promote CTL differentiation. Cytokines produced by NK cells might also regulate antitumor Ab production by B cells. Ab, antibody; ADCC, antibody-dependent cellular cytotoxicity; CTL, cytotoxic T lymphocyte; DC, dendritic cell; IFN, interferon; NK, natural killer.
Direct tumor clearance by NK-mediated cytotoxicity
Upon cellular transformation, surface MHC-I expression on tumor cells is often reduced or lost to evade recognition by antitumor T cells. In parallel, cellular stress and DNA damage lead to upregulated expression of ligands on tumor cells for NK cell-activating receptors. Human tumor cells that have lost self MHC-I expression or bear ‘altered-self’ stress-inducible proteins are ideal NK cell targets, as NK cells are activated by initially recognizing certain ‘stress’ or ‘danger’ signals.46 The ‘missing-self’ model of tumor cell recognition by NK cells was first demonstrated by observing that MHC-I-deficient syngeneic tumor cells were selectively rejected by NK cells; additionally, NK cell inhibitory receptors were shown to detect this absence of MHC-I expression.47,48,49 NK cells can also kill certain MHC-I-sufficient tumor cells by detecting stress-induced self ligands through their activating receptors. Broad MICA/B expression has been detected on epithelial tumors, melanoma, hepatic carcinoma and some hematopoetic malignancies, representing a counter-measure by the immune system to combat tumor development.31 NK cell-mediated cytotoxicity is also important against tumor initiation and metastasis in vivo.50,51,52
NK cells directly kill target tumor cells through several mechanisms: (i) by releasing cytoplasmic granules containing perforin and granzymes that leads to tumor-cell apoptosis by caspase-dependent and -independent pathways.53,54 Cytotoxic granules reorient towards the tumor cell soon after NK–tumor cell interaction and are released into the intercellular space in a calcium-dependent manner; granzymes are allowed entry into tumor cells by perforin-induced membrane perforations, leading to apoptosis; (ii) by death receptor-mediated apoptosis. Some NK cells express tumor-necrosis factor (TNF) family members, such as FasL or TNF-related apoptosis-inducing ligand (TRAIL), which can induce tumor-cell apoptosis by interacting with their respective receptors, Fas and TRAIL receptor (TRAILR), on tumor cells.55,56,57,58,59 TNF-α produced by activated NK cells can also induce tumor-cell apoptosis;60 (iii) by secreting various effector molecules, such as IFN-γ, that exert antitumor functions in various ways, including restricting tumor angiogenesis and stimulating adaptive immunity.61,62 Cytokine activation or exposure to tumor cells is also associated with nitric oxide (NO) production, where NK cells kill target tumor cells by NO signaling;63,64 (iv) through antibody-dependent cellular cytotoxicity (ADCC) by expressing CD16 to destroy tumor cells.40 The antitumor activity of NK cells can be further enhanced by cytokine stimulation, such as by IL-2, IL-12, IL-18, IL-15 or those that induce IFN production.40,65,66,67,68,69,70
Indirect NK-mediated antitumor immunity
NK cells act as regulatory cells when reciprocally interact with DCs, macrophages, T cells and endothelial cells by producing various cytokines (IFN-γ, TNF-α and IL-10), as well as chemokines and growth factors.26,71 By producing IFN-γ, activated NK cells induce CD8+ T cells to become cytotoxic T lymphocytes (CTLs), and also help to differentiate CD4+ T cells toward a Th1 response to promote CTL differentiation.72,73 NK cell-derived cytokines might also regulate antitumor antibody (Ab) production by B cells.40 In addition, cancer cells killed by NK cells could provide tumor antigens for DCs, inducing them to mature and present antigen.74By lysing surrounding DCs that have phagocytosed and processed foreign antigens, activated NK cells also could provide additional antigenic cellular debris for other DCs. Thus, activated NK cells promote antitumor immunity by regulating DC activation and maturation,75 as these DCs can facilitate the generation of antigen-specific CTL responses through their ability to cross-present tumor-specific antigens (derived from NK cell-mediated tumor lysis) to CD8+ T cells.76,77
During tumor progression, tumor cells develop several mechanisms to either escape from NK-cell recognition and attack or to induce defective NK cells. These include losing expression of adhesion molecules, costimulatory ligands or ligands for activating receptors, upregulating MHC class I, soluble MIC, FasL or NO expression, secreting immunosuppressive factors such as IL-10, TGF-β and indoleam ine 2,3-d ioxygense (IDO) and resisting Fas- or perforin-mediated apoptosis.31,78,79 In cancer patients, NK cell abnormalities have been observed, including decreased cytotoxicity, defective expression of activating receptors or intracellular signaling molecules, overexpression of inhibitory receptors, defective proliferation, decreased numbers in peripheral blood and in tumor infiltrate, and defective cytokine production.60Given that NK cells play critical roles in the first-line of defense against malignancies by direct and indirect mechanisms, the therapeutic use of NK cells in human cancer immunotherapy has been proposed and followed in a clinical context (Table 1).
For NK cell immunotherapy, obtaining a sufficient number of functional NK cells is critical in clinical protocols. Therefore, the number, purity and state of NK cell proliferation and activation are considered as the key factors.151 In Table 2, the purification/expansion of clinical-grade NK cells developed in recent years is summarized. They can be produced from cord blood, bone marrow, peripheral blood and embryonic stem cells. Overall, the summarized methods suggest that long-term ex vivoexpansion of NK cells may present a clinical benefit, but not the short-term activation which is not sufficient for augmenting the functions of NK cells.152
Results from treating hematological malignancies demonstrated a critical role for NK cells in clinical immunotherapy, as alloreactive NK cells highlighted the graft-vs.-leukemia effect in AML patients.172 The graft-vs.-tumor effect of alloreactive NK cells was also strengthened by mismatched IL-2-activated lymphocytes in patients with solid tumors or hematological malignancies.173 As discussed above, autologous NK cells, allogeneic NK cells, NK cell lines and genetically modified NK cells were investigated for effectiveness as tumor immunotherapies. The clinical study designs evaluating the efficacy of these various NK cell-mediated tumor therapies are summarized in Table 3.
NK cell-based immunotherapy holds great promise for cancer treatment. However, only modest clinical success has been achieved thus far using NK cell-based therapies in cancer patients. Progress in the field of understanding NK cell biology and function is therefore needed to assist in developing novel approaches to effectively manipulate NK cells for the ultimate benefit of treating cancer patients.
Present and Future of Allogeneic Natural Killer Cell Therapy
Natural killer (NK) cells are innate lymphocytes that are capable of eliminating tumor cells and are therefore used for cancer therapy. Although many early investigators used autologous NK cells, including lymphokine-activated killer cells, the clinical efficacies were not satisfactory. Meanwhile, human leukocyte antigen (HLA)-haploidentical hematopoietic stem cell transplantation revealed the antitumor effect of allogeneic NK cells, and HLA-haploidentical, killer cell immunoglobulin-like receptor ligand-mismatched allogeneic NK cells are currently used for many protocols requiring NK cells. Moreover, allogeneic NK cells from non-HLA-related healthy donors have been recently used in cancer therapy. The use of allogeneic NK cells from non-HLA-related healthy donors allows the selection of donor NK cells with higher flexibility and to prepare expanded, cryopreserved NK cells for instant administration without delay for ex vivo expansion. In cancer therapy with allogeneic NK cells, optimal matching of donors and recipients is important to maximize the efficacy of the therapy. In this review, we summarize the present state of allogeneic NK cell therapy and its future directions.
Cancer is a major threat for humans worldwide, with approximately 14 million new cases and 8.2 million cancer-related deaths in 2012 (1). Although most common cancer treatments include surgery, chemotherapy, and radiotherapy, unsatisfactory cure rates require new therapeutic approaches, especially for refractory cancers. For this purpose, cancer immunotherapies with various cytokines, antibodies, and immune cells have been clinically applied to patients to encourage their own immune system to help fight the cancer (2).
Adoptive cellular immunotherapies have employed several types of immune cells, including dendritic cells (DCs), cytotoxic T lymphocytes (CTLs), lymphokine-activated killer (LAK) cells, cytokine-induced killer (CIK) cells, and natural killer (NK) cells. Although there has been recent progress in DC therapy and CTL therapy, clinical applications are somewhat limited because cancer antigens must first be characterized and autologous cells must be used. By contrast, LAK cells, CIK cells, and NK cells have antigen-independent cytolytic activity against tumor cells. In particular, NK cells can be used from not only autologous sources but also allogeneic sources and, recently, allogeneic NK cells have been employed more often in cancer treatment. Whereas autologous NK cells from cancer patients may have functional defects (3), allogeneic NK cells from healthy donors have normal function and can be safely administered to cancer patients (4). Allogeneic NK cell therapy is particularly beneficial because it can enhance the anti-cancer efficacy of NK cells via donor–recipient incompatibility in terms of killer cell immunoglobulin-like receptors (KIRs) on donor NK cells and major histocompatibility complex (MHC) class I on recipient tissues.
Natural killer cells are innate lymphocytes that provide a first line of defense against viral infections and cancer (5). Human NK cells are recognized as CD3−CD56+ lymphocytes. They can be further subdivided into two subsets based on the surface expression level of CD56. The CD56dim population with low-density expression of CD56 comprises approximately 90% of human blood NK cells and has a potent cytotoxic function, whereas the CD56bright population (approximately 10% of blood NK cells) with high-density expression of CD56 displays a potent cytokine producing capacity and has immunoregulatory functions (6). The CD56dim NK cell subset also expresses high levels of the Fc receptor for IgG (FcγRIII, CD16), which allows them to mediate antibody-dependent cellular cytotoxicity (ADCC) (7). NK cells comprise 5–15% of circulating lymphocytes and are also found in peripheral tissues, including the liver, peritoneal cavity, and placenta. Activated NK cells are capable of extravasation and infiltration into tissues that contain pathogens or malignant cells while resting NK cells circulate in the blood (8).
The NK cell activity is regulated by signals from activating and inhibitory receptors (9, 10). The activating signal is mediated by several NK receptors including NKG2D and natural cytotoxicity receptors (NCRs) (9–11). By contrast, NK cell activity is suppressed by inhibitory receptors, including KIRs, which bind to human leukocyte antigen (HLA) class I molecules on target cells (9, 10, 12). NKG2A is also an important inhibitory receptor binding to non-classical HLA molecule, HLA-E (13). If target cells lose or downregulate HLA expression (14), the NK inhibitory signal is abrogated, allowing NK cells to become activated and kill malignant targets. However, NK cell function is impaired in cancer patients by various mechanisms, particularly in tumor microenvironment (15).
Although NK cell activity is determined by the summation of signals from activating and inhibitory receptors, the inhibitory signal through KIRs is a main regulator of NK cell function particularly in allogeneic settings. Inhibitory KIRs have long cytoplasmic tails containing two immunoreceptor tyrosine-based inhibition motifs (ITIMs). Each KIR has its cognate ligand and consists of two (KIR2DL) or three (KIR3DL) extracellular Ig-domains. KIR2DL1 and KIR2DL2/3 recognize group 2 HLA-C (called C2, Lys80) and group 1 HLA-C (called C1, Asn80), respectively. KIR3DL1 recognizes HLA-Bw4 (16). The KIR repertoire on human NK cells is randomly determined and independent of the number and allotype of HLA class I ligands (17).
The antitumor activity of allogeneic NK cells has been demonstrated in the setting of hematopoietic stem cell transplantation (HSCT). Allogeneic HSCT is an established curative treatment for hematologic malignancies. In allogeneic HSCT, donor T cells contribute to graft-versus-host disease (GVHD) and graft-versus-tumor (GVT) effects (18). In T cell-depleted HSCT, however, donor NK cells are the major effector cells responsible for controlling residual cancer cells before T cell reconstitution (19, 20).
Natural killer cells are the first lymphoid population to recover after allogeneic HSCT. In the first month of transplantation, reconstituted NK cells represent the predominant lymphoid cells and play a crucial role in controlling the host immune system. Allogeneic NK cells prevent viral infections and restrain residual cancer cells in the early phase of transplantation (21). Of note, the GVT activity of donor NK cells is significantly improved when KIRs of donor and HLA class I of the recipient are incompatible, and consequently when inhibitory signals are absent, as observed in HLA-haploidentical HSCT (22). Therefore, increased GVT activity of NK cells with KIR-HLA incompatibility is the underlying rationale for the development of allogeneic NK cell therapy.
Following the discovery of inhibitory KIRs and the understanding that they play a role in preventing NK cell killing of self MHC class I-expressing tumor cells, investigators began to research the possibility of using allogeneic donor NK cells instead of autologous NK cells for cancer therapy. Several groups have infused activated, expanded donor NK cells to patients early after allogeneic HSCT to provide antitumor effects (23). In Table Table1,1, clinical trials with allogeneic NK cells as therapeutics are summarized.
Table 1Selected clinical trials with expanded allogeneic NK cells
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As summarized in Table Table2,2, two clinical trials are investigating the use of CAR-expressing allogeneic NK cells. The aim of both studies is to assess the safety, feasibility, and efficacy of expanded, activated, and CD19-redirected haploidentical NK cells in ALL patients who have persistent disease after intensive chemotherapy or HSCT (NCT00995137, NCT01974479). Further, other tumor antigens, such as CS1, CEA, CD138, and CD33, are targeted by CARs expressed by NK cells, although NK-92, YT, or NKL cell lines were used (48–51).
Table 2Genetically modified, expanded allogeneic NK cells.
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Therapeutic regimens
In allogeneic NK cell therapy, optimal therapeutic regimens for clinical applications should be considered because adoptively transferred NK cells not only target tumor cells but also interact with the immunological environment. To potentiate the therapeutic efficacy of allogeneic NK cells, proper strategies, including pre-conditioning or combination therapy, could be applied (34).
Upregulation of NKG2D ligands by spironolactone (63) or histone deacetylase inhibitors (64, 65) and upregulation of TRAIL-R2 by doxorubicin (66) result in enhanced antitumor efficacy of NK cells. Proteasome inhibitors also sensitize tumor cells to NK cell-mediated killing via TRAIL and FasL pathways. In addition, c-kit tyrosine kinase inhibitor (67) and JAK inhibitors (68) increase the susceptibility of tumor cells to NK cytotoxicity and enhance antitumor responses by increased IFN-γ production from NK cells. However, protein kinase inhibitors should be used cautiously because some protein kinase inhibitors, such as sorafenib, inhibit the effector function of NK cells (69).
Immunomodulatory drugs can augment NK cell function. Lenalidomide enhances rituximab-induced killing of non-Hodgkin’s lymphoma and B-cell chronic lymphocytic leukemia through NK cell and monocyte-mediated ADCC mechanisms (70). Combination therapy using IL-2 and anti-CD25 shows anti-leukemic effects by depletion of regulatory T cells in addition to activation and expansion of NK cells (71). Alloferon, an immunomodulatory peptide, enhances the expression of NK-activating receptor 2B4 and granule exocytosis from NK cells against cancer cells (72).
Therapeutic antibodies can be combined with allogeneic NK cell therapy (73). Antibodies against tumor antigens (e.g., CD20 and CS1) can induce ADCC of NK cells (74, 75). Antibodies to activating NK receptors (e.g., 4-1BB, GITR, NKG2D, DNAM-1, and NCRs) can enhance NK activation (74, 76–79). In addition, inhibitory receptors (e.g., KIR2DL, PD-1, PD-L1, and NKG2A) can be blocked by antibodies (80–85). Bispecific and trispecific killer cell engagers directly activate NK cells through CD16 signaling and thus, induce cytotoxicity and cytokine production against tumor targets (86, 87).
Conclusion
Antitumor activity of allogeneic NK cells was first observed in a setting of HLA-haploidentical HSCT. Allogeneic NK cell therapy was tried mostly using HLA-haploidentical NK cells with or without allogeneic HSCT and, recently, allogeneic NK cells from unrelated, random donors have been used in a non-HSCT setting. The efficacy of allogeneic NK cell therapy can be enhanced by optimal donor selection in terms of the KIR genotype of donors and donor KIR-recipient MHC incompatibility. Furthermore, efficacy can be increased by genetic modification of NK cells and optimized therapeutic regimens. In the future, allogeneic NK cell therapy can be an effective therapeutic modality for cancer.
δγ T cells for immune therapy of patients with lymphoid malignancies
http://dx.doi.org:/10.1182/blood-2002-12-3665 Prepublished online Blood March 6, 2003; 2003 102: 200-206
Martin Wilhelm, Volker Kunzmann, Susanne Eckstein, Peter Reimer, Florian Weissinger, Thomas Ruediger and Hans-Peter Tony
There is increasing evidence that gammadelta T cells have potent innate antitumor activity. We described previously that synthetic aminobisphosphonates are potent gammadelta T cell stimulatory compounds that induce cytokine secretion (ie, interferon gamma [IFN-gamma]) and cell-mediated cytotoxicity against lymphoma and myeloma cell lines in vitro. To evaluate the antitumor activity of gammadelta T cells in vivo, we initiated a pilot study of low-dose interleukin 2 (IL-2) in combination with pamidronate in 19 patients with relapsed/refractory low-grade non-Hodgkin lymphoma (NHL) or multiple myeloma (MM). The objectives of this trial were to determine toxicity, the most effective dose for in vivo activation/proliferation of gammadelta T cells, and antilymphoma efficacy of the combination of pamidronate and IL-2. The first 10 patients (cohort A) who entered the study received 90 mg pamidronate intravenously on day 1 followed by increasing dose levels of continuous 24-hour intravenous (IV) infusions of IL-2 (0.25 to 3 x 106 IU/m2) from day 3 to day 8. Even at the highest IL-2 dose level in vivo, gammadelta T-cell activation/proliferation and response to treatment were disappointing with only 1 patient achieving stable disease. Therefore, the next 9 patients were selected by positive in vitro proliferation of gammadelta T cells in response to pamidronate/IL-2 and received a modified treatment schedule (6-hour bolus IV IL-2 infusions from day 1-6). In this patient group (cohort B), significant in vivo activation/proliferation of gammadelta T cells was observed in 5 patients (55%), and objective responses (PR) were achieved in 3 patients (33%). Only patients with significant in vivo proliferation of gammadelta T cells responded to treatment, indicating that gammadelta T cells might contribute to this antilymphoma effect. Overall, administration of pamidronate and low-dose IL-2 was well tolerated. In conclusion, this clinical trial demonstrates, for the first time, that gammadelta T-cell-mediated immunotherapy is feasible and can induce objective tumor responses.
Despite significant improvement in the treatment of low-grade non-Hodgkin lymphoma (NHL) and multiple myeloma (MM), most patients relapse or become resistant to conventional treatment strategies such as chemotherapy or radiation. Therefore, there is need for alternative tumor therapies. One possibility is manipulating the immune system to target and eliminate neoplastic cells. Most current immunotherapeutic approaches aim at inducing antitumor response via stimulation of the adaptive immune system, which is dependent on major histocompatibility complex (MHC)– restricted T cells. Despite major advances in our understanding of the adaptive immunity toward tumors and the introduction of vaccine-based strategies, durable responses are rare, and active immunotherapy is still not an established treatment modality. Adaptive immunotherapeutic approaches have several disadvantages: T cells need specific tumor-associated antigens (TAAs) and appropriate costimulatory molecules for activation. Failure or loss of TAAs, MHC molecules, and/or costimulatory molecules renders tumor cells resistant to T-cell–mediated cytotoxicity or induces anergy of specific T cells.1
Mice deficient in innate effector cells such as natural killer (NK) cells, NK T cells, or T cells show a significantly increased incidence of tumors and provide clear evidence for an immune surveillance function of the innate immune system.2-4 Recognition of transformed cells by the innate immune system seems to be dependent on expression of stress-induced ligands and/or loss of MHC class I molecules on tumor cells.5 Several studies have demonstrated a role for human T cells in recognition of transformed cells.6,7 T cells exhibit a potent MHC-unrestricted lytic activity against different tumor cells in vitro.8-10 In addition, T cells have been found with increased frequency in disease-free survivors of acute leukemia following allogeneic bone marrow transplantation.11 Adoptive transfer of ex vivo–expanded human T cells in a mouse tumor model further supports the in vivo antitumor effects of T cells.12 V9V2 T cells, which represent most of the human circulating T cells, recognize small nonpeptide compounds with an essential phosphate residue (ie, microbial metabolites) or alkylamines.13-17 As we have shown previously, also synthetic aminobisphosphonates such as pamidronate are potent T-cell– stimulatory compounds.18 In addition, we could demonstrate that pamidronate-activated T cells produce cytokines (ie, interferon [IFN-]), exhibit specific cytotoxicity against lymphoma or myeloma cell lines, and lead to reduced survival of autologous myeloma cells.8
The aim of this pilot study is to evaluate the feasibility of activation and/or expansion of T cells in vivo using the combination of pamidronate and interleukin 2 (IL-2) in patients with refractory/relapsed lymphoma or myeloma, to determine the most effective IL-2 dose, to assess the toxicity of this regimen, and to evaluate its ability to exert antitumor effects. …..
There has been no study published so far on in vivo stimulation of T cells in humans, and the consequences of a selective activation of T cells in vivo were not known. Therefore, evaluation of toxicity was one major end point of this study. We started with a low IL-2 dose of 0.25 106 IU IL-2/m2 and subsequently increased the IL-2 dose to 3 106 IU IL-2/m2 in cohort A and to 2 106 IU IL-2/m2 in cohort B. Overall, the combination of pamidronate and IL-2 was well tolerated, and no dose-limiting toxicity was observed. Most of the patients developed self-limiting fever and thrombophlebitis at the infusion site. Local thrombophlebitis has been described as a rare side effect in
patients receiving pamidronate alone.20,21 The high frequency of local thrombophlebitis in patients receiving pamidronate in combination with IL-2 might reflect immune-mediated effects on endothelial cells. It has also been recently shown that aminobisphosphonates have dose-dependent effects on proliferation-inhibition and apoptosis-induction of human endothelial cells in vitro.22
Next we asked whether the combination of pamidronate and IL-2 induces activation and proliferation of T cells in vivo. None of the first 10 patients included in this pilot study (cohort A, Table 1) developed a measurable T-cell response in vivo. The inability to induce T-cell proliferative response in vivo correlated with the negative in vitro proliferation of T cells in response to pamidronate/IL-2 in 4 of 5 analyzable patients. Therefore, extensive prior in vitro testing was initiated for all further eligible patients. Using this strategy, we found that a much lower proportion of patients with hematologic malignancies showed positive in vitro proliferation of T cells in response to pamidronate/IL-2 compared with a control group of healthy donors (49% versus 88%). Although the exact mechanisms of this defect are currently under investigation, a severe immunodeficiency caused by extensive prior chemotherapy in these relapsed/ refractory patients and/or the underlying disease itself may account for this observation. Indeed, the type of underlying disease seems to influence the in vitro proliferative response to pamidronate/IL-2 (Table 2). The failure of patients with B-CLL to develop a measurable T-cell proliferative response may be a result of the very small number of T cells in peripheral blood, which were often below the detection limit in our series. However, a larger number of patients with distinct disease entities and at different disease stages (eg, untreated versus treated) need to be evaluated to support this observation and to identify additional clinical parameters influencing T-cell reactivity. Furthermore, extensive prior in vitro testing in eligible patients revealed that T-cell proliferation in response to pamidronate can be significantly enhanced by concomitant addition of IL-2 to PBMC cultures on day 1 instead of day 3 (as previously done).
Thus, for all further patients the treatment schedule was changed (concomitant administration of IL-2 on day 1), and only patients with significant in vitro proliferation of T cells in the presence of pamidronate and IL-2 were included (cohort B, Table 1). After these modifications, significant in vivo expansion of T cells could be observed in 5 of 9 patients (55%) (Table 1). In vivo proliferation of T cells was associated with a robust up-regulation of early (CD69) and late (HLA-DR) activation markers, whereas pamidronate and IL-2 failed to induce comparable effects on T cells and NK cells (Table 3). These data support in vitro findings that the action of pamidronate is highly specific and, except for V9V2 T cells, it does not activate other immune effector cells.8,23,24 However, at higher IL-2 doses unspecific stimulation effects of IL-2 became more evident because a proportion of patients showed a moderate up-regulation of activation markers on T cells and NK cells at the highest dose level of IL-2 tested in this study. On the basis of the analysis of activation marker expression and proliferation we conclude that 1 106 IU IL-2/m2 IL-2 per day seems to be the most effective dose with respect to specific and effective T-cell stimulation in vivo.
Another aim of our study was to assess the clinical response. None of the 9 analyzable patients of cohortA(Table 1) achieved an objective tumor response. After change of protocol and inclusion criteria (cohort B, Table 1) 3 of 9 patients (33%) achieved an objective tumor response (3 PR). Clinical response could be associated with T-cell proliferation in vivo, because all 4 patients from cohort B without T-cell proliferation in vivo did not experience an objective tumor response, and 4 of 5 patients with T-cell proliferation in vivo responded (3 PR, 1 stable disease [SD]). These results suggest that the observed tumor regression in our patients is dependent on T-cell activation and proliferation. The relevance of this correlation is underlined by the fact that pamidronate-stimulated T cells possess an increased capacity for killing tumor cells in vitro.8,10 It is still open which mechanisms may have been responsible for the clinical responses. Several other antitumor effects have been attributed to aminobisphosphonates. However, at pharmacologically achievable concentrations in vivo, only the specific stimulation of V9V2T cells can be observed.8 Alternatively, the occurrence of clinical remissions may be attributed to an IL-2–mediated effect on other immune effector cells. However, our immunologic monitoring indicates that the combination of pamidronate and low-dose IL-2 does not induce specific activation and expansion of T cells or NK cells compared with the effect on T cells. In addition, the concentrations of IL-2 used here are much lower than the doses required in other immunotherapeutic approaches for these malignancies.25-27
The important question of what precise mechanisms are involved in tumor recognition and eradication by T cells is out of the scope of this study and will require further in vitro and in vivo studies. However, tumor cell recognition by T cells seems to be modulated by a balance of positive and negative signals.28 Although killer inhibitory receptors (KIRs) are obviously involved in the mediation of negative signals, the positive signals are only incompletely understood. One example of such a positive signal is the NKG2D-DAP10 receptor complex, which is known to interact with stress-induced ligands on tumor cells such as MICA and Rae-1.29 The very slow response profiles of most of the patients in our series strongly argue for an indirect influence on lymphoma cells rather than a sole cytotoxic effect. One possible mechanism may be secretion of cytokines, which influence tumor cells or their microenvironment.30 We have already shown that IFN- is the major cytokine secreted by pamidronate-activated T cells.8,31 IFN- has multiple antitumor effects such as direct inhibition of tumor growth, blocking angiogenesis, or stimulation of macrophages.32 Recently, a significant negative correlation between angiogenetic factors (ie, VEGF) and IFN- serum levels was described in patients treated with pamidronate.33 Therefore, IFN- might be one of the key cytokines involved in the T-cell– mediated antitumor response.
In conclusion, this study indicates for the first time that in vivo T-cell stimulation by pamidronate and low-dose IL-2 is a safe and promising immunotherapy approach in the treatment of
patients with low-grade B-NHL and MM. Further studies are necessary to confirm the clinical efficacy of this novel strategy. Our immunologic and clinical monitoring data provide further insight into the capacity of T cells to induce an antitumor immune response. However, this study also reveals that the function of T cells can be impaired in some patients with lymphoid malignancies. Therefore, the results of this study provide principles relevant to the design of future trials, including appropriate prior in vitro testing.
EXPANSION OF HIGHLY CYTOTOXIC HUMAN NATURAL KILLER CELLS FOR CANCER CELL THERAPY
Infusions of natural killer (NK) cells are an emerging tool for cancer immunotherapy. The development of clinically applicable methods to produce large numbers of fully functional NK cells is a critical step to maximize the potential of this approach. We determined the capacity of the leukemia cell line K562 modified to express a membrane-bound form of interleukin-15 and 4-1BB ligand (K562-mb15-41BBL) to generate human NK cells with enhanced cytotoxicity. Seven-day coculture with irradiated K562-mb15-41BBL induced a median 21.6-fold expansion of CD56+CD3– NK cells from peripheral blood (range, 5.1-86.6-fold; n = 50), which was considerably superior to that produced by stimulation with interleukin (IL)-2, IL-12, IL-15 and/or IL-21 and caused no proliferation of CD3+ lymphocytes. Similar expansions could also be obtained from the peripheral blood of patients with acute leukemia undergoing therapy (n = 11). Comparisons of the gene expression profiles of the expanded NK cells and of their unstimulated or IL-2-stimulated counterparts demonstrated marked differences. The expanded NK cells were significantly more potent than unstimulated or IL-2-stimulated NK cells against acute myeloid leukemia (AML) cells in vitro. They could be detected for more than one month when injected into immunodeficient mice and could eradicate leukemia in murine models of AML. We therefore adapted the K562-mb15-41BBL stimulation method to large-scale clinical-grade conditions, generating large numbers of highly cytotoxic NK cells. The results that we report here provide rationale and practical platform for clinical testing of expanded and activated NK cells for cell therapy of cancer.
Natural killer (NK) cells can kill cancer cells in the absence of prior stimulation and hold considerable potential for cell-based therapies targeting human malignancies (1–4). This notion is corroborated by the observation that, among patients with leukemia undergoing hematopoietic stem cell transplantation, the antileukemic effect of the transplant was significantly greater when the donor NK cells exhibited a killer inhibitory receptor (KIR) profile that predicted a higher cytotoxicity against the leukemic cells of the recipient (3;5–7). Moreover, allogeneic NK cells might be beneficial when directly infused into patients, a procedure that was shown to induce clinical remission in patients with high-risk acute myeloid leukemia (AML) (8). Infusions of NK cells have also been proposed as a means to improve the treatment of other cancers (9).
Because NK cells represent a small fraction of peripheral blood mononuclear cells, generating them in numbers sufficient to meet clinical requirements, especially if multiple infusions are planned, is problematic. Hence, NK cell-based therapies would greatly benefit from reliable methods to produce large numbers of fully functional NK cells ex vivo. Unlike T and B lymphocytes, which readily respond to a variety of stimuli, NK cells typically do not undergo sustained proliferation. Indeed, their reported proliferative responses to cytokines with or without coculture with other cells have generally been modest and of short duration in most studies (10–16).
We previously found that the K562 leukemia cell line genetically modified to express membrane-bound interleukin (IL)-15 and 41BB ligand specifically activates NK cells, drives them into the cell cycle and allows their genetic modification (17). In this study, we determined the capacity of NK cells stimulated by contact with K562-mb15-41BBL cells to exert anti-AML cytotoxicity.
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We found that K562-mb15-41BBL cells induce sustained and specific proliferation of human NK cells. NK cell expansion was observed in all donors tested, including patients with acute leukemia undergoing therapy, with no apparent proliferative advantage of any particular NK cell subset. Gene expression of NKAES-NK cells was markedly different than that of unstimulated and IL-2-stimulated cells, not only in regards to their expression of cell proliferation-associated genes but also in that of molecules that might regulate NK-cell function and their interaction with other cell types. NKAES-NK cells had powerful cytotoxicity against AML cell lines and AML cells from patients, and were more potent than unstimulated or IL-2-activated NK cells from the same donors. Based on these findings, and on the effectiveness of NKAES-NK cells in murine models of AML, we developed a Master Cell Bank of K562-mb15-41BBL cells under cGMP guidelines, and demonstrated that large-scale expansion and activation of human NK cells for clinical studies was feasible, producing expansions of CD56+CD3– cells that were even higher than those observed in the initial small-scale experiments while maintaining high anti-AML cytotoxicity.
IL-2 can induce proliferative responses in human NK cells but only a minor fraction sustains continued growth (10;26;27). Conceivably, some NK-cell subsets might be more responsive, as suggested by early reports of up to 50-fold expansion after culture with IL-2 for 2 weeks of an NK subset that adheres to plastic (28–31). It is unclear, however, whether some CD3+ cells might have had, at least in part, contributed to the increased cell numbers (29;30). More recently, anti-CD3 and IL-2 reportedly induced 190-fold NK expansions after 21 days from the blood of healthy individuals (32) and, surprisingly, 1600-fold expansions after 20 days from that of patients with myeloma (25). However, these cells’ cytotoxicity against K562 cells was <10% at 1 : 1 E : T (25), a ratio at which NKAES-NK cells from healthy donors or leukemia patients had a median cytotoxicity of 69% cells. Our results with IL-2 alone or in combination with other cytokines are in line with those of earlier reports (10;26;27;33). Indeed, most investigators have indicated that sustained expansions of CD56+CD3– cells require additional signals (14;16), such as the presence of B-lymphoblastoid cells (26;34;35). B-lymphoblastoid cells, however, also induce vigorous expansions of T lymphocytes, whereas NKAES cultures do not stimulate T-cell proliferation. In the setting of allogeneic NK-cell therapy, this could be an important practical advantage as it would facilitate the complete removal of residual T cells at the end of the cultures (to avoid the risk of graft-versus-host disease). Because K562-mb15-41BBL cells are lethally-irradiated before culture and they are lysed by the expanding NK cells, the risk of infusing viable K562-mb15-41BBL is negligible. Nevertheless, we have incorporated safeguards in our clinical protocol. We prepare cultures of irradiated K562-mb15-41BBL cells, and monitor their growth and DNA-synthesis rate. We also test for the presence of viable K562-mb15-41BBL cells at the end of the culture by flow cytometry, using GFP as a marker. The clinical product is released only if there is no cell growth and no viable of K562-mb15-41BBL cell at the end of the cultures.
Most patients with AML respond to initial treatment and achieve remission, but occult resistant leukemia persists in approximately half of the patients, leading to overt (and usually fatal) relapse (36;37). NK cell infusions have shown to be clinically effective in patients with high-risk AML (8); they are being considered for the therapy of other hematological malignancies (9;38). Conceivably, NK-cell therapy will be most powerful when the number of NK cells infused is sufficiently high to produce a high E : T ratio. In our murine models of AML, multiple injections of NKAES-derived cells were required to eradicate leukemia and achieve long-term remissions. The number of NK cells that can be generated with the method that we describe should meet the requirement for a high E : T ratio, particularly in the setting of minimal residual disease, and allow multiple NK cell infusions. We found that administration of IL-2 significantly prolonged the survival of NKAES-NK cells in immunodeficient mice. It is possible that other cytokines not yet available for clinical studies, such as IL-15, might prove to be superior for this purpose. Of note, it was shown in clinical studies that lymphodepletion of the recipients, a procedure essential to ensure prolonged engraftment of the infused cells (39), resulted in high levels of serum IL-15 (8).
Although infusion of allogeneic unstimulated or IL-2-stimulated NK cells has proven to be safe, with no significant graft-versus-host disease detected, the safety of NKAES-NK cell infusions must be established. To this end, we have begun a Phase I dose-escalation clinical study of haploidentical NKAES-NK cells in patients with refractory leukemia. In addition to AML and other hematologic malignancies, some solid tumors should also be susceptible to NK cell cytotoxicity (9). Therefore, patients with these malignancies could also be eligible for clinical studies of NK cell therapy.
ADOPTIVE T CELL THERAPY: HARNESSING THE IMMUNE SYSTEM TO FIGHT CANCER
Immunotherapy — using one’s immune system to treat a disease — has been long lauded as the “magic bullet” of cancer treatments, one that can be more effective than the conventional therapies of surgery, radiation or chemotherapy. One specific type of immunotherapy, called adoptive T cell therapy, is demonstrating promising results for blood cancers and may have potential against other types of cancers, too.
In adoptive T cell therapy, T cells (in blue, above) are extracted from the patient and modified to recognize unique cancer markers and attack the cells carrying those markers. They are then reinfused back into the patient, where they can kill cancer cells throughout the body. (Photo credit: Lawrence Berkeley Laboratory)
What is adoptive T cell therapy and how does it work to treat cancer?
Every day, our immune system works to recognize and destroy abnormal, mutated cells. But the abnormal cells that eventually become cancer are the ones that slip past this defense system. The idea behind this therapy is to make immune cells (specifically, T lymphocytes) sensitive to cancer-specific abnormalities so that malignant cells can be targeted and attacked throughout the body.
Who would be good candidates for this type of therapy?
Currently, adoptive T cell therapy is mostly used to treat lymphoma and lymphoid leukemia, because these cancer cells have unique surface markers that we can reprogram T cells to recognize and attack. However, we also studying how to adapt this approach to treat other cancers as well, including myeloid leukemia, multiple myeloma and solid tumors.
What happens to the patient during this therapy?
First, we collect the patient’s own T cells from the bloodstream, which takes about four hours. The cells are then modified to recognize the patient’s cancer; a two- to three-week process in our laboratories. They are then frozen for later use as needed.
While the T cells are being modified, the patient undergoes an autologous stem cell transplant. Afterward, the re-engineered T cells are infused back into the patient so that they can kill any residual cancer cells that remained after the transplant. Depending on the type of cancer, its stage, the patient’s health and other factors, some patients may receive the modified T cell infusions shortly after their transplant; others may get their infusions later on, when tests showed that the cancer has relapsed.
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The Application of Natural Killer Cell Immunotherapy for the Treatment of Cancer
Natural killer (NK) cells are essential components of the innate immune system and play a critical role in host immunity against cancer. Recent progress in our understanding of NK cell immunobiology has paved the way for novel NK cell-based therapeutic strategies for the treatment of cancer. In this review, we will focus on recent advances in the field of NK cell immunotherapy, including augmentation of antibody-dependent cellular cytotoxicity, manipulation of receptor-mediated activation, and adoptive immunotherapy with ex vivo-expanded, chimeric antigen receptor (CAR)-engineered, or engager-modified NK cells. In contrast to T lymphocytes, donor NK cells do not attack non-hematopoietic tissues, suggesting that an NK-mediated antitumor effect can be achieved in the absence of graft-vs.-host disease. Despite reports of clinical efficacy, a number of factors limit the application of NK cell immunotherapy for the treatment of cancer, such as the failure of infused NK cells to expand and persist in vivo. Therefore, efforts to enhance the therapeutic benefit of NK cell-based immunotherapy by developing strategies to manipulate the NK cell product, host factors, and tumor targets are the subject of intense research. In the preclinical setting, genetic engineering of NK cells to express CARs to redirect their antitumor specificity has shown significant promise. Given the short lifespan and potent cytolytic function of mature NK cells, they are attractive candidate effector cells to express CARs for adoptive immunotherapies. Another innovative approach to redirect NK cytotoxicity towards tumor cells is to create either bispecific or trispecific antibodies, thus augmenting cytotoxicity against tumor-associated antigens. These are exciting times for the study of NK cells; with recent advances in the field of NK cell biology and translational research, it is likely that NK cell immunotherapy will move to the forefront of cancer immunotherapy over the next few years.
Natural killer (NK) cell-mediated cytotoxicity contributes to the innate immune response against various malignancies, including leukemia (1, 2). The antitumor effect of NK cells is a subject of intense investigation in the field of cancer immunotherapy. In this review, we will focus on recent advances in NK cell immunotherapy, including
augmentation of antibody-dependent cytotoxicity,
manipulation of receptor-mediated activation, and
adoptive immunotherapy with ex vivo-expanded,
chimeric antigen receptor (CAR)-engineered, or
engager-modified NK cells.
Biology of NK Cells Relevant to Adoptive Immunotherapy
Natural killer cells are characterized by the lack of CD3/TCR molecules and by the expression of CD16 and CD56 surface antigens. Around 90% of circulating NK cells are CD56dim, characterized by their distinct ability to mediate cytotoxicity in response to target cell stimulation (3, 4). This subset includes the alloreactive NK cells that play a central role in targeting leukemia cells in the setting of allogeneic hematopoietic stem cell transplant (HSCT) (5). The remaining NK cells, predominantly housed in lymphoid organs, are CD56bright, and although less mature (“unlicensed”) (3, 6, 7), they have a greater capability to secrete and respond to cytokines (8, 9). CD56bright and CD56dim NK cells are also distinguished by their differential expression of FcγRIII (CD16), an integral determinant of NK-mediated antibody-dependent cellular cytotoxicity (ADCC), with CD56dim NK cells expressing high levels of the receptor, while CD56bright NK cells are CD16 dim or negative (6). In contrast to T and B lymphocytes, NK cells do not express rearranged, antigen-specific receptors; rather, NK effector function is dictated by the integration of signals received through germ-line-encoded receptors that can recognize ligands on their cellular targets. Functionally, NK cell receptors are classified as activating or inhibitory. NK cell function, including cytotoxicity and cytokine release, is governed by a balance between signals received from inhibitory receptors, notably the killer Ig-like receptors (KIRs) and the heterodimeric C-type lectin receptor (NKG2A), and activating receptors, in particular the natural cytotoxicity receptors (NCRs) NKp46, NKp30, NKp44, and the C-type lectin-like activating immunoreceptor NKG2D (9).
The inhibitory KIRs (iKIRs) with known HLA ligands include KIR2DL2 and KIR2DL3, which recognize the HLA-C group 1-related alleles characterized by an asparagine residue at position 80 of the α-1 helix (HLA-CAsn80); KIR2DL1, which recognizes the HLA-C group 2-related alleles characterized by a lysine residue at position 80 (HLA-CLys80); and KIR3DL1, which recognizes the HLA-Bw4 alleles (9, 10). NK cells also express several activating receptors that are potentially specific for self-molecules. KIR2DS1 has been shown to interact with group 2 HLA-C molecules (HLA-C2), while KIR2DS2 was recently shown to recognize HLA-A*11 (10, 11). Hence, these receptors require mechanisms to prevent inadvertent activation against normal tissues, processes referred to as “tolerance to self.” Engagement of iKIR receptors by HLA class I leads to signals that block NK-cell triggering during effector responses. These receptors explain the “missing self” hypothesis, which postulates that NK cells survey tissues for normal levels of the ubiquitously expressed MHC class I molecules (12, 13). Upon cellular transformation or viral infection, surface MHC class I expression on the cell surface is often reduced or lost to evade recognition by antitumor T cells. When a mature NK cell encounters transformed cells lacking MHC class I, their inhibitory receptors are not engaged, and the unsuppressed activating signals, in turn, can trigger cytokine secretion and targeted attack of the virus-infected or transformed cell (13, 14). In parallel, cellular stress and DNA damage (occurring in cells during viral or malignant transformation) results in upregulation of “stress ligands” that can be recognized by activating NK receptors. Thus, human tumor cells that have lost self-MHC class I expression or bear “altered-self” stress-inducible proteins are ideal targets for NK recognition and killing (14–16). NK cells directly kill tumor cells through several mechanisms, including release of cytoplasmic granules containing perforin and granzyme (16–18), expression of tumor necrosis factor (TNF) family members, such as FasL or TNF-related apoptosis-inducing ligand (TRAIL), which induce tumor cell apoptosis by interacting with their respective receptors Fas and TRAIL receptor (TRAILR) (16–19) as well as ADCC (9).
Interaction Between Natural Killer Cells and Other Immune Subsets
Increasing understanding of NK cell biology and their interaction with other cells of the immune system has led to several novel immunotherapeutic approaches as discussed in this review. NK cells produce cytokines that can exert regulatory control of downstream adaptive immune responses by influencing the magnitude of T cell responses, specifically T helper-1 (TH1) function (20). NK cell function, in turn, is regulated by cytokines, such as IL-2, IL-15, IL-12, and IL-18 (21), as well as by interactions with other cell types, such as dendritic cells, macrophages, and mesenchymal stromal cells (10, 22, 23). IL-15 has emerged as a pivotal cytokine required for NK cell development and maintenance. Whereas mice deficient in IL-2 (historically the cytokine of choice to expand and activate NK cells) have normal NK cells, IL-15-deficient mice lack NK cells (24).
Several cytokines are also known to inhibit NK cell activation and function, thus playing a crucial role in tumor escape from NK immune surveillance. Recently, considerable attention has been paid to the inhibitory effects of transforming growth factor-beta (TGF-β) and IL-10 on NK cell cytotoxicity (12, 25, 26). Several groups have shown that secretion of TGF-β by tumor cells results in downregulation of activating receptors, such as NKp30 and NKG2D, with resultant NK dysfunction (25,26). Similarly, IL-10 production by acute myeloid leukemia (AML) blasts induces upregulation of NKG2A with significant impairment in NK function (3).
Modulation of Antibody-Dependent Cellular Cytotoxicity
The CD56dim subset of NK cells expresses the Fcγ receptor CD16, through which NK cells mount ADCC, providing opportunities for its modulation to augment NK effector function (27, 28). In fact, a number of clinically approved therapeutic antibodies targeting tumor-associated antigens (such as rituximab or cetuximab) function at least partially through triggering NK cell-mediated ADCC. Several studies using mouse tumor models have established that efficient antibody–Fc receptor (FcR) interactions are essential for the efficacy of monoclonal antibody (mAb) therapy, a mainstay of cancer therapy (28, 29). Based on this premise, Romain et al. successfully engineered the Fc region of the IgG mAb, HuM195 targeting the AML leukemia antigen CD33, by introducing the triple mutation S293D/A330L/I332E (DLE). Using timelapse imaging microscopy in nanowell grids (TIMING, a method of analyzing kinetics of thousands of NK cells and mAb-coated targets), they demonstrated that the DLE-HuM195 antibody increased both the quality and quantity of NK cell-mediated ADCC by recruiting NK cells to participate in cytotoxicity via CD16-mediated signaling. NK cells encountering DLE-HuM195-coated targets induced rapid target cell apoptosis by promoting conjugation to multiple target cells (leading to increased “serial killing” of targets), thus inducing apoptosis in twice the number of targets as the wild-type mAb (27).
Additional approaches under investigation to enhance NK cell-mediated ADCC include antibody engineering and therapeutic combination of antibodies predicted to have synergistic activity. For example, mogamulizumab (an anti-CCR4 mAb recently approved in Japan) is defucosylated to increase binding by FcγRIIIA and thereby enhances ADCC. Mogamulizumab successfully induced ADCC activity against CCR4-positive cell lines and inhibited the growth of EBV-positive NK-cell lymphomas in a murine xenograft model (30). These findings suggest that mogamulizumab may be a therapeutic option against EBV-associated T and NK-lymphoproliferative diseases (30). Obinutuzumab (GA101) is a novel type II glycoengineered mAb against CD20 with increased FcγRIII binding and ADCC activity. In contrast to rituximab, GA101 induces activation of NK cells irrespective of their inhibitory KIR expression, and its activity is not negatively affected by KIR/HLA interactions (31). These data show that modification of the Fc fragment to enhance NK-mediated ADCC can be an effective strategy to augment the efficacy of therapeutic mAbs (31).
Although enhanced NK-mediated ADCC occurs in the presence of certain mAbs, in the case of non-engineered mAbs (such as rituximab), this NK-mediated cytotoxicity is typically still under the jurisdiction of KIR-mediated inhibition. However, ADCC responses can be potentiated in vitro in the presence of antibodies that block NK cell inhibitory receptor interaction with MHC class I ligands (32). These include the use of anti-KIR Abs to block the interaction of iKIRs with their cognate HLA class I ligands. To exploit this pathway pharmacologically, a fully humanized anti-KIR mAb 1-7F9 (IPH2101) (33) with the ability to block KIR2DL1/L2/L3 and KIR2DS1/S2 was generated. In vitro, anti-KIR mAbs can augment NK cell-mediated lysis of HLA-C-expressing tumor cells, including autologous AML blasts and autologous CD138+ multiple myeloma (MM) cells (34). Additionally, in a dose-escalation phase 1 clinical trial in elderly patients with AML, 1-7F9 mAb was reported to be safe and could block KIRs for prolonged periods (35). A recombinant version of this mAb with a stabilized hinge (lirilumab) was recently developed. Lirilumab is a fully humanized IgG4 anti-KIR2DL1, -L2, -L3, -S1, and -S2 mAb. The iKIRs targeted by lirilumab collectively recognize virtually all HLA-C alleles, and the blockade of the three KIR2DLs allows targeting of every patient without the need for prior HLA or KIR typing (33, 34). Furthermore, the combination of an anti-KIR mAb with the immunomodulatory drug lenalidomide was shown to potentiate ADCC and is being tested in a phase 1 clinical trial in patients with MM [NCT01217203 (35)]. A potential concern is related to how inhibitory KIR blockade may impact on the ability of NK cells to discriminate self, healthy cells from abnormal virally infected or cancerous cells. Preliminary in vitro data suggest that Ab blockade of iKIRs will preferentially augment the ADCC response, without increasing cytotoxicity against self healthy cells (32). It is reassuring that in the IPH2101 phase 1 studies, no alterations in the expression of major inhibitory or activating NK receptors or frequencies of circulating peripheral lymphocytes were reported, indicating that the Ab does not induce clinically significant targeting of normal cells by NK cells (35). Lin et al. recently reported on the application of an agonistic NK cell-targeted mAb to augment ADCC (36). Following FcR triggering during ADCC, expression of the activation marker CD137 is increased. Agonistic antibodies targeting CD137 have been reported to augment NK-cell function, including degranulation, secretion of IFN-γ, and antitumor cytotoxicity in in vitro and in vivo preclinical models of tumor (36–39). The combination of the agonistic anti-CD137 antibody with rituximab is currently being evaluated in a phase 1 trial in patients with lymphoma [NCT01307267 (35–37)].
Other factors, such as specific CD16 polymorphisms and NKG2D engagement, can also influence ADCC, with certain polymorphisms (such as FcγRIIIa-V158F polymorphism) resulting in a stronger IgG binding (40). These findings are clinically relevant, as supported by the observation that patients with non-Hodgkin lymphoma (NHL) with the FcγRIIIa-V158F polymorphism experienced improved clinical response to rituximab (41, 42). In summary, several antibody combinations designed to boost ADCC have shown promising results in preclinical and early clinical trials, thus warranting further study of this strategy to enhance NK cell activity against tumor cells.
Adoptive Transfer of Autologous NK Cells
The early studies of adoptive NK cell therapy focused on enhancing the antitumor activity of endogenous NK cells (43). Initial trials of adoptive NK therapy in the autologous setting involved using CD56 beads to select NK cells from a leukapheresis product and subsequently infusing the bead-selected autologous NK cells into patients (43, 44). Infusions were followed by administration of systemic cytokines (most commonly IL-2) to provide additional in vivo stimulation and support their expansion. This strategy met with limited success due to a combination of factors (44). Although cytokine stimulation promoted NK cell activation and resulted in greater cytotoxicity against malignant targets in vitro, only limited in vivoantitumor activity was observed (43–45). Similar findings were observed when autologous NK cells and systemic IL-2 were given as consolidation treatment to patients with lymphoma who underwent autologous BMT (46). The poor clinical outcomes observed with adoptive transfer of ex vivo activated autologous NK cells followed by systemic IL-2 were attributed to three factors: (1) development of severe life-threatening side effects, such as vascular leak syndrome as a result of IL-2 therapy; (2) IL-2-induced expansion of regulatory T cells known to directly inhibit NK cell function and induce activation-induced cell death (47–49); and (3) lack of antitumor effect related to the inhibition of autologous NK cells by self-HLA molecules. Strategies to overcome this autologous “checkpoint,” thus redirecting autologous NK cells to target and kill leukemic blasts are the subject of intense investigation (33–35). These include the use of anti-KIR Abs (such as the aforementioned lirilumab) to block the interaction of inhibitory receptors on the surface of NK cells with their cognate HLA class I ligand.
Exploiting the Alloreactivity of Allogeneic NK Cells – Adoptive Immunotherapy and Beyond
An alternative strategy is to use allogeneic instead of autologous NK cells, thus taking advantage of the inherent alloreactivity afforded by the “missing self” concept (13). Over the past decade, adoptive transfer of ex vivo-activated or -expanded allogeneic NK cells has emerged as a promising immunotherapeutic strategy for cancer (24, 50–52). Allogeneic NK cells are less likely to be subject to the inhibitory response resulting from NK cell recognition of self-MHC molecules as seen with autologous NK cells. A number of studies have shown that infusion of haploidentical NK cells to exploit KIR/HLA alloreactivity is safe and can mediate impressive clinical activity in some patients with AML (50–52). In fact, algorithms have been developed to ensure selection of stem cell donors with the greatest potential for NK cell alloreactivity for allogeneic HSCT (50).
Promising results in the HSCT setting suggest that the application of this strategy in the non-transplant setting may be a plausible option. Miller et al. were among the first to show that adoptive transfer of ex vivo-expanded haploidentical NK cells after lymphodepleting chemotherapy is safe, and can result in expansion of NK cells in vivo without inducing graft-vs.-host disease (GVHD) (50). In a phase I dose-escalation trial, 43 patients with either hematologic malignancies (poor prognosis AML or Hodgkin lymphoma) or solid tumor (metastatic melanoma or renal cell carcinoma) received up to 2 × 107cells/kg of haploidentical NK cells following either low intensity [low-dose cyclophosphamide (Cy) and methylprednisolone or fludarabine (Flu)] or high intensity regimens (Hi-Cy/Flu). All patients received subcutaneous IL-2 after NK cell infusion. Whereas adoptively infused NK cells persisted only transiently following low intensity regimens, AML patients who received the more intense Hi-Cy/Flu regimen had a marked rise in endogenous IL-15 associated with expansion of donor NK cells and induction of complete remission (CR) in five of 19 very high-risk patients. The superior NK expansion observed after high-dose compared to low-dose chemotherapy was attributed to a combination of factors including prevention of host T cell-mediated rejection and higher levels of cytokines, such as IL-15. These findings provided the first evidence that haploidentical NK cells are safe and can persist and expand in vivo, supporting the proof of concept that NK cells may be applied for the treatment of selected malignancies either alone or as an adjunct to HSCT (50).
Another pivotal pilot study, the NKAML trial (Pilot Study of Haploidentical NK Transplantation for AML), reported that infusion of KIR-HLA-mismatched donor NK cells can reduce the risk of relapse in childhood AML (51). Ten pediatric patients with favorable or intermediate risk AML in first CR were enrolled following completion of 4–5 cycles of chemotherapy. All patients received a low-dose conditioning regimen consisting of Cy/Flu prior to infusion of NK cells (median, 29 × 106/kg NK cells) from a haploidentical donor, followed by six doses of IL-2. NK infusions were well tolerated with limited non-hematologic toxicity. All patients had transient engraftment of NK cells for a median of 10 days (range 2–189 days) with significant expansion of KIR-mismatched NK cells. With a median follow-up of 964 days, all patients remained in remission, suggesting that donor-recipient HLA-mismatched NK cells may reduce the risk of relapse in childhood AML (51).
Other strategies currently under investigation include the infusion of KIR-ligand-mismatched haploidentical NK cells as part of the pre-HSCT conditioning regimen (NCT00402558), and NK cell infusion to prevent relapse or as therapy for minimal residual disease in patients after haploidentical HSCT (NCT01386619).
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Human NK Cell Lines as a Source of NK Immunotherapy
The adoptive transfer of NK cell lines has several theoretical advantages over the use of patient- or donor-derived NK cells. These are primarily related to the lack of expression of iKIRs, presumed lack of immunogenicity, ease of expansion and availability as an “off-the-shelf” product (85). Several human NK cell lines, such as NK-92 and KHYG-1, have been documented to exert antitumor activity in both preclinical and clinical settings (86–88). NK-92, the most extensively characterized NK-cell line, was established in 1994 from the PB of a male Caucasian patient with NHL. NK-92 cells are IL-2-dependent, harbor a CD2+CD56+CD57+ phenotype and exert potent in vitro cytotoxicity (86). Infusion of up to 1010 cells/m2NK-92 cells into patients with advanced lung cancer and other advanced malignancies was well tolerated and the cells persisted for a minimum of 48 h with encouraging clinical responses (86, 88–91). However, potential limitations of using NK cell lines, such as NK-92 cells, include the requirement for irradiation to reduce the risk of engrafting cells with potential in vivo tumorigenicity, and the need for pre-infusion conditioning to avoid host rejection. Furthermore, infusion of allogeneic NK cell lines may induce T and B cell alloimmune responses, limiting their in vivo persistence and precluding multiple infusions. A number of studies are testing NK-92 cells (Neukoplast®) in patients with solid tumors, such as Merkel cell cancer and renal cell carcinoma, as well as in hematological malignancies (85).
While results from clinical studies of NK cell adoptive therapy are encouraging (48–52, 70), significant gaps remain in our understanding of the optimal conditions for NK cell infusion. Based on the pioneering work from Rosenberg et al. demonstrating the importance of lymphodepletion to support the expansion of tumor-infiltrating T cells (92) and given its emergence as a key determinant of efficacy with CAR therapy, several groups are actively investigating the ideal preparative regimen to promote the expansion and persistence of adoptively infused NK cells (53, 69, 70, 75). Available data support the use of high-dose Cy/Flu regimen as the frontrunner, considering it is reasonably well tolerated and shown to support the in vivo expansion of NK cells (51, 70). IL-15 is an ideal candidate cytokine for the expansion of NK cells in vivo, especially since it does not promote expansion of regulatory T cells (66), which have been shown to suppress NK cell effector function in IL-2-based trials (69, 70). In a recent phase 1 study in patients with metastatic melanoma or renal cell carcinoma, rhIL-15 was shown to activate NK cells, monocytes, γδ, and CD8 T cells (93). However, as an intravenous bolus dose, rhIL-15 proved too difficult to administer because of significant clinical toxicities (93). Based on these promising data, alternative dosing strategies are being investigated, including continuous intravenous infusions. To this effect, systemic IL-15 along with infusion of donor NK cells are currently being tested in a phase I clinical trial for AML (NCT01385423).
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Bispecific and Trispecific Engagers
An innovative immunoglobulin-based strategy to redirect NK cytotoxicity towards tumor cells is to create either bispecific or trispecific antibodies (BiKE, TriKE) (113). BiKEs are constructed by joining a single-chain Fv against CD16 and a single-chain Fv against a tumor-associated antigen (BiKE), or two tumor-associated antigens (TriKE). Gleason et al. showed that bispecific (bscFv) CD16/CD19 and trispecific (tscFv) CD16/CD19/CD22 engagers directly trigger NK cell activation through CD16, significantly increasing NK cell cytolytic activity and cytokine production against various CD19-expressing B cell lines. The same group also developed and tested a CD16 × 33 BiKE in refractory AML and demonstrated that the potent killing by NK cells could overcome the inhibitory effect of KIR signaling (113, 114).
Notably, activated NK cells lose CD16 (FcRγIII) and CD62L through a metalloprotease called ADAM17, which is expressed on NK cells, which may in turn impact on the efficacy of Fc-mediated cytotoxicity (115). Romee et al. recently showed that selective inhibition of ADAM17 enhances CD16-mediated NK cell function by preserving CD16 on the NK cell surface, thus enhancing ADCC (115). Additionally, Fc-induced production of cytokines by NK cells exposed to rituximab-coated B cell targets can be further enhanced by ADAM17 inhibition. These findings support a role for targeting ADAM17 to prevent CD16 shedding and to improve the efficacy of therapeutic mAbs. The same group subsequently discovered that ADAM17 inhibition enhances CD16 × 33 BiKE responses against primary AML targets (114).
NK Cells – What Does the Future Hold?
Recent advances in the understanding of NK cell immunobiology have paved the way for novel and innovative anti-cancer therapies. Here, we have discussed a representation of these novel immunotherapeutic strategies to potentiate NK cell function and enhance antitumor activity including ADCC-inducing mAbs, ex vivo activated or genetically modified NK cells and bi- or trispecific engagers (Figure 1).
For the first time, treatment with genetically engineered T-cells has used CD4 T-cells instead of the CD8 T-cells, which are used in the chimeric antigen receptor (CAR) T-cell approach. Early data suggest that this CD4 T-cell approach has activity against solid tumors, whereas the CAR T-cell approach so far has achieved dramatic success in hematologic malignancies.
In the new approach, CD4 T-cells were genetically engineered to target MAGE-A3, a protein found on many tumor cells. The treatment was found to be safe in patients with metastatic cancers, according to data from a phase 1 clinical study presented here at the American Association for Cancer Research (AACR) 2016 Annual Meeting.
“This is the first trial testing an immunotherapy using genetically engineered CD4 T-cells,” senior author Steven A. Rosenberg, MD, PhD, chief of the Surgery Branch at the National Cancer Institute (NCI), told Medscape Medical News.
Most approaches use CD8 T-cells. Although CD8 T-cells are known be cytotoxic and CD4 T-cells are normally considered helper cells, CD4 T-cells can induce tumor regression, he said.
Louis M. Weiner, MD, director of the Lombardi Comprehensive Cancer Center at Georgetown University, in Washington, DC, indicated that in contrast with CAR T-cells, these CD4 T-cells target proteins on solid tumors. “CAR T-cells are not tumor specific and do not target solid tumors,” he said.
Engineering CD4 Cells
Immunotherapy with engineered CD4 T-cells was personalized for each patient whose tumors had not responded to or had recurred following treatment with least one standard therapy. The immunotherapy was specific for patients in whom a specific human leukocyte antigen (HLA) — HLA-DPB1*0401 — was found to be expressed on their cells and whose tumors expressed MAGE-A3.
MAGE-A3 belongs to a class of proteins expressed during fetal development. The expression is lost in normal adult tissue but is reexpressed on tumor cells, explained presenter Yong-Chen William Lu, PhD, a research fellow in the Surgery Branch of the NCI.
Targeting MAGE-A3 is relevant, because it is frequently expressed in a variety of cancers, such as melanoma and urothelial, esophageal, and cervical cancers, he pointed out.
Researchers purified CD4 T-cells from the peripheral blood of patients. Next, the CD4 T-cells were genetically engineered with a retrovirus carrying the T-cell receptor (TCR) gene that recognizes MAGE-A3. The modified cells were grown ex vivo and were transferred back into the patient.
Clinical Results
Dr Lu presented data for 14 patients enrolled into the study: eight patients received cell doses from 10 million to 30 billion cells, and six patients received up to 100 billion cells.
This was similar to a phase 1 dose-finding study, except the researchers were seeking to determine the maximum number of genetically engineered CD4 T-cells that a patient could safely receive.
One patient with metastatic cervical cancer, another with metastatic esophageal cancer, and a third with metastatic urothelial cancer experienced partial objective responses. At 15 months, the response is ongoing in the patient with cervical cancer; after 7 months of treatment, the response was durable in the patient with urothelial cancer; and a response lasting 4 months was reported for the patient with esophageal cancer.
Dr Lu said that a phase 2 trial has been initiated to study the clinical responses of this T-cell receptor therapy in different types of metastatic cancers.
In his discussion of the paper, Michel Sadelain, MD, of the Memorial Sloan Kettering Cancer Center, New York City, said, “Although therapy with CD4 cells has been evaluated using endogenous receptor, this is the first study using genetically engineered CD4 T-cells.”
Although the study showed that therapy with genetically engineered T-cells is safe and efficacious at least in three patients, the mechanism of cytotoxicity remains unclear, Dr Sadelain indicated.
Comparison With CAR T-cells
CAR T-cells act in much the same way. CARs are chimeric antigen receptors that have an antigen-recognition domain of an antibody (the V region) and a “business end,” which activates T-cells. In this case, CD8 T-cells from the patients are used to genetically engineer T-cells ex vivo. In the majority of cases, dramatic responses have been seen in hematologic malignancies.
CARs, directed against self-proteins, result in on-target, off-tumor effects, Gregory L. Beatty, MD, PhD, assistant professor of medicine at the University of Pennsylvania, in Philadelphia, indicated when he reported the first success story of CAR T-cells in a solid pancreatic cancer tumor.
Side effects of therapy with CD4 T-cells targeting MAGE-A3 were different and similar to side effects of chemotherapy, because patients received a lymphodepleting regimen of cyclophosphamide and fludabarine. Toxicities included high fever, which was experienced by the majority of patients (12/14). The fever lasted 1 to 2 weeks and was easily manageable.
High levels of the cytokine interleukin-6 (IL-6) were detected in the serum of all patients after treatment. However, the elevation in IL-6 levels was not considered to be a cytokine release syndrome, because no side effects occurred that correlated with the syndrome, Dr Liu indicated.
He also indicated that future studies are planned that will employ genetically engineered CD4 T-cells in combination with programmed cell death protein 1–blocking antibodies.
This study was funded by Intramural Research Program of the National Institutes of Health. The NCI’s research and development of T-cell receptor therapy targeting MAGE-A3 are supported in part under a cooperative research and development agreement between the NCI and Kite Pharma, Inc. Kite has an exclusive, worldwide license with the NIH for intellectual property relating to retrovirally transduced HLA-DPB1*0401 and HLA A1 T-cell receptor therapy targeting MAGE-A3 antigen. Dr Lu and Dr Rosenberg have disclosed no relevant financial relationships.
American Association for Cancer Research (AACR) 2016 Annual Meeting: Abstract CT003, presented April 17, 2016.
Searches Related to immunotherapy using genetically engineered CD4 T-cells
To be effective for the treatment of cancer and infectious diseases, T cell adoptive immunotherapy requires large numbers of cells with abundant proliferative reserves and intact effector functions. We are achieving these goals using a gene therapy strategy wherein the desired characteristics are introduced into a starting cell population, primarily by high efficiency lentiviral vector-mediated transduction. Modified cells are then expanded using ex vivo expansion protocols designed to minimally alter the desired cellular phenotype. In this article, we focus on strategies to (1) dissect the signals controlling T cell proliferation; (2) render CD4 T cells resistant to HIV-1 infection; and (3) redirect CD8 T cell antigen specificity.
Adoptive T cell therapy is a form of transfusion therapy involving the infusion of large numbers of T cells with the aim of eliminating, or at least controlling, malignancies or infectious diseases. Successful applications of this technique include the infusion of CMV-or EBVspecific CTLs to protect immunosuppressed patients from these transplantation-associated diseases [1,2]. Furthermore, donor lymphocyte infusions of ex vivo-expanded allogeneic T cells have been used to successfully treat hematological malignancies in patients with relapsed disease following allogeneic hematopoietic stem cell transplant [3]. However, in many other malignancies and chronic viral infections such as HIV-1, adoptive T cell therapy has achieved inconsistent and/or marginal successes. Nevertheless, there are compelling reasons for optimism on this strategy. For example, the existence of HIV-positive elite non-progressors [4], as well as the correlation between the presence of intratumoral T cells and a favorable prognosis in malignancies such as ovarian [5,6] and colon carcinoma [7,8], provides in vivo evidence for the critical role of the immune system in controlling both HIV and cancer.
The key to successful adoptive immunotherapy strategies appears to consist of (1) using the “right” T cell type(s) and (2) obtaining therapeutically effective numbers of these cells without compromising their effector functions or their ability to engraft within the host. This article is focused on strategies employed in our laboratory to generate the “right” cell through genetic engineering approaches, with an emphasis on redirecting the antigen specificity of CD8 T cells, and rendering CD4 T cells resistant to HIV-1 infection. The article by Paulos et al. describes the evolving process of how to best obtain therapeutically effective numbers of the “right” cells by optimizing ex vivo cell expansion strategies.
Our laboratory’s overall strategy and flow plan for development and evaluation of engineered T cells is depicted in Fig. 1. We work almost exclusively with primary human T cells; little or no work is performed with conventional established cell lines. Thus, we benefit substantially from our close association with the UPenn Human Immunology Core. The Core performs leukaphereses on healthy donors 2–3 times a week, and provides purified peripheral blood mononuclear cell subsets, ensuring a constant influx of fresh human T cells into our laboratory. We have extensive experience in developing both bead- and cell-based artificial antigen presenting cells (aAPCs), as described in detail in the article by Paulos et al. The ability to genetically modify T cells at high efficiency is critical for virtually every project within the laboratory. We have adapted the lentiviral vector system described by Dull [15] for most, but not all, of the engineering applications in our laboratory.
CD4 T cells are the primary target of HIV-1, and decreasing CD4 T cell numbers is a hallmark of advancing HIV-1 disease [34]. Thus, strategies that protect CD4 T cells from HIV-1 infection in vivo would conceivably provide sufficient immunological help to control HIV-1 infection. Our early observations that CD3/CD28 costimulation resulted in improved ex vivo expansion of CD4 T cells from both healthy and HIV-infected donors, as well as enhanced resistance to HIV-1 infection [35,36], ultimately led to the first-in-human trial of lentiviral vector-modified CD4 T cells [37]. In this trial, CD4 T cells from HIV-positive subjects who had failed antiretroviral therapy were transduced with a lentiviral vector encoding an antisense RNA that targeted a 937 bp region in the HIV-1 envelope gene. Preclinical studies demonstrated that this antisense region, directed against the HIV-1NL4-3 envelope, provided robust protection from a broad range of both R5-and X4-tropic HIV-1 isolates [38]. One year after administration of a single dose of the gene-modified cells, four of the five enrolled patients had increased peripheral blood CD4 T cell counts, and in one subject, a 1.7 log decrease in viral load was observed. Finally, in two of the five patients, persistence of the gene-modified cells was detected one year post-infusion.
Since its identification as the primary co-receptor involved in HIV transmission, CCR5 has attracted considerable attention as a target for HIV therapy [42,43]. Indeed, “experiments of nature” have shown that individuals with a homozygous CCR5 Δ32 deletion are highly resistant to HIV-1 infection. Thus, we hypothesized that knocking out the CCR5 locus would generate CD4 T cells permanently resistant to infection by R5 isolates of HIV-1. To test this hypothesis we took advantage of zinc-finger nuclease (ZFN) technology [44]. ZFNs introduce sequencespecific double-strand DNA breakage, which is imperfectly repaired by non-homologous endjoining. This results in the permanent disruption of the genomic target, a process termed genome editing (Fig. 3).
Genetic modification of T cells to redirect antigen specificity is an attractive strategy compared to the lengthy process of growing T cell lines or CTL clones for adoptive transfer. Genetically modified, adoptively transferred T cells are capable of long-term persistence in humans [37, 46,47], demonstrating the feasibility of this approach. When compared to the months it can take to generate an infusion dose of antigen-specific CTL lines or clones from a patient, a homogeneous population of redirected antigen-specific cells can be expanded to therapeutically relevant numbers in about two weeks [3]. Several strategies are being explored to bypass the need to expand antigen-specific T cells for adoptive T cell therapy. The approaches currently studied in our laboratory involve the genetic transfer of chimeric antigen receptors and supraphysiologic T cell receptors.
Chimeric antigen receptors (CARs or T-bodies) are artificial T cell receptors that combine the extracellular single-chain variable fragment (scFv) of an antibody with intracellular signaling domains, such as CD3ζ or Fc(ε)RIγ [48–50]. When expressed on T cells, the receptor bypasses the need for antigen presentation on MHC since the scFv binds directly to cell surface antigens. This is an important feature, since many tumors and virus-infected cells downregulate MHCI, rendering them invisible to the adaptive immune system. The high-affinity nature of the scFv domain makes these engineered T cells highly sensitive to low antigen densities. In addition, new chimeric antigen receptors are relatively easy to produce from hybridomas. The key to this approach is the identification of antigens with high surface expression on tumor cells, but reduced or absent expression on normal tissues. Since one can redirect both CD4 and CD8 T cells, the T-body approach to immunotherapy represents a near universal “off the shelf” method to generate large numbers of antigen-specific helper and cytotoxic T cells.
Many T-bodies targeting diverse tumors have been developed [51], and four have been evaluated clinically [52–55]. Three of the four studies were characterized by poor transgene expression and limited T-body engraftment. However, in a study of metastatic renal cell carcinoma using a T-body directed against carbonic anhydrase IX [55], T-body-expressing cells were detectable in the peripheral blood for nearly 2 months post-administration.
The major goals in the T-body field currently are to optimize their engraftment and maximize their effector functions. Our laboratory is addressing both problems simultaneously through an in-depth study of the requirements for T-body activation. We hypothesize that their limited persistence is due to incomplete cell activation due to the lack of costimulation. While naïve T cells depend on costimulation through CD28 ligation to avoid anergy and undergo full activation in response to antigen, it is recognized that effector cells also require costimulation to properly proliferate and produce cytokines [56]. Previous studies have shown that providing CD28 costimulation is crucial for the antitumoral function of adoptively transferred T cells and T-bodies [57–59]. Unlike conventional T cell activation, which requires two discrete signals, T-bodies can be engineered to provide both costimulation and CD3 signaling through one binding event.
A different approach for redirecting specificity to T cells for adoptive immunotherapy involves the genetic transfer of full-length TCR genes. A T cell’s specificity for its cognate antigen is solely determined by its TCR. Genes encoding the α and β chains of a T cell receptor (TCR) can be isolated from a T cell specific for the antigen of interest and restricted to a defined HLA allele, inserted into a vector, and then introduced into large numbers of T cells of individual patients that share the restricting HLA allele as well as the targeted antigen. In 1999, Clay and colleagues from Rosenberg’s group at the National Cancer Institute were the first to report the transfer of TCR genes via a retroviral vector into human lymphocytes and to show that T cells gained stable reactivity to MART-1 [67]. To date, many others have shown that the same approach can be used to transfer specificity for multiple viral and tumor associated antigens in mice and human systems. These T cells gain effector functions against the transferred TCR’s cognate antigen, as defined by proliferation, cytokine production, lysis of targets presenting the antigen, trafficking to tumor sites in vivo, and clearance of tumors and viral infection.
In 2006, Rosenberg’s group redirected patients’ PBLs with the naturally occurring, MART-1- specific TCR reported in 1999 by Clay. In the first clinical trial to test TCR-transfer immunotherapy, these modified T cells were infused into melanoma patients [68]. While the transduced T cells persisted in vivo, only two of the 17 patients had an objective response to this therapy. One issue revealed by the study was the poor expression of the transgenic TCRs by the transferred T cells. Nonetheless, the results from this trial showed the potential of TCR transfer immunotherapy as a safe form of therapy for cancer and highlighted the need to optimize such therapy to attain maximum potency.
The adoptive immunotherapy field is advancing by a tried-and-true method: learning from disappointments and moving forward. Our ability to fully realize the therapeutic potential of adoptive T cell therapy is tied to a more complete understanding of how human T cells receive signals, kill targets, and modulate effective immune responses. Our goal is to perform labbased experiments that provide insight into how primary T cells function in a manner that will facilitate and enable adoptive T cell therapy clinical trials. Our ability to efficiently modify (and expand) T cells ex vivo provides the opportunity to deliver sufficient immune firepower where it has heretofore been lacking. Sustained transgene expression, coupled with enhanced in vivo engraftment capability, will move adoptive immunotherapy into a realm where longterm therapeutic benefits are the norm rather than the exception.
Genetic Modification of T Lymphocytes for Adoptive Immunotherapy
Adoptive transfer of T lymphocytes is a promising therapy for malignancies—particularly of the hemopoietic system—and for otherwise intractable viral diseases. Efforts to broaden the approach have been limited by the physiology of the T cells themselves and by a range of immune evasion mechanisms developed by tumor cells. In this review we show how genetic modification of T cells is being used preclinically and in patients to overcome these limitations, by incorporation of novel receptors, resistance mechanisms, and control genes. We also discuss how the increasing safety and effectiveness of gene transfer technologies will lead to an increase in the use of gene-modified T cells for the treatment of a wider range of disorders.
That gene transfer could be used to improve the effectiveness of T lymphocytes was apparent from the beginning of clinical studies in the field. T cells were the very first targets for genetic modification in human gene transfer experiments. Rosenberg’s group marked tumor-infiltrating lymphocytes ex vivo with a Moloney retroviral vector encoding neomycin phosphotransferase before reinfusing them and attempting to demonstrate selective accumulation at tumor sites. Shortly thereafter, Blaese and Anderson led a group that infused corrected T cells into two children with severe combined immunodeficiency due to ADA deficiency. While neither study was completely successful in terms of outcome, both showed the feasibility of ex vivo gene transfer into human cells and set the stage for many of the studies that followed. More recently, a second wave of interest in adoptive T cell therapies has developed, based on their success in the prevention and treatment of viral infections such as EBV and cytomegalovirus (CMV) and on their apparent ability to eradicate hematologic and perhaps solid malignancies1,2,3,4,5,6. There has been a corresponding increase in studies directed toward enhancing the antineoplastic and antiviral properties of the T cells. In this article we will review how gene transfer may be used to produce the desired improvements focusing on vectors and genes that have had clinical application.
Currently available viral and nonviral vector systems lack a pattern of biodistribution that would favor T cell transduction in vivo—as occurs, for example, with adenovectors and the liver or liposomal vectors and the lung. This lack of favorable biodistribution cannot yet be compensated for by the introduction of specific T-cell-targeting ligands into vectors. Hence, all T cell gene transfer studies conducted to date have used ex vivo transduction followed by adoptive transfer of gene-modified cells. This approach is inherently less attractive for commercial development than directin vivo gene transfer and has probably restricted interest in developing clinical applications using these cells. On the other hand, ex vivo transduction may be more readily controlled, characterized, and standardized than in vivo efforts and may ultimately produce a better defined final product (the transduced cell).
The gene products of suicide and coexpressed resistance genes are highly immunogenic and may induce immune-mediated rejection of the transduced cells. In one study, the persistence of adoptively transferred autologous CD8+ HIV-specific CTL clones modified to express the hygromycin phosphotransferase (Hy) gene and the herpesvirus thymidine kinase gene as a fusion gene was limited by the induction of a potent CD8+ class I MHC-restricted CTL response specific for epitopes derived from the Hy-tk protein126. Less immunogenic suicide and selection marker genes, preferably of human origin, may reduce the immunological inactivation of genetically modified donor lymphocytes. Human-derived prodrug-activating systems include the human folylpolyglutamate synthetase/methotrexate127, the deoxycytidine/cytosine arabinoside128, or the carboxylesterase/irinotecan129 systems. These systems do not activate nontoxic prodrugs but are based on enhancement of already potent chemotherapeutic agents. The administration of methotrexate to treat severe GVHD may not only kill transduced donor lymphocytes but may also have additional inhibitory activity on nontransduced but activated T cells.
Finally, endogenous proapoptotic molecules have been proposed as nonimmunogenic suicide genes. A chimeric protein that contains the FK506-binding protein FKBP12 linked to the intracellular domain of human Fas130 was recently introduced. Addition of the dimerizing prodrug induces Fas crosslinking with subsequent triggering of an apoptotic death signal.
Genetic engineering of T lymphocytes should help deliver on the promise of immunotherapies for cancer, infection, and autoimmune disease. Improvements in transduction, selection, and expansion techniques and the development of new viral vectors incapable of insertional mutagenesis will reduce the risks and further enhance the integration of T cell and gene therapies. Nonetheless, successful application of the proposed modifications to the clinical setting still requires many iterative studies to allow investigators to optimize the individual components of the approach.
Genetically modified T cells in cancer therapy: opportunities and challenges
Tumours use many strategies to evade the host immune response, including downregulation or weak immunogenicity of target antigens and creation of an immune-suppressive tumour environment. T cells play a key role in cell-mediated immunity and, recently, strategies to genetically modify T cells either through altering the specificity of the T cell receptor (TCR) or through introducing antibody-like recognition in chimeric antigen receptors (CARs) have made substantial advances. The potential of these approaches has been demonstrated in particular by the successful use of genetically modified T cells to treat B cell haematological malignancies in clinical trials. This clinical success is reflected in the growing number of strategic partnerships in this area that have attracted a high level of investment and involve large pharmaceutical organisations. Although our understanding of the factors that influence the safety and efficacy of these therapies has increased, challenges for bringing genetically modified T-cell immunotherapy to many patients with different tumour types remain. These challenges range from the selection of antigen targets and dealing with regulatory and safety issues to successfully navigating the routes to commercial development. However, the encouraging clinical data, the progress in the scientific understanding of tumour immunology and the improvements in the manufacture of cell products are all advancing the clinical translation of these important cellular immunotherapies.
The immune system is split into two arms: the innate and adaptive immune systems (Fig. 1). Through immune surveillance, any molecules that are identified as non-self are eliminated. Targets include not only virally infected cells but also transformed (tumour) cells, which can acquire antigenicity (see Box 1for a glossary of terms) and hence immunogenicity through the expression of neo-antigens that can be recognised as non-self. However, cancer cells have developed strategies to escape and suppress the immune system (Pinzon-Charry et al., 2005; Blankenstein et al., 2012), which results in a failure to initiate and maintain adequate antitumour immunity, and consequently facilitates tumour survival and progression. Strategies include tumour antigens being only weakly immunogenic. Alternatively, the tumour might downregulate or modulate the expression of antigens, thus evading immune-cell detection. In addition, tumours can suppress the immune response through the synthesis of various immune suppressants that have roles in maintaining self-tolerance, or that inhibit effector immune cell function. Tumour immune suppression affects all branches of the immune system and can result in tumour escape from the immune system.
Fig. 1. Cells of the innate and adaptive immune systems. The innate immune system provides an immediate response to foreign targets, with responses typically within minutes to hours. It consists of a number of soluble factors and proteins as well as a diverse set of cells, including granulocytes, macrophages, dendritic cells and natural killer cells. The second branch of the immune system is the adaptive or acquired immune system, which provides specific, long-lasting immune responses. The adaptive and innate immune systems are linked; for example dendritic cells are important adaptive immune system cell activators. The adaptive immune system consists of antibodies, B cells, and CD4+ and CD8+ T cells, and these enable a highly specific response against a particular target. Natural killer T cells and γδ T cells are cytotoxic lymphocytes that overlap both innate and adaptive immunity. Cells from both arms of the immune system are in development as potential cellular immunotherapies.
The concept of transferring T cells to patients (adoptive T-cell transfer) to treat disease has been established over many years through the ex vivo manipulation, expansion and reinfusion of T cells that target specific viruses, for example to treat viral infections, such as cytomegalovirus or Epstein Barr virus infections following haematopoietic stem cell transplantation (Walter et al., 1995; Heslop et al., 2010; Rooney and Leen, 2012). As described above, rare populations of tumour-antigen-specific T cells do exist and specifically can be isolated at the site of the tumour, and these are known as tumour infiltrating lymphocytes (TILs) (Kawakami et al., 1994; Robbins et al., 2013). TILs can be isolated from excised tumour tissue, cultivated, activated and expanded ex vivo, and, on reinfusion, have shown promising efficacy in the clinic, particularly in the treatment of melanoma (Rosenberg et al., 1988,Besser et al., 2013; Kvistborg et al., 2012; Dudley et al., 2013), supporting the therapeutic potential of tumour-specific T cells.
An alternative option to these approaches that is now starting to generate compelling clinical data is based on the premise that the antigen specificity of T cells can be manipulated by genetic modification and redirected to successfully target antigens that are expressed by tumours. In particular, T cells can be engineered to express modified TCRs (so-called TCR therapies) or protein-fusion-derived chimeric antigen receptors (CARs) that have enhanced antigen specificity (Fig. 3). These approaches could overcome the fundamental limitations associated with central and peripheral tolerance, and generate T cells that will be more efficient at targeting tumours without the requirement for de novo T-cell activation in the patient.
Fig. 3. Genetically modified TCRs for cancer immunotherapy. (A) T-cell response can be manipulated and redirected against cancer, with improved specificity and affinity for tumour antigens, via genetic engineering of the endogenous TCR. (B) Genetically modified TCR: gene sequences are transferred to the T cell to encode new TCR α and β chains with different peptide specificity. In addition, there can also be transmembrane changes (red bars). To minimise interchain mispairing with the endogenous TCR, modifications such as the addition of a disulphide bridge (ss) are made. (C) Alternatively, a fusion receptor can be generated, a chimeric antigen receptor (CAR). Typically, these consist of three parts: a recognition sequence [represented here by an antibody-derived single-chain variable fragment (scFv)], a transmembrane element and an intracellular bespoke signalling domain (CD3ζ), which also contains co-stimulatory molecules, such as CD28 and tumour necrosis factor receptors (TNFr) such as OX-40.
The feasibility of T-cell adoptive transfer was first reported nearly 20 years ago (Walter et al., 1995) and the field of T-cell therapies is now poised for significant clinical advances. Recent clinical trial successes have been achieved through multiple small advances, improved understanding of immunology and emerging technologies. As the key challenges of T-cell avidity, persistence and ability to exert the desired anti-tumour effects as well as the identification of new target antigens are addressed, a broader clinical application of these therapies could be achieved. As the clinical data emerges, the challenge of making these therapies available to patients shifts to implementing robust, scalable and cost-effective manufacture and to the further evolution of the regulatory requirements to ensure an appropriate but proportionate system that is adapted to the characteristics of these innovative new medicines.
Targeting Epithelial To Mesenchymal Transition (EMT) As A Therapy Strategy For Pancreatic Cancer
Curator: David Orchard-Webb, PhD
Epithelial to mesenchymal transition (EMT) is a mechanism by which cells of an epithelial phenotype dedifferentiate to a plastic mesenchymal phenotype. The epithelial cell rearranges its actin cytoskeleton from a cortical tight junction associated ring to form elongated stress fibres, redistributes and down regulates its cell-cell contacts, loses its polarity, and upregulates mesenchymal markers including α-smooth muscle actin (α-SMA) and vimentin [1]. The cell changes the composition of its extracellular matrix (ECM) contacts and secretes matrix metalloproteinases [2]. EMT has a role during development [3], chronic fibrotic disorders [4], and a postulated role in epithelial cancer metastasis [5].
Mouse mammary cell line induced to EMT with TGFβ1. Image Source: David Orchard-Webb.
Inflammatory signalling associated with pancreatitis is a driver of both pancreatic cancer and EMT [4,8]. Pancreatic cancer has a large stromal component that has therapeutic implications such as reduced drug tumour penetrance [9]. EMT is a mechanism of pancreatic stroma generation and may generate cancer stem-like cells [10]. This suggests a strategy for success in pancreatic cancer therapy. Cancer stem cells and stroma are major impediments to current therapeutics therefore targeting EMT is strategically viable to enhance their effectiveness.
A number of drug candidates have entered clinical trial which target EMT pathways. Curcumin which can reverse the EMT phenotype in vitro, has been shown to enhance the effectiveness of gemcitabine, the first FDA approved chemotherapeutic for pancreatic cancer [11]. Prism Pharma Co., Ltd. has developed a Wnt pathway inhibitor that may be effective in pancreatic cancer, however the associated phase I trial had to be terminated in 2015 due to low enrolment [14]. There are ongoing clinical trials targeting the hedgehog pathway which plays a role in EMT, in combination with gemcitabine and nab-paclitaxel (Abraxane) [12, 13].
STATs are transcription factors which are normally present in the cytoplasm and activated by inflammatory signalling associated with EMT which leads to their nuclear import [15]. STAT3 expression is maintained and constitutive activation has been reported in at least 30% of pancreatic cancers [6]. STAT3 is not active in normal pancreatic tissue but its activation is required in the early stages of pancreatic cancer progression. A means to eliminate STAT3 has been developed by Astrazeneca – stable systemically delivered siRNA which has completed phase I clinical trials [7]. This may prove beneficial in combination with standard chemotherapeutics.
In summary a number of EMT pathway targeting therapeutics are in development which have the potential to target pancreatic cancer stem cells, which could reduce cancer recurrence, and deplete the cancer associated stroma which should improve the penetrance of existing therapeutics and may help relieve suppression of the immune system by pancreatic tumours.
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Oncolytic viruses are novel anticancer agents, currently under investigation in Phase I–III clinical trials. Until recently, most studies have focused on the direct antitumor properties of these viruses, although there is now an increasing body of evidence that the host immune response may be critical to the efficacy of oncolytic virotherapy. This may be mediated via innate immune effectors, adaptive antiviral immune responses eliminating infected cells or adaptive antitumor immune responses. This report summarizes preclinical and clinical evidence for the importance of immune interactions, which may be finely balanced between viral and tumor elimination. On this basis, oncolytic viruses represent a promising novel immunotherapy strategy, which may be optimally combined with existing therapeutic modalities.
The anticancer activity of viruses has been reported throughout the 20th century. Developments in virology, genetic manipulation and molecular biology have led to a surge of research investigating viruses with oncolytic or antitumor properties over the last 15 years. Several oncolytic viruses are currently in Phase I–III clinical trials [1]. Until recently, despite the multitude of studies investigating direct viral effects upon cancer cells, relatively little attention had been paid to the interaction between oncolytic viruses and the immune system. We discuss the evidence supporting the view that the host immune response is critical to the efficacy of oncolytic virotherapy. The potential of oncolytic viruses to break immunological tumor tolerance, generating antitumor immunity, represents a novel avenue of immunotherapy.
Oncolytic viruses are self-replicating, tumor selective and directly lyze cancer cells [2]. They may be tumor selective in wild-type or attenuated forms or may be engineered to provide tumor selectivity. Naturally occurring oncolytic viruses include the double-stranded RNA reovirus and single-stranded RNA Newcastle disease virus (NDV) and vesicular stomatitis virus (VSV). By contrast, human DNA viruses, including adenoviruses, vaccinia and herpes simplex viruses (HSV) have been genetically modified in a variety of ways to provide tumor selectivity. A diverse range of mechanisms provide tumor specificity, including inactivation of antiviral defences, such as type I IFN responses in many cancer cells, viral deletions permitting replication only in tumor cells that can substitute for viral defects, tumor-selective uptake via upregulated or mutated receptors, and targeting to tumor promoters.
In the majority of clinical trials performed so far, oncolytic viruses have been administered via intratumoral injection. A smaller number of studies have examined regional or intravenous delivery. Clinical experience has demonstrated a favorable toxicity and safety profile and a number of tumor responses, although overall antitumor efficacy has been limited [1]. For example, ONYX-015, a modified adenovirus, has been used in clinical trials with response rates of 0–14% following intratumoral administration [3]. In view of the short history of oncolytic virotherapy, along with recent scientific advances in methods of viral delivery and enhancing antitumor potency, these low levels of single-agent clinical responses provide encouragement for the future.
An increasingly powerful body of evidence supports the ability of the immune system to modify the immunogenicity and behavior of tumors [4]. A host of tumor-associated antigens (TAA) have been characterized [5] and in a single tumor, tumor-infiltrating lymphocytes directed towards multiple TAAs can be identified [6]. Despite these antigenic differences, the antitumor immune response is commonly ineffectual. Tumors can subvert antitumor immunity, generating an immunosuppressive tumor microenvironment by a multitude of mechanisms. These include the induction of Treg cells, secretion of soluble immunosuppressive mediators including nitric oxide, IL-10 and TGF-β and recruitment of myeloid suppressor cells [4]. Matzinger’s ‘danger’ hypothesis proposes that the prime role of the immune system is to respond to cellular or tissue distress as opposed to nonself per se [7]. Several danger signals have been identified, including RNA, DNA, IFN-α, heat-shock proteins, uric acid and hyaluron, providing a mechanistic basis for this hypothesis [8]. On this basis, tumor-associated danger signals are critical to the generation of effective antitumor immunity. In addition to their ability to disrupt immune responses, tumors commonly lack such signals and successful tumor immunotherapy will probably to depend upon their provision. Oncolytic virotherapy represents a potent approach to cancer immunotherapy, combining the enhanced release of TAA via tumor cell death, in the context of danger signals (FIGURE 1).
Figure 1Concept of how oncolytic viral infection of tumor cells may lead to the generation of antitumor immune responses
The role of the innate immune response to cancer is double-edged. Chronic inflammatory changes can promote tumor progression via proliferative and proangiogenic signals [9], while by contrast, the infiltration of activated innate inflammatory cells can mediate tumor regression in vivo [10]. Manipulation of the immune environment within a tumor is a potentially critical strategy towards successful tumor immunotherapy [11].
Oncolytic viruses represent prime candidates to enhance the immunogenicity of the tumor microenvironment. As detailed below, oncolytic virotherapy may be immunomodulatory via tumor cell death, production of endogenous danger signals, the release of tumor-derived cytokines and direct effects upon cells of the innate immune system. Evidence from preclinical models suggests that an early influx of immune cells, including macrophages and natural killer (NK) cells, occurs in response to tumor viral therapy [12–14]. These changes within the tumor hold the potential to alter the pre-existing immunosuppressive microenvironment, in favor of the generation of therapeutic immune responses. Dendritic cells (DC), the prime antigen-presenting cells and a component of the innate immune response are critical for the subsequent generation of antigen-specific or adaptive immune responses. However, as discussed later, the outcome of the innate response is finely balanced between promotion of tumor clearance and viral clearance limiting efficacy.
Virally induced cell death would be expected to enhance the availability of TAA for uptake by DC. Indeed, viral infection of tumors has been reported to enhance the phagocytosis of tumor-derived material [15,16]. The relationship between the mode of cell death and tumor immunogenicity has, however, been controversial; the immunogenicity of tumors has been reported not to be affected by whether tumor cells are alive, apoptotic or necrotic [17]. Even if the mode of cell death is not an immunogenic determinant, the release of intrinsic cell factors, including heat-shock protein [18], uric acid [19] and bradykinin [20], can be identified as danger signals by DC. Oncolytic viral infection may mediate production of these factors. For example, tumor cell infection by a modified oncolytic adenovirus increases intracellular uric acid levels, activating DC [19].
An array of cytokines provides costimulation for T-cell responses, while by contrast, tumor-derived cytokines, including TGF-β and IL-10, have immunosuppressive properties. In addition, the tumor-derived proinflammatory cytokines VEGF, TNF-α and several chemokines have been linked to promotion of tumor growth [21]. Oncolytic viral infection is likely to alter the balance of cytokines produced and the nature of the subsequent immune response. We have investigated the release of cytokines following infection of melanoma cells with reovirus, a naturally occurring double-stranded RNA virus currently in clinical trials [22]. Reovirus was found to induce secretion of IL-8, RANTES and MIP-1α/β, which play a role in the recruitment of DC, neutrophils and monocytes [23], and of IL-6, which can inhibit the immunosuppressive function of Treg cells [24]. Reovirus additionally reduced tumor secretion of the immunosuppressive cytokine IL-10. The immunogenic property of tumor-conditioned media from reovirus-infected tumor cells (filtered to remove viral particles) was confirmed by their ability to activate DC.
DC & the response to viral infection
The immune system is adept at pathogen recognition and a host of receptors specific for pathogen-associated molecular patterns, including the toll-like receptors (TLR), have been identified [25]. Innate viral recognition can center around viral nucleic acids or viral proteins [25]. DC play a critical role in the early innate immune responses, reciprocally interacting with other innate immune cells, including NK cells [26]. In this context, oncolytic viruses can influence the nature of the innate tumor response. Reovirus-infected DC, for example, enhance NK cytotoxicity towards tumor cells [27].
The effect of viruses upon DC is virus specific: measles and a vaccinia virus strain impair DC phenotype and function [28,29], an oncolytic adenovirus has a neutral effect [30], while reovirus is directly stimulatory to DC [27]. Although the immunomodulatory effects of oncolytic viruses have been investigated to a limited degree, it follows that the immune consequences of therapy with different viruses will vary widely. In addition, the genetic modification of viruses to confer oncolytic specificity may involve interference with virulence genes whose function is to modify the antiviral immune response, including type I interferon response genes [2,31]; alteration of such immunomodulatory genes will alter the consequences of the immune interactions of these modified viruses.
Oncolytic viruses & adaptive antitumor immunity
The innate immune response is thought to provide an important link to the generation of adaptive immune responses. DC are key to this link, taking up TAA, integrating danger signals and presenting antigen in an appropriate costimulatory context to the adaptive arm of the immune system. An adaptive antitumor immune response requires activation of cytotoxic CD8 T cells by DC presenting tumor antigen on MHC class I molecules. The presentation of exogenous antigen in a MHC class I context is termed ‘cross-presentation’. Critically, virally infected cells have been shown to be superior at delivering nonviral antigen for cross-presentation and cross-priming adaptive immune responses in vivo [32]. Intriguingly, recent work has defined a role for TLR-4 receptor ligands (bacterially derived lipopolysaccharide) in enhancing cross-presentation [33]; a similar effect of viral as opposed to bacterial TLR ligands has yet to be explored. Inflammatory stimuli have additionally been shown to enhance antigen processing and the generation of MHC class II complexes, required for CD4+ T-cell help in adaptive immune responses [34,35]; such inflammatory stimuli could be provided by viral tumor infection. Oncolytic virotherapy may therefore enhance immune priming via multiple effects upon DC. There is an emerging body of data from murine and human preclinical research supporting the concept that the efficacy of oncolytic virotherapy is at least partially immune mediated and that antitumor immunity can be generated.
Overall, the antiviral humoral and cellular immune responses may have contrasting consequences. Methods of enhancing viral delivery to tumors or immunomodulation provide an opportunity to alter this balance in favor of therapeutic benefit.
Clinical trials & the immune response
Although preclinical studies have provided support for the concept that the efficacy of oncolytic virotherapy may be dependent upon the host immune response, there are limited data on the immune response following virotherapy from early clinical trials.
Studies of intratumoral administration have provided direct evidence of a cellular immunological response. In a Phase I trial of a second-generation oncolytic HSV expressing GM–CSF injected into subcutaneous metastases from a variety of tumor types, post-treatment biopsies revealed an extensive immune cell infiltrate [54]. Additionally, suggestive of an immune-mediated antitumor effect, was the observation of inflammation in uninjected tumor deposits in four of 30 treated patients. Similarly, in a study of intratumoral administration of a recombinant vaccinia–GM–CSF virus in patients with melanoma deposits, treated lesions were shown to have a dense immune cell infiltrate. The generation of antitumor immunity was implied by the regression of noninjected regional dermal metastases in association with an immune infiltrate in four of seven treated patients [55]. A Phase I study of injection of JX-594, a targeted poxvirus armed with GM–CSF, into primary and metastatic liver tumors has recently been reported with encouraging evidence of activity, with a partial response in three and stable disease in six of ten evaluable patients by Response Evaluation Criteria in Solid Tumors (RECIST) [56]. Consistent with a possible antitumor immune response was the durability of tumor responses. Notably, there was evidence of functional response in noninjected tumors in three of seven evaluable patients by Choi criteria for reduction in Hounsfield units (n = 2) and by reduced 18F-fluorodeoxyglucose (18FDG)-PET signal (n = 1). There was evidence of viral dissemination to noninjected tumor tissue. The responses in injected and noninjected tumor tissue could therefore have been mediated by direct viral oncolysis, antiviral immune responses towards virally infected cells or antitumor immune responses established in the injected lesions.
Oncolytic viruses have been combined with tumor vaccines in an attempt to exploit viral danger signals. Vaccinia virus–melanoma cell lysate vaccines were used in an adjuvant Phase III study of 700 patients following melanoma resection, with no improvement in recurrence or overall survival [57]. A series of clinical studies has been performed by Schirrmacher et al. using a live autologous tumor vaccine infected by NDV irradiated to render tumor cells nonviable [58]. A significant proportion of patients developed antitumor immune responses as assessed by a delayed-type hypersensitivity response to skin prick tests. Phase II studies have been performed in glioblastoma multiforme, melanoma, breast and colorectal cancer with improvements in overall survival by 20–36% at 2–5-year follow-up compared with historical controls. These studies suggest that oncolytic viruses can break immunological tumor tolerance, although Phase III studies are needed to confirm these findings.
Combination therapy may be the optimal context in which to exploit the immunotherapeutic potential of oncolytic viruses. A rationale exists for combination with existing immunotherapy strategies, along with conventional therapy.
Adoptive cellular therapy & viral delivery
The use of cell carriers to chaperone viral particles to the tumor is a promising innovation [51]. Cells of the immune system have proven particularly adept, including cytokine-activated killer cells [52] and T lymphocytes [36]. Adoptive cellular therapy has met with some clinical success, but has been limited by the trafficking to and survival of T cells in the tumor microenvironment [62]. In a mouse model, the combination of oncolytic virus delivery with antigen-specific adoptive T-cell therapy has been shown to improve upon either treatment modality alone [63]. Although yet to be tested in clinical trials, these findings are of significant translational potential.
Immunotherapy combinations
Immunotherapy approaches may be logically combined with virotherapy to enhance antitumor responses.
The host immune response will probably be critical to the efficacy of oncolytic virotherapy, although it is a fine balance between rapid viral elimination and innate and adaptive responses, which may mediate tumor regression. The rational design of combination therapy, modulating the immunological outcome, may hold the key to fulfilling the potential of these novel agents. Clinical trials should be designed to include specific assessment of immune responses to both tumor and viral antigens, and recognize the immunotherapeutic potential of virotherapy in terms of clinical end points and patient selection.
Oncolytic Viruses and Their Application to Cancer Immunotherapy
Oncolytic virus (OV) therapy is based on selective replication of viruses in cancer cells and their subsequent spread within a tumor without causing damage to normal tissue (1, 2). It represents a unique class of cancer therapeutics with distinct mechanisms of action. The activity of OVs is very much a reflection of the underlying biology of the viruses from which they are derived and the host–virus interactions that have evolved in the battle between pathogenesis and immunity. This provides a diverse set of activities that can be harnessed and manipulated. Typically, OVs fall into two classes: (i) viruses that naturally replicate preferentially in cancer cells and are nonpathogenic in humans often due to elevated sensitivity to innate antiviral signaling or dependence on oncogenic signaling pathways. These include autonomous parvoviruses, myxoma virus (MYXV; poxvirus), Newcastle disease virus (NDV; paramyxovirus), reovirus, and Seneca valley virus (SVV; picornavirus); and (ii) viruses that are genetically manipulated for use as vaccine vectors, including measles virus (MV; paramyxovirus), poliovirus (PV; picornavirus), and vaccinia virus (VV; poxvirus), and/or those genetically engineered with mutations/deletions in genes required for replication in normal but not in cancer cells including adenovirus (Ad), herpes simplex virus (HSV), VV, and vesicular stomatitis virus (VSV; rhabdovirus; refs. 1,3). Genetic engineering has facilitated the rapid expansion of OVs in the past two decades, enabling a broad range of potentially pathogenic viruses to be manipulated for safety and targeting (3). Many of the hallmarks of cancer described by Hanahan and Weinberg (4) provide a permissive environment for OVs; they include sustained proliferation, resisting cell death, evading growth suppressors, genome instability, DNA damage stress, and avoiding immune destruction. In addition, insertion of foreign sequences can endow further selectivity for cancer cells and safety, as well as altering virus tropism through targeting of translation with internal ribosome entry sites (IRES) or microRNAs (PV and VSV), transcription with cell-specific promoter/enhancers (Ad, HSV), or transduction with altered virus receptors (HSV, Ad, MV, and VSV; refs.1, 3). These strategies are also being used to target replication-deficient viral vectors for gene therapy applications in cancer immunotherapy.
OVs have many features that make them advantageous and distinct from current therapeutic modalities: (i) there is a low probability for the generation of resistance (not seen so far), as OVs often target multiple oncogenic pathways and use multiple means for cytotoxicity; (ii) they replicate in a tumor-selective fashion and are relatively nonpathogenic and, in fact, only minimal systemic toxicity has been detected; (iii) virus dose in the tumor increases with time due to in situ virus amplification, as opposed to classical drug pharmacokinetics that decrease with time; and (iv) safety features can be built in, such as drug and immune sensitivity. These features should result in a very high therapeutic index. An important issue for OV therapy is delivery. Although systemic intravenous administration is simpler than intratumoral injection and can target multiple tumors, it has drawbacks, including nonimmune human serum, anti-OV antibodies that preexist for human viruses or can be induced by multiple administrations, lack of extravasation into tumors, and sequestration in the liver (1). Cell carriers [i.e., mesenchymal stromal cells, myeloid-derived suppressor cells (MDSC), neural stem cells, T cells, cytokine-induced killer cells, or irradiated tumor cells] can shield virus from neutralization and facilitate virus delivery to the tumor (5). The effectiveness will vary depending upon the cell phenotype, permissiveness to virus infection, tumor-homing ability, and transfer of infectious virus to tumor cells. To block virus neutralization and extend vascular circulation, viruses can also be coated in nanoparticles (i.e., PEGylation; ref. 1).
OV Immunotherapy
Virus infection and pathogenicity have been major drivers in the evolution of the human immune system, and vaccination against viruses is the quintessential exploitation of adaptive immunity. A major goal of OV-mediated immunotherapy is to activate and redirect functional innate and adaptive immune responses toward the tumor. Interactions between innate and adaptive immune cells and signaling factors (i.e., cytokines and chemokines), often involved in virus infections, play a large role in antitumor immunity or lack thereof, as well as successful immunotherapies (Fig. 1). Virus infection induces an inflammatory response leading to adaptive antivirus immunity. Thus, the immune system was seen initially as a negative factor in OV therapy for limiting virus infection/delivery because of preexisting or therapy-induced immunity, virus replication because of innate antiviral responses, and virus spread because of the infiltration of innate immune cells (6). In addition, most early studies were performed in human xenograft tumor models in immunodeficient mice lacking adaptive immune responses because some viruses were species selective or replicated better in human cells, and because there was availability of a broad diversity of human cancer cell lines. With the use of syngeneic tumor models in immunocompetent mice, it became clear that the consequences of the immune system were complex, but that the induction of antitumor immunity was feasible and efficacious (6). In particular, many OVs act asin situ vaccines, inducing robust, long lasting, and specific adaptive antitumor responses, often CD8+ T cell–mediated (7, 8). Interestingly, adaptive antiviral immunity can enhance antitumor immunity for HSV, but not for VSV (8, 9).
Figure 1.
Cartoon of OV-mediated effects in tumor. First phase, OV delivered intratumorally or systemically, infects tumor cells (can be blocked by humoral defense systems; antibodies). After infection, OV replicates (can be blocked by innate responses; i.e., IFN-α/β), kills cells often by ICD, and spreads throughout the tumor (can be blocked by innate immune cells, i.e., NK cells and macrophages), eliciting an inflammatory response. When an armed OV is used, the immunomodulatory transgene is expressed (transgene product). Second phase, ICD and inflammation recruit DCs to the tumor, where they take up TAAs and induce an adaptive immune response (T and B cells), which targets the tumor (can be blocked by Tregs and MDSCs). Innate cells such as NK cells also have antitumor activities. Antitumor immune responses can be further enhanced by transgene products. CPA, cyclophosphamide.
The inflammatory cascade and immunogenic cell death (ICD) induced by OV infection of tumors makes OVs particularly powerful inducers of antitumor immunity (8, 10). Among the many different types of cell death, some are immunogenic and characterized by the release of danger-associated molecular patterns (DAMP), such as calreticulin, high-mobility group protein B1 (HMGB1), and ATP, along with tumor-associated antigens (TAA; ref. 10). Multiple forms of ICD have been observed after OV (Ad, VV, HSV, MV, and coxsackievirus) infection of cancer cells, and there is a suggestion that ICD occurs in patients after treatment with oncolytic Ad and temozolomide (11). However, much remains to be learned about the mechanisms of OV-mediated cell death and how it can be exploited to enhance immunogenicity. Inflammation, typically chronic, can also promote tumorigenesis and inhibit T-cell antitumor activity (12). Restraining antiviral immune responses and minimizing pathology, while promoting antitumor immune responses, is a complex and poorly understood balancing act that will dictate OV therapy outcomes. In some cases, where minimal OV replication occurs in mouse tumors (i.e., HSV) or no replication is required (i.e., reovirus; ref. 13), antitumor efficacy is principally due to OV-induced immune responses. Understanding, harnessing, modulating, and/or enhancing OV-mediated immune responses for effective antitumor immunity are major areas in current research that intersect with other immunotherapeutic strategies.
Many viruses express immune evasion genes that enable them to establish infections and spread within their host (14). Mutations in these genes (i.e., HSV Us11, VV E3L, MYXV M156R, Ad VAI, and reovirus σ2/σ3, inhibitors of PKR; HSV ICP0, VV N2, NDV V, and MV V, inhibitors of IRF3; HSV ICP0, MYXV M13L, MV V, PV 3C, and VSV M, inhibitors of NF-κB; VV B8R and MYXV MT-7, inhibitors of IFN-γ; HSV ICP47 and AdE3-19K, inhibitors of MHC class I presentation; MV gp, inhibitor of T cells; and MYXV M128L and MV H, inhibitors of CD46) are likely to enhance the induction of immunity and possibly cross-presentation of TAAs. Such mutations should improve the safety of OVs by making them more visible to the immune system, as well as increasing antitumor immune responses. Conversely, they may diminish virus replication and spread. An additional problem not as easily addressed is OV infection of immune cells, especially dendritic cells (DC), that interferes with their function (15, 16).
Innate Immunity
Although adaptive immunity seems to provide and, in fact, represent even the major mode of anticancer action for OVs, it is also evident that an initial host response against an administered OV could destroy it along with the infected cells before the OV has a chance to replicate and induce cytotoxicity of a magnitude that is sufficient to set up an effective vaccination response (17). Location and site of OV administration is an important determinant of the characteristics of these initial host responses against the OV. For instance, intravenous or intra-arterial administration of OVs, such as recombinant HSV1, leads to its rapid recognition and elimination by the circulating complement and antibodies of the humoral defense system (18, 19). This has also been shown for VV (20), NDV (21), MV (22), and Ad (23, 24). Intratumoral administration can also lead to complement- and antibody-mediated destruction of the OV. In addition, intracellular and microenvironmental antiviral defense responses in infected tumor cells can also greatly limit the magnitude of OV replication (25–31). Finally, innate immune cells can rapidly respond to an administered OV, further limiting its survival and that of OV-infected tumor cells (32–35). In all these models, circumvention of such responses using pharmacologic agents, such as histone deacetylase (HDAC) inhibitors or immunomodulating drugs, or genes that block antiviral defense mechanisms, has led to improved OV replication and tumor cytotoxicity (reviewed in ref. 36). When pharmacologic agents are used, the interference of antiviral responses can be applied in a transient fashion usually right before or at the time of OV administration. This should lead to an initial burst of OV replication leading to tumor cell lysis. As the pharmacologic effects against host innate immunity wane, a large debris field of OVs and tumor antigens could be more promptly recognized by the antiviral host response, leading to a secondary long-term vaccination effect responsible for effective tumor immunity (Fig. 1). However, quantification of responses to OV therapy is a sorely needed area of investigation. For instance, the number of OV-replicative rounds, the tumor cell-OV burst size, the number of OV-replicative tumor foci, and the temporal kinetics of innate response suppression that are needed for an efficient lytic and vaccination effect are still undetermined. In fact, current applications of innate immunity modulation with OV administration remain to be determined in an empirical manner.
Enhancing OV Immunotherapy
Many OVs can accommodate gene insertions and thus can be “armed” with therapeutic transgenes, combining local gene delivery with oncolytic activity (42). Local expression in the tumor obviates toxicity arising from systemic administration of potent immune modulators. GM-CSF, based on its effects in cytokine-transduced cancer cell vaccines (i.e., clinically approved Sipuleucel-T), has been incorporated into a number of OVs [HSV T-Vec, VV JX-594, Ad Ad5/3-D24-GMCSF (43), and CG0070 (44)] that have entered clinical trials (8). GM-CSF–expressing OVs demonstrated only moderate activity in preclinical studies (45, 46), while JX-594 was not compared with a VV lacking GM-CSF (47). Other therapeutic transgenes include interleukin (IL)-2 (NDV, HSV, and parvovirus), IL-12 (Ad and HSV), IL-15 (VSV), IL-18 (HSV), IFN-α/β (Ad, VSV, and VV), soluble CD80 (Ad and HSV), 4-1BB (VV), CD40L (Ad, and no effect with VSV), Flt3L (Ad and HSV), CCL3 (Ad), CCL5 (Ad and VV), and combinations thereof (2). In addition to transgenes that enhance adaptive immune responses, cytokines/chemokines directed at the tumor microenvironment can alter the immune cell balance toward productive therapeutic immunity (Fig. 1). IL-12, a potent antitumor cytokine with antiangiogenic activities, when expressed from oncolytic HSV, reduced neovasculature and tumor regulatory T cells (Treg) and induced T cell–mediated immunity in an immunocompetent cancer stem cell model (48). Expression of a CXCR4 antagonist from oncolytic VV reduced tumor vasculature and accumulation of bone marrow–derived epithelial and myeloid cells and induced antitumor humoral responses (49).
Like many cancer vaccine strategies, OVs expressing TAAs can be used to induce tumor-selective adaptive immune responses. The combination of TAA expression in the tumor and OV-mediated cell killing induces enhanced T-cell migration and activation compared with OV-infected tumor cells expressing the TAA (50). This can be coupled to a prime (replication-deficient Ad or oncolytic Semliki Forest virus expressing a TAA)–boost (oncolytic VSV or VV expressing the same TAA) vaccine strategy, in which the boosted secondary response to the tumor dominates the primary anti-OV response (6, 8). To expand the antigenic repertoire, cDNA libraries from normal tissue (e.g., prostate for prostate tumors) or recurrent tumors have been inserted into VSV, and induced therapeutic immunity (51). Further enhancement was obtained by expressing xenogeneic TAAs (51, 52). The ability of oncolytic VSV expressing TAAs to induce IL-17 in the context of tumor immunity has been exploited to screen tumor cDNA libraries for individual TAAs and optimal TAA combinations, limiting potentially inappropriate responses of whole-cell or cDNA vaccines (53). Developing a similar strategy in a human setting would be a major advance.
A number of immunomodulatory agents have been examined to restrain antiviral immune responses and promote OV replication and spread. Cyclophosphamide can increase OV replication and inhibit tumor growth by suppressing innate immune cell (34) and antibody responses (54), depleting Tregs, and enhancing the antitumor activity of CTLs (Fig. 1; ref.8). A challenge is to identify immunosuppressive strategies that can blunt acute innate cells from blocking virus replication and spread, while permitting sufficient inflammation and cross-priming for robust antitumor immunity. Conversely, it will be of interest to combine OV with chemotherapies that induce ICD (e.g., cyclophosphamide, oxaloplatin, or anthracyclines such as doxorubicin and mitoxantrone), increase tumor cell antigenicity (e.g., gemcitabine, cisplatin, or etoposide) or susceptibility to immune cells (e.g., HDAC inhibitors, paclitaxel, or doxorubicin), or suppress MDSCs (e.g., gemcitabine and paclitaxel) and Tregs (e.g., cyclophosphamide or sunitinib; ref. 55) in immunocompetent preclinical models.
In conclusion, the field of virotherapy is becoming mature in its knowledge of effective anticancer mechanisms in animal tumor models with OVs that are also safe in human clinical trials. It seems that there may soon be a first-in-humans OV approved for use in the United States, which will further stimulate laboratory and clinical endeavors with this therapeutic strategy.
Oncolytic viruses: a new class of immunotherapy drugs.
Oncolytic viruses represent a new class of therapeutic agents that promote anti-tumour responses through a dual mechanism of action that is dependent on selective tumour cell killing and the induction of systemic anti-tumour immunity. The molecular and cellular mechanisms of action are not fully elucidated but are likely to depend on viral replication within transformed cells, induction of primary cell death, interaction with tumour cell antiviral elements and initiation of innate and adaptive anti-tumour immunity. A variety of native and genetically modified viruses have been developed as oncolytic agents, and the approval of the first oncolytic virus by the US Food and Drug Administration (FDA) is anticipated in the near future. This Review provides a comprehensive overview of the basic biology supporting oncolytic viruses as cancer therapeutic agents, describes oncolytic viruses in advanced clinical trials and discusses the unique challenges in the development of oncolytic viruses as a new class of drugs for the treatment of cancer.
MC Speranza, K Kasai, SE Lawler – ILAR Journal, 2016 – ilarjournal.oxfordjournals.org
Abstract After more than two decades of research and development, oncolytic herpes
viruses (oHSVs) are moving into the spotlight due to recent encouraging clinical trial data.
oHSV and other oncolytic viruses function through direct oncolytic cancer cell–killing …
THOUSAND OAKS, Calif., Oct. 27, 2015/PRNewswire/–Amgen (AMGN) today announced
that the US Food and Drug Administration (FDA) has approved the Biologics License
Application for IMLYGIC™(talimogene laherparepvec), a genetically modified oncolytic …
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