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.

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.

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.

Cell therapy is poised to play a larger role in medicine, most notably for immuno-oncology. Despite the recent success of CAR-T therapeutics in the treatment of blood tumors and the rapid progress toward improved versions of both CAR- and TCR-Ts, important analytical aspects of preclinical development and manufacturing of engineered T cells remain immature. One limiting factor is the absence of robust multivariate assays to disentangle key parameters related to function of engineered effector cells, especially in the peptide-MHC (pMHC) target realm, the natural ligand for TCRs. Here we describe an imaging-based primary T cell assay that addresses several of these limitations. To our knowledge, this assay is the first quantitative, high-content assay that separates the key functional parameters of time- and antigen-dependent T cell proliferation from cytotoxicity. We show that the assay sheds light on relevant biology of CAR- and TCR-T cells, including response kinetics and the influence of effector:target ratio.

Author(s): Nampe, Daniel Pareza | Advisor(s): Tsutsui, Hideaki | Abstract: The success of human pluripotent stem cells (hPSCs) as a source of future cell therapies hinges in part on the availability of a robust scalable culture system that can readily produce clinically relevant number of cells and their derivatives. Stirred suspension culture has been identified as one of such promising platforms due to its ease of use, scalability, and widespread use in the pharmaceutical industry (e.g., CHO cell-based production of therapeutic proteins) among others. However, culture of undifferentiated hPSCs in stirred suspension is a relatively new development in the past several years, and little is known beyond empirically optimized culture parameters. The goal of this study was to elucidate the impact of fluidic agitation on hPSCs in stirred suspension culture. In particular, we systematically investigated various agitation rates to characterize their impact on cell yield, viability, and maintenance of pluripotency. Additionally, we closely examined the distribution of cell aggregates and how the observed culture outcomes are attributed to their size distribution. Our results showed that moderate agitation maximized the propagation of hPSCs by controlling the cell aggregates below the critical size, beyond which the cells suffer from diffusion limitation, while limiting cell death caused by excessive fluidic forces. Furthermore, we observed that fluidic agitation could regulate not only cell aggregation, but also expression of some key signaling proteins in hPSCs. Upon discovering this mechanosensitive effector enabled a novel approach for linking expansion and cardiac differentiation to generate over 90% cardiomyocytes. In addition, these cardiomyocytes displayed highly organized sarcomere structure which suggests an improved maturation in their morphology. Altogether, results presented in this study indicate the new possibility of guiding stem cell fate determination by fluidic agitation in stirred suspension cultures.