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Regulatory T cells (Tregs) contribute to immunosuppression within the tumor microenvironment and have been associated with poor outcomes in a range of cancers. Targeted depletion of tumor-infiltrating Tregs (TITRs) is an attractive therapeutic strategy with the potential to enhance antitumor immunity in patients who do not respond to current treatments. However, such approaches have been limited to date by a lack of markers that are highly specific for TITRs. CCR8 is a chemokine G protein-coupled receptor (GPCR) that has recently been shown to be specifically expressed on TITRs as compared to Tregs in the periphery or other T cells within tumors. Here, we report the development and characterization of HFB101110, a humanized monoclonal antibody against CCR8 with potent and specific antibody-dependent cellular cytotoxicity (ADCC) activity. HFB101110 specifically recognizes an epitope on the N-terminus extracellular domain of CCR8 and does not recognize the closely related chemokine receptor CCR4. HFB101110 acts through a dual mechanism of action, by both depleting CCR8+ cells via ADCC and blocking binding of the CCL1 chemokine to its receptor CCR8. Blockade by HFB101110 inhibited calcium flux and chemotaxis induced by the interaction between CCL1 and CCR8. Furthermore, HFB101110 showed potent single agent anti-tumor activity associated with depletion of intratumoral Tregs in a human CCR8 knock-in mouse model. HFB101110 mediated specific ex vivo killing of Tregs from primary patient tumor samples both in the presence, and in some cases the absence, of allogeneic NK cells. HFB101110 showed favorable pharmacokinetic properties and a favorable developability profile, and was well-tolerated in both wild-type mice and cynomolgus monkeys. HFB101110 is currently being developed as a novel immunotherapy for the treatment of solid tumors coupled with a patient biomarker strategy derived from HiFiBiO’s Drug Intelligent Science (DIS™) single-cell immune profiling platform. Citation Format: Roshan M. Kumar, Julie Prigent, Hombline Poullain, Ayrin Kok, Carine George, Sami Ellouze, Yun-Yueh Lu, Rebecca Silver, Clarisse Monchecourt, Ross B. Fulton, Qian Zhang, Nicola Beltraminelli, Bernhard Moser, Francisco Adrian, Liang Schweizer. Targeting regulatory T cells with HFB101110, a novel anti-human CCR8 antibody for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2899.

Despite the success of immune check point inhibition, identification of other pathways capable of modulating the immune response against the tumor remains challenging. T-cell co-stimulation has been investigated with limited clinical success so far due in part to the fine tuning required for agonist antibodies against those co-stimulatory receptors and to the lack of biomarkers to facilitate the selection of patients likely to benefit from T-cell co-stimulation. TNFR2 belongs to the TNFR family of costimulatory molecules, and its expression on tumor infiltrating lymphocytes across a wide range of tumors make it an attractive target for T-cell co-stimulation. Recently, we identified HFB200301, an anti-TNFR2 antibody with Fc-independent agonist activity that does not block TNFR2 interaction with TNFα. HFB200301 activates CD4+, CD8+ T cells, and NK cells in vitro. In vivo, HFB200301 demonstrated potent single agent anti-tumor activity in syngeneic tumor models and can further increase the antitumor activity in combination with PD-1 blockade. To understand the immunological basis for the anti-tumor efficacy of HFB200301, we investigated the pharmacodynamic effects of HFB200301 in syngeneic mouse tumor models, including immuno-phenotyping and receptor occupancy of tumor infiltrating cells. In hTNFR2 knock-in mice bearing MC38 tumors, HFB200301 induces expansion of CD4+ and CD8+ T cells, and NK cells in the tumor micro-environment without affecting regulatory T cell numbers. We also demonstrate that the anti-tumor efficacy of HFB200301 is correlated with receptor occupancy and circulating soluble TNFR2 in a dose-dependent manner in this model. To discover predictive biomarkers of response to HFB200301, we used primary tumor samples and our proprietary Drug Intelligent Science (DIS™) single-cell platform to establish an immune-related signature. Single-cell RNA sequencing and clonotype barcoding of ex-vivo tumor cultures treated with HFB200301 were used to identify unique T cell profiles with a T cell centric gene panel. These unique T cell profiles may help identifying patients more likely to respond to HFB200301 treatment. In summary, HFB200301 exhibits a unique mechanism of action mainly relying on its agonistic activity on several effector cell types in tumor micro-environment that we expect will benefit a patient population selected with a unique biomarker signature. HFB200301 is currently in preclinical development and a biomarker-driven Phase 1 clinical study is projected for 2021. Citation Format: Shuo Wei, Ross Fulton, Yun-Yueh Lu, Qian Zhang, He Zhou, Andreas Raue, Mingjie Chen, Wenhua Xu, Xing Cai, Juliana Crivello, Zachary Duda, Zhiyuan Wang, Rebecca Silver, Alexandra Staskus, Charina Ortega, Sami Ellouze, Carine George, Sophie Foulon, Dean Lee, Monika Manne, Nicola Beltraminelli, Jinping Gan, Francisco Adrian, Liang Schweizer, Jennifer Watkins-Yoon. Mechanism of action and biomarker strategy for HFB200301, an anti-TNFR2 agonist antibody for the treatment of cancer [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 1883.

CXCR5 is a seven transmembrane protein coupled to protein G(i) expressed mainly on mature, resting B cells, follicular helper T cells (TFH), tonsillar B cells, and B cell lymphoma cells. CXCR5 is activated by its ligand CXCL13 and involved in cell migration. Dysregulated CXCL13 and/or CXCR5 expression and signaling have been associated with cancer promotion, as well as autoimmune and chronic inflammatory disorders. Overexpression of CXCL13 in autoimmune and lymphoproliferative disorders has been shown to correlate with disease progression. We therefore hypothesized that an antibody that selectively depletes CXCR5+ malignant B cells through antibody dependent cell-mediated cytotoxicity (ADCC) could be developed to treat B cell lymphomas. Here, we present preclinical data supporting the rationale for indication selection, biomarker identification, dose selection, and phase I clinical trial design. Using our single-cell screening platform technology, CelliGo™, we identified a panel of monoclonal antibodies against human CXCR5. We selected HFB2-4, which binds to CXCR5 expressing cells, blocks CXCL13-induced intracellular signaling and B cell migration, and induces ADCC of Raji B cell lymphoma cells at sub-nM concentrations. HFB2-4 demonstrates good PK behavior in mice and exhibits potent anti-tumor activity in a Raji xenograft murine model. HFB2-4 efficacy in this model is comparable to that of rituximab, despite a lower number of huCXCR5 receptors compared to CD20 on the surface of Raji cells. Our data shows that HFB2-4 holds promise as a therapeutic agent for B cell lymphoproliferative diseases by depleting CXCR5+ tumor B cells in refractory B cell lymphoma and lymphomas derived from severe Sjogren syndrome. Compared to the emerging treatments for refractory B cell lymphomas such as Antibody-Drug-Conjugates, bi-specific antibodies or cell therapies, HFB2-4 may represent a well-tolerated, efficacious, cost-efficient and easy to manufacture alternative. Citation Format: Ayrin Kok, Germain Margallducos, Stephanie Beq, Clarisse Monchecourt, Hombline Poullain, Muriel David, Carine George, Sami Ellouze, Mingjie Chen, Yun-Yueh Lu, Juying Li, Qian Zhang, Jean Wang, Francisco Adrian, Liang Schweizer, Nicola Beltraminelli. Dicovery and characterization of a novel anti-human CXCR5 antibody for the treatment of B cell lymphomas [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 1843.

Agonist OX40 antibodies have shown promising pre-clinical activities, but their clinical activities have been limited thus far. Several reasons may account for this limited clinical activity, including sub-optimal antibody design, dose selection, and lack of a biomarker strategy for indication selection and patient enrichment. Previous clinical trials selected doses that maximized receptor occupancy, but some patients responded at lower doses, indicating nuances to choosing the correct therapeutic doses for an agonist antibody. In addition, preclinical work has demonstrated a so-called “hook” effect whereby agonist activity decreases at higher concentrations, which further emphasizes the need to develop a novel anti-OX40 therapeutic antibody that addresses the previously encountered challenges. HFB301001 is a novel human IgG1 agonist antibody that binds to a unique epitope on OX40. This allows for agonistic activity that does not compete with the endogenous OX40 ligand. Relative to other clinical stage OX40 antibodies, HFB301001 has reduced OX40 downregulation following co-stimulation of T cells, and it has demonstrated superior in vivo anti-tumor activity and pharmacodynamic immune modulation in a human OX40 knock-in mouse model. HFB301001 is well tolerated in cynomolgus monkeys. To progress into clinical studies, we have determined human dose projections for clinical evaluation of HFB301001 using PKPD modeling, serum exposure in non-human primates, antitumor efficacy in mouse models and immune cell pharmacodynamics. We also took advantage of Fc variants to delineate the relative contributions of Treg depletion versus enhancing T cell activity by agonism to efficacy of HFB301001. To further enhance probability of success (POS) in clinical studies, we are applying our single-cell Drug Intelligent Science (DIS™) platform to rationally identify cancer indications and to define novel predictive response biomarkers. We have used single-cell profiling to identify unique tumor-infiltrating T cell signatures that may help identify patients more likely to response to HFB301001 treatment, inform indication selection, and establish a patient stratification biomarker strategy. Finally, we show here our phase I trial design for HFB301001 that implements these findings. In conclusion, HFB301001 is a highly differentiated therapeutic antibody which is well positioned to enter a global, multi-center Phase I clinical trial to explore optimal biologically active dose and evaluate predictive biomarker hypotheses. Here, we present results supporting the rationale for indication selection, biomarker identification, dose selection, and phase I clinical trial design. Citation Format: Ross Fulton, Jinping Gan, Yun-Yueh Lu, Julianna Crivello, Zachery Duda, Zhiyuan Wang, Rebecca Silver, Alexandra Staskus, Charina Ortega, Sami Ellouze, Carine George, Sophie Foulon, Wenhua Xu, Xing Cai, Joyce Pi, Dean Lee, Monika Manne, Ruina Jin, Yuan Wang, Hongkai Zhang, Nicola Beltraminelli, Francisco Adrian, Robert Petit, Liang Schweizer, Andreas Raue. Clinical approach and biomarker strategy for HFB301001, a novel OX40 agonistic antibody [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 1882.