Emerging Cell Therapies: Realizing the Vision of NextGen Cell Therapeutics | EK15

Jan 25, 2021 ‐ Jan 27, 2021



Sessions

Antibody:CD47 ratio regulates macrophage phagocytosis through competitive receptor phosphorylation

Jan 25, 2021 12:00am ‐ Jan 25, 2021 12:00am

Antibody:CD47 ratio regulates macrophage phagocytosis through competitive receptor phosphorylation Emily C. Suter, Eva M. Schmid, Erik Voets, Brian Francica, Daniel A. Fletcher Department of Bioengineering, University of California Berkeley, Berkeley CA, USA UC Berkeley/UC San Francisco Graduate Group in Bioengineering, Berkeley, CA, USA Aduro Biotech Europe, Oss, Netherlands Aduro Biotech Inc., Emeryville, CA, USA Division of Biological Systems and Engineering, Lawrence Berkeley National Laboratory, Berkeley, CA, USA Chan Zuckerberg Biohub, San Francisco, CA, USA Cancer immunotherapies often modulate macrophage effector function by introducing either targeting antibodies that activate Fc gamma receptors or blocking antibodies that disrupt inhibitory SIRPα-CD47 engagement. Yet how these competing signals are integrated is poorly understood mechanistically, raising questions about how to effectively titrate immune responses. Here we find that macrophage phagocytic decisions are regulated by the ratio of activating ligand to inhibitory ligand on targets over a broad range of absolute molecular densities. Using endogenous as well as chimeric receptors, we show that activating:inhibitory ligand ratios of at least 10:1 are required to promote phagocytosis of model antibody-opsonized CD47-inhibited targets and that lowering this ratio reduces FcγR phosphorylation due to inhibitory phosphatases recruited to CD47-bound SIRPα. We demonstrate that ratiometric signaling is critical for phagocytosis of tumor cells and can be modified by blocking SIRPα in vitro, indicating that balancing targeting and blocking antibodies may be important for controlling macrophage phagocytosis in cancer immunotherapy.

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Dual targeting of cancer and suppressive myeloid cells by tumor-redirected iNKT cells and antigen-carrying microparticles

Jan 25, 2021 12:00am ‐ Jan 25, 2021 12:00am

Dual targeting of cancer and suppressive myeloid cells by tumor-redirected iNKT cells and antigen-carrying microparticles G. Delfanti1, F. Cortesi1,3, G. Antonini1, C.Garavaglia1, M.Consonni1, H. Shen2, G. Casorati1, P. Dellabona1 1Experimental Immunology Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milan 20132, Italy 2 Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX 77030,USA 3 present address: University Medical Center Hamburg-Eppendorf, Department of General, Visceral and Thoracic Surgery Campus Forschung N27, Hamburg, Germany Background Adoptive immunotherapy with T cells engineered with tumor-specific TCRs or CARs hold promise for the treatment of hematological and solid malignancies. However, suppressive cues generated by the tumor microenvironment (TME) can dampen the efficacy of engineered T cells. Hence, reprograming the TME is considered critical to optimize the current cell therapy approaches. CD1d-restricted invariant natural killer T (iNKT) cells are active component of the TME and participate in the tumor immunosurveillace by restraining cancer-supporting myeloid populations. Retargeting iNKT cells against cancer cells, by transducing tumor-specific TCR genes, may produce enhanced effectors able to concurrently kill malignant cells and modulate detrimental myeloid cells in TME. Methods Mouse iNKT cells were expanded in vitro, engineered with TCRs specific for MHC-restricted tumor-associate peptide antigens and assessed either in vitro or upon transfer in vivo against tumors expressing the nominal tumor associate antigens. Moreover, the adoptive iNKT cell transfer was combined with their local restimulation with the strong agonist aGalactosylCeramide (αGalCer) delivered using porous silicon microparticle-based nanotherapeutics, which sequentially overcome biological barriers and accumulate at the tumor site. Results iNKT cells engineered with MHC-restricted TCRs specific for tumor-associate peptide antigens are indeed bi-specific for CD1d- and MHC-restricted antigens in vitro. Upon adoptive transfer in vivo, TCR-engineered iNKT cells effectively delay the progression of tumors expressing the cognate antigens and remodel the local myeloid components. These dual anti-tumor functions are further sustained by delivering in vivo αGalCer using porous silicon microparticles resulting in enhanced tumor control. Conclusions Collectively, these results support the use of tumor-retargeted iNKT cells plus local restimulation to enhance adoptive cell transfer efficacy, suggesting a rational for future therapeutic strategies in cancer patients.

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Chimeric antigen receptors have different downstream signal transduction compared to T cell receptors that may be tunable for optimal efficacy

Jan 25, 2021 12:00am ‐ Jan 25, 2021 12:00am

Chimeric antigen receptors have different downstream signal transduction compared to T cell receptors that may be tunable for optimal efficacy Title: Chimeric antigen receptors have different downstream signal transduction compared to T cell receptors that may be tunable for optimal efficacy Authors: Katherine J Carpenter, PhD 1; Jeremy Bjelajac 1,2; Ashley Wilson, PhD 1; Joshua Gustafson, PhD 1; James Matthaei, PhD 1,3; Michael Jensen, MD 1,4,5; Stephen EP Smith, PhD 1,5 Affiliations: 1 Seattle Children’s Research Institute 2 Stanford University School of Medicine 3 Sonoma Biotherapeutics 4 Fred Hutchinson Cancer Research Center 5 University of Washington School of Medicine Abstract: Chimeric antigen receptors (CARs) are synthetic constructs containing extracellular antigen-specific scFv (single-chain variable immunoglobulin) fragments linked to intracellular signaling domains. These intracellular domains most commonly include CD3z subunits and either CD28 or 4-1BB domains. This complex differs drastically from the endogenous T cell receptor (TCR), which is comprised of 2 TCR chains and 6 intracellular CD3 chains. TCR signaling is a well characterized molecular circuit, but the CAR signal transduction network (STN) has not been well studied. Another unique aspect of CAR design is antigen binding affinity. CARs have been designed to behind strongly to antigen (Kd 106-109 M-1) compared to endogenous TCRs (104-106 M-1). Despite their different components, when CARs are activated by antigen they are able to stimulate potent anti-tumor T cell responses. CAR T cell therapy has been shown to be effective at killing tumors in vivo, particularly in the case of B cell lymphomas and leukemias. These CARs are the epitome of personalized medicine as they are created specifically for each patient’s individual needs. However, as with many other immuno-oncology approaches, CARs have been shown to cause negative side effects such as cytokine release syndrome (CRS) and neurotoxicity (NT). We hypothesize differential STNs downstream of CAR activation promote either optimal (tumor clearance) or negative (CRS/NT) outcomes in patients. We stimulated human clinical-grade anti-CD19 CAR T cells with antigen presenting cells expressing either anti-CD3 (TCR stimulation) or CD19 (CAR stimulation). We then utilized Quantitative Multiplex co-Immunoprecipitation (QMI) to analyze protein-protein interactions downstream of either TCR or CAR engagement. Using weighted correlation network analysis (CNA), we found 2 distinct modules correlating with CD3 or CD19 stimulation. We also used anti-fluorescein CARs with different ligand affinities (low, medium, or high) to assess maximum responses. We found, surprisingly, that medium affinity anti-fluorescein CARs had the highest response intensity compared to low and high affinity CARs. Lastly, we produced CARs from 3 different healthy donors and stimulated them with CD19. Through Hierarchical Clustering by Principal Components (HCPC) we saw distinct clustering of Donor 3 compared to Donor 1 and 2. This leads us to believe there is detectable inter-individual differences in STNs between CARs from different donors. Using these results, we believe CAR functionality could be optimized by tuning scFv affinity and modifying downstream STNs to promote clearance of tumors potentially without causing negative side effects.

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High-throughput screening for rare antigen-reactive TCRs using natively-paired TCRab expression libraries generated from millions diverse primary T cells.

Jan 25, 2021 12:00am ‐ Jan 25, 2021 12:00am

High-throughput screening for rare antigen-reactive TCRs using natively-paired TCRab expression libraries generated from millions diverse primary T cells. Matthew J. Spindler1, Ayla L. Nelson1, James M. Heather2, Ellen K. Wagner1, Adam S. Adler1, David S. Johnson1 1 GigaMune, Inc., 1 Tower Place, Suite 750, South San Francisco, CA 94080, USA 2 Massachusetts General Hospital, 275 Cambridge St., Cambridge, MA 02114, USA ABSTRACT Engineered TCR-T cells can provide potent cancer therapies by targeting HLA presented peptides derived from tumor associated antigens, oncoviral antigens, and neoantigens. Current clinical trials are limited to a handful of antigen-reactive TCRs with the vast majority targeting HLA-A0201 presented peptides. The efficient identification of clinical candidate TCRs is needed to broaden the treatable patient population. Single cell sorting of pHLA-binding T cells using multimers and ex vivo antigen expansion are the current gold standards for identifying antigen-reactive primary T cells. However, functional validation of the TCRs identified by these approaches requires resource intensive cloning of each individual TCRab pair. Additionally, primary T cells, especially tumor infiltrating lymphocytes (TILs), are a limited resource, which restricts the number of antigens that can be screened. To address this, we developed a microfluidic approach to capture and functionally express natively-paired TCRab libraries from millions of single T cells. Unlike DNA barcoding approaches that mark single cells by adding a sequence tag, we physically link the TCRa-TCRb chains to generate sequencing and full-length expression libraries which we introduce into Jurkat cells for functional testing. Using these methods, we are building expression libraries from healthy and cancer patient samples; these libraries include over 2.9 million TCRab clonotypes from six healthy PBMC donors and over 0.5 million from expanded melanoma TIL samples. We applied pMHC binding and cellular activation screens to identify and validate 14 TCRs reactive to common viral and tumor associated antigens with starting frequencies of

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Donor-unrestricted targeting of CD1c-expressing leukemia by T cells engineered with a lipid-specific TCR

Jan 25, 2021 12:00am ‐ Jan 25, 2021 12:00am

Donor-unrestricted targeting of CD1c-expressing leukemia by T cells engineered with a lipid-specific TCR Acute leukemia is currently treated by chemotherapy and allogeneic hematopoietic stem cell transplantation (HSCT). The major unmet clinical need of this approach remains the frequent post-transplant leukemia recurrence. Adoptive immunotherapy with allogeneic donor-derived T cells can control disease recurrence by inducing a beneficial graft-versus-leukemia (GVL) reaction. However, grafted alloreactive T cells can also attack patients’ non-hematopoietic tissues, resulting in detrimental graft-versus-host-disease (GVHD), prompting the search for more specific T-cell targets on malignant cells. We have shown that primary acute myelogenous (AML) and B-lymphoblastic (B-ALL) leukemia blasts express the CD1c molecule, and that a group of human CD1c-restricted, self-reactive T cell clones kills acute leukemia blasts by recognizing the leukemia-associated lipid antigen methyl-lysophosphatidic acid (mLPA). Because CD1c is identical in all individuals and expressed only by mature leukocytes, these results suggest a donor-unrestricted adoptive immunotherapy approach with reduced risk of GvHD induction. To assess the feasibility of ACT for acute leukemia with mLPA-specific T cells, we generated a library of lentiviral vectors encoding a panel of human mLPA-specific TCRs. Upon TCR transduction, either Jurkat T cells or human primary T cells are specifically retargeted against CD1c-expressing malignant cells in vitro, defining a lead mLPA-specific TCR suitable for adoptive immunotherapy. Indeed, we can engineer total T lymphocytes from any donor with the lead mLPA-specific TCR to kill any CD1c-expressing leukemia cell in vitro and in immunodeficient mouse xenografts. These results highlight a novel approach for ACT of acute leukemia with T cells engineered to recognize malignant cells by the transfer of a lipid-specific TCR that works across MHC-barriers like a CAR. Michela Consonni1, Claudio Garavaglia1, Andrea Grilli2, Claudia de Lalla1, Alessandra Mancino1, Lucia Mori3,Gennaro De Libero3, Daniela Montagna4, Angelo Lombardo5, Monica Casucci6, Marta Serafini7, Chiara Bonini8, Daniel Häussinger9, Fabio Ciceri10, Massimo Bernardi10, Sara Mastaglio10, Silvio Bicciato2, Paolo Dellabona1, Giulia Casorati1 1Experimental Immunology Unit, Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan 20132, Italy; 2Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy; 3Experimental Immunology, Department of Biomedicine, University of Basel and University Hospital, Basel, Switzerland; 4Foundation IRCCS Policlinico San Matteo; Department of Sciences Clinic-Surgical, Diagnostic and Pediatric, University of Pavia, Pavia, Italy; 5San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy; 6Innovative Immunotherapies Unit, Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy; 7M. Tettamanti Research Center, University of Milano-Bicocca, Monza, Italy; 8Experimental Hematology Unit, Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy; 9NMR-Laboratory, Department of Chemistry, University of Basel; 10Hematology and Bone Marrow Transplant Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy

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Engineered Chimeric Antigen Receptors for Nano-optogenetic Control of Therapeutic T-cells (LiCAR T-cells)

Jan 25, 2021 12:00am ‐ Jan 25, 2021 12:00am

Engineered Chimeric Antigen Receptors for Nano-optogenetic Control of Therapeutic T-cells (LiCAR T-cells) Summary: Chimeric antigen receptor (CAR) T-cell immunotherapy has demonstrated high potential for elimination of tumors, particularly in patients with CD19-positive lymphoma and leukemia. CARs are synthetic receptors engineered onto the surface of T cells, where they can engage specific tumor antigens in a major histocompatibility complex (MHC)-independent manner. The recognition of antigen allows T cells to be activated and subsequently perform their killing/effector activities toward tumor cells. Despite the tremendous success of CAR T-cell therapy in cancer treatment, this type of immunotherapy imposes significant safety challenges, as most notably exemplified by the cytokine release syndrome (CRS) and the “on-target, off-tumor” cytotoxicity due to the lack of control over the dose, location, and timing of T cell activity. As CAR T-cells are activated, they will exponentially proliferate and eventually reach precarious levels where a cytokine response exceeds tolerability. The recently FDA approved CAR T-cell therapies are designed for CD19-positive tumors, but they cannot discriminate between normal CD19+ cells and cancerous CD19+ cells. As such, CAR T-cells will likely attack normal cells/tissues and lead to B cell aplasia or even more devastating consequences in certain patients, thereby posing limitations on the use of the current CAR T-cell therapy. We present herein the design of light-switchable CAR T-cells (designated “LiCAR”), which enables phototunable activation of therapeutic T cells to inducibly kill tumor cells. When coupled with upconversion nanoparticles (UCNPs), LiCAR permits the timing, location, and dosage of T cell-mediated therapeutic activity to be tailored in the dual presence of tumor antigen and light. This nano-optogenetic device sets the stage for the future biomedical application of optogenetic immunotherapy to deliver personalized anti-cancer therapy. Authors and Affiliations: Nhung T. Nguyen1, Kai Huang2, Hongxiang Zeng3, Ji Jing1, Joyce Chen4, Zixian Huang1, Xin Liu5, Anjana Rao4, Yun Huang3,*, Gang Han2,*, Yubin Zhou1,6* 1Center for Translational Cancer Research, Institute of Biosciences and Technology, Texas A&M University, Houston, TX 77030, 2Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, 3Center for Epigenetics and Disease Prevention, Institute of Biosciences and Technology, Texas A&M University, Houston, TX 77030, 4Division of Signaling and Gene Expression, La Jolla Institute for Immunology, La Jolla, CA, USA, 5Center for Inflammation and Epigenetics, Houston Methodist Research Institute, Houston, Texas 77030, 6Department of Medical Physiology, College of Medicine, Texas A&M University, Temple, Texas 76504, USA.; *Email: yubinzhou@tamu.edu

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Computational Protein Design for Next-Generation Cellular Therapeutics

Jan 25, 2021 10:00am ‐ Jan 25, 2021 10:40am

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Welcoming Remarks and Keynote Address

Jan 25, 2021 10:00am ‐ Jan 25, 2021 10:40am

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Next-Generation CART Approaches Exploiting Natural Killer Receptor Targeting

Jan 25, 2021 10:40am ‐ Jan 25, 2021 11:05am

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The Current State of Cellular Therapies for Cancer: Lessons from the Clinic

Jan 25, 2021 10:40am ‐ Jan 25, 2021 1:00pm

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