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In 1987, researchers identified cytotoxic T-lymphocyte antigen 4, or CTLA-4. Allison found that CTLA-4 prevents T cells from attacking tumor cells. He wondered whether blocking CTLA-4 would allow the immune system to make those attacks. In 1996, Allison showed that antibodies against CTLA-4 allowed the immune system to destroy tumors in mice.[2] In 1999, biotech firm Medarex acquired rights to the antibody. In 2010, Medarex acquirer Bristol-Myers Squibb reported that patients with metastatic melanoma lived an average of 10 months on the antibody, versus 6 months without it. It was the first time any treatment had extended life in advanced melanoma in a randomized trial.[2]
In the early 1990s, a biologist discovered a molecule expressed in dying T cells, which he called programmed death 1, or PD-1 and which he recognized as another disabler of T cells. An antibody that targeted PD-1 was developed and by 2008 produced remission in multiple subjects across multiple cancer types. In 2013, clinicians reported that across 300 patients tumors shrunk by about half or more in 31% of those with melanoma, 29% with kidney cancer and 17% with lung cancer.[2]
In 1997 rituximab, the first antibody treatment for cancer, was approved by the FDA for treatment of follicular lymphoma. Since this approval, 11 other antibodies have been approved for cancer; alemtuzumab (2001), ofatumumab (2009) and ipilimumab (2011).
In 2003 cytokines such as interleukin were administered.[3] The adverse effects of intravenously administered cytokines[4] led to the extraction, in vitro expansion against a tumour antigen and reinjection of the cells[5] with appropriate stimulatory cytokines.
However, with both anti–CTLA-4 and anti–PD-1, some tumors continued to grow before vanishing months later. Some patients kept responding after the antibody had been discontinued. Some patients, developed side effects including inflammation of the colon or of the pituitary gland.[2]
After success harvesting T cells from tumors, expanding them in the lab and reinfusing them into patients reduced tumors, in 2010, Steven Rosenberg announced chimeric antigen receptor therapy, or CAR therapy. This technique is a personalized treatment that involves genetically modifying each patient’s T cells to target tumor cells. It produced complete remission in a majority of leukemia patients, although some later relapsed.[2]
Some of the most promising advances in cancer research in recent years involve treatments known as immunotherapy. These advances are spurring billions of dollars in investment by drug companies, and are leading to hundreds of
July 31, 2016 – By DENISE GRADY and ANDREW POLLACK – Health – Print Headline: “A Guide to Immunotherapy, an Evolving but Promising Field”
declared him in remission. It was a result that put him at the vanguard of a new generation of cancer treatment called immunotherapy that casts into sharp relief the harshness of how we have long treated cancer and the less grueling
August 01, 2016 – By MATT RICHTEL – Health – Print Headline: “As Death Lurked, Tumors Melted”
The hot new field of immunotherapy got a shock on Friday when a best-selling new drug failed as an initial treatment for lung cancer in a clinical trial. Bristol-Myers Squibb said Friday that the drug, Opdivo, had not slowed the
August 06, 2016 – By ANDREW POLLACK – Business Day – Print Headline: “Immunotherapy Drug Fails Lung Cancer Trial”
The Food and Drug Administration on Wednesday approved a newimmunotherapy drug from Roche to treat bladder cancer, a form of cancer for which there have been no significant new medicines in years. The drug, called Tecentriq, is the
May 19, 2016 – By ANDREW POLLACK – Business Day – Print Headline: “F.D.A. Approves Immunotherapy Drug for Treatment of Bladder Cancer”
media as the early president of Facebook. Now he wants to pioneer in a field that is already jumping with activity: cancer immunotherapy. Mr. Parker is announcing Wednesday that he is donating $250 million to a new effort that will
April 13, 2016 – By ANDREW POLLACK – Business Day – Print Headline: “Facebook and Napster Pioneer to Push Innovation in Cancer Treatment”
Sloan Kettering Cancer Center in New York, recommended an experimental treatment: immunotherapy. Rather than attacking the cancer directly, as chemo does, immunotherapy tries to rally the patient’s own immune
July 31, 2016 – By DENISE GRADY – Health – Print Headline: “A Sickened Body as Cancer Weapon”
She also took part in a clinical trial at Johns Hopkins for Opdivo, an immunotherapy drug made by the pharmaceutical company Bristol-Myers Squibb. Briefly stated, immunotherapy is a recently developed, highly
August 09, 2016 – By MATT JABLOW – Opinion – Print Headline: “Drug Ads vs. Drug Reality”
family and many friends. Contributions in his memory may be made to Memorial Sloan Kettering Cancer Center, Melanoma and Immunotherapy Research under Dr. Jedd Wolchok. 1/3
July 17, 2016 – – Paid Death Notices – Print Headline: “Paid Notice: Deaths SPRAYREGEN, NICHOLAS (NICK)”
St. and Amsterdam Ave. Contributions in his memory may be made to Memorial Sloan Kettering Cancer Center, Melanoma and Immunotherapy Research under Dr. Jedd Wolchok. 1/3
July 14, 2016 – – Paid Death Notices – Print Headline: “Paid Notice: Deaths SPRAYREGEN, NICHOLAS (NICK)”
Sloan Kettering Cancer Center. This radical, science-fictionlike therapy differs sharply from the more established type of immunotherapy, developed by other researchers. Those off-the-shelf drugs, known as checkpoint inhibitors,
August 02, 2016 – By ANDREW POLLACK – Health – Print Headline: “Setting Body’s ‘Serial Killers’ Loose on Cancer”
Below, we report on the State of the Science for Overcoming Obstacles in Oncolytic Virus Delivery and provide the source for all the references used
ONCOLYTIC VIROTHERAPY FOR PANCREATIC CANCER
Adenovirus
ONYX-015 was the first TOV used in a clinical trial for pancreatic cancer. ONYX-015 was administered intratumourally under endoscopic ultrasound-guidance into patients with locally advanced adenocarcinoma of the pancreas or metastatic disease in phase I/II trials[132]. The treatment was well-tolerated in most patients, however no objective responses were seen with ONYX-015 as a single agent and only 2/21 patients experienced mild responses when combined with gemcitabine[132]. A second adenovirus vector carries a deletion in the E1A gene[133]. E1A normally binds to the retinoblastoma protein, forcing cells to prematurely enter the S phase of the cell cycle. Since most pancreatic cancers harbor a mutation in CDKN2A[134], the E1A protein is unnecessary for entry of the TOV into cancer cells. Furthermore a double-deleted (E1A and E1B19) adenovirus demonstrated increase potency and selectivity in pancreatic cancer models[135,136]. This demonstrates that TOVs can be genetically engineered to increase selectivity and efficacy while maintaining their potency. Adenovirus selectivity has also been improved by engineering tumour-specific promoters such as a human CEA promoter[137] or by substituting the adenovirus serotype 5 fiber knob with the fiber knob from serotype 3[138]. The potency of TOVs can also be improved further by engineering them with therapeutic genes that stimulate the immune system and/or improve direct oncolysis. Adenovirus ZD55-IL-24 expressing IL-24 locally in pancreatic tumours in immune competent mice inhibited tumour growth and induced a stronger T cell response compared to its backbone virus, as measured by IL-6 and IFN-γ levels[139].
HSV
Two oncolytic HSV-1 vectors are currently in clinical trials for the treatment of pancreatic cancer. HF10 is a non-engineered, naturally occurring oncolytic HSV that demonstrated regression in 1/6 of the patients treated[140,141]. OncoVex GM-CSF is a ∆34.5 and ICP47-deleted mutant expressing GM-CSF, whereby the deletions allow for tumour-selective replication and inhibition of protein-kinase R activation, respectively[142]. Phase I/II trials in various solid tumours demonstrated OncoVex GM-CSF to be well-tolerated at high and repeated doses[143,144]. A phase I clinical trial with OncoVex GM-CSF in patients with unresectable pancreatic cancer is underway.
Poxviruses
The most widely studied poxvirus is VV, which is highly immunogenic and produces a strong cytotoxic T cell response[145] and circulating neutralizing antibodies which can be detected decades later[146]. For its crucial role in the eradication of smallpox, much has been learned about its potential role in immunotherapy today. The Lister strain of vaccinia remarkably showed no replication degradation even under the hypoxic conditions of PDAC[147]. A second Lister strain, thymidine kinase-deleted replicating VV armed with IL-10 demonstrated superior and long-lasting antitumour immunity in both a subcutaneous pancreatic cancer model and a Kras-p53 mutant-transgenic pancreatic cancer model after systemic delivery compared to its unarmed backbone virus[148]. Myxoma virus, a rabbit-specific poxvirus combined with gemcitabine resulted in 100% long-term survival in Pan02-engrafted immunocompetent intraperitoneal dissemination models of pancreatic cancer[149]. The only poxvirus to be tested in clinical trials is a non-replicative VV that expresses the pancreatic TAAs CEA and MUC-2[150]. The vaccine also includes a triad of costimulatory molecules, B7.1 (CD80), ICAM-1 (intra-cellular adhesion molecule-1) and LFA-3 (leukocyte function-associated antigen-3) (TRICOM) (PANVAC-VF)[150]. GM-CSF was also used as an adjuvant following each vaccination of PANVAC-VF. Phase I trials demonstrated antigen-specific antitumour responses in 62.5% of patients enrolled and antibody responses against VV was observed in all ten patients, which was associated with an increase in survival (15.1 mo vs 3.9 mo)[48]. A phase III clinical trial for the treatment of metastatic pancreatic cancer after failing treatment with gemcitabine, however, was terminated after failing to reach its primary efficacy endpoint[151].
Other pre-clinical TOVs for pancreatic cancer therapy
Parvovirus, measles virus and reovirus have also demonstrated pre-clinical activity in pancreatic cancer models. Parvoviruses particularly demonstrated enhanced IL-2-activated NK responses against PDAC cells[152,153]. An armed measles virus (MV), MV-purine nucleoside phosphorylase (PNP)-anti-prostate stem cell antigen, that expresses the prodrug convertase PNP, which then activates the prodrug fludarabine, was shown to enhance the oncolytic efficacy of the virus in gemcitabine-resistant PDAC cells[154]. Reovirus is another promising TOV for pancreatic cancer therapy, particularly because its selectivity depends on the cellular activity of Ras, which is constitutively active in pancreatic cancer[155]. Reolysin® (Oncolytics Biotech Inc., Calgary, AB, Canada) a reovirus administered intraportally resulted in decreased metastatic tumour volumes in the liver of immunocompetent animal models[156,157]. A phase II study of Reolysin® in combination with gemcitabine in patients with advanced PDAC has been completed (clinicaltrials.gov: NCT00998322). A two-armed randomized phase II study of carboplatin and paclitaxel plus Reolysin®vs carboplatin and paclitaxel alone in recurrent or metastatic pancreatic cancer is currently being conducted by the United States National Cancer Institute (NCI-8601/OSU-10045).
A understanding how antitumour immunity is regulated allows us to recognize barriers against effective immunotherapy delivery and furthermore, allow for the development of rational combination therapies aiming targeting these mechanisms[108,158,159]. This approach allows therapies to work synergistically and also has the potential to benefit a broader patient population[108]. Tumours have evolved to avoid immune recognition and/or destruction at every stage in the antitumour response, therefore targeting more than one immune resistance mechanism will enhance antitumour immunity.
An important immunological barrier in cancer immunotherapy is the tolerance towards self-antigens. Tumours downregulate their antigenicity through various mechanisms in response to selective pressure by the immune system, a process called “immunoediting”[37]. Therefore, in order to raise an effective antitumour response, the immunological tolerance must be broken to allow tumour antigen-specific cytotoxic T cell responses[158]. This can be achieved by increasing the tumour load and/or enhance antigen presentation[108]. TOVs can initiate selective infection and replication in the tumour bed, exposing TAA, disrupting the immunotolerance employed by the tumour while re-engaging adaptive immune effector responses[39]. Combining an agent that can cause disruption to the tumour bed i.e., an oncolytic virus, with a novel antitumour immunomodulating agent such as anti-PD-1/PD-L1 antibodies can maximize immune-stimulating and immune-recruiting inflammatory responses[39]. Specifically, TOV lysis induces the release of tumour antigens into the microenvironment, which are then cross-presented to T cells in the draining lymph nodes by APCs[159] (Figure (Figure1).1). This allows T cell infiltration to the tumour bed. Next, T cell dysfunction must be reversed[108,158]. Immune checkpoint inhibitors alleviate immunosuppression, allowing the elimination of the tumour by the adaptive immune system[70]. TOVs in combination with immune checkpoint inhibitors can therefore potentiate and activate the immune system synergistically, ultimately creating a pro-inflammatory environment. Pre-existing TILs are strong prognostic predictors in cancer[106]. This is extremely relevant for tumours with poor immune-cell infiltration, such as pancreatic cancer, which would depend on TOV-infection mediated lymphocyte infiltration for an enhanced response to immune checkpoint blockade. Zamarin et al[160] demonstrated constrained replication of an intratumoural-injected Newcastle disease virus in a B16 melanoma model. Lymphocytic infiltrates, however, were detected in both TOV-injected and non-TOV-injected tumours, and rendered the tumours sensitive to CTLA-4 blockade. The antitumour activity was dependent on CD8+ T cells, NK cells and type I and II IFNs[160]. Ipilimumab with or without talimogene laherparapvec, is in early clinical testing in patients with unresected melanoma (clinicaltrials.org: NCT01740297). Interestingly, an MV engineered to express CTLA-4 or PD-L1 antibodies delayed tumour progression and prolonged median OS in B16 melanoma models[161]. Finally, TOVs have demonstrated a tolerable toxicity profile, whereby flu-like symptoms are the most common adverse events, and in fact, most of the side effects seen so far in the combination regiment are related to the immune checkpoint blockade inhibitor[162]. Dias et al[163] suggested an oncolytic adenovirus expressing CTLA-4 locally might reduce systemic side effects normally induced with anti-CTLA-4 antibodies alone.
OVERCOMING OBSTACLES IN ONCOLYTIC VIRUS DELIVERY
The main issue with virotherapy is systemic delivery for targeting metastatic cancer cells. Intravenous administration is more practical, especially for treatment of a tumour in a hard-to-reach location such as the pancreas, and with the majority of patients presenting with advanced or metastatic disease. However, nonimmune human serum and existing anti-TOV antibodies may neutralize the TOV in the bloodstream. Furthermore, non-specific hepatic and splenic sequestration of the TOV and ineffective extravasation into the tumours are important issues[164]. Currently, studies in pre-clinical models aim to overcome these obstacles. These include chemical modification of viral coat proteins by conjugation of biocompatible polymers e.g. polyethylene glycosylation[165,166], using mesenchymal stem cell carrier systems to deliver the TOV to the tumour bed[167–169], and increasing vessel permeabilization[170,171].
In PDAC, however, the biggest hurdle may not be the host immune system, but the TME. The TME has played a significant role in not only acting as a physical barrier to deliver treatments, but it also in the development of resistance to conventional drugs. The TME remains a problem for successful TOV treatment. The TOV must be able to spread in the hypoxic and densely stromal-rich TME in order to attract enough attention to induce antitumour immunity[172]. Breaching the stromal barrier in PDAC is needed for TOVs to access the cancer cells[173]. Paradoxically, a recent study by Ilkow et al[174] demonstrated that the cross-talk between CAFs and cancer cells actually lead to increased permissibility of TOV-based therapeutics. Tumour cells producing TGF-α reprogrammed CAFs, dampening levels of anti-viral transcripts. This allowed the cells to be more sensitive to VV, vesicular stomatitis virus and maraba MG1 TOVs. The reprogrammed CAFs produced fibroblast growth factor (FGF)-2 which suppressed levels of retinoic acid-inducible gene I and increased the susceptibility of the tumour cells to virus[175]. This study also demonstrated that an FGF2-expressing TOV has improved therapeutic efficacy by sensitizing the tumour cells to virotherapy and is particularly relevant to pancreatic cancers, where CAFs are a major component of the tumour stroma[175]. It is important to note that not only the patient’s existing immune system may impede successful TOV therapy, but that the enhanced antitumour response by combinatory approaches (e.g., the inclusion of immune-checkpoint inhibitors) may also impede successful TOV infection, spread and engagement of the immune system. This stresses the importance of determining strategic combinations, dosing and timing schedules in future studies.
CONCLUSION
The poor prognosis of pancreatic cancer due in part to the limited efficacy of conventional and targeted therapies, appeals for a novel strategy to treat this disease. It has become very clear that the immune system has the greatest potential to selectively destroy tumours, and when it is strategically induced, a durable benefit can be achieved. Past and present studies have defined means for tumour escape from immune surveillance and have developed immunotherapies to counteract these mechanisms. However, with the various escape strategies leading to low immunogenicity and highly immunosuppressive tumour beds, a successful control of tumour growth by immunotherapy does not come without various obstacles and challenges. Future steps include the development of immune-monitoring strategies for the identification of biomarkers, to establishment guidelines to assess clinical end points of immunotherapy and finally to evaluate combination therapeutic strategies to maximize clinical benefit[176]. The ability of TOVs to stimulate inflammation, deliver genes and immunomodulatory agents as well as reduce tumour burden by direct cell lysis, allows them to be important therapeutic vectors for a highly immunosuppressed tumour such as PDAC. Immune checkpoint blockade agents can then reverse T cell anergy and further boost OV-induced responses. As this combinatory approach may exist as a double-edged sword, it is crucial to determine appropriate timing, dosing and sequence schedules of each agent.
SOURCE & REFERENCES
World J Gastroenterol. 2016 Jan 14; 22(2): 748–763.
AGENDA for Oncolytic Virus Immunotherapy Unlocking Oncolytic Virotherapies: From Science to Commercialization CHI’S 4TH ANNUAL IMMUNO-ONCOLOGY SUMMIT – AUGUST 29-30, 2016 | Marriott Long Wharf Hotel – Boston, MA
Oncolytic Virus Immunotherapy Unlocking Oncolytic Virotherapies: From Science to Commercialization
MONDAY, AUGUST 29 & 30
MONDAY, AUGUST 29
7:30 am Registration & Morning Coffee
REALIZING THE POTENTIAL OF ONCOLYTIC VIRUS IMMUNOTHERAPY
8:25 Chairperson’s Opening Remarks
Brian Champion, Ph.D., Senior Vice President, R&D, PsiOxus Therapeutics Ltd
8:30 T-Vec: From Market Approval to Future Plans
Jennifer Gansert, Ph.D., Executive Director, Global Development Lead, Amgen, Inc.
9:00 Oncolytic Virotherapies as a Single Shot Cure?
Stephen J. Russell, M.D, Ph.D., Professor, Mayo Clinic
Oncolytic virotherapy is increasingly used as a cancer immunotherapy. However, certain oncolytic viruses can also mediate wholesale tumor destruction independently of an antitumor immune response. This is the oncolytic paradigm, where a cytolytic virus with preferential tumor tropism spreads extensively at sites of tumor growth and directly kills the majority of the tumor cells in the body leaving only a few uninfected tumor cells to be controlled by the concomitant antitumor immune response.
9:30 Coffee Break
UNDERSTANDING MECHANISMS OF ACTION
9:55 Chairperson’s Remarks Fares Nigim, M.D., Massachusetts General Hospital and Harvard Medical School
10:00 Designing Clinical Trials to Elucidate Oncolytic Virus Mechanisms-of-Action
Caroline Breitbach, Ph.D., Vice President, Translational Development, Turnstone Biologics Oncolytic viruses have been shown to target tumors by multiple complementary mechanisms-of-action, including direct oncolysis, tumor vascular targeting and induction of anti-tumor immunity. Phase I/II clinical trials can be designed to validate these mechanisms. Development experience of an oncolytic vaccinia virus and a novel rhabdovirus oncolytic vaccine will be summarized.
10:30 Seprehvir, an Icp34.5 Deleted OHSV with Both Direct and Covert Modes of Action
Joe Conner, Ph.D., CSO, Virttu Biologics
Seprehvir, an oncolytic HSV, is a complex biologic with multi-mechanistic modes of action. Lytic cytotoxicity, induction of Th1 cytokines/chemokine responses, recruitment of innate and adaptive immune cells and changes in the tumor microenvironment can enhance therapeutic efficacy in combination with other anti-cancer agents. How these modes of action intersect with PD-1 checkpoint inhibitors, CAR T cells and small molecule targeted therapies will be discussed.
11:00 The Mechanism of Action of Oncos-102, Oncolytic Adenovirus-Based Immune Activator
Antti Vuolanto, Ph.D., Executive Vice President, Targovax
ONCOS-102 is an engineered human serotype 5 adenovirus expressing GMCSF. A total of 12 cancer patients were treated with repeated intratumoral injections of ONCOS-102 in a Phase I study. Eleven
out of twelve patients showed a post-treatment increase in tumor-infiltrating immune cells including CD8+ T cells and two patients showed systemic induction of tumor specific CD8+ T cells.
11:30 Moving Toward MultiFunctionality in PoxvirusBased Oncolytic Virotherapy
Eric Quemeneur, Ph.D., Pharm.D., Executive VP and CSO, Transgene
Poxviruses are powerful immunotherapeutics and tumor-targeting platforms. We recently expanded Transgene’s portfolio of armed oncolytic Vaccinia Viruses (oVV) by engineering a vector that targets anti-PD1 IgG expression into the tumor. Local concentration of virus-encoded antibody was ~10-50 times higher than the reference mAb, leading to significant improvement of survival in a sarcoma preclinical model. Such results announce the next-generation oVVs, combining immunogenic oncolysis with the capacity to deliver complex therapeutic modalities in the tumor micro-environment.
12:00 pm Luncheon Presentation (Sponsorship Opportunity Available) or Enjoy Lunch on Your Own
12:30 Session Break
BIOMARKERS AND IMPROVING VIRUS ACTIVITY
1:25 Chairperson’s Remarks
David Kirn, M.D., CEO & Co-Founder, 4D Molecular Therapeutics & Adjunct Professor of Bioengineering, UC Berkeley
1:30 New Biomarkers that Predict Response to Oncolytic Virus Immunotherapy
Howard L. Kaufman, M.D., FACS, Associate Director, Clinical Sciences, Rutgers Cancer Institute of New Jersey; Professor and Chief, Division of Surgical Oncology, Rutgers Robert Wood Johnson Medical School
T-VEC is the first oncolytic virus approved for the treatment of melanoma, and will soon enter clinical trials for treatment of other cancers. Further studies using T-VEC in combination with T cell checkpoint inhibitors are underway and showing promising early results. The identification of predictive biomarkers of response would be helpful for improving patient selection and optimizing therapeutic outcomes. We have recently focused on HSV-1 entry receptors and oncogenic signaling pathways within cancer cells as potential biomarkers of T-VEC response.
2:00 Therapeutic Viral Vector Evolution: A Robust Platform for the Discovery of Optimized Vectors
David Kirn, M.D., CEO & Co-Founder, 4D Molecular Therapeutics; Adjunct Professor, Bioengineering, University of California, Berkeley
Therapeutic virus vectors hold great promise for cancer gene and immunotherapy. However, novel vectors with improved efficacy are needed. Therapeutic Vector Evolution is a discovery platform from which optimized and proprietary viral vectors can be identified with beneficial characteristics of interest.
2:30 Enhancing Oncolytic Virus Activity by Engineering of Artificial MicroRNAs
John Bell, Ph.D., Senior Scientist, Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Professor, Departments of Medicine and Biochemistry, Microbiology & Immunology, University of Ottawa
We have devised a novel strategy to enhance the ability of oncolytic viruses to infect malignant cells by expressing artificial microRNAs (amiRNAs) from the oncolytic virus genome. We have screened a variety of amiRNAs and identified a number that enhance virus replication within tumour but not normal cells. The characterization of these miRNAs and their targets will be discussed.
3:00 Refreshment Break
3:30 KEYNOTE PRESENTATION: ONCOLYTIC IMMUNOTHERAPY IN THE ERA OF CHECKPOINT BLOCKADE
Robert Coffin, Ph.D., CEO, Replimmune
Oncolytic immunotherapy treats cancer by virus-mediated tumor cell lysis and generation of a patient specific cancer vaccine, including to neo-antigens, in situ directly in the patient. Both are likely important for the clinical efficacy seen with single agent use, and also for the clinical synergy observed with immune checkpoint blockade. Background and data supporting single agent and combination use will be discussed, and future directions described.
4:00 Arming the Oncolytic Virus Enadenotucirev to Develop Tumor-Localized Combination Immunotherapeutics
Brian Champion, Ph.D., Senior Vice President, R&D, PsiOxus Therapeutics Ltd.
We have developed a systemically deliverable, oncolytic adenoviral platform for directing efficient and selective local production of a combination of biotherapeutic agents selectively within the tumor. This has
the potential for enhanced efficacy while reducing side effects by limiting systemic exposure. Up to three separate biomolecules can be encoded in the same virus without affecting oncolytic properties of the virus.
4:30 Immuno-Oncolytic Viruses as Cancer Therapies
Stephen Thorne, Ph.D., Professor and Scientific Advisor, Inventor, Western Oncolytics
Oncolytic viruses primarily act as immunotherapies, yet most vectors still rely on the virus’ inherent immune activation, often coupled to single cytokine transgene expression. However, for optimal activity they will need to overcome the tumor¹s immunosuppressive microenvironment, to raise anti-tumor CTL and allow repeated systemic delivery. Approaches to achieve all of these activities in a single vector are being developed.
5:00 End of Day
TUESDAY, AUGUST 30
8:00 amMorning Coffee
TRANSLATIONAL AND CLINICAL UPDATES
8:25 Chairperson’s Opening Remarks
8:30 Phase I of Intravenous Vcn-01 in Patients with Advanced Cancer: Update on Clinical & Biologic Data
Manel Cascallo, Ph.D., Co-Founder, President and CEO, VCN Biosciences
A first-in-human Phase I dose escalation study of intravenous administration of VCN-01 (an oncolytic adenovirus with RB tumour-targeting properties and expressing hyaluronidase) with or without gemcitabine and Abraxane is ongoing for patients with advanced solid tumours including pancreatic cancer. Dose dependent tolerability data and VCN-01 levels in different biological samples (including blood and tumour biopsies) are available.
8:50 T-Stealth Technology Mitigates Antagonism between Oncolytic Viruses and the Immune System through Viral Evasion of Anti-Viral T-Cells
Matthew Mulvey, Ph.D., CEO, BeneVir
Simultaneous combination of OV and immune checkpoint inhibitors (ICI) are antagonistic because ICI enhance anti-viral T-cell effector activity and speed OV clearance. BeneVir’s T-StealthTM armed OV mitigate antagonism between OV and ICI because they evade anti-viral T-cells. This allows OV and ICI to be administered simultaneously over several treatment cycles to maximize the likelihood of a synergistic clinical response
9:10 Reolsyin: A Clinical Update of a Directed Cytotoxic Agent and Immune Modulator
Brad Thompson, Ph.D., CEO, Oncolytics Biotech
REOLYSIN was initially investigated for its potential as a selective cytotoxin. However, recent research shows that it also functions as an immune modulator. This dual mechanism of action for a single viral agent suggests that the potential of viral therapies may be broader than previously anticipated.
9:30 Retroviral Replicating Vectors for Cancer-Selective Immuno/Gene Therapy: Translational and Clinical Update
Noriyuki Kasahara, M.D., Ph.D., Professor, Departments of Cell Biology and Pathology, CoLeader, Viral Oncology Program, University of Miami
Pro-drug activator gene therapy with retroviral replicating vectors is tumor-selective, and can lead to development of anti-tumor immunity. Ascending dose Phase I trials by Tocagen Inc. in recurrent high-grade glioma demonstrated favorable safety and tolerability, intratumoral virus spread, radiographic responses, and survival surpassing historical benchmarks. Based on these results, a randomized controlled Phase II/III trial is now underway.
10:30 Grand Opening Coffee Break in the Exhibit Hall with Poster Viewing
11:15 Virus Manufacturing Comes of Age: Turning Bugs into Features Anthony Davies, Ph.D., COO, 4D Molecular Therapeutics
Viruses destroy the host in which you’re trying to produce them and then must be separated from all components of those cells. Many solutions to these challenges have been invented since the earliest production of viral vaccines in primary cells obtained directly form animals. But few have proven amenable to cost-effective, compliant and scaleable operation.
11:45 Manufacturing Large Enveloped Oncolytic Viruses for Human Clinical Trials
Mark J. Federspiel, Ph.D., Professor and Director, Viral Vector Production Laboratory, Mayo Clinic
The large-scale production and purification of larger enveloped oncolytic viruses are particularly challenging. We have developed enveloped virus GMP production processes using suspension cells in combination with gentle but effective purification using hollow fiber tangential flow filtration that result in greater than 99.5% removal of contaminants and greater than 100-fold increases in final infectious virus titers.
AGENDA for Adoptive T Cell Therapy Delivering CAR, TCR, and TIL from Research to Reality, CHI’S 4TH ANNUAL IMMUNO-ONCOLOGY SUMMIT – SEPTEMBER 1-2, 2016 | Marriott Long Wharf Hotel – Boston, MA
Adrian Bot, M.D., Ph.D., Vice President, Translational Sciences, R&D, Kite Pharma Inc.
8:30 KEYNOTE PRESENTATION: CLINICAL DEVELOPMENT OF TUMOR-INFILTRATING LYMPHOCYTE THERAPY FOR SOLID TUMORS: THE MYTHS AND THE REALITY
Laszlo Radvanyi, Ph.D., Senior Vice President and Head, ImmunoOncology Translational Innovation Platform (TIP), EMDSerono
Many of the patients treated with TILs have progressed after multiple therapies, including checkpoint blockade with anti-CTLA-4 and anti-PD-1/PD-L1, making TIL also an ultimate salvage therapy option. In this presentation, we will discuss common misconceptions about TIL therapy and current efforts that can realistically bring TIL therapy into the mainstream as an approved product for melanoma care as well as its promise as a cellular therapy for other solid cancers.
9:00 Engineering Better T Cells
Daniel Williams, Head, UK Research Operations, Adaptimmune
Adaptimmune is a clinical-stage biopharmaceutical company developing engineered TCRs for adoptive T cell therapy. Engineering TCRs specific for tumor antigens which are self-antigens, requires balancing the need for increasing affinity to the target peptide to allow recognition of immune-selected tumors, with the risk of cross-reactivity, which increases at higher affinities. Target identification and validation, together with a broad and robust preclinical safety testing strategy are critical in the development of safe and efficacious affinity-enhanced TCRs.
9:30 Development of an Anti-BCMA Specific CAR with Potent Anti-Multiple Myeloma Activity
Richard A. Morgan, Vice President, Immunotherapy, bluebird bio
B cell maturation antigen (BCMA) is expressed on most multiple myeloma (MM) cells, yet normal tissue expression is limited to plasma and some B cells. Here we demonstrate that a potent, antigen-dependent, memory-like BCMA CAR T cells can be produced with a scalable manufacturing process that mediated robust tumor regressions in animal models. A Phase I clinical trial to test this approach in patients with relapse refractor MM is underway.
10:00 Coffee Break with Exhibit and Poster Viewing
TARGETS FOR T CELL INTERVENTIONS
10:45 CD19 as a Prototypic B Cell Lineage Target for CAR T Cells Adrian Bot, M.D., Ph.D., Vice President, Translational Sciences, R&D, Kite Pharma Inc. Anti-CD19 CAR T cells could be the first marketed products in this space. Prolonged B cell deficiency may not be desirable since B cells are required to combat major pathogens. Product candidates capable to mediate rapid and profound tumor debulking, can yield durable clinical responses without persisting B cell deficiency. Novel evidence will be presented, linking product characteristics, conditioning, and biomarkers, to the clinical outcome afforded by CAR T cells.
11:15 CAR-T Cells for Hematological Malignancies: Exploring Alternative Targets
Gianpietro Dotti, M.D., Professor, Microbiology and Immunology, University of North Carolina
Adoptive transfer of CD19-specific CAR-T cells showed remarkable anti-tumor activity in patients with B-cell derived acute lymphoblastic leukemia and lymphomas. Alternative targets have been explored in Phase I studies in hematological malignancies. Specifically, in the effort to reduce the B cell aplasia associated with effective and long-term persisting CD19-specific CAR T cells, we implemented at Baylor College of Medicine a Phase I study with CAR-T cells targeting the k-light chain of human immunoglobulin to selectively eliminate k+ tumors whilst sparing normal l+ B lymphocytes. To target Hodgkin’s Lymphoma and other CD30+ lymphomas, we also implemented a Phase I study with CAR-T cells targeting the CD30 antigen. Outcome of these studies and future directions will be reported.
11:45 PANEL: CAR T Cell Therapy: Target Antigen Discovery and Clinical Translation
Panelists: Adrian Bot, M.D., Ph.D., Vice President, Translational Sciences, R&D, Kite Pharma Inc. Gianpietro Dotti, M.D., Professor, Microbiology and Immunology, University of North Carolina Richard A. Morgan, Vice President, Immunotherapy, bluebird bio
12:45 Luncheon Presentation (Sponsorship Opportunity Available) or Enjoy Lunch on Your Own
1:15 Session Break
2:00 Chairperson’s Remarks
Adrian Bot, M.D., Ph.D., Vice President, Translational Sciences, R&D, Kite Pharma Inc.
Adoptive T Cell Therapy Delivering CAR, TCR, and TIL from Research to Reality Cover Conference at-a-Glance Faculty Keynotes
2:05 Human Papillomavirus (HPV)-Targeted T Cells for HPV-Associated Cancers
Christian S. Hinrichs, M.D., Investigator, Experimental Transplantation and Immunology Branch, Lasker Clinical Research Scholar, NIH
Human papillomavirus (HPV) causes cancers of the uterine cervix, oropharynx, anus, vulva, vagina and penis. These cancers express the HPV E6 and E7 oncoproteins, which are attractive immunotherapeutic targets. Recent work has sought to target these antigens using tumor-infiltrating lymphocytes and genetically engineered T cells.
2:35 XPRESIDENT®-Guided Target Identification and Profiling of On-and Off Target Toxicity for Safer Adoptive Cell Therapy
A major constraint for the broad and safe application of Adoptive Cellular Therapy is the limited number of validated tumor targets and TCRs. We used a proprietary target discovery engine (XPRESIDENT®) combining highly sensitive quantitative mass spectrometry, transcriptomics, immunology and bioinformatics to characterize the immunopeptidome directly on human normal and tumor tissues. We show how this approach can be used to predict on- and off-target toxicities in TCR-based immunotherapies.
4:05 Engineering Designer T Cells for Immunotherapy
Wolfgang Uckert, Associate Professor, Department of Cell Biology and Gene Therapy, Humboldt University Berlin
Successful immunotherapy using T cell receptor (TCR) gene-modified T cells to treat cancer, viral infections or autoimmune diseases is depending on the careful selection of: (i) the target antigen, (ii) a TCR with optimal affinity, (iii) an efficient gene delivery system, and (iv) a safety switch to interrupt therapy in case of severe adverse side effects. Examples of these different areas to generate designer T cells for successful TCR gene therapy are given and discussed.
4:35 KEYNOTE PRESENTATION: A COMPARISON OF TCRS AND CARS: SENSITIVITIES AND SPECIFICITIES
David M. Kranz, Phillip A. Sharp Professor, Biochemistry, University of Illinois, Urbana-Champaign
T cells, via their T-cell receptors (TCRs), evolved to target intracellular peptides as low-density, cell-surface complexes with MHC products. Synthetic constructs known as chimeric antigen receptors (CARs) contain an anti-tumor antigen scFv and recognize higher density antigens. We have designed high-affinity human TCRs in conventional heterodimer format and in CAR-like formats to directly compare features of both systems. These features include T cell surface levels, antigen sensitivities and other properties.
5:05 PANEL: Designing T Cells for Immunotherapies Panelists: Christian S. Hinrichs, M.D., Investigator, Experimental Transplantation and Immunology Branch, Lasker Clinical Research Scholar, NIH Steffen Walter, Ph.D., CSO, Immatics Biotechnologies GmbH Wolfgang Uckert, Ph.D., Associate Professor, Cell Biology and Gene Therapy, Humboldt University Berlin David M. Kranz, Phillip A. Sharp Professor, Biochemistry, Biochemistry, University of Illinois
Strategies for antigen selection and targeting
Increasing sensitivity and specificities
Utilizing efficient gene delivery systems
Dealing with toxicities
5:35 End of Day
FRIDAY, SEPTEMBER 2
8:00 am Morning Coffee
TARGETING SOLID TUMORS
8:25 Chairperson’s Opening Remarks
Armon Sharei, Ph.D., CEO, SQZ Biotechnologies
8:30 Vector-Free Engineering of Immune Cells for Enhanced Antigen Presentation
Armon Sharei, Ph.D., CEO,
SQZ Biotechnologies Robust engineering of immune cell function is critical to realizing the therapeutic potential of cell therapies in cancer. Our vector-free CellSqueeze technology has demonstrated novel capabilities in diverse areas including engineering antigen presentation to drive powerful and significant T-cell responses. We will present recent developments in our cell-based immunotherapy programs aimed at using patient-derived antigens to target a variety of cancer indications, including solid tumors.
9:00 Design of a Highly Efficacious CAR Targeting Mesothelin in Solid Tumors Boris Engels, Ph.D., Investigator, Exploratory ImmunoOncology, Novartis Institutes for Biomedical Research
The treatment of solid tumors with CAR T cells remains challenging. We describe the design of a human CAR targeting mesothelin, a tumor associated antigen overexpressed in many cancers. A pooled screen identified scFvs, which show enhanced efficacy, superior to the CARs currently used in the clinic. We performed in-depth characterization of the scFvs and CARs to gain insight into structure-activity relationships, which may influence CAR efficacy and future design.
9:30 Overcoming CAR T Cell Checkpoint Blockade in Solid Tumors Prasad S. Adusumilli, M.D., FACS, FCCP, Deputy Chief, Translational and Clinical Research, Thoracic Surgery; Associate Attending, Thoracic Surgery; Member, Center for Cell Engineering, Memorial Sloan-Kettering
Cancer Center CAR T-cell therapy for solid tumors is prone to the checkpoint blockade inhibition similar to innate tumor-infiltrating lymphocytes. Strategies to overcome this ‘Adaptive Resistance’ of infused CAR
Immuno-Oncology Summit.com T cells can promote their anti-tumor efficacy and functional persistence. Understanding solid tumor type-specific immune microenvironment can guide both cell-intrinsic and extrinsic strategies that can modulate the solid tumor microenvironment in addition to promoting CAR T-cell efficiency.
10:00 Coffee Break MANUFACTURE AND SCALE-UP
10:30 Challenges and Opportunities for Scale-up of CAR T Cells Máire Quigley, Ph.D., Research Investigator I, Cell and Gene Therapies Unit, Novartis
The response rates of autologous CAR T cell therapies in early clinical trials give hope that these treatments can be developed and widely distributed to patients with unmet need. To successfully evolve from small scale production to commercial manufacturing, multiple challenges must be overcome. Lessons learned from the process scale up of CD19 CAR T cell production will be discussed.
11:00 Cell Therapy: Quality and Good Manufacturing Practices
Yeong “Christopher” Choi, Ph.D., Director, TCPF and Assistant Professor, Oncology, Center for Immunotherapy, Roswell Park Cancer Institute
To bring ground breaking new cell therapies into the clinic, a massive amount of infrastructure is needed. One of the key components is a drug manufacturing facility, which is compliant with US FDA GMP regulations. One of the major challenges of operating a cell therapy cGMP facility, is maintaining a robust Quality Management System. Fortunately, there are a number of voluntary accrediting organizations to promote excellence in the laboratory.
This talk will provide an introduction of the concept of Cellectis’ allogeneic UCART product candidates and our technological platform. It will describe and explain the challenges during the process of bringing allogeneic UCART product candidates from R&D development phase to the GMP manufacturing phase, in order to have CTM (clinical trial material) available for clinical studies, and afterwards to plan commercial manufacturing of Cellectis’ allogeneic UCART GMP cellular gene therapy products.
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.
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.
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
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.
TUMOR NEOANTIGENS FOR PERSONALIZED IMMUNOTHERAPY
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
PERSONALIZED IMMUNOTHERAPY WITH CANCER VACCINES
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
PERSONALIZED CELL THERAPY
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.
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Cancer Vaccines: Targeting Cancer Genes for Immunotherapy – – A Conference by Keystone Symposia on Molecular and Cellular Biology – Late publishing
Organizer(s) A. Karolina Palucka, Hyam I. Levitsky and Laurence Zitvogel
March 6—10, 2016
Fairmont Chateau Whistler • Whistler, British Columbia Canada
Reporter: Aviva Lev-Ari, PhD, RN
Though the Conference took place in March 6—10, 2016, I am posting its Agenda on 7/4/2016.
It was 4/3/2016 when we launched “DrugDiscovery @LPBI Group”with a focus on Cancer Vaccines: Targeting Cancer Genes for Immunotherapy. The academic nature of this conference is of particular interest to our Team at present time while the Team is working on Biologics for Pancreatic Cancer: New MOA in Immunotherapy.
In addition, of interest are the following two resources:
Three Methods for Design of a Novel Immune Therapy for Cancer: Conceptual Foundation for Development of a Novel Mechanism of Action for a Combination Therapy of Biologics
Keystone Symposia on Molecular and Cellular Biology: Mission
Keystone Symposia will serve as a catalyst for the advancement of biomedical and life sciences by connecting scientists within and across disciplines at conferences and workshops held at venues that create an environment conducive to information exchange, generation of new ideas and acceleration of applications that benefit society.
Background
Keystone Symposia on Molecular and Cellular Biology is a 501(c)(3) nonprofit organization headquartered in Silverthorne, Colorado, USA that convenes open, peer-reviewed conferences across a broad range of the life sciences. Approximately 50-60 conferences take place each year. More than half the symposia are held in mountain venues across the American and Canadian West, with the remainder in generally North American cities and various global locations. We have now convened conferences on six continents: Africa, Asia, Australia, Europe, North America and South America.
Scientific content for each conference is organized by volunteer scientists who are experts in their respective fields and who also select program speakers, with guidelines from Keystone Symposia to encourage fresh and diverse participation. The conferences are typically three to four full days in length and consist of two daily plenary sessions complemented by workshops and poster sessions. The meeting format is designed to maximize informal networking among participants. Scholarships and travel awards help make possible the participation of graduate students and postdoctoral fellows, who typically account for 40% of attendees each year. Keystone Symposia has a range of Diversity in Life Science Programs and actively encourages participation of underrepresented investigators and scientists from developing countries.
Keystone Symposia’s Chief Executive Officer reports to the Board of Directors and, along with the Chief Scientific Officer, acts on the advice of a Scientific Advisory Board comprised of more than 75 leading scientists from academia, industry and government worldwide. This Board meets twice a year to determine conference topics, identify potential scientific organizers and review proposed programs.
We receive revenue from two sources: registration fees (approximately 65-70%) and generous support from corporations, foundations, government entities and individuals (approximately 30-35%). This support provides funding for scholarships as well as speaker travel expenses (no honoraria are paid), allowing registration fees to be kept as low as possible.
A staff of 40 full-time, part-time or seasonal employees handles all aspects of administration, meeting management/logistics, attendee services, fundraising and marketing.
History
Founded in 1972 in Los Angeles as the ICN-UCLA Symposium on Molecular Biology by Professor C. Fred Fox, the organization evolved into UCLA Symposia before relocating to Silverthorne, Colorado in 1990. At that time we became a free-standing division of a nonprofit called The Keystone Center and were renamed Keystone Symposia on Molecular and Cellular Biology. We separated from The Keystone Center and became an entirely independent nonprofit in a phased transition beginning in 1995 and ending in 1997.
The first meeting organized was on Membrane Research in Squaw Valley, California in March 1972. While still known as UCLA Symposia, the organization convened the first-ever open, international meeting on AIDS in 1984, which was widely credited with catalyzing a consensus that AIDS was caused by a retrovirus now known as the Human Immunodeficiency Virus.
We proudly celebrated our 40th anniversary in 2012!
Timeline of Key Milestones
1972: Keystone Symposia was founded as the ICN-UCLA Symposium on Molecular Biology and held an initial conference on membrane research in Squaw Valley, California, March 13-17, 1972.
1990: Keystone Symposia relocated to Silverthorne, Colorado, became a division of The Keystone Center and was renamed Keystone Symposia on Molecular and Cellular Biology.
1995-97: Keystone Symposia became an independent 501(c)(3) nonprofit organization.
2001: First conference outside of the US in Canada (“Hematopoiesis” in Whistler, British Columbia, Canada).
2005: First conference in Asia (“Stem Cells, Senescence and Cancer” in Singapore).
2006: First conference in Europe (“Multi-Protein Complexes Involved in Cell Regulation” in Cambridge, UK).
2007: First conference in Africa (“Challenges of Global Vaccine Development” in Cape Town, South Africa).
2009: First conference in Australia (“Telomere Biology and DNA Repair” in Ashmore).
2013: First conference in South America (“The Innate Immune Response in the Pathogenesis of Infectious Disease” in Ouro Preto, Brazil).
Organizer(s) A. Karolina Palucka, Hyam I. Levitsky and Laurence Zitvogel
March 6—10, 2016
Fairmont Chateau Whistler • Whistler, British Columbia Canada
Summary of Meeting: The past decade has seen tremendous developments in novel cancer therapies, through targeting of tumor cell-intrinsic pathways whose activity is linked to genetic alterations, as well as the targeting of tumor cell-extrinsic factors such as growth factors. Remarkable clinical success of checkpoint inhibitors as well as adoptively transferred genetically engineered T cells further demonstrates the critical role of T cells in cancer control and rejection. However, many patients still do not respond to checkpoint inhibitor therapies, possibly due to the lack of T cell repertoire with specificity against cancer antigens. This creates the need for effective means of expanding the T cells in patients via immunization, i.e., cancer vaccines. Much progress has been made in recent years in this regard, and several phase III clinical trials testing various approaches to therapeutic vaccination are ongoing. The challenge for next-generation vaccines is to resolve the discrepancy between the immune and clinical efficacy measured by the rate of cancer rejection. The future immunotherapy of cancer lies in combination approaches targeting T cells as well as cancer genes to combat underlying inflammation. Spectacular progress has been made in these two parallel fields, i.e., cancer genomics and genetics, and tumor immunology. It is time now to link these fields to enable the linking of genetic alterations with the type of immune response. The meeting will therefore host cancer geneticists, cancer biologists, experts in cancer antigen presentation, tumor immunologists and vaccinologists. It will discuss the immunological basis for therapeutic cancer vaccines and how the current understanding of cancer genomics, antigen presentation and T cell biology might enable development of next-generation curative therapies for patients with cancer.
The meeting will begin on Sunday, March 6 with registration from 16:00 to 20:00 and a welcome mixer from 18:00 to 20:00. Conference events conclude on Thursday, March 10 with a closing plenary session from 17:00 to 19:00, followed by a social hour and entertainment. We recommend return travel on Friday, March 11 in order to fully experience the meeting.
SUNDAY, MARCH 6
16:00—20:00
Arrival and Registration
Macdonald Foyer
18:00—20:00
Welcome Mixer
No registration fees are used to fund alcohol served at this function.
Rakesh K. Jain, Massachusetts General Hospital and Harvard Medical School, USA Reengineering the Tumor Microenvironment to Enhance Cancer Treatment: Bench to Bedside
Andreas G. Plückthun, University of Zürich, Switzerland Protein Engineering Approaches to New Biologicals
* A. Karolina Palucka, Tha Jackson Laboratory for Genomic Medicine, USA
Cornelis J. M. Melief, Leiden University Medical Center, Netherlands Synergy of Therapeutic Vaccination against HPV16 Oncogenic Proteins and Standard Chemotherapeutics
Victor H. Engelhard, University of Virginia, USA Phosphopeptides Displayed by MHC Molecules as New-Generation Cancer Vaccine Targets
The session will cover some of the major underlying challenges facing the field of antibody based therapeutics.
Macdonald ABC
* Peter D. Senter, Seattle Genetics Inc., USA
James M. Olson, Fred Hutchinson Cancer Research Center, USA Micro Needle Technologies for Intratumoral Drug Delivery and Combination Screening
Alison M. Betts, Pfizer, Inc., USA PK/PD Modeling for Macromolecular Drug Delivery
Richard Tavaré, University of California, Los Angeles, USA Short Talk: Noninvasive Detection of Tumor-Infiltrating Lymphocytes using Anti-CD8 Immuno-PET: Impact of Protein Dose
11:30—12:30
Lunch
Frontenac
12:00—14:30
Poster Session 1
Frontenac
14:30—16:30
Workshop 1: Molecular Vaccines
Macdonald DEF
* Darrell J. Irvine, Massachusetts Institute of Technology, USA
* Yvette van Kooyk, Vrije University Medical Center, Netherlands
Tomasz Ahrends, Netherlands Cancer Institute, Netherlands Combined CD27-Costimulation and PD-1-Inhibition Recapitulates CD4+ T-cell Help in Therapeutic Vaccination Against Cancer
Suresh de Silva, Heat Biologics Inc., USA Combination Immunotherapy: T-cell Costimulation (OX40L, TL1A, 4-1BBL and ICOSL) Secreted Locally by Gp96-Ig Vaccines, Elicits Robust Antigen-Specific, Memory T Cell Responses and Tumor Elimination
Christa I. DeVette, University of Oklahoma Health Sciences Center, USA Development of Tools to Evaluate Anti-tumor Immunity
James Moon, University of Michigan, USA Nanodisc-Based Peptide Vaccines for Personalized Cancer Immunotherapy
Geoffrey Lynn, National Institutes of Health, USA Self-Assembling Nanoparticles Codelivering Peptide Neoantigens and TLR-7/8 Agonists Enhance the Breadth and Potency of Anti-Tumor Immunity
Naveen Mehta, Massachusetts Institute of Technology, USA Utilizing Protein Carriers to Engineer Cancer Vaccines with Improved Potency
Kristen Radford, University of Queensland, Australia Targeting Antigen to Human CD141+ Dendritic Cells via CLEC9A in vitro and in vivo Confers Effective Cross-Presentation to CD8+ T Cells
Jonathan H. Davis, Bristol-Myers Squibb, USA Synergy Generates Picomolar Potency and a High Resistance Barrier in Combinectin, a Novel Trispecific HIV-1 Entry Inhibitor with Clinical Promise
Eric Hatterer, NovImmune.SA, Switzerland Characterizing the Preclinical Candidate, NI-1701, a CD47 Targeting Bispecific Antibody in Development for the Treatment of B Cell Lymphomas and Leukemias
Paul A. Moore, MacroGenics, USA MGD009, a B7-H3 x CD3 Bispecific Dual-Affinity Re-Targeting (DART®) Molecule Directing T Cells to Solid Tumors
Marijn A. Gillissen, Aimm Therapeutics / AMC, Netherlands Donor-Derived B Cells Produce Potent AML-Specific Antibodies that Recognize a Novel Tumor-Specific Antigen and Mediate Graft-Versus- Leukemia Immunity
Yu-Sang Sabrina Yang, Massachusetts Institute of Technology, USA Targeting Immunomodulators to Lymphocytes Via Amphiphilic Gold Nanocarriers
Travis Young, California Institute for Biomedical Research, USA Antibody-based Control of CAR-T Cell Therapy
* Jacques F. Banchereau, The Jackson Laboratory for Genomic Medicine, USA
Julie Magarian Blander, Mount Sinai School of Medicine, USA Regulation of Antigen Cross-presentation in Dendritic Cells
A. Karolina Palucka, Tha Jackson Laboratory for Genomic Medicine, USA Dendritic Cells as Cancer Vaccines
Matthew F. Krummel, University of California, San Francisco, USA T Cell-Antigen Presenting Dynamics in the Tumor Microenvironment
Brian J. Francica, Johns Hopkins University School of Medicine, USA Short Talk: Radio-resistant Stromal Cells Initiate the Innate Response to Cyclic Dinucleotides
Margaret Ellen Ackerman, Dartmouth College, USA Evolution of Antibodies for Optimized Effector Function Activities
Jeanette H.W. Leusen, University Medical Center Utrecht, Netherlands IgA as Alternative Therapeutic Antibody: Combination with IgG, Half-Life Extension and More
Sarel J. Fleishman, Weizmann Institute of Science, Israel De novo Design of Novel Protein Functions
Paul W. H. I. Parren, Genmab B.V., Netherlands Functional Aspects of Antigen- and Fc-Dependent IgG Hexamer Formation
Victor Appay, INSERM U1135, France Impaired Induction of High-Quality Effector CD8+ T-Cells in Old Humans
Luca Gattinoni, NCI, National Institutes of Health, USA Generation of Long-Lived Cancer Antigen-Specific CD8+ T Cell Memory
Pamela S. Ohashi, Princess Margaret Cancer Centre, Canada Novel Subsets Involved in T Cell Anti-Tumor Immunity
Ugur Sahin, TRON – Translationale Oncology, Germany Exploiting Antiviral Immune Mechanisms for Cancer Immunotherapy
Dixon Dorand, Case Western Reserve University, USA Short Talk: Cyclin Dependent Kinase 5 (CDK5) Controls PD-L1 Expression via Phosphorylation of IRF2BP2 in Murine Medulloblastoma and CDK5 Deficiency Results in CD4+ Dependent T-Cell Rejection
Alison Taylor, University of Cambridge, UK Short Talk: Glycogen Synthase Kinase 3 Inhibition Blocks PD-1 Expression via Increased Tbet Expression for Enhanced CD8+ Cytolytic T-Cell Responses
How the immune system adapts to infection and other challenges, and how antibody combinations can be used to treat diseases.
Macdonald ABC
* David A. Scheinberg, Memorial Sloan-Kettering Cancer Center, USA
George Georgiou, University of Texas at Austin, USA Systematic Characterization and Comparative Analysis of Antibody Repertoires
Pavel Tolar, Francis Crick Institute, UK EMBO Young Investigator Lecture: Regulation of Antibody Responses by B Cell Mechanical Activity
Laura M. Walker, Adimab, USA Rapid and Large-Scale Isolation of Potent Neutralizing Antibodies from a Survivor of the 2014 Ebola Virus Outbreak
Paul A. Ramsland, RMIT University, Australia Short Talk: Unanticipated Global Changes in IgG1 from Mutation of FcRn Binding Site Residues
Sai T. Reddy, ETH Zurich, Switzerland Short Talk: Correlations of Antibody Response Phenotype to Genotype Revealed by Molecular Amplification Fingerprinting
09:20—09:40
Coffee Break
Macdonald Foyer
11:15—13:00
Poster Setup
Frontenac
13:00—22:00
Poster Viewing
Frontenac
11:15—14:30
On Own for Lunch
14:30—16:30
Workshop 2: Cell-Based Vaccines
Macdonald DEF
* Robert A. Seder, NIAID, National Institutes of Health, USA
* Masaki Terabe, NCI, National Institutes of Health, USA
Andres Alloatti, Institut Curie, France Dissecting Cross-Presentation in Dendritic Cells: Who, How and Why?
Anja C. Bloom, NCI, National Institutes of Health, USA INT230-6 Administered Intratumorally Converts Tumor to an Endogenous Vaccine in Mouse Colon Cancer
Agamemnon Antoniou Epenetos, Imperial College London, UK Creation of Biological Microparticles from Mesenchymal Stem Cell (MSC) to Deliver Vaccines and other Therapeutics such as Proteins or Nucleic Acids
Simon Heidegger, Klinikum Rechts der Isar, Technical University Munich, Germany Activation of the Cytosolic RNA Receptor RIG-I in Tumor and Immune Cells Triggers Efficient Anti-Tumor Immunity and Synergizes with Checkpoint Blockade
Kavya Rakhra, Massachusetts Institute of Technology, USA Combination Immunotherapy of an Autochthonous Murine Lung Cancer Model Expressing Human CEA as a Tumor-Associated Self-Antigen
Yvette van Kooyk, Vrije University Medical Center, Netherlands Vaccines Facilitating Cross-Presentation for the Induction of Tumor-Immunity Overcoming Tumor Induced Suppression
Megan C. Wise, University of Pennsylvania, USA Various forms of CD40L Encoded as an Immune Plasmid Adjuvant Generate Unique Anti-HPV DNA Vaccine Induced Responses
Rafi Ahmed, Emory University School of Medicine, USA Features of Memory CD8+ T Cells in Chronic Infection
Nicholas P. Restifo, NCI, National Institutes of Health, USA Lineage Relationship of Effector and Memory T Cells
Jonathan D. Powell, Johns Hopkins University School of Medicine, USA Targeting Immunometabolism as a Means of Enhancing Immunotherapy
Madhusudhanan Sukumar, NCI, National Institutes of Health, USA Short Talk: Mitochondrial Membrane Potential Identifies Cells with Enhanced Stemness for TCR and CAR based Adoptive Immunotherapy of Cancer
* Anna M. Wu, University of California, Los Angeles, USA
David A. Scheinberg, Memorial Sloan-Kettering Cancer Center, USA Drugging Intracellular Targets with Vaccines and Human Therapeutic TCR Mimic Antibodies
Dennis R. Benjamin, Seattle Genetics Inc., USA Antibody Drug Conjugates for Cancer Therapy
Birgit Schoeberl, Merrimack Pharmaceuticals, USA Multispecific Antibody Therapeutics Derived from Systems Biology and Computational Modeling
John C. Williams, Beckman Research Institute at City of Hope, USA Functionalization of MAbs Using Mechanical Bonds
19:00—20:00
Social Hour with Lite Bites
No registration fees are used to fund alcohol served at this function.
* Cornelis J. M. Melief, Leiden University Medical Center, Netherlands
Laurence Zitvogel, Institut Gustave Roussy, France Molecular Mechanisms of Cancer Antigen Presentation at the Tumor Site
Thomas W. Dubensky, Jr., Aduro Biotech, USA Insights from Listeria monocytogenes for the Development of Vector- and Small Molecule-Based Cancer Immunotherapy Strategies
Howard L. Kaufman, Rutgers Cancer Institute of New Jersey, USA Advances in Oncolytic Virus Immunotherapy
Sandra Demaria, Weill Cornell Medical College, USA In situ Vaccination by Radiation Therapy
Rupert Kenefeck, Immunocore Ltd., UK Short Talk: Overcoming Immune Regulation to Kill Cancers: ImmTAC Targeting and Checkpoint Inhibition
Hamid Bassiri, Children’s Hospital of Philadelphia, USA Novel Invariant Natural Killer T Cell-Based Immunotherapies for Cancer
Hanke Matlung, Sanquin Research and Landsteiner Laboratory, Netherlands Neutrophils Kill Antibody-Opsonized Cancer Cells by Trogoptosis
Sheena N. Smith, University of Zurich, Switzerland Targeted Adenoviral Delivery of Protein-Based Therapeutics to the Tumor Microenvironment
Michael W. Munks, Oregon Health & Science University, USA Major Histocompatibility Class I – Antibody Fusion Proteins (pMHCI-IgGs) that Link B16 Melanoma Cells to Cytomegalovirus-Specific CD8 T Cells Control B16 Melanoma in vivo
Huafeng Xu, D.E. Shaw Research LLC, USA Divergent Sequences Stabilize Similar Antigen-Binding Conformations in the Affinity Maturation of a Broadly Neutralizing Influenza Antibody Lineage
David P. Humphreys, UCB Pharma, UK, UK Statistical Design for Effector Function Engineering of Hexameric Fc Domains
Andre BH Choo, Bioprocessing Technology Institute, Singapore Glycan: A Sweet Opportunity for Antibody Discovery
* Laurence Zitvogel, Institut Gustave Roussy, France
Madhav V. Dhodapkar, Yale School of Medicine, USA Harnessing Host Response to Premalignancy in Prevention of Cancer
Carl G. Figdor, Radboud University Medical Center, Netherlands Dendritic Cell Based Cancer Immunotherapy
Ira Mellman, Genentech, Inc., USA The Mechanistic Basis of Cancer Immunotherapy
Nina Bhardwaj, Mt. Sinai Hospital, USA Modulation of Innate Immunity in Cancer
Kelly D. Moynihan, Massachusetts Institute of Technology, USA Short Talk: Eradication of Large Established Tumors by Combination Immunotherapy Engaging Innate and Adaptive Immunity
Rony Dahan, Rockefeller University, USA Short Talk: Improved Therapeutic Activity of Agonistic, Human Anti-CD40 Monoclonal Abs by Selective FcgammaR-Engagement
09:20—09:40
Coffee Break
Macdonald Foyer
11:15—14:30
On Own for Lunch
14:30—16:30
Workshop 3: Peptides as Vaccines
Macdonald DEF
* Robert D. Schreiber, Washington University School of Medicine, USA
Anne-Mette Bjerregaard, Technical University of Denmark, Denmark MuPeXI: A Tool for Prediction of Neo-Epitopes from Tumor Sequencing Data
Gustav Gaudernack, Ultimovacs AS/Norwegian Radium Hospital, Norway UV1 – A Second-Generation, Peptide-Based, Therapeutic Cancer Vaccine (TCV) Targeting the Reverse Transcriptase Subunit of Human Telomerase (hTERT
Matthew M. Gubin, Washington University in St. Louis, USA Tumor-Specific Mutant Antigens in Cancer Immunotherapy
Bruno Laugel, Adaptimmune Ltd, UK Safety Assessment of DNA Methyl Transferase Inhibitors in Combination with NY-ESO T-cell Therapy.
Aizea Morales Kastresana, NCI, National Institutes of Health, USA Optimization of Extracellular Vesicle Labels for NanoFACS
Hideho Okada, University of California, San Francisco, USA Novel and Shared Neoantigen for Glioma T Cell Therapy Derived from Histone 3 Variant H3.3 K27M Mutation
Jeffrey Ward, Washington University in St. Louis, USA Tumor Mutant Antigen-Specific CD8+ T-cells Require CD4+ T-cell Help to Induce Tumor Regression
Klaus Früh, Oregon Health & Science University, USA CMV-induced MHC-II and MHC-E-Restricted CD8+ T Cells: A Role in Cancer Immunotherapy
16:30—17:00
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Macdonald Foyer
17:00—18:45
Linking Cancer Genetics and Genomics with Immunity
Meeting Wrap-Up: Outcomes and Future Directions (Organizers)
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Outsource a part of the T cell’s immune value chain, propose cancer immunotherapy researchers, from patient T cells to donor T cells. The novel allogeneic approach could rely on T-cell receptor gene transfer to generate broad and tumor-specific T-cell immune responses. [NIAID]
A new cancer immunotherapy approach could essentially outsource a crucial T-cell function. This function, T-cell reactivity to specific cancer antigens, is sometimes lacking in cancer patients. Yet, according to a new proof-of-principle study, these patients could benefit from T cells provided by healthy donors. Specifically, the healthy donors’ T cells could be used to broaden the T-cell receptor repertoires of the cancer patients’ T cells.
Ultimately, this approach relies on a cancer immunotherapy technique called T-cell receptor (TCR) transfer, or the genetic transfer of TCR chains. TCR transfer can be used to outsource the T cell’s learning function, the process by which a T cell acquires the ability to recognize foreign antigens—in this case, the sort of proteins that can be expressed on the surface of cancer cells. Because cancer cells harbor faulty proteins, they can also display foreign protein fragments, also known as neoantigens, on their surface, much in the way virus-infected cells express fragments of viral proteins.
The approach was detailed in a paper that appeared May 19 in the journal Science, in an article entitled, “Targeting of Cancer Neoantigens with Donor-Derived T Cell Receptor Repertoires.” This article, by scientists based at the Netherlands Cancer Institute and the University of Oslo, describes a novel strategy to broaden neoantigen-specific T-cell responses. Such a strategy would be useful in overcoming a common limitation seen in the immune response to cancer: Neoantigen-specific T-cell reactivity is generally limited to just a few mutant epitopes, even though the number of predicted epitopes is large.
“We demonstrate that T cell repertoires from healthy donors provide a rich source of T cells that specifically recognize neoantigens present on human tumors,” the study’s authors wrote. “Responses to 11 epitopes were observed, and for the majority of evaluated epitopes, potent and specific recognition of tumor cells endogenously presenting the neoantigens was detected.”
First, the researchers mapped all possible neoantigens on the surface of melanoma cells from three different patients. In all three patients, the cancer cells seemed to display a large number of different neoantigens. But when the researchers tried to match these to the T cells derived from within the patient’s tumors, most of these aberrant protein fragments on the tumor cells went unnoticed.
Next, the researchers tested whether the same neoantigens could be seen by T cells derived from healthy volunteers. Strikingly, these donor-derived T cells could detect a significant number of neoantigens that had not been seen by the patients’ T cells.
“Many of the T cell reactivities [among donor T cells] involved epitopes that in vivo were neglected by patient autologous tumor-infiltrating lymphocytes,” the authors of the Science article continued. “T cells re-directed with T cell receptors identified from donor-derived T cells efficiently recognized patient-derived melanoma cells harboring the relevant mutations, providing a rationale for the use of such ‘outsourced’ immune responses in cancer immunotherapy.”
“In a way, our findings show that the immune response in cancer patients can be strengthened; there is more on the cancer cells that makes them foreign that we can exploit. One way we consider doing this is finding the right donor T cells to match these neoantigens,” said Ton Schumacher, Ph.D., a principal investigator at the Netherlands Cancer Institute. “The receptor that is used by these donor T cells can then be used to genetically modify the patient’s own T cells so these will be able to detect the cancer cells.”
“Our study shows that the principle of outsourcing cancer immunity to a donor is sound,” added Johanna Olweus, M.D., Ph.D., who heads a research group at the University of Oslo. “However, more work needs to be done before patients can benefit from this discovery. Thus, we need to find ways to enhance the throughput.”
“We are currently exploring high-throughput methods to identify the neoantigens that the T cells can ‘see’ on the cancer and isolate the responding cells. But the results showing that we can obtain cancer-specific immunity from the blood of healthy individuals are already very promising.”
Targeting of cancer neoantigens with donor-derived T cell receptor repertoires
Accumulating evidence suggests that clinically efficacious cancer immunotherapies are driven by T cell reactivity against DNA mutation-derived neoantigens. However, among the large number of predicted neoantigens, only a minority is recognized by autologous patient T cells, and strategies to broaden neoantigen specific T cell responses are therefore attractive. Here, we demonstrate that naïve T cell repertoires of healthy blood donors provide a source of neoantigen-specific T cells, responding to 11/57 predicted HLA-A2-binding epitopes from three patients. Many of the T cell reactivities involved epitopes that in vivo were neglected by patient autologous tumor-infiltrating lymphocytes. Finally, T cells re-directed with T cell receptors identified from donor-derived T cells efficiently recognized patient-derived melanoma cells harboring the relevant mutations, providing a rationale for the use of such “outsourced” immune responses in cancer immunotherapy.
Metabolic maintenance of cell asymmetry following division in activated T lymphocytes.
Asymmetric cell division, the partitioning of cellular components in response to polarizing cues during mitosis, has roles in differentiation and development. It is important for the self-renewal of fertilized zygotes in Caenorhabditis elegans and neuroblasts in Drosophila, and in the development of mammalian nervous and digestive systems. T lymphocytes, upon activation by antigen-presenting cells (APCs), can undergo asymmetric cell division, wherein the daughter cell proximal to the APC is more likely to differentiate into an effector-like T cell and the distal daughter is more likely to differentiate into a memory-like T cell. Upon activation and before cell division, expression of the transcription factor c-Myc drives metabolic reprogramming, necessary for the subsequent proliferative burst. Here we find that during the first division of an activated T cell in mice, c-Myc can sort asymmetrically. Asymmetric distribution of amino acid transporters, amino acid content, and activity of mammalian target of rapamycin complex 1 (mTORC1) is correlated with c-Myc expression, and both amino acids and mTORC1 activity sustain the differences in c-Myc expression in one daughter cell compared to the other. Asymmetric c-Myc levels in daughter T cells affect proliferation, metabolism, and differentiation, and these effects are altered by experimental manipulation of mTORC1 activity or c-Myc expression. Therefore, metabolic signalling pathways cooperate with transcription programs to maintain differential cell fates following asymmetric T-cell division.
T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy associated with Notch pathway mutations. While both normal activated and leukemic T cells can utilize aerobic glycolysis to support proliferation, it is unclear to what extent these cell populations are metabolically similar and if differences reveal T-ALL vulnerabilities. Here we show that aerobic glycolysis is surprisingly less active in T-ALL cells than proliferating normal T cells and that T-ALL cells are metabolically distinct. Oncogenic Notch promoted glycolysis but also induced metabolic stress that activated 5′ AMP-activated kinase (AMPK). Unlike stimulated T cells, AMPK actively restrained aerobic glycolysis in T-ALL cells through inhibition of mTORC1 while promoting oxidative metabolism and mitochondrial Complex I activity. Importantly, AMPK deficiency or inhibition of Complex I led to T-ALL cell death and reduced disease burden. Thus, AMPK simultaneously inhibits anabolic growth signaling and is essential to promote mitochondrial pathways that mitigate metabolic stress and apoptosis in T-ALL.
Obesity and diabetes are associated with excessive inflammation and impaired wound healing. Increasing evidence suggests that macrophage dysfunction is responsible for these inflammatory defects. In the setting of excess nutrients, particularly dietary saturated fatty acids (SFAs), activated macrophages develop lysosome dysfunction, which triggers activation of the NLRP3 inflammasome and cell death. The molecular pathways that connect lipid stress to lysosome pathology are not well understood, but may represent a viable target for therapy. Glutamine uptake is increased in activated macrophages leading us to hypothesize that in the context of excess lipids glutamine metabolism could overwhelm the mitochondria and promote the accumulation of toxic metabolites. To investigate this question we assessed macrophage lipotoxicity in the absence of glutamine using LPS-activated peritoneal macrophages exposed to the SFA palmitate. We found that glutamine deficiency reduced lipid induced lysosome dysfunction, inflammasome activation, and cell death. Under glutamine deficient conditions mTOR activation was decreased and autophagy was enhanced; however, autophagy was dispensable for the rescue phenotype. Rather, glutamine deficiency prevented the suppressive effect of the SFA palmitate on mitochondrial respiration and this phenotype was associated with protection from macrophage cell death. Together, these findings reveal that crosstalk between activation-induced metabolic reprogramming and the nutrient microenvironment can dramatically alter macrophage responses to inflammatory stimuli.
Immunoregulatory Protein B7-H3 Reprograms Glucose Metabolism in Cancer Cells by ROS-Mediated Stabilization of HIF1α
CD8(+) T cells can respond to unrelated infections in an Ag-independent manner. This rapid innate-like immune response allows Ag-experienced T cells to alert other immune cell types to pathogenic intruders. In this study, we show that murine CD8(+) T cells can sense TLR2 and TLR7 ligands, resulting in rapid production of IFN-γ but not of TNF-α and IL-2. Importantly, Ag-experienced T cells activated by TLR ligands produce sufficient IFN-γ to augment the activation of macrophages. In contrast to Ag-specific reactivation, TLR-dependent production of IFN-γ by CD8(+) T cells relies exclusively on newly synthesized transcripts without inducing mRNA stability. Furthermore, transcription of IFN-γ upon TLR triggering depends on the activation of PI3K and serine-threonine kinase Akt, and protein synthesis relies on the activation of the mechanistic target of rapamycin. We next investigated which energy source drives the TLR-induced production of IFN-γ. Although Ag-specific cytokine production requires a glycolytic switch for optimal cytokine release, glucose availability does not alter the rate of IFN-γ production upon TLR-mediated activation. Rather, mitochondrial respiration provides sufficient energy for TLR-induced IFN-γ production. To our knowledge, this is the first report describing that TLR-mediated bystander activation elicits a helper phenotype of CD8(+) T cells. It induces a short boost of IFN-γ production that leads to a significant but limited activation of Ag-experienced CD8(+) T cells. This activation suffices to prime macrophages but keeps T cell responses limited to unrelated infections.
The bidirectional interaction between the immune system and whole-body metabolism has been well recognized for many years. Via effects on adipocytes and hepatocytes, immune cells can modulate whole-body metabolism (in metabolic syndromes such as type 2 diabetes and obesity) and, reciprocally, host nutrition and commensal-microbiota-derived metabolites modulate immunological homeostasis. Studies demonstrating the metabolic similarities of proliferating immune cells and cancer cells have helped give birth to the new field of immunometabolism, which focuses on how the cell-intrinsic metabolic properties of lymphocytes and macrophages can themselves dictate the fate and function of the cells and eventually shape an immune response. We focus on this aspect here, particularly as it relates to regulatory T cells.
Figure 1: Proposed model for the metabolic signatures of various Treg cell subsets.
(a) Activated CD4+ T cells that differentiate into the Teff cell lineage (green) (TH1 or TH17 cells) are dependent mainly on carbon substrates such as glucose and glutamine for their anabolic metabolism. In contrast to that, pTreg cells…
T-bet is a key modulator of IL-23-driven pathogenic CD4+ T cell responses in the intestine
IL-23 is a key driver of pathogenic Th17 cell responses. It has been suggested that the transcription factor T-bet is required to facilitate IL-23-driven pathogenic effector functions; however, the precise role of T-bet in intestinal T cell responses remains elusive. Here, we show that T-bet expression by T cells is not required for the induction of colitis or the differentiation of pathogenic Th17 cells but modifies qualitative features of the IL-23-driven colitogenic response by negatively regulating IL-23R expression. Consequently, absence of T-bet leads to unrestrained Th17 cell differentiation and activation characterized by high amounts of IL-17A and IL-22. The combined increase in IL-17A/IL-22 results in enhanced epithelial cell activation and inhibition of either IL-17A or IL-22 leads to disease amelioration. Our study identifies T-bet as a key modulator of IL-23-driven colitogenic responses in the intestine and has important implications for understanding of heterogeneity among inflammatory bowel disease patients.
Th17 cells are enriched at mucosal sites, produce high amounts of IL-17A, IL-17F and IL-22, and have an essential role in mediating host protective immunity against a variety of extracellular pathogens1. However, on the dark side, Th17 cells have also been implicated in a variety of autoimmune and chronic inflammatory conditions, including inflammatory bowel disease (IBD)2. Despite intense interest, the cellular and molecular cues that drive Th17 cells into a pathogenic state in distinct tissue settings remain poorly defined.
The Th17 cell programme is driven by the transcription factor retinoid-related orphan receptor gamma-t (RORγt) (ref. 3), which is also required for the induction and maintenance of the receptor for IL-23 (refs 4, 5). The pro-inflammatory cytokine IL-23, composed of IL-23p19 and IL-12p40 (ref. 6), has been shown to be a key driver of pathology in various murine models of autoimmune and chronic inflammatory disease such as experimental autoimmune encephalomyelitis (EAE)7, collagen induced arthritis8 and intestinal inflammation9, 10, 11, 12. Several lines of evidence, predominantly derived from EAE, suggest that IL-23 promotes the transition of Th17 cells to pathogenic effector cells9, 10, 11, 12. Elegant fate mapping experiments of IL-17A-producing cells during EAE have shown that the majority of IL-17A+IFN-γ+ and IL-17A−IFN-γ+ effector cells arise from Th17 cell progeny13. This transition of Th17 cells into IFN-γ-producing ‘ex’ Th17 cells required IL-23 and correlated with increased expression of T-bet. The T-box transcription factor T-bet drives the Th1 cell differentiation programme14 and directly transactivates the Ifng gene by binding to its promoter as well as multiple enhancer elements15. Indeed, epigenetic analyses have revealed that the loci for T-bet and IFN-γ are associated with permissive histone modifications in Th17 cells suggesting that Th17 cells are poised to express T-bet which could subsequently drive IFN-γ production16, 17.
A similar picture is emerging in the intestine where IL-23 drives T-cell-mediated intestinal pathology which is thought to be dependent on expression of T-bet18 and RORγt (ref. 19) by T cells. In support of this we have recently shown that IL-23 signalling in T cells drives the emergence of IFN-γ producing Th17 cells in the intestine during chronic inflammation20. Collectively these studies suggest a model whereby RORγt drives differentiation of Th17 cells expressing high amounts of IL-23R, and subsequently, induction of T-bet downstream of IL-23 signalling generates IL-17A+IFN-γ+ T cells that are highly pathogenic. Indeed, acquisition of IFN-γ production by Th17 cells has been linked to their pathogenicity in several models of chronic disease13, 21, 22, 23, 24 and a population of T cells capable of producing both IL-17A and IFN-γ has also been described in intestinal biopsies of IBD patients25, 26.
However, in the context of intestinal inflammation, it remains poorly defined whether the requirement for RORγt and T-bet reflects a contribution of Th17 and Th1 cells to disease progression or whether Th17 cells require T-bet co-expression to exert their pathogenic effector functions. Here, we use two distinct models of chronic intestinal inflammation and make the unexpected finding that T-bet is dispensable for IL-23-driven colitis. Rather the presence of T-bet serves to modify the colitogenic response restraining IL-17 and IL-22 driven pathology. These data identify T-bet as a key modulator of IL–23-driven colitogenic effector responses in the intestine and have important implications for understanding of heterogeneous immune pathogenic mechanisms in IBD patients.
Figure 1: IL-23 signalling is required for bacteria-driven T-cell-dependent colitis and the emergence of IL-17A+IFN-γ+ T cells.
C57BL/6 WT and Il23r−/− mice were infected orally with Hh and received weekly i.p. injections of IL-10R blocking antibody. Mice were killed at 4 weeks post infection and assessed for intestinal inflammation. (a) Colitis scores. (b) Typhlitis sores. (c) Representative photomicrographs of colon and caecum (× 10 magnification; scale bars, 200μM). (d) Representative flow cytometry plots of colonic lamina propria gated on viable CD4+ T cells. (e) Frequencies of IL-17A+ and/or IFN-γ+ CD4+ T cells present in the colon. Data represent pooled results from two independent experiments (n=12 for WT, n=10 for Il23r−/−). Bars are the mean and each symbol represents an individual mouse. *P<0.05, ***P<0.001 as calculated by Mann–Whitney U test.
C57BL/6 Rag1−/− mice were injected i.p. with 4 × 105 CD4+CD25−CD45RBhi T cells from C57BL/6 WT,Rorc−/− or Tbx21−/− donors. Mice were killed when recipients of Tbx21−/− T cells developed clinical signs of disease (4–6 weeks) and assessed for intestinal inflammation. (a) Colitis scores. (b) Representative photomicrographs of proximal colon sections (× 10 magnification; scale bars, 200μM). (c) Concentration of cytokines released from colon explants into the medium after overnight culture. Data represent pooled results from two independent experiments (n=14 for WT, n=11 for Rorc−/−, n=14 forTbx21−/−). Bars are the mean and each symbol represents an individual mouse. Bars are the mean and error bars represent s.e.m. *P<0.05, **P<0.01, ***P<0.001 as calculated by Kruskal–Wallis one-way ANOVA with Dunn’s post-test.
C57BL/6 Rag1−/− mice were injected i.p. with 4×105 CD4+CD25−CD45RBhi T cells from C57BL/6 WT,Rorc−/− or Tbx21−/− donors. Mice were killed when recipients of Tbx21−/−T cells developed clinical signs of disease (4–6 weeks). (a) Representative plots of IL-17A and IFN-γ expression in colonic CD4+ T cells. (b) Frequencies of IL-17A+ and/or IFN-γ+ cells among colonic CD4+ T cells. (c) Total numbers of IL-17A+and/or IFN-γ+ CD4+ T cells present in the colon. Data represent pooled results from three independent experiments (n=20 for WT, n=18 for Tbx21−/−, n=12 for Rorc−/−). Bars are the mean and each symbol represents an individual mouse. *P<0.05, **P<0.01, ***P<0.001 as calculated by Kruskal–Wallis one-way ANOVA with Dunn’s post-test.
T-bet deficiency promotes an exacerbated Th17-type response
Our transfer of Tbx21−/− T cells revealed a striking increase in the frequency of IL-17A+IFN-γ−cells (Fig. 3) and we reasoned that T-bet-deficiency could impact on Th17 cell cytokine production. Therefore, we transferred WT or Tbx21−/− CD4+ T cells into Rag1−/− recipients and measured the expression of RORγt, IL-17A, IL-17F and IL-22 by CD4+ T cells isolated from the colon. In agreement with our earlier findings, Tbx21−/− T cells gave rise to significantly increased frequencies of RORγt-expressing T cells capable of producing IL-17A (Fig. 4a). Furthermore, T-bet deficiency also led to a dramatic expansion of IL-17F and IL-22-expressing cells, which constituted only a minor fraction in WT T cells (Fig. 4a,b). By contrast, the frequency of granulocyte-macrophage colony-stimulating factor (GM-CSF) and IFN-γ producing cells was significantly reduced in T-bet-deficient T cells as compared with WT T cells. When analysed in more detail we noted that the production of IL-17A, IL-17F and IL-22 increased specifically in T-bet-deficient IL-17A+IFN-γ+ T cells as compared with WT T cells whereas IFN-γ production decreased overall in the absence of T-bet as expected (Supplementary Fig. 4A). Similarly, GM-CSF production was also generally reduced in Tbx21−/− CD4+ T cells further suggesting a shift in the qualitative nature of the T cell response.
Figure 4: T-bet-deficient CD4+ T cells promote an exacerbated Th17-type inflammatory response.
C57BL/6 Rag1−/− mice were injected i.p. with 4×105 CD4+CD25−CD45RBhi T cells from C57BL/6 WT orTbx21−/− donors. Mice were killed when recipients of Tbx21−/−T cells developed clinical signs of disease (4–6 weeks). (a) Representative plots of cytokines and transcription factors in WT or Tbx21−/− colonic CD4+ T cells. (b) Frequency of IL-17A+, IL-17F+, IL-22+, GM-CSF+ or IFN-γ+ colonic T cells in WT orTbx21−/−. (c) quantitative reverse transcription PCR (qRT-PCR) analysis of mRNA levels of indicated genes in colon tissue homogenates. (d) Total number of neutrophils (CD11b+ Gr1high) in the colon. (e) Primary epithelial cells were isolated from the colon of steady state C57BL/6 Rag1−/− mice and stimulated with 10ngml−1 cytokines for 4h after which cells were harvested and analysed by qRT-PCR for the indicated genes. Data in b–d represent pooled results from two independent experiments (n=14 for WT, n=11 for Tbx21−/−). Bars are the mean and error bars represent s.e.m. Data in e are pooled results from four independent experiments, bars are the mean and error bars represent s.e.m. *P<0.05, **P<0.01,***P<0.001 as calculated by Mann–Whitney U test.
T-bet-deficient colitis depends on IL-23, IL-17A and IL-22
In the present study we show that bacteria-driven colitis is associated with the IL-23-dependent emergence of IFN-γ-producing Th17 cells co-expressing RORγt and T-bet. Strikingly, while RORγt is required for the differentiation of IFN-γ-producing Th17 cells and induction of colitis, T-bet is dispensable for the emergence of IL-17A+IFN-γ+ T cells and intestinal pathology. Our results show that instead of a mandatory role in the colitogenic response, the presence of T-bet modulates the qualitative nature of the IL-23-driven intestinal inflammatory response. In the presence of T-bet, IL-23-driven colitis is multifunctional in nature and not functionally dependent on either IL-17A or IL-22. By contrast, in the absence of T-bet a highly polarized colitogenic Th17 cell response ensues which is functionally dependent on both IL-17A and IL-22. T-bet-deficient T cells are hyper-responsive to IL-23 resulting in enhanced STAT3 activation and downstream cytokine secretion providing a mechanistic basis for the functional changes. These data newly identify T-bet as a key modulator of IL-23-driven colitogenic CD4+ T cell responses.
Contrary to our expectations T-bet expression by CD4 T cells was not required for their pathogenicity. In keeping with the negative effect of T-bet on Th17 differentiation40, 41, 42, we observed highly polarized Th17 responses in T-bet-deficient intestinal T cells. Early studies demonstrated that IFN-γ could suppress the differentiation of Th17 cells40 and thus the reduced IFN-γ production by Tbx21−/−T cells could facilitate Th17 cell generation. However, our co-transfer studies revealed unrestrained Th17 differentiation of Tbx21−/− T cells even in the presence of WT T cells, suggesting a cell autonomous role for T-bet-mediated suppression of the Th17 programme. Indeed, the role of T-bet as a transcriptional repressor of the Th17 cell fate has been described recently. For example, T-bet physically interacts with and sequesters Runx1, thereby preventing Runx1-mediated induction of RORγt and Th17 cell differentiation43. In addition, T-bet binds directly to and negatively regulates expression of many Th17-related genes15, 34 and we identified IL23r to be repressed in a T-bet-dependent manner. In line with this we show here that T-bet-deficient intestinal T cells express higher amounts of Il23r as well as Rorc. This resulted in enhanced IL-23-mediated STAT3 activation and increased production of IL-17A and IL-22. It has also been suggested that T-bet activation downstream of IL-23R signalling is required for pathogenic IL-23-driven T cell responses43, 44. However, we did not find a role for IL-23 in the induction and/or maintenance of T-bet expression and colitis induced by T-bet-deficient T cells was IL-23 dependent. Collectively, these findings demonstrate that T-bet deficiency leads to unrestrained expansion of colitogenic Th17 cells, which is likely mediated through enhanced activation of the IL-23R-STAT3 pathway.
The observation that T-bet-deficient T cells retain their colitogenic potential is in stark contrast to earlier studies. Neurath et al.18 convincingly showed that adoptive transfer of Tbx21−/− CD4+ T cells into severe combined immunodeficiency (SCID) recipients failed to induce colitis and this correlated with reduced IFN-γ and increased IL-4 production. Another study revealed that IL-4 plays a functional role in inhibiting the colitogenic potential of Tbx21−/− T cells, as recipients ofStat6−/−Tbx21−/− T cells developed severe colitis37. Importantly, the intestinal inflammation that developed in recipients of Stat6−/−Tbx21−/− T cells could be blocked by administration of IL-17A neutralizing antibody, suggesting that the potent inhibitory effect of IL-4/STAT6 signals on Th17 differentiation normally prevent colitis induced by Tbx21−/− T cells37. Various explanations could account for the discrepancy between our study and those earlier findings. First, in contrast to the published reports, we used naïve Tbx21−/− CD4+ T cells from C57BL/6 mice instead of BALB/c mice. An important difference between Tbx21−/− CD4+ T cells from these genetic backgrounds appears to be their differential susceptibility to suppression by IL-4/STAT6 signals. We found that transfer of Tbx21−/− T cells induced IL-17A-dependent colitis despite increased frequencies of IL-4-expressing cells in the intestine. This discrepancy may be due to higher amounts of IL-4 produced by activated CD4+ T cells from BALB/c versus C57BL/6 mice45, leading to the well-described Th2-bias of the BALB/c strain45. Second, differences in the composition of the intestinal microbiota between animal facilities can have a substantial effect on skewing CD4+ T cells responses. In particular, the Clostridium-related segmented filamentous bacteria (SFB) have been shown to drive the emergence of IL-17 and IL-22 producing CD4+ T cells in the intestine46. Importantly, the ability of naïve CD4+ T cells to induce colitis is dependent on the presence of intestinal bacteria, as germ-free mice do not develop pathology upon T cell transfer47. In line with this, we previously described that colonization of germ-free mice with intestinal microbiota containing SFB was necessary to restore the development of colitis47. Since our Rag1−/− colony is SFB+ and the presence of SFB was not reported in the previous studies, it is possible that differences in SFB colonization status contributed to the observed differences in pathogenicity ofTbx21−/− T cells.
It is important to note that T-bet-deficient T cells did not induce more severe colitis than WT T cells but rather promoted a distinct mucosal inflammatory response. Colitis induced by WT T cells is characterized by a multifunctional response with high amounts of IFN-γ and GM-CSF and a lower IL-17A and IL-22 response. Consistent with this, we have shown that blockade of GM-CSF abrogates T cell transfer colitis48 as well as bacteria-driven intestinal inflammation49 in T-bet sufficiency whereas blockade of IL-17A or IL-22 fails to do so. By contrast T-bet deficiency leads to production of high amounts of IL-17A and IL-22 in the colon and neutralization of either was sufficient to reduce intestinal pathology. Our in vitro experiments suggest that IL-17A and IL-22 synergise to promote intestinal epithelial cell responses, which may in part explain the efficacy of blocking IL-17A or IL-22 in colitis induced by T-bet-deficient T cells. A similar synergistic interplay has been described in the lung where IL-22 served a tissue protective function in homeostasis but induced airway inflammation in the presence of IL-17A (ref. 50). This highlights the complexity of the system in health and disease, and the need for a controlled production of both cytokines. We describe here only one mechanism of how IL-17A/IL-22 induce a context-specific epithelial cell response that potentially impacts on the order or composition of immune cell infiltration. Overall, these results provide a new perspective on T-bet, revealing its role in shaping the qualitative nature of the IL-23-driven colitogenic T cell response.
We also describe here the unexpected finding that a substantial proportion of T-bet-deficient intestinal T cells retain the ability to express IFN-γ. To investigate the potential mechanisms responsible for T-bet-independent IFN-γ production by intestinal CD4+ T cells we focused on two transcription factors, Runx3 and Eomes. Runx3 has been shown to promote IFN-γ expression directly through binding to the Ifng promoter38 and Eomes is known to compensate for IFN-γproduction in T-bet-deficient Th1 cells37. We found IL-23-mediated induction of Runx3 protein in WT and Tbx21−/− T cells isolated from the intestine, thus identifying Runx3 downstream of IL-23R signalling. By contrast, we could only detect Eomes protein and its induction by IL-23 in T-bet-deficient but not WT T cells. Thus, Runx3 and Eomes are activated in response to IL-23 in T-bet-deficient cells and are likely to be drivers of T-bet-independent IFN-γ production. In support of this we found that the majority of T-bet-deficient IL-17A−IFN-γ+ T cells expressed Eomes. However, only a minor population of IL-17A+IFN-γ+ T cells stained positive for Eomes, suggesting the existence of alternative pathways for IFN-γ production by Th17 cells. Intriguingly, a recent study identified Runx3 and Runx1 as the transcriptional regulators critical for the differentiation of IFN-γ-producing Th17 cells51. The author’s demonstrated that ectopic expression of Runx transcription factors was sufficient to induce IFN-γ production by Th17 cells even in the absence of T-bet. These findings, combined with our data on Runx3 activation downstream of IL-23R signalling strongly suggest that Runx3 rather than Eomes is driving IFN-γ expression by intestinal Th17 cells.
We have not formally addressed the role of IFN-γ in colitis driven by T-bet-deficient T cells. A recent report by Zimmermann et al.52 found that antibody-mediated blockade of IFN-γ ameliorates colitis induced by WT or T-bet-deficient T cells suggesting IFN-γ also contributes to the colitogneic response mediated by T-bet-deficient T cells as originally described for WT T cells53, 54. By contrast with our results the Zimmerman study found that IL-17A blockade exacerbated colitis following transfer of Tbx21−/− T cells. The reason for the differential role of IL-17A in the two studies is not clear but it is notable that the Zimmerman study was performed in the presence of co-infection with SFB and Hh, and this strong inflammatory drive may alter the pathophysiological role of particular cytokines. Together the data indicate that T-bet deficiency in T cells does not impede their colitogenic activity but that the downstream effector cytokines of the response are context dependent.
In conclusion, our data further underline the essential role for IL-23 in intestinal inflammation and demonstrate that T-bet is an important modulator of the IL–23-driven effector T cell response. The colitogenic T cell response in a T-bet sufficient environment is multifunctional with a dominant GM-CSF and IFN-γ response. By contrast T-bet-deficient colitogenic responses are dominated by IL-17A and IL-22-mediated immune pathology. These results may have significant bearing on human IBD where it is now recognized that differential responsiveness to treatment may reflect considerable disease heterogeneity. As such, identification of suitable biomarkers such as immunological parameters, that allow stratification of patient groups, is becoming increasingly important55. Genome-wide association studies have identified polymorphisms in loci related to innate and adaptive immune arms that confer increased susceptibility to IBD. Among these are Th1 (STAT4, IFNG and STAT1) as well as Th17-related genes (RORC, IL23R and STAT3) (refs56, 57). Thus, detailed profiling of the T cell response in IBD patients may help identify appropriate patient groups that are most likely to benefit from therapeutic blockade of certain effector cytokines. Finally, our studies highlight the importance of IL-23 in the intestinal inflammatory hierarchy and suggest that IL-23 could be an effective therapeutic target across a variety of patient groups.
Yale study: How antibodies access neurons to fight infection
Yale scientists have solved a puzzle of the immune system: how antibodies enter the nervous system to control viral infections. Their finding may have implications for the prevention and treatment of a range of conditions, including herpes and Guillain-Barre syndrome, which has been linked to the Zika virus.
Many viruses — such as West Nile, Zika, and the herpes simplex virus — enter the nervous system, where they were thought to be beyond the reach of antibodies. Yale immunobiologists Akiko Iwasaki and Norifumi Iijima used mice models to investigate how antibodies could gain access to nerve tissue in order to control infection.
In mice infected with herpes, they observed a previously under-recognized role of CD4 T cells, a type of white blood cell that guards against infection by sending signals to activate the immune system. In response to herpes infection, CD4 T cells entered the nerve tissue, secreted signaling proteins, and allowed antibody access to infected sites. Combined, CD4 T cells and antibodies limited viral spread.
“This is a very elegant design of the immune system to allow antibodies to go to the sites of infection,” said Iwasaki. “The CD4 T cells will only go to the site where there is a virus. It’s a targeted delivery system for antibodies.”
Access of protective antiviral antibody to neuronal tissues requires CD4 T-cell help
Circulating antibodies can access most tissues to mediate surveillance and elimination of invading pathogens. Immunoprivileged tissues such as the brain and the peripheral nervous system are shielded from plasma proteins by the blood–brain barrier1 and blood–nerve barrier2, respectively. Yet, circulating antibodies must somehow gain access to these tissues to mediate their antimicrobial functions. Here we examine the mechanism by which antibodies gain access to neuronal tissues to control infection. Using a mouse model of genital herpes infection, we demonstrate that both antibodies and CD4 T cells are required to protect the host after immunization at a distal site. We show that memory CD4 T cells migrate to the dorsal root ganglia and spinal cord in response to infection with herpes simplex virus type 2. Once inside these neuronal tissues, CD4 T cells secrete interferon-γ and mediate local increase in vascular permeability, enabling antibody access for viral control. A similar requirement for CD4 T cells for antibody access to the brain is observed after intranasal challenge with vesicular stomatitis virus. Our results reveal a previously unappreciated role of CD4 T cells in mobilizing antibodies to the peripheral sites of infection where they help to limit viral spread.
T Cells Help Reverse Ovarian Cancer Drug Resistance
T cells (red) attack ovarian cancer cells (green). [University of Michigan Health System]
Researchers at the University of Michigan have recently published the results from a new study that they believe underscores why so many ovarian tumors develop resistance to chemotherapy. The tumor microenvironment is made up of an array of cell types, yet effector T cells and fibroblasts constitute the bulk of the tissue. The investigators believe that understanding the interplay between these two cell types holds the key to how ovarian cancer cells develop resistance.
The new study suggests that the fibroblasts surrounding the tumor work to block chemotherapy, which is why nearly every woman with ovarian cancer becomes resistant to treatment. Conversely, the scientists published evidence that T cells in the microenvironment can reverse the resistance phenotype—suggesting a whole different way of thinking about chemotherapy resistance and the potential to harness immunotherapy drugs to treat ovarian cancer.
“Ovarian cancer is often diagnosed at late stages, so chemotherapy is a key part of treatment,” explained co-senior study author J. Rebecca Liu, M.D., associate professor of obstetrics and gynecology at the University of Michigan. “Most patients will respond to it at first, but everybody develops chemoresistance. And that’s when ovarian cancer becomes deadly.”
Dr. Liu continued, stating that “in the past, we’ve thought the resistance was caused by genetic changes in tumor cells. But we found that’s not the whole story.”
The University of Michigan team looked at tissue samples from ovarian cancer patients and separated the cells by type to study the tumor microenvironment in vitro and in mice. More importantly, the scientists linked their findings back to actual patient outcomes.
The results of this study were published recently in Cell through an article entitled “Effector T Cells Abrogate Stroma-Mediated Chemoresistance in Ovarian Cancer.”
Ovarian cancer is typically treated with cisplatin, a platinum-based chemotherapy. The researchers found that fibroblasts blocked platinum. These cells prevented platinum from accumulating in the tumor and protected tumor cells from being killed off by cisplatin.
Diagram depicting how T cells can reverse chemotherapeutic resistance. [Cell, Volume 165, Issue 5, May 19, 2016]
“We show that fibroblasts diminish the nuclear accumulation of platinum in ovarian cancer cells, resulting in resistance to platinum-based chemotherapy,” the authors wrote. “We demonstrate that glutathione and cysteine released by fibroblasts contribute to this resistance.”
T cells, on the other hand, overruled the protection of the fibroblasts. When researchers added the T cells to the fibroblast population, the tumor cells began to die off.
“CD8+ T cells abolish the resistance by altering glutathione and cystine metabolism in fibroblasts,” the authors explained. “CD8+ T-cell-derived interferon (IFN)γ controls fibroblast glutathione and cysteine through upregulation of gamma-glutamyltransferases and transcriptional repression of system xc−cystine and glutamate antiporter via the JAK/STAT1 pathway.”
By boosting the effector T cell numbers, the researchers were able to overcome the chemotherapy resistance in mouse models. Moreover, the team used interferon, an immune cell-secreted cytokine, to manipulate the pathways involved in cisplatin.
“T cells are the soldiers of the immune system,” noted co-senior study author Weiping Zou, M.D., Ph.D., professor of surgery, immunology, and biology at the University of Michigan. “We already know that if you have a lot of T cells in a tumor, you have better outcomes. Now we see that the immune system can also impact chemotherapy resistance.”
The researchers suggest that combining chemotherapy with immunotherapy may be effective against ovarian cancer. Programmed death ligand 1 (PD-L1) and PD-1 pathway blockers are currently FDA-approved treatments for some cancers, although not ovarian cancer.
“We can imagine re-educating the fibroblasts and tumor cells with immune T cells after chemoresistance develops,” Dr. Zou remarked.
“Then we could potentially go back to the same chemotherapy drug that we thought the patient was resistant to. Only now we have reversed that, and it’s effective again,” Dr. Liu concluded.
Effector T Cells Abrogate Stroma-Mediated Chemoresistance in Ovarian Cancer
•Fibroblasts diminish platinum content in cancer cells, resulting in drug resistance
•GSH and cysteine released by fibroblasts contribute to platinum resistance
•T cells alter fibroblast GSH and cystine metabolism and abolish the resistance
•Fibroblasts and CD8+ T cells associate with patient chemotherapy response
Summary
Effector T cells and fibroblasts are major components in the tumor microenvironment. The means through which these cellular interactions affect chemoresistance is unclear. Here, we show that fibroblasts diminish nuclear accumulation of platinum in ovarian cancer cells, resulting in resistance to platinum-based chemotherapy. We demonstrate that glutathione and cysteine released by fibroblasts contribute to this resistance. CD8+ T cells abolish the resistance by altering glutathione and cystine metabolism in fibroblasts. CD8+ T-cell-derived interferon (IFN)γ controls fibroblast glutathione and cysteine through upregulation of gamma-glutamyltransferases and transcriptional repression of system xc− cystine and glutamate antiporter via the JAK/STAT1 pathway. The presence of stromal fibroblasts and CD8+ T cells is negatively and positively associated with ovarian cancer patient survival, respectively. Thus, our work uncovers a mode of action for effector T cells: they abrogate stromal-mediated chemoresistance. Capitalizing upon the interplay between chemotherapy and immunotherapy holds high potential for cancer treatment.
Activation of effect or T cells leads to increased glucose uptake, glycolysis, and lipid synthesis to support growth and proliferation. Activated T cells were identified with CD7, CD5, CD3, CD2, CD4, CD8 and CD45RO. Simultaneously, the expression of CD95 and its ligand causes apoptotic cells death by paracrine or autocrine mechanism, and during inflammation, IL1-β and interferon-1α.. The receptor glucose, Glut 1, is expressed at a low level in naive T cells, and rapidly induced by Myc following T cell receptor (TCR) activation. Glut1 trafficking is also highly regulated, with Glut1 protein remaining in intracellular vesicles until T cell activation. CD28 co-stimulation further activates the PI3K/Akt/mTOR pathway in particular, and provides a signal for Glut1 expression and cell surface localization. Mechanisms that control T cell metabolic reprogramming are now coming to light, and many of the same oncogenes importance in cancer metabolism are also crucial to drive T cell metabolic transformations, most notably Myc, hypoxia inducible factor (HIF)1a, estrogen-related receptor (ERR) a, and the mTOR pathway. The proto-oncogenic transcription factor, Myc, is known to promote transcription of genes for the cell cycle, as well as aerobic glycolysis and glutamine metabolism. Recently, Myc has been shown to play an essential role in inducing the expression of glycolytic and glutamine metabolism genes in the initial hours of T cell activation. In a similar fashion, the transcription factor (HIF)1a can up-regulate glycolytic genes to allow cancer cells to survive under hypoxic conditions
UPDATE 6/11/2021
Bispecific Antibodies Emerging as Effective Cancer Therapeutics
Science 28 May 2021: Vol. 372, Issue 6545, pp. 916-917 DOI: 10.1126/science.abg1209
Bispecific antibodies (bsAbs) bind two different epitopes on the same or different antigens. Through this dual specificity for soluble or cell-surface antigens, bsAbs exert activities beyond those of natural antibodies, offering numerous opportunities for therapeutic applications. Although initially developed for retargeting T cells to tumors, with a first bsAb approved in 2009 (catumaxomab, withdrawn in 2017), exploring new modes of action opened the door to many additional applications beyond those of simply combining the activity of two different antibodies within one molecule. Examples include agonistic “assembly activities” that mimic the activity of natural ligands and cofactors (for example, factor VIII replacement in hemophilia A), inactivation of receptors or ligands, and delivery of payloads to cells or tissues or across biological barriers. Over the past years, the bsAb field transformed from early research to clinical applications and drugs. New developments offer a glimpse into the future promise of this exciting and rapidly progressing field.
Monoclonal antibodies (mAbs) comprise antigen-binding sites formed by the variable domains of the heavy and light chain and an Fc region that mediates immune responses. BsAbs, produced through genetic engineering, combine the antigen-binding sites of two different antibodies within one molecule, with a plethora of formats available (1). Conceptually, one can discriminate between bsAbs with combinatorial modes of action where the antigen-binding sites act independently from each other, and bsAbs with obligate modes of action where activity needs binding of both, either in a sequential (temporal) way or dependent on the physical (spatial) linkage of both (see the figure) (2). BsAbs approved as drugs are so far in the obligate dual-binding category: A T cell recruiter (blinatumomab) against cancer and a factor VIIIa mimetic to treat hemophilia A (emicizumab). Most but not all of the more than 100 bsAbs in clinical development address cancers. Some are in late stage (such as amivantamab, epcoritamab, faricimab, and KNO46), but most are still in early stages (2). Most of these entities enable effector cell retargeting to induce target cell destruction.
An increasing number of programs also explore alternative modes of action. This includes bsAbs that target pathways involved in tumor proliferation (such as amivantamab), invasion, ocular angiogenesis (such as faricimab), or immune regulation by blocking receptors and/or ligands, mainly in a combinatorial manner. Challenges for all of these entities are potential adverse effects, toxicity in normal tissues, and overshooting and systemic immune responses, especially with T cell retargeting or immune-modulating or activating entities. Such issues need to be carefully addressed.
Most of the bispecific T cell engagers comprise a binding site for a tumor-associated antigen and CD3 [a component of the T cell receptor (TCR) activation complex] as trigger molecule on T cells. To prevent or ameliorate “on-target, off-tumor” effects of T cell recruiters, approaches currently investigated include the modulation of target affinities and mechanisms to allow conditional activation upon target cell binding. Thus, a reduced affinity for CD3 increased tolerability by reducing peripheral cytokine concentrations that are associated with nonspecific or overshooting immune reactions (3). Similarly, reduced affinity for the target antigen was shown to ameliorate cytokine release and damage of target-expressing tissues (4). Tumor selectivity can be further increased by implementing avidity effects—for example, by using 2+1 bsAb formats with two low-affinity binding sites for target antigens and monovalent binding to CD3 (4).
In further approaches, binders to CD3 were identified that efficiently trigger target cell destruction without inducing undesired release of cytokines, demonstrating the importance of epitope specificity to potentially uncouple efficacy from cytokine release (5). Complementing these T cell–recruiting principles, the nonclassical T cell subset of γ9d2 T cells with strong cytotoxic activity emerged as potent effectors, which can be retargeted with bsAbs binding to the γ9d2 TCR. Thereby, global activation of all T cells, including inhibitory regulatory T cells (Treg cells), through CD3 binding, may be avoided (6). However, even these approaches might result in a narrow therapeutic window to treat solid tumors because of T cell activation in normal tissues.
Consequently, there are several approaches to conditionally activate T cells within tumors, including a local liberation of the CD3-binding sites or triggering local assembly of CD3-binding sites from two half-molecules. For example, CD3-binding sites have been masked by fusing antigen binding or blocking moieties—such as peptides, aptamers, or anti-idiotypic antibody fragments—to one or both variable domains. These moieties are released within the tumor by tumor-associated proteases, or through biochemical responses to hypoxia or low pH (7). This approach can also be applied to confer specific binding of antibody therapeutics, including bsAbs, to antigens on tumor cells (8).
An on-target restoration of CD3-binding sites requires application of two target-binding entities, each comprising parts of the CD3-binding site, which assemble into functional binding sites upon close binding of both half-antibodies. The feasibility of this approach was recently shown, for example, for a split T cell–engaging antibody derivative (Hemibody) that targets a cell surface antigen (9). Such approaches can also be applied to half-antibodies that recognize two different targets expressed on the same cell, further increasing tumor selectivity.
Regarding T cell engagers, increasing efforts are made to target not only cell-surface antigens expressed on tumor cells but also human leukocyte antigen (HLA)–presented tumor-specific peptides. This expands the target space of bsAbs toward tumor-specific intracellular antigens and can be achieved by using either recombinant TCRs or antibodies with TCR-like specificities combined with, for example, CD3-binding arms to engage T cell responses. A first TCR–anti-CD3 bispecific molecule is in phase I and II trials to treat metastatic melanoma (10). A challenge of this approach is the identification of TCRs or TCR-like antibodies that bind the peptide in the context of HLA with high affinity and specificity, without cross-reacting with related peptides to reduce or avoid off-target activities. Comprehensive screening tools and implementation of computational approaches are being developed to achieve this task.
A rapidly growing area of bsAbs in cancer therapy is their use to foster antitumor immune responses. Here, they are especially applied for dual inhibition of checkpoints that prevent immune responses—for example, programmed cell death protein 1 (PD-1) and its ligand (PD-L1), cytotoxic T lymphocyte–associated antigen 4 (CTLA-4), or lymphocyte activation gene 3 (LAG-3; for example, KNO46). Tumor-targeted bsAbs can also target costimulatory factors such as CD28 or 4-1BB ligand (4-1BBL) to enhance T cell responses when combined with PD-1 blockade or to provide an activity-enhancing costimulatory signal in combination with CD3-based bsAbs (11). Furthermore, bsAbs are being developed for local effects by targeting one arm to antigens that are expressed by tumor cells or cells of the tumor microenvironment (2).
Clinical application of bsAbs now expands to other therapeutic areas, including chronic inflammatory, autoimmune, and neurodegenerative diseases; vascular, ocular, and hematologic disorders; and infections. In contrast to mAbs, bsAbs can inactivate the signaling of different cytokines with one molecule to treat inflammatory diseases (12). Simultaneous dual-target binding is not essential to elicit activity for bsAbs against combinations of proinflammatory cytokines, such as tumor necrosis factor (TNF), interleukin-1α (IL-1α), IL-1β, IL-4, IL-13, IL-17, inducible T cell costimulator ligand (ICOSL), or B cell–activating factor (BAFF). This presumably also applies to blockade of immune cell receptors, although dual targeting might confer increased efficacy due to avidity effects and increased selectivity through simultaneous binding of two different receptors.
A further application of combinatorial dual targeting is in ophthalmology. Loss of vision in wet age-related macular degeneration (AMD) results from abnormal proliferation and leakiness of blood vessels in the macula. This can be treated with antibodies that bind and inactivate factors that stimulate their proliferation (13). In contrast to mAbs or fragments that recognize individual factors, bsAbs bind two such factors. For example, faricimab that binds vascular endothelial growth factor A (VEGF-A) and angiopoietin-2 (ANG2), demonstrated dual efficacy in preclinical studies, and is currently in phase 3 trials.
BsAbs with obligate modes of action that mandate simultaneous dual-target binding are “assemblers” that replace the function of factors necessary to form functional protein complexes. One of these bsAbs with an assembly role (emicizumab, approved in 2018) replaces factor VIIIa in the clotting cascade. Deficiency of factor VIII causes hemophilia A, which can be overcome by substitution with recombinant factor VIII. However, a proportion of patients develop factor VIII–neutralizing immune responses and no longer respond to therapy. To overcome this, a bsAb was developed with binding sites that recognize and physically connect factors IXa and X, a process normally mediated by factor VIIIa. Extensive screening of a large set of bsAbs was required to identify those that combine suitable epitopes with optimized affinities and geometry to serve as functional factor VIIIa mimetics (14). This exemplifies the complexity of identifying the best bsAb for therapeutic applications.
A mode of action requiring sequential binding of two targets is the transport of bsAbs across the blood-brain barrier (BBB). This is a tight barrier of brain capillary endothelial cells that controls the transport of substances between the blood and the cerebrospinal fluid—the brain parenchyma. Passage of large molecules, including antibodies, across the BBB is thereby restricted. Some proteins, such as transferrin or insulin, pass through the BBB by way of transporters on endothelial cells. Antibodies that bind these shuttle molecules, such as the transferrin receptor (TfR), can hitchhike across the BBB. BsAbs that recognize brain targets (such as β-amyloid for Alzheimer’s disease) and TfR with optimized affinities, epitopes, and formats can thereby enter the brain. Such bsAbs are currently in clinical evaluation to treat neurodegenerative diseases (15).
In the past years, there has been a transition from a technology-driven phase, solving hurdles to generate bsAbs with defined composition, toward exploring and extending the modes of action for new therapeutic options. The challenge of generating bsAbs is not only to identify suitable antigen pairs to be targeted in a combined manner. It is now recognized that the molecular composition has a profound impact on bsAb functionality (13). That more than 30 different bsAb formats are in clinical trials proves that development is now driven by a “fit for purpose” or “format defines function” rationale. Many candidates differ in their composition, affecting valency, geometry, flexibility, size, and half-life (1). Not all members of this “zoo of bsAb formats” qualify to become drugs. Strong emphasis is therefore on identifying candidates that exhibit drug-like properties and fulfill safety, developability, and manufacturability criteria. There is likely to be an exciting new wave of bsAb therapeutics available in the coming years.
Researchers have hijacked a defense system normally used by bacteria to fend off viral infections and redirected it against the human papillomavirus (HPV), the virus that causes cervical, head and neck, and other cancers.
Using the genome editing tool known as CRISPR, the Duke University researchers were able to selectively destroy two viral genes responsible for the growth and survival of cervical carcinoma cells, causing the cancer cells to self-destruct.
The findings, published in the Journal of Virology, give credence to an approach only recently attempted in mammalian cells, and could pave the way toward antiviral strategies targeted against other DNA-based viruses like hepatitis B and herpes simplex.
“Because this approach is only going after viral genes, there should be no off-target effects on normal cells,” said Bryan R. Cullen, Ph.D., senior study author and professor of molecular genetics and microbiology at Duke University School of Medicine. “You can think of this as targeting a missile that will destroy a certain target. You put in a code that tells the missile exactly what to hit, and it will only hit that, and it won’t hit anything else because it doesn’t have the code for another target.”
In this study, Cullen decided to target the human papillomavirus (HPV), which causes almost all cervical cancers and about half of head and neck cancers. Specifically, he and his colleagues went after the viral genes E6 and E7, two “oncogenes” that block the host’s own efforts to keep cancer cells at bay.
To run CRISPR against the virus, the researchers needed two ingredients. First, they needed the target code for E6 or E7, consisting of a short strip of RNA sequence, the chemical cousin of DNA. To this “guide RNA” they added the Cas9 protein, which would cut any DNA that could line up and bind to that RNA sequence.
The carcinoma cells that received the anti-HPV guide RNA/Cas9 combination immediately stopped growing. In contrast, cells that had received a control virus, containing a random guide RNA sequence, continued on their path to immortality. The researchers then dug down to the molecular level to investigate the consequences of destroying E6 or E7 in cancer cells. E6 normally blocks a protein called p53, known as the guardian of the genome because it can turn on suicide pathways in the cell when it senses that something has gone awry. In this study, targeting E6 enabled p53 to resume its normal function, spurring death of the cancer cell.
E7 works in a similar way, blocking another protein called retinoblastoma or Rb that can trigger growth arrest and senescence, another form of cell death. As expected, the researchers found that targeting E7 also set this second “tumor suppressor” back in motion.
“As soon as you turn off E6 or E7, the host defense mechanisms are allowed to come back on again, because they have been there this whole time, but they have been turned off by HPV,” Cullen said. “What happens is the cell immediately commits suicide.”
Cullen and his colleagues are now working on developing a different viral vector, based on the adeno-associated virus, to deliver their CRISPR cargo into cancer cells. Once they are happy with their delivery system, they will begin to test this approach in animal models.
“What we would hope to see in an HPV-induced cancer is rapid induction of tumor necrosis caused by loss of E6 or E7,” Cullen said. “This method has the potential to be a single hit treatment that will dramatically reduce tumor load without having any effect on normal cells.”
The researchers are also targeting other viruses that use DNA as their genetic material, including the hepatitis B virus and herpes simplex virus.
Reference: “Inactivation of the human papillomavirus E6 or E7 gene in cervical carcinoma cells using a bacterial CRISPR/Cas RNA-guided endonuclease,” Edward M. Kennedy, Anand V. R. Kornepati, Michael Goldstein, Hal P. Bogerd, Brigid C. Poling, Adam W. Whisnant, Michael B. Kastan and Bryan R. Cullen.Journal of Virology, August 6, 2014. DOI 10.1128/JVI.01879-14.
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