Induced pluripotent stem cell (iPSC)-derived effector cells offer distinct advantages for immune therapy over existing patient- or donor- derived platforms, both in terms of scalable manufacturing from a renewable starting cellular material and precision genetic engineering that is performed at the single-cell level. iPSC derived natural killer (iNK) cells offer the further advantage of innate reactivity to stress ligands and MHC downregulation and the potential to recruit downstream adaptive responses. These unique features form the basis of our multi-antigen targeted chimeric antigen receptor (CAR) CAR-iNK cell product candidate, termed FT596, which is further combined with additional functionality to enhance effector function. FT596 is consistently manufactured from a master iPSC line engineered to uniformly express an NK cell-calibrated CD19-targeting CAR (CD19-CAR), an enhanced functioning high-affinity, non-cleavable CD16 (hnCD16) and a recombinant fusion of IL-15 and IL-15 receptor alpha (IL-15RF) for cytokine-autonomous persistence. The design of the CD19-CAR involved exploiting the intrinsic polyfunctionality of NK cells, which function by engaging multiple signaling pathways activated through combinations of distinct germline encoded receptors. Using this approach, the transmembrane region of activating receptor NKG2D, combined with the intracellular signaling domains of SLAM co-receptor 2B4 and CD3ζ, proved the most effective in triggering antigen specific functional responses in NK cells. Chimerization of an anti-CD19 scFv onto this NKG2D-2B4-CD3ζ signaling platform produced specific in vitro recognition of CD19+ B cell lymphoma cells in short-term and long-term NK cytotoxicity assays (>80% and <40% clearance of tumor cells at 60H, p<0.001 respectively). The functionality of the CD19-CAR was further enhanced in combination with autonomous IL-15 signaling. Introduction of the IL-15RF enabled expansion of iNK cells without addition of soluble cytokine and greatly improved longevity and functional persistence of iNK cells both in vitro and in animal models. Moreover, iNK cells modified with IL-15RF showed enhanced functional maturation, including upregulated expression of effector molecules such as granzyme B. iNK cells with both CD19-CAR and IL-15RF resulted in enhanced CAR functionality in vitro, and mouse models for B cell malignancy demonstrated that treatment with iNK cells engineered with CD19-CAR and IL-15RF were curative against B cell lymphoma (p<0.002), when compared with iNK cells alone or iNK cells modified with CD19-CAR alone. In combination with hnCD16, co-expression of CD19-CAR and IL15-RF culminates in iNK cells capable of dual-specificity through combinatorial use with monoclonal antibodies to tackle antigen escape. In long term killing assays, FT596 alone demonstrated equivalent levels of CD19 targeted anti-tumor activity as primary CD19-targeted CAR (CAR19) T cells when tested against CD19+ CD20+ B lymphoblast target cells and demonstrated enhanced levels of activity when used in combination with anti-CD20 (rituximab). When targeting CD19- CD20+ B lymphoblast target cells and used in combination with rituximab, only FT596 was able to effectively eliminate the CD19 antigen escaped target cell (64% vs 30% clearance of tumor cells at 36H vs rituximab alone). In vivo FT596 showed equivalent levels of tumor cell clearance as primary CAR19 T cells against the CD19+ acute lymphoblastic leukemia cell line NALM6 and CD19+CD20+ Burkitts lymphoma cell line RAJI, and enhanced clearance of RAJI tumor cells in combination with rituximab (p=0.0002). Furthermore, utilizing an allogenic human CD34 engrafted NSG mouse model, FT596 demonstrated improved survival and safety over primary CAR19 T cells, either as a monotherapy or as a combination therapy with rituximab versus RAJI tumor cells. Together, these studies demonstrate FT596 provides a multi-antigen targeting, potent and persistent engineered immune cell that is derived from a master iPSC line which utilizes the intrinsic versatility of NK cells to enable a highly effective combination therapy in a single, standardized, scalable, off-the-shelf platform and supports the rational for a first-of-kind Phase I Study as a monotherapy and in combination with CD20-targeted mAbs including rituximab in subjects with relapsed/refractory B-cell lymphoma and leukemia. Figure Disclosures Goodridge: FATE THERAPEUTICS: Employment. Mahmood:Fate Therapeutics, Inc: Employment. Gaidarova:Fate Therapeutics, Inc: Employment. Bjordahl:Fate Therapeutics, Inc.: Employment. Cichocki:Fate Therapeutics, Inc: Research Funding. Chu:FATE THERAPEUTICS: Employment. Bonello:Fate Therapeutics, Inc.: Employment. Lee:Fate Therapeutics, Inc.: Employment. Groff:FATE THERAPEUTICS: Employment. Meza:FATE THERAPEUTICS: Employment. Malmberg:Vycellix: Consultancy, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc.: Consultancy, Research Funding. Miller:Moderna: Membership on an entity's Board of Directors or advisory committees; Dr. Reddys Laboratory: Membership on an entity's Board of Directors or advisory committees; CytoSen: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc: Consultancy, Research Funding; OnKImmune: Membership on an entity's Board of Directors or advisory committees; GT BioPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Kaufman:FATE Therapeutics: Consultancy, Research Funding. Valamehr:Fate Therapeutics, Inc: Employment.
Substantial numbers of B cell leukemia and lymphoma patients relapse due to antigen loss or heterogeneity after anti-CD19 chimeric antigen receptor (CAR) T cell therapy. To overcome antigen escape and address antigen heterogeneity, we engineered induced pluripotent stem cell (iPSC)-derived NK cells to express both an NK cell-optimized anti-CD19 CAR for direct targeting and a high affinity, non-cleavable CD16 to augment antibody-dependent cellular cytotoxicity (ADCC). In addition, we introduced a membrane-bound IL-15/IL-15R fusion (IL-15RF) protein to promote in vivo persistence. These engineered cells, termed iDuo NK cells, displayed robust CAR-mediated cytotoxic activity that could be further enhanced with therapeutic antibodies targeting B cell malignancies. In multiple in vitro and xenogeneic adoptive transfer models, iDuo NK cells exhibited robust anti-lymphoma activity. Furthermore, iDuo NK cells effectively eliminated both CD19+ and CD19- lymphoma cells and displayed a unique propensity for targeting malignant cells over healthy cells that expressed CD19, features not achievable with anti-CAR19 T cells. iDuo NK cells combined with therapeutic antibodies represents a promising approach to prevent relapse due to antigen loss and tumor heterogeneity in patients with B cell malignancies.
Multiple redundancy within the spectrum of an immune response is required to prevent antigen escape or adaptation of the targeted population to host defenses. As adoptive cell therapies continue to evolve, multi-modal engineering of effector cells offers the prospect of tackling increasingly complex disease settings such as multiple myeloma (MM), where targeting of a single tumor associated antigen is frequently confounded by antigen shedding and escape variation resulting in the inability to develop a curative therapy. There are multiple advantages in expanding treatment options beyond autologous primary T and NK cells, including the use of induced pluripotent stem cells (iPSC) to derive effector cells that can be uniformly manufactured at scale from renewable starting cellular material and where precision genetic engineering can be achieved at the clonal level which can be applied sequentially in order to build multiple specificities and functional modalities. To create a platform targeted toward MM, a multiplexed edited base iPSC-derived NK (iNK) cell configuration consisting of a CD38 KO iPSC modified to overexpress a recombinant IL-15 signaling complex (IL15RF) for autonomous persistence and a functionally enhanced high-affinity, non-cleavable CD16 (hnCD16) was developed. Introduction of IL15RF enabled expansion of iNK cells without additional exogenous cytokine support during the manufacturing process and greatly improved functional persistence of iNK cells both in vitro and in various xenograft mouse models (Figure 1). To target MM in a broad and comprehensive manner, we tested our novel BCMA-CAR in combination with different myeloma targeted antibodies. In combination with hnCD16, co-expression of BCMA-CAR and IL15RF culminates in an iNK cell therapeutic, termed FT576, capable of multiantigen-specificity through combinatorial use of CAR and hnCD16 with monoclonal antibodies to tackle antigen escape. Chimerization of an anti-BCMA scFv shown to elicit higher affinity onto the CAR platform produced specific in vitro recognition of BCMA+ myeloma cells in short-term and long-term NK cell cytotoxicity assays. Specificity of the BCMA-CAR was demonstrated using NALM6 overexpressing BCMA using a short range 4H caspase assay (NALM6_BCMA EC50 14.4, NALM6wt EC50 39.1, p*<0.0001). Utilizing a long range clearance assay, serial restimulation by repeated rounds of exposure to fresh MM1S MM target cells was tested, showing remarkable persistence and antigen-mediated expansion of CAR function in isolation or combined with antibody through 3 rounds of stimulation in the absence of exogenous cytokine support (Figure 2). Continuous long-range clearance assays demonstrated levels of BCMA targeting activity of FT576 alone was equivalent to primary BCMA-targeted CAR-T cells against a panel of BCMA+ target cells. Utilizing hnCD16, BCMA-CAR was tested in combination with anti-CD38 (daratumumab), anti-SLAMF7 (elotuzumab), or anti-CD19, showing synergistic increase in tumor targeting through various tumor associated antigens (TAAs). Polyfunctionality of FT576 stimulated either through CAR or ADCC was similarly measured by both Isoplexis and single cell RNA sequencing. Specificity for plasma cells was confirmed using primary bone marrow samples from either healthy donors or patients. In animal models, as a monotherapy, FT576 achieved sustained tumor control against disseminated MM1s with persistence profile suggestive of antigen mediated expansion (Figure 3). In combination with daratumumab, FT576 was able to achieve complete clearance of MM1S. Combination with other monoclonal antibodies displayed a similar response demonstrating the unique ability of FT576 to be directed to target multiple TAAs. Together, these studies demonstrate the versatility of FT576 as a highly effective multi-antigen targeting and cost-effective off-the-shelf BCMA-CAR iNK cell product and supports the rational for a first-of-kind Phase I Study as a monotherapy or in combination with therapeutic mAbs targeted to MM-associated surface antigens, driving a path towards a curative therapeutic in MM. Disclosures Goodridge: Fate Therapeutics, Inc: Current Employment. Bjordahl:Fate Therapeutics: Current Employment. Mahmood:Fate Therapeutics, Inc: Current Employment. Reiser:FATE THERAPEUTICS: Current Employment. Gaidarova:Fate Therapeutics, Inc: Current Employment. Blum:Fate Therapeutics: Current Employment. Cichocki:Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding. Chu:Fate Therapeutics, Inc: Current Employment. Bonello:Fate Therapeutics, Inc: Current Employment. Lee:Fate Therapeutics, Inc.: Current Employment. Groff:Fate Therapeutics, Inc: Current Employment. Meza:Fate Therapeutics, Inc: Current Employment. Chu:Roche Holding AG: Current equity holder in publicly-traded company; Fate Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Walcheck:Fate Therapeutics: Consultancy, Research Funding. Malmberg:Vycellix: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Consultancy, Patents & Royalties. Miller:Vycellix: Consultancy; Onkimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding; GT Biopharma: Consultancy, Patents & Royalties, Research Funding. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company.
Multiple myeloma (MM) is a B cell neoplasm that originates from the malignant transformation of plasma cells, with treatment strategies that include chemotherapeutic agents and immunomodulatory drugs. Recently, significant effort has been applied to the development of monoclonal antibody (mAb) and chimeric antigen receptor (CAR) T cell therapies for the treatment of advanced MM. Anti-CD38 mAb therapy is at the forefront of these efforts, with clearly demonstrated clinical benefit and availability of a FDA-approved mAb in daratumumab. Antibody-dependent cellular cytotoxicity (ADCC) is a key mechanism of action of CD38-targeted mAbs; however, high CD38 expression on natural killer (NK) cells results in fratricide, which depletes the NK cells necessary for ADCC. In addition to CD38, targeting of other MM-associated cell-surface proteins has been explored. Of these antigens, the TNF-superfamily member BCMA is among the most researched and is under development by multiple groups as a CAR target. Several clinical trials in MM have shown promising initial results targeting BCMA with CAR T cells, however there remains significant opportunity to improve both relapse rates and treatment of relapsed patients. Collectively, clinical data would suggest that combinatorial targeting of both CD38 and BCMA may improve clinical efficacy compared with targeting either antigen alone. We have developed a multiple-target, adoptive NK cell immunotherapy approach for the treatment of MM. The strategy utilizes our off-the-shelf NK cell platform with four engineered attributes: 1) an anti-BCMA CAR for direct MM targeting, 2) high affinity non-cleavable CD16 (hnCD16) for enhanced ADCC in combination with anti-CD38 mAbs, 3) CD38 deletion for resistance to anti-CD38 mAb induced NK cell depletion, and 4) IL-15/IL-15 receptor α fusion protein (IL-15RF; IL-15 fused to IL-15Rα) for enhanced NK cell persistence. The anti-BCMA CAR consists of a unique single chain variable fragment (scFv) targeting domain with a BCMA binding affinity in the low nanomolar range, providing high functional avidity and efficacy in disease settings where BCMA antigen density is low. Our approach utilizes NK cells derived from a genetically engineered, clonally-derived master pluripotent stem cell line with uniform expression of anti-BCMA CAR, IL-15RF, hnCD16, and CD38 bi-allelic knockout. The engineered master pluripotent stem cell line serves as the starting material for consistent and repeatable manufacture of off-the-shelf NK cells that contain all described attributes in a homogenous manner (termed FT576) and that can be produced at a scale to support multi-dose treatment strategies and on-demand dose availability. In preclinical studies, FT576 NK cells exhibited uniform expression of CD16, CAR, and IL15-RF and did not express CD38 (<1%). In an in vitro fratricide assay, FT576 NK cells were entirely resistant to daratumumab-induced fratricide, with no detectable specific cytotoxicity when exposed to increasing concentrations of daratumumab. Conversely, peripheral blood NK cells were sensitive to daratumumab-induced fratricide (up to 33% cytotoxicity within 3 hrs of daratumumab exposure). FT576 NK cells demonstrated enhanced cytotoxicity against the MM1.S MM cell line during a long-term cytotoxicity assay compared with control NK cells that lacked CAR expression (62% cytotoxicity for FT576 vs 26% for control). In addition, cellular persistence was greater than NK cells lacking the IL-15RF protein, and FT576 NK cells demonstrated the unique ability to expand in vitro absent of exogenous cytokine support (61-fold expansion vs. 4-fold for IL-15RF negative). Importantly, FT576 NK cells remained ADCC competent, as combination with daratumumab enhanced cytotoxicity against MM cell lines in a 2D cytotoxicity assay. Additionally, FT576 mediated direct cytotoxicity against RPMI-8226 MM spheroids, leading to >99% cytotoxicity in a 3D-spheroid culture model. Preclinical studies are ongoing to support the advancement of FT576 as the first-of-kind cellular therapeutic for the combination of anti-BCMA CAR and mAb-directed targeting of MM. Disclosures Bjordahl: Fate Therapeutics, Inc.: Employment. Gaidarova:Fate Therapeutics, Inc: Employment. Goodridge:FATE THERAPEUTICS: Employment. Mahmood:Fate Therapeutics, Inc: Employment. Bonello:Fate Therapeutics, Inc.: Employment. Robinson:Fate Therapeutics, Inc.: Employment. Ruller:Fate Therapeutics, Inc.: Employment. Pribadi:Fate Therapeutics, Inc.: Employment. Lee:Fate Therapeutics, Inc.: Employment. Abujarour:Fate Therapeutics, Inc.: Employment. Dinella:Fate Therapeutics, Inc.: Employment. Huffman:Fate Therapeutics, Inc.: Employment. Chu:FATE THERAPEUTICS: Employment. Valamehr:Fate Therapeutics, Inc: Employment.
Chimeric antigen receptor (CAR) T-cell therapy has proven highly effective in patients with hematological malignancies. However, resistance to CAR-T cell therapy arising from target protein shedding and other forms of antigen downregulation can lead to CAR-resistant disease relapse. Tumor escape may be successfully prevented through the simultaneous targeting of multiple tumor antigens. The ability to target multiple antigens with a single therapeutic modality offers the potential for anti-tumor responses, broader coverage of heterogeneous tumor populations, and the potential to prevent antigen escape, potentially inducing durable clinical remission. Multiple myeloma (MM) presents an ideal case to employ a dual-CAR approach, as BCMA-targeting cell therapies have shown impressive efficacy to date, but curative treatment remains elusive. Additionally, the oligoclonal nature of MM may contribute to antigen escape and clonal resistance. Here, we demonstrate the application of a unique dual-CAR approach simultaneously targeting two tumor associated antigens (TAA) for the treatment of MM. We further demonstrate the efficacy in an induced pluripotent stem cell (iPSC) platform, where a master engineered iPSC line is used as the starting material for mass production of off-the-shelf, dual-CAR immune effector cells. We selected B cell maturation antigen (BCMA), a well-defined TAA in MM, as the first antigen target. To develop the CAR-BCMA motif, we utilized our previously published high-affinity binding sequence shown to exhibit high selectivity to BCMA with enhanced recognition of low-BCMA expressing myeloma cells (Bluhm et al., Molec Ther 2018). As shown previously, the designed CAR-BCMA demonstrates potent and selective cellular cytotoxicity against MM (Figure 1a, left panel). BCMA has been observed to be actively cleaved from the surface of MM cells though, resulting in reduced efficacy and clinical relapse. To circumvent BCMA antigen escape, we developed a companion CAR targeting the pan-TAAs, MICA and MICB, which are expressed on MM plasma cells as well as monoclonal gammopathy of undetermined significance (MGUS) plasma cells. The CAR binding sequence targets the conserved α3 domain of MICA/MICB, which we have previously shown to inhibit MICA/B shedding and drive anti-tumor immunity (Andrade et al., Science 2018). The designed anti-MICA/B-α3 CAR exhibits selective targeting potential against an array of cancers, including the MM.1S cancer cell line (Figure 1a, right panel). To determine the suitability of co-targeting BCMA and MICA/B in MM, we surveyed surface expression patterns of BCMA and MICA/B antigens on a variety of MM cancer cell lines and observed a complimentary pattern of co-expression compatible with a dual-CAR to broaden targeting approach of malignant plasma cells (Figure 1b). Initial studies to evaluate the dual CAR approach in MM were performed by generating anti-BCMA and anti-MICA/B-α3 dual-CAR (MM dual-CAR) T-cells. MM dual-CAR T cells showed antigen-specific activation, degranulation and cytotoxicity against both antigens in an additive manner, consistent with the initial antibody staining on target cells and illustrating that co-targeting MICA/B and BCMA may increase the activity against MM (Figure 1c). Similar trends were observed in a series of cytotoxicity assays against several MM lines. Preliminary studies are ongoing in induced pluripotent stem cell (iPSC)-derived NK (iNK) cells expressing MM dual-CARs as a unique off-the-shelf cell therapy targeting both BCMA and MICA/B. Since MM dual-CAR iNK cells also express CD16, which mediates antibody-dependent cellular cytotoxicity, combination with therapeutic antibodies, such as anti-CD38 antibodies, can be deployed to target three TAAs for a complete therapeutic approach in MM. The data highlights the applicability of a multi-targeted approach in MM patients, whereby MM dual-CAR NK and/or T cells maintain responsiveness to malignant cells that shed or downregulate tumor antigens to evade treatment. Figure 1 Figure 1. Disclosures Lee: Fate Therapeutics, Inc.: Current Employment. Wucherpfennig: Novartis: Research Funding; SQZ Biotech: Membership on an entity's Board of Directors or advisory committees; TScan Therapeutics: Membership on an entity's Board of Directors or advisory committees; Immunitas Therapeutics: Current holder of individual stocks in a privately-held company; Nextechinvest: Membership on an entity's Board of Directors or advisory committees; TCR2 Therapeutics: Membership on an entity's Board of Directors or advisory committees. Bjordahl: Fate Therapeutics: Current Employment. Valamehr: Fate Therapeutics, Inc.: Current Employment.
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