CD3-zeta Signaling Domains Research Articles

CCR1-Targeting CAR T Cells for Acute Myeloid Leukemia

BACKGROUND Chimeric antigen receptor (CAR) T cell therapy is highly effective in patients with B cell malignancies and multiple myeloma, but initial clinical trials with CARs targeting acute myeloid leukemia (AML) have shown less successful results. Challenges inherent to using CAR T cells for AML include lack of a leukemia-specific target antigen due to heterogeneous antigen expression and potential on-target/off-tumor toxicity due to co-expression of target antigens on normal myeloid cells. C-C chemokine receptor 1 (CCR1, also known as CD191), a G protein-coupled receptor that binds to members of the C-C chemokine family, is a promising AML-associated antigen that is reported to be expressed on over 75% of AML samples and minimally expressed on hematopoietic stem/progenitor cells and T cells. The present study aimed to explore the potential of CCR1 as a novel CAR target antigen using a syngeneic mouse model that allows investigation of both efficacy and toxicity of the CAR T cells. METHODS AND RESULTS We developed a CAR based on the single chain variable fragment (scFv) KM5907 targeting both mouse and human CCR1. We constructed a second-generation CCR1-CAR containing a CD28 hinge, a CD28 transmembrane domain, a CD3 zeta signaling domain, and a CD28 co-stimulatory domain. We mutated the immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 zeta chain to retain only a single active membrane-proximal (1XX) ITAM, based on a prior study showing that the 1XX ITAM format enhanced CAR T cell activity by reducing T cell exhaustion. Primary murine T cells were retrovirally transduced and CCR1-CAR expression was assessed by flow cytometry. CCR1-CAR T cells promoted effective elimination of BM185 leukemia cells overexpressing murine CCR1 in vitro. Similarly, primary human T cells transduced with the CCR1-CAR exhibited potent cytotoxicity against C1498 leukemia cells overexpressing human CCR1 in vitro. To evaluate anti-leukemia activity of the CCR1-CAR T cells in vivo, BALB/c mice were sublethally irradiated and engrafted with 1x10 5 BM185 leukemia cells modified to overexpress murine CCR1. Two days later, mice were treated with 2x10 6 CCR1-CAR T cells and monitored for weight changes and survival. Growth of BM185 leukemia cells (expressing firefly luciferase) and expansion of CAR T cells (expressing gaussia luciferase) were assessed via two-color bioluminescence imaging (BLI). On day 6, mice were bled retro-orbitally and serum was analyzed for cytokine levels. The CCR1-CAR T cells demonstrated potent anti-leukemia activity and T cell expansion in vivo, promoting improved overall survival compared with control mice that received only tumor cells (Figure 1). CCR1-CAR T cell activity was accompanied by mild, self-limited toxicity, characterized by transient weight loss (Figure 2), but not significant elevation of serum levels of cytokines associated with cytokine release syndrome (CRS) such as IL-6 and TNF-α. BLI of the CCR1-CAR T cells revealed enhanced trafficking into the bone marrow and spleen followed by proliferation in irradiated mice even in the absence of tumor, suggesting that CCR1-CAR T cells are stimulated by CCR1 antigen expressed on non-leukemic host cells in addition to leukemia cells. CONCLUSIONS In summary, CCR1-CAR T cells represent a promising therapeutic strategy for AML that to our knowledge has not yet been evaluated in preclinical or clinical studies. We generated CCR1-CAR T cells that demonstrated potent anti-leukemia activity in vitro and in vivo in a syngeneic mouse model. Clinical signs of self-limited toxicity were not associated with elevated levels of inflammatory cytokines and might be caused by an on-target/off-tumor effect, which warrants further investigations to better characterize the safety profile of CCR1-CAR T cells. The CCR1-CAR T cells also displayed potent cytotoxicity in vitro via human CCR1, which will facilitate clinical translation of the findings. A CCR1-CAR may also offer benefit in other hematologic malignancies with reported CCR1 expression such as multiple myeloma or Hodgkin's lymphoma.

CT-167 Using the Sodium Iodide Symporter to Visualize CAR-T Cell Trafficking

Chimeric antigen receptor T (CAR-T) cell therapy revolutionized the treatment of refractory B cell malignancies, but relapses occur through a variety of incompletely understood mechanisms. Currently, tracking CAR-T expansion and persistence in patients is limited to peripheral blood sampling, but this approach provides no insights into how and where CAR-T cells traffic beyond the peripheral blood or how cellular localization may impact persistence and efficacy. Furthermore, costimulatory endodomains that are known to influence CAR-T cell phenotype have unclear downstream effects on how CAR-T cells traffic. The sodium iodide symporter (NIS) is a plasma membrane glycoprotein that actively uptakes iodide and its analogs, and it can be transduced into various cell types in preclinical models allowing sensitive and specific PET/SPECT imaging. Coexpression of NIS with CAR on T cells would allow real-time, in vivo imaging of CAR-T cells and aid in understanding CAR-T cell biology and trafficking. Using Golden Gate modular vector construction, multiple anti-CD20 CAR constructs with specificity for both human and non-human primate (NHP) CD20 antigens were generated. Each construct included one of several costimulatory domains (4-1BB, CD28, or CD27), a standard CD3-zeta signaling domain, and either the rhesus-NIS reporter or truncated EGFR following a short ribosomal skip sequence peptide, allowing transduction under control of the same EF1a promoter. CAR was effectively cotransduced with NIS in both human and NHP primary T cells with no effect of NIS on CAR-T cell-mediated cytotoxicity against CD20-expressing Raji cells in vitro compared to control. Cytotoxicity was antigen-specific as no difference was seen against CD20- control cells. Functional iodide and [18F]-TFB, an iodide analog, uptake via NIS was specific to NIS-transduced cells and was appropriately inhibited by coincubation with KClO4. An NSG mouse xenograft model will be used to track the localization, expansion, efficacy, and persistence of CAR-T constructs against Raji lymphoma cells. Different costimulatory domains appear to elicit only slight differences in cytokine production in vitro but will be further studied for differences in efficacy and trafficking in vivo in mice and later in a syngeneic NHP model.

Abstract CT522: Feasibility and safety of a novel CD19 CAR T cell therapy in adults with R/R B-NHL

Abstract Genetically engineered chimeric antigen receptor (CAR) T cells have exhibited distinct effectiveness against chemotherapy refractory CD19 expressing B cell malignancies in both adults and children. This phase I clinical trial tests a novel anti-CD19 CAR T cell product in adults with relapsed/refractory (R/R) B-cell Non-Hodgkin’s Lymphoma (B-NHL). The CAR construct is comprised of the short chain variable regions of the anti-CD19 monoclonal antibody FMC63, the TNFRSF19-derived transmembrane domain, the 4-1BB costimulatory domain, and the CD3-zeta signaling domain. CD19 CAR T cells were manufactured utilizing the CliniMACS Prodigy® T Cell Transduction Process (CD3/CD28 TransAct™ reagent) allowing for highly automated production, with IL-7 and IL-15 used for T cell expansion for 8-12 days. To date, 7 patients have been treated with an average dose of 1.2 ± 0.2 x 108 CAR T cells. The histology includes marginal zone lymphoma (n=1), follicular lymphoma Grade IIIA (n=1), transformed lymphoma (n=3), follicular lymphoma low grade (n=2), and diffuse large B-cell lymphoma (n=1). One patient required a second apheresis due to poor cell expansion. Despite heterogeneity in disease subtype and leukapheresis product quality, CAR T production and expansion have been consistent with final transduction efficiencies between 14-45%, cell viability between 88-91%, and an overall average yield of 3.2 ± 0.3 x 109 cells before harvest, allowing for product banking. No safety-related out of specifications (OOS) events have occurred, however, 2 patients had OOS product infused due to low transduction efficiency (both at 14% rather than the ≥ 20% release criteria). Two patients experienced Grade 2 CRS, 1 patient experienced Grade 2 neurotoxicity; otherwise, no new safety signals were detected. Disease response was assessed on Days 90, 180, 270, and 360 post-infusion. The assessments were based on 2014 Lugano criteria. Even with 2 OOS products, Day 90 scans showed a complete metabolic remission (CMR) in 6 evaluable patients to date. Of the 6 patients with CMR, 1 patient progressed at Day 180 and the others remain in remission (median f/u = 12 months). Flow cytometry was utilized to measure CAR T cell peak expansion and persistence in 5 patients. Peak CAR T cell expansion (2.9-44.4% of CD3 cells) ranged from Day 5 to 15. Cell persistence was detected for the 5 patients through at least Day 180. ddPCR is currently in development to perform persistence testing in parallel. Additionally, cytokine concentrations including INFγ, IL-10, IL-12p70, IL-13, IL1β, IL-2, IL-4, IL-6, IL-8, and TNFα were evaluated over the first 30 days. Overall, 7 patients diagnosed with 5 different B-NHL subtypes have been treated with the CD19 CAR T cell product. Manufacturing was successful for all patients with no safety related OOS, and no new post-infusion safety signals detected. To date, 6 out of 7 patients are alive, 5 with CMR and with CD19 CAR T cell persistence through at least 180 days. Citation Format: Manali Kamdar, Cheri Adams, Steven Bair, Boro Dropulic, Jonathon Gutman, Bradley Haverkos, Kimberly Jordan, Rebecca Mallo, Russell Marians, Felicia Mast, Lindsey Murphy, Andrew Roth, Matthew Seefeldt, Andrew Worden, Mike Kadan, Ying Xiong, Dina Schneider, Rimas Orentas, Terry Fry, Michael Verneris. Feasibility and safety of a novel CD19 CAR T cell therapy in adults with R/R B-NHL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT522.

Anti-FLT3 CAR T Cells in Acute Myeloid Leukemia

INTRODUCTIONIn patients with AML who are eligible for intensive therapy, the goal of treatment is the achievement of complete response followed by consolidation chemotherapy (in favorable risk disease) or hematopoietic stem cell transplantation (in intermediate or adverse risk disease). Patients who do not attain this initial goal lack effective therapeutic options. Extensive experience with chimeric antigen receptor (CAR) T cells in B-ALL has shown that CART cells can deliver potent and durable antigen-specific leukemia control, and that targeting a single antigen (CD19 for B-ALL) is associated with antigen-negative relapse. In this context, we sought to expand the existing preclinical CART armamentarium in AML by developing FLT3-specific CART cells and comparing them to our existing gold standard CD123-specific CART cells. Since activating mutations in FLT3 occur commonly in AML, we reasoned that this molecule would serve as an “Achilles heel” in AML immunotherapy.METHODSNovel fully humanized anti-human FLT3 receptor single chain variable fragments (scFV) were fused to CD28 and CD137 (41BB) costimulatory molecules and the CD3zeta signaling domain and cloned into a lentiviral expression vector. Based on recently published data, we tested linker lengths ranging from 5 to 20 amino acids between the light and heavy chains of the CAR. We used a FLT3-ITD mutated AML cell line (MOLM14) expressing luciferase for in vitro function studies including an exhaustion assay. For in vivo function studies, we engrafted MOLM14 expressing luciferase into NSG mice and treated with CART-FLT3 or untransduced T cells (negative control).RESULTSAll FLT3 and CD123-specific CART cells degranulated and produced the effector cytokines IL-2, INFg, TNF and GM-CSF in an antigen-specific manner, with some variability between the different linker lengths and with some superiority of the CAR123 likely resulting from the higher expression of CD123 compared with FLT3 in this model (p < 0.0001, one way ANOVA) (Figure 1). Short-term killing assays (24 hours) revealed that all CART cells killed MOLM14 with equivalent efficiency at low effector:target ratios (Figure 2A). Since short-term killing assays likely do not replicate the physiological situation in vivo wherein CART cells encounter cancer cells repeatedly over many days, we next developed an in vitro exhaustion assay. We incubated MOLM14 cells with CAR T cells at 1:10 E:T ratio and added MOLM 14 tumor cells along with fresh media every other day. Killing was quantified every 48 hours. Interestingly, all CAR constructs showed equivalently efficient cytotoxicity from days 5-15. However, after day 15 there was progressive dysfunction and loss of cytotoxic activity. This exhaustion “stress test” revealed some superiority of the FLT3 CAR 10AA construct (p = 0.042 on day 17, two way ANOVA) (Figure 2B).NOD/SCID gamma chain KO (NSG) mice were then engrafted with 1x10 6 luciferized MOLM14 cells and treated with 0.5x10 6 CAR T cells 7 days later, randomized to treatment groups based on tumor burden. CAR T cells expansion was monitored in peripheral blood by flow cytometry. (Fig 3A). Serial BLI revealed prompt and durable leukemia remissions and survival (Figure 3B,C).CONCLUSIONSWe have developed CART-FLT3 for AML using novel human anti-FLT3 targeting domains and demonstrated preclinical efficacy similar to that of CART-123 in an AML model with substantially lower expression of FLT3 compared to CD123 (data not shown). Since inhibition of FLT3 leads to upregulation of surface FLT3 expression, future experiments will explore combinatorial FLT3 inhibition with CART-FLT3. If successful, these experiments will provide a strong rationale for a combination clinical trial in AML where leukemia control by small molecules is followed by a coup-de-grace delivered by CART cells. [Display omitted] DisclosuresSleiman: Hemogenyx Pharmaceuticals LLC: Research Funding. Shestova: Hemogenyx Pharmaceuticals LLC: Research Funding. Santiago: Hemogenyx Pharmaceuticals LLC: Research Funding. Shrestha: Hemogenyx Pharmaceuticals LLC: Current Employment. Liang: Hemogenyx Pharmaceuticals LLC: Current Employment. Ben Jehuda: Hemogenyx Pharmaceuticals LLC: Current Employment. Sandler: Hemogenyx Pharmaceuticals LLC: Current Employment, Current equity holder in publicly-traded company. Gill: Novartis: Other: licensed intellectual property, Research Funding; Interius Biotherapeutics: Current holder of stock options in a privately-held company, Research Funding; Carisma Therapeutics: Current holder of stock options in a privately-held company, Research Funding.

In Vivo Delivery of a CD20 CAR Using a CD8-Targeted Fusosome in Southern Pig-Tail Macaques (M. nemestrina) Results in B Cell Depletion

Introduction: Ex vivo manufactured chimeric antigen receptor (CAR) T cell therapies are highly effective for treating B cell malignancies. However, the complexity, cost and time required to manufacture CAR T cells limits access. To overcome conventional ex vivo CAR T limitations, a novel gene therapy platform has been developed that can deliver CAR transgenes directly to T cells through systemic administration of a fusosome, an engineered, target-directed novel paramyxovirus-based integrating vector that binds specific cell surface receptors for gene delivery through membrane fusion. Here, we demonstrate that systemic administration of a CD8a-targeted, integrating vector envelope (i.e., fusogen) encoding an anti-CD20 CAR into Southern pig-tail macaques (M. nemestrina), which is a species permissive to the integrating vector-mediated transduction, results in T cell transduction and B cell depletion with no treatment-related toxicities.Methods: CD8a-specific single chain variable fragments (scFvs) were generated and measured for target specificity versus non-CD8-expressing cells in vitro. Cross-reactivity of the CD8a-specific fusogen for human and nemestrina T cells was confirmed in vitro. Targeted fusogens were then used to pseudotype integrating vector expressing an anti-CD20 CAR containing the 4-1BB and CD3zeta signaling domains (CD8a-anti-CD20CAR). Transduction and B cell killing was confirmed on human and nemestrina PBMCs. To evaluate in vivo activity, normal, healthy nemestrina macaques were treated with a single dose of CD8a-targeted anti-CD20 CAR fusosome (n=6) or saline (n=2) via intravenous infusion at 10mL/kg/hr for 1-hour and evaluated for up to 52 days for evidence of adverse effects, B cell depletion, CAR-mediated cytokine production, CAR T cell persistence and vector biodistribution using ddPCR and anti-CD20CAR transgene by RT-ddPCR to detect transgene levels. Histopathology of several organs and immunohistochemistry for CD3 and CD20 on lymph nodes, spleen, and bone marrow were performed at termination (days 35 and 52). Tolerability of the treatment was assessed by body weight, body temperature, neurological exams, serum chemistry panel, and complete blood counts pre-dose and post-dose up to 52 days.Results: The CD8a-targeted fusogen demonstrated CD8a-specificity versus human CD8 negative cell lines, and cross-reactivity and transduction efficiency in nemestrina PBMCs in vitro. Compared to a control vector (GFP), anti-CD20CAR-modified T cells showed a dose-dependent depletion of B cells using in vitro assays. Following infusion of CD8a-anti-CD20CAR fusosomes into macaques, pharmacological activity in peripheral blood was detected by a reduction of B cells in 4 of 6 animals after 7 to 10 days. Two animals showed persistent B cell depletion until study termination, with two others showing a temporary response. The presence of vector copy could be detected in the peripheral blood of all treated animals between days 3 and 10, and in isolated spleen cells in 5 of 6 animals. All control animals (saline) were negative for vector. RT-ddPCR mRNA expression similarly revealed the presence of anti-CD20CAR transcripts in isolated spleen cells from treated animals; no expression was detected in tissues from control animals. Elevations in inflammatory cytokines could be detected in the serum of treated animals between days 3 and 14. Fusosome treatment was well-tolerated in all animals with no evidence of adverse effects. Moreover, T cell transduction and B cell depletion was not associated with cytokine-related toxicities, and blood chemistry and histopathology were within normal limits.Conclusion: These data obtained in an immunologically competent animal demonstrate the proof-of-concept that systemic administration of a CD8a-anti-CD20CAR fusosome can specifically transduce T cells in vivo without pre-conditioning or T cell activation, resulting in B cell depletion in the absence of vector- or CAR T-related toxicities. Therefore, fusosome technology represents a novel therapeutic opportunity to treat patients with B cell malignancies and potentially overcome some of the treatment barriers that exist with conventional CAR T therapies. DisclosuresCunningham: Sana Biotechnology: Current Employment. Chandra: Sana Biotechnology: Current Employment. Emmanuel: Sana Biotechnology: Current Employment. Mazzarelli: Sana Biotechnology: Current Employment. Passaro: Sana Biotechnology: Current Employment. Baldwin: Sana Biotechnology: Current Employment. Nguyen-McCarty: Sana Biotechnology: Current Employment. Rocca: Sana Biotechnology: Current Employment. Joyce: Sana Biotechnology: Current Employment. Kim: Sana Biotechnology: Current Employment. Vagin: Sana Biotechnology: Current Employment. Kaczmarek: Sana Biotechnology: Current Employment. Chavan: Sana Biotechnology: Current Employment. Jewell: Sana Biotechnology: Current Employment. Lipsitz: Sana Biotechnology: Current Employment. Shamashkin: Sana Biotechnology: Current Employment. Hlavaty: Sana Biotechnology: Current Employment. Rodriguez: Sana Biotechnology: Current Employment. Co: Sana Biotechnology: Current Employment. Cruite: Sana Biotechnology: Current Employment. Ennajdaoui: Sana Biotechnology: Current Employment. Duback: Sana Biotechnology: Current Employment. Elman: Sana Biotechnology: Current Employment. Amatya: Sana Biotechnology: Current Employment. Harding: Sana Biotechnology: Current Employment. Lyubinetsky: Sana Biotechnology: Current Employment. Patel: Sana Biotechnology: Current Employment. Pepper: Sana Biotechnology: Current Employment. Ruzo: Sana Biotechnology: Current Employment. Iovino: Sana Biotechnology: Current Employment. Varghese: Sana Biotechnology: Current Employment. Foster: Sana Biotechnology: Current Employment. Gorovits: Sana Biotechnology: Current Employment. Elpek: Sana Biotechnology: Current Employment. Laska: Sana Biotechnology: Current Employment. McGill: Sana Biotechnology: Current Employment. Shah: Sana Biotechnology: Current Employment. Fry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Dambach: Sana Biotechnology: Current Employment.

Targeting the Membrane-Proximal C2-Set Domain of CD33 for Improved CD33-Directed CAR T Cell Therapy

Background: There is increasing interest in targeting CD33 in malignant and non-malignant disorders, but available drugs are ineffective in many patients. As one limitation, therapeutic CD33 antibodies typically recognize the membrane-distal V-set domain. Likewise, currently tested CD33-directed chimeric antigen receptor (CAR) T cells likewise target the V-set domain and have thus far shown limited clinical activity. We have recently demonstrated that binding closer to the cell membrane enhances the effector functions of CD33 antibodies. We therefore raised antibodies against the membrane-proximal C2-set domain of CD33 and identified antibodies that bound CD33 regardless of the presence/absence of the V-set domain (“CD33 PAN antibodies”). Here, we tested their properties as targeting moiety in CD33 PAN CAR T cell constructs, using a clinically validated lentiviral backbone.Methods: To generate CAR T cells, negatively selected CD8 + T cells were transduced with an epHIV7 lentivirus encoding the scFv from a CD33 PAN antibody (clone 1H7 or 9G2) linked to either a short (IgG 4 hinge only), intermediate (hinge plus IgG 4 CH3 domain), or long (hinge plus IgG 4 CH3 domain plus IgG 4 CH2 domain) spacer, the CD28-transmembrane domain, CD3zeta and 4-1BB intracellular signaling domains, and non-functional truncated CD19 (tCD19) as transduction marker. Similar constructs using scFvs from 2 different V-set domain-targeting CD33 antibodies, including hP67.6 (My96; used in gemtuzumab ozogamicin), were generated for comparison. CAR-T cells were sorted, expanded in IL-7 and IL-15, and used in vitro or in vivo against human AML cell lines endogenously expressing CD33 and cell lines engineered to lack CD33 (via CRISPR/Cas9) with/or without forced expression of different CD33 variants.Results: CD33 V-set-directed CAR T cells exerted significantly more cytolytic activity against AML cells expressing an artificial CD33 variant lacking the C2-set domain (CD33 ΔE3-4) than cells expressing full-length CD33 at similar or higher levels, consistent with the notion that CD33 CAR T cell efficacy is enhanced when targeting an epitope that is located closer to the cell membrane. CD33 PAN CAR T cells were highly potent against human AML cells in a strictly CD33-dependent fashion, with constructs containing the short and intermediate-length spacer demonstrating robust cytokine secretion, cell proliferation, and in vitro cytolytic activity, as determined by 51Cr release cytotoxicity assays. When compared to optimized CD33 V-set CAR T cells, optimized CD33 PAN CAR T cells were significantly more potent in cytotoxicity, proliferation, and cytokine production without appreciably increased acquisition of exhaustion markers. In vivo, CD33 PAN CAR T cells extended survival in immunodeficient NOD.SCID. IL2rg -/- (NSG) mice bearing significant leukemic burdens from various cell line-derived xenografts (HL-60, KG1α and MOLM14) with efficient tumor clearance demonstrated in a dose-dependent fashion.Conclusion: Targeting the membrane proximal domain of CD33 enhances the anti-leukemic potency of CAR T cells. Our data provide the rationale for the further development of CD33 PAN CAR T cells toward clinical testing. DisclosuresFiorenza: Link Immunotherapeutics: Consultancy; Bristol Myers Squibb: Research Funding. Godwin: Pfizer: Research Funding; Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Turtle: Allogene: Consultancy; Amgen: Consultancy; Arsenal Bio: Consultancy; Asher bio: Consultancy; Astrazeneca: Consultancy, Research Funding; Caribou Biosciences: Consultancy, Current holder of individual stocks in a privately-held company; Century Therapeutics: Consultancy, Other; Eureka therapeutics: Current holder of individual stocks in a privately-held company, Other; Juno therapeutics/BMS: Patents & Royalties, Research Funding; Myeloid Therapeutics: Current holder of individual stocks in a privately-held company, Other; Nektar therapeutics: Consultancy, Research Funding; PACT Pharma: Consultancy; Precision Biosciences: Current holder of individual stocks in a privately-held company, Other; T-CURX: Other; TCR2 Therapeutics: Research Funding. Walter: Kite: Consultancy; Janssen: Consultancy; Genentech: Consultancy; BMS: Consultancy; Astellas: Consultancy; Agios: Consultancy; Amphivena: Consultancy, Other: ownership interests; Selvita: Research Funding; Pfizer: Consultancy, Research Funding; Jazz: Research Funding; Macrogenics: Consultancy, Research Funding; Immunogen: Research Funding; Celgene: Consultancy, Research Funding; Aptevo: Consultancy, Research Funding; Amgen: Research Funding.

Abstract 1545: Development of FGFR4-targeted chimeric antigen receptors (CARs) for the treatment of rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is a soft tissue cancer commonly arising in muscle, and is the most common soft tissue cancer in children. Patients with metastatic RMS have a poor prognosis that has not improved in decades, highlighting the need for novel therapies. Pediatric tumors, and especially the fusion-gene driven alveolar RMS (aRMS) subset, have among the lowest mutational burden of any tumor, suggesting that checkpoint therapies alone will be insufficient to control tumors. CAR-T cells are genetically engineered T lymphocytes expressing an extracellular binding domain and intracellular T cell signaling domains. Upon engagement of the binding domain (often antibody-derived) to the antigenic target on a tumor cell, activation and degranulation occurs resulting in cell-mediated toxicity induced death of the tumor cells. The key to CAR specificity is to guide engineered T cells to a molecular target that is tumor specific, expressed on the cell surface, and expressed at high enough levels for CAR-T activation. Previous work identified fibroblast growth factor receptor 4 (FGFR4, CD334) as specifically upregulated in RMS tumors compared to normal tissue, making it a candidate for the selective targeting of CAR T cells. A previous CAR designed to target FGFR4 (m410) showed in vitro efficacy, and some control of tumor in a metastatic (intravenous) RMS model but failed to control orthotopic (intramuscular) RMS tumors in vivo. We have developed a new generation of FGFR4-binding moieties to be tested as new components of CARs. Our new binders were derived from both human F(ab)- and human VH-only (single chain domain antibodies, dAbs) phage display libraries. Binders were cloned into a CAR featuring a CD8 hinge and transmembrane domain, 4-1BB signaling domain, and CD3zeta signaling domain. FGFR4 CARs were screened for surface expression by flow cytometry, cytotoxicity against target and non-target cells, and cytokine production in response to RMS cell lines in vitro. Two F(ab)-based FGFR4-binders engineered into CARs were selected for further development based on high cellular cytotoxicity and tumor specificity. These will be tested for in vivo efficacy against metastatic and intramuscular RMS tumor models in NSG mice. Additionally, we have tested RMS tumor cell lines, with and without exposure to IFN-gamma, to model induced mechanisms of tumor defense against immune cell attack. Pathway analysis identified several potential mechanisms of tumor resistance to CAR T therapy which will be exploited to help armor engineered CAR-T cells to control RMS tumors. This data will determine whether our new FGFR4 CARs are sufficient for targeting and eliminating RMS tumors, provide insight into which armoring approaches are best suited to control RMS tumors, and thus develop a potent new therapeutic approach for rhabdomyosarcoma.​ Citation Format: Peter Sullivan, Rajesh Kumar, Wei Li, Lingyang Wang, Yue Zhang, Adam Cheuk, Nityashree Shivaprasad, Javed Khan, Dimiter S. Dimitrov, Rimas J. Orentas. Development of FGFR4-targeted chimeric antigen receptors (CARs) for the treatment of rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1545.

Development of CAR-T Cell Constructs with Broad Anti-Tumor Tropism

Chimeric antigen receptor T (CAR-T) cell therapy is a transformative approach to cancer eradication. CAR-T is expensive in part due to the restricted use of each CAR construct for a specific set of tumors such as B cell lymphoma targeted with CD19 and multiple myeloma targeted with BCMA. A CAR construct with broad anti-tumor activity can be advantageous due to wide applicability and scalability of production. We show that CD126, the IL-6 receptor alpha, is an antigen that is expressed by many hematologic and solid malignancies including multiple myeloma, non-Hodgkin lymphoma, acute myeloid leukemia, pancreatic and prostate adenocarcinoma, non-small cell lung cancer and malignant melanoma amongst others. High CD126 expression is a negative prognostic marker in many malignancies. The two CD126 targeting CAR-T cell constructs contain the CD28 anchoring domain followed by 4-1BB and CD3 zeta signaling domain. Lentiviral vectors were generated with triple plasmid (CAR, psPAX2 and pMD2.G) transfection of 293T cells and the vector concentrated by ultracentrifugation and used to transduce human T cells. T cells were isolated from leuko-reduction cones using negative selection with magnetic beads. The transduction efficiency was around 60%. The T cells were activated with anti-CD3/CD28 beads and expanded for two weeks before using for downstream experiments. CD126 CAR-T cells are able to kill many tumor cells in an antigen specific manner and with an efficiency that is directly proportional to the cell surface expression of CD126 expression (rho = 0.6, p = 0.0019). The presence of soluble CD126 in the culture media did not interfere with CAR-T cell killing. The CAR-T constructs bind murine CD126. However, injection of CD126 targeting CAR-T cells in NSG mice did not lead to any evidence of hepatotoxicity and weight loss despite possible expression of this antigen on hepatocytes. In vivo studies in NSG mice with multiple myeloma (RPMI-8226) and prostate adenocarcinoma (DU-145) xenograft models (n=10 tumors per group) showed that the intravenously injected CD126 targeted CAR-T cells (107) infiltrated the tumors, expanded, produced human interferon gamma and killed the tumor cells (p&amp;lt;0.001). Bioluminescence imaging showed control of tumor growth in the actively treated tumors compared to the controls (p&amp;lt;0.05). At post mortem, mice injected with CD126 targeted CAR-T cells had smaller residual tumors compared to controls injected with non-engineered human T cells from the same donor. Binding of sIL-6R by CAR-T cells could mitigate cytokine release syndrome. In support of this, murine SAA-3 levels (the equivalent of human CRP) were lower in mice injected with CD126 CAR-T compared to controls (p&amp;lt;0.05), suggesting that binding of sIL-6R by CAR-T cells could mitigate cytokine release syndrome. CD126 provides a novel therapeutic for CAR-T cells in a broad variety of tumors with low risk of toxicity. Disclosures Dingli: Apellis: Consultancy; Millenium: Consultancy; Janssen: Consultancy; Bristol Myers Squibb: Research Funding; Sanofi-Genzyme: Consultancy; Alexion: Consultancy; Rigel: Consultancy; Karyopharm Therapeutics: Research Funding.

Glucuronoxylomannan in the Cryptococcus species capsule as a target for CAR+ T-cell therapy

Abstract The genus Cryptococcus comprises two major fungal species that cause clinical infections in humans: C. gattii and C. neoformans. Each year, about 1 million cases of Cryptococcus infection are reported worldwide, and the infection’s mortality rate ranges from 20% to 70%. To combat the cryptococcosis, we proposed the redirection of CD8+ T cells to target glucuronoxylomannan (GXM), a sugar present in the Cryptococcus species capsule, via expression of a GXM-specific chimeric antigen receptor (GXMR-CAR) for treatment of cryptococcosis. GXMR-CAR has an anti-GXM single-chain variable fragment followed by an IgG4 stalk, a CD28 transmembrane domain, and CD3-zeta and CD28 signaling domains. After lentiviral transduction of human T cells with the GXMR-CAR construct, flow cytometry demonstrated that 82.4% of the cells expressed GXMR-CAR on their surface. To determine whether the GXMR-CAR+ T cells exhibited GXM-specific recognition, these cells were incubated with GXM for 24 h and examined using bright-field microscopy. Large clusters of proliferating GXMR-CAR+ T cells were observed, while no clusters were present in the control cells. The ability of GXMR-CAR T cells to bind to the yeast form of C. neoformans was detected by flow cytometry and fluorescent microscopy, but no binding was detected in mock-transduced control T cell (NoDNA T cells). Furthermore, when GXMR-CAR+ T cells were administered to immunocompromised NSG mice infected with C. neoformans their C. neoformans burden was significantly lower than mock-transduced control T cell treated mice as shown via immunofluorescence using an anti-GXM antibody and Gomori methenamine-silver (GMS) staining of Titan cells in lung tissue.

EMA Review of Axicabtagene Ciloleucel (Yescarta) for the Treatment of Diffuse Large B-Cell Lymphoma.

On June 28, 2018, the Committee for Advanced Therapies and the Committee for Medicinal Products for Human Use adopted a positive opinion, recommending the granting of a marketing authorization for the medicinal product Yescarta for the treatment of adult patients with relapsed or refractory diffuse large B-cell lymphoma and primary mediastinal large B-cell lymphoma, after two or more lines of systemic therapy. Yescarta, which was designated as an orphan medicinal product and included in the European Medicines Agency's Priority Medicines scheme, was granted an accelerated review timetable. The active substance of Yescarta is axicabtagene ciloleucel, an engineered autologous T-cell immunotherapy product whereby a patient's own T cells are harvested and genetically modified ex vivo by retroviral transduction using a retroviral vector to express a chimeric antigen receptor (CAR) comprising an anti-CD19 single chain variable fragment linked to CD28 costimulatory domain and CD3-zeta signaling domain. The transduced anti-CD19 CAR T cells are expanded ex vivo and infused back into the patient, where they can recognize and eliminate CD19-expressing cells. The benefits of Yescarta as studied in ZUMA-1 phase II (NCT02348216) were an overall response rate per central review of 66% (95% confidence interval, 56%-75%) at a median follow-up of 15.1 months in the intention to treat population and a complete response rate of 47% with a significant duration. The most common adverse events were cytokine release syndrome, neurological adverse events, infections, pyrexia, diarrhea, nausea, hypotension, and fatigue. IMPLICATIONS FOR PRACTICE: Yescarta (axicabtagene ciloleucel) was the first chimeric antigen receptor T-cell therapy to be submitted for evaluation to the European Medicines Agency and admitted into the "priority medicine" scheme; it was granted accelerated assessment on the basis of anticipated clinical benefit in relapsed/refractory diffuse large B-cell lymphoma, a condition of unmet medical need. Indeed, Yescarta showed an overall response rate of 66% and a complete response rate of 47% with a significant duration and a manageable toxicity that compared very favorably with historical controls. Here the analysis of benefits and risks is presented, and specific challenges with this important novel product are highlighted, providing further insights and reflections for future medical research.

Efficacy and Safety of Fully Human Bcma CAR T Cells in Combination with a Gamma Secretase Inhibitor to Increase Bcma Surface Expression in Patients with Relapsed or Refractory Multiple Myeloma

Efficacy and Safety of Fully Human Bcma CAR T Cells in Combination with a Gamma Secretase Inhibitor to Increase Bcma Surface Expression in Patients with Relapsed or Refractory Multiple Myeloma

Phase 1 Trial of the Safety and Efficacy of Fully Human Anti-Bcma CAR T Cells in Relapsed/Refractory Multiple Myeloma

Background: B cell maturation antigen (BCMA) is a potential therapeutic target in multiple myeloma. CT053 CAR-BCMA, which is a BCMA-specific chimeric antigen receptor (CAR) T cell, consists of autologous T cells genetically modified with a second-generation CAR incorporating a fully human anti-BCMA single chain fragment variant, a 4-1BB co-stimulatory domain and a CD3-zeta signaling domain. CT053 was studied in a single-arm, open-label phase I program (NCT03716856, NCT03302403, and NCT03380039) in eastern China. Methods: This investigation is a 3-site study in adult subjects with relapsed/refractory multiple myeloma (rrMM) who had received at least 2 prior myeloma regimens. Subjects received one cycle of CT053 CAR-BCMA after fludarabine/cyclophosphamide infusion. The primary objective was the assessment of subject safety. The secondary objectives were pharmacokinetics and efficacy. Efficacy was assessed according to IMWG 2016 criteria. The data cutoff date was June 30, 2019. Results: A total of 24 subjects with median age of 60.1 years (range, 38.5-69.9) were enrolled from Sep 10, 2017 to Sep 22, 2018 (Table 1). The subjects had a median of 4.5 (range, 2-11) prior regimens of therapy, and 41.7% (10/24) underwent autologous stem cell transplantation. At baseline, eleven out of 24 subjects (45.8%) had concomitant extramedullary involvement. Eight subjects (33.3%) had ECOG score 2-3, and 9 subjects (37.5%) reported ISS grade III. All subjects received lymphodepletion preconditioning of fludarabine/cyclophosphamide for 2-4 days, followed by one cycle of CT053 at a dose of 1.5 x 108 T cells except 3 subjects who received 0.5 x 108, 1 x 108, and 1.8 x 108 cells, respectively. The overall response rate was 87.5% (21/24) including 79.2% (19/24) with complete responses or stringent complete response (5 CR, 14 sCR). As shown in Figure 1, P1 who received the lowest dose of 0.5 x 108 experienced partial response (PR) at M1 and very good partial response (VGPR) at M2. P1 then converted to CR at M14 and sCR at M16. Among 20 subjects who underwent the evaluation of minimal residual disease (MRD) status, 17 achieved MRD-negative (≤10−4 nucleated cells) and reported a tumor response (17 CR/sCR). In 13 subjects with ongoing CR/sCR, the median follow-up after CT053 infusion was 383 days (range, 301-467). Nine subjects progressed with median progression-free period of 281 days (range, 57-573); among them, 5 progressed within 6-12 months, 1 at 13 months and 1 at 19 months. Compared to 13 subjects with persistent CR/sCR, the 9 progressed subjects were observed to have a higher percentage of ECOG score 2-3 (66.7% vs 15.4%), ISS Grade III (55.6% vs 15.4%), and concomitant extramedullary diseases (66.7% vs 38.5%) and a decreased hemoglobin (70g/L vs 92g/L) at baseline. Three subjects died of disease progression at the time of analysis. Hematologic toxic effects were the most common treatment-related adverse events of grade (G) 3 or higher, including white blood cell count decreased (87.5%), neutrophil count decreased (66.7%), lymphocyte count decreased (79.2%) and thrombocytopenia (25%). No dose limiting toxicities were observed. Low-grade cytokine release syndrome (CRS) was reported in 15 of 24 (62.5%) subjects. All CRS events (3 G1, 12 G2) recovered within 2-8 days; among them 8 received tocilizumab. Three subjects (12.5%) had neurotoxicity (2 G1, 1 reversible G3). One subject died of bone marrow failure and neutropenic infection. CAR-BCMA T cell expansion was detected as early as Day 1-7 after infusion and reached peak values on Day 7-21 with the highest at 4.5×105 copies/µg genomic DNA. Median T cell persistence was 172 days. The longest persistence of CAR-BCMA copies was measured at 341 days and continues. Conclusion: This study demonstrated that CT053 had an excellent efficacy and a good safety profile in subjects with rrMM. Disclosures Li: CARsgen Therapeutics Co. Ltd: Employment, Equity Ownership. Xiao:CARsgen Therapeutics Co. Ltd: Employment. WANG:CARsgen Therapeutics Co. Ltd: Employment. Yuan:CARsgen Therapeutics Co. Ltd: Employment. Ma:CARsgen Therapeutics Co. Ltd: Employment.

Development of a Genetically-Engineered Allogeneic Anti-CD38 T Cell Therapy Utilizing a Novel Antigen Receptor Structure

Background: Autologous Chimeric Antigen Receptor (CAR) T cell therapy has shown great promise as a treatment modality for a variety of hematological malignancies. But autologous cell therapies still face several practical hurdles, including reliance on patient immune cells and manufacturing difficulties. Sorrento has pioneered an allogeneic T cell therapy approach utilizing genetic engineering of donor-derived T cells to express a Dimeric Antigen Receptor (DAR). The first DAR-T cell therapy being developed is targeted against CD38, a clinically-validated antigen in multiple myeloma. Preclinical data demonstrate potent anti-tumor activity in both in vitro assays and in vivo studies against CD38-expressing lymphoma and multiple myeloma (MM) cell lines. Methods: Anti-CD38 DAR-T cells were generated through genetic engineering of T cells derived from healthy donors inserting the anti-CD38 DAR construct into the TRAC gene locus resulting in loss of endogenous TCR expression while expressing the DAR. Three distinct DAR constructs were utilized differing only in the intracellular signaling components, namely CD28/CD3zeta, 4-1BB/CD3zeta and CD28/4-1BB/CD3zeta. The CD38 DAR-T were expanded and purified for subsequent preclinical studies. Using in vitro assays, the 3 different CD38 DAR-T cells were evaluated against multiple myeloma and lymphoma cell lines for specific cytotoxicity as well as stimulus-induced cytokine secretion and cell expansion. The in vivo anti-tumor activity was assessed using luciferase-expressing RPMI8226 cells in NSG mice in a model of disseminated disease. A single dose of anti-CD38 DAR-T cells or relevant control cells was administered and tumor burden was assessed weekly using bioluminescence imaging. Results: An anti-CD38 DAR gene was efficiently integrated into TRAC locus of T cells from healthy donors by one step knock out/knock in (KOKI) methodology with high efficiency (~40-80% CD38 DAR expression and ~90% TCR knock out). Following a CD3-depletion step, the TCR-positive T cells were less 1%. When co-cultured with CD38-positive tumor cells, anti-CD38 DAR T cells killed as effectively as retroviral anti-CD38 CAR-T cells with similar cytokine secretion profiles while no cytotoxicity was observed against CD38-negative cancer cells. Moreover, in vivo DAR-T cells showed better killing activity against multiple myeloma cell lines than CAR-T cell with anti-CD38 4-1BB/CD3zeta DAR demonstrating the best anti-tumor activity in an NSG mouse model. The anti-CD38 DAR-T cells with 41BB/CD3 zeta internal signals have been selected for clinical development. Conclusions: All tested anti-CD38 DAR-T cells exhibited potent in vitro and in vivo anti-tumor activity. Direct comparison of three different cytoplasmic signaling compositions of the DAR allowed for selection of the most potent construct, namely the anti-CD38 DAR utilizing 4-1BB and CD3zeta signaling domains. Based on these data, further development of CD38 DAR-T therapy for hematological malignancies is warranted. GMP manufacturing of the allogeneic anti-CD38 DAR-T cells has been initiated. Disclosures Ding: Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents &amp; Royalties. Gray:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents &amp; Royalties. Krapf:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Zhang:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents &amp; Royalties. Zhang:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Deng:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Wei:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Knight:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Zeldis:Sorrento Therapeutics Inc: Employment, Equity Ownership. Kaufmann:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents &amp; Royalties. Ji:Sorrento Therapeutics Inc: Employment, Equity Ownership, Patents &amp; Royalties; Celularity, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Guo:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents &amp; Royalties.

Development of an Allogeneic Anti-Bcma T Cell Therapy Utilizing a Novel Dimeric Antigen Receptor (DAR) Structure

Background: Multiple myeloma remains an incurable malignancy of plasma cells. Adoptive transfer of chimeric antigen receptor (CAR)-expressing T cells is a promising new therapy for hematologic malignancies. B-cell maturation antigen (BCMA) is a protein that is selectively expressed by B-lineage cells, including Multiple Myeloma (MM) cells, and represents a suitable target for T cell therapy. We have developed an allogeneic T cell therapy approach utilizing genetic engineering of donor-derived T cells to express an anti-BCMA Dimeric Antigen Receptor (DAR) using a proprietary non-viral vector Knock out/knock in (KOKI) technology. Preclinical data demonstrate potent anti-tumor activity both vitro and in vivo against BCMA-expressing MM cell lines. Methods: Anti-BCMA DAR-T cells were generated through genetic engineering of T cells derived from healthy donors by inserting the anti-BCMA DAR construct into the TRAC gene locus, resulting in loss of endogenous TCR expression while expressing the DAR. Distinct DAR constructs were utilized differing only in their intracellular signaling components, namely combinations of 4-1BB, CD28, and CD3zeta. The anti-BCMA DAR-T cells were expanded and purified for subsequent preclinical studies. Using in vitro assays, the different anti-BCMA DAR-T cells were evaluated against multiple myeloma cell lines for specific cytotoxicity as well as stimulus-induced cytokine secretion and cell expansion. The in vivo anti-tumor activity was assessed using luciferase-expressing RPMI8226 cells in NSG mice in a model of disseminated disease. A single dose of anti-BCMA DAR-T cells or relevant control cells was administered, and tumor burden was assessed weekly using bioluminescence imaging. Results: After purification, the anti-BCMA DAR T cells population contained less than 1% TCR-expressing ab T cells. The DAR-positive T cell population was between 20-50%. All anti-BCMA DAR-T cells exhibited BCMA-specific activation, including cytokine production, proliferation, cytotoxicity, and in vivo tumor eradication. The DAR-T cells using a third generation signaling configuration containing components from 4-1BB, CD28 and CD3zeta signaling domains performed best overall. Conclusions: All tested anti-BCMA DAR-T cells exhibited effective anti-tumor activity. Direct comparison of different cytoplasmic signaling compositions of the DAR allowed for selection of the most potent construct, namely the anti-BCMA DAR utilizing a 3rd generation signaling domain configuration. Based on these data, further development of anti-BCMA DAR-T therapy for hematological malignancies is warranted. These allogeneic abTCR-negative anti-BCMA DAR-T cells have been selected for clinical development. Disclosures Ding: Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents &amp; Royalties. Gray:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents &amp; Royalties. Zhang:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Zhang:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents &amp; Royalties. Cao:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents &amp; Royalties. Krapf:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Deng:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Wei:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Zeldis:Sorrento Therapeutics Inc: Employment, Equity Ownership. Knight:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Kaufmann:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents &amp; Royalties. Ji:Sorrento Therapeutics Inc: Employment, Equity Ownership, Patents &amp; Royalties; Celularity, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Guo:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents &amp; Royalties.

CT053, Anti-BCMA CAR T-cell therapy for Relapsed/Refractory Multiple Myeloma: Proof of Concept results from a Phase I Study

B-cell maturation antigen (BCMA)-specific chimeric antigen receptor (CAR) T cells (CT053) are autologous T cells genetically modified with a second-generation CAR incorporating a fully human anti-BCMA single chain fragment variant, a 4-1BB co-stimulatory domain and a CD3-zeta signaling domain. CT053 was studied in a single-arm, open-label, 3-site phase I study (NCT03716856, NCT03302403, and NCT03380039).

T cell antigen discovery using signaling and antigen presenting bifunctional receptors (SABRs)

Checkpoint inhibitors, cancer vaccines, and adoptive cell therapy exploit T cell mediated immune responses to cancers. Discovering the exact antigens targeted by T cell responses is important for their efficacy. Antigen discovery for ‘orphan’ T cells or TCRs has been a challenging prospect due to high number of possible pMHC specificities. Several current approaches to decipher antigen specificities require prior knowledge of antigen sequences, are unable to scale up, or require production of soluble TCRs. To overcome these drawbacks, we have developed chimeric receptors called Signaling and Antigen-presenting Bifunctional Receptors (SABRs) that allow identification of antigen-presenting cells. SABRs present display pMHC on their extracellular domain, which is recognized by an orphan TCR. Upon recognition, SABRs initiate signaling in the presenting cell using a CD3zeta signaling domain. We transduced reporter cells with SABRs presenting HLA-A2-restricted epitopes from MelanA and NY-ESO-1, and co-incubated them with target cells expressing their cognate TCRs, which resulted in signal transduction only upon correct pMHC-TCR pairing, allowing the presenting cells to express GFP. Second, we showed that SABRs displaying independently expressed peptide and MHC could function similarly. These receptors could present pulsed peptides or endogenously expressed proteins, allowing the uncoupling of peptide and MHC, while retaining their signaling capability. We are currently testing the use of SABR-based antigen libraries to identify novel antigenic specificities targeted by T cells in cancers, infectious diseases, and autoimmune diseases.

Chimeric Antigen Receptor T Cells Specific for CLL-1 for Treatment of Acute Myeloid Leukemia

Chimeric antigen receptor (CAR) T cells are a novel therapeutic approach which have shown good clinical outcomes in patients receiving CD19 CAR T cells for B cell acute lymphoblastic leukemia. CAR T cells are made to express a CAR that recognizes a specific surface antigen on a cell upon which they can then exert cytotoxic effects. We aim to extend the success of this therapy to acute myeloid leukemia (AML), a disease with generally poor clinical outcomes. However, due to the genetic heterogeneity characteristic of AML and the limited number of distinctive tumor markers, it has been difficult to find effective targets for CAR T cells on AML.C-type lectin like molecule-1 (CLL-1), also known as CD371, is a transmembrane glycoprotein that is expressed on about 90% of AML patient samples. CLL-1 may function as an inhibitory signaling receptor, as it contains an intracellular immunoreceptor tyrosine based inhibitory motif (ITIM). CLL-1 is primarily expressed on myeloid lineage cells in the bone marrow and in peripheral blood. While CLL-1 has been shown to be expressed on some granulocytes in the spleen, it is not reported to be expressed in non-hematopoietic tissues or on hematopoietic stem cells, which make CLL-1 a potential therapeutic target for AML.We generated two types of CLL-1 CARs, termed A and B, by using two different single chain variable fragments (scFvs) recognizing CLL-1. We used second generation CARs containing the scFvs, CD8 hinge and transmembrane domain, 4-1BB co-stimulatory domain, and CD3 zeta signaling domains. Using a lentiviral vector, we transferred the CAR gene into healthy donor human T cells and detected CAR expression by flow cytometry. We then tested the specific cytotoxic effects of CLL-1 CART-A and B on a CLL-1-expressing AML cell line, U937, by conducting a 4-hour chromium release assay. We found that both CAR T cells exhibited a dose-dependent killing of U937 (CLL-1 positive), while the untransduced (UTD) T cells had no cytotoxic effect (Figure 1A). We also found that U937 induces degranulation of CLL-1 CAR T cells as measured by CD107a expression by flow cytometry, while Ramos, a CLL-1 negative cell line, does not (Figure 1B).We then proceeded to investigate the in vivo efficacy of the CAR T cells. We injected NOD/SCID/IL2RG-null (NSG) mice with 1 x 106 THP-1 cells, a CLL-1 positive cell line. We confirmed engraftment by bioluminescent imaging (BLI) after 7 days and then injected 4 x 106 UTD, CLL-1 CART-A or CLL-1 CART-B. Surprisingly, only one of the CAR constructs, CLL-1 CART-A, showed significant activity in vivo, although both CARs had shown comparable activity in vitro. CLL-1 CART-A treated mice had delayed tumor progression and significantly increased length of survival (85 days vs. 63 days, p = 0.0021) compared to mice injected with UTD (Figure 1C and D). While CLL-1 CART-B treated mice also exhibited slower tumor growth and a trend towards better survival (72 days vs. 63 days, p=0.0547) this was not statistically significant. Post-mortem analysis showed that human T cells that continued to express CAR were present in the tumor, bone marrow and spleen of mice treated with CLL-1 CART-A only, while the UTD and CLL-1 CART-B treated mice showed tumor in all organs and no T cells.In summary, we show that CLL-1 CAR T cells can selectively eliminate CLL-1 positive target cells in vitro and in vivo, albeit with different degrees of efficacy modulated by the scFv. Studies are ongoing to investigate the mechanism behind the differential activity of these CAR constructs and to increase the long-term antitumor efficacy. Our results demonstrate that targeting CLL-1 using CAR T cell therapy holds promise for the treatment of AML. [Display omitted] DisclosuresCooper:WUGEN: Consultancy, Equity Ownership.

Fully Human Bcma Targeted Chimeric Antigen Receptor T Cells Administered in a Defined Composition Demonstrate Potency at Low Doses in Advanced Stage High Risk Multiple Myeloma

Fully Human Bcma Targeted Chimeric Antigen Receptor T Cells Administered in a Defined Composition Demonstrate Potency at Low Doses in Advanced Stage High Risk Multiple Myeloma

Bioengineered Autologous Dendritic Cells Enhance CAR T Cell Cytotoxicity By Providing Cytokine Stimulation and Intratumoral Dendritic Cells

▪Background: The combination of antigen recognition, costimulatory ligands, and dendritic cells (DC)-derived cytokines (IL-12 or type I IFNs) stimulate T cells upon antigen presentation of DC. Chimeric antigen receptor (CAR) T cells induce anti-tumor cytotoxicity independent of DC by employing antigen recognition portion (single chain variable fragment)/CD3zeta and costimulatory signaling domain. However, the clinically available CARs are not engineered to provide DC-derived cytokine stimulation to T cells. This deficiency may prevent the CAR T cells from developing optimal effector functions, surviving, and forming a responsive memory T cell population. DC can enhance CAR T cell functionality by producing T cell stimulating cytokines. Intratumoral DC, marked by the expression of CD141/CLEC9A, play a critical role in recruiting T cells into the intratumoral area and inducing T cell cytotoxicity against the tumor. IRF8 is an essential transcription factor in developing intratumoral DCs. A co-stimulatory protein, 4-1BB is expressed on activated T cells and is a part of a CAR construct. 4-1BB has been suggested to stimulate IRF8 through the NF-kB signaling and could participate in the generation of intratumoral DC. Therefore, we hypothesized that autologous DCs transduced with 4-1BB CAR would enhance the efficacy of anti-CD33 CAR T cell therapy against acute myelogenous leukemia (AML) by providing DC-derived cytokines and recruiting CAR T cells in bone marrow microenvironment.Methods: We sorted bone marrow CD34+ progenitors and T cells. Cells were transduced with an anti-CD33 41BBz CAR lentivector (pCCL-HP67.6-4-1BB-CD3z). We sorted transduced T (CAR T), and CD34+ progenitors three days after transduction. While expanding transduced CAR T cells further, we induced the differentiation of transduced CD34+ cells to DC (CAR-DC) in vitro by incubating cells with Flt3L/GM-CSF/IL-4 and AML cell lysate. After an additional four days of culture, we analyzed CAR-DC using flow cytometry. We co-cultured a human AML cell line, Kasumi-1 cells with CAR T +/- CAR-DC (E/T ratio=1), or mock control, and quantified cell death in different CAR T to Kasumi-1 ratios (10, 5, and 2) using CytoTox 96 NonRadioactive Cytotoxicity Assay and Annexin V. We also utilized multiplex cytokine immunoassays to quantify cytokine production. For in vivo studies, we injected luciferase-GFP tagged Kasumi-1 cells (10X106) into NSG mice, followed by injection of CAR T (5X105) +/- CAR-DC (1.5X105) or control T cells (5X105). We monitored the NSG mice using serial bioluminescence imaging and compared the survival of each group.Results: On phenotypic analysis using flow cytometry, we found that frequencies of cells expressing CD141/CLEC9A+ were significantly higher in CAR-DC vs. control DC (35.2 +/- 4.1 % vs. 9.0 +/- 1.7 % of HLA-DR+ cells), which suggest 4-1BB activation induce CD34+ progenitors to intratumoral DCs. The cytotoxicity assay showed 63.2 +/- 0.6 % Kasumi-1 death with CAR T/CAR-DC compared to 46.5 +/- 3.5 % with CAR T cells alone. CAR T/CAR-DC also demonstrated more Annexin V positive Kasumi-1 cells compared to CAR T and control T cells (78.4 +/- 5.1 % vs 39.9 +/- 7.7 % vs 17.6 +/- 2.2 %). These cytotoxicity assays demonstrated that CAR-DC enhanced the anti-Kasumi-1 cytotoxicity of anti-CD33 CAR T cells. CAR T cells co-cultured with CAR-DC produced a two-fold higher IFN-gamma and TNF-alpha than CAR T cells alone (p<0.01). The IFN-gamma and TNF-alpha production increases in correlation with the counts of CAR T cells. However, CAR T/CAR-DC group produced a four-fold higher IL-12 throughout different E/T ratios compared to CAR T alone group (p<0.01), which suggest DCs are the major source of IL-12 production and CAR T cells produce a higher level of IFN-gamma and TNF-alpha in response to DCs. In vivo NSG mice experiments demonstrated that CAR T/CAR-DC group had increased survival (p<0.01) and decreased AML burden than CAR T alone group.Conclusions: Our data show that 1) in vitro differentiation of DCs with 4-1BB stimulation increases intratumoral CD141/CLEC9A+ DCs, 2) interaction between CAR-DC and CAR T cells enhances cytotoxic cytokine production in response to DC-derived IL-12. These combined effects resulted in improved anti-CD33 CAR T cytotoxicity in vitro and in vivo NSG AML mice model. Our findings implicate the development of a new strategy of CAR T therapy combined to CAR-DC to increase the efficacy of cancer immunotherapy. DisclosuresNo relevant conflicts of interest to declare.

Abstract A62: NKG2D RNA CAR is effective in treating peritoneal carcinomatosis in a mouse model

Abstract Peritoneal carcinomatosis (PC) is widely shown in end-stage neoplastic disease, especially in recurrent colorectal cancer, and has become a significant threat to the overall survival of patients. Since NKG2D ligands are commonly up-regulated on cell surface of carcinomas but rarely present on most healthy tissues, they can be the ideal targets in the treatment of NKG2D ligand-overexpressing colorectal PC. In this study, we developed a chimeric antigen receptor (CAR)-T cell therapy approach by arming human T lymphocytes with mRNA coding a chimeric NKG2D receptor to target NKG2D ligands. We constructed first, second, and third generation CARs specific for NKG2D ligands by linking the extracellular domain of the human NKG2D receptor to the CD3-zeta signaling domain and costimulatory moieties. mRNAs of those chimeric NKG2D CARs were electroporated into human T lymphocytes and the anti-tumor activities of engineered T cells were examined afterwards. The NKG2D CAR expression was detectable for at least 6 days and the modified T cells exhibited efficient cytotoxic immune activity against NKG2D ligand-positive tumor cells, but not NKG2D ligand-negative cells. Multiple infusions of the RNA CAR modified T cells in immunodeficient mice bearing established peritoneal human colorectal tumors led to a significant reduction in the tumor burden. Here, we showed that T cells engineered with NKG2D CAR by RNA electroporation can efficiently control the colorectal peritoneal carcinomatosis in the mouse model. Transfection of mRNA encoding a CAR is an economical way to benefit the test of new CARs and it holds great promise in controlling on-target/off-tumor toxicity and cytokine storms. The possibility of using NKG2D ligand-specific RNA CARs to treat colorectal PC warrants further investigation. Citation Format: Zhendong Li, Shijun Zha, Shu Wang. NKG2D RNA CAR is effective in treating peritoneal carcinomatosis in a mouse model [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A62.