Chimeric Antigen Receptor Design Research Articles

Beyond CAR Tcells: exploring alternative cell sources for CAR-like cellular therapies.

Chimeric antigen receptor (CAR)-Tcell therapy has led to remarkable clinical outcomes in the treatment of hematological malignancies. However, challenges remain, such as limited infiltration into solid tumors, inadequate persistence, systemic toxicities, and manufacturing insufficiencies. The use of alternative cell sources for CAR-based therapies, such as natural killer cells (NK), macrophages (MΦ), invariant Natural Killer T (iNKT) cells, γδTcells, neutrophils, and induced pluripotent stem cells (iPSC), has emerged as a promising avenue. By harnessing these cells' inherent cytotoxic mechanisms and incorporating CAR technology, common CAR-T cell-related limitations can be effectively mitigated. We herein present an overview of the tumoricidal mechanisms, CAR designs, and manufacturing processes of CAR-NK cells, CAR-MΦ, CAR-iNKT cells, CAR-γδTcells, CAR-neutrophils, and iPSC-derived CAR-cells, outlining the advantages, limitations, and potential solutions of these therapeutic strategies.

Current understanding and management of CAR T cell-associated toxicities.

Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of several haematological malignancies and is being investigated in patients with various solid tumours. Characteristic CAR T cell-associated toxicities such as cytokine-release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) are now well-recognized, and improved supportive care and management with immunosuppressive agents has made CAR T cell therapy safer and more feasible than it was when the first regulatory approvals of such treatments were granted in 2017. The increasing clinical experience with these therapies has also improved recognition of previously less well-defined toxicities, including movement disorders, immune effector cell-associated haematotoxicity (ICAHT) and immune effector cell-associated haemophagocytic lymphohistiocytosis-like syndrome (IEC-HS), as well as the substantial risk of infection in patients with persistent CAR T cell-induced B cell aplasia and hypogammaglobulinaemia. A more diverse selection of immunosuppressive and supportive-care pharmacotherapies is now being utilized for toxicity management, yet no universal algorithm for their application exists. AsCAR T cell products targeting new antigens are developed, additional toxicities involving damage to non-malignant tissues expressing the target antigen are a potential hurdle. Continued prospective evaluation of toxicity management strategies and the design of less-toxic CAR T cell products are both crucial for ongoing success in this field. In this Review, we discuss the evolving understanding and clinical management of CAR T cell-associated toxicities.

Toll-like receptor 4 signaling activation domains promote CAR T cell function against solid tumors

Chimeric antigen receptor (CAR) Tcell therapy has emerged as a powerful therapeutic approach against a range of hematologic malignancies. While the incorporation of CD28 or 4-1BB costimulatory signaling domains into CARs revolutionized immune responses, there is an exciting prospect of further enhancing CAR functionality. Here, we investigated the design of CD19 CARs enriched with distinct Toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), or Toll/IL-1 domain-containing adaptor-inducing interferon (IFN)-β (TRIF) costimulatory domains. Screening of various designs identified several candidates with no tonic activity but with increased CD19 target cell-dependent interleukin (IL)-2 production. Human Tcells transduced with the selected CAR construct exhibited augmented hIL-2 and hIFN-γ induction and cytotoxicity when cocultured with CD19-positive lymphoma and solid-tumor cell lines. RNA sequencing (RNA-seq) analysis demonstrated the upregulation of some genes involved in the innate immune response and Tcell activation and proliferation. In experiments on a xenogeneic solid-tumor mice model, MyD88 and TLR4 CAR Tcells exhibited prolonged remission. This study demonstrates that the integration of a truncated TLR4 signaling costimulatory domain could provide immunotherapeutic potential against both hematologic malignancies and solid tumors.

Chimeric Antigen Receptor (CAR) T-Cell Therapy in Hematologic Malignancies: Clinical Implications and Limitations.

Chimeric antigen receptor (CAR) T-cell therapy has become a powerful treatment option in B-cell and plasma cell malignancies, and many patients have benefited from its use. To date, six CAR T-cell products have been approved by the FDA and EMA, and many more are being developed and investigated in clinical trials. The whole field of adoptive cell transfer has experienced an unbelievable development process, and we are now at the edge of a new era of immune therapies that will have its impact beyond hematologic malignancies. Areas of interest are, e.g., solid oncology, autoimmune diseases, infectious diseases, and others. Although much has been achieved so far, there is still a huge effort needed to overcome significant challenges and difficulties. We are witnessing a rapid expansion of knowledge, induced by new biomedical technologies and CAR designs. The era of CAR T-cell therapy has just begun, and new products will widen the therapeutic landscape in the future. This review provides a comprehensive overview of the clinical applications of CAR T-cells, focusing on the approved products and emphasizing their benefits but also indicating limitations and challenges.

Abstract LB070: In vitro modeling of antigen-driven exhaustion in human CAR T cells

Abstract A major hurdle for chimeric antigen receptor (CAR) T cells to function in solid tumors is chronic antigen exposure leading to terminal lymphocyte exhaustion and impaired anti-tumor activity. Therefore, it is necessary to establish improved in vitro models of antigen-driven CAR T cell exhaustion to evaluate new CAR design candidates prior to validation with costly in vivo studies. Using the human tumor antigen mesothelin (MSLN) and MSLN-targeting CAR T cells as a model, we developed two in vitro assays that drive an exhausted phenotype in human CAR T cells and can be used to predict anti-tumor responses in vivo: 1) a chronic stimulation assay with plate-bound recombinant antigen (MSLN), ICAM-1, and CD58 and 2) a high antigen-expressing 3D tumor spheroid co-culture assay. In the chronic antigen stimulation assay, repetitive exposure to plate-bound MSLN (alongside co-stimulation signals provided by recombinant ICAM-1 and CD58) reprogrammed the transcriptomic profile of MSLN CAR T cells leading to increased surface expression of inhibitory checkpoint molecules and reduced cytokine production. Moreover, chronically stimulated MSLN CAR T cells demonstrated a drastically impaired ability to kill MSLN+ tumor cells compared to their unstimulated or acutely stimulated counterparts. A similar exhausted phenotype was observed in the high-antigen tumor spheroid model: when MSLN CAR T cells were co-cultured with MSLN-high human NCI-H226 spheroids for 7 days in a 96-well plate, expression of inhibitory checkpoint molecules markedly increased on the surface of CAR T cells. This increased expression of exhaustion markers coincided with impairment of NCI-H226 spheroid clearance. By contrast, other tumor spheroid models expressing low to intermediate levels of MSLN (NCI-H1650, NCI-H2052, NCI-H292, and SKOV-3 tumor cells) did not substantially induce expression of inhibitory checkpoint molecules and the spheroids were successfully cleared by MSLN CAR T cells. Importantly, the ability (or inability) of a CAR construct to clear NCI-H226 high-antigen tumor spheroids in vitro accurately predicted CAR T cell performance in in vivo solid tumor studies: the CAR construct that showed effective killing in the in vitro spheroid model was the only condition to provide durable regression of NCI-H1650 tumors in a NSG xenograft mouse model. Additionally, the rank order of CAR efficacy for a series of MSLN CAR binders obtained using the tumor spheroid model mirrored in vivo efficacy. As the CAR T cell field develops novel strategies to combat antigen-driven exhaustion for use in solid tumors, these new antigen-dependent in vitro models of exhaustion can enable more cost-efficient and effective evaluation of those strategies than using in vivo models alone. Citation Format: Maria Steele, Tina Tan, Jason Yokoyama, Allan Wang, Ty Crowl, Jared Hammer, Jerry Chen, Kevin Haworth, Howell Moffett, Aaron Foster, Marc Lajoie, Scott Boyken, Rupesh Amin. In vitro modeling of antigen-driven exhaustion in human CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB070.

Augmenting the Antitumor Efficacy of Natural Killer Cells via SynNotch Receptor Engineering for Targeted IL-12 Secretion.

Natural killer (NK) cells are crucial components of innate immunity, known for their potent tumor surveillance abilities. Chimeric antigen receptors (CARs) have shown promise in cancer targeting, but optimizing CAR designs for NK cell functionality remains challenging. CAR-NK cells have gained attention for their potential to reduce side effects and enable scalable production in cancer immunotherapy. This study aimed to enhance NK cell anti-tumor activity by incorporating PD1-synthetic Notch (synNotch) receptors. A chimeric receptor was designed using UniProt database sequences, and 3D structure models were generated for optimization. Lentiviral transduction was used to introduce PD1-Syn receptors into NK cells. The expression of PD1-Syn receptors on NK cell surfaces was assessed. Engineered NK cells were co-cultured with PDL1+ breast cancer cells to evaluate their cytotoxic activity and ability to produce interleukin-12 (IL-12) and interferon-gamma (IFNγ) upon interaction with the target cells. This study successfully expressed the PD1-Syn receptors on NK cells. CAR-NK cells secreted IL-12 and exhibited target-dependent IFNγ production when engaging PDL1+ cells. Their cytotoxic activity was significantly enhanced in a target-dependent manner. This study demonstrates the potential of synNotch receptor-engineered NK cells in enhancing anti-tumor responses, especially in breast cancer cases with high PDL1 expression.

Abstract 1321: In vivo immune cell programming using mRNA-LNP chimeric antigen receptors

Abstract Introduction: The tumor microenvironment (TME) is a dynamic and multifaceted system that comprises largely myeloid lineage immune cells including monocytes, neutrophils, macrophages, and dendritic cells. Reprogramming the myeloid compartment within the context of cancer therapy presents an attractive strategy to overcome current limitations in solid tumor treatment. We have developed a novel approach that harnesses the natural ability of Myeloid cells to take up mRNA loaded lipid nanoparticles, to reprogram these cells with chimeric antigen receptors (CAR’s) that arm these cells to recognize and kill tumor cells. Methods: Current LNPs technology is sufficient to deliver significant mRNA cargo to numerous cell types, including B cells, T cells and myeloid cells. Leveraging this phenomenon we have developed a new class of chimeric antigen receptors (CARs), using protein biology that imparts targeted immune cell expression and functionality, specifically in Myeloid cells. These CARs harness Immunoreceptor Tyrosine-based Activation Motif (ITAM) signaling adaptors expressed exclusively in immune cells, achieving immune cell-type specific CAR-directed tumor lysis and cytokine responses. With a FcaR I (CD89) based CAR design, we have developed a CAR that is dependent on the common Fc receptor gamma chain, primarily restricted to myeloid cells, for stable expression and function. Results: We have demonstrated specific expression of CD89-based CAR in myeloid cells in mouse and monkey following systemic mRNA/LNP delivery, and in human myeloid cells following in vitro treatment with mRNA/LNPs. Systemic mRNA/LNP delivery of CD89-based CAR has demonstrated robust anti-tumor efficacy against multiple tumor antigens in various xenograft mouse models of human breast cancer, hepatocellular carcinoma, and ovarian cancer. Furthermore, in syngeneic mouse tumor models, robust anti-tumor immune responses, characterized by tumoral infiltration of activated CD8+ T cells, diminished tumor-associated Tregs, systemic enhancement of dendritic cells and anti-tumor IgG production, were observed for treated animals. Conclusions: Taken together, we present CAR designs that achieve expression and function in targeted immune cell populations, capable of eliciting anti-tumor efficacy against multiple target antigens by leveraging innate and adaptive immune responses, and demonstrating a class of novel CAR designs to overcome limitations faced by current in vivo mRNA/LNP delivery strategy. A phase I clinical trial is currently on-going to assess the safety and preliminary efficacy of CD89-based CAR against TROP2+ epithelial malignancies. Citation Format: Shannon Argueta, Franciele K. Melber, Michael Gorgievski, Neha Divangi, Edward Cochran, Thach Chu, Yuxiao Wang, Jian Ding, Daniel Getts. In vivo immune cell programming using mRNA-LNP chimeric antigen receptors [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 1321.

Abstract 5228: Engineering CD123-targeting vδ1-enriched γδT cells with enhanced preclinical efficacy and safety profile

Abstract Acute myeloid leukemia (AML) is an aggressive hematological malignancy that, despite advances in treatment, has an overall 5-year survival rate of less than 30%. There is an urgent unmet need for new and effective therapies for AML. Chimeric antigen receptor (CAR)-modified cell therapies are emerging as promising options. However, the selection of suitable antigens is challenging, due to the heterogeneous expression of potential antigen targets in AML. Most antigens, such as CD123, are expressed on both leukemic cells and normal hematopoietic stem and progenitor cells. In addition, conventional autologous or allogeneic T cell therapies present many limitations, such as cytokine release syndrome (CRS) and graft-vs-host disease (GvHD). We therefore evaluated CD123-targeting CAR designs in vδ1-enriched γδT cells, an innate immune cell platform that is inherently non-MHC restricted, has low risks of CRS and GvHD, and is capable of discriminating between malignant and healthy cells in preclinical models. We engineered vδ1-enriched γδT cells from peripheral blood of healthy individuals to express various CD123-targeting CAR constructs. The CD123-targeting CAR-engineered vδ1-enriched γδT cells expressed high levels of natural cytotoxicity receptors, suggesting capability to target innate receptor ligands on AML cell surface. CD123-targeting CAR-expressing vδ1-enriched γδT cells demonstrated enhanced cytotoxicity in vitro against multiple AML cell lines, in both short-term co-culture and repeated antigen stimulation assays. CD123-targeting CAR-expressing vδ1-enriched γδT cells also showed enhanced efficacy and survival in vivo against mouse models of human AML. Interestingly, attenuation of CAR activity via novel designs preserved the inherent selectivity of vδ1-enriched γδT cells against AML cells relative to normal hematopoietic stem or progenitor cells, which also express CD123. Together, our results demonstrated through rational CAR designing and engineering in vδ1-enriched γδT cells, an allogeneic cell therapy targeting CD123 can be generated with selective cytotoxicity against AML cells while sparing normal hematopoietic stem cells. Such product designs have the potential to be a transformative therapeutic option for a broad population of AML patients. Citation Format: Mei Rosa Ng, Sarah L. Hayward, Di Zhang, Gary Gu, Bri Paulhus, Donjeta Gjuka, Collin Crowley, Sophie McLaughlin, Anya Lublinsky, Angel Mathew, Evans Berreondo Giron, Doanh Mai, Madisen Lee, Megha Wal, Rebecca Alade, Andre Simoes, Robin Lytle, James Gavin, Shira Cramer, Zhong-hua Yan, Taylor Hickman, Rashmi Choudhary, Cori Abikoff, Yana Wang, Istvan Kovacs, Nandhu M. Sobhana, Lan Cao. Engineering CD123-targeting vδ1-enriched γδT cells with enhanced preclinical efficacy and safety profile [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 5228.

Aptamer-Based Nongenetic Reprogramming of CARs Enables Flexible Modulation of T Cell-Mediated Tumor Immunotherapy.

Innovating the design of chimeric antigen receptors (CARs) beyond conventional structures would be necessary to address the challenges of efficacy, safety, and applicability in T cell-based cancer therapy, whereas excessive genetic modification might complicate CAR design and manufacturing, and increase gene editing risks. In this work, we used aptamers as the antigen-recognition unit to develop a nongenetic CAR engineering strategy for programming the antitumor activity and specificity of CAR T cells. Our results demonstrated that aptamer-functionalized CAR (Apt-CAR) T cells could be directly activated by recognizing target antigens on cancer cells, and then impart a cytotoxic effect for cancer elimination in vitro and in vivo. The designable antigen recognition capability of Apt-CAR T cells allows for easy modulation of their efficacy and specificity. Additionally, multiple features, e.g., tunable antigen-binding avidity and the tumor microenvironment responsiveness, could be readily integrated into Apt-CAR design without T cell re-engineering, offering a new paradigm for developing adaptable immunotherapeutics.

The Art of Bioimmunogenomics (BIGs) 5.0 in CAR-T Cell Therapy for Lymphoma Management

Purpose: Lymphoma, the most predominant neoplastic disorder, is divided into Hodgkin and Non-Hodgkin Lymphoma classifications. Immunotherapeutic modalities have emerged as essential methodologies in combating lymphoid malignancies. Chimeric Antigen Receptor (CAR) T cells exhibit promising responses in chemotherapy-resistant B-cell non-Hodgkin lymphoma cases. Methods: This comprehensive review delineates the advancement of CAR-T cell therapy as an immunotherapeutic instrument, the selection of lymphoma antigens for CAR-T cell targeting, and the conceptualization, synthesis, and deployment of CAR-T cells. Furthermore, it encompasses the advantages and disadvantages of CAR-T cell therapy and the prospective horizons of CAR-T cells from a computational research perspective. In order to improve the design and functionality of artificial CARs, there is a need for TCR recognition investigation, followed by the implementation of a quality surveillance methodology. Results: Various lymphoma antigens are amenable to CAR-T cell targeting, such as CD19, CD20, CD22, CD30, the kappa light chain, and ROR1. A notable merit of CAR-T cell therapy is the augmentation of the immune system's capacity to generate tumoricidal activity in patients exhibiting chemotherapy-resistant lymphoma. Nevertheless, it also introduces manufacturing impediments that are laborious, technologically demanding, and financially burdensome. Physical, physicochemical, and physiological limitations further exacerbate the challenge of treating solid neoplasms with CAR-T cells. Conclusion: While the efficacy and safety of CAR-T cell immunotherapy remain subjects of fervent investigation, the promise of this cutting-edge technology offers valuable insights for the future evolution of lymphoma treatment management approaches. Moreover, CAR-T cell therapies potentially benefit patients, motivating regulatory bodies to foster international collaboration.

Chimeric Antigen Receptor Design in Human Mesenchymal Stromal Cells (CAR-MSCs) Mediates Homing to Target Sites and Downstream Immunosuppression for Immune Disease Treatment

Chimeric Antigen Receptor Design in Human Mesenchymal Stromal Cells (CAR-MSCs) Mediates Homing to Target Sites and Downstream Immunosuppression for Immune Disease Treatment

Peptide-scFv antigen recognition domains effectively confer CAR T cell multiantigen specificity

The emergence of immune escape is a significant roadblock to developing effective chimeric antigen receptor (CAR) Tcell therapies against hematological malignancies, including acute myeloid leukemia (AML). Here, we demonstrate feasibility of targeting two antigens simultaneously by combining a GRP78-specific peptide antigen recognition domain with a CD123-specific scFv to generate a peptide-scFv bispecific antigen recognition domain (78.123). To achieve this, we test linkers with varying length and flexibility and perform immunophenotypic and functional characterization. We demonstrate that bispecific CAR Tcells successfully recognize and kill tumor cells that express GRP78, CD123, or both antigens and have improved antitumor activity compared to their monospecific counterparts when both antigens are expressed. Protein structure prediction suggests that linker length and compactness influence the functionality of the generated bispecific CARs. Thus, we present a bispecific CAR design strategy to prevent immune escape in AML that can be extended to other peptide-scFv combinations.

Systemically administered low-affinity HER2 CAR T cells mediate antitumor efficacy without toxicity

BackgroundThe paucity of tumor-specific targets for chimeric antigen receptor (CAR) T-cell therapy of solid tumors necessitates careful preclinical evaluation of the therapeutic window for candidate antigens. Human epidermal growth factor...

Chimeric Antigen Receptor-T (CAR-T) Cells as “Living Drugs”: A Clinical Pharmacist Perspective

Background. Chimeric antigen receptor (CAR) T cell therapy, a “living drug” immunotherapy, harnesses the power of T-cells from a patient (autologous) or healthy donor (allogeneic) to target and kill cancer cells and has shown unprecedented outcomes in patients with relapsed and refractory malignancies. Treatment with CAR-T cells requires the application of unique skillsets in recognised specialist centres for successful outcomes and requires management by the multidisciplinary team incorporating the specialist pharmacist. Method. A multimodal research strategy was employed for this literature review whereby PubMed, Google Scholar, Embase, Stella Library Search, EMA website, and EBMT website were sources of information. The search was limited from 2020 onwards with key terms referring to CAR-T cell therapy. Results and Discussion. There are six CAR-T cell products currently approved by the European Medicines Agency (EMA) and Food and Drug Administration (FDA) which target haematological malignancies with abundant clinical trials underway exploring new and improved CAR designs and antigen targets. As CAR-T cell therapy is an advanced therapy medicinal product (ATMP), there is need for an extensive regulatory framework underpinning its safety and efficacy. The clinical pharmacist plays an integral role in the provision of safe and effective CAR-T cell therapy including governance, operational and clinical aspects of treatment. Pharmacists may also be involved through provision of “Qualified Person” (QP) expertise in clinical trials and for release within hospitals under certain circumstances. There is a need for harmonised and accessible guidance on the clinical delivery of ATMPs such as CAR-T cells, with fully delineated responsibilities of pharmacists involving the oversight and supervision of CAR-T cell treatment. Conclusion. There is an unmet need to provide suitable and applicable literature for clinical pharmacists who are involved in the delivery of CAR-T cells. We have provided an overview of T-cell biology and an explanation of CAR-T cell design and the biomanufacturing process. We reviewed the complex and multifaceted treatment cycle requiring considerable logistics, and described the involvement of the clinical pharmacist in each part of this cycle from patient selection to postinfusion care. Finally, we look to the challenges and future opportunities that will require the involvement of the clinical pharmacist.

Endogenous Signaling Molecule Activating (ESMA) CARs: A Novel CAR Design Showing a Favorable Risk to Potency Ratio for the Treatment of Triple Negative Breast Cancer.

As chimeric antigen receptor (CAR) T cell therapy continues to gain attention as a valuable treatment option against different cancers, strategies to improve its potency and decrease the side effects associated with this therapy have become increasingly relevant. Herein, we report an alternative CAR design that incorporates transmembrane domains with the ability to recruit endogenous signaling molecules, eliminating the need for stimulatory signals within the CAR structure. These endogenous signaling molecule activating (ESMA) CARs triggered robust cytotoxic activity and proliferation of the T cells when directed against the triple-negative breast cancer (TNBC) cell line MDA-MB-231 while exhibiting reduced cytokine secretion and exhaustion marker expression compared to their cognate standard second generation CARs. In a NOD SCID Gamma (NSG) MDA-MB-231 xenograft mouse model, the lead candidate maintained longitudinal therapeutic efficacy and an enhanced T cell memory phenotype. Profound tumor infiltration by activated T cells repressed tumor growth, further manifesting the proliferative capacity of the ESMA CAR T cell therapy. Consequently, ESMA CAR T cells entail promising features for improved clinical outcome as a solid tumor treatment option.

In vitro re-challenge of CAR T cells.

Chimeric antigen receptor (CAR) T cells (CAR T) have emerged as a potential therapy for cancer patients. CAR T cells are capable of recognizing membrane proteins on cancer cells which initiates a downstream signaling in T cells that ends in cancer cell death. Continuous antigen exposure over time, activation of inhibitory signaling pathways and/or chronic inflammation can lead to CAR T cell exhaustion. In this context, the design of CARs can have a great impact on the functionality of CAR T cells, on their potency and exhaustion. Here, using CD19CAR as model, we provide a re-challenge protocol where CAR T cells are cultured weekly with malignant lymphoid cell lines BL-41 and Nalm-6 to simulate them with continuous antigen pressure over a four-week period. This protocol can be value for assessing CAR T cell functionality and for the comparison of different CAR constructs.

CAR NK Cell Therapy for the Treatment of Metastatic Melanoma: Potential & Prospects.

Melanoma is among the most lethal forms of cancer, accounting for 80% of deaths despite comprising just 5% of skin cancer cases. Treatment options remain limited due to the genetic and epigenetic mechanisms associated with melanoma heterogeneity that underlie the rapid development of secondary drug resistance. For this reason, the development of novel treatments remains paramount to the improvement of patient outcomes. Although the advent of chimeric antigen receptor-expressing T (CAR-T) cell immunotherapies has led to many clinical successes for hematological malignancies, these treatments are limited in their utility by their immune-induced side effects and a high risk of systemic toxicities. CAR natural killer (CAR-NK) cell immunotherapies are a particularly promising alternative to CAR-T cell immunotherapies, as they offer a more favorable safety profile and have the capacity for fine-tuned cytotoxic activity. In this review, the discussion of the prospects and potential of CAR-NK cell immunotherapies touches upon the clinical contexts of melanoma, the immunobiology of NK cells, the immunosuppressive barriers preventing endogenous immune cells from eliminating tumors, and the structure and design of chimeric antigen receptors, then finishes with a series of proposed design innovations that could improve the efficacy CAR-NK cell immunotherapies in future studies.

Role of Cell Therapy Product Characteristics in Patient Response to TAK-940 (CD19(T2)28z1xx Chimeric Antigen Receptor (CAR) T): Biomarker Analyses from a Phase I Study in Adult Patients with Relapsed or Refractory B-Cell Malignancies

TAK-940 (CD19(T2)28z1XX Chimeric Antigen Receptor (CAR) T) is an autologous CD19 CAR T cell therapy utilizing an optimized 1XX signaling domain substituting for the natural CD3ζ immunoreceptor tyrosine-based activation motifs. This 1XX design extends the functional persistence of CAR T cells with increased potency in mice (Feucht J et al. Nat Med 2019). TAK-940 is being evaluated in a Ph1 trial in patients with Relapsed or Refractory B-cell malignancies (NCT04464200) with demonstrated early signals of clinical efficacy (Park J et al. ASH 2022). The present study aims to provide a better understanding of the clinical performance of CD19(T2)28z1XX CAR T cells by in depth characterization of the cellular products and patient samples from this Ph1 trial. A total of 28 subjects were enrolled and treated in the dose escalation and expansion cohorts of the Ph1 trial. Responses were observed across all dose levels (25x10 6 to 200x10 6 CAR T cells), achieving ORR 82%, CR 71%. Immunophenotyping by flow cytometry was conducted on patient apheresis material, un-transduced cells, transduced cellular drug product and peripheral blood samples from infused subjects. Circulating cytokine profiling was performed on patient serum samples collected pre and post treatment with TAK-940. In the transduced cellular drug products, CAR+ and CAR- CD8+ central memory and transitional memory T cells were more abundant than naïve, stem cell memory or terminal effector populations. This elevation in central and transitional memory T cells seen in the drug product was not observed in the patient apheresis material, indicating that this effect is likely driven by the production process. CD57 and TIM3 expression in both CD4+ and CD8+ CAR+ T cells in the drug product showed a positive association with clinical response, while CTLA4 or LAG3 expression independently was negatively associated with response. The drug product also contained low numbers of dysregulated/exhausted T cells, with no observable difference between responders and non-responders. Interestingly, in the starting apheresis material, increased expression of TIM3 as well as decreased expression of either LAG3 or KLRG1 in CD8 T cells, independently associated with responses. Current analysis is based on associations with complete response. Additional analyses to be reported will include associations with ORR and durability of responses. These findings suggest potential utility of these T cell markers in predicting patient response and warrant evaluation in a larger population. Although CD57 expression has been associated with a senescent phenotype, we hypothesize that the observed increase in CD57 expression in CD4+ and CD8+ CAR+ T cells in the product may indicate higher cytotoxic potential of those cells. Increase in CD57+ CAR+ T cell subsets has been previously associated with durable CRs in large B cell lymphoma patients treated with axi-cel (Good Z et al. Nat Med 2022). Additional analyses to further characterize the CD57+ cell populations and establish their role in clinical responses to CAR T cell therapy with the 1XX CAR design are in progress. The increased expression of CD57 and the activation marker TIM3, and decreased expression of inhibitory receptors CTLA4 and LAG3 in the drug product from responders, together with the robust clinical responses observed with doses as low as 25x10 6 CAR T cells, suggest that the product consists of highly potent T cells, in accordance with the preclinical results obtained with the 1XX CAR. Low levels of circulating cytokines were observed following treatment with TAK-940, including IFNg, IL-6, IL-10, indicating a safe and tolerable CAR T product consistent with the observed clinical safety profile of TAK-940. Correlative analysis of T cell immunophenotypes observed in the product to clinical parameters including durability of responses, metabolic tumor volume/tumor burden, and induction of cytokines and chemokines is ongoing and will be reported. This will further contribute to our understanding of CD19 CAR T cells endowed with a 1XX signaling domain and potentially support the identification of product and/or apheresis biomarkers that may predict patient response.

Targeting Normal Myelopoiesis Negatively Affects CAR T Cell Activity

Chimeric antigen receptor (CAR) T cells have not been successful for the treatment of acute myeloid leukemia (AML) to date, and the reason for this is not clear. While CAR T cells have been traditionally studied as independent agents targeting tumor, in reality they work in the context of the endogenous host immune system, which may influence outcomes of therapy. We hypothesized that targeting normal myelopoiesis is detrimental to CAR T cell activity, and this creates a unique obstacle to effective CAR T cell therapy for AML. We generated prototypical CAR T cells targeting B cell (CD19) and myeloid (CD33) antigens, using a standard 2 nd generation CAR design with 4-1BB and CD3ζ costimulatory domains. In parallel we created tumor cell lines expressing both targets by adding CD33 expression to a CD19+ B cell line (Ramos +33), and by adding CD19 expression to a CD33+ myeloid cell line (THP1 +19). For ease of tracking tumor cells were labeled with GFP, and CAR T cells were labeled with RFP. CD19-targeting CAR T cells (CART19) and CD33-targeting CAR T cells (CART33) exhibited similar anti-tumor activity in standard in vitro cytotoxicity assays and in vivo NSG mouse models. We then proceeded to add PBMCs to CAR T cell and tumor co-culture and measured tumor cell clearance and CAR T cell expansion using flow cytometry and the Incucyte Live-Cell analysis system. Addition of PBMCs enhanced the tumor clearance and CAR T cell expansion of both CART19 and CART33 in short-term co-culture assays (7-10 days). Fractionation of PBMCs revealed that CD33+ classical monocytes were able to replicate the effect of PBMCs, while the presence of CD19+ B cells did not have any effect. Monocytes are viable in vitro for less than 7 days. To assess the long-term effects of monocytes on CAR T cells, we performed serial replating assays, in which fresh tumor cells and monocytes were added to CAR T cells every 7 days. Ongoing monocyte support further improved CART19 expansion and tumor clearance. However, the continuous presence of monocytes was detrimental to CART33 activity upon serial replating, as evidenced by suboptimal T cell expansion and loss of tumor control. To study the effects of normal myelopoiesis on CAR T cell activity in vivo, NSGS mice (NOD/SCID/IL2Rg null mice with human SCF, IL-3 and GM-CSF expression) were injected with human cord blood CD34+ cells to engraft human hematopoiesis. Mice were then injected with THP1 +19 tumor cells, followed by administration of untransduced (UTD) control T cells, CART19 or CART33. To prevent the confounding effect of allogeneic killing, CAR T cells were generated from the same cord blood product as the CD34+ cells. Consistent with our in vitro data, CART19 and CART33 showed better anti-tumor activity in the presence of human hematopoiesis. Notably, while the anti-tumor activity of CART19 and CART33 was equivalent in non-humanized mice, CART19 had better long-term activity in humanized mice, as mice treated with CART33 had late tumor relapses. To directly compare the effects of myeloid cells as bystanders versus targets, we lentivirally transduced cord blood CD34+ cells with human CD19 so that they would be targets for CART19. We then differentiated these cells into myeloid progenitors (MP) by culturing in IMDM with 20% FBS and the following cytokines: SCF (100ng/ml), FLT3L (50ng/ml), IL-3 (10ng/ml), and GM-CSF (10ng/ml). After 10 days of culture >95% of MP expressed CD33, and were selected for >95% CD19 expression (Fig A). Addition of non-transduced MP to CAR T cell and Ramos +33 co-culture enhanced CART19-mediated tumor clearance without affecting CART19 expansion. However, the presence of MP CD19+ had a detrimental effect on both tumor control and expansion of CART19, confirming that CAR T cells are less effective when targeting myeloid cells. In contrast, both MP and MP CD19+ had similar effects on CART33 that mirrored the negative effects of MP CD19+ on CART19 (Fig B). To summarize, we show that normal myeloid cells have beneficial effects on CAR T cell function, and targeting these same myeloid cells is detrimental to CAR T cell activity. These findings provide insight into the reason for suboptimal CAR T cell activity in AML, and show that independent of target antigen and CAR construct, the nature of the cells being targeted can change the effectiveness of this therapy.

Harnessing TCR/CAR Antagonism to Enhance Immunotherapeutic Precision

Chimeric Antigen Receptor (CAR) T cell immunotherapy represents a conceptual breakthrough in the treatment of hematological malignancies. However, the rarity of cell surface protein targets specific to cancerous but not vital tissue has hindered its broad application to solid tumor treatment. While new logic-gated CAR designs have shown reduced toxicity against healthy tissues, the generalizability of such approaches across tumors remains unclear. Here, we harness a universal characteristic of endogenous T cell receptors (TCRs), their ability to discriminate between self and non-self ligands through inhibition of response against self (weak) antigens, to develop a broadly applicable method of enhancing immunotherapeutic precision. We hypothesized that this discriminatory mechanism, known as antagonism, would apply across receptors, allowing for a transfer of specificityfrom TCRs onto CARs. We therefore systematically mapped out the responses of CAR T cells to joint TCR and CAR stimulations. We transduced ovalbumin-specific TCR T cells with mouse CD19 CAR to produce T cells expressing both TCR and CAR and evaluated the response of TCR/CAR T cells using in vivo and in vitro leukemia models ( Figure 1A). We discovered that strong TCR antigen enhanced CAR T killing of CD19 + leukemia, while weak TCR antigen antagonized CAR T responses as assessed in vivo cytotoxicity and in vitro multiplexed dynamic profiling ( Figure 1B). We developed a mathematical model based on cross-receptor inhibitory coupling that accurately predicted the extent of TCR/CAR antagonism across a wide range of immunological settings. This model was validated in a CD19 + B16 mouse melanoma model showing that TCR/CAR antagonism decreased the infiltration of a tumor-reactive T cell cluster (cluster 1), while TCR/CAR agonism enhanced infiltration of this cluster1 ( Figure 1C). We then applied our quantitative knowledge of TCR/CAR crosstalk to design an Antagonism-Enforced Braking System (AEBS) for CAR T cell therapy. This was assessed in a model system using a CAR targeting the tyrosine-protein kinase erbB-2 (HER2), expressed on a subset of patients with both B-ALL and AML together with a hedgehog acyltransferase (HHAT) specific TCR, which responds strongly to mutated peptides presented on tumor cells and weakly to wild-type peptides presented on healthy tissue. Consistent with our discovery of the TCR/CAR antagonism, TCR signals against healthy cells expressing wild-type HHAT peptide antigen antagonized HER2 CAR T cell responses, minimizing on-target/off-tumor cytotoxicity against healthy cells. Notably though, AEBS-CAR T cells exhibited high anti-tumor cytotoxicity against tumor cells expressing HER2 and mutated HHAT peptides ( Figure 1D). AEBS CAR T cells sharpen the discriminatory power of synthetic anti-tumor lymphocytes, laying the groundwork for future studies to engineer complex logic into cells with minimal numbers of receptors ( Figure 1E). Our work highlights a novel mechanism by which TCRs can enforce CAR T cell specificity, with practical implications for the rational design of future anti-leukemia immunotherapies.