Abstract Autologous chimeric antigen receptor (CAR) T cell therapies have revolutionized treatment of hematological malignancies and are showing promising results in autoimmune settings. Despite these successes, widespread accessibility to autologous CAR T-cell therapy is challenged by manufacturing complexities, high cost, lack of on-demand drug product availability and the requirement for intensive conditioning-chemotherapy. Moreover, single antigen targeting often fails to address the complex and heterogenous nature of such diseases. FT839 is an off-the-shelf, uniformly engineered, 13-point edited iPSC-derived CAR T cell therapy that overcomes these challenges, delivering potent and flexible multi-antigen targeting with broad patient accessibility.Mediated by its dual CAR system targeting CD19 and CD38 to eliminate malignant and aberrant immune cells, in preclinical studies FT839 exhibited selective elimination of autoimmune disease drivers like B cells, plasma cells and activated T cells as well as hematological cancer cells of lymphoma/leukemia and myeloma origin with profound potency (p<0.01). The comprehensive targeting strategy and the depth of activity was further extended when combined with the monoclonal antibody (mAb) rituximab or the T cell engager (TCE) epcoritamab facilitated through the expression of high affinity/non-cleavable Fc receptor (hnCD16) and CD3ε fusion receptor (CD3FR), respectively (p<0.01). Furthermore, FT839 features Sword and ShieldTM technology, designed to promote functional persistence by directly targeting and evading alloreactive host immune cells. In allogeneic settings with HLA-mismatched donor PBMCs or primed allogeneic T cells designed to quickly eliminate non-host cells, FT839 showed enhanced persistence (21x, p<0.0001 vs control), limited alloreactive cell expansion (0.25x, p<0.0005 vs control), and durable tumor control (>18x greater cytotoxicity vs control). FT839 has also been engineered with the chemokine receptor CXCR2, and a TGFβ signal redirection receptor to improve trafficking to sites of pathological activity and to counter the suppressive effect of TGFβ, respectively (6.5x greater tissue potency, p<0.005). With Sword and ShieldTM technology, FT839 obviates the need for conditioning chemotherapy, reducing patient burden and maximizing access. Armed with CD19 and CD38 targeting CARs and the ability to functionally combine with approved therapeutic mAbs and TCEs, FT839 selectively and uniquely eliminates heterogeneously populated disease-driving immune cells. Collectively, FT839 is a scalable, cost-effective, and uniform off-the-shelf CAR T-cell therapy for the broad treatment of hematological malignancies and autoimmune diseases. Citation Format: Alex J. Garcia, Shilpi Chandra, Soheila Shirinbak, Mark Jelcic, Brian Groff, Spas Markov, Alma Gutierrez, Angela Gentile, Miguel Meza, Karina Palomares, Carissa Dege, Bjoern Gaertner, Ramzey Abujarour, John Goulding, Tom Lee, Karl-Johan Malmberg, Maksim Mamonkin, Jode Goodridge, Martin Hosking. FT839: A next-generation, off-the-shelf CAR T cell uniquely engineered with a dual CAR system targeting CD19 and CD38 for the treatment of hematological malignancies and autoimmune diseases without conditioning chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4284.

Induced pluripotent stem cell (iPSC)-derived natural killer (iNK) cells offer a promising platform for off-the-shelf immunotherapy against hematological malignancies. NK cell function is dynamically regulated through education driven by inhibitory receptors, including CD94/NKG2A and killer cell immunoglobulin-like receptors (KIR). However, the acquisition of inhibitory receptors in iNK cells and their role during differentiation and education remains poorly defined. In this study, we monitored receptor repertoires, transcriptional states, and functional responses in a range of genetically engineered iNK cell lines. Transcriptional reference mapping placed iNK cells close to cytokine-activated NKG2A+ CD56dim peripheral blood (PB) NK cells. Despite their early differentiation stage, iNK cells displayed a well-developed cytotoxic effector program, which was also reflected in high protein expression of Eomes, granzyme B, and activating receptors DNAM-1 and NKG2D. Acquisition of NKG2A by iNK cells was associated with a more differentiated transcriptional state and superior functional responses against a broad range of targets, including those expressing low to moderate levels of HLA-E, suggesting attenuated inhibitory signaling through NKG2A in iNKs. CRISPR knockout of β2-microglobulin (B2M) in iNK cells revealed that the functional potency of NKG2A+ iNK cells was independent of educating interactions with HLA-E in cis or trans. Finally, CRISPR-mediated ablation of NKG2A led to a spontaneous compensatory surface expression of CD94/NKG2C heterodimers, associated with enhanced IFN-γ production and cytotoxic activity against target cells with forced high expression of single-chain β2m-HLA-E-peptide trimers. Our results indicate an education-independent functional maturation of iNK cells, characterized by potent effector programs coupled with a favorable early-stage transcriptional profile.

Clinical administration of chimeric antigen receptor (CAR) T cell and CAR NK cell therapies rely on conditioning chemotherapy (CCT) to deplete the host immune system, maximize access to homeostatic cytokines, and promote cell expansion and functional persistence. However, CCT also elicits pan-immune cell cytopenia, increases susceptibility to infection and malignancy, and requires costly patient hospitalization and administration of therapy in tertiary healthcare settings. Indeed, the administration of off-the-shelf CAR T cell and CAR NK cell therapies to patients without requiring CCT has the potential to significantly reduce treatment-related toxicities and cost of care, and, importantly, extend patient reach by enabling treatment in primary or outpatient community healthcare settings. To eliminate the need for CCT, we established a unique approach through the cooperative action of a novel alloimmune defense receptor (ADR), a synthetic receptor that targets cell surface expression of 4-1BB (CD137) to eliminate alloreactive immune cells, and the knock-out of CD58 (CD58KO), a unidirectional synapse-stabilizing ligand where disruption attenuates the ability of alloreactive immune cells to properly function. To enable the distinct ability for consistent manufacture and uniform production of an off-the-shelf available, multiplexed-edited CAR T cell, we generated iPSC-derived T cells (iT cells), produced from a clonal master iPSC line engineered to homogeneously express ADR, to be completely deficient for the expression of CD58, and to express a CAR targeting B cell maturation antigen (BCMA) (ADR+/CD58KO CAR iT cells; ADR and CAR surface expression >95%, CD58 not detected). In an MLR assay containing saturated levels of allogeneic peripheral blood mononuclear cells (PBMCs) from an unmatched donor, ADR+/CD58KO CAR iT cells exhibited resistance to host-mediated rejection and uniquely demonstrated expansion in cell number over the duration of the assay, while the co-culture with control CAR iT cells (lacking ADR and with intact CD58) were eliminated. To further increase the stringency of our assessment and generate an environment to promote supraphysiological allogeneic rejection by immune cells, we substituted allogeneic PBMCs with T cells from an unmatched donor that were primed and enriched for alloreactivity (primed-T cells) against iT cells. As observed with allogeneic PBMCs, control CAR iT cells co-cultured with primed-T cells were depleted within a few days and expression of 41BB was detected on a large subset of the primed-T cell population in culture. In contrast, ADR+/CD58KO CAR iT cells durably persisted over the entire culture period, with the primed-T cells in the co-culture significantly reduced in number (p<0.05) and without detectable 41BB expression (not detected vs 47% 41BB+ in control). To confirm ADR+/CD58KO CAR iT cells retain potent anti-tumor efficacy under alloreactive pressure, we performed a rigorous tri-culture MLR study that combined anti-BCMA CAR iT cells with daily-dosing of tumor cells expressing BCMA in the presence of primed-T cells. In the absence of primed-T cells, co-cultures with repeat restimulation of BCMA+ tumor cells were equally eliminated by either CAR iT cells or ADR+/CD58KO CAR iT cells over the course of the assay. In the presence of primed-T cells, the tri-culture assay with control CAR iT cells showed rapid loss of tumor control with associated expansion of 41BB+ primed-T cells. In contrast, ADR+/CD58KO CAR iT cells demonstrated tumor growth inhibition equivalent to the control culture lacking primed-T cells, durably persisted through the end of the assay, and completely suppressed the 41BB+ primed-T cell population. Similar results were observed in allogeneic xenograft tumor models assessing the control of lymphoma and leukemia burden in vivo in the presence of alloreactive PBMCs and T cells. Collectively, we demonstrate the potential of CAR iT cells incorporating the novel combination of ADR-arming and CD58KO to elicit a robust cytotoxic response against diseased cells in the presence of an intact endogenous immune compartment. The effective use of cell-based immunotherapies without requiring administration of CCT to patients facilitates broad clinical application across multiple lines of therapy, including in combination with standard-of-care agents that are used for treatment of patients with newly-diagnosed disease.

Chimeric antigen receptor (CAR) T cell therapies have shown clinical success in treating hematologic malignancies. However, heterogeneous target antigen expression can impair the durability of response. Combining CAR and T cell engagers (TCEs) targeting additional tumor antigens can address tumor heterogeneity and antigen escape. In allogeneic settings, eliminating the T cell receptor (TCR) of the adoptive T cell therapy prevents graft-versus-host disease. However, the absence of TCR leads to loss of surface CD3 expression, preventing cooperative activity with CD3-directed TCEs. We utilized induced pluripotent stem cells (iPSCs) to support the required multiplexed editing, establish a renewable starting material for off-the-shelf manufacture, and create the desired TCR-less CAR+ CD3+ T cells. Here, we illustrate surface expression of a CD3ε fusion receptor (CD3FR) in iPSC-derived CAR T (CAR iT) cells, enabling TCE-mediated targeting of diverse antigens. In vitro and in vivo, CD3FR+ CAR iT cells demonstrated potent cytotoxic response and cooperative activity against mixed tumor lines and multiple antigens. CD3FR+ iT cells were further engineered to secrete TCEs, eliminating the need for extra supplementation with TCEs. Collectively, the data highlight the ability to integrate TCEs with allogeneic CAR iT cells for multi-antigen targeting, overcoming tumor relapse, and supporting off-the-shelf therapy for patient access.

Following autologous chimeric antigen receptor (CAR) T-cell therapy, approximately 50% of multiple myeloma (MM) patients relapse within two years of treatment. Relapses can occur for many reasons, but ultimately it is the result of tumor re-growth caused by the underlying attribute of tumor heterogeneity. FT839 is an induced pluripotent stem cell (iPSC) derived CAR T cell product specifically designed to address tumor heterogeneity while enabling broad patient access. To uniquely address tumor heterogeneity, FT839 was engineered to contain two unique CARs, i) a lineage-antigen CD19 shown to be associated with MM initiating cancer stem cells and ii) an activated state-antigen CD38 broadly associated with MM. To further enable tertiary antigen targeting and synergies with other therapeutic agents, FT839 can also be combined with monoclonal antibodies (mAbs) through a modified high affinity and protease resistant CD16 receptor (hnCD16), and T cell engagers (TCEs) through a novel CD3-chimeric fusion receptor (CD3-CFR). In vitro cytotoxicity assays against MM cell lines RPMI-8226, OPM2, MM1.s and H929 demonstrated durable and potent cell cytotoxicity at low effector:target ratios, as a monotherapy with further deepening of response when combined with mAbs such as daratumumab or sarclisa, or T cell engagers such as teclistamab or talquetamab (exceeding 90% cytotoxicity in each case). These data demonstrate that FT839, via combinatorial dual-CAR, hnCD16, and/or CD3-CFR targeting, can circumvent antigen loss to successfully eliminate MM target cells with diverse and heterogenous antigen expression. FT839 has also been specifically engineered to support functional persistence without the need for intense conditioning chemotherapy through Sword and ShieldTM engineering which confers resistance to potent pre-existing and de novo host immune cell-mediated rejection, at the same time preserving anti-tumor responses. In mixed lymphocyte co-cultures, FT839 maintained functional persistence and prevented expansion of pre-existing alloreactive T cells by 20.7x compared to control. Notably, FT839 demonstrated durable anti-tumor activity over multiple rounds of tumor challenge with repeat exposure to alloreactive T cells primed to specifically target FT839 and create a supraphysiological allogeneic environment. Collectively, FT839 is engineered to eliminate cancer cells with broad and heterogenous antigen expression by a unique dual-CAR system and in combination with standard of care therapeutics, including mAbs and TCEs via hnCD16 and CD3-CFR transgenes, to overcome multiple challenges that have limited autologous CAR T-cell therapies in treating MM. Moreover, as an off-the-shelf CAR T-cell therapy, FT839 is intended for broad and on-demand access without the need for complicated and variable manufacturing processes or intense conditioning chemotherapy.

CD19-targeting chimeric antigen receptor (CAR) T-cell therapies have demonstrated profound clinical efficacy in the treatment of a growing list of autoimmune diseases through their ability to target and reset pathogenic B cell immune compartments. The extension of CAR T-cell therapy to additional autoimmune indications offers substantial therapeutic potential, especially for patients with refractory disease and limited treatment options. Currently approved autologous CAR T-cell therapies face several important limitations including their narrow, single antigen targeting (e.g., CD19) mechanism of action that is unlikely to eliminate all disease-causing immune cell types in complex autoimmune diseases such as multiple sclerosis (MS) and rheumatoid arthritis (RA). Moreover, broad patient access to these therapies is limited due to inconsistencies associated with the manufacturing of heterogeneously edited cells, production costs, the inability to be available on-demand, and the need for intense conditioning chemotherapy which requires hospitalization and treatment at specialized centers to monitor adverse events, including the risk of severe infection. FT839, a next generation off-the-shelf CAR T-cell therapy, is designed to overcome these limitations by expanding CAR T-cell therapy access to a broader pool of autoimmune disease patients. Derived from a clonal, multiplex precision-engineered induced pluripotent stem cell that allows for routine and scaled manufacture, FT839 incorporates novel synthetic functional elements that enable multi-antigen targeting to effectively eliminate a range of pathogenic immune cell types while simultaneously resisting immune rejection in allogeneic patient settings. FT839 co-expresses two distinct CARs: the first targets the B-cell lineage marker CD19, and the second targets the immune activation marker CD38. Combined with genomic deletion of CD38 to avoid fratricide and T-cell receptor to eliminate the risk of graft-versus-host disease, this multi-antigen targeting approach enables selective elimination across a comprehensive range of disease-causing immune cell subsets. FT839 also expresses a high-affinity, non-cleavable CD16 receptor and a chimeric CD3-fusion receptor, enabling synergistic combinations with standard-of-care monoclonal antibodies and clinically approved T-cell engagers to achieve potentially enhanced therapeutic activity and functional versatility through engagement of one or more activating receptors on separate lineage markers. In vitro cytotoxicity assays exhibited CAR-mediated targeting of CD19 and/or CD38, resulting in potent elimination of antigen-expressing target cells, including CD19+ B cells, CD38+ plasma cells, and CD38+ alloreactive immune cells. In vivo, FT839 demonstrated potent and specific elimination of xenografts consisting of CD19+ and CD38+ target cells. Notably, FT839 simultaneously eliminated autoimmune disease driving cell types in vitro, such as B cells, plasma cells, activated Th1 and Th17 CD4+ T cells, activated CD8+ T cells, and inflammatory macrophages (>90% depletion of all CD19+ and CD38+ targets), underscoring the broad suitabilityof FT839 for the control of autoreactive immune subsets. To eliminate the current requirement to administer conditioning chemotherapy alongside CAR T-cell therapy, FT839 incorporates Sword and Shield™ technology engineering with a novel allo-immune defense receptor (ADR), designed to eliminate 4-1BB+ alloreactive immune cells, and genetic disruption of the immune synapse adhesion ligand CD58 (CD58KO) to enable passive evasion from host allogeneic immune cells. In the presence of alloreactive peripheral blood mononuclear cells, FT839 demonstrated sustained cytotoxicity upon serial rechallenge in vitro and maintained tumor growth inhibition and functional persistence in vivo, consistently displaying superior durability and potency compared to controls that lack Sword and Shield™ (p<0.001). These results demonstrate the unique ability of FT839 to functionally persist in an allogeneic and mismatched setting without the need for intensive conditioning chemotherapy. In summary, FT839 enables the simultaneous and selective elimination of multiple disease-driving immune cells without the need for supportive conditioning chemotherapy. Its scalable, cost-effective manufacturing and off-the-shelf delivery supports broad clinical accessibility across a range of autoimmune disease settings.

Introduction: Recently approved autologous chimeric antigen receptor (CAR) T-cell therapies (Abecma® and Carvykti®) have demonstrated clear clinical benefit for patients with relapsed/refractory multiple myeloma (MM) with initial response rates ranging between 73-98%. Unfortunately, many of these patients ultimately relapse, often as the result of antigen shedding and tumor heterogeneity, tumor microenvironment suppression, and poor CAR T-cell functional persistence, highlighting the need for alternative therapies that can simultaneously mitigate and overcome these tumor-intrinsic and -extrinsic challenges. Furthermore, broad patient access of patient- and donor-derived CAR T cells are limited by manufacturing challenges and the use of conditioning chemotherapy. Methods FT836 is an iPSC-derived CAR T cell that uniquely targets the conserved α3 domain of the inducible stress ligands MICA/B, enabling broad recognition of both hematologic and solid tumors. The unique engineered elements of FT836 further enable (i) multi-antigen targeting by antibody-dependent cellular cytotoxicity (ADCC) in combination with the high-affinity non-cleavable CD16a Fc receptor (hnCD16) and therapeutic monoclonal antibodies (e.g. sarclisa and daratumumab), (ii) functional persistence in an allogeneic setting without the reliance on conditioning chemotherapy using dual Sword and ShieldTM engineering, incorporating a synthetic alloimmune defense receptor (ADR) that selectively eliminates 4-1BB+ alloreactive immune cells and genetic deletion of CD58 to avoid recognition by host immune cells, and (iii) improved tumor homing and resistance to immunosuppression via expression of the chemokine receptor CXCR2 and the TGFβ signal redirection receptor, respectively. Results Analysis of MM at both the mRNA and protein level revealed MICA/B and CD38 to be highly enriched with comprehensive disease coverage, as demonstrated by i) the Cancer Cell Line Encyclopedia (median Log2(TPM+1) MICA/B: 4.39 and CD38:5.022), ii) primary multiple myeloma patient samples (MMRF-CoMMpass, Skerget et al. 2024; median Log2(TPM+1) MICA/B: 4.23 and CD38: 7.64), and iii) surface expression analysis of a panel of multiple myeloma cancer cell lines, including RPMI-8226, NCI-H929, OPM2, and MM.1s. In vitro cytotoxicity assays exhibited dose-dependent killing in combination with CD38 antibodies (daratumumab and sarclisa), uniquely enabling multi-antigen targeting through both innate and adaptive immunity (CAR+ADCC). Importantly, unlike NKG2D-mediated cytotoxicity, the presence of soluble MICA/B did not impact the anti-tumor efficacy of FT836. In a xenograft model of solid tumor using RPMI-8226, the tumor burden was effectively controlled by FT836 and the depth of response was extended when combined with daratumumab, underscoring the potential for potent and effective control of MM using FT836 by simultaneously targeting MICA/B and CD38 antigens. In a two-way MLR assay against a panel of HLA-mismatched PBMC donors, FT836 limited the generation of alloreactive immune cells (4x, p<0.0005) and demonstrated significantly enhanced functional persistence (21x, p<0001), displaying the utility of Sword and ShieldTM engineering compared to control. Similarly, in the presence of HLA-mismatched PBMCs previously primed to react to product material to represent a supraphysiological allogeneic environment, FT836 demonstrated improved functional persistence (3x, p<0.01), eliminated reactive allogeneic cells (3x, p<0.05), and maintained potent anti-MICA/B CAR-mediated anti-tumor activity compared to control. Conclusion The data demonstrates that FT836 mediates potent and comprehensive control of MM without the need for intense conditioning chemotherapy, and that its depth of coverage can be uniquely enhanced in combination with therapeutics antibodies. As an off-the-shelf CAR T-cell therapy derived from a multiplex-engineered iPSC master cell bank, FT836 is scalable and cost-effective, with the potential to broadly and effectively treat patients with MM.

Although chimeric antigen receptor (CAR) T cells have demonstrated therapeutic activity in hematopoietic malignancies, tumor heterogeneity has impeded the efficacy of CAR T cells and their extension into successful solid tumor treatment. To address these challenges, induced pluripotent stem cell (iPSC)-derived T (iT) cells are engineered to uniformly express CAR and T cell receptor (TCR), enabling targeting of both surface and intracellular antigens, respectively, along with a high-affinity, non-cleavable variant of CD16a (hnCD16) to support antibody-dependent cellular cytotoxicity (ADCC) when combined with therapeutic antibodies. Co-expression of each antitumor strategy on engineered iT cells enables independent and antigen-specific targeting across a diverse set of liquid and solid tumors. In heterogeneous tumor models, coactivation of these modalities is required for measurable antitumor efficacy, with activation of all three modalities displaying maximal efficacy. These data highlight the therapeutic potential of an off-the-shelf engineered iPSC-derived trimodal T cell expressing CAR, TCR, and hnCD16 to combat difficult-to-treat heterogeneous tumors.

Autologous chimeric antigen receptor (CAR) T-cell therapy has had tremendous success in the treatment of hematological malignancies, yet its clinical application remains hindered by several significant limitations. Major challenges include the high cost, complex manufacturing process, the requirement for intense lymphodepleting chemotherapy prior to infusion, and the limited accessibility and scalability of the therapy. FT839 is an induced pluripotent stem cell (iPSC) derived CAR T cell designed to overcome these limitations and provide potent and versatile therapy against lymphocytic cancers. FT839 is a multiplex-engineered CAR T cell equipped with anti-CD19 and anti-CD38 dual-CAR system to target lineage specific or activated pathological cell subsets, including cells of hematologic malignancies. FT839 also incorporates Sword and ShieldTM technology, the synergistic action of a novel Alloimmune Defense Receptor (ADR), which eliminates 4-1BB+ alloreactive immune cells, and the genetic ablation of CD58 (CD58KO), which limits synapse formation with alloreactive cells to promote functional persistence and evade host alloreactive immune responses, designed to eliminate the need for intensive conditioning chemotherapy. Co-targeting of CD19 and CD38 on B cell acute lymphoblastic leukemia cell line NALM6, both in combination or individually through engineered isogenic lines (CD19+CD38+, CD19-CD38+ and CD19+CD38-) to represent cancer heterogeneity, showed enhanced and durable efficacy (91.6%, 92.8%, & 82.6% vs 52.8%, 0%, & 50%) respectively, when comparing FT839 and CD19 primary CAR T cells at low effector: target ratio. FT839 CAR T cells are also uniquely engineered to express a novel CD3-chimeric fusion receptor (CD3CFR) and a high-affinity, non-cleavable CD16 (hnCD16). These engineered attributes allow for flexible multi-antigen targeting in combination with clinically approved T cell engagers (TCEs) or monoclonal antibodies (mAbs). Indeed, in combination with the CD20-specific mAb rituximab or the CD38-specific mAb daratumumb, FT839 potently eliminated the mantle cell lymphoma cell line Jeko-1 (97.8% CD20+, 54,888 rMFI) and Burkitt's lymphoma cell line RAJI (91.6% CD38+, 101,129 rMFI), respectively, highlighting the flexible and broad multi-antigen targeting of FT839 via CAR and hnCD16 activation. FT839 also demonstrated potent control of NALM-6 xenografts in vivo, as a monotherapy, and with further deepening response in combination with mAb, compared to control. With Sword and ShieldTM engineering (ADR & CD58 KO), FT839 demonstrated resistance to potent pre-existing as well as de-novo generated host vs graft allogeneic immune responses, maintaining functional anti-tumor activity. In a 2-way MLR with HLA-mismatched donor peripheral blood mononuclear cells (PBMCs), FT839 demonstrated enhanced persistence (21x, p<0001) and reduced alloreactive expansion (4x, p<0.0005), compared to control CAR-T cells. FT839 also showed higher resistance (113% of base wells) to pre-existing allogeneic responses (primed allogeneic T cells), as compared to CD58-sufficient, ADR-negative CAR-T cells (2.4% of base wells). Finally, FT839 maintained durable anti-tumor activity against repeat challenges with NALM6 tumor cells 1.17x p=ns) even in the presence of primed allogeneic PBMCs that elicit potent reaction against mismatched cells. Compared to traditional CAR T-cells, FT839 demonstrates versatility in targeting cancer cells via multiple antigen-receptor activation pathways, enabling potent and flexible multi-antigen targeting for the successful treatment of relapsed/refractory B cell lymphomas that is otherwise challenging to treat because of its heterogenous cellular composition. Furthermore, unlike autologous and allogeneic CAR T cells, expression of ADR and deletion of CD58 ensures the functional persistence of FT839, potentially eliminating the need for intense conditioning chemotherapy regimen and ensuring broad and on-demand access for patients with relapsed/refractory B-cell leukemia/lymphoma.

Abstract Immune cell therapy has proven highly effective for the treatment of multiple myeloma (MM). However, key challenges remain that include disease relapse, limited patient access, and inability to effectively combine with existing standard-of-care therapies. Rapid progress in the development of off-the-shelf, multiplexed-engineered, induced pluripotent stem cell (iPSC)-derived cell therapies enables large-scale manufacture of immune cells incorporating multiple novel synthetic controls of cell function to improve cell fitness, enhance cell function, and enable synergistic combination with existing effective therapies such as CD38-targeted antibody (mAb) therapy. We have developed an iPSC-derived chimeric antigen receptor T (CAR-iT) cell therapy that uniquely leverages elements of both adaptive and innate immunity by incorporating a BCMA-targeted CAR (BCMA-CAR) derived from a scFv domain exhibiting high-binding affinity in the low nanomolar range, and a high-affinity, non-cleavable CD16 (hnCD16) Fc receptor to enable antibody-dependent cellular cytotoxicity with mAb therapy. The genetic deletion of CD38 gene was also incorporated to eliminate the possibility for CD38-mediated fratricide, and genetic deletion of TRAC gene was introduced to remove the potential risk of graft-versus-host disease in an allogeneic setting. These CAR iT cells, which were generated from a clonally-derived iPSC line, demonstrated homogenous expression of each genetic edit (>95% BCMA-CAR and hnCD16; <1% CD38 and TCR surface expression). Using a stringent, disseminated xenograft mouse model of multiple myeloma, MM.1S, which was allowed to achieve complete systemic engraftment during the initial 4 days, treatment with BCMA-CAR iT cells demonstrated comparable tumor cell clearance (p=0.0024 vs. vehicle at Day 17) to primary BCMA-CAR T cells (p=0.002 vs. vehicle). In combination with a CD38-targeted mAb to exploit hnCD16 and biallelic CD38 KO, BCMA-CAR iT cells are capable of dual-antigen targeting to address antigen escape and promote durable tumor control. To this end, a single dose of BCMA-CAR iT cells combined with daratumumab exhibited near complete TGI for the duration of the study (p<0.0001 vs. vehicle at Day 37). Together, these studies demonstrate CAR iT cells incorporating a high-avidity BCMA CAR and high-affinity, non-cleavable CD16 Fc receptor can uniquely leverage elements of both adaptive and innate immunity and can be combined with CD38-targeted mAb to potentially outcompete primary BCMA CAR T cells. As these CAR iT cells can be administered off-the-shelf, key challenges associated with current immune cell therapy, such as patient access and inability to synergize with standard-of-care therapies, can be addressed for the treatment of relapsed/refractory MM. Citation Format: John Reiser, Alison O'Connor, Bryan Hancock, Spas Markov, Brian Groff, Alma Gutierrez, Miguel Meza, Mark Jelcik, Yijia Pan, Alex Garcia, Bobby Goulding, Matthew Denholtz, Tom Lee, Ramzey Abujarour, Ryan Bjordahl, Armin Rehm, Raedun Clarke, Jode Goodridge, Bahram Valamehr. High-avidity BCMA CAR and high-affinity, non-cleavable CD16 Fc receptor incorporated in off-the-shelf CAR T cells promote multi-antigen targeting and durable anti-tumor cytotoxicity in the treatment of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3618.

The production of autologous T cells expressing a chimaeric antigen receptor (CAR) is time-consuming, costly and occasionally unsuccessful. T-cell-derived induced pluripotent stem cells (TiPS) are a promising source for the generation of 'off-the-shelf' CAR T cells, but the in vitro differentiation of TiPS often yields T cells with suboptimal features. Here we show that the premature expression of the T-cell receptor (TCR) or a constitutively expressed CAR in TiPS promotes the acquisition of an innate phenotype, which can be averted by disabling the TCR and relying on the CAR to drive differentiation. Delaying CAR expression and calibrating its signalling strength in TiPS enabled the generation of human TCR- CD8αβ+ CAR T cells that perform similarly to CD8αβ+ CAR T cells from peripheral blood, achieving effective tumour control on systemic administration in a mouse model of leukaemia and without causing graft-versus-host disease. Driving T-cell maturation in TiPS in the absence of a TCR by taking advantage of a CAR may facilitate the large-scale development of potent allogeneic CD8αβ+ T cells for a broad range of immunotherapies.