There is significant debate regarding whether B cells and their antibodies contribute to effective anti-cancer immune responses. Here we show that patients with metastatic but non-progressing melanoma, lung adenocarcinoma, or renal cell carcinoma exhibited increased levels of blood plasmablasts. We used a cell-barcoding technology to sequence their plasmablast antibody repertoires, revealing clonal families of affinity matured B cells that exhibit progressive class switching and persistence over time. Anti-CTLA4 and other treatments were associated with further increases in somatic hypermutation and clonal family size. Recombinant antibodies from clonal families bound non-autologous tumor tissue and cell lines, and families possessing immunoglobulin paratope sequence motifs shared across patients exhibited increased rates of binding. We identified antibodies that caused regression of, and durable immunity toward, heterologous syngeneic tumors in mice. Our findings demonstrate convergent functional anti-tumor antibody responses targeting public tumor antigens, and provide an approach to identify antibodies with diagnostic or therapeutic utility.
Autologous chimeric antigen receptor (CAR)-T cell therapy has shown great promise in various hematologic malignancies. However, the complexities associated with immune cell evasion are prevalent causes of disease relapse in many cancers. With the advent of pluripotent stem cell (iPSC)-derived CAR-T cells, many factors that hamper therapeutic efficacy of CAR-T cells can be addressed through multiplexed engineering at the clonal level. This includes enhanced potency, increased capacity for multi-antigen targeting, and the consistency of a clonally derived engineered cellular product for off-the-shelf patient administration. In particular, strategies to mitigate antigen escape and address tumor heterogeneity may help promote durable responses. To combine the potent targeted therapy of the CAR with universal targeting of secondary and tertiary antigens, we expressed an MR1 clonal T cell receptor (TCR) and a high-affinity, non-cleavable CD16 Fc receptor (hnCD16) in our iPSC-derived CAR19 T cells (CAR19-iT cells) directed to leukemia and lymphoma and CAR-MICA/B T cells directed to solid tumors. The MR1-TCR allows highly specific recognition of tumor associated antigen presented by the MR1 protein. The non-polymorphic MHC class I-related protein MR1 is widely expressed with minimal variability among patients and enables the unique prospect to be a universal cancer immunotherapy by using the cognate MR1-TCR. The hnCD16 Fc receptor has been shown to improve antibody-dependent cellular cytotoxicity (ADCC) leveraging the broad range of available therapeutic monoclonal antibodies to target clinically validated tumor antigens. A preliminary assessment demonstrated that MR1-TCR overexpressed in T cells allowed for enhanced recognition of multiple hematological and solid tumor cell lines. Notably, prominent target specific killing was seen in A549 lung carcinoma cells (>75% reduction in total viable cells) with the directed cytotoxicity specifically inhibited by an MR1 blocking antibody. Next, in vitro functional testing was performed on the engineered CAR19-iT cells in co-culture assays where we measured killing of tumor cells via MR1-TCR engagement and via hnCD16 mediated ADCC. Specifically, we show that CAR19-iT cells expressing hnCD16 can be efficiently directed to lyse CD20+ Raji cells in the presence of rituximab or HER2+ SKOV3 cells in the presence of Herceptin, demonstrating the potential to target both hematological malignancies and solid tumors with one target modality in combination with various monoclonal antibodies. Moreover, CAR19-iT cells expressing either MR1-TCR or hnCD16 show the ability to control growth of CD19 KO lymphoma cells in the co-culture assays, further highlighting the unique ability to elicit multiple ways to target antigen escape. Further in vitro and in vivo combinatorial targeting studies focused on antigen escape and tumor heterogeneity are ongoing and will be discussed. In summary, the advances presented here demonstrate that both the MR1-TCR and hnCD16 modalities synergize with CAR-iT cells as an off-the-shelf therapeutic that can provide durable responses and enable broad applicability for targeting of additional tumor antigens where single-agent therapeutics fail to provide clinical benefit for patients. Disclosures Nguyen: Fate Therapeutics, Inc.: Current Employment. Peralta:Fate Therapeutics, Inc.: Current Employment. Lu:Fate Therapeutics, Inc.: Current Employment. Sung:Fate Therapeutics, Inc.: Current Employment. Lee:Fate Therapeutics, Inc.: Current Employment.
The development of chimeric antigen receptor (CAR) T cell therapeutics is widely recognized as a significant advancement for the treatment of cancer. However, several obstacles currently impede the broad use of CAR T cells, including the inherent process variability, cost of manufacturing, the absolute requirement for precise and uniform genetic editing in the allogeneic setting, and the challenge to keep pace with clonal heterogeneity and tumor growth. Utilizing our previously described induced pluripotent stem cell (iPSC)-derived T (iT) cell platform, we illustrate here the unique ability to address these challenges by creating a consistent CAR iT cell product that can be repeatedly manufactured in large quantities from a renewable iPSC master cell bank that has been engineered to mitigate the occurrence of graft versus host disease (GvHD), antigen escape and tumor relapse. Utilizing our proprietary cellular reprogramming and engineering platform and stage-specific T cell differentiation protocol, we demonstrate that iPSC can be engineered at the single cell level to generate a fully characterized clonal iPSC line, which can then be accessed routinely to yield CAR iT cells in a highly scalable manufacturing process (>100,000 fold expansion). Through bi-allelic targeting of a CAR into the T cell receptor alpha constant (TRAC) region, we generated CAR iT cells with uniform CAR expression (99.0 ± 0.5% CAR+) and complete elimination of T cell receptor (TCR) expression to avoid GvHD in the allogeneic setting. We elected to utilize the 1XX-CAR configuration, which has demonstrated superior anti-tumor performance relative to other CAR designs and when introduced into iT cells displayed enhanced antigen specificity (% specific cytotoxicity at E:T=10:1, antigen positive group: 86.4 ± 7.8; antigen null group: 8.9 ± 3.5). To enhance persistence without reliance on exogenous cytokine support, we engineered signaling-fusion complexes, including IL-7 receptor fusion (RF), into iPSC and studied its impact on iT phenotype, persistence, and efficacy. In vitro, IL-7RF clones demonstrated improved anti-tumor activity in a serial antigen dependent tumor challenge assay (Day 10, relative tumor counts, IL-7RF group: 1.95 ± 0.01; control group: 57.56 ± 4.55, P<0.000001). In a preclinical in vivo model of disseminated leukemia, IL-7RF clones demonstrate enhanced tumor growth inhibition (Day 34, Log [BLI], IL-7RF group: 6.68 ± 1.93; control group: 9.99 ± 0.23, P=0.0143). We next investigated a unique strategy to incorporate multi-antigen targeting potential into anti-CD19 1XX CAR iT cells with the addition of a high-affinity non-cleavable CD16 (hnCD16) Fc receptor. The combination of hnCD16 with anti-CD19 1XX CAR culminated in iT cells capable of multi-antigen specificity through combinatorial use with monoclonal antibodies to tackle antigen escape. Utilizing CD19 negative leukemia cells as targets, superior antibody-dependent cellular cytotoxicity (ADCC) was demonstrated by the combination of hnCD16 CAR iT and Rituximab (% specific cytotoxicity at E:T=1:1, hnCD16 group + Rituximab: 75.64 ± 2.12; control group + Rituximab: 16.98 ± 3.87, P<0.001). To address T cell fitness, the role of CD38 knockout (KO) in T cells was investigated, which we have previously shown to mediate NK cell resistance to oxidative stress induced apoptosis. CD38 gene was disrupted at the iPSC stage to generate 1XX-CAR T cells that lack CD38 expression (% CD38+ population, CD38WT group: 69.67 ± 24.34; CD38KO group: 0.12 ± 0.11) and upon antigen mediated stimulation, CD38KO CAR iT cells showed higher percentages of degranulation (2.3-fold increase in CD107a/b), and IFNγ (4.1-fold increase) and TNFα (2.5-fold increase) production. Antigen specific in vitro tumor killing also was enhanced in CD38KO CAR iT cells (EC50, 3.2-fold decrease). Lastly, to avoid the potential host-mediated rejection, the inclusion of allogeneic defense receptor (ADR) which has been shown to significantly reduce host-mediated rejection will be discussed. Collectively, the described studies demonstrate that iPSCs are an ideal cellular source to generate large-quantities of uniformly multi-edited off-the-shelf CAR T cell products that include a best-in-class CAR design, enhanced product modalities, and complete elimination of TCR expression to avoid the potential of GvHD while maintaining high anti-tumor efficacy in allogeneic setting. Disclosures Hsia: Fate Therapeutics Inc.: Current Employment. Clarke:Fate Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Lee:Fate Therapeutics, Inc.: Current Employment. Robbins:Fate Therapeutics, Inc.: Current Employment. Denholtz:Fate Therapeutics, Inc: Current Employment. Hanok:Fate Therapeutics, Inc.: Current Employment. Carron:Fate Therapeutics, Inc.: Current Employment. Navarrete:Fate Therapeutics, Inc.: Current Employment. ORourke:Fate Therapeutics, Inc.: Current Employment. Sung:Fate Therapeutics, Inc.: Current Employment. Gentile:Fate Therapeutics, Inc.: Current Employment. Nguyen:Fate Therapeutics, Inc.: Current Employment. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company.
The full potential of cell therapy has yet to be realized in solid tumor indications and new approaches are urgently required. Macrophages are innate immune cells that can kill tumor cells and orchestrate the anti-tumor immune response. Macrophage cell therapy is an exciting new approach to treat cancer with the aim to harness the powerful activity of macrophages to reignite the immune system. Patient derived macrophages, however, are difficult to genetically engineer and do not proliferate, which makes generating high-quality cells at clinically relevant scales challenging. To address this, we have developed an induced pluripotent stem cell (iPSC) approach to macrophage cell therapy. These cells can be genetically engineered at the iPSC stage and then differentiated into billions of highly functional iPSC derived macrophages (iMACs). This allogenic cell therapy product can then be cryopreserved and stored for immediate use in the clinic when the patient is ready. Here we show that iMACs function like normal macrophages. They migrate towards tumor cells, respond to challenges through innate immune receptors, and produce immune recruiting and activating cytokines and chemokines. iMACs also express high levels of antibody receptors and we show that these cells can be directed to kill tumors in vitro and in vivo via antibody dependent cellular phagocytosis. Furthermore, we have conducted a robust screen to develop a novel CAR that is optimized for use in iMACs. These receptors can be engineered into iMACs at the iPSC stage and trigger robust tumor cell killing. In summary, these data highlight the therapeutic potential of iMACs in oncology and support the further development of this technology for clinical use. Citation Format: Huafeng Wang, May Sumi, Christine Huh, Fereshteh Parviz, Jessica Mastroianni, Jane Healy, Nicole Stevens, Leah Mitchell, Susanne Lang, Dan Kaufman, Robert Hollingsworth, David T. Rodgers. Developing an allogeneic iPSC derived macrophage cell therapy for oncology. [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 4059.
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