Adoptive T cell therapies have achieved significant clinical responses, especially in hematopoietic cancers. Two types of receptor systems have been used to redirect the activity of T cells, normal heterodimeric T-cell receptors (TCRs) or synthetic chimeric antigen receptors (CARs). TCRs recognize peptide-HLA complexes whereas CARs typically use an antibody-derived scFv (single-chain fragments variable) that recognizes cancer-associated cell-surface antigens. While both receptors mediate diverse effector functions, a quantitative comparison of the sensitivity and signaling capacity of TCRs and CARs has been limited due to their differences in affinities and ligands. Here we describe their direct comparison by using TCRs that could be formatted either as conventional αβ heterodimers, or as scFv constructs linked to CD3ζ and CD28 signaling domains or to CD3ζ only. Two high-affinity TCRs (KD values of approximately 50 and 250 nM) against MART1/HLA-A2 or WT1/HLA-A2 were used, allowing MART1 or WT1 peptide titrations to easily assess the impact of antigen density. Although CARs were expressed at higher surface levels than TCRs, they were 10 to 100-fold less sensitive, even in the absence of the CD8 co-receptor. Mathematical modeling demonstrated that lower CAR sensitivity could be attributed to less efficient signaling kinetics. Furthermore, reduced cytokine secretion observed at high antigen density for both TCRs and CARs suggested a role for negative regulators in both systems. Interestingly, at high antigen density, CARs also mediated greater maximal release of some cytokines, such as IL-2 and IL-6. These results have implications for next-generation design of receptors used in adoptive T cell therapies.

Glucagon-like peptide-1 (GLP-1) is a natural agonist for GLP-1R, a G protein-coupled receptor (GPCR) on the surface of pancreatic β cells. GLP-1R agoinsts are attractive for treatment of type 2 diabetes, but GLP-1 itself is rapidly degraded by peptidases in vivo. We describe a design strategy for retaining GLP-1-like activity while engendering prolonged activity in vivo, based on strategic replacement of native α residues with conformationally constrained β-amino acid residues. This backbone-modification approach may be useful for developing stabilized analogues of other peptide hormones.

Activation of a G protein-coupled receptor (GPCR) causes recruitment of multiple intracellular proteins, each of which can activate distinct signaling pathways. This complexity has engendered interest in agonists that preferentially stimulate subsets among the natural signaling pathways (“biased agonists”). We have examined analogues of glucagon-like peptide-1 (GLP-1) containing β-amino acid residues in place of native α residues at selected sites and found that some analogues differ from GLP-1 in terms of their relative abilities to promote G protein activation (as monitored via cAMP production) versus β-arrestin recruitment (as monitored via BRET2 assays). The α→β replacements generally cause modest declines in stimulation of cAMP production and β-arrestin recruitment, but for some replacement sets cAMP production is more strongly affected than is β-arrestin recruitment. The central portion of GLP-1 appears to be critical for achieving bias toward β-arrestin recruitment. These results suggest that backbone modification via α→β residue replacement may be a versatile source of agonists with biased GLP-1R activation profiles.

The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor that is a major therapeutic target for the treatment of type 2 diabetes. Activation of this receptor promotes insulin secretion and blood glucose regulation. The GLP-1R can initiate signaling through several intracellular pathways upon activation by GLP-1. GLP-1R ligands that preferentially stimulate subsets among the natural signaling pathways (“biased agonists”) could be useful as tools for elucidating the consequences of specific pathways and might engender therapeutic agents with tailored effects. Using HEK-293 cells recombinantly expressing human GLP-1R, we have previously reported that backbone modification of GLP-1, via replacement of selected α-amino acid residues with β-amino acid residues, generates GLP-1 analogues with distinctive preferences for promoting G protein activation versus β-arrestin recruitment. Here, we have explored the influence of cell background across these two parameters and expanded our analysis to include affinity and other key signaling pathways (intracellular calcium mobilization and ERK phosphorylation) using recombinant human GLP-1R expressed in a CHO cell background, which has been used extensively to demonstrate biased agonism of GLP-1R ligands. The new data indicate that α/β-peptide analogues of GLP-1 exhibit a range of distinct bias profiles relative to GLP-1 and that broad assessment of signaling endpoints is required to reveal the spectrum of behavior of modified peptides. These results support the view that backbone modification via α→β amino acid replacement can enable rapid discovery of peptide hormone analogues that display substantial signal bias at a cognate GPCR.

Mutated peptides (neoantigens) from a patient’s cancer genome can serve as targets for T-cell immunity, but identifying which peptides can be presented by an MHC molecule and elicit T cells has been difficult. Although algorithms that predict MHC binding exist, they are not yet able to distinguish experimental differences in half-lives of the complexes (an immunologically relevant parameter, referred to here as kinetic stability). Improvement in determining actual neoantigen peptide/MHC stability could be important, as only a small fraction of peptides in most current vaccines are capable of eliciting CD8+ T-cell responses. Here, we used a rapid, high-throughput method to experimentally determine peptide/HLA thermal stability on a scale that will be necessary for analysis of neoantigens from thousands of patients. The method combined the use of UV-cleavable peptide/HLA class I complexes and differential scanning fluorimetry (DSF) to determine the Tm values of neoantigen complexes. Measured Tm values were accurate and reproducible and were directly proportional to the half-lives of the complexes. Analysis of known HLA-A2–restricted immunogenic peptides showed that Tm values better correlated with immunogenicity than algorithm-predicted binding affinities. We propose that temperature stability information can be used as a guide for the selection of neoantigens in cancer vaccines in order to focus attention on those mutated peptides with the highest probability of being expressed on the cell surface.

Family BGprotein-coupled receptors play importantp hysiological roles and possess large extracellular domains (ECDs) that aid in binding the long polypeptide hormones that are their natural agonists. We have previously shown that agonist analogues in which subsets of native a-amino acid residues are replaced with b-aminoa cid residues can retain activity while avoiding proteolytic degradation. This study focuses on eight new a/b analogues of glucagon-like peptide 1( GLP-1) that each contain five a-to-b replacements in the C-terminal half of the peptide. This portion of GLP-1 is known to adopt an a-helicalc onformation and contact the ECD. All four registries of the aaab backbone pattern weree valuated;p revious work has shown that the aaab pattern supports adoption of an ahelix-like conformation. Two a-to-b replacement formats were employed, one involving b 3 homologues of the native residues replaced and the other involving ac yclic b residue. GLP-1R response was characterizedi nt erms of stimulation of cAMP production and b-arrestin recruitment.S ome of the backbone-modifiedG LP-1 analogues display biased agonism of the GLP-1R. This study helps to establish the scope of the a!b backbone modification strategy.

BackgroundTreatment of some blood cancers with T cells that express a chimeric antigen receptor (CAR) against CD19 have shown remarkable results. In contrast, CAR-T cell efficacy against solid tumors has been difficult to achieve.MethodsTo examine the potential of CAR-T cell treatments against ovarian cancers, we used the mouse ovarian cancer cell line ID8 in an intraperitoneal model that exhibits disseminated solid tumors in female C57BL/6J mice. The CAR contained a single-chain Fv from antibody 237 which recognizes a Tn-glycopeptide-antigen expressed by ID8 due to aberrant O-linked glycosylation in the absence of the transferase-dependent chaperoneCosmc. The efficacy of four Tn-dependent CARs with varying affinity to Tn antigen, and each containing CD28/CD3ζ cytoplasmic domains, were compared in vitro and in vivo in this study.ResultsIn line with many observations about the impact of aberrant O-linked glycosylation, the ID8Cosmcknock-out (ID8Cosmc-KO) exhibited more rapid tumor progression compared with wild-type ID8. Despite the enhanced tumor growth in vivo, 237 CAR and a mutant with 30-fold higher affinity, but not CARs with lower affinity, controlled advanced ID8Cosmc-KO tumors. Tumor regression could be achieved with a single intravenous dose of the CARs, but intraperitoneal administration was even more effective. The CAR-T cells persisted over a period of months, allowing CAR-treated mice to delay tumor growth in a re-challenge setting. The most effective CARs exhibited the highest affinity for antigen. Antitumor effects observed in vivo were associated with increased numbers of T cells and macrophages, and higher levels of cleaved caspase-3, in the tumor microenvironment. Notably, the least therapeutically effective CAR mediated tonic signaling leading to antigen-independent cytokine expression and it had higher levels of the immunosuppressive cytokine interleukin10.ConclusionThe findings support the development of affinity-optimized CAR-T cells as a potential treatment for established ovarian cancer, with the most effective CARs mediating a distinct pattern of inflammatory cytokine release in vitro. Importantly, the most potent Tn-dependent CAR-T cells showed no evidence of toxicity in tumor-bearing mice in a syngeneic, immunocompetent system.

Adoptive T cell therapy using T cell receptors (TCRs) or chimeric antigen receptors (CARs) requires careful selection of the receptor so as to target only the specific cancer antigen, without cross-reactivity toward structurally similar antigens. In this study, we used two TCRs (T1 and RD1) that bind to the HLA-A2 restricted melanoma antigen MART-1 (ELAGIGILTV). TCR-T1 was initially isolated from a melanoma-reactive T cell line specific for MART-1 whereas TCR-RD1 was derived completely by in vitro engineering, switching the specificity of the well-studied A6 TCR from its cognate viral peptide (Tax) to MART-1 by directed evolution. Despite their distinct origins, engineered high-affinity forms of both TCRs had remarkably similar fine-specificities as determined using a panel of MART-1 variant peptides. This binding signature allowed us to conduct a bioinformatic scan of the human and mouse proteomes for peptides that were homologous to MART-1. Our analysis resulted in identification of a self-peptide (TM74B: IIAGLGILTV) that also bound to both T1 and RD1 TCRs. T1-transduced T cells (CD4+) from HLA-A2/Db transgenic (AAD) mice were potently activated by MART-1, and cross-reacted (at 1000-fold higher doses) with the TM74B peptide. CD8+ T cells transduced with the T1 TCR were activated by AAD+ antigen presenting cells, indicating recognition of an endogenous peptide, possibly TM74B. To extend these studies, we have shown that T1-transduced AAD T cells (CD4+) are also stimulated by murine MART-1 (EAAGIGILIV) and that transfer of T1-transduced T cells into AAD mice did not result in overt toxicity. Thus, the AAD mouse could be used to study efficacy and toxicity of T1 TCR in syngeneic models. We are currently developing a B16/AAD tumor model to compare recognition of B16 melanoma by MART-1 directed TCRs and CARs. In summary, the convergence of specificity of T1 and RD1 suggests that TCRs against a cancer peptide antigen are focused on the same exposed features of the peptide. Furthermore, the results with RD1 suggest that it is possible to engineer TCRs completely in vitro, and that these TCRs can recapitulate the fine-specificities of conventional TCRs isolated from T cell clones. Citation Format: Preeti Sharma, Daniel T. Harris, Marlies V. Hager, David M. Kranz. Convergence of the specificity and cross-reactivity of multiple T cell receptors isolated against a single cancer peptide/HLA complex [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3581.