The development of checkpoint antibodies for cancer therapy has been guided by the principle of blocking T cell inhibitory signals. Recognition of the role of the Fc domain in therapeutic activities, through the depletion of immunosuppressive populations and myeloid cell activation, prompts a shift toward the development of optimized Fc-engineered checkpoint antibodies.

Testing of candidate monoclonal antibody therapeutics in preclinical models is an essential step in drug development. Identification of antibody therapeutic candidates that bind their human targets and cross-react to mouse orthologs is often challenging, especially for targets with low sequence homology. In such cases, surrogate antibodies that bind mouse orthologs must be used. The antibody 9D9, which binds mouse CTLA-4, is a commonly used surrogate for CTLA-4 checkpoint blockade studies in mouse cancer models. In this work, we reveal that 9D9 has significant biophysical dissimilarities to therapeutic CTLA-4 antibodies. The 9D9-mCTLA4 complex crystal structure was determined and shows that the surrogate antibody binds an epitope distinct from ipilimumab and tremelimumab. In addition, while ipilimumab has pH-independent binding to hCTLA-4, 9D9 loses binding to mCTLA-4 at physiologically relevant acidic pH ranges. We used phage and yeast display to engineer ipilimumab to bind mouse CTLA-4 with single-digit nM affinity from an initial state with no apparent binding. The engineered variants showed pH-independent and cross-reactive binding to both mouse and human CTLA-4. Crystal structures of a variant in complex with both mouse and human CTLA-4 confirmed that it targets an equivalent epitope as ipilimumab. These cross-reactive ipilimumab variants may facilitate improved translatability and future mechanism-of-action studies for anti-CTLA-4 targeting in murine models.

The tumor vasculature acts as the gatekeeper to the tumor microenvironment, thus regulating the ability of immune cells to roll, anchor, and cross the endothelium from blood to tissue. While the role of endothelial cells (EC) in T Cell migration and extravasation has been extensively studied, the ability of ECs to control T Cell function is less well appreciated. Several well-characterized regulators of T Cell function are expressed by ECs, while others are induced by soluble factors required for EC development and differentiation. Targeting the molecules expressed by immunomodulatory ECs may provide approaches to improve tumor immunotherapy.

The nonpolymorphic major histocompatibility complex E (MHC-E) molecule is up-regulated on many cancer cells, thus contributing to immune evasion by engaging inhibitory NKG2A/CD94 receptors on NK cells and tumor-infiltrating T cells. To investigate whether MHC-E expression by cancer cells can be targeted for MHC-E–restricted T cell control, we immunized rhesus macaques (RM) with rhesus cytomegalovirus (RhCMV) vectors genetically programmed to elicit MHC-E–restricted CD8 + T cells and to express established tumor-associated antigens (TAAs) including prostatic acidic phosphatase (PAP), Wilms tumor-1 protein, or Mesothelin. T cell responses to all three tumor antigens were comparable to viral antigen-specific responses with respect to frequency, duration, phenotype, epitope density, and MHC restriction. Thus, CMV-vectored cancer vaccines can bypass central tolerance by eliciting T cells to noncanonical epitopes. We further demonstrate that PAP-specific, MHC-E–restricted CD8 + T cells from RhCMV/PAP-immunized RM respond to PAP-expressing HLA-E + prostate cancer cells, suggesting that the HLA-E/NKG2A immune checkpoint can be exploited for CD8 + T cell–based immunotherapies.

Abstract Introduction: Blockade of the CTLA-4 pathway with ipilimumab (IPI), alone and in combination with nivolumab (anti-programmed death-1 antibody), has shown clinical benefit in multiple tumor types. However, not all tumors respond to IPI ± nivolumab, and peripheral effects can lead to immune-related adverse events. To enhance the therapeutic index of CTLA-4-directed therapy, a proprietary Probody® therapeutics technology platform was used to develop a modified version of IPI. BMS-986288 is an anti-CTLA-4 antibody that combines a Probody® therapeutic masking peptide and protease-cleavable linker, designed to localize anti-CTLA-4 activity to the tumor microenvironment, with an NF Fc region hypothesized to enhance antigen-presenting cell (APC)-mediated T-cell priming and regulatory T cell (Treg) modulation via increased binding to the FcγRIIIA (CD16) receptor. Here, we present the characterization of the mechanism of action and pharmacodynamic (PD) response of BMS-986288 in preclinical models. Methods: APC-mediated T-cell priming and antigen-specific responses were evaluated in a superantigen model using peripheral blood mononuclear cells (PBMCs) and human CTLA-4 knock-in (KI) mice. Antitumor activity and immune cell population changes were assessed in an MC38 tumor model implanted in human CTLA-4 KI mice. Intratumoral Treg depletion was further investigated using patient-derived dissociated tumor samples as a physiologically relevant model. Peripheral PD effects and tolerability of BMS-986288 were evaluated in a non-human primate (NHP) Ad5 vaccine model. Results: Protease-cleaved BMS-986288 demonstrated enhanced APC-mediated T-cell priming compared with IPI in superantigen-stimulated PBMCs. Similarly, administration of superantigen staphylococcal enterotoxin B (SEB) peptide elicited an increased antigen-specific T-cell receptor (Vβ8+) T-cell response after treatment with cleaved BMS-986288 vs IPI in human CTLA-4 KI mice. In the MC38 tumor-bearing mouse model, BMS-986288 showed enhanced antitumor activity as compared to IPI, with tumor clearance in all mice. Although BMS-986288 enhanced intratumoral CTLA-4+ Treg depletion in the MC38 model, limited Treg depletion was observed in the patient-derived dissociated tumor model with an NF anti-CTLA-4 antibody without the masking peptide, potentially reflecting differences in CTLA-4 expression on human vs mouse Tregs. BMS-986288 reduced PD effects in the peripheral blood compartment and increased tolerability in NHP relative to IPI. Conclusion: BMS-986288 leverages unique characteristics to differentiate it from IPI, enhancing APC-mediated T-cell priming and anti-tumor activity while also reducing peripheral activity in preclinical models. These data support an ongoing phase 1/2 clinical trial of BMS-986288 (NCT03994601) in patients with advanced solid tumors. Citation Format: Amy Jhatakia, Mohammed Nasser, Anandaroop Mukhopadhyay, Kyeongah Kang, Courtni Newsome, Neha Gupta, Remie Gail Z. Mandawe, Felix Findeisen, Jack A. Lohre, Leslie Leung, Yun Wei, Joshua Dobroff, Karen Price, John Engelhardt, Mark Selby, Alan J. Korman, Nicholas Wilson. Preclinical characterization of BMS-986288, a novel non-fucosylated (NF) anti-cytotoxic T lymphocyte antigen-4 (anti-CTLA-4) Probody® therapeutic [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 1351.

<p>Legends to supplementary figures</p>

<p>hCD137 and hPD1 expression in human T-lymphocytes infiltrating spleen and liver.</p>

Figure S5 shows human splenic and skin expressions of CCR8

PD-L1/B7-H1 expression on tumor cells is preferentially observed in tumors of the Combo group mice.

Urelumab and Nivolumab bind to hCD137 and hPD-1 expressed on human T-lymphocytes engrafted in Rag2-/-IL2Rgcnull mice.

<p>Anti-mCD137 and anti-mPD-1 combined antitumor effects are solely CD8 T-cells dependent in a murine colon cancer model.</p>

Figure Legends for Supplementary Data

Supplementary Table S1. Staining panels for comprehensive analysis of cell types expressing PD-L1.

Anti-hCD137 (Urelumab) and anti-hPD-1 (Nivolumab) treatment as single agents or in combination show antitumor effects against a xenografted human gastric cancer transferred with autologous lymphocytes drawn from the same patient.

Urelumab and nivolumab do not exert antitumor effects in the absence of hPBMCs.

CCR Translation on this article from Anti–Programmed Death-1 Synergizes with Granulocyte Macrophage Colony-Stimulating Factor–Secreting Tumor Cell Immunotherapy Providing Therapeutic Benefit to Mice with Established Tumors

Supplementary Figures S1-S3 from Anti–Programmed Death-1 Synergizes with Granulocyte Macrophage Colony-Stimulating Factor–Secreting Tumor Cell Immunotherapy Providing Therapeutic Benefit to Mice with Established Tumors

Combined treatment with radiation and tumor-specific mAb or IC does not impact mouse weight.

Figure S4 shows donor immune cell reconstitution in the blood post Ccr8-/- donor bone marrow transplant

PDF file - 206K, Supplemental Figure S1. Equivalent binding of anti-CTLA-4 isotypes to cell surface CTLA-4. Supplemental Figure S2. Serum concentrations of anti-CTLA-4 isotypes in C57BL/6 mice. Supplemental Figure S3. Representative FACS plots for peripheral regulatory T cells. Supplemental Figure S4. A. Representative FACS plots for intratumoral regulatory T cells. B. Kinetics of intratumoral Treg reduction following anti-CTLA-4 treatment. Supplemental Figure S5. Isotype-dependent recruitment of MDSCs and IL-1α production.