Posts Tagged ‘Cancer immunotherapy’

Lectures by The 2017 Award Recipients of Warren Alpert Foundation Prize in Cancer Immunology, October 5, 2017, HMS, 77 Louis Paster, Boston

Top, from left: James Allison and Lieping Chen. Bottom, from left: Gordon Freeman, Tasuku Honjo, Arlene Sharpe.

The 2017 Warren Alpert Foundation Prize has been awarded to five scientists for transformative discoveries in the field of cancer immunology.

Collectively, their work has elucidated foundational mechanisms in cancer’s ability to evade immune recognition and, in doing so, has profoundly altered the understanding of disease development and treatment. Their discoveries have led to the development of effective immune therapies for several types of cancer.

The 2017 award recipients are:

  • James Allison, professor of immunology and chair of the Department of Immunology, The University of Texas MD Anderson Cancer Center
  • Lieping Chen, United Technologies Corporation Professor in Cancer Research and professor of immunobiology, of dermatology and of medicine, Yale University
  • Gordon Freeman, professor of medicine, Dana-Farber Cancer Institute, Harvard Medical School
  • Tasuku Honjo, professor of immunology and genomic medicine, Kyoto University
  • Arlene Sharpe, the George Fabyan Professor of Comparative Pathology, Harvard Medical School; senior scientist, department of pathology, Brigham and Women’s Hospital

The honorees will share a $500,000 prize and will be recognized at a day-long symposium on Oct. 5 at Harvard Medical School.

The Warren Alpert Foundation, in association with Harvard Medical School, honors trailblazing scientists whose work has led to the understanding, prevention, treatment or cure of human disease. The award recognizes seminal discoveries that hold the promise to change our understanding of disease or our ability to treat it.

“The discoveries honored by the Warren Alpert Foundation over the years are remarkable in their scope and potential,” said George Q. Daley, dean of Harvard Medical School. “The work of this year’s recipients is nothing short of breathtaking in its profound impact on medicine. These discoveries have reshaped our understanding of the body’s response to cancer and propelled our ability to treat several forms of this recalcitrant disease.”

The Warren Alpert Foundation Prize is given internationally. To date, the foundation has awarded nearly $4 million to 59 scientists. Since the award’s inception, eight honorees have also received a Nobel Prize.

“We commend these five scientists. Allison, Chen, Freeman, Honjoand Sharpe are indisputable standouts in the field of cancer immunology,” said Bevin Kaplan, director of the Warren Alpert Foundation. “Collectively, they are helping to turn the tide in the global fight against cancer. We couldn’t honor more worthy recipients for the Warren Alpert Foundation Prize.”

The 2017 award: Unraveling the mysterious interplay between cancer and immunity

Understanding how tumor cells sabotage the body’s immune defenses stems from the collective work of many scientists over many years and across multiple institutions.

Each of the five honorees identified key pieces of the puzzle.

The notion that cancer and immunity are closely connected and that a person’s immune defenses can be turned against cancer is at least a century old. However, the definitive proof and demonstration of the steps in this process were outlined through findings made by the five 2017 Warren Alpert prize recipients.

Under normal conditions, so-called checkpoint inhibitor molecules rein in the immune system to ensure that it does not attack the body’s own cells, tissues and organs. Building on each other’s work, the five award recipients demonstrated how this normal self-defense mechanism can be hijacked by tumors as a way to evade immune surveillance and dodge an attack. Subverting this mechanism allows cancer cells to survive and thrive.

A foundational discovery made in the 1980s elucidated the role of a molecule on the surface of T cells, the body’s elite assassins trained to seek, spot and destroy invaders.

A protein called CTLA-4 emerged as a key regulator of T cell behavior—one that signals to T cells the need to retreat from an attack. Experiments in mice lacking CTLA-4 and use of CTLA-4 antibodies demonstrated that absence of CTLA-4 or blocking its activity could lead to T cell activation and tumor destruction.

Subsequent work identified a different protein on the surface of T cells—PD-1—as another key regulator of T cell response. Mice lacking this protein developed an autoimmune disease as a result of aberrant T cell activity and over-inflammation.

Later on, scientists identified a molecule, B7-H1, subsequently renamed PD-L1, which binds to PD-1, clicking like a key in a lock. This was followed by the discovery of a second partner for PD-1—the molecule PD-L2—which also appeared to tame T-cell activity by binding to PD-1.

The identification of these molecules led to a set of studies showing that their presence on human and mouse tumors rendered the tumors resistant to immune eradication.

A series of experiments further elucidated just how tumors exploit the interaction between PD-1 and PD-L1 to survive. Specifically, some tumor cells appeared to express PD-L1, essentially “wrapping” themselves in it to avoid immune recognition and destruction.

Additional work demonstrated that using antibodies to block this interaction disarmed the tumors, rendering them vulnerable to immune destruction.

Collectively, the five scientists’ findings laid the foundation for antibody-based therapies that modulate the function of these molecules as a way to unleash the immune system against cancer cells.

Antibody therapy that targets CTLA-4 is currently approved by the FDA for the treatment of melanoma. PD-1/PD-L1 inhibitors have already shown efficacy in a broad range of cancers and have been approved by the FDA for the treatment of melanoma; kidney; lung; head and neck cancer; bladder cancer; some forms of colorectal cancer; Hodgkin lymphoma and Merkel cell carcinoma.

In their own words

“I am humbled to be included among the illustrious scientists who have been honored by the Warren Alpert Foundation for their contributions to the treatment and cure of human disease in its 30+ year history.  It is also recognition of the many investigators who have labored for decades to realize the promise of the immune system in treating cancer.”
        -James Allison

“The award is a great honor and a wonderful recognition of our work.”
         Lieping Chen

I am thrilled to have made a difference in the lives of cancer patients and to be recognized by fellow scientists for my part in the discovery of the PD-1/PD-L1 and PD-L2 pathway and its role in tumor immune evasion.  I am deeply honored to be a recipient of the Alpert Award and to be recognized for my part in the work that has led to effective cancer immunotherapy. The success of immunotherapy has unleashed the energies of a multitude of scientists to further advance this novel strategy.”
                                        -Gordon Freeman

I am extremely honored to receive the Warren Alpert Foundation Prize. I am very happy that our discovery of PD-1 in 1992 and subsequent 10-year basic research on PD-1 led to its clinical application as a novel cancer immunotherapy. I hope this development will encourage many scientists working in the basic biomedical field.”
-Tasuku Honjo

“I am truly honored to be a recipient of the Alpert Award. It is especially meaningful to be recognized by my colleagues for discoveries that helped define the biology of the CTLA-4 and PD-1 pathways. The clinical translation of our fundamental understanding of these pathways illustrates the value of basic science research, and I hope this inspires other scientists.”
-Arlene Sharpe

Previous winners

Last year’s award went to five scientists who were instrumental in the discovery and development of the CRISPR bacterial defense mechanism as a tool for gene editing. They were RodolpheBarrangou of North Carolina State University, Philippe Horvath of DuPont in Dangé-Saint-Romain, France, Jennifer Doudna of the University of California, Berkeley, Emmanuelle Charpentier of the Max Planck Institute for Infection Biology in Berlin and Umeå University in Sweden, and Virginijus Siksnys of the Institute of Biotechnology at Vilnius University in Lithuania.

Other past recipients include:

  • Tu Youyou of the China Academy of Chinese Medical Science, who went on to receive the 2015 Nobel Prize in Physiology or Medicine with two others, and Ruth and Victor Nussenzweig, of NYU Langone Medical Center, for their pioneering discoveries in chemistry and parasitology of malaria and the translation of their work into the development of drug therapies and an anti-malarial vaccine.
  • Oleh Hornykiewicz of the Medical University of Vienna and the University of Toronto; Roger Nicoll of the University of California, San Francisco; and Solomon Snyder of the Johns Hopkins University School of Medicine for research into neurotransmission and neurodegeneration.
  • David Botstein of Princeton University and Ronald Davis and David Hogness of Stanford University School of Medicine for contributions to the concepts and methods of creating a human genetic map.
  • Alain Carpentier of Hôpital Européen Georges-Pompidou in Paris and Robert Langer of MIT for innovations in bioengineering.
  • Harald zur Hausen and Lutz Gissmann of the German Cancer Research Center in Heidelberg for work on the human papillomavirus (HPV) and cancer of the cervix. Zur Hausenand others were honored with the Nobel Prize in Physiology or Medicine in 2008.

The Warren Alpert Foundation

Each year the Warren Alpert Foundation receives between 30 and 50 nominations from scientific leaders worldwide. Prize recipients are selected by the foundation’s scientific advisory board, which is composed of distinguished biomedical scientists and chaired by the dean of Harvard Medical School.

Warren Alpert (1920-2007), a native of Chelsea, Mass., established the prize in 1987 after reading about the development of a vaccine for hepatitis B. Alpert decided on the spot that he would like to reward such breakthroughs, so he picked up the phone and told the vaccine’s creator, Kenneth Murray of the University of Edinburgh, that he had won a prize. Alpert then set about creating the foundation.

To award subsequent prizes, Alpert asked Daniel Tosteson (1925-2009), then dean of Harvard Medical School, to convene a panel of experts to identify scientists from around the world whose research has had a direct impact on the treatment of disease.



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Meeting report: Cambridge Healthtech Institute’s 4th Annual Immuno-Oncology SUMMIT: Oncolytic Virus Immunotherapy Stream – 2016

Reporter: David Orchard-Webb, PhD


Cambridge Healthtech Institute’s 4th Annual Immuno-Oncology SUMMIT took place August 29-September 2, 2016 at the Marriott Long Wharf Boston, MA. The following is a synthesis of the Oncolytic Virus Immunotherapy stream.




Biomarkers for patient selection in clinical trials is an important consideration for developing cancer therapeutics and immunotherapeutics such as oncolytic viruses in particular. Howard L. Kaufman, M.D., discussed the development of biomarkers for oncolytic virus efficaciousness and patient selection focusing on Imlygic (HSV-1). An important consideration for any viral therapy is the presence or absence of the receptors that the virus uses to gain entry to the cell. For example HSV-1 utilises Nectin and HVEM cell surface receptors and their expression levels on a patient’s tumour will influence whether Imlygic can gain entry and replicate in tumours. In addition he reported that B-RAF mutation facilitates Imlygic infection and that MEK inhibitors sensitise melanoma cell lines to Imlygic. Stephen Russell also presented data on the mathematical modelling of Vesicular Stomatitis Virus (VSV) tumour spread and the development of a companion diagnostic based on gene expression profiling to predict patients whose tumours will be readily infected.


The immune reaction triggered by oncolytic viruses is important to monitor. Howard L. Kaufman discussed immunogenic cell death and stated that oncolytic viruses trigger immunity through the release of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). He reported that immunosuppressive Tregs, PDL1 and IDO expression were associated with anti-cancer CD8+ T cell infiltration. Imlygic also promoted the tumour infiltration of monocytes which depending on the context may either be immunosuppressive or beneficial through recruiting natural killer (NK) cells. This highlights the importance of combining Imlygic with other immune modulating therapeutics that can modulate the immunosuppressive cells and messengers that are present in the tumour environment. He discussed the finding that high mutation burden is a marker for response to immune checkpoint inhibition (such as CTLA and PD1) and suggested that due to the fact that oncolytic viruses release tumour associated antigens (TAA) during cell lysis this may also be a predictive marker for oncolytic viral therapy immune response. Supporting this notion Stephen Russell reported that a patient that underwent complete remission of multiple myeloma plasmacytomas in response to a measles virus oncotherapy had a very high mutational burden.


Targeting the tumour stroma with adenoviral vectors


VCN Biosciences SL is a privately-owned company focused in the development of new therapeutic approaches for tumors that lack effective treatment”. Manel Cascalló presented data from an ongoing phase I, multi-center, open-label dose escalation study of intravenous administration of VCN-01 oncolytic adenovirus with or without intravenous gemcitabine and Abraxane® in advanced solid tumors. Patients were selected based on low anti-Ad levels. Manel highlighted the problems of the pancreatic cancer matrix which limit intratumoral virus spread and also reduces chemotherapy uptake and tumour lymphocyte infiltration. VCN-01 expresses hyaluronidase to degrade the extracellular matrix and is administered intravenously. Liver tropism is reduced by replacement of the heparan sulfate glycosaminoglycan putative-binding site KKTK of the fiber shaft with an integrin-binding motif RGDK. VCN-01 replicates only in Rb tumour suppressor pathway dysregulated cancers, achieved through genetic modification of the E1A protein. In previous mouse xenograft studies of pancreatic and melanoma tumours VCN-01 showed efficaciousness in intratumoral spread, degradation of hyaluronan, and evidence of sensitisation to chemotherapy. The mouse models suggested that strategies that further target other major components of the ECM such as collagen and stromal cells may increase VCN-01 efficaciousness further [1]. The phase I trial supported safety and demonstrated that when administered intravenously VCN-01 reached the pancreatic tumour and replicated. In combination with gemcitabine and Abraxane® neutropenia was observed earlier than with chemotherapy alone. This is suggestive of increased efficaciousness of the chemotherapeutics as would be expected if a greater effective concentration reached the tumour. Biopsies suggested that VCN-01 shifted the balance of immune cells towards CD8+ T cells and away from immunosuppressive Treg.


Adenovirus tumor-specific immunogene (T-SIGn) Therapy


PsiOxus Therapeutics Ltd develops novel therapeutics for serious diseases with a particular focus upon cancer”. Brian Champion discussed the application EnAd a chimeric Ad11p/Ad3 adenovirus which retains the Ad11 receptor usage (CD46 and DSG2). PsiOxus are developing Membrane-integrated T-cell Engagers (MiTe) proteins delivered via EnAd. These MiTe proteins are expressed at the cancer cell surface and engage with and activate T-cells. Their lead candidate NG-348 showed promising T-cell activation in vitro.


Vaccinia virus – overcoming the immunosuppressive cancer microenvironment


David Kirn provided a recent history of the oncolytic virus field and provided an overview of the validation of vaccinia virus over the period 2007-14 stating that it can produce cancer oncolysis, induce an immune response, and result in angiogenic ablation.


Western Oncolytics develops novel therapies for cancer”. Steve Thorne discussed strategies to mitigate the immunosupressive environment encountered by oncolytic viruses. He presented data from models of tumours resistant to vaccinia oncolytic virus that Treg, and myeloid-derived suppressor cell (MDSC) numbers were higher whereas CD8+ T-cell levels were lower than in a sensitive model. He elaborated on a strategy of targeting the PGE2 pathway in order to reduce MDSC numbers entering the tumour microenvironment. He demonstrated that vaccinia virus expressing HPGD has reduced levels of MDSC in target tumours.


Transgene (Euronext: TNG), part of Institut Mérieux, is a publicly traded French biopharmaceutical company focused on discovering and developing targeted immunotherapies for the treatment of cancer and infectious diseases”. Eric Quéméneur presented preclinical data on Transgene’s oncolytic vaccinia virus TG6002 which expresses a chimeric bifunctional enzyme which converts the nontoxic prodrug 5‐FC into the toxic metabolites 5‐FU and 5‐FUMP. This allows systemic delivery of the non-toxic prodrug chemotherapy with activation at tumours infected with the Vaccinia oncolytic virus. The virus plus prodrug combination was effective against all of the solid tumour cell lines tested. In addition the combination was effective against glioblastoma cancer stem-like cells. In pancreatic and colorectal cancer cell line models the vaccinia prodrug combination was synergistic or additive when combined with additional chemotherapeutics. In immunocompetent mouse models TG6002 increased the Tumour Teff/Treg ratio indicative of a shift from an immunosuppressive to an immunocompetent microenvironment. Furthermore in mouse models TG6002 induced an abscopal response.


Vesicular Stomatitis Virus (VSV) – A single shot cure for cancer?


Vyriad strives to develop potent, safe and cost-effective cancer therapies in areas of unmet need”. Stephen Russell presented his position that oncolytic viruses could be a single shot cure for cancer. He emphasised the point that in oncolytic viral therapy the initial dose will be the most effective due to the relatively low levels of neutralising antibodies present and therefore defining the optimal dose is critical. The trend is for increased initial dose. Two IND’s have been accepted by the FDA, one for measles virus and the other for VSV.


John Bell described using VSV to deliver Artificial microRNAs (amiRNAs) to tumours. It was demonstrate that a VSV delivering ARID1A amiRNA was synthetic lethal when combined with EZH2 (methyl transferase) inhibition. He postulated that oncolytic viruses can be used to create factories of therapeutic amiRNAs transmitted throughout the tumour by exosomes.


HSV-1 an update on immune checkpoint combinations


Amgen was the first company to launch an FDA approved (October 2015) oncolytic virus, trade name Imlygic, which was developed by the UK based company Biovex. Jennifer Gansert gave a background on Imlygic and presented new data on combination with the CTLA4 inhibitor Ipilimumab. In mouse models abscopal response in contralateral tumours was 100% when a single tumour was treated with Imlygic combined with systemic delivery of anti-CTLA4. A Phase 1b clinical trial to test the combination in unresectable melanoma patients was completed and published in 2016. Fifty percent of the patients had durable response for greater than 6 months and 20% of the patients had ongoing complete response after a year of follow-up. Overall 72% of patients has controlled disease (no progression). In addition Amgen is recruiting for a phase III trial of the anti-PD1 Pembrolizumab in combination with Imlygic for unresectable stage IIIB to IVM1c melanoma.


Virttu is a privately held biotechnology company, which has pioneered the development of oncolytic viruses for treating cancer”. Joe Connor discussed Seprehvir an oncolyic virus based on HSV-1 like Imlygic which is in clinical trials for which 100 patients have been treated to date. The trial data indicate that Seprehvir induces CD8+ T cell infiltration and activity as well as a novel anti-tumour immune response against select antigens such as Mage A8/9. Preclinical investigations focus on combination with checkpoint inhibitor antibodies, CAR-T targeted to GD2, and synergies with targeted therapies on the mTOR/VEGFR signalling axes.


Reovirus – an update


Oncolytics Biotech Inc. is a clinical-stage oncology company focused on the development of oncolytic viruses for use as cancer therapeutics in some of the most prevalent forms of the disease”. Brad Thompson provided an update on REOLYSIN®, Oncolytics Biotech’s proprietary T3D reovirus. Highlights included concluding the first checkpoint inhibitor and REOLYSIN® study in patients with pancreatic cancer and preparing for registration study in multiple myeloma.


Maraba virus – privileged antigen presentation in splenic B cell follicles


Turnstone Biologics is developing “a first-in-class oncolytic viral immunotherapy that combines a bioselected and engineered oncolytic virus to directly lyse tumors with a potent vaccine technology to drive tumor-antigen specific T-cell responses of unprecedented magnitude”. Caroline Breitbach described Maraba MG1 Oncolytic Virus which was isolated from Brazilian sand flies. Their lead candidate is an MG1 virus expressing the tumour antigen MAGE-A3. In mouse models a combination of adenovirus-MAGE-A3 and MG1-MAGE-A3 in a prime-boost regimen produced extremely robust CD8+ T cell responses. It is thought that a privileged antigen presentation in splenic B cell follicles maximizes the T cell responses. A phase I/II trial is enrolling patients to test the adenovirus-MAGE-A3 and MG1-MAGE-A3 prime-boost regimen in patients with MAGE‐A3 positive solid tumours for which there is no life prolonging standard therapy.


Oncolytic virus manufacturing


Anthony Davies of Dark Horse Consulting Inc. reviewed the manufacturing hurdles facing oncolytic viruses and pointed out that thus far adenovirus is the gold standard. He discussed isoelectric focusing for virus manufacturing, process flow and the procurement of key raw materials. He emphasized the importance of codifying analytical methods, and the statistical design of experiments (DOE) for optimal use of finite resources.


Mark Federspiel described the difficulties associated with measles virus manufacturing which include the large pleomorphic size (100-300nm) which cannot be filter sterilized efficiently due to shear stress. As a result aseptic conditions must be maintained throughout the manufacturing process. There are also issues with genomic contamination from infected cells. He described improved manufacturing bioprocesses to overcome these limitations using the HeLa S3 cell line. Using this cell line resulted in less residual genomic DNA than the standard however it was still relatively high compared to vaccine production. There is still much room for improvement.


Rodríguez-García A, Giménez-Alejandre M, Rojas JJ, Moreno R, Bazan-Peregrino M, Cascalló M, Alemany R. Safety and efficacy of VCN-01, an oncolytic adenovirus combining fiber HSG-binding domain replacement with RGD and hyaluronidase expression. Clin Cancer Res. 2015 Mar 15;21(6):1406-18. Doi: 10.1158/1078-0432.CCR-14-2213. Epub 2014 Nov 12. PubMed PMID: 25391696.


Other Related Articles Published In This Open Access Online Journal Include The Following:

Real Time Coverage and eProceedings of Presentations on August 29 and August 30, 2016 CHI’s 4th IMMUNO-ONCOLOGY SUMMIT – Oncolytic Virus Immunotherapy Track

LIVE Tweets via @pharma_BI and by @AVIVA1950 for August 29 and August 30, 2016 of CHI’s 4th IMMUNO-ONCOLOGY SUMMIT – Oncolytic Virus Immunotherapy Track, Marriott Long Wharf Hotel – Boston

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Deep Learning for In-silico Drug Discovery and Drug Repurposing: Artificial Intelligence to search for molecules boosting response rates in Cancer Immunotherapy: Insilico Medicine @John Hopkins University

Reporter: Aviva Lev-Ari, PhD, RN

Insilico Medicine –>>> transcriptome-based pathway perturbation analysis

Insilico Medicine, Inc. is a bioinformatics company located at the Emerging Technology Centers at the Johns Hopkins University Eastern campus in Baltimore with R&D resources in Belgium, Russia, and Poland hiring talent through hackathons and competitions. It utilizes advances in genomics, big data analysis and deep learning for in silico drug discovery and drug repurposing for aging and age-related diseases. The company pursues internal drug discovery programs in cancer, Parkinson’s, Alzheimer’s, sarcopenia and geroprotector discovery. Through its Pharmaceutical Artificial Intelligence division the company provides advanced machine learning services to biotechnology, pharmaceutical, and skin care companies.


Brief company video:

Insilico Medicine develops a new approach to concomitant cancer immunotherapy

Artificial intelligence to search for molecules boosting response rates in cancer immunotherapy





  • Some of the most promising drugs for cancer therapy called checkpoint inhibitors often result in complete remissions, however, a majority of patients fail cancer immunotherapy with antibodies targeting immune checkpoints, such as CTLA-4 or programmed death-1 (PD-1).
  • Insilico Medicine developed a set of pathway-based signatures of response to popular checkpoint inhibitors
  • Using these markers and a deep learned drug scoring engine Insilico Medicine identified 12 leads that may help increase response to cancer immunotherapy and is seeking industry partnerships to test these leads

Thursday, July 14, 2016, Baltimore, MD — Recent advances in cancer immunotherapy demonstrated complete remission in multiple tumor types including melanoma and lung cancers. Almost every major pharmaceutical company operating in oncology space started multiple programs in immuno-oncology with thousands of clinical trials underway. Immuno-oncology is now a very broad field ranging from treatment of a patient with an engineered antibody to genome editing of patient’s immune cells. Genetic mutations accruing from the inherent genomic instability of tumor cells present neo-antigens that are recognized by the immune system. Cross-presentation of tumor antigens at the immune synapse between antigen-presenting dendritic cells and T lymphocytes can potentially activate an adaptive antitumor immune response, however, tumors continuously evolve to counteract and ultimately defeat such immune surveillance by co-opting and amplifying mechanisms of immune tolerance to evade elimination by the immune system. This prerequisite for tumor progression is enabled by the ability of cancers to produce negative regulators of immune response.

Cancer immunotherapy is currently focused on targeting immune inhibitory checkpoints that control T cell activation, such as CTLA-4 and PD-1. Monoclonal antibodies that block these immune checkpoints (commonly referred to as immune checkpoint inhibitors) can unleash antitumor immunity and produce durable clinical responses in a subset of patients with advanced cancers, such as melanoma and non-small-cell lung cancer. However, these immunotherapeutics are currently constrained by their inability to induce clinical responses in the vast majority of patients and the frequent occurrence of immune-related adverse events. A key limitation of checkpoint inhibitors is that they narrowly focus on modulating the immune synapse but do not address other key molecular determinants that may also be responsible for immune dysfunction.

Immunoresistance often ensues as a result of the concomitant activation of multiple, often overlapping signaling pathways. Therefore, inhibition of multiple, cross-talking pathways involved in survival control with combination therapy is usually more effective in decreasing the likelihood that cancer cells will develop therapeutic resistance than with single agent therapy. While research efforts are now focused on identifying new inhibitory mechanisms that could be targeted to achieve responses in patients with refractory cancers and provide durable and adaptable cancer control, there are outstanding challenges in determining what combination of immunotherapies and conventional therapies will prove beneficial against each tumor type.

“Immunotherapy is the most promising area in oncology resulting in cures, but we need to identify effective combinations of both established methods and new drugs developed specifically to boost response rates. At Insilico Medicine we developed a new method for screening, scoring and personalizing small molecules that may boost response rates to PD-1, PD-L1, CTLA4 and other checkpoint inhibitors. We can identify effective combinations of both established methods and new drugs developed specifically to boost response rates to immunotherapy”, said Artem Artemov, director of computational drug repurposing at Insilico Medicine.

Insilico Medicine, Inc. is one of the leaders in transcriptome-based pathway perturbation analysis. It is also a pioneer in applying cutting edge artificial intelligence techniques to biological and medical data analysis, particularly focused on in silico screening for new compounds against cancer and known drugs which can be repurposed against different cancers. One of the major programs currently ongoing at Insilico Medicine is evaluation of the transcriptional responses to multiple checkpoint inhibitors and analyzing the pathway-level differences in patients who respond and fail to respond to clinically approved checkpoint inhibitors. This novel computational approach is aimed at identifying new drug candidates which can be used in combination with immunotherapy to unleash durable antitumor effect against several types of cancers.

Recently, scientists at Insilico Medicine performed a large in silico screening of compounds that can be administered in combination with anti-PD1 immunotherapy to increase response rates. The researchers collected transcriptomic data from tumors of patients who either responded or failed to respond to standard immunotherapy, using both publically available and internally generated data. Next, they used differential pathway activation analysis and deep learning based approaches to identify transcriptomic signatures predicting the success of immunotherapy in a particular tumor type.

Finally, they analyzed drug-induced transcriptomic effects to screen for the drugs that can robustly drive transcriptomes of tumor cells from non-responsive state to the state responsive to immunotherapy. In other words, researchers developed approach that can predict whether drug of interest would induce a transcriptional signature that characterizes those patients that respond to cancer immunotherapy in non-respondents. This method allows personalizing these drugs to individual patients and specific checkpoint inhibitors. Among the top-scoring drugs, they found several compounds known to increase response rates in combination with cancer immunotherapy. One of the top-scoring compounds included a naturally-occurring substance marketed as a natural product.

The current list of top-scoring leads that may increase response rates to checkpoint inhibitors included 12 small molecules identified using signaling pathway perturbation analysis and annotated using a deeply learned drug scoring system. Insilico Medicine is currently open for partnerships which will allow further testing and validation of the discovered compounds ex vivo on cell cultures established from tumors which respond and failed to respond to immunotherapy, as well as in mice with patient-derived tumor xenografts. This approach may greatly reduce the costs of preclinical trials and significantly shorten the timeframe from a drug prediction to validation and marketing. The compounds, after preclinical and clinical validation, may improve cancer care and dramatically increase the lifespan of cancer patients.

A panel of leads for concomitant immunotherapy is part of a large number of leads developed using DeepPharma™, artificially-intelligent drug discovery engine, which includes a large number of molecules predicted to be effective antineoplastic agents, metabolic regulators, CVD and CNS lead, senolytics and ED drugs. Recently Insilico Medicine published several seminal papers demonstrating proof of concept of utilizing deep learning techniques to predict pharmacological properties of small molecules using transcriptional response data utilizing deep neural networks for biomarker development. “Deep Learning Applications for Predicting Pharmacological Properties of Drugs and Drug Repurposing Using Transcriptomic Data,” a paper published in Molecular Pharmaceuticals received the American Chemical Society Editors’ Choice Award. Another recent collaboration with Biotime, Inc resulted in a launch of Embryonic.AI, deep learned predictor of differentiation state of the sample.



Disclaimer: AAAS and EurekAlert! are not responsible for the accuracy of news releases posted to EurekAlert! by contributing institutions or for the use of any information through the EurekAlert system.


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Personalized Immunotherapy: The Immuno-Oncology Summit August 30-31 2016 Boston MA

Reporter: Stephen J Williams, PhD




Leaders in Pharmaceutical Business intelligence (LPBI) Group will cover in Real Time using Social Media

The CHI’S 4TH ANNUAL IMMUNO-ONCOLOGY SUMMIT – Personalized Immunotherapy

Personalized Oncology in the Genomic Era: Expanding the Druggable Space

Aviva Lev-Ari, PhD, RN

will be streaming LIVE from the Marriott Long Wharf Hotel in Boston, MA





Plenary Keynotes


Matthew Goldstein

4:00 A New Era of Personalized Therapy: Using Tumor Neoantigens to Unlock the Immune System

Matthew J. Goldstein, M.D., Ph.D., Director, Translational Medicine, Neon Therapeutics, Inc.

Neon Therapeutics, Inc. launched in 2015 to focus on advancing neoantigen biology to improve cancer patient care. A neoantigen-based product engine will allow Neon to develop further treatment modalities including next-generation vaccines and T cell therapies targeting both personalized as well as shared neoantigens. The company’s first trial will launch later this year investigating the combination of a personalized, vaccine with nivolumab in advanced Melanoma, NSCLC, and Bladder Cancer.

Michael Rosenzweig

4:30 Emerging Innate Immune Targets for Enhancing Adaptive Anti-Tumor Responses

Michael Rosenzweig, Ph.D., Executive Director, Biology-Discovery, IMR Early Discovery, Merck Research Laboratories

Novel cancer immunotherapies targeting T cell checkpoint proteins have emerged as powerful tools to induce profound, durable regression and remission of many types of cancer. Despite these advances, multiple studies have demonstrated that not all patients respond to these therapies, and the ability to predict which patients may respond is limited. Harnessing the innate immune system to augment the adaptive anti-tumor response represents an attractive target for therapy, which has the potential to enhance both the percentage and rate of response to checkpoint blockade.


Morganna Freeman

5:00 Reading Tea Leaves:
The Dilemma of Prediction and Prognosis in Immunotherapy

Morganna Freeman, D.O., Associate Director, Melanoma & Cutaneous Oncology Program, The Angeles Clinic and Research Institute

With the rapid expansion of immunotherapeutics in oncology, scientifically significant advances have been made with both the depth and duration of antitumor responses. However, not all patients benefit, or quickly relapse, thus much scientific inquiry has been devoted to appropriate patient selection and how such obstacles might be overcome. While more is known about potential biomarkers, accurate prognostication persists as a knowledge gap, and efforts to bridge it will be discussed here.

Personalized Immunotherapy | The Immuno-Oncology Summit
August 30-31, 2016 | Marriott Long Wharf Hotel – Boston, MA

Personalized Immunotherapy
Personalized Oncology in the Genomic Era: Expanding the Druggable Space
August 30-31, 2016 | Learn More | Sponsorship & Exhibit Opportunities | Register by July 29 & SAVE up to $200!

Fueled with advances in genomic technologies, personalized oncology promises to innovate cancer therapy and target the previously undruggable space. Developments in immune checkpoint inhibitors, cancer vaccines, and adoptive T-cell therapies, as well as biomarker-driven immuno-oncology clinical trials, are enabling the next generation of cancer therapy. Cambridge Healthtech Institute’s Inaugural Personalized Immunotherapy meeting brings together clinical immuno-oncologists and thought leaders from pharmaceutical and biotech companies, and leading academic teams to share research and case studies in implementing patient-centric approaches to using the immune system to beat cancer.


Basics of Personalized Immunotherapy: What Is a Good Antigen?
Pramod K. Srivastava, M.D., Ph.D., Professor, Immunology and Medicine, Director, Carole and Ray Neag Comprehensive Cancer Center, University of Connecticut School of Medicine

Novel Antibodies against Immunogenic Neoantigens
Philip M. Arlen, M.D., President & CEO, Precision Biologics, Inc.

PD-1 Blockade in Tumors with Mismatch-Repair Deficiency
Luis Alberto Diaz, M.D., Associate Professor, Oncology, Johns Hopkins Sidney Kimmel Comprehensive Cancer Center


Cancer Vaccines in the Era of Checkpoint Inhibitors
Keith L. Knutson, Ph.D., Professor, Immunology, Mayo Clinic

Developing Therapeutic Cancer Vaccine Strategies for Prostate Cancer
Ravi Madan, M.D., Clinical Director, Genitourinary Malignancies Branch, National Cancer Institute, National Institutes of Health

Getting Very Personal: Fully Individualized Tumor Neoantigen-Based Vaccine Approaches to Cancer Therapy
Karin Jooss, Ph.D., CSO, Gritstone Oncology

Approaches to Assess Tumor Mutation Load for Selecting Patients for Cancer Immunotherapy
John Simmons, Ph.D., Manager, Research Services, Personal Genome Diagnostics

In situ Vaccination for Lymphoma
Joshua Brody, M.D., Director, Lymphoma Immunotherapy Program, Icahn School of Medicine at Mount Sinai

Immunotherapy Using Ad5 [E1-, E2b-] Vector Vaccines in the Cancer MoonShot 2020 Program
Frank R. Jones, Ph.D., Chairman & CEO, Etubics Corporation


Integration of Natural Killer-Based Therapy into the Treatment of Lymphoma
Andrew M. Evens, D.O., Professor and Chief, Hematology/Oncology, Tufts University School of Medicine; Director, Tufts Cancer Center

Dendritic Cells: Personalized Cancer Vaccines and Inducers of Multi-Epitope-Specific T Cells for Adoptive Cell Therapy
Pawel Kalinski, M.D., Ph.D., Professor, Surgery, Immunology, and Bioengineering, University of Pittsburgh School of Medicine, University of Pittsburgh Cancer Institute

Mesothelin-Targeted CAR T-Cell Therapy for Solid Tumors
Prasad S. Adusumilli, M.D., FACS, Deputy Chief of Translational & Clinical Research, Thoracic Surgery, Memorial Sloan-Kettering Cancer Center

Synthetic Regulation of T Cell Therapies Adds Safety and Enhanced Efficacy to Previously Unpredicted Therapies
David M. Spencer, Ph.D., CSO, Bellicum Pharmaceuticals

Long-Term Relapse-Free Survival of Patients with Acute Myeloid Leukemia (AML) Receiving a Telomerase- Engineered Dendritic Cell Immunotherapy
Jane Lebkowski, Ph.D., President & CSO, Research and Development, Asterias Biotherapeutics

Activated and Exhausted Tumor Infiltrating B Cells in Non-Small Cell Lung Cancer Patients Present Antigen and Influence the Phenotype of CD4 Tumor Infiltrating T Cells
Tullia Bruno, Ph.D., Research Assistant Professor, Immunology, University of Pittsburgh

About the Immuno-Oncology Summit

CHI’s 4th Annual Immuno-Oncology Summit has been designed to support a coordinated effort by industry players to bring commercial immunotherapies and immunotherapy combinations through clinical development and into the market. This weeklong, nine-meeting set will include topics ranging from early discovery through clinical development as well as emerging areas such as oncolytic virotherapy. Overall, this event will provide a focused look at how researchers are applying new science and technology in the development of the next generation of effective and safe immunotherapies.

Monday, August 29 –
Tuesday, August 30
Tuesday, August 30 –
Wednesday, August 31
Thursday, September 1 –
Friday, September 2
Immunomodulatory Antibodies Combination Immunotherapy Adoptive T Cell Therapy
Oncolytic Virotherapy Personalized Immunotherapy Biomarkers for Immuno-Oncology
Training Seminar: Immunology for Drug Discovery Scientists Preclinical & Translational Immuno-Oncology Clinical Trials for Cancer Immunotherapy

For more info about sponsorship opportunities, including podium presentations and 1-2-1 meetings, please contact:

Companies A-K
Ilana Quigley
Sr Business Development Manager
Companies L-Z:
Joe Vacca
Associate Director, Business Development

For conference updates please visit

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

This email is being sent to This email communication is for marketing purposes. If it is not of interest to you, please disregard and we apologize for any inconvenience this may have caused.
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Aduro Biotech Phase II Pancreatic Cancer Trial CRS-207 plus cancer vaccine GVAX Fails

Reporter: Stephen J. Williams, Ph.D

From Biospace News

May 16, 2016
By Alex Keown, Breaking News Staff

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

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

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

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

For full story please see

Also from FierceBiotech

UPDATED: Aduro combo fails in a key pancreatic cancer study

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Cyclic Dinucleotides and Histone deacetylase inhibitors

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



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

Reporter: Aviva Lev-Ari, PhD, RN

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

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

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

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

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

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


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


Histone deacetylase inhibitors: molecular mechanisms of action

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

Oncogene (2007) 26, 5541–5552;

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

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

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

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

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

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

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

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

Table 2 – HDACi in clinical trials.  Full table

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

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

Figure 1.  Full figure and legend (90K)

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

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

HDACi selectively alters gene expression

HDACi-induced antitumor pathways

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

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


Combination of HDACi with other antitumor agents

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

Clinical development of HDACi

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

The resistance to HDACi

Conclusions and perspectives

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

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

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

References  ….

NEWS AND VIEWS   Blocking HDACs boosts regulatory T cells

Nature Medicine News and Views (01 Nov 2007)


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

Nature Biotechnology Research (01 Jan 2007)

Comments of reviewer:


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


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


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


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


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


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






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

Curator: Larry H. Bernstein, MD, FCAP



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

Jeffrey S. Miller, Yvette Soignier, Angela Panoskaltsis-Mortari, …, Todd E. Defor, Linda J. Burns, Paul J. Orchard, Bruce R. Blazar, John E. Wagner, Arne Slungaard, Daniel J. Weisdorf, Ian J. Okazaki, and Philip B. McGlave
Blood. 2005;105:3051-3057

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.

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 CD3CD56+ lymphocytes, are distinguished as CD56bright and CD56dim subsets. Approximately 90% of peripheral blood and spleen NK cells belong to the CD56dimCD16+ subset with marked cytotoxic function upon interacting with target cells.19,20In contrast, most NK cells in lymph nodes and tonsils belong to the CD56brightCD16 subset and exhibit predominantly immune regulation properties by producing cytokines such as interferon (IFN)-γ in response to IL-12, IL-15 and IL-18 stimulation.19,21

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

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

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

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

Figure 1.

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

Full figure and legend (96K)

Direct tumor clearance by NK-mediated cytotoxicity

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

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

Indirect NK-mediated antitumor immunity

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

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


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

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


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

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


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

Front Immunol. 2015; 6: 286.  Published online 2015 Jun 3.    doi:  10.3389/fimmu.2015.00286

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

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

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

Natural killer cells are innate lymphocytes that provide a first line of defense against viral infections and cancer (5). Human NK cells are recognized as CD3CD56+ 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) (911). By contrast, NK cell activity is suppressed by inhibitory receptors, including KIRs, which bind to human leukocyte antigen (HLA) class I molecules on target cells (9, 10, 12). NKG2A is also an important inhibitory receptor binding to non-classical HLA molecule, HLA-E (13). If target cells lose or downregulate HLA expression (14), the NK inhibitory signal is abrogated, allowing NK cells to become activated and kill malignant targets. However, NK cell function is impaired in cancer patients by various mechanisms, particularly in tumor microenvironment (15).

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

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

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

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

Table 1   Selected clinical trials with expanded allogeneic NK cells
….. more
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 (4851).
Table 2  Genetically modified, expanded allogeneic NK cells.

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, 7679). In addition, inhibitory receptors (e.g., KIR2DL, PD-1, PD-L1, and NKG2A) can be blocked by antibodies (8085). 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).


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                      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.


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

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

Natural killer (NK) cells can kill cancer cells in the absence of prior stimulation and hold considerable potential for cell-based therapies targeting human malignancies (14). 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;57). 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 (1016).

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


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

IL-2 can induce proliferative responses in human NK cells but only a minor fraction sustains continued growth (10;26;27). Conceivably, some NK-cell subsets might be more responsive, as suggested by early reports of up to 50-fold expansion after culture with IL-2 for 2 weeks of an NK subset that adheres to plastic (2831). 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.


August 15, 2014 | by Hiu Chung So

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

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

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

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

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

Who would be good candidates for this type of therapy?

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

What happens to the patient during this therapy?

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

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



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

Katayoun Rezvani1* and Rayne H. Rouce2,3
THIS ARTICLE IS PART OF THE RESEARCH TOPIC   NK cell-based cancer immunotherapy
Front. Immunol., 17 November 2015 |


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 (1416). NK cells directly kill tumor cells through several mechanisms, including release of cytoplasmic granules containing perforin and granzyme (1618), 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) (1619) 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 (3639). The combination of the agonistic anti-CD137 antibody with rituximab is currently being evaluated in a phase 1 trial in patients with lymphoma [NCT01307267 (3537)].

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 (4345). 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 (4749); 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 (3335). 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, 5052). 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 (5052). 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).



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 (8688). 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, 8891). 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 (4852, 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).


Bispecific and Trispecific Engagers

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

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


NK Cells – What Does the Future Hold?

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




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