Clinical applications of CAR-T cells are limited by the scarcity of tumor-specific targets and are often afflicted with the same on-target/off-tumor toxicities that plague other cancer treatments. A new promising strategy to enforce tumor selectivity is the use of logic-gated, two-receptor systems. One well-described application is termed Tmod™, which originally utilized a blocking inhibitory receptor directed towards HLA-I target antigens to create a protective NOT gate. Here we show that the function of Tmod blockers targeting non-HLA-I antigens is dependent on the height of the blocker antigen and is generally compatible with small, membrane-proximal targets. We compensate for this apparent limitation by incorporating modular hinge units to artificially extend or retract the ligand-binding domains relative to the effector cell surface, thereby modulating Tmod activator and blocker function. By accounting for structural differences between activator and blocker targets, we developed a set of simple geometric parameters for Tmod receptor design that enables targeting of blocker antigens beyond HLA-I, thereby broadening the applications of logic-gated cell therapies.

The CEACAM5 gene product [carcinoembryonic antigen (CEA)] is an attractive target for colorectal cancer because of its high expression in virtually all colorectal tumors and limited expression in most healthy adult tissues. However, highly active CEA-directed investigational therapeutics have been reported to be toxic, causing severe colitis because CEA is expressed on normal gut epithelial cells. Here, we developed a strategy to address this toxicity problem: the Tmod dual-signal integrator. CEA Tmod cells use two receptors: a chimeric antigen receptor (CAR) activated by CEA and a leukocyte Ig-like receptor 1 (LIR-1)-based inhibitory receptor triggered by human leukocyte antigen (HLA)-A*02. CEA Tmod cells exploit instances of HLA heterozygous gene loss in tumors to protect the patient from on-target, off-tumor toxicity. CEA Tmod cells potently killed CEA-expressing tumor cells in vitro and in vivo. But in contrast to a traditional CEA-specific T cell receptor transgenic T cell, Tmod cells were highly selective for tumor cells even when mixed with HLA-A*02-expressing cells. These data support further development of the CEA Tmod construct as a therapeutic candidate for colorectal cancer.

We set out to examine the behavior in preclinical models of a dual-receptor cell therapy approach (Tmod࣪ system) designed to exploit instances of somatic LOH in cancer that can be readily identified through molecular diagnostics. We utilized quantitative pharmacologic assays in vitro and dual-flank efficacy/selectivity models in vivo to test the activity of a variety of constructs comprised of: (i) CAR “activators” that target tumor-associated antigens (TAAs) such as CEA, MSLN and others; and, (ii) LIR-1-based “blockers” that target HLA class I proteins. We generated substantial datasets around multiple targets, including well known tumor-associated antigens (TAAs). In the presence of the HLA class I blocker antigen, Tmod cells were OFF within a wide range of activator antigen expression levels on the “normal” target cells. They were ON when tumor cells expressed the TAA without the HLA blocker antigen, and killed tumor cells selectively even in mixed tumor-normal cell populations. The Tmod system transformed imperfect TAAs into true tumor-selective targets, where the activity of the engineered T cells is controlled by the presence/absence of the HLA blocker antigen. The system was sufficiently modular to use with autologous and allogeneic products. Several of these Tmod constructs are advancing to the clinic where a robust genetic selection tool is being used to screen patients for treatment and match them with a suitable Tmod therapy. In summary, Tmod is a robust system with quantitative pharmacologic behavior in preclinical models consistent with efficacy and safety across a range of targets. Citation Format: Han Xu, Agnes Hamburger, Yuta Ando, Grace Asuelime, Kristian Bolanos, Mark Daris, Breanna DiAndreth, Maria C. Imun, Wen-Hua Lee, Chuck Z. Li, Breanna Luna, Diane Manry, Aaron D. Martin, Michele E. McElvain, Jee-Young Mock, Martin Naradikian, Mark L. Sandberg, Julyun Oh, Sanam Shafaattalab, Shruti Sharma, Talar Tokatlian, Xueyin Wang, Lu Min Wong, Alexander Kamb. The modular Tmod dual_receptor system exploits tumor deletions to achieve robust tumor-selective cytotoxicity [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 2751.

Background Nearly all colorectal and most pancreatic and lung cancers express carcinoembryonic antigen (CEA). However, due to its expression in normal gut epithelial cells, CEA-targeted therapies have resulted in on-target, off-tumor toxicity. To overcome this, we have developed Tmod™, a logic-gated T-cell therapy platform. Tmod constructs are composed of an activating CAR or T-cell receptor that targets a tumor antigen and an inhibitory receptor recognizing an antigen expressed on normal healthy tissues, but not on tumor cells due to loss of heterozygosity (LOH).1,2 A2B530 is a CEA-directed Tmod construct utilizing an LIR-1-based inhibitory receptor (blocker) targeting human leukocyte antigen A*02 (HLA-A*02). Methods To generate CEA Tmod, T cells from HLA-A*02(+) donors were transduced with a single lentivirus to express i) the CAR, ii) the blocker, and iii) an shRNA targeting β2M. Cytotoxicity was measured by culturing CEA(+) target cell line pairs (A*02[-] and A*02[+]), expressing either GFP or RFP, with engineered T cells and quantifying live target cells over time. In vivo activity was examined using NSG mice subcutaneously implanted with "normal" (CEA[+]A*02[+]) and tumor cells (CEA[+]A*02[-]), in the right and left flanks. Mice were treated intravenously with CEA Tmod cells or control T cells. Results Control CEA CAR T cells killed CEA(+) target cell lines in vitro irrespective of HLA-A*02 expression. In contrast, CEA Tmod cells selectively killed tumor cells (CEA[+]A*02[-]) while sparing "normal" cells (CEA[+]A*02[+]). In mixed target cell cultures, CEA Tmod cells killed only the A*02(-) target cells, whereas the CEA CAR T cells killed both the A*02(-) and A*02(+) cell lines. Further, CEA Tmod cells exhibited bidirectional control between the activated and blocked states. While mice treated with control CEA CAR T cells experienced a reduction in volume and bioluminescence of both normal and tumor grafts, CEA Tmod cells specifically cleared A*02(-) tumors in mice (table 1). Finally, although expansion of Tmod cells in peripheral blood trended lower than CAR and TCR controls, anti-tumor activity was comparable in these groups. Conclusions A2B530 is an autologous CEA Tmod cell product that exploits common LOH at the HLA locus in cancer cells, enabling these engineered T cells to discriminate between normal and tumor cells. BASECAMP-1 (NCT04981119), an observational study identifying patients with somatic HLA LOH, is recruiting. Eligible patients with metastatic colorectal, pancreatic, or non-small cell lung cancer will be apheresed for a future A2B530 EVEREST-1 interventional study. References Hamburger A, DiAndreth B, Cui J, et al. Engineered T cells directed at tumors with defined allelic loss. Mol Immunol. 2020;128:298-310. DiAndreth B, Hamburger AE, Xu H, Kamb A. The Tmod cellular logic gate as a solution for tumor-selective immunotherapy. Clin Immunol. 2022;241:109030.

BackgroundMesothelin (MSLN) and carcinoembryonic antigen (CEA) are classic tumor-associated antigens that are expressed in many solid tumors including the majority of lung, colorectal and pancreatic cancers. However, both MSLN and CEA are also expressed in vital normal organs. This normal expression creates risk of serious inflammation for CEA- or MSLN-directed therapeutics. To date all active CEA- or MSLN-targeted investigational therapeutics have been toxic when administered systemically.MethodsWe have developed a safety mechanism to protect normal tissues without abrogating sensitivity of cytotoxic T cells directed at MLSN(+) or CEA(+) tumors in a subset of patients with defined loss of heterozygosity (LOH) in their tumors (figure 1). This dual-receptor (Tmod< sup >TM) system exploits common LOH at the HLA locus in cancer cells, allowing T cells to recognize the difference between tumor and normal tissue.1 2 T cells engineered with specific Tmod constructs contain: (i) a MSLN- or CEA-activated CAR; and, (ii) an inhibitory receptor gated by HLA-A*02. HLA-A*02 binding blocks T cell cytotoxicity, even in the presence of MSLN or CEA. The Tmod system is designed to treat heterozygous HLA class I patients, selected for HLA LOH. When HLA-A*02 is absent from tumors selected for LOH, the CARs are predicted to mediate potent killing of the A*02(-) malignant cells.ResultsThe Tmod system robustly protects surrogate normal cells even in mixed-cell populations in vitro while mediating robust cytotoxicity of tumor cells in xenograft models (see example in figure 2). The MSLN CAR can also be paired with other blockers, supporting scalability of the approach to patients beyond HLA-A*02 heterozygotes.Abstract 122 Figure 1Illustration of the Tmod T cell engaging with tumor cells with somatic loss of HLA-A*02 and with normal cells.Abstract 122 Figure 2Bioluminescence measurements show the average difference between the size of the MSLN(+)A*02(+) ‘normal’ graft compared to the MSLN(+)A*02(-) tumor graft on the two flanks of mice after T cell infusion. Both tumor and normal grafts are destroyed by CAR-Ts (CAR-3 and M5 benchmark) while the MSLN Tmod cells kill the tumor but not the normal graft.ConclusionsThe Tmod mechanism may provide an alternative route to leverage solid-tumor antigens such as MSLN and CEA in safer, more effective ways than previously possible.ReferencesHamburger AE, DiAndreth B, Cui J, et al. Engineered T cells directed at tumors with defined allelic loss. Mol Immunol 2020;128:298–310.Hwang MS, Mog BJ, Douglass J, et al. Targeting loss of heterozygosity for cancer-specific immunotherapy. Proc Natl Acad Sci U S A 2021;118(12):e2022410118.

Next-generation T-cell therapies will likely continue to utilize T-cell receptors (TCRs) and chimeric antigen receptors (CARs) because each receptor type has advantages. TCRs often possess exceptional properties even when tested unmodified from patients' T cells. CARs are generally less sensitive, possibly because their ligand-binding domains are grafted from antibodies selected for binding affinity or avidity and not broadly optimized for a functional response. Because of the disconnect between binding and function among these receptor types, the ultimate potential of CARs optimized for sensitivity and selectivity is not clear. Here, we focus on a thoroughly studied immuno-oncology target, the HLA-A*02/HPV-E629-38 complex, and show that CARs can be optimized by a combination of high-throughput binding screens and low-throughput functional assays to have comparable activity to clinical TCRs in acute assays in vitro. These results provide a case study for the challenges and opportunities of optimizing high-performing CARs, especially in the context of targets utilized naturally by TCRs.

Though TCRs have been subject to limited engineering in the context of therapeutic design and optimization, they are used largely as found in nature. On the other hand, CARs are artificial, composed of different segments of proteins that function in the immune system. This characteristic raises the possibility of altered response to immune regulatory stimuli. Here we describe a large-scale, systematic comparison of CARs and TCRs across 5 different pMHC targets, with a total of 19 constructs examined in vitro. These functional measurements include CAR- and TCR-mediated activation, proliferation, and cytotoxicity in both acute and chronic settings. Surprisingly, we find no consistent difference between CARs and TCRs as receptor classes with respect to their relative sensitivity to major regulators of T cell activation: PD-L1, CD80/86 and IL-2. Though TCRs often emerge from human blood directly as potent, selective receptors, CARs must be heavily optimized to attain these properties for pMHC targets. Nonetheless, when iteratively improved and compared head to head in functional tests, CARs appear remarkably similar to TCRs with respect to immune modulation.

Background Recurrent cancer-specific targets are rare. Given the pace of genomic research over the past three decades, few are likely to lie yet undiscovered. In 2013 an innovative MAGE-A3-directed cancer therapeutic of great potential value was terminated in the clinic because of neurotoxicity.1 The safety problems were hypothesized to originate from off-target TCR activity against a closely related MAGE-A12 peptide. Methods A combination of published and new data led us to test this hypothesis with current technology, including RNA hybridization in situ and further analysis of the clinical TCR's specificity to MAGE-A12 and other antigens. Results We find that a key prediction of the MAGE-A12 toxicity hypothesis, the existence of rare, high-MAGE-A12-expressing cells in the brain, is not supported by the data. Our results imply that an alternative related peptide from the EPS8L2 protein is more likely responsible for the toxicity. Therefore, it may be valuable to reconsider MAGE-A3 as a cancer target using HLA-A*02-restricted-TCRs or CARs. As a step in this direction, we isolated MAGE-A3 pMHC-directed CARs, targeting the same peptide as the clinical TCR. These CARs have high selectivity, and avoid cross-reaction with the EPS8L2 peptide that represents a significant risk for MAGE-A3-targeted therapeutics. Conclusions Given the qualities of MAGE-A3 as an onco-testis antigen widely expressed in tumors and largely absent from normal adult tissues, our findings suggest that MAGE-A3 may deserve further consideration as a cancer target. We have identified CARs with selectivity profiles consistent with a cell therapeutic directed against HLA-A*02-positive, MAGE-A3-expressing cancers. The relative merits of TCRs and CARs for this target will be discussed. Reference Morgan RA, Chinnasamy N, Abate-Daga D, Gros A, Robbins PF, Zheng Z, Dudley ME, Feldman SA, Yang JC, Sherry RM, et al. Cancer regression and neurological toxicity following anti-MAGE-A3 TCR gene therapy. J Immunother 2013;36:133–151, doi:10.1097/CJI.0b013e3182829903.

Chimeric antigen receptors (CARs) and their parent signaling molecule, the T cell receptor (TCR), are fascinating proteins of increasing relevance to disease therapy. Here we use a collection of 1221 pMHC-directed CAR constructs representing 10 pMHC targets to study aspects of CAR structure-activity relationships (SAR), with particular focus on the extracellular and transmembrane structural components. These experiments that involve pMHC targets whose number/cell can be manipulated by peptide dosing in vitro enable systematic analysis of the SAR of CARs in carefully controlled experimental situations (Harris and Kranz, 2016). We find that CARs tolerate a wide range of structural variation, with the ligand-binding domains (LBDs) dominating the SAR of CAR antigen sensitivity. Notwithstanding the critical role of the LBD, CAR antigen-binding on the cell surface, measured by pMHC tetramer staining, is not an effective predictor of functional sensitivity. These results have important implications for the design and testing of CARs aimed toward the clinic.

In 2013, an innovative MAGE-A3-directed cancer therapeutic of great potential value was terminated in the clinic because of neurotoxicity. The safety problems were hypothesized to originate from off-target T-cell receptor activity against a closely related MAGE-A12 peptide. A combination of published and new data led us to test this hypothesis with current technology. Our results call into question MAGE-A12 as the source of the neurotoxicity. Rather, the data imply that an alternative related peptide from EPS8L2 may be responsible. Given the qualities of MAGE-A3 as an onco-testis antigen widely expressed in tumors and largely absent from normal adult tissues, these findings suggest that MAGE-A3 may deserve further consideration as a cancer target. As a step in this direction, the authors isolated 2 MAGE-A3 peptide-major histocompatibility complex-directed chimeric antigen receptors, 1 targeting the same peptide as the clinical T-cell receptor. Both chimeric antigen receptors have improved selectivity over the EPS8L2 peptide that represents a significant risk for MAGE-A3-targeted therapeutics, showing that there may be other options for MAGE-A3 cell therapy.

To accelerate the cardiac drug discovery pipeline, we set out to develop a platform that would be capable of quantifying tissue-level functions such as contractile force and be amenable to standard multiwell-plate manipulations. We report a 96-well-based array of 3D human pluripotent stem cell (hPSC)-derived cardiac microtissues - termed Cardiac MicroRings (CaMiRi) - in custom 3D-print-molded multiwell plates capable of contractile force measurement. Within each well, two elastomeric microcantilevers are situated above a circumferential ramp. The wells are seeded with cell-laden collagen, which, in response to the gradual slope of the circumferential ramp, self-organizes around tip-gated microcantilevers to form contracting CaMiRi. The contractile force exerted by the CaMiRi is measured and calculated using the deflection of the cantilevers. Platform responses were robust and comparable across wells, and we used it to determine an optimal tissue formulation. We validated the contractile force response of CaMiRi using selected cardiotropic compounds with known effects. Additionally, we developed automated protocols for CaMiRi seeding, image acquisition, and analysis to enable the measurement of contractile force with increased throughput. The unique tissue fabrication properties of the platform, and the consequent effects on tissue function, were demonstrated upon adding hPSC-derived epicardial cells to the system. This platform represents an open-source contractile force screening system useful for drug screening and tissue engineering applications.

Despite key advances in cancer therapies, malignant tumors, such as melanoma, continue to be one of the leading causes of mortality. Recent debate on whether cancer can originate from a tumor-initiating subpopulation has permeated oncology and stem cell research. It has been well established that primary and immortalized tumor cells consist of heterogeneous cell populations. The profound effect of tumor heterogeneity on tumor growth and drug resistance remains elusive, but it is highly likely that subpopulations of cancer cells have different capabilities of self-renewal and drug resistance. Discrepancies between excellent in vitro potency and efficacy and poor patient response have been observed on multiple cancer therapeutics. Although this observation can be attributed to many factors, a better understanding of the contribution from subpopulations within a cancer will help bridge the gap between in vitro assay results and patient prognosis. To comprehend this impact, it is critical to isolate and characterize cancer subpopulations that possess higher growth and drug resistance properties so that novel therapeutics can be developed to eventually eradicate all cancer cells. In this article, we describe a method to enrich a subpopulation, CB4, from the melanoma cell line WM115. CB4 exhibited higher anchorage-independent growth, higher survival under serum starvation condition, and lower drug sensitivity to commonly used melanoma treatment compared with WM115. Details of functional properties and gene expression of CB4 compared with WM115 are reported. Our study demonstrates that it is feasible to isolate and enrich a subpopulation that exhibits higher growth capacity and treatment resistance from an immortalized tumor cell line.

The EBV latent membrane protein 1 (LMP1) is an integral membrane protein that acts like a constitutively activated receptor. LMP1 interacts with members of the tumor necrosis factor receptor-associated factor family, as well as with tumor necrosis factor receptor-associated death domain, resulting in induction of nuclear factor-kappaB, the p38 mitogen-activated protein kinase pathway, and the c-Jun NH(2)-terminal kinase activator protein 1-signaling cascade. The binding of Janus kinase 3 results in activation of signal transducers and activators of transcription. The domain structure of LMP1 has been mapped extensively, but the quantitative contribution of distinct LMP1 domains to the efficiency of B-cell proliferation by EBV has not been determined. On the basis of the maxi-EBV system, which allows us to introduce and study mutations in the context of the complete EBV genome, a panel of 10 EBV mutants with alterations in the LMP1 gene locus was established. The mutant EBVs were tested for their efficiency to induce and maintain proliferation of clonal B-cell lines in vitro. Surprisingly and with reduced frequency, EBV mutants which deleted LMP1's COOH terminus, transmembrane domains, or the entire open reading frame were able to generate proliferating B-cell clones that were dependent on the presence of human fibroblast feeder cells. A B-cell clone carrying the LMP1-null mutant EBV genome was also analyzed for oncogenicity in severe combined immunodeficiency mice. Our results demonstrate that LMP1 is critical but not mandatory for the generation of proliferating B cells in vitro. LMP1 functions greatly contribute to EBV's transformation potential and appear essential for its oncogenicity in severe combined immunodeficiency mice.