Hoffmann, Michele M.

Abstract TCR mediated antigen recognition results in a signaling cascade leading to T cell activation. Antigen binding to its cognate TCR drives receptor aggregation and conformational changes that trigger signaling from the receptor. Conformational change in the CD3 complex (CD3Δc) is required for robust signaling and productive T cell responses. In contrast, weakly recognized antigens do not induce CD3Δc and the resulting T cell response is poor. Thus, T cell responses to weak antigens may be improved by induction of CD3Δc. We previously described an anti-mouse CD3ɛ monovalent Fab (Mono-7D6-Fab) that induced CD3Δc and synergized with weak antigens to enhance the T cell response, a process we termed co-potentiation. However, Mono-7D6-Fab did not induce classical T cell signaling in the absence of antigenic ligation. Mono-7D6-Fab effectively co-potentiated T cells against murine B16-F10 melanoma cells and reduced tumor burden in vivo in a T cell dependent manner. We exploited a model of human cytomegalovirus (CMV) to generate proof of concept that co-potentiation may enhance T cell antigen recognition in humans. Anti-human CD3ɛ monovalent Fab (Mono-OKT3-Fab) improved the CD8 T cell response to the CMV pp65495–503 peptide as measured by increased T cell proliferation, cytokine production, and the ability to kill target cells. Co-potentiation was dependent on antigen specificity, as 1) the effects were dampened when antigen recognition was impaired and 2) sequencing of the TCRs revealed a restricted number of expanded TCR clones. Our results suggest that Mono-OKT3-Fab may have therapeutic clinical applications to achieve a more robust antigen-specific T cell response in the context of diverse human disease, from cancer to chronic infection.

Abstract TCR interactions with agonist pMHCs induce a conformational change in the tails of the associated CD3 complex (CD3Δc) that contributes to efficient TCR/CD3 triggering and productive T cell responses. In contrast, TCR engagement by antagonistic pMHCs does not induce either CD3Δc or full TCR/CD3 signaling in mature T cells. Here, we have used a monovalent Fab fragment (Mono-Fab) specific for the murine CD3 complex to provide induction of CD3Δc to mature T cells engaging pMHC ligands. This Mono-Fab does not interfere with TCR capacity to bind pMHC and fails to elicit signaling in the absence of TCR/MHC interactions. Using OT-I Tg T cells and a collection of altered peptide ligands derived of the agonist peptide OVA, we show that addition of CD3Δc to OT-I T cells using Mono-Fab enables T cell responses to antagonist ligands that fail to induce CD3Δc on their own. Moreover, using a mouse model of metastatic melanoma, we show that Mono-Fab promotes in vivo T cell dependent anti-melanoma responses that are antigen-specific. This Mono-Fab can also be combined with established therapies targeting signals 2 and 3 involved in T cell activation for a synergistic effect. Our data suggests that in-trans CD3Δc provision decreases the activation threshold of T cells. We propose that targeting the CD3 complex for the induction of CD3Δc could be exploited to achieve efficient TCR/CD3 triggering by poorly immunogenic antigens in the context of vaccination against cancer and infectious diseases.

Abstract Malignant melanoma is the most aggressive form of skin cancer, accounting for 10,000 deaths annually. Largely resistant to conventional cytotoxic chemotherapy and radiation, immune checkpoint blockade (ICB) therapies have revolutionized patient care, accounting for partial or complete responses in up to 70% of patients. An important aspect of cancer elimination is activation of the cytotoxic T lymphocyte (CTL) response. Recent data suggest that ICB mediated broadening of the peripheral blood CTL repertoire correlates with good clinical outcomes. Thus, recent studies in cancer immunotherapy have focused on targeting tumor neoantigens (tNeoAg) with cancer vaccines for use in combination with ICB. Computational algorithm-driven predictions of immunogenic tNeoAgs yield vast numbers of potential peptide targets, only a fraction of which may be immunogenic in patients. This likely contributed to the modest success of cancer vaccines targeting predicted, high-avidity tNeoAgs, emphasizing the importance of identifying true tumor rejection antigens, including sub-dominant antigens, outside the scope of predictive models. In addition to promoting T cell-mediated tumor rejection, ICB often comes at the expense of treatment-induced immune-related adverse effects (irAE) that frequently require discontinuation of treatment. Modulation of ICB towards antitumor immunity and away from autoimmunity may dramatically improve the therapeutic index of modern cancer therapy. This appears feasible considering the clinical observation that antitumor efficacy does not correlate with type/severity of irAE, suggesting that the mechanisms of both processes, though likely related (CTL mediated), may not be identical. We aim to identify and separate non-cross-reacting antigenic targets mediating tumor rejection from those mediating irAEs to enable therapeutic interventions that maximize the efficacy of ICB, expanding tumor-specific CTLs with vaccines while minimizing irAEs through desensitization. We have access to samples from responding/nonresponding melanoma patients subjected to ICB with varying degrees of irAEs. We have designed an experimental approach that combines established mass spectrometry and sequencing techniques to identify peptides and matching TCR clones with a novel strategy that targets the TCR-associated CD3 complex to allow inclusion of subdominant antigens in our studies. T-cell “co-potentiation” is achieved when anti-CD3 monovalent Fabs induce a conformational change in the CD3 complex that sustains the T-cell response to weak antigenic stimulation. We have successfully used anti-human CD3 Fabs to co-potentiate in vitro the activation and subsequent response to weak peptide-HLA/TCR interactions of human T cells found in PBMCs isolated from healthy donors. Our preliminary data suggest T-cell co-potentiation may allow identification of ICB-induced CTL clones specific for subdominant tNeoAgs and irAE targets in patients with melanoma undergoing active immunotherapy. Citation Format: Laura Elsbernd, Alfreda Nelson, Hien Huynh, Michele Hoffmann, Christopher Parks, Wendy Nevala, Shari Sutor, Larry Pease, Adam Schrum, Diana Gil, Svetomir N. Markovic. Co-potentiation of human T cells to identify subdominant tumor neoantigens from melanoma patients responding to immune checkpoint blockade [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B78.

Abstract Human cytomegalovirus (HCMV) induces long-lasting T-cell immune responses that control but do not clear infection. Typical responses involve private T-cell clones, expressing T-cell antigen receptors (TCRs) unique to a person, and public T-cell clones with identical TCRs active in different people. Here, we report the development of a pretherapeutic immunostimulation modality against HCMV for human T cells, CD3 copotentiation, and the clonal analysis of its effects in recall assays at single-cell resolution. CD3 copotentiation of human T cells required identification of an intrinsically inert anti-CD3 Fab fragment that conditionally augmented signaling only when TCR was coengaged with antigen. When applied in recall assays, CD3 copotentiation enhanced the expansion of both public and private T-cell clones responding to autologous HLA-A2(+) antigen-presenting cells and immunodominant NLVPMVATV (NLV) peptide from HCMV pp65 protein. Interestingly, public vs private TCR expression was associated with distinct clonal expansion signatures in response to recall stimulus. This implied that besides possible differences in their generation and selection in an immune response, public and private T cells may respond differently to pharmacoimmunomodulation. Furthermore, a third clonal expansion profile was observed upon CD3 copotentiation of T-cell clones from HLA-A2(−) donors and 1 HLA-A2(+) presumed-uninfected donor, where NLV was of low intrinsic potency. We conclude that human T-cell copotentiation can increase the expansion of different classes of T-cell clones responding to recall antigens of different strengths, and this may be exploitable for therapeutic development against chronic, persistent infections such as HCMV.

Fab binding to CD3 couples with weak antigen recognition to enhance T cell responses and immunotherapy.