Synthetic Antigen Receptors Research Articles

Electrophilic proximity-inducing synthetic adapters enhance universal T cell function by covalently enforcing immune receptor signaling

Proximity-induction of cell-cell interactions via small molecules represents an emerging field in basic and translational sciences. Covalent anchoring of these small molecules represents a useful chemical strategy to enforce proximity; however, it remains largely unexplored for driving cell-cell interactions. In immunotherapeutic applications, bifunctional small molecules are attractive tools for inducing proximity between immune effector cells like Tcells and tumor cells to induce tumoricidal function. We describe a two-component system composed of electrophilic bifunctional small molecules and paired synthetic antigen receptors (SARs) that elicit Tcell activation. The molecules, termed covalent immune recruiters (CIRs), were designed to affinity label and covalently engage SARs. We evaluated the utility of CIRs to direct anti-tumor function of human Tcells engineered with three biologically distinct classes of SAR. Irrespective of the electrophilic chemistry, tumor-targeting moiety, or SAR design, CIRs outperformed equivalent non-covalent bifunctional adapters, establishing a key role for covalency in maximizing functionality. We determined that covalent linkage enforced early Tcell activation events in a manner that was dependent upon each SARs biology and signaling threshold. These results provide a platform to optimize universal SAR-T cell functionality and more broadly reveal new insights into how covalent adapters modulate cell-cell proximity-induction.

Identification of Core Techniques That Enhance Genome Editing of Human T Cells Expressing Synthetic Antigen Receptors.

Genome editing technologies have seen remarkable progress in recent years, enabling precise regulation of exogenous and endogenous genes. These advances have been extensively applied to the engineering of human T lymphocytes, leading to the development of practice changing therapies for patients with cancer and the promise of synthetic immune cell therapies for a variety of nonmalignant diseases. Many distinct conceptual and technical approaches have been used to edit T-cell genomes, however targeted assessments of which techniques are most effective for manufacturing, gene editing, and transgene expression are rarely reported. Through extensive comparative evaluation, we identified methods that most effectively enhance engineering of research-scale and preclinical T-cell products at critical stages of manufacturing.

Abstract 4021: The development of CAR T cell therapeutics targeting tumor specific CD146

Abstract Chimeric antigen receptor (CAR) T cells represent a promising class of "living drugs" for cancer treatment. These primary T cells are genetically engineered to express a synthetic antigen receptor, incorporating various domains to enhance their effector functions. While CAR T cells have shown success against liquid cancers, their application to solid cancers is hindered by significant "on-target/off-tumor" toxicities due to the absence of specific target proteins. In this study, we aim to enhance the tumor specificity of CAR T cells by targeting a tumor-specific version of CD146. CD146 is a glycoprotein widely expressed in several solid tumors, particularly melanoma and ovarian cancer. However, it is also expressed on benign cells, such as endothelial cells, pericytes, and smooth muscle cells. Nevertheless, a CD146 isoform exists that is enriched in cancer cells. We assessed CAR activity using flow cytometry to measure surface CD69 expression (an early activation marker) on CAR-expressing Jurkat cells co-cultured with various CD146-positive and CD146-negative human melanoma and ovarian cancer cell lines. The killing ability of primary CAR-T cells was evaluated by measuring the relative luciferase activity of target cells, and their effector functions were further analyzed by ELISA for the secretion of IFN-g and TNF-a. Through multiple rounds of protein engineering, we have developed a CAR construct capable of reprogramming T cells to respond to a tumour-enriched CD146 isoform. Our CAR demonstrated antigen-dependent activation of Jurkat cells when co-cultured with CD146-positive human melanoma and ovarian cancer cell lines, comparable to a CAR targeting pan-CD146. Moreover, primary CAR T cells exhibited proportional killing activity and other effector functions. We have successfully engineered a CAR with specificity for a tumour-enriched isoform of CD146. Our future work will involve rigorously evaluating the tumor selectivity of our CAR, as well as assessing the ability of primary CAR T cells expressing this CAR to control tumor growth in vivo. Citation Format: Erez Uzuner, Holly Mole, Chun W. Wong, Macarena L. Fernandez Carro, Rotem Leshem, Kieran Sefton, Helen Tovey, Andrew Picken, Marcel Blot-Chabaud, Adam F. Hurlstone. The development of CAR T cell therapeutics targeting tumor specific CD146 [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 4021.

Photothermal nanofibers enable macromolecule delivery in unstimulated human T cells

Cell therapies such as adoptive T cell transfer require ex vivo modification of cells with exogenous cargo to modulate their phenotype (e.g., to express a synthetic antigen receptor) for optimal therapeutic efficacy upon reinfusion in a patient. Several studies have shown superior anti-tumor activity of minimally differentiated T cell subsets over their activated counterparts. Therefore, developing techniques for safe and efficient manipulation of these quiescent cells is important for both clinical applications and fundamental studies of T cell biology. Photoporation with photothermal electrospun nanofibers (PEN) is an efficient and minimally perturbing non-viral intracellular delivery technique for activated and expanded T cells. However, the technique has not yet been applied to unstimulated T cells. Here, we investigated the potential of PEN photoporation for delivery of macromolecules into these cells. First, we confirmed with inductively coupled plasma tandem mass spectrometry that there was no significant iron release from fibers after laser activation of PEN substrates for laser fluences up to 0.36 J/cm². Next, we demonstrated successful intracellular delivery of 150 kDa FITC-dextran as model macromolecule in resting and pre-activated lymphocytes with 55–60 % delivery efficiency. By analyzing metabolic activity, activation surface marker presentation and extracellular cytokine release, we found that PEN treatment had no effect on cell proliferation and limited impact on T cell activation propensity for all tested irradiation energies. Thus, our findings show that PEN photoporation holds promise as a safe and efficient delivery strategy, paving the way for its use in genetic modification of minimally differentiated T cells.

Harnessing CD3 diversity to optimize CAR T cells

Current US Food and Drug Administration-approved chimeric antigen receptor (CAR) T cells harbor the T cell receptor (TCR)-derived ζ chain as an intracellular activation domain in addition to costimulatory domains. The functionality in a CAR format of the other chains of the TCR complex, namely CD3δ, CD3ε and CD3γ, instead of ζ, remains unknown. In the present study, we have systematically engineered new CD3 CARs, each containing only one of the CD3 intracellular domains. We found that CARs containing CD3δ, CD3ε or CD3γ cytoplasmic tails outperformed the conventional ζ CAR T cells in vivo. Transcriptomic and proteomic analysis revealed differences in activation potential, metabolism and stimulation-induced T cell dysfunctionality that mechanistically explain the enhanced anti-tumor performance. Furthermore, dimerization of the CARs improved their overall functionality. Using these CARs as minimalistic and synthetic surrogate TCRs, we have identified the phosphatase SHP-1 as a new interaction partner of CD3δ that binds the CD3δ–ITAM on phosphorylation of its C-terminal tyrosine. SHP-1 attenuates and restrains activation signals and might thus prevent exhaustion and dysfunction. These new insights into T cell activation could promote the rational redesign of synthetic antigen receptors to improve cancer immunotherapy.

Enhancing Anticancer Efficacy of Chemotherapeutics Using Targeting Ligand-Functionalized Synthetic Antigen Receptor-Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) have been studied for their potential in facilitating tumor-targeted delivery of chemotherapeutics due to their tumor-homing characteristics. We hypothesized that targeting effectiveness of MSCs can be further enhanced by incorporating tumor-targeting ligands on MSC surfaces that will allow for enhanced arrest and binding within the tumor tissue. We utilized a unique strategy of modifying MSCs with synthetic antigen receptors (SARs), targeting specific antigens overexpressed on cancer cells. MSCs were surface-functionalized by first incorporating recombinant protein G (PG) on the surface, followed by binding of the targeting antibody to the PG handle. We functionalized MSCs with antibodies targeting a tyrosine kinase transmembrane receptor protein, epidermal growth factor receptor (EGFR), overexpressed in non-small-cell lung cancer (NSCLC). The efficacy of MSCs functionalized with anti-EGFR antibodies (cetuximab and D8) was determined in murine models of NSCLC. Cetuximab-functionalized MSCs demonstrated improved binding to EGFR protein and to EGFR overexpressing A549 lung adenocarcinoma cells. Further, cetuximab-functionalized MSCs loaded with paclitaxel nanoparticles were efficient in slowing orthotopic A549 tumor growth and improving the overall survival relative to that of other controls. Biodistribution studies revealed a six-fold higher retention of EGFR-targeted MSCs than non-targeted MSCs. Based on these results, we conclude that targeting ligand functionalization could be used to enhance the concentration of therapeutic MSC constructs at the tumor tissue and to achieve improved antitumor response.

Constitutive expression of interleukin-27 diminishes proinflammatory cytokine production without impairing effector function of engineered T cells

Immunomodulatory cytokines can alter the tumor microenvironment and promote tumor eradication. Interleukin (IL)-27 is a pleiotropic cytokine that has potential to augment anti-tumor immunity while also facilitating anti-myeloma activity. We engineered human T cells to express a recombinant single-chain (sc)IL-27 and a synthetic antigen receptor targeting the myeloma antigen, B-cell maturation antigen, and evaluated the anti-tumor function of T cells bearing scIL-27 in vitro and in vivo. We discovered that T cells bearing scIL-27 sustained anti-tumor immunity and cytotoxicity yet manifested a profound reduction in pro-inflammatory cytokines granulocyte-macrophage colony-stimulating factor and tumor necrosis factor alpha. IL-27–expressing T cells therefore present a potential avenue to avert treatment-related toxicities commonly associated with engineered T-cell therapy due to the reduced pro-inflammatory cytokine profile.

T-cell engineered with a fully humanized B-cell maturation antigen-specific T-cell antigen coupler receptor effectively target multiple myeloma

B-cell maturation antigen (BCMA) is a clinically validated target for multiple myeloma. T-cell engineered with chimeric antigen receptors (CARs) directed against BCMA have demonstrated robust therapeutic activity in clinical trials, but toxicities remain a significant concern for a subset of patients, supporting continued investigation of other engineered T-cell platforms that may offer equal efficacy with an improved toxicity profile. The authors recently described a BCMA-specific, T-cell-centric synthetic antigen receptor, the T-cell antigen coupler (TAC) receptor, that can be used to engineer T-cell with robust anti-myeloma activity. Here the authors describe the creation of a fully humanized BCMA-specific TAC receptor. Single-chain variable fragments (scFvs) were developed from BCMA-specific F(ab)s that were identified in a fully human phage display library. Twenty-four configurations of the F(ab)s were evaluated in a medium-throughput screening using primary T-cell, and a single F(ab), TRAC 3625, emerged as the most robust following in vitro and in vivo evaluation. An optimized BCMA-specific TAC receptor was developed through iterations of the BCMA-TAC design that evaluated a next-generation TAC scaffold sequence, different domains connecting the TAC to the 3625 scFv and different orientations of the TRAC 3625 heavy and light variable regions.

Chimeric Antigen Receptor T-Cell Therapy (Car T-Cells) in Solid Tumors, Resistance and Success

CARs are chimeric synthetic antigen receptors that can be introduced into an immune cell to retarget its cytotoxicity toward a specific tumor antigen. CAR T-cells immunotherapy demonstrated significant success in the management of hematologic malignancies. Nevertheless, limited studies are present regarding its efficacy in solid and refractory tumors. It is well known that the major concerns regarding this technique include the risk of relapse and the resistance of tumor cells, in addition to high expenses and limited affordability. Several factors play a crucial role in improving the efficacy of immunotherapy, including tumor mutation burden (TMB), microsatellite instability (MSI), loss of heterozygosity (LOH), the APOBEC Protein Family, tumor microenvironment (TMI), and epigenetics. In this minireview, we address the current and future applications of CAR T-Cells against solid tumors and their measure for factors of resistance and success.

Abstract 6255: Augmented lung tumor infiltration of chemotherapeutics using synthetic antigen receptor- mesenchymal stem cells (SAR-MSCs)

Abstract Targeted delivery can improve drug accumulation in tumor tissue and treatment outcomes. However, currently available options suffer from a lack of selectivity for tumor cells. Mesenchymal Stem cells (MSCs) can be engineered with chemotherapeutic-loaded polymeric nanoparticles (PNPs) for tumor-targeted delivery, owing to their characteristic tumor tropic properties. Although nano-engineered MSCs demonstrated significant anticancer activity in multiple ovarian cancer models, their non-specific accumulation in clearance organs such as lungs and liver remains a concern. Parallel to the concept of chimeric antigen receptor (CAR)-T cell therapy, we advance here a strategy of modifying MSCs with synthetic antigen receptors (SARs) that target specific antigens overexpressed on cancer cells. Our approach consists of stably incorporating recombinant protein G (PG) on the surface of MSCs, followed by binding of a full-length IgG to the PG handle. Because protein G binds to the Fc region of IgG, antigen-binding affinity of the antibody is conserved. Epidermal growth factor receptor (EGFR) has been reported to be overexpressed and implicated in the pathogenesis of several malignancies especially lung cancer. Cetuximab (Cmab) is an anti-EGFR monoclonal antibody used in the treatment of metastatic lung cancer. In the current study, SAR-MSCs were developed by coating the nano-engineered MSCs with palmitated PG, followed by incorporating Cmab on the cell surface. Flow cytometry and confocal microscopy were used to confirm the incorporation of fluorescently labeled PAPG handle on MSC surface. Similarly, the installation of fluorescently labeled antibody on PAPG-functionalized MSCs was confirmed by flow cytometry. Preliminary efficacy studies in a mouse orthotopic A549-luc human lung adenocarcinoma model demonstrated significantly (P<0.05) improved survival of mice treated with Cmab-modified nano-engineered MSCs compared to isotype IgG-modified nano-engineered MSCs control group. These results suggest that the SAR technology could further improve the effectiveness of MSCs as tumor targeted drug delivery vehicles. Citation Format: Susheel Kumar Nethi, Drishti Sehgal, Jayanth Panyam, Swayam Prabha. Augmented lung tumor infiltration of chemotherapeutics using synthetic antigen receptor- mesenchymal stem cells (SAR-MSCs) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6255.

Non-toxic cross-reactivity with healthy tissues supports in vivo expansion and therapeutic efficacy of T cells engineered with synthetic antigen receptors

Background & Aim Clinical success of engineered T cell therapeutics against blood cancers has fueled rapid evolution of the field of T cell-based therapies and created demand for new antigen-binding domains. Among different tumor-targeting domains, single-chain antibodies (scFvs) remain the most prevalent. Extensive screening of scFvs for unwanted cross-reactivities with healthy tissues can reduce the likelihood of selecting ligands reactive to host tissues but not completely eliminate the possibility. Here we describe our experience with two BCMA-specific scFvs and the effect of non-toxic tissue cross-reactivity on the performance of engineered T cells. Methods, Results & Conclusion BCMA-specific scFvs derived from the monoclonal antibodies C11D5.3 and J22.9-xi were used as antigen-binding domains on two synthetic antigen receptor frameworks, T cell antigen coupler (TAC) receptor and chimeric antigen receptor (CAR), and tested against multiple myeloma xenografts. When T cells were engineered with the TAC framework, both scFvs yielded TAC T cells with equivalent in vitro performance; however, the J22.9-xi TAC T cells were more therapeutically robust in vivo. Further analysis revealed superior in vivo expansion of J22.9-xi TAC T cells, compared to C11D5.3 TAC T cells. Surprisingly, J22.9-xi TAC T cells expanded equally well in tumor-free and tumor-bearing mice in the absence of toxicity, suggesting that cross-reactivity to the host supported in vivo expansion and efficacy of J22.9-xi TAC T cells. In the context of a CD28-based CAR, T cells engineered with the J22.9-xi CAR displayed markedly greater levels of exhaustion, compared with T cells engineered with the C11D5.3 CAR, as evidenced by upregulation of checkpoint receptors, higher basal cytokine production, and reduced ability to upregulate cytokine production upon stimulation. Despite functional in vitro impairments, the J22.9-xi-28ζ CAR T cells proved to be the most efficacious and persistent in vivo when compared to the other engineered T cell populations; again, therapeutic efficacy was not associated with overt toxicities. Taken together, these observations suggest that non-lethal cross-reactivity of engineered T cells can augment in vivo potency and enable therapeutic efficacy of T cell products with evidence of functional exhaustion.

Immunotherapy Deriving from CAR-T Cell Treatment in Autoimmune Diseases.

Chimeric antigen receptor T (CAR-T) cells are T cells engineered to express specific synthetic antigen receptors that can recognize antigens expressed by tumor cells, which after the binding of these antigens to the receptors are eliminated, and have been adopted to treat several kinds of malignancies. Autoimmune diseases (AIDs), a class of chronic disease conditions, can be broadly separated into autoantibody-mediated and T cell-mediated diseases. Treatments for AIDs are focused on restoring immune tolerance. However, current treatments have little effect on immune tolerance inverse; even the molecular target biologics like anti-TNFα inhibitors can only mildly restore immune balance. By using the idea of CAR-T cell treatment in tumors, CAR-T cell-derived immunotherapies, chimeric autoantibody receptor T (CAAR-T) cells, and CAR regulatory T (CAR-T) cells bring new hope of treatment choice for AIDs.

Molecular Design, Optimization, and Genomic Integration of Chimeric B Cell Receptors in Murine B Cells.

Immune cell therapies based on the integration of synthetic antigen receptors comprise a powerful strategy for the treatment of diverse diseases, most notably T cells engineered to express chimeric antigen receptors (CAR) for targeted cancer therapy. In addition to T lymphocytes, B lymphocytes may also represent valuable immune cells that can be engineered for therapeutic purposes such as protein replacement therapy or recombinant antibody production. In this article, we report a promising concept for the molecular design, optimization, and genomic integration of a novel class of synthetic antigen receptors, chimeric B cell receptors (CBCR). We initially optimized CBCR expression and detection by modifying the extracellular surface tag, the transmembrane regions and intracellular signaling domains. For this purpose, we stably integrated a series of CBCR variants using CRISPR-Cas9 into immortalized B cell hybridomas. Subsequently, we developed a reliable and consistent pipeline to precisely introduce cassettes of several kb size into the genome of primary murine B cells also using CRISPR-Cas9 induced HDR. Finally, we were able to show the robust surface expression and antigen recognition of a synthetic CBCR in primary B cells. We anticipate CBCRs and our approach for engineering primary B cells will be a valuable tool for the advancement of future B cell- based immune cell therapies.

Abstract 2175: Synthetic antigen receptor mesenchymal stem cells (SAR-MSCs) targeting perlecan for drug delivery to ovarian cancer

Abstract Mesenchymal stem cells (MSCs) can be engineered with polymeric nanoparticles for tumor-targeted delivery of small molecule drugs. Such nano-engineered MSCs have demonstrated exciting anticancer activity in multiple ovarian cancer models. Despite significantly improved delivery of chemotherapeutics to tumor tissues, non-specific accumulation of MSCs in clearance organs remains a concern. Paralleling the concept of CAR-T cells, we advance here a strategy for synthetic modification of MSCs with antibodies targeting specific antigens overexpressed on cancer cells. Our approach consists of stably incorporating recombinant protein G (PG) on the surface of MSCs, followed by binding of a full-length IgG to the PG handle. Because protein G binds to the Fc region of IgG, antigen-binding affinity of the antibody is conserved. We have previously shown the overexpression of perlecan (HSPG2) on ovarian cancer cells and its correlation to poor patient survival. In the current study, we investigated the incorporation of anti-perlecan antibody on the surface of nano-engineered MSCs. The anti-perlecan IgG antibody was first derivatized with palmitic acid (PA), which was then used to insert PG on MSCs cell membrane. We characterized the PAPG derivative by LC/Q-TOF/MS. Flow cytometry and confocal microscopy were used to confirm the incorporation of fluorescently labeled PAPG handle on MSC surface. Similarly, the binding of fluorescently labeled anti-perlecan IgG to PAPG-functionalized MSCs was confirmed by flow cytometry. We are currently investigating the anticancer efficacy of paclitaxel-loaded, anti-perlecan SAR-MSCs using in vitro and mouse models of ovarian cancer. Citation Format: Susheel Kumar Nethi, Dristhi Sehgal, Shen Cheng, Jayanth Panyam, Swayam Prabha. Synthetic antigen receptor mesenchymal stem cells (SAR-MSCs) targeting perlecan for drug delivery to ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2175.

CAR T cell trogocytosis and cooperative killing regulate tumour antigen escape.

Chimeric antigen receptors (CARs) are synthetic antigen receptors that reprogram T cell specificity, function and persistence1. Patient-derived CAR T cells have demonstrated remarkable efficacy against a range of B-cell malignancies1-3, and the results of early clinical trials suggest activity in multiple myeloma4. Despite high complete response rates, relapses occur in a large fraction of patients; some of these are antigen-negative and others are antigen-low1,2,4-9. Unlike the mechanisms that result in complete and permanent antigen loss6,8,9, those that lead to escape of antigen-low tumours remain unclear. Here, using mouse models of leukaemia, we show that CARs provoke reversible antigen loss through trogocytosis, an active process in which the target antigen is transferred to T cells, thereby decreasing target density on tumour cells and abating T cell activity by promoting fratricide T cell killing and T cell exhaustion. These mechanisms affect both CD28- and 4-1BB-based CARs, albeit differentially, depending on antigen density. These dynamic features can be offset by cooperative killing and combinatorial targeting to augment tumour responses to immunotherapy.