ALBERT

Description: Autologous chimeric antigen receptor (CAR) T cells targeting B-Cell Maturation Antigen (BCMA) have demonstrated promising clinical activity, inducing durable responses in patients with relapsed/refractory multiple myeloma (MM). Development of autologous CAR T therapies is however limited by logistical challenges and the time required for manufacturing, which has to be done for each patient. In addition, manufacturing may not be feasible in some patients. An allogeneic approach that utilizes engineered cells from a healthy donor could potentially expand patient access to these therapies by providing a readily available off-the-shelf product. We have previously described the screening of a library of single chain variable fragments (scFvs) with high affinity to human BCMA and the identification of candidate BCMA CARs with potent antitumor activity. Here we sought to further characterize ALLO-715, our lead allogeneic BCMA CAR T cell product, for its specificity to human BCMA, antitumor efficacy in vitro using a long-term killing assay and in xenograft mouse models with physiologic levels of human IL-7 and IL-15, and suitability for scale-up manufacturing. Allogeneic ALLO-715 CAR T cells were generated by lentiviral transduction with a second generation CAR construct incorporating a novel scFv derived from a fully-human antibody with high affinity to BCMA (KD value ~ 5 nM, determined at 37°C) and featuring a rituximab-driven off-switch. Transduced T cells were then transfected with mRNAs encoding Transcription Activator-Like Effector Nucleases (TALEN®) designed to specifically disrupt the T cell receptor alpha chain and CD52 loci. These modifications result in a cell product with a lower risk of TCR-mediated graft-versus-host disease and resistance to the CD52 antibody alemtuzumab, a lymphodepleting agent. BCMA CAR T cells exhibited robust cell expansion, with low levels of tonic signaling that resulted in minimal differentiation (〉 50% Tscm/Tcm phenotype). In in vitro assays, ALLO-715 CAR T cells displayed potent cytotoxic activity when co-cultured with the target cell lines MM.1S, Molp-8, and BCMA-REH but negligible cytotoxicity against BCMA-negative REH cells. The high proliferative potential indicated by the high frequency of memory T cells was validated in long-term killing assays, where ALLO-715 CAR T cells showed substantial expansion in the presence of MM.1S cells with no evidence of exhaustion or diminished cytolytic activity after seven days of continuous exposure to target. The potency of ALLO-715 CAR T cells was unaffected by high concentrations of soluble BCMA (〉10 ug/mL), which has been shown previously to interfere with the activity of some BCMA-specific CARs. In MM xenograft mouse models, ALLO-715 CAR T cells were highly efficacious at single dose. High serum IL-15 levels have been associated with CAR T cell expansion in clinical trials. To evaluate the impact of homeostatic cytokines on CAR T cell survival and antitumor activity in our xenograft models, mice were administered adeno-associated viruses (AAV) for the expression of human IL-7 and IL-15. In the presence of physiological concentrations of these cytokines, enhanced BCMA CAR T cell expansion and anti-tumor activity were observed. To assess potential off-target interactions of ALLO-715 CAR, tissue cross-reactivity studies were carried out on standard human tissue panels using a scFv-human IgG fusion protein. Consistent with the limited expression pattern of BCMA, reactivity was seen on scattered cells in lymphoid tissues such as tonsil and abundantly on BCMA-expressing cell lines, but no appreciable staining was detected in other tissues. We examined BCMA CAR T cells manufactured following a proprietary GMP-like clinical scale process and found that cell expansion and viability, T cell phenotype and in vivo antitumor efficacy were preserved. These results demonstrate the potential of ALLO-715 as a novel allogeneic BCMA CAR T therapy for the treatment of relapsed/refractory MM and other BCMA-positive malignancies. Disclosures Sommer: Allogene Therapeutics: Employment, Equity Ownership, Patents & Royalties. Boldajipour:Pfizer Inc.: Employment, Patents & Royalties. Valton:Cellectis.Inc: Employment, Equity Ownership, Patents & Royalties. Galetto:Cellectis SA: Employment, Equity Ownership, Patents & Royalties. Bentley:Allogene Therapeutics: Employment, Equity Ownership. Sutton:Allogene Therapeutics: Employment, Equity Ownership. Ni:Allogene Therapeutics: Employment, Equity Ownership. Leonard:Allogene Therapeutics: Employment, Equity Ownership. Van Blarcom:Allogene Therapeutics: Employment, Equity Ownership. Smith:Cellectis. Inc: Employment, Patents & Royalties. Chaparro-Riggers:Pfizer Inc.: Employment, Patents & Royalties. Sasu:Allogene Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Description: Autologous chimeric antigen receptor (CAR) T cells have achieved unprecedented clinical responses in patients with B-cell leukemias, lymphomas and multiple myeloma, raising interest in using CAR T cell therapies in AML. These therapies are produced using a patient's own T cells, an approach that has inherent challenges, including requiring significant time for production, complex supply chain logistics, separate GMP manufacturing for each patient, and variability in performance of patient-derived cells. Given the rapid pace of disease progression combined with limitations associated with the autologous approach and treatment-induced lymphopenia, many patients with AML may not receive treatment. Allogeneic CAR T (AlloCAR T) cell therapies, which utilize cells from healthy donors, may provide greater convenience with readily available off-the-shelf CAR T cells on-demand, reliable product consistency, and accessibility at greater scale for more patients. To create an allogeneic product, the TRAC and CD52 genes are inactivated in CAR T cells using Transcription Activator-Like Effector Nuclease (TALEN®) technology. These genetic modifications are intended to minimize the risk of graft-versus-host disease and to confer resistance to ALLO-647, an anti-CD52 antibody that can be used as part of the conditioning regimen to deplete host alloreactive immune cells potentially leading to increased persistence and efficacy of the infused allogeneic cells. We have previously described the functional screening of a library of anti-FLT3 single-chain variable fragments (scFvs) and the identification of a lead FLT3 CAR with optimal activity against AML cells and featuring an off-switch activated by rituximab. Here we characterize ALLO-819, an allogeneic FLT3 CAR T cell product, for its antitumor efficacy and expansion in orthotopic models of human AML, cytotoxicity in the presence of soluble FLT3 (sFLT3), performance compared with previously described anti-FLT3 CARs and potential for off-target binding of the scFv to normal human tissues. To produce ALLO-819, T cells derived from healthy donors were activated and transduced with a lentiviral construct for expression of the lead anti-FLT3 CAR followed by efficient knockout of TRAC and CD52. ALLO-819 manufactured from multiple donors was insensitive to ALLO-647 (100 µg/mL) in in vitro assays, suggesting that it would avoid elimination by the lymphodepletion regimen. In orthotopic models of AML (MV4-11 and EOL-1), ALLO-819 exhibited dose-dependent expansion and cytotoxic activity, with peak CAR T cell levels corresponding to maximal antitumor efficacy. Intriguingly, ALLO-819 showed earlier and more robust peak expansion in mice engrafted with MV4-11 target cells, which express lower levels of the antigen relative to EOL-1 cells (n=2 donors). To further assess the potency of ALLO-819, multiple anti-FLT3 scFvs that had been described in previous reports were cloned into lentiviral constructs that were used to generate CAR T cells following the standard protocol. In these comparative studies, the ALLO-819 CAR displayed high transduction efficiency and superior performance across different donors. Furthermore, the effector function of ALLO-819 was equivalent to that observed in FLT3 CAR T cells with normal expression of TCR and CD52, indicating no effects of TALEN® treatment on CAR T cell activity. Plasma levels of sFLT3 are frequently increased in patients with AML and correlate with tumor burden, raising the possibility that sFLT3 may act as a decoy for FLT3 CAR T cells. To rule out an inhibitory effect of sFLT3 on ALLO-819, effector and target cells were cultured overnight in the presence of increasing concentrations of recombinant sFLT3. We found that ALLO-819 retained its killing properties even in the presence of supraphysiological concentrations of sFLT3 (1 µg/mL). To investigate the potential for off-target binding of the ALLO-819 CAR to human tissues, tissue cross-reactivity studies were conducted using a recombinant protein consisting of the extracellular domain of the CAR fused to human IgG Fc. Consistent with the limited expression pattern of FLT3 and indicative of the high specificity of the lead scFv, no appreciable membrane staining was detected in any of the 36 normal tissues tested (n=3 donors). Taken together, our results support clinical development of ALLO-819 as a novel and effective CAR T cell therapy for the treatment of AML. Disclosures Sommer: Allogene Therapeutics, Inc.: Employment, Equity Ownership. Cheng:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Yeung:Pfizer Inc.: Employment, Equity Ownership. Nguyen:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Sutton:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Melton:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Valton:Cellectis, Inc.: Employment, Equity Ownership. Poulsen:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Djuretic:Pfizer, Inc.: Employment, Equity Ownership. Van Blarcom:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Chaparro-Riggers:Pfizer, Inc.: Employment, Equity Ownership. Sasu:Allogene Therapeutics, Inc.: Employment, Equity Ownership.

Description: Multiple myeloma (MM) is a debilitating disease characterized by the abnormal accumulation of malignant plasma cells in the bone marrow. Despite recent advances in myeloma therapy, including proteasome inhibitors, immunomodulatory drugs, and targeted antibody therapies, patients relapse and the disease remains incurable and one of high unmet need. T cell redirecting therapies are a new and exciting class of therapeutics that harness the potent cytotoxic activity of T cells and redirect it to target tumor cells. T cell redirecting therapies are only as good as their targeted tumor associated antigen (TAA) and the potent nature of the therapy requires a lack of TAA expression in essential normal tissue. B-cell Maturation Antigen, BCMA, is a tumor necrosis factor superfamily member highly expressed on the surface of myeloma cells. Detectable normal BCMA tissue expression appears limited to plasmablasts and mature plasma cells, making it an ideal T cell redirecting target for the treatment of MM. Other groups have developed T cell redirecting therapies against BCMA, including CAR T and BiTE therapy (a short half-life CD3 bispecific). Here we present preclinical studies on a fully-human IgG CD3 bispecific molecule targeting BCMA (half-life in mice of ~3 days). This molecule utilizes anti-BCMA and anti-CD3 targeting arms paired through hinge mutation technology and placed in an IgG2A backbone. The molecule binds to BCMA-expressing myeloma cell lines and to T cells with affinities of 20pM and ~40nM, respectively. T cells co-cultured with MM cell lines were activated and de-granulated in the presence of BCMA bispecific. In vitro cytotoxicity assays revealed the high potency of the molecule, as it was able to drive lysis of MM target cells with an EC50 of 6± 8 pM (mean ± SD). We also observed strong in vitro potency with the BCMA bispecific in four different MM primary patient samples, EC50 =0.093±0.1 nM (mean ± SD). When the same four samples were targeted with a BCMA antibody drug conjugate (ADC), 3 of the samples gave EC50 values of 1.25±0.7 nM (mean ± SD) - i.e. a 43 fold decrease in potency compared to the CD3 bispecific. The fourth patient did not respond to the ADC. Together, these results illustrate the potential advantages of a CD3 bispecific over an ADC for targeting BCMA. In orthotopic, established, tumor mouse models utilizing three different MM cell lines, (OPM2, MM.1S and MOLP8), a single injection of BCMA bispecific effectively treated tumors in a dose-dependent manner. Re-dosing the bispecific was able to provide additional and prolonged efficacy. The extreme potency of T cell redirecting therapies results in outstanding efficacy, but can also lead to lysis of normal cells expressing even minute levels of target. The species cross-reactivity of the BCMA bispecific allowed for exploratory toxicity studies in cynomologus monkeys. The molecule was able to effectively deplete normal plasma B cells expressing low levels of BCMA, providing evidence of activity. Activity was accompanied by a cytokine spike following initial dosing. No cytokine release was observed following a second bispecific dose. Encouragingly, animals experienced no additional adverse events (AEs), confirming the favorable safety profile of BCMA as a target for MM. In summary, we report on a fully human IgG CD3 bispecific molecule targeting BCMA for the treatment of multiple myeloma. Our BCMA bispecific is expected to have an antibody-like half-life in humans and, taken together, our findings support that the molecule has the potential to be both a potent and safe therapeutic. Disclosures Panowski: Pfizer Inc.: Employment. Kuo:Alexo Therapeutics: Employment. Chen:Alexo Therapeutics: Employment. Geng:Kodiak Sciences: Employment. Van Blarcom:Pfizer Inc.: Employment. Lindquist:Pfizer Inc.: Employment. Chen:Pfizer Inc.: Employment. Chaparro-Riggers:Pfizer Inc.: Employment. Sasu:Pfizer Inc.: Employment.

Description: Adoptive transfer of T cells expressing chimeric antigen receptors (CARs) is an exciting new therapy showing great promise in hematologic malignancies. Recent approval of two CD19-targeting CAR Ts, Kymriah® and Yescarta®, has been followed with promising results from BCMA clinical trials, showing that activity can extend to other targets. While these treatments show excellent potential for patients with an otherwise poor prognosis, a number of patients are still subject to relapse. These data suggest the potential utility of expanding the number of targets available for hematological malignancies. Autologous chimeric antigen receptor (CAR) T therapies use a patient's own T cells to produce the therapy. This approach has inherent challenges, including requiring significant time for production, complex supply chain logistics, separate GMP manufacturing for each patient, and variability in performance of patient-derived cells. Given rapid disease progression, combined with limitations associated with the autologous approach, many patients may not be able to receive CAR T therapy. Allogeneic CAR T (AlloCAR T) cell therapy, or "off-the-shelf" therapy which utilizes cells from healthy donors, may provide greater convenience with readily available CAR T therapy on-demand, reliable product consistency, and accessibility at greater scale for more patients. To create an allogeneic product, mRNAs encoding Transcription Activator-Like Effector Nucleases (TALEN®) designed to specifically disrupt the TRAC and CD52 genes are introduced to CAR Ts. These modifications are intended to minimize the risk of graft-versus-host disease and to confer resistance to ALLO-647, an anti-CD52 antibody that can be used as part of the conditioning regimen to deplete host alloreactive immune cells potentially leading to increased persistence and efficacy of the infused allogeneic cells. CD70 is a TNF superfamily member expressed on subsets of activated lymphocytes and is being explored as a CAR T target for renal cell carcinoma but may also be suitable for targeting hematological malignancies. To evaluate the potential of CD70 as a heme target, expression of CD70 RNA was compared to that of three known targets, CD19, BCMA, and FLT3, across a range of hematological malignancies by interrogating The Cancer Genome Atlas (TCGA) database. CD19 was expressed in lymphomas and leukemias, BCMA was expressed primarily in MM, and FLT3 expression was largely limited to AML. CD70 expression was observed across all 4 cancer types, indicating the potential broad utility CD70 CAR T cells. To determine cell-surface expression of these four targets, flow cytometry and receptor quantification was performed on a panel of heme cell lines. Good correlation was seen between RNA and the cell-surface protein expression of CD70. CAR T cells against each of the 4 targets were generated and evaluated in vitro. All CAR T cells exhibited robust specific activity against cells expressing their corresponding antigens. To ensure clinical relevance of results with cell lines, studies are currently ongoing to evaluate CD70 expression on primary patient samples and activity of CD70 CAR T cells against these same samples. Based on our studies, CD70 has a broad expression profile across a range of hematological malignancies and potent and selective CD70 CAR T activity has been demonstrated. Given these results, an allogeneic CD70 CAR T is expected to be clinically useful against a range of hematological tumor types either as a single agent or in combination with other CAR Ts. Disclosures Panowski: Allogene Therapeutics: Employment, Equity Ownership. Srinivasan:Allogene Therapeutics: Employment, Equity Ownership. Van Blarcom:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Sommer:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Cheng:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Pertel:Allogene Therapeutics: Employment, Equity Ownership. Santaguida:Notable Labs: Employment. Galetto:Cellectis Inc: Employment. Sasu:Allogene Therapeutics, Inc.: Employment, Equity Ownership.

Description: Multiple myeloma (MM) is a hematological disease of plasma B cells that remains incurable despite the availability of numerous therapies. The plasma cell-specific expression of the TNF superfamily receptor BCMA may allow targeting of normal and malignant plasma cells. Genetically engineered chimeric antigen-receptor T cells (CAR T) have shown tremendous promise in the treatment of several hematological diseases, including MM. However, conventional autologous CAR T therapies use patient-derived T cells and the logistics of on-demand CAR T manufacture limits their availability to a broad patient pool. Here we describe the preclinical evaluation of an allogeneic CAR T therapy targeting BCMA that has the potential for a readily available, off-the-shelf therapy for MM and other malignancies expressing BCMA. Human T cells were transduced with recombinant lentiviral vectors encoding three BCMA CAR candidates designed with fully human anti-BCMA scFvs, CD8a transmembrane domains and the intracellular signaling domains of 4-1BB and CD3zeta. All CAR T efficiently killed BCMA-expressing multiple myeloma cell lines (KMS12BM, MM1.S, Molp-8 and OPM-2), but not BCMA-negative REH cells in vitro and in vivo. Whereas 2 of the 3 candidates exhibited target-independent cytokine production, accelerated T cell differentiation and reduced target cell-induced expansion in vitro, the third candidate did not exhibit this scFv-induced autoactivation and was chosen as the lead molecule. Due to the allogeneic nature of this T cell therapy, the possibility of graft-versus-host (GvH) reactions can be a safety concern. We applied Cellectis' know-how and TALEN® technology for the gene inactivation of the T cell receptor (TCR) alpha chain to significantly reduce the probability for TCR-mediated GvH reactions and found that TCR knockout did not affect CAR T activity in vitro or in vivo. Furthermore, we incorporated intra-CAR rituximab-recognition domains into the CAR molecule to enable depletion of CAR T cells from patients when necessary. We found that this modified CAR retained anti-BCMA CAR T activity and enabled CAR T depletion by rituximab. Another aspect of allogeneic CAR T therapies is the rejection of the CAR T by host-versus-graft (HvG) reactions. Lymphodepletion prior to CAR T infusion enhances CAR T efficacy in autologous CAR T trials and may also prevent anti-CAR HvG reactions in allogeneic therapy settings. Engineering lymphodepletion resistance into CAR T cells could therefore enable sustained lymphodepletion for enhanced allogeneic CAR T persistence and efficacy. CD52 is expressed on all lymphocytes and administration of the anti-CD52 antibody alemtuzumab for prolonged lymphodepletion is an approved treatment for multiple sclerosis. TALEN®-mediated knockout of CD52 protected BCMA CAR T from alemtuzumab-induced cytotoxicity and did not alter BCMA CAR T anti-tumor activity. Taken together these results support allogeneic BCMA CAR T as an off-the-shelf adoptive immunotherapy for the treatment of multiple myeloma and other BCMA-positive malignancies. Disclosures Boldajipour: Pfizer: Employment. Galetto:Cellectis SA: Employment. Sommer:Pfizer Inc.: Employment. Pertel:Pfizer Inc.: Employment. Valton:Cellectis Inc.: Employment. Park:Pfizer Inc.: Employment. Gariboldi:Cellectis SA: Employment. Chen:Alexo Therapeutics: Employment. Geng:Kodiak Sciences: Employment. Dong:Pfizer Inc.: Employment. Boucher:Pfizer Inc.: Employment. Van Blarcom:Pfizer Inc.: Employment. Chaparro-Riggers:Pfizer Inc.: Employment. Rajpal:Pfizer Inc.: Employment. Smith:Cellectis SA: Employment. Kuo:Pfizer Inc.: Employment. Sasu:Pfizer Inc.: Employment.