Allogeneic CAR Research Articles

Phase 1 results with anti-CD19 allogeneic CAR T ALLO-501/501A in relapsed/refractory large B-cell lymphoma (r/r LBCL).

2517 Background: Approximately 50% to 60% of pts with r/r LBCL will either not achieve a complete response (CR) or will relapse with current treatments and require additional therapy. Autologous anti-CD19 chimeric antigen receptor T (CAR T) therapies have revolutionized the care of patients whose disease progresses following standard therapies, but patient-specific manufacturing processes and long wait times to treatment present challenges for their broad clinical use. Allogeneic (healthy-donor-derived), off-the-shelf, anti-CD19 (CAR T) treatment promises broader and more rapid access to one-dose treatment with curative intent. Initial phase 1 data for the anti-CD19 AlloCAR T cell product ALLO-501 (NCT03939026) and successor, ALLO-501A (NCT04416984), demonstrated a manageable safety profile with no dose-limiting toxicities (DLTs), and efficacy comparable to autologous CAR T therapy in pts with r/r LBCL who were autologous CAR T-naïve. This update provides additional follow up and a focus on participants who received the conditioning regimen and cells made with an optimized manufacturing process currently under study in the first potentially pivotal trial for an allogeneic CAR T product. Methods: In these two multicenter, single-arm, open-label, phase 1 trials, a cohort of autologous CAR T-naïve pts with r/r LBCL underwent 3-day lymphodepletion (LD) with FCA90, fludarabine (F, 30 mg/m2/day), cyclophosphamide (C, 300 mg/m2/day), and ALLO-647 (A [anti-CD 52 mAb] 30 mg/day; total dose: 90 mg) followed by a single dose of ALLO-501 or ALLO-501A produced by the Alloy manufacturing process. Results: As of Jan 26, 2023, 12 pts received the FCA90 LD regimen and CAR T therapy with 100% receiving product per specifications with a median time from enrollment to LD of 3 days. Median follow-up time (actual) was 7.1 months (range: 1.4, 36.0). No DLTs, severe (Gr3+) cytokine-release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), or graft-versus-host disease (GvHD) events occurred. An overall response rate and a CR rate of 66.7% and 58.3%, respectively, were observed. Median DOR was 23.1 months. Among 8 pts with the opportunity to be followed for 6 months, 5 (62.5%) had achieved CR and 4 (50.0%) sustained CR through 6 months. Analyses of vector copy number pharmacokinetics at Days 28 and 56 documented substantial and sustained expansion of CAR T cells. Conclusions: A one-time dose of allogeneic CAR T therapy following LD with FCA90 provided durable responses with a manageable safety profile in patients with r/r/ LBCL comparable to those treated with autologous CAR T cells. This treatment enables rapid access to off-the-shelf treatment with a median time from trial enrollment to treatment of 3 days. These findings support broader evaluation of ALLO-501A in the ongoing, first potentially pivotal phase 2 trial (ALPHA2) of off-the-shelf allogeneic CAR T cells. Clinical trial information: NCT03939026 .

NATHALI‐01: A PHASE 1/2A TRIAL OF UCART20X22, AN ALLOGENEIC DUAL CAR T‐CELL THERAPY FOR PATIENTS WITH RELAPSED/REFRACTORY B‐CELL NON‐HODGKIN LYMPHOMA (NHL)

Introduction: Autologous CAR T-cell therapies have been transformative in the treatment of selected blood cancers. Despite this remarkable success, long term studies on patients treated with CD19 or CD22 CAR T-cells revealed the emergence of relapses often due to antigen loss. The therapeutic options after CAR T-cell relapses are limited, underscoring the urgent need to develop novel therapies to improve current survival rates. In addition, there is a need to develop allogeneic “off-the-shelf” therapies that are readily available at the time of treatment decision and that overcome the development limitations of current autologous approaches. To address these challenges, UCART20x22, the first allogeneic dual CAR T-cell product candidate targeting two well-validated antigens in B-cell malignancies, CD20 and CD22, was generated from normal healthy donor T cells using a bicistronic lentiviral construct to express both CARs. TALEN® gene editing technology was used to inactivate the TRAC and CD52 genes to both minimize Graft-vs-Host Disease and allow for the use of alemtuzumab (an anti-CD52 monoclonal antibody) in the lymphodepleting regimen. UCART20x22 displays strong activity against tumor cell lines with diverse CD20/CD22 antigen combinations, as well as increased activity against cells presenting both targets simultaneously using in vitro cytotoxic and proliferation assays. The activity of UCART20x22 cells persists against tumor cells expressing both antigens (CD20, CD22) or only one. In a pre-clinical in vivo model carrying subcutaneous lymphoma tumors expressing different antigen combinations in a single mouse, UCART20x22 cells provide efficient in vivo clearance of tumor cells expressing one or two antigens in a dose dependent manner. Furthermore, in primary NHL patient samples expressing diverse CD22 and CD20 antigen levels, UCART20x22 displays robust and specific cytotoxic activity and IFN-γ release in all tested combinations. Methods: NatHaLi-01 (NCT05607420) is a Phase1/2a open-label dose-finding and dose-expansion study to evaluate the safety, expansion, persistence, and clinical activity of UCART20x22 in subjects with relapsed or refractory B-Cell NHL. Primary endpoints are safety, tolerability, and determining the MTD/RP2D of UCART20x22. Additional endpoints are anti-lymphoma activity and describing the expansion and trafficking of UCART20x22. Eligibility criteria include age 18‒80y, lymphoma cell expression of either or both CD20 and CD22, and ≥ 2 prior treatment regimens including autologous CD19 CAR T-cell therapy if eligible. After lymphodepletion with FCA (fludarabine 30 mg/m2 × 3d, cyclophosphamide 0.5g/m2 × 3d, alemtuzumab 12 mg on D1, 24 mg on D2, D3), patients will receive a single infusion of UCART20x22 at a flat dose level ([DL]; DL1-50 × 106 cells, DL2-150 × 106 cells, and DL3-450x106 cells). The trial is currently open for enrollment. The research was funded by: Cellectis, S.A. Keywords: aggressive B-cell non-Hodgkin lymphoma, cellular therapies, ongoing trials Conflicts of interests pertinent to the abstract. E. Bachy Honoraria: Roche, Takeda, Kite/Gilead, BMS, Novartis, Pfizer, Incyte, ADC Therapeutics Research funding: Amgen, BMS Other remuneration: Kite/Gilead, BMS, Novartis, Pfizer A. Ramakrishnan Consultant or advisory role Novartis, AztraZeneca, Genmab, Janssen A. Alfonso Consultant or advisory role BMS, Syros, Jazz Pharma, Astellas Honoraria: Novartis, BMS, Abbvie, Jazz Pharma, Janssen Research funding: Astra Zeneca P. A. Riedell Consultant or advisory role AbbVie, Genmab, ADC Therapeutics, Pharmacyclics, Novartis, BMS, Kite/Gilead, Nurix Therapeutics, Nektar Therapeutics, Takeda, Intellia Therapeutics, Sana Biotechnology, BeiGene, Janssen, and CVS Caremark Honoraria: Novartis Research funding: BMS, Kite/Gilead, Novartis, MorphoSys, CRISPR Therapeutics, Calibr, Xencor, Fate Therapeutics, Roche, and Tessa Therapeutics Other remuneration: Kite Pharma G. Cartron Consultant or advisory role Roche, BMS, Abbvie, Onwards therapeutics, MedXcell, MabQi Honoraria: Roche, BMS, Jansen, Gilead, Novartis, Abbvie Educational grants: Roche P. Barba Honoraria: Allogene, Amgen, BMS, Kit/Gilead, Incyte, Jazz Pharmaceuticals, Miltenyi Biomedicine, Nektar Novartis and Pierre Fabre S. Meadows Employment or leadership position: Cellectis, Inc. Stock ownership: Cellectis, Inc. A. LaCroce Employment or leadership position: Cellectis, Inc. Stock ownership: Cellectis, Inc. D. Thomas Employment or leadership position: Cellectis, Inc. Stock ownership: Cellectis, Inc. C. Poirot Employment or leadership position: Cellectis, S.A. Stock ownership: Cellectis, S.A. K. J. Newhall Employment or leadership position: Cellectis, Inc. Stock ownership: Cellectis, Inc. D. J. Lee Employment or leadership position: Cellectis, Inc. Stock ownership: Cellectis, Inc. M. G. Frattini Employment or leadership position: Cellectis, Inc. Stock ownership: Cellectis, Inc. J. Abramson Consultant or advisory role AbbVie, Astra-Zeneca, BeiGene, BMS, Caribou Biosciences, Cellectar, Century Therapeutics, Epizyme, Genentech, Genmab, Incyte, Interius, Janssen, Kite Pharma, Kymera, Lilly, Morphosys, Mustang Bio, Ono Pharma, Regeneron, Takeda

Multicenter experience with radiotherapy for relapse after chimeric antigen receptor T cell therapy in non‐Hodgkin lymphoma

Introduction: Approximately half of patients relapse following chimeric antigen receptor T-cell therapies (CAR T), a challenging clinical scenario without established treatment paradigms. Salvage radiotherapy (SRT) may be an important strategy for this population with rapidly growing, often chemotherapy-refractory disease. Limited data are available regarding SRT utilization and outcomes. Methods: We reviewed all patients with Non-Hodgkin Lymphoma treated with SRT for any intent post CAR T from 2016 to 2022 at four US cancer centers. Comprehensive SRT was defined as including all sites with PET avidity > liver max. Failure sites were classified as pre-existing (present pre SRT) versus new, and as in field, marginal (within 1 cm of), or distant with respect to SRT prescription dose region. Toxicities were graded by CTCAE v5.0. Event free and overall survival (EFS/OS) were measured from SRT start by Kaplan Meier. EFS was defined as freedom from progression, initiation of subsequent therapy, or death. Clinicodemographic associations with outcomes were assessed by Cox univariate proportional hazards. Results: 121 patients with diffuse large B cell lymphoma (DLBCL; n = 110), mantle cell lymphoma (n = 8), and primary mediastinal B cell lymphoma (n = 3) were included. Patients received axicabtagene (n = 62), tisagenlecleucel (n = 27), lisocabtagene (n = 17), experimental CAR T (n = 12), and brexucabtagene (n = 3). For 49%, SRT was the first post-CAR T therapy. SRT indications included palliation (n = 72), consolidation (n = 5), bridging to allogeneic transplant (allo; n = 5) or 2nd CAR T (n = 3), and other (n = 33). At SRT, 29 (24%) had localized disease of which 28 were treated comprehensively; overall 37% of SRT was comprehensive. Median dose was 30 Gy (4–50.4). 27% received concurrent systemic therapy. SRT was well tolerated: 8% (n = 10) had ≥ grade 2 (G2) acute adverse events (G3 = 1, no G4/5). Objective response rate was 74% (41% complete) within the SRT field and 38% (22% complete), overall. With a median follow up of 6 mo, 78 patients (64%) progressed. First failure sites were 64% pre-existing/mixed and 38% in field/marginal. For DLBCL patients, median EFS was 4.2 mo in limited stage and 1.2 mo in advanced (p < 0.001). Median OS was 59.6 mo and 3.4 mo in localized and advanced DLBCL relapse (p < 0.001). Median OS for RT as bridging to allo/CAR T, consolidation, and palliation was unreached, 6.7 mo, and 3.0 mo (p < 0.001). Localized relapse and receipt of comprehensive SRT were associated with improved EFS (p < 0.001) while primary refractory disease to CAR T (p = 0.02), elevated LDH pre SRT (p = 0.02), and SRT for palliation (p = 0.03) or for ≥2ndsalvage post CAR T (0.04) were associated with shorter EFS. Keywords: Aggressive B-cell non-Hodgkin lymphoma, Cellular therapies, Radiation Therapy Conflicts of interests pertinent to the abstract. C. Ladbury Research funding: Reflexion G. Shah Consultant or advisory role: ArcellX Research funding: Janssen, Amgen, Beyond Spring, BMS M. D. Jain Consultant or advisory role: Kite/Gilead, Novartis, BMS, MyeloidTx Research funding: Kite/Gilead, Incyte S. Dandapani Research funding: Reflexion, Bayer B. S. Imber Honoraria: GT Medical Technologies Inc.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: MZL is a heterogeneous disease comprising 3 subtypes, extra-nodal MZL of mucosa-associated lymphoid tissue, nodal MZL, and splenic MZL. Treatment of R/R MZL is similar to other indolent B-cell non-Hodgkin lymphoma (B-NHL) subtypes (e.g., follicular lymphoma [FL]), comprising rituximab-based immunochemotherapy regimens that achieve an objective response rate (ORR) of ∼45%–80%. Options after progression on rituximab-based regimens include the BTK inhibitor ibrutinib, which demonstrated an ORR of 46%, but a compete response (CR) rate of only 3%, in its pivotal trial in R/R MZL. Overall, limited options are available after progression on current therapies, and a need remains for more effective treatments for R/R MZL. Odronextamab is a bispecific antibody that binds CD20-expressing B-NHL cells and CD3 on T cells. In the Phase 1 ELM-1 study, odronextamab monotherapy showed antitumor activity and a manageable safety profile in a range of R/R B-NHL subtypes, including MZL (n = 6; ORR 67%) (Bannerji et al. Lancet Haematol, 2022). In the Phase 2 ELM-2 study, odronextamab elicited an ORR of 82% and CR rate of 75% in patients with heavily pretreated R/R FL (Kim, et al. ASH, 2022). These positive data in an indolent B-NHL subtype support evaluation of odronextamab in R/R MZL. Methods: ELM-2 (NCT03888105) is an open-label study of odronextamab in patients with R/R B-NHL comprising 5 disease-specific cohorts: FL, diffuse large B-cell lymphoma, mantle cell lymphoma, MZL, and other B-NHLs. Each cohort will be evaluated separately, and patients recruited from sites across North America, Europe, and Asia-Pacific regions. The MZL cohort is planned to include 78 patients, who will receive IV odronextamab monotherapy until disease progression or other protocol-defined reason for treatment discontinuation. Odronextamab is administered according to a step-up regimen during the first 21-day cycle (C), consisting of 0.7 mg split over C1 Day (D) 1 (0.2 mg) and C1D2 (0.5 mg), 4 mg split over C1D8 and C1D9, and 20 mg split over C1D15 and C1D16. The full 80 mg dose is given QW during C2 to C4, then 160 mg Q2W from C5 onwards. If a patient achieves a CR and has a durable response for ≥9 months after initial determination of the CR, then dosing interval will be decreased from Q2W to Q4W. Patients eligible for the MZL cohort will be ≥18 years of age; relapsed/refractory to ≥2 prior lines of systemic therapy; ECOG performance status ≤1; and have adequate bone marrow and hepatic functions. Patients with prior allogeneic stem cell transplant or CAR T treatment will be excluded. Primary endpoint is ORR (Lugano classification; assessed by independent central review). Key secondary endpoints include ORR (investigator evaluation); CR rate; progression-free survival; overall survival; duration of response; safety; pharmacokinetics; and patient-reported quality of life outcomes. As of January 2023, 19 patients with MZL had enrolled. Encore Abstract—previously submitted to ASCO 2023 and EHA 2023 The research was funded by: Regeneron Pharmaceuticals, Inc. Keywords: indolent non-Hodgkin lymphoma, immunotherapy, ongoing trials Conflicts of interests pertinent to the abstract. T. M. Kim Consultant or advisory role Boryung, AstraZeneca/MedImmune, Takeda, Novartis, Janssen, Hanmi, and Regeneron Pharmaceuticals, Inc Honoraria: Takeda, Janssen, Regeneron Pharmaceuticals, Inc, Samsung Bioepis, and AstraZeneca Research funding: Celgene, Boryung, Boehringer-Ingelheim, BMS, Bayer, AstraZeneca/MedImmune, Takeda, Roche/Genentech, Novartis, Janssen, Hanmi, Genmab, Merck Serono, Merck Sharp & Dohme, Regeneron Pharmaceuticals, Inc, and Sanofi M. Taszner Consultant or advisory role Roche and Takeda Honoraria: Roche, Takeda, Novartis, and Servier Educational grants: Roche A. Chaudhry Employment or leadership position: Regeneron Pharmaceuticals, Inc Stock ownership: Regeneron Pharmaceuticals, Inc M. Ufkin Employment or leadership position: Regeneron Pharmaceuticals, Inc Stock ownership: Regeneron Pharmaceuticals, Inc A. Uppala Employment or leadership position: Regeneron Pharmaceuticals, Inc Stock ownership: Regeneron Pharmaceuticals, Inc A. Sabir Employment or leadership position: Regeneron Pharmaceuticals, Inc Stock ownership: Regeneron Pharmaceuticals, Inc H. Mohamed Employment or leadership position: Regeneron Pharmaceuticals, Inc Stock ownership: Regeneron Pharmaceuticals, Inc S. Ambati Employment or leadership position: Regeneron Pharmaceuticals, Inc Stock ownership: Regeneron Pharmaceuticals, Inc E. Bachy Consultant or advisory role Roche, Gilead, ADC Therapeutics, Takeda, Novartis, and Incyte Honoraria: Kite, Gilead, Novartis, Roche, Incyte, Miltenyi Biotech, Takeda, and Sanofi Research funding: Amgen and BMS

First in human data of NKX019, an allogeneic CAR NK for the treatment of relapsed/refractory (R/R) B‐cell malignancies

First in human data of NKX019, an allogeneic CAR NK for the treatment of relapsed/refractory (R/R) B‐cell malignancies

Abstract ND02: CTX112 and CTX131: Next-generation CRISPR/Cas9-engineered allogeneic (allo) CAR T cells incorporating novel edits that increase potency and efficacy in the treatment of lymphoid and solid tumors

Abstract Allo CAR T cells represent an “off-the-shelf” treatment option that addresses the unmet need for immediately available, potentially curative therapies. CRISPR Therapeutics’ first generation allo CAR T cell products, CTX110® (CD19-directed) and CTX130TM (CD70-directed), have shown encouraging efficacy in ongoing clinical trials (NCT04035434, NCT04502446, NCT04438083), including complete and durable responses in B- and T-cell lymphoma and renal cell carcinoma (RCC). However, T cell exhaustion, occurring even prior to CAR T cell clearance by the immune system, can result in diminished efficacy and loss of response, especially in patients with high tumor burden. To address this challenge, we performed a CRISPR screen to discover potency edits to incorporate into next-generation CAR T programs. Herein, we describe two novel allo CAR T cell therapies, CTX112TM and CTX131TM, that incorporate the synergistic potency edits Regnase-1 and TGFBR2. CTX112 (CD19-directed) and CTX131 (CD70-directed) are produced from healthy donor T cells modified ex vivo using CRISPR/Cas9 gene editing. Regnase-1 and TGFBR2 have been added to edits in our first-generation programs: TRAC (T-cell receptor to prevent GvHD), B2M (MHC class I to reduce T-cell mediated rejection), and CD70 (to eliminate fratricide and increase potency), and site-specific CAR insertion at the TRAC locus using an AAV template. These modifications result in next-generation CAR-T therapies that have demonstrated increased potency, prolonged persistence, and sustained antitumor activity. Disruption of Regnase-1 improves functional persistence by increasing cellular expansion potential, correlating with central memory phenotype. TGFBR2 disruption allows CAR T cells to avoid tumor microenvironment suppression. In combination, these edits increase potency at least 10-fold. In addition, long-term maintenance of CM phenotype enables efficient manufacturing. CTX112 and CTX131 demonstrated increased expansion, resistance to exhaustion, and elevated effector cytokine release when tested using in vitro and in vivo models. CTX112 increased survival and tumor regression in CD19+ Nalm6-Luciferase leukemia and Jeko-1 lymphoma models relative to control CAR T cells with only Regnase-1 or TGFBR2 disruption. Similarly, in a “rechallenge model” in which xenografts were serially implanted following a single dose of CAR T cells, CTX131 caused complete regression of NCI-H1975 lung, A498 RCC, and Caki-2 RCC tumors. No evidence of clonal expansion nor cytokine independent growth occurred, suggesting multiplex edited CTX112 and CTX131 cells are safe for clinical use. In summary, CTX112 and CTX131 are the first CAR T programs to incorporate novel Regnase-1 and TGFBR2 edits, with the potential to increase expansion and functional persistence, which may translate clinically to deeper, more durable responses. CTX112 will be developed in patients with B cell malignancies, and CTX131 will be developed in patients with advanced solid tumors, including RCC. Citation Format: Jonathan A. Terrett, Demetrios Kalaitzidis, Mary-Lee Dequeant, Sushant Karnik, Mohammed Ghonime, Changan Guo, Robert Chain, Heidi Heath, Nivedita Jaishankar, Rachel Yuen, Davis Settipane, Zinkal Padalia, Lauren Zakas, Meghna Kuppuraju, Paul Tetteh, Chandirasegaran Massilamany, Brigid McEwan, Glenn Leary, Henia Dar, Daniel Ferulo, Melanie Allen, Kimberley Tipton, Laura Serwer, Thao Nguyen, Melanie Butler-Gauthier, Parin Sripakdeevong, Shashwat D. Nagar, Yin Tang, Hemangi Chaudhari, Nicole Flanagan, Elaine Huang, Shashwant Phuyal, Elizabeth Koch, Andrew Dunn, Erisa Sula, Jacob Waldman, Cristian Loaiza, Maria Lei Zhang, Erin Thorstensen, Keith Steiger, Katie Schum, Kayla Urbaez, Mark Benton, Anna Ma, Sarah Cohen, Annie Weaver, Christopher Finch, Phuong Khanh Morrow. CTX112 and CTX131: Next-generation CRISPR/Cas9-engineered allogeneic (allo) CAR T cells incorporating novel edits that increase potency and efficacy in the treatment of lymphoid and solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr ND02.

Abstract CT011: A phase 1 multicenter study (TRAVERSE) evaluating the safety and efficacy of ALLO-316 following conditioning regimen in pts with advanced or metastatic clear cell renal cell carcinoma (ccRCC)

Abstract Background: While doublet therapies provide important first-line treatment options in metastatic renal cell carcinoma (mRCC), novel and mechanistically distinct agents are needed for pts who are not cured by/intolerant of such therapies. Due to the high incidence (~80%) of CD70 antigen expression in primary and mRCC, yet limited expression in normal tissue, ccRCC is an attractive proof-of-concept tumor for CD70 directed allogeneic CAR T. TRAVERSE (NCT04696731), a first-in-human trial, seeks to identify a maximum tolerated dose (MTD) of ALLO-316 after conditioning with fludarabine/cyclophosphamide with/without ALLO-647 in pts with advanced or metastatic ccRCC. ALLO-316 is an anti-CD70 allogeneic CAR T cell product that utilizes TALEN® gene editing to knock out TCRα constant gene to reduce the risk of graft-versus-host disease (GvHD) and knock out CD52 gene to permit use of ALLO-647 (a humanized anti-CD52 mAb) to selectively deplete host T cells without affecting allogeneic CAR T cells. Methods: This multicenter, single-arm, open-label, 3+3 dose-escalation trial enrolls adults with advanced or metastatic ccRCC and ≥1 measurable lesion and ECOG Performance Status 0 or 1. Prior treatment with an immune checkpoint inhibitor and a vascular endothelial growth-factor targeted therapy was required, with evidence of progression on/after treatment or discontinuation due to toxicity. ALLO-316 is administered at escalating doses (40 - 240 × 106 allogeneic CAR+ cells IV) on Day 0 after conditioning. The primary endpoint is a target incidence rate for dose-limiting toxicities (DLTs) &amp;lt;33% in the first 28 days after infusion of ALLO-316. Results: By 12/3/2022, 18 pts with ccRCC (median age: 63 yrs; 82% male) were enrolled; all (100%) 17 pts who received ALLO-316, had metastatic disease with 3 lines (median) of prior therapy. Eleven (65%) of these pts experienced CRS, all low Gr except one (6%) Gr 3. No ICANS or GVHD was observed. One (6%) DLT (elevated LFT) was observed and required dose expansion. MTD has not yet been reached. Three pts achieved best overall response of PR at all time points with two PRs confirmed at subsequent visits; ORR = 12% and disease control rate (DCR) = 71%. In pts with confirmed CD70+ tumors (n=9), confirmed ORR = 22% (unconfirmed ORR = 33%) and DCR = 100%. High CAR T cell expansion was observed in peripheral blood (median Cmax &amp;gt; 35,000 copies/μg) and high VCN in available tumor aspirates (n=3). Conclusions: ALLO-316, an allogeneic CAR T cell product targeting CD70 in advanced mRCC, is demonstrating encouraging antitumor activity and a manageable safety profile. A single administration of ALLO-316 could be an effective treatment for pts with CD70+ solid tumors, including RCC, and hematologic malignancies. The MTD for ALLO-316 in TRAVERSE will support Phase 2 trial design. Enrollment of pts with CD70+ tumors is ongoing. Citation Format: Samer Srour, Ritesh Kotecha, Brendan Curti, Jad Chahoud, Alexandra Drakaki, Lily Tang, Lovely Goyal, Sacha Prashad, Victoria Szenes, Kevin Norwood, Sumanta Pal. A phase 1 multicenter study (TRAVERSE) evaluating the safety and efficacy of ALLO-316 following conditioning regimen in pts with advanced or metastatic clear cell renal cell carcinoma (ccRCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT011.

Abstract 899: Deciphering the benefits of variable delivery routes and molecular armoring to enhance efficacy of MUC1-CAR T-cells in targeting triple-negative breast cancer

Abstract As immune-therapies continue to develop for solid tumors, the tumor microenviroment (TME) poses many challenges that CAR T-cells need to overcome for efficient tumor cell clearance. Tumor immune evasion mechanisms, presence of immunosuppressive factors and cellular resistance to tumor infiltration are key mechanisms that the TME uses to reduce efficacy of T-cells against the tumor. Therapeutic options for triple negative breast cancer (TNBC) remain limited to date despite it being the most aggressive subtype of breast cancers and carrying the poorest prognosis. Tumor-associated MUC1 antigen is overexpressed in a large number of TNBC patients offering an effective discriminatory target for CAR T-cell therapy. Here, we present innovative strategies exploring alternative delivery routes and purposeful armoring of CAR T-cells to boost efficiency while preserving safety. To maintain anti-tumor CAR T-cell cytotoxic and proliferation in the hostile TME of solid tumors, we developed state-of-the-art multiplexed gene editing using high precision TALEN® technology. With that aim, we armored allogeneic CAR T-cells with specific attributes to release immune inflammatory cytokines and protect from inhibitory effects of the TGFB1 and PD1 pathways. A combination of strategies using allogeneic CAR T-cells with diverse attributes were evaluated against TNBC tumors in various in vivo pre-clinical models. Local CAR T-delivery resulted in effective control of tumor growth and robust antigen specific recognition. Furthermore, we assessed how the delivery route contributes to CAR-T cell distribution. CAR T-cell activity could also be increased systemically upon tumor-specific release of immunostimulatory cytokines without compromising safety. Our results demonstrate not only superior activity of armored MUC1-CAR T-cells resulting in tumor clearance but also in the recovery of normal glands. Thus, innovative strategies can be used to allow CAR T-cell efficiency in the hostile tumor microenvironment while preserving safety. Overall, our pre-clinical results underline the capability of armored allogenic MUC1-CAR T-cells to excel in the immune suppressive tumor micro-environment, suggesting that allogenic MUC1-CAR T-cell therapy could be an effective option in treating relapsed/refractory TNBC patients with limited therapeutic options. Citation Format: Piril Erler, Hana Cho, Jordan Skinner, Laurent Poirot, Beatriz Aranda-Orgilles. Deciphering the benefits of variable delivery routes and molecular armoring to enhance efficacy of MUC1-CAR T-cells in targeting triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 899.

Abstract 4091: Engineered hypoimmune CAR T cells provide lasting tumor control in immunocompetent allogeneic humanized mice even with re-challenge

Abstract Off-the-shelf CAR T cells potentially offer advantages over autologous strategies such as ease of manufacturing, quality control, off-the-shelf availability, and lack of T cell dysfunction, as well as the ability to generate a more consistent CAR T product from healthy T cells. However, the vigorous host-versus-graft immune response against histoincompatible T cells prevents expansion and persistence of allogeneic CAR T cells and mitigates the efficacy of this approach. A major challenge is that, while HLA deletion can result in adaptive immune evasion, innate reactivity is enhanced with this approach. CD47 overexpression can block both NK cell and macrophage killing (J Exp Med 2021;218(3):e20200839), and we hypothesized that T cells would lose their immunogenicity when human leukocyte antigen (HLA) class I and II genes are disrupted and CD47 is over-expressed. We describe here the engineering of human immune evasive CAR T cells building on our previously described hypoimmune technology (Nat Biotechnol 2019;37(3):252-258 and Proc Natl Acad Sci U S A 2021;118(28):e2022091118).Human T cells from healthy donors were obtained by leukapheresis. CRISPR/Cas12b technology was used to disrupt the B2M, CIITA, and TCR genes, and lentiviral transduction was used to overexpress CD47 and to express a CD19 CAR to generate hypoimmune (HIP) CD19 CAR T cells. Control T cells were unmanipulated except for overexpression of the CD19 CAR (unmodified).For 3 months persistence studies, allogeneic SGM3 humanized mice were injected with 1 × 106 Luc+ Nalm6 cells and received 7 × 106 control CD19 CAR T cells or HIP CD19 CAR T cells.In the mice treated with either unmodified CD19 CAR T cells and HIP CD19 CAR T cells, tumor control was initially rapidly achieved. However, unmodified CD19 CAR T cells were eventually rejected by the host and the loss of these cells resulted in re-growth of tumor. By contrast, in HIP CD19 CAR T injected mice, tumor control was maintained throughout the study, including following a rechallenge at day 83 with NALM6 cells without further administration of HIP CD19 CAR T cell. Flow cytometry at endpoint from bone marrow and spleen confirmed persistence of HIP CD19 CAR T cells.These findings show that HIP CD19 CAR T cells are immune evasive in allogeneic recipients and data suggest that HIP CD19 CAR T cells are able to persist and maintain efficacy without immunosuppression. Citation Format: Xiaomeng Hu, Pascal Beauchesne, Karl Manner, Corie Gattis, Priscilla Ngo, Rowena De Jesus, Ramya Ankala, Chi Young, Frank Wells, Lindong Weng, Kathy White, William E. Dowdle, Aaron Foster, Terry J. Fry, Sonja Schrepfer. Engineered hypoimmune CAR T cells provide lasting tumor control in immunocompetent allogeneic humanized mice even with re-challenge. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4091.

Cellular Therapies in Chronic Lymphocytic Leukemia and Richter’s Transformation: Recent Developments in Chimeric Antigen Receptor T-Cells, Natural Killer Cells, and Allogeneic Stem Cell Transplant

Cellular therapies can be viewed as both the newest and oldest techniques for treating chronic lymphocytic leukemia (CLL) and Richter's transformation (RT). On one hand, allogeneic hematopoietic stem cell transplantation (alloHSCT) has been available for decades, though its use is diminishing with the increasing availability of effective novel targeted agents, especially in CLL. Among newer techniques, chimeric antigen receptor T-cells (CAR-T) have demonstrated astounding efficacy in several hematologic malignancies, leading to FDA approval and use in clinical practice. However, though CLL is the earliest disease type for which CAR-T were studied, development has been slower and has yet to lead to regulatory approval. Owing partially to its rarity but also due to the aggressive behavior of RT, CAR-T in RT have only been minimally explored. Here, we will focus on the applications of cellular therapies in CLL and RT, specifically reviewing more recent data related to alloHSCT in the novel-agent era and CAR-T cell development in CLL/RT, focusing on safety and efficacy successes and limitations. We will review strategies to improve upon CAR-T efficacy and discuss ongoing trials utilizing CAR-T in CLL/RT, as well as emerging technologies, such as allogeneic CAR-T and natural killer CAR (CAR NK) cells.

Early data indicate safety of allogeneic CAR T cells.

Early data indicate safety of allogeneic CAR T cells.

Allogeneic CAR T Cells Targeting DLL3 Are Efficacious and Safe in Preclinical Models of Small Cell Lung Cancer.

Small cell lung cancer (SCLC) is an aggressive disease with limited treatment options. Delta-like ligand 3 (DLL3) is highly expressed on SCLC and several other types of neuroendocrine cancers, with limited normal tissue RNA expression in brain, pituitary, and testis, making it a promising CAR T-cell target for SCLC and other solid tumor indications. A large panel of anti-DLL3 scFv-based CARs were characterized for both in vitro and in vivo activity. To understand the potential for pituitary and brain toxicity, subcutaneous or intracranial tumors expressing DLL3 were implanted in mice and treated with mouse cross-reactive DLL3 CAR T cells. A subset of CARs demonstrated high sensitivity for targets with low DLL3 density and long-term killing potential in vitro. Infusion of DLL3 CAR T cells led to robust antitumor efficacy, including complete responses, in subcutaneous and systemic SCLC in vivo models. CAR T-cell infiltration into intermediate and posterior pituitary was detected, but no tissue damage in brain or pituitary was observed, and the hormone-secretion function of the pituitary was not ablated. In summary, the preclinical efficacy and safety data presented here support further evaluation of DLL3 CAR T cells as potential clinical candidates for the treatment of SCLC.

Tvt CAR7: Phase 1 Clinical Trial of Base-Edited "Universal” CAR7 T Cells for Paediatric Relapsed/Refractory T-ALL

Background Genome editing can overcome HLA barriers to generate 'off-the-shelf’ CAR T cell therapies. Despite the success of CAR-T cell therapies in B-cell malignancies, the expression of shared T cell antigens has constrained the development of CAR T cells targeting T-cell malignancies, due to T cell fratricide. Targeted base editing using CRISPR guided cytidine deamination mediates highly precise C→U→T conversion which can directly disrupt gene expression without DNA breaks. This Phase I study will investigate the feasibility and safety of base edited (BE) allogeneic CAR T cells against CD7, disrupted for TCR, CD7 and CD52. The cells are used to secure remission ahead of allogeneic stem cell transplantation (allo-SCT). Investigational medicinal product (IMP) Two allogeneic healthy donor derived BE-CAR7 T cell banks were manufactured from steady state apheresis harvests using a semi-automated process under compliant conditions. Cells were activated with an anti-CD3/CD28 reagent (Transact) and electroporated with codon optimised base editor (coBE) mRNA and three single guide RNAs targeting TRBC, CD52 and CD7. Subsequent lentiviral transduction delivered CAR7 (59-62% efficiency) and magnetic bead processing depleted residual TCRαβ+ T cells (residual 0.1-0.3%). Highly efficient disruption of CD7 and CD52 (98-99%) was confirmed by sequencing. Cells were cryopreserved and QC tested by flow cytometry, ddPCR for copy number (3.1-3.6) and replication competent lentivirus was excluded. Anti-leukemic potency was confirmed in vitro and in human:murine chimera experiments. Trial sponsorship & approvals The TvT CAR7 study (ISRCTN15323014) is an open label, single centre, single arm and non-randomised clinical trial. The trial is sponsored by Great Ormond Street Hospital NHS trust and is supported by MRC, Wellcome Trust and NIHR. Clinical trial authorisation was provided by MHRA and ethical approval by a gene therapy advisory committee (GTAC) ahead of site initiation. Study aims and Objectives The study aims to establish the safety and feasibility of BE-CAR7 to induce molecular remission in children with relapsed/refractory (r/r) CD7-positive T-ALL, ahead of a planned allo-SCT. Assessments include incidence of adverse events, duration of remission, disease-free survival and overall survival. Expansion, persistence and elimination of BE-CAR7 cells and immune recovery after allo-SCT are monitored. Eligibility, treatment and recruitment The study opened on 14th April 2022 at Great Ormond Street Hospital recruiting children across the UK aged between 6 months and 16 years with r/r CD7+ T-cell malignancies quantifiable in bone marrow (>10-4 by flow or PCR). Exclusions include active uncontrolled infections, pre-existing GvHD or the detection of anti-HLA antibodies against the IMP. Eligible patients receive lymphodepletion with fludarabine (150 mg/sqm), cyclophosphamide (120 mg/kg) and alemtuzumab (1 mg/kg), followed by infusion of 0.2-2.0x106 BE-CAR7 T cells (maximum 5x104/kg TCRαβ T cells). Patients in molecular remission at day +28 proceed to allo-SCT, to remove BE-CAR 7 cells and promote immune reconstitution. The first subject was infused on 4th May 2022. Grade 2 CRS and grade 1 ICANS were observed, but no GvHD. Bone marrow assessment at d+28 revealed morphological remission without count recovery and with PCR-MRD <10-4 and the patient proceeded to reduced intensity allo-SCT as planned. This Phase 1 study aims to treat 10 children in the UK. Conclusion The study has demonstrated the feasibility of manufacturing "off-the-shelf” GMP-compliant base edited CAR7 T cells and is investigating their safety and efficacy against paediatric r/r T-ALL.

Efficient and Specific Multi-Locus Editing of Allogeneic CAR T Cells for Hypoimmunity during Large Scale Manufacture Using Cas12b

Introduction: Allogeneic CAR T cell therapies may offer key advantages over autologous therapies including choice of starting cells, better manufacturing control, and lower cost. However, the manufacture of these cells imposes a high demand on genome engineering specificity and efficiency, given the dual needs for product safety and yield. This challenge is amplified for newer generations of allogeneic CAR T cell designs that may offer improved performance but require more edits to produce, which may increase the potential of unintended genome modification and insufficient yield of fully engineered product. Here we have tested a novel editing platform, Cas12b, for the ability to mediate efficient and specific disruption of three genes (B2M, CIITA, and TRAC) during large scale manufacturing of CAR T cells with immune evasive properties and abrogated graft-vs-host response. We have characterized the resulting cell product for on-target modification efficiency, type and levels of unintended genome modification, and potency for tumor clearance in a Nalm6 humanized mouse model, with the aim of demonstrating suitability for translation to human studies. Methods: Our manufacturing strategy to generate hypoimmune CAR T cells introduces five genetic modifications, using Cas12b for disruption of B2M, CIITA, and TCR expression, and lentiviral transduction to overexpress CD47 and introduce a CD19 CAR. T cells obtained from healthy donors were first transduced with lentiviral vector and then edited simultaneously to disrupt the B2M, CIITA and TRAC genes via electroporation of Cas12b mRNA and appropriate guides. Edited and purified cells were characterized via flow cytometry for efficiency of each genetic modification. Cells were further characterized for knockout levels via next generation sequencing (NGS) as well as vector copy number per diploid genome (VCN). Cells were characterized for viability, proliferation, and efficacy in a Nalm6 mouse model of tumor clearance. An initial assessment of chromosomal translocations was performed by G banding karyotype analysis. To assess editing specificity, each Cas12b guide was evaluated by Cas-OFFinder and an unbiased oligonucleotide duplex capture assay in T cells from multiple donors under conditions of high on-target modification. Candidate off-target sites were then examined for evidence of cleavage via PCR amplification from the manufactured cell product followed by NGS. Results: Our manufacturing process yielded clinically relevant scales of cell product having > 85% B2M knockout, > 90% CIITA knockout, and < 1% of residual TCR expressing cells. VCN levels were < 5. Cell viability and proliferation were minimally impacted by the gene editing process. Cells exhibited robust anti-tumor properties with doses of 7 x 106 hypoimmune CD19 CAR T cells (per animal) providing efficient killing in a Nalm6 mouse model. Initial karyotype analysis identified low levels of translocation between on-target sites with overall translocation levels otherwise not significantly affected compared to unedited cells. Our characterization of Cas12b in edited T cells across multiple donors revealed high specificity, consistent with previous reports (Nat Commun 10 212), with studies to date having yielded no evidence of cleavage at any candidate off-target site. Summary: Cas12b-mediated multi-locus editing of allogeneic CAR T cells during large scale manufacture resulted in a high yield product with potent tumor clearance capabilities. Initial analysis of editing specificity across multiple donors and engineering runs identified no off-target sites, and early karyotype assessments indicated an overall level of chromosomal translocations consistent with prior experiences in the field. In companion in vivo studies, these hypoimmune CD19 CAR T cells have been shown to efficiently evade host immune response (see posters by Hu and Duback). Together, these data demonstrate a profile for potency, genome integrity, and manufacturing yield that supports progressing these cells into human studies.

Single and Combinational Multiplex Base-Edited 'Universal' CAR T Cells in a Humanised Model of Primary CD7+CD33+ AML

Introduction Universal allogeneic donor CAR T cells are being developed as part of a 'deep conditioning' strategy against AML to secure disease clearance ahead of allogeneic stem cell transplantation (Allo-SCT). CD33 is widely expressed on AML, sometimes on the entire leukemia-associated populations, but is also present on healthy precursors; CD7 may be expressed on AML subpopulations and is present on normal T and NK cells, resulting in problematic fratricide effects during CAR T manufacture. Healthy donor derived CAR T cell banks generated using cytidine deamination base-editing and lentiviral transduction with CARs against both CD33 and CD7 circumvent such issues and could offer cell based anti-leukemic therapy alongside preparative effects ahead of allo-SCT. Here we investigated single and combinational BE-CAR T cells against primary human AML in vivo. Methods: Healthy donor lymphocytes were activated by anti-CD3/28 stimulation ahead of electroporation to deliver codon optimised base editor mRNA and guide RNAs against CD7, CD52 and TRBC, and lentiviral transduction to express either an anti-CD7 or anti-CD33 CAR. Magnetic bead depletion removed residual TCRab T cells, and disruption of CD7 ensured cells avoided fratricide. In vitro, antigen specific function was verified in cytotoxicity and cytokine release assays, and in vivo potency was confirmed against luciferase modified Molm14 and Kasumi lines in NSG mice. Responses against primary AML were investigated in a patient derived xenograft model (PDX) from a KIT mutated AML patient, established by serial transplantation, with full expression of CD33 (100% of blasts) and partial expression of CD7 (30% of blasts). Cells were injected into irradiated NSG mice (n=10 per group). On day 24 at a mean leukemic engraftment of 15% human CD45 positive cells in peripheral blood, mice were treated with either 10x106 BE-CAR33, BE-CAR7 or a combination of both or with anti-CD19-CAR-T cells (CAR19) as a control group (in total 20x106 T cells per mouse). AML engraftment, T cell persistence and expression of checkpoint markers were monitored by multicolour flow cytometric analysis. Results: On days 14 and 28 after CAR-T cell infusion, AML cells were readily detected in peripheral blood of control CAR19 animals (42.8% and 90% respectively) and the BE-CAR7 group (27.8% and 67.6%), but were notably reduced in the BE-CAR33 alone group (2.3% and 8.3%) and the BE-CAR33 and BE-CAR7 combination group (2.5% and 3.4%) (Figure 1A). Survival was significantly prolonged for BE-CAR33 and BE-CAR33/BE-CAR7 treated mice compared to the other groups (P<0.001) (Figure 1B). CD7+ AML cells were depleted to a mean of 1% and 4.5% in the BE-CAR33 group, to 0.6% and 0.7% in the BE-CAR33/BE-CAR7 and to 2.6% and 2.9% in the BE-CAR7 group, while CD7+ AML cells increased to 12% and 30% in the CAR19 group at day 14 and 28 after CAR-T cell infusion. Total CD2+CAR-T cell detection in peripheral blood ranged from 1.6 to 9.8% at day 14, 1 to 3.7% at day 28, and 0.8 to 2.7% at day 42, indicating durable persistence over the investigation period. Interestingly, PD-L1 on AML cells was present in all groups while checkpoint markers PD1, GAL9, TIM3, and LAG3 were significantly upregulated in BE-CAR33 cell treated mice, but not BE-CAR7 or CAR19 cell treated mice (P<0.001 for all comparisons). Conclusions: Lentiviral transduction and multiplexed base editing to remove TCR, CD52 and the shared AML/T lineage antigen CD7, enabled the generation of universal donor CAR T cells for combinational use. This data demonstrates robust activity of BE-CAR33 alone and in combination with BE-CAR7 against human CD7+CD33+AML in mice and highlights the importance of strategies that ensure complete targeting of leukemic populations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Functional T Cell Assays Are Predictive of Pre-Clinical Potency to Generate Allogeneic, Hypoimmune CD19 CAR T Cells

Introduction: Allogeneic, hypoimmune (HIP) T cells may provide universal CAR T cells that persist in patients and deliver therapeutic efficacy without the need for immunosuppression to limit graft rejection. Healthy donor T cells possess a spectrum of biological activities that contribute to final CAR T cell functionality. Thus, identifying characteristics that are predictive of CAR T cell potency is essential for generating high-quality, allogeneic, HIP CAR T cells for clinical studies. Here, we define a series of T cell functional assays that differentiate T cell potency that may be used to select high-quality CAR T cell donors for clinical trials. Methods: Human T cells from healthy donors were obtained by leukapheresis and used to generate allogeneic HIP CD19 CAR T cells by disrupting B2M, CIITA, and TCRa using Cas12b gene editing, and lentiviral transduction to overexpress CD47 and to express the CD19 CAR. Generated CAR T cells were evaluated in vitro by repetitive tumor challenge (3x) performed over a 14-day period using the IncuCyte platform. Bulk cytokine analysis and single-cell cytokine levels were evaluated for polyfunctionality (Isoplexis IsoLight). For bulk cytokine profiling, 10 cytokines were measured (GM-CSF, GzmA, GzmB, IFN-g, TNF-a, IL-2, IL-6, IL-17A, IL-1b, IL-1RA). Gene expression profiling was assessed on pre-production, post-CAR manufacturing, and post-serial tumor challenge CD4+ and CD8+ T cells using the nCounter CAR T panel. In vivo efficacy was evaluated using a systemic NALM-6 challenge in immune-deficient NSG mice at HIP CD19 CAR T cell doses of 5e5 and 5e6 cells per animal. Results: We have defined assays that produce single, quantitative indices to enable the classification of CAR T cells as exceptional, good, or poor based on functional performance. To do this, we assessed differences in CAR T cell performance when placed under 'stress' (i.e., low effector to target (E:T) ratios or repetitive tumor challenge) and defined indices that exceed (exceptional), meet (good), or trail (poor) a described median value. For serial tumor challenge, median CAR T cell expansion was 4-fold during each restimulation cycle, with exceptional donors exceeding this value (expansion index >10% above median). Bulk cytokine values produced from CAR T cells ranged from 1.08-2.08 pg/CAR T cell with exceptional donors ascending 10% above the median; while donors scored as exceptional produced a polyfunctionality index >15% via Isoplexis analysis. During in vivo challenge, we observed a median area under the curve (AUC) value of 29 at the high (5e6) CAR T dose, and an AUC value of 10548 at the low (5e5) CAR T dose. Metrics from the serial tumor challenge, bulk cytokine quantification, Isoplexis analysis, and in vivo challenge were compared in an unbiased way to collectively rank CAR T cell donors as exceptional, good, or poor. In addition, gene expression profiling of ranked donors identified that exceptional donors are less activated and have a reduced NK-like signature at the pre-production state. Additionally, exceptional donors have an activated phenotype at a resting CAR T (post-CAR manufacturing) state. CAR T cells skew towards Th1/Tc1 and Th17/Tc17 states at resting (post-CAR manufacturing) and activated (tumor challenge) stages for exceptional donors. Exceptional donors showed a higher amount of mucosal-associated invariant T (MAIT) cells at the pre-production, resting (post-CAR manufacturing), and early activation (tumor challenge) steps. Summary: We have evaluated four assays that could identify CAR T cell variability and segregate CAR T cells into exceptional, good, or poor-performing donors. These assays provide single, quantitative readouts on T cell quality when CAR T cells are placed under 'stress' and may be used to predictively measure T cell quality differences between healthy donors. The information collected across these CAR T productions will be potentially useful for allogeneic donor selection for HIP CAR T manufacturing.

BCMA-Targeted, Hypoimmune Allogeneic CAR T Cells Exhibit Potent Anti-Tumor Activity Together with the Ability to Evade Innate and Adaptive Immune Rejection in Pre-Clinical Tumor Models

BCMA-Targeted, Hypoimmune Allogeneic CAR T Cells Exhibit Potent Anti-Tumor Activity Together with the Ability to Evade Innate and Adaptive Immune Rejection in Pre-Clinical Tumor Models

Combined Genetic Ablation of CD54 and CD58 in CAR Engineered Cytotoxic Lymphocytes Effectively Averts Allogeneic Immune Cell Rejection

Allogeneic cell therapies hold promise to be cost effective with scaled manufacturing for multi-dosing and on-demand off-the-shelf availability. A critical consideration for allogeneic cell products is their ability to persist, maintain function and avoid rejection by the patient's immune system. Genetic knockout (KO) of beta-2-microglobulin (B2M) leads to complete loss of cell-surface human leukocyte antigen (HLA) class I expression and efficiently abrogates CD8+ T-cell reactivity. However, loss of HLA class I triggers NK cell-mediated missing-self recognition and manipulation of B2M must therefore be combined with other immune-modulating strategies to limit recipient NK cell reactivity. We hypothesized that rejection by the patient's immune system can be diminished in primary CAR T cells, iPSC-derived T (iT) and NK (iNK) cells by reverse-engineering common tumor escape mechanisms. The adhesion molecules CD54 and CD58 are both present at the target cell side of the immune synapse, and loss of either of these molecules have previously been reported to elicit immune escape. Here, we show that the combined deletion of CD54 and CD58 in allogeneic immune effector cells makes them resistant to rejection by recipient immune cells through unidirectional reduced synapse formation (Figure 1A). HLA class I down-regulation by B2M silencing in primary T and NK cells triggered potent cytotoxicity by resting allogeneic NK cells. This response was mostly driven by educated NK cells expressing either NKG2A or killer cell immunoglobulin-like receptors (KIR) binding to HLA-E and HLA-C, respectively. However, over-expression of HLA-E or single HLA-C ligands in a K562 screening model only shut down the specific response of the NK cell subset carrying the cognate inhibitory receptor, resulting in only partial resistance to NK cells at the bulk level. Notably, the introduction of HLA-E was particularly detrimental in donors with expanded NKG2C+ NK cell subsets, due to its stimulatory effect through the activating NKG2C receptor. In contrast, combined deletion of CD54 and CD58 in target cells uniquely decreased the response of all tested NK cell subsets and showed universal reduction across NK cell populations from 18 healthy donors (Figure 1B). To delineate the mechanisms behind the increased resistance of target cells carrying these edits, we studied NK cell-target cell interactions at the single cell level by confocal microscopy in microchips. Allogeneic NK cells formed fewer conjugates and failed to form productive immune synapses with CD54-/-CD58-/- target cells, supporting the notion that they are more resistant to NK-cell mediated killing by unidirectional altered adhesion. We next introduced these edits in primary B2M-/- T cells engineered to express a second generation CAR19 from the TRAC locus. Corroborating the K562 screen, CD54-/-CD58-/-B2M-/- CAR-T cells had a selective survival advantage over B2M-/- CAR T cells and HLA-E-over-expressing B2M-/- CAR T cells in conventional mixed lymphocyte reaction (MLR) assays in vitro. Furthermore, we established an in vivo model to probe the effect of different genetic edits on the persistence of allogeneic cell therapy products. To this end, a mixed population of B2M-/- CAR T cells additionally bearing either CD54 and/or CD58 KO, HLA-E over-expression, or no further edits were infused into mice harboring allogeneic healthy donor PBMC. We found that CD54-/-CD58-/-B2M-/- CAR T cells had significantly better in vivo persistence compared to both B2M-/- CAR T cells and HLA-E+B2M-/- CAR T cells in the presence of PBMC from healthy donors (Figure 1B). Although multiplexed editing is feasible in primary CAR T cells, the iPSC platform has an unmatched capacity for homogenously introducing multiple immune-evasion strategies for off-the-shelf cell therapy. Similar to primary CAR T cells, multiplexed edited CD54-/-CD58-/-B2M-/-CIITA-/- iNK cells showed normal growth kinetics and were resistant to rejection by activated allogeneic NK cells in MLR assays. Together, these data demonstrate that reverse-engineering of common tumor escape mechanisms, which render target cells less susceptible to immune synapse formation, is an effective strategy to avert immune rejection of allogeneic CAR T and iPSC-derived CAR NK cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Engineered Hypoimmune CAR T Cells Provide Lasting Tumor Control in Fully Immunocompetent Allogeneic Humanized Mice

Off-the-shelf CAR T cells potentially offer advantages over autologous strategies such as ease of manufacturing, quality control, avoidance of malignant contamination and T cell dysfunction, as well as the ability to generate a more consistent CAR T product from healthy T cells. However, the vigorous host-versus-graft immune response against histoincompatible T cells prevents expansion and persistence of allogeneic CAR T cells and mitigates the efficacy of this approach. A major challenge is that, while HLA deletion can result in adaptive immune evasion, innate reactivity is enhanced with this approach. CD47 overexpression can block both NK cell and macrophage killing (J Exp Med 2021;218(3):e20200839), and we hypothesized that T cells would lose their immunogenicity when human leukocyte antigen (HLA) class I and II genes are disrupted and CD47 is over-expressed. We describe here the engineering of human immune evasive CAR T cells building on our previously described hypoimmune technology (Nat Biotechnol 2019;37(3):252-258 and Proc Natl Acad Sci U S A 2021;118(28):e2022091118). Human T cells from healthy donors were obtained by leukapheresis. CRISPR/Cas12b technology was used to disrupt the B2M, CIITA, and TCR genes, and lentiviral transduction was used to overexpress CD47 and to express a CD19 CAR to generate hypoimmune (HIP) CD19 CAR T cells. Control T cells were unmanipulated except for overexpression of the CD19 CAR. When transplanted into allogeneic humanized mice, hypoimmune CAR T cells evade immune recognition by T cells using ELISPOT analysis. In contrast, transplantation of control CD19 CAR T cells generated from the same human donor resulted in a significant T cell activation (p<0.0001 unpaired t-test). Innate immune cell assays show that CD47 overexpression protects HLA-I/II deficient CD19 CAR T cells from NK cell and macrophage killing in vitro and blocking of CD47 results in killing by innate allogeneic immune cells. For persistence studies, allogeneic humanized mice were injected with 1´106 Luc+ Nalm6 cells and received 7´106 control CD19 CAR T cells or HIP CD19 CAR T cells. However, CD19 CAR T cells are recognized and rejected by the allogeneic immune system of the humanized mice, leading to tumor grow in this group. Flow cytometry on Day 56 from bone marrow and spleen confirms persistence of HIP CD19 CAR T cells, but not control CD19 CAR T cells (see Figure). These findings show that HIP CD19 CAR T cells are functionally immune evasive in allogeneic recipients with cytotoxic anti-tumor capacity and suggest they could provide universal HIP CAR T cells that are able to persist without immunosuppression. Furthermore, these data suggest that HIP CD19 CAR T cells can be used for re-dosing strategies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Dual-Antigen Targeting, Hypoimmune Allogeneic CAR T to Evade Innate and Adaptive Immune Rejection and Overcome Antigen Escape

A Dual-Antigen Targeting, Hypoimmune Allogeneic CAR T to Evade Innate and Adaptive Immune Rejection and Overcome Antigen Escape