Current Chimeric Antigen Receptor Research Articles

Chimeric antigen receptor T cells targeting the GM3(Neu5Gc) ganglioside.

Chimeric antigen receptor (CAR) T cell technology has ushered in a new era of immunotherapy, enabling the targeting of a broad range of surface antigens, surpassing the limitations of traditional T cell epitopes. Despite the wide range of non-protein tumor-associated antigens, the advancement in crafting CAR T cells for these targets has been limited. Owing to an evolutionary defect in the CMP-Neu5Ac hydroxylase (CMAH) that abolishes the synthesis of CMP-Neu5Gc from CMP-Neu5Ac, Neu5Gc is generally absent in human tissues. Despite this, Neu5Gc-containing antigens, including the ganglioside GM3(Neu5Gc) have consistently been observed on tumor cells across a variety of human malignancies. This restricted expression makes GM3(Neu5Gc) an appealing and highly specific target for immunotherapy. In this study, we designed and evaluated 14F7-28z CAR T cells, with a targeting unit derived from the GM3(Neu5Gc)-specific murine antibody 14F7. These cells exhibited exceptional specificity, proficiently targeting GM3(Neu5Gc)-expressing murine tumor cells in syngeneic mouse models, ranging from B cell malignancies to epithelial tumors, without compromising safety. Notably, human tumor cells enhanced with murine Cmah were effectively targeted and eliminated by the 14F7 CAR T cells. Nonetheless, despite the detectable presence of GM3(Neu5Gc) in unmodified human tumor xenografts, the levels were insufficient to trigger a tumoricidal T-cell response with the current CAR T cell configuration. Overall, our findings highlight the potential of targeting the GM3(Neu5Gc) ganglioside using CAR T cells across a variety of cancers and set the stage for the optimization of 14F7-based therapies for future human clinical application.

Oncolytic viruses improve cancer immunotherapy by reprogramming solid tumor microenvironment.

Immunotherapies using immune checkpoint inhibitors (ICIs) and chimeric antigen receptor (CAR) T-cell therapy have achieved successful results against several types of human tumors, particularly hematological malignancies. However, their clinical results for the treatment of solid tumors remain poor and unsatisfactory. The immunosuppressive tumor microenvironment (TME) plays an important role by interfering with intratumoral T-cell infiltration, promoting effector T-cell exhaustion, upregulating inhibitory molecules, inducing hypoxia, and so on. Oncolytic viruses are an encouraging biocarrier that could be used in both natural and genetically engineered platforms to induce oncolysis in a targeted manner. Oncolytic virotherapy (OV) contributes to the reprogramming of the TME, thus synergizing the functional effects of current ICIs and CAR T-cell therapy to overcome resistant barriers in solid tumors. Here, we summarize the TME-related inhibitory factors affecting the therapeutic outcomes of ICIs and CAR T cells and discuss the potential of OV-based approaches to alleviate these barriers and improve future therapies for advanced solid tumors.

Charging CAR by electrostatic power.

Chimeric antigen receptor (CAR)-T cell therapy has emerged as a promising approach for cancer treatment. CAR is a synthetic immune receptor that recognizes tumor antigen and activates T cells through multiple signaling pathways. However, the current CAR design is not as robust as T cell receptor (TCR), a natural antigen receptor with high sensitivity and efficiency. TCR signaling relies on specific molecular interactions, and thus electrostatic force, the major force of molecular interactions, play critical roles. Understanding how electrostatic charge regulates TCR/CAR signaling events will facilitate the development of next-generation T cell therapies. This review summarizes recent findings on the roles of electrostatic interactions in both natural and synthetic immune receptor signaling, specifically that in CAR clustering and effector molecule recruitments, and highlights potential strategies for engineering CAR-T cell therapy by leveraging charge-based interactions.

Hypoimmune anti-CD19 chimeric antigen receptor T cells provide lasting tumor control in fully immunocompetent allogeneic humanized mice

Manufacturing autologous chimeric antigen receptor (CAR) T cell therapeutics is complex, and many patients experience treatment delays or cannot be treated at all. Although current allogeneic CAR products have the potential to overcome manufacturing bottlenecks, they are subject to immune rejection and failure to persist in the host, and thus do not provide the same level of efficacy as their autologous counterparts. Here, we aimed to develop universal allogeneic CAR T cells that evade the immune system and produce a durable response. We generated human hypoimmune (HIP) T cells with disrupted B2M, CIITA, and TRAC genes using CRISPR-Cas9 editing. In addition, CD47 and anti-CD19 CAR were expressed using lentiviral transduction. These allogeneic HIP CD19 CAR T cells were compared to allogeneic CD19 CAR T cells that only expressed the anti-CD19 CAR (allo CAR T). In vitro assays for cancer killing and exhaustion revealed no differences between allo CAR T and HIP CAR T cells, confirming that the HIP edits did not negatively affect T cell performance. Clearance of CD19+ tumors by HIP CAR T cells in immunodeficient NSG mice was comparable to that of allo CAR T cells. In fully immunocompetent humanized mice, HIP CAR T cells significantly outperformed allo CAR T cells, showed improved persistence and expansion, and provided lasting cancer clearance. Furthermore, CD47-targeting safety strategies reliably and specifically eliminated HIP CAR T cells. These findings suggest that universal allogeneic HIP CAR T cell-based therapeutics might overcome the limitations associated with poor persistence of allogeneic CAR T cells and exert durable anti-tumor responses.

Therapeutic implication of chimeric antigen receptor T-cell therapy for head and neck squamous cell carcinoma.

Therapeutic implication of chimeric antigen receptor T-cell therapy for head and neck squamous cell carcinoma.

Engineered exosome-mediated messenger RNA and single-chain variable fragment delivery for human chimeric antigen receptor T-cell engineering

Background aimsMost current chimeric antigen receptor (CAR) T cells are generated by viral transduction, which induces persistent expression of CARs and may cause serious undesirable effects. Messenger RNA (mRNA)-based approaches in manufacturing CAR T cells are being developed to overcome these challenges. However, the most common method of delivering mRNA to T cells is electroporation, which can be toxic to cells. MethodsThe authors designed and engineered an exosome delivery platform using the bacteriophage MS2 system in combination with the highly expressed protein lysosome-associated membrane protein 2 isoform B on exosomes. ResultsThe authors’ delivery platform achieved specific loading and delivery of mRNA into target cells and achieved expression of specific proteins, and anti-CD3/CD28 single-chain variable fragments (scFvs) expressed outside the exosomal membrane effectively activated primary T cells in a similar way to commercial magnetic beads. ConclusionsThe delivery of CAR mRNA and anti-CD3/CD28 scFvs via designed exosomes can be used for ex vivo production of CAR T cells with cancer cell killing capacity. The authors’ results indicate the potential applications of the engineered exosome delivery platform for direct conversion of primary T cells to CAR T cells while providing a novel strategy for producing CAR T cells in vivo.

CAR immune cells: design principles, resistance and the next generation.

The remarkable clinical activity of chimeric antigen receptor (CAR) therapies in B cell and plasma cell malignancies has validated the use of this therapeutic class for liquid cancers, but resistance and limited access remain as barriers to broader application. Here we review the immunobiology and design principles of current prototype CARs and present emerging platforms that are anticipated to drive future clinical advances. The field is witnessing a rapid expansion of next-generation CAR immune cell technologies designed to enhance efficacy, safety and access. Substantial progress has been made in augmenting immune cell fitness, activating endogenous immunity, arming cells to resist suppression via the tumour microenvironment and developing approaches to modulate antigen density thresholds. Increasingly sophisticated multispecific, logic-gated and regulatable CARs display the potential to overcome resistance and increase safety. Early signs of progress with stealth, virus-free and in vivo gene delivery platforms provide potential paths for reduced costs and increased access of cell therapies in the future. The continuing clinical success of CAR T cells in liquid cancers is driving the development of increasingly sophisticated immune cell therapies that are poised to translate to treatments for solid cancers and non-malignant diseases in the coming years.

Human VH-based chimeric antigen receptor T cells targeting glypican 3 eliminate tumors in preclinical models of HCC.

Efficacy of chimeric antigen receptor (CAR) T cells for treating solid tumors, including HCC, remains a challenge. Nanobodies are emerging building blocks of CAR T cells due to their small size and high expression. Membrane proximal sites have been shown as attractive epitopes of CAR T cells. However, current CAR formats are not tailored toward nanobodies or targeting membrane distal epitopes. Using hYP7 Fv (membrane proximal) and HN3 VH nanobody (membrane distal) as GPC3 targeting elements, we sought to determine how hinges and transmembrane portions of varying structures and sizes affect CAR T-cell function. We generated multiple permutations of CAR T cells containing CD8, CD28, IgG4, and Fc domains. We show that engineered HN3 CAR T cells can be improved by 2 independent, synergistic changes in the hinge and transmembrane domains. The T cells expressing the HN3 CAR which contains the hinge region of IgG4 and the CD28 transmembrane domain (HN3-IgG4H-CD28TM) exhibited high cytotoxic activity and caused complete HCC tumor eradication in immunodeficient mice. HN3-IgG4H-CD28TM CAR T cells were enriched for cytotoxic-memory CD8+ T cells and NFAT signals, and reduced β catenin levels in HCC cells. Our findings indicate that altering the hinge and transmembrane domains of a nanobody-based CAR targeting a distal GPC3 epitope, in contrast to a membrane proximal epitope, lead to robust T-cell signaling and induce swift and durable eradication of HCC tumors.

Chimeric Antigen Receptor (CAR) T-cell Treatment in Renal Cell Carcinoma: Current Clinical Trials and Future Directions

Renal cell carcinoma (RCC) has long been found to be responsive to immunotherapy. While high dose interleukin-2 resulted in some durable remissions, this treatment has largely been replaced by immune checkpoint inhibitor therapy, due to the safer toxicity profile and emerging evidence for long term remissions. However, the majority of patients continue to face disease progression and death from metastatic RCC. Chimeric antigen receptor T-cells (CAR T) represent the next step in immunotherapy for this malignancy and hold promise for a higher rate of durable remissions. The realization of this therapeutic strategy for RCC will require identification of the best tumor antigen and T cell modifications and will depend on achieving remissions with an acceptable toxicity profile. This review summarizes current CAR T-cell treatment targets and clinical trials for metastatic RCC, highlighting the potential therapeutic impact as well as obstacles to successful development.

Utilization of the Tapestri Platform for Quantification of Single Cell Vector Copy Number in CAR T Cell Products

Chimeric antigen receptor (CAR) T cell immunotherapies have been transformative solutions to treat both adult and pediatric patients with a variety of cancers. Given that CAR T cell therapies involve genetic alterations of a patient's T cells by introduction of the CAR into host cells using lentiviral vectors, the quality of CAR T cell therapies are extensively regulated to warrant safety. Key safety and efficacy attributes need to be accurately measured prior to reintroducing modified T cells back into patients, including CAR transgene copy number or viral vector copy number (VCN). We have previously compared digital PCR (dPCR) strategies to the historical standard release assay of quantitative PCR (qPCR) for VCN quantification given that dPCR is known to provide absolute quantification of target nucleic acid sequences with many advantages over qPCR in regards to simplicity, reproducibility, lower limit of detection, and definitive quantification. We were able to demonstrate that dPCR produces accurate, precise, and reproducible CAR T cell VCN measurements when compared to qPCR. However, these conventional PCR methods measuring vector copy number are either population averaged or involve labor-intensive and time-consuming single cell colony picking and outgrowth, with respective drawbacks in resolution and sample representation. Here, we introduce the Tapestri platform, to measure thousands of single cell level VCN with accuracy and precision. The Tapestri platform workflow is enabled by its two droplet steps. First, thousands of cells are each microfluidically encapsulated in a first droplet where lysis of the cells occur, releasing DNA from its heterochromatin state for uniform DNA interrogation. Then, the individual lysates are uniquely barcoded and target sequences amplified via multiplexed PCR inside each droplet. The final products are sequenced on an Illumina sequencing instrument. Single cell VCN Tapestri data was generated and orthogonally compared with dPCR and matches with the bulk measurement on clonal Jurkat cell lines (VCN=2) mixed with control cell lines (VCN=0) at a range of different ratios with accuracy and precision. We demonstrate that the Tapestri platform provides absolute quantification of CAR transgenes at the single cell level and has the potential to provide more precise measurements of CAR T cell therapeutic product attributes prior to release to patients. Future studies will evaluate the utility of this Tapestri platform in measuring both CAR transgene copies and surface expression/density of the CAR at the single cell level, further improving upon current CAR T cell product release assays.

Recent Advances in the Development of Anti-FLT3 CAR T-Cell Therapies for Treatment of AML.

Following the success of the anti-CD19 chimeric antigen receptor (CAR) T-cell therapies against B-cell malignancies, the CAR T-cell approach is being developed towards other malignancies like acute myeloid leukemia (AML). Treatment options for relapsed AML patients are limited, and the upregulation of the FMS-like tyrosine kinase 3 (FLT3) in malignant T-cells is currently not only being investigated as a prognostic factor, but also as a target for new treatment options. In this review, we provide an overview and discuss different approaches of current anti-FLT3 CAR T-cells under development. In general, these therapies are effective both in vitro and in vivo, however the safety profile still needs to be further investigated. The first clinical trials have been initiated, and the community now awaits clinical evaluation of the approach of targeting FLT3 with CAR T-cells.

In This Issue

In This Issue

Overcoming resistance to anti-CD19 CAR T-cell therapy in B-cell malignancies.

Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy has rapidly changed current treatment pattern, providing a better option for individuals with primary refractory or relapsed B-cell non-Hodgkin lymphoma (r/r B-NHL) and B-cell acute lymphoblastic leukemia (r/r B-ALL). However, despite the outstanding efficacy, a high relapse rate is still found in some B-cell malignancies after anti-CD19 CAR T-cell therapy, which emerges as a main barrier for improving the overall response and long-term outcomes. Understanding the resistance mechanism is crucial to improve current CAR T products, better incorporate them into the current therapy system and develop novel CAR approaches. Herein, we discuss the latest advances in understanding the mechanisms limiting efficacy of CAR T-cell therapy, resulting in CD19 negative (CD19- ) and CD19 positive (CD19+ ) relapses. We also provide a whole scenario of current potential strategies to overcome these barriers.

Abstract 554: Development of an iPSC-derived NK cell screening platform for discovery of NK cell optimized Chimeric Antigen Receptors (CARs) for next-generation CAR-NK cell immunotherapies

Abstract Chimeric Antigen Receptor (CAR) immunotherapy has transformed cancer therapeutics and has shown great success in treating hematopoietic malignancies. To date, autologous CAR-T-cell therapy has been the main clinical approach. However, autologous CAR-T cells have several major limitations including high-cost and complexity of production, donor-to-donor variability, and potentially severe toxicities. Allogenic NK cell-based therapies have also been shown to mount potent responses against hematopoietic malignancies (i.e., AML). However, unlike T-cell therapies, allogeneic NK cells do not cause toxicities such as cytokine release syndrome, neurotoxicity, or graft-vs-host-disease. Therefore, this approach provides an important platform for allogeneic CAR-enabled therapies. Several groups have demonstrated the clinical efficacy of allogeneic CAR-expressing NK cells that utilize CARs developed for T-cells. However, recent studies using NK cell-based activating receptor signaling modules such as NKG2D and 2B4 as CAR co-stimulatory domains proved increased anti-tumor activity compared to T-cell CARs. The goal of this study was to develop next-generation NK cell-optimized CARs to improve targeting and potency of our iPSC-derived NK cell therapies. To achieve this, we developed a platform to comprehensively screen immune cell signaling modules in iPSC-derived NK cells. First, we constructed a library of 44 novel CAR constructs containing signaling modules from diverse NK cell specific signaling receptors. Next, we developed a viral-based CAR expression protocol in mature iPSC-derived NK cells that yields high CAR expression (>75% CAR+) while maintaining high viability (>90%). We then screened for CAR activity using two co-culture target cell killing assays (eSight impedance assay and caspase 3/7 killing assay) and two NK cell-resistant CD19+ target cell lines (Raji and SupB15). Our screen identified 7 novel CAR signaling modules that performed better than both a second-generation T-cell CAR (CD28-CD28-zeta) and 3 previously described NK cell-CARs (NKG2D-2B4-zeta, CD28-OX40-zeta, and CD28-OX40L-zeta). These results were consistent across both functional assays and both target cell lines tested adding support to our findings. Overall, we have successfully developed an efficient CAR screening platform in iPSC-derived NK cells and have identified NK optimized CARs that enable increased potency of NK cells compared to current CAR solutions. The most potent CARs identified in our screen are being engineered into CISH-knockout iPSC-derived NK cells which were previously developed by us that demonstrate improved in vivo anti-tumor activity, in vivo persistence, metabolic fitness, polyfunctionality and resistance to cell exhaustion to create next-generation CAR-NK cell-based immunotherapies. Citation Format: Lee Swanson, Gabrielle Allen, Kenyon Lyon, Wael Tadros, Max Shabla, Dan S. Kaufman, Huang Zhu. Development of an iPSC-derived NK cell screening platform for discovery of NK cell optimized Chimeric Antigen Receptors (CARs) for next-generation CAR-NK cell immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 554.

Chimeric antigen receptor clustering via cysteines enhances T-cell efficacy against tumor.

Chimeric antigen receptor (CAR) T-cell therapy achieves great success for hematological malignancies. However, clinical trials have revealed some limitations in both improving the efficacy and reducing the relapse, which calls for innovative strategies to engineer more powerful CAR-T cells. Promoting the formation of CAR clusters provides an alternative approach and potentially improves current CAR T-cell therapy against cancers. Here, we generated CARCys-T cells using a 4-1BB-derived hinge region including 11 cysteines residues. The cysteines in the hinge were found to facilitate CARCys clustering upon antigen stimulation and promote the antitumor activity of CAR-T cells. Compared with most conventionally used CAR-T cells with CD8α-derived hinge (CARconv-T cells), CARCys-T cells exhibited larger diameter of CAR clusters and enhanced antigen-specific tumor lysis both in vitro and in vivo. In addition, the CARCys-mediated enhancement could be applied to HER2, CD19 as well as GPC3-targeted CAR-T cells. More importantly, CARCys-T cells showed potent antitumor efficacy in clinically relevant patient-derived primary tumor cells and organoids. Thus, the novel hinge containing 11 cysteines provides a promising strategy to facilitate CAR clustering and maximize anti-tumor activity of CAR-T cells, which emphasizes the importance of CAR clustering to improve CAR T-cell therapy in the clinic.

Characterizing piggyBat—a transposase for genetic modification of T cells

Chimeric antigen receptor (CAR) T cells targeting CD19 have demonstrated remarkable efficacy in the treatment of B cell malignancies. Current CAR T cell manufacturing protocols are complex and costly due to their reliance on viral vectors. Non-viral systems of genetic modification, such as with transposase and transposon systems, offer a potential streamlined alternative for CAR T cell manufacture and are currently being evaluated in clinical trials. In this study, we utilized the previously described transposase from the little brown bat, designated piggyBat, for production of CD19-specific CAR T cells. PiggyBat demonstrates efficient CAR transgene delivery, with a relatively low variability in integration copy number across a range of manufacturing conditions as well as a similar integration site profile to super-piggyBac transposon and viral vectors. PiggyBat-generated CAR T cells demonstrate CD19-specific cytotoxic efficacy in vitro and in vivo. These data demonstrate that alternative, naturally occurring DNA transposons can be efficiently re-tooled to be exploited in real-world applications.

CAR T Cell Therapy in Primary Brain Tumors: Current Investigations and the Future.

Chimeric antigen receptor T cells (CAR T cells) are engineered cells expressing a chimeric antigen receptor (CAR) against a specific tumor antigen (TA) that allows for the identification and elimination of cancer cells. The remarkable clinical effect seen with CAR T cell therapies against hematological malignancies have attracted interest in developing such therapies for solid tumors, including brain tumors. Glioblastoma (GBM) is the most common primary brain tumor in adults and is associated with poor prognosis due to its highly aggressive nature. Pediatric brain cancers are similarly aggressive and thus are a major cause of pediatric cancer-related death. CAR T cell therapy represents a promising avenue for therapy against these malignancies. Several specific TAs, such as EGFR/EGFRvIII, IL13Rα2, B7-H3, and HER2, have been targeted in preclinical studies and clinical trials. Unfortunately, CAR T cells against brain tumors have showed limited efficacy due to TA heterogeneity, difficulty trafficking from blood to tumor sites, and the immunosuppressive tumor microenvironment. Here, we review current CAR T cell approaches in treating cancers, with particular focus on brain cancers. We also describe a novel technique of focused ultrasound controlling the activation of engineered CAR T cells to achieve the safer cell therapies. Finally, we summarize the development of combinational strategies to improve the efficacy and overcome historical limitations of CAR T cell therapy.

Efficient Human Acute Myeloid Leukemia Targeting By Universal Chimeric Antigen Receptor T-Cells Via Combinatorial Use of Linkers

Chimeric antigen receptor (CAR) T-cells are genetically engineered T-cells with potent biocidal activity against respective target-expressing cells. Recently, CAR T-cells have been successfully used clinically to eradicate B cell-derived malignancies by targeting B-cell lineage specific surface antigens (e.g. CD19, BCMA). However, several limitations of current single-antigen targeting CAR T-cell therapies are becoming evident: a) low target tumor-antigen expression might lead to low CAR T-cell targeting efficacy; b) single antigen-targeting might lead to rapid selection of tumor cells with low or loss of antigen expression; c) single antigen-targeting does frequently not generate a high tumor-selectivity as tumor-antigens are frequently also expressed on healthy tissues; d) production of single-antigen CAR T-cells is time- and resource-consuming but results in effectors that target only one antigen; e) on-target off-tumor as well as off-target side-effects are difficult to control without terminally eliminating CAR T-cells in the recipient. While a-c) might be overcome by combinatorial tumor-antigen targeting, d-e) might be addressed by production of Universal CAR T-cells that recognize a specific tag on selective bridging molecules with short half-life.To address some of these limitations in principle, we have here developed universal CAR T-cells targeting fluorescein, as well as fluorescein-labeled antibody-constructs directed against several cell surface antigens, that would serve as versatile, combinatorial selective linkers and, upon target binding, also CAR T-cells activators. We then tested the system on human acute myeloid leukemia (AML) cell lines and primary patient AML blasts. Specifically, we engineered CAR T-cells, termed FluA-CAR T-cells, to display the anti-fluorescein engineered lipocalin FluA, which mediated recognition of randomly or site-specifically fluorescein-labeled antibodies in IgG or short half-life diabody (Db) format, directed against the frequently AML expressed antigens CD33, CD117 and CD371. Site-specific chemical modification methods and cysteine-tagged Db mediated the strongest AML killing results in vitro over a broad range of antibody concentrations.We then hypothesized that FluA-CAR T-cells, targeting AML cells via combinatorial use of linker molecules adapted to specific AML antigen-expression profiles, would allow to most efficiently eliminate AML cells in a dose- and timely-regulated fashion. To this end, we tested single and combinatorial use of fluoresceinated anti-CD117 Db and anti-CD371 Db on MOLM13 AML cell lines, engineered to be either CD117 highCD371 neg, CD117 negCD371 high, or CD117 highCD371 high. Indeed, combination of anti-CD117 and anti-CD371 Db-linkers, resulted in significantly improved MOLM13 cell lysis compared to equimolar concentrations of single agents in vitro (example figure). We then tested the same approach, targeting CD117 +CD371 + primary AML cells from two different patients, using either patient-derived or allogeneic FluA CAR T-cells. Again, the combinatorial use of linkers generated a significantly higher AML cell lysis than the use of single Db linkers.We thus here provide proof-of-concept for the generation of highly potent universal targeting FluA CAR T-cells from healthy donors and AML patients. By choosing suitable CAR-adaptors with respect to their conjugation chemistry and size, it is possible to tightly regulate CAR-T cell activity against CD33, CD117 and CD371 expressing AML cells and likely any tumor cell expressed antigen. Short half-life small molecule linkers will allow to control FluA CAR T-cell on-off activity and combinatorial use of linkers will allow to maximize anti-tumor activity and to minimize on-target off-tumor toxicity. [Display omitted] DisclosuresMyburgh: University of Zurich: Patents & Royalties: CD117xCD3 TEA. Neri: Philogen S.p.A.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Multiple patents on vascular targeting; ETH Zurich: Patents & Royalties: CD117xCD3 TEA. Manz: CDR-Life Inc: Consultancy, Current holder of stock options in a privately-held company; University of Zurich: Patents & Royalties: CD117xCD3 TEA.

Development of CMV-CD19 bi-specific CAR T cells with post-infusion in vivo boost using an anti-CMV vaccine.

Adoptive transfer of in vitro expanded, chimeric antigen receptor (CAR)-redirected CD19-specific T cells can induce dramatic disease regression in patients with leukemia and lymphomas. However, the full potential of this emerging modality is hampered in some cancer settings by a significant rate of therapeutic failure arising from the attenuated engraftment and persistence of CAR-redirected T cells, and tumor relapse following adoptive transfer. Here, we discuss an advanced strategy that facilitates post-infusion in vivo boosting of CAR T cells via CMV vaccination, to mediate durable remission of B cell malignancies by engrafting a CAR molecule onto a CMV-specific T cell. We also discuss a feasible and unique platform for the generation of the CMV-CD19CAR T cells for clinical application. This new approach would overcome multiple challenges in current CAR T cell technology including: short T cell persistence, limited duration of response, and inability to re-stimulate T cells after relapse or persistent disease.

Adenosine Deaminase 1 Overexpression Enhances the Antitumor Efficacy of Chimeric Antigen Receptor-Engineered T Cells.

Chimeric antigen receptor (CAR) T cell therapy mediates unprecedented benefit in certain leukemias and lymphomas, but has yet to achieve similar success in combating solid tumors. A substantial body of work indicates that the accumulation of adenosine in the solid tumor microenvironment (TME) plays a crucial role in abrogating immunotherapies. Adenosine deaminase 1 (ADA) catabolizes adenosine into inosine and is indispensable for a functional immune system. We have, for the first time, engineered CAR T cells to overexpress ADA. To potentially improve the pharmacokinetic profile of ADA, we have modified the overexpressed ADA in two ways, through the incorporation of a (1) albumin-binding domain or (2) collagen-binding domain. ADA and modified ADA were successfully expressed by CAR T cells and augmented CAR T cell exhaustion resistance. In a preclinical engineered ovarian carcinoma xenograft model, ADA and collagen-binding ADA overexpression significantly enhanced CAR T cell expansion, tumor tissue infiltration, tumor growth control, and overall survival, whereas albumin-binding ADA overexpression did not. Furthermore, in a syngeneic colon cancer solid tumor model, the overexpression of mouse ADA by cancer cells significantly reduced tumor burden and remodeled the TME to favor antitumor immunity. The overexpression of ADA for enhanced cell therapy is a safe, straightforward, reproducible genetic modification that can be utilized in current CAR T cell constructs to result in an armored CAR T product with superior therapeutic potential.