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Background: One cause for the failure of checkpoint inhibitors is the immunosuppressive nature of the tumor microenvironment. LILRB1 (ILT2) and LILRB2 (ILT4) are ITIM-containing inhibitory receptors that recognize HLA Class 1 and nonclassical ligands (e.g., HLA-A, HLA-G, etc.). LILRB1 is expressed on myeloid cells and subsets of B, NK, and T cells, while LILRB2 expression is mostly restricted to myeloid cells. Interaction of LILRB1 and LILRB2 receptors with HLA ligands promotes an inhibitory milieu that prevents T cells from attacking cancer cells. The distinct pattern of expression and function of these lymphoid and myeloid checkpoints suggests complementary targeting approaches for cancer immunotherapy. Dual blockade of LILRB1 and LILRB2 receptors by a single antibody that restores both innate and adaptive immune responses is a promising strategy to enhance efficacy of checkpoint inhibitors. Methods: Dual LILRB1 and LILRB2 targeting antibodies were cloned from B cells derived from rabbits immunized with human LILRB2 recombinant protein, and subsequently humanized. Antibodies were evaluated for binding to human LILRB1 and LILRB2 proteins. Dual targeting antibodies were evaluated in a panel of functional and phenotypic assays. Selected antibodies were further tested for efficacy in a humanized NSG-SGM3 tumor model. Results: Dual antibodies were selected based on binding to recombinant human LILRB1 and LILRB2 protein, as well as blocking of HLA-G binding. These antibodies demonstrated binding to cells expressing LILRB1 and LILRB2, with no appreciable binding to other family members. Lead antibodies demonstrated activity in functional cell-based assays modeling LILRB1- or LILRB2-mediated immunosuppression. Dual antibodies also enhanced IFN-γ production by LPS-stimulated human PBMC. Selected clones restored T-cell function from M2c macrophage-mediated suppression in coculture with CD8+ T cell, and enhanced the tumoricidal activity of NK cells. Importantly, the lead antibody demonstrated in vivo efficacy with significant tumor growth inhibition and tumor regression in an SK-MEL-5 tumor model in humanized NSG-SGM3 mice. Conclusions: We have identified dual antagonist antibodies targeting both LILRB1 and LILRB2 antibodies that restore both innate and adaptive immune responses. Additionally, dual antibodies restored CD8+ T cell activation from macrophage-mediated suppression and enhanced NK cell cytotoxic activity. These data provide a strong rationale for further development of dual antibodies as an anti-cancer immunotherapy. Citation Format: Meghan Zuck, Myriam Bouchlaka, Huyen Dinh, Kevin Green, Francisco Zapata, Ramya Chandrasekaran, Tatyana Pisarenko, Lauren Loh, Gajendra S. Naika, Meilyn Sylvestre, Jacob Heit, Raymond Fox, Darbie Whitman, Tom Graddis, Kamal D. Puri, Peter Probst. Discovery and preclinical characterization of dual antagonist antibodies targeting both LILRB1 and LILRB2 that enhance innate and adaptive anti-cancer immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6376.

BackgroundOR2805 antibody was discovered using B cells derived from an elite responder to checkpoint inhibitor (CPI) therapy. It is a fully human IgG1 antibody that binds to CD163, an immune-suppressive receptor highly expressed on tumor associated macrophages (TAMs). High numbers of CD163-expressing TAMs generally predict an unfavorable prognosis in solid tumors. These CD163-expressing TAMs contribute to an immune-suppressive tumor microenvironment and inhibit an anti-tumor T-cell response by engaging immune checkpoints and secreting immune-suppressive cytokines. Relieving the immune suppression of CD163-expressing TAMs to improve anti-tumor T-cell responses is a rational therapeutic strategy as monotherapy and in combination with CPI therapy.MethodsCocultures of immunosuppressive primary human polarized M2c macrophages with autologous CD8+ T cells or phytohemagglutinin (PHA)-T cell blasts (exhausted T cells) were used to interrogate OR2805-dependent immunomodulatory responses as single agent and in combination with pembrolizumab, an anti-PD1 antibody. The anti-tumor activity of OR2805 was evaluated in humanized mouse models. Safety and pharmacokinetics (PK) profile of OR2805 was evaluated in cynomolgus monkeys and human whole blood for cytokine release assessment.ResultsIn coculture assays, OR2805-treatment relieved the suppressive effect of M2c macrophages as demonstrated by increased T-cell proliferation and the release of IFN-γ and perforin. OR2805 restored the IFN-γ production of exhausted T cells and showed a synergistic effect on cocultures treated in combination with pembrolizumab. OR2805-treatment demonstrated significant anti-tumor activity in lung cancer xenograft models in humanized NSG-SGM3 mice. In cynomolgus monkeys, OR2805 demonstrated a typical IgG1 PK profile and good serum exposure. Furthermore, OR2805 did not trigger the release of IL-1β, IL-2, IL-4, IL-6, IL-10, IFN-γ, or TNF-α cytokines in whole blood from either healthy donors or NSCLC patients.ConclusionsOR2805 reduced M2c-mediated immunosuppression and enhanced T cell effector functions. OR2805-treatment resulted in significant anti-tumor activity in lung cancer xenograft models in humanized mice. The pharmacology, PK, and toxicokinetic data support further development of OR2805 as an anti-cancer therapy, both as a monotherapy and in combination with CPI therapy.

BackgroundSiglec-15 is an immunosuppressive sialic acid-binding Ig-like lectin expressed by myeloid cells, tumor associated macrophages (TAMs), and some human tumors. Interactions between Siglec-15 on TAMs and sialoglycans found on cancer cells contribute to the immunosuppressive tumor microenvironment. Furthermore, Siglec-15 expressed by TAMs inhibits anti-tumor immune responses by engaging unknown immune checkpoint(s) on T cells. Notably, the mutually exclusive expression of Siglec-15 and the checkpoint ligand PD-L1 in the tumor tissue emphasizes Siglec-15 as an attractive target for combination immunotherapy. Anti-Siglec-15 antibody is currently being evaluated in clinical trials for the treatment of cancer.MethodsAnti-Siglec-15 antibodies were cloned from B cells derived from rabbits immunized with human Siglec-15 protein. The antibodies were evaluated for binding to human and cynomolgus Siglec-15 by enzyme-linked immunosorbent assay (ELISA). Top clones were selected based on activity in a panel of functional and phenotypic assays using primary human macrophages and T cells and were subsequently fully humanized. The humanized anti-Siglec-15 IgG1 antibodies were screened for binding to human and cynomolgus Siglec-15 by ELISA, binding to cells expressing Siglec-15, and ability to rescue T cell functional activity (proliferation and IFN-γ) from M2c-mediated immune suppression in vitro. The pharmacokinetics of lead humanized Siglec-15 IgG1 antibodies and their anti-tumor activity were evaluated in humanized mouse models.ResultsWe have identified a panel of fully humanized anti-Siglec-15 antibodies that bind to recombinant human and cynomolgus Siglec-15 proteins, to Siglec-15-expressing cell lines and immunosuppressive M2c macrophages without appreciable binding to other Siglec family members. Lead antibodies were identified using functional screens modeling Siglec-15-mediated immune suppression by M2c macrophages. These antibodies restored T cell immune responses in two different M2c/CD8 T cell coculture assays. In the first model, lead antibodies rescued the proliferative and IFN-γ responses of anti-CD3-activated human T cells from the inhibitory activity of M2c macrophages. In the second model, these antibodies restored the ability of exhausted CD8 T cells to secrete IFN-γ in the presence of M2c macrophages. Lead antibodies demonstrated a half-life of 6–11 days in humanized FcRn mice, and tumor growth inhibition in humanized NSG-SGM3 mice.ConclusionsWe have identified novel humanized anti-Siglec-15 antibodies that restore effector function of activated and exhausted T cells from M2c-mediated immune suppression, with excellent half-life and anti-tumor activity in humanized mouse models. These data provide a strong rationale for further development of these antibodies for anti-cancer immunotherapy.

Background: OR2805 is a fully human IgG1 antibody that binds to CD163, an immune-suppressive receptor highly expressed on tumor associated macrophages (TAMs). High numbers of CD163-expressing TAMs generally predict an unfavorable prognosis in solid tumors. CD163-expressing TAMs contribute to an immune-suppressive tumor microenvironment (TME) and inhibit an anti-tumor T-cell response by engaging immune checkpoints, producing immune-suppressive cytokines, and promoting T-cell skewing towards a pro-cancer Th2 phenotype. Relieving the immune suppression of CD163-expressing TAMs in the TME to improve T-cell-mediated responses is a rational adjunct to immune checkpoint inhibitor (CPI) therapy. Methods: OR2805 was discovered using OncoResponse's discovery platform by cell-based functional and phenotypic assays. B cells derived from CPI elite responders were cultured at clonal density, and IgG antibodies in supernatants were evaluated for binding to myeloid-derived suppressor cells. Variable-regions from positive hits were sequenced, cloned, and expressed as recombinant IgG1. OR2805 was identified as one of the top hits that relieved myeloid cell-mediated immune suppression. Co-cultures of immunosuppressive primary human M2 macrophages and autologous T cells were used to interrogate OR2805-dependent immunomodulatory responses in vitro. The anti-tumor activity of OR2805 in vivo was evaluated in a humanized mouse model. Results: OR2805 binds to CD163 expressed on TAMs and M2-like macrophages. OR2805 does not bind to other hematopoietic cells nor to a panel of human primary non-immune cells. OR2805 treatment reduces expression of cell-surface markers associated with tumor-promoting M2c-like macrophages. In co-culture assays, OR2805 relieves the suppressive effect of M2 macrophages and results in increased T-cell activation and proliferation, upregulation of T-cell activation markers, and enhanced T-cell-mediated tumor cell killing. Administration of OR2805 in humanized NSG-SGM3 mouse tumor models resulted in approximately 55% and 75% reduction in A549 tumor growth and NCI-H1975 tumor growth, respectively. In this model, OR2805 treatment significantly increased the proportions of human CD8+ T cells and human CD11b+ myeloid cells, as well as significantly enhanced expression of activation markers by human CD8+ T cells. Conclusions: OR2805 reduces M2 macrophage-mediated immunosuppression and enhances anti-tumor immune responses. OR2805 treatment induces anti-tumor activity in lung cancer xenograft models in humanized mice. These data support further development of OR2805 as an anti-cancer therapy, both as a monotherapy and an addition to current CPI therapy. Citation Format: Peter Probst, Randi Simmons, Valerie Wall, Meghan Zuck, Myriam Bouchlaka, Sam Lam, Raymond Fox, Darbie Whitman, Tom Graddis, Kamal D. Puri. OR2805, an anti-CD163 antibody derived from an elite responder to checkpoint inhibitor therapy relieves immunosuppression caused by tumor associated macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1719.

Influenza A continues to be a serious public health threat as it continuously evolves to evade and escape immune surveillance through genetic drift, shift, and re-assortment. Hemagglutinin (HA) is the main glycoprotein target of the humoral immune response and elicits protective, neutralizing, antibodies during infection. However, these antibodies are usually specific for the influenza subtype causing infection and exhibit a narrow spectrum of protectiveness. To date, many of the influenza-neutralizing monoclonal antibodies described are cross-specific for subtypes H1 and H5 but do not neutralize subtypes H3 or H7. Therefore, we have designed an approach to survey the human B cell repertoire to identify monoclonal antibodies that neutralize influenza viruses of both the H1 and H3 subtypes. Our strategy is to identify humans that have broadly reactive serum antibodies, culture IgG+ memory B cells from these individuals at near clonal density, and then assay the culture supernatants for the presence of antibodies that neutralize both H1 and H3 influenza subtypes. By combining functional and binding assays we will determine if there exist in nature highly conserved neutralizing epitopes that are represented on most or all of the influenza HA subtypes tested.

A serum titer of autoantibodies to IFN-α has been reported in several autoimmune conditions and in many cases has been shown to neutralize IFN-α. The IFN-α antibody repertoire from 10 patients with autoimmune myasthenia gravis and associated thymoma (MG/T) was characterized. Surface IgG positive memory B-cells from peripheral blood mononuclear cells (PBMC) were cultured in 384-well plates to produce assayable quantities of antibody. Sensitive antibody microarray and AlphaScreen™ binding assays were used to identify antibodies reactive with IFN-α(2a). A range of IFN-α subtype specificity profiles was observed in competition assays for binding to the 12 subtypes of IFN-α. A subset of these antibodies was expressed recombinantly after recovery of heavy and light chain variable regions by RT-PCR and these recombinant antibodies recapitulated the activities demonstrated in the B-cell culture supernatants. These antibodies fell into 2 groups, blocking binding of IFN-α(2a) to either the IFNAR2 or IFNAR1 subunit of the Type 1 interferon receptor. The ability of these antibodies to neutralize the activity of IFN-α(2a) in a Daudi cell proliferation assay was confirmed. The antibody repertoires of MG/T patients include high affinity antibodies with both broad and narrow specificities for the different subtypes of IFN-α. These function-blocking human antibodies may have therapeutic utility in IFN-α dependent diseases such as SLE.