BackgroundCancer immunotherapy has revolutionized cancer treatment. However, considering the limited success of immunotherapy to only some cancer types and patient cohorts, there is an unmet need for developing new treatments that will result in higher response rates in patients with cancer. Immunoglobulin-like transcript 2 (ILT2), a LILRB family member, is an inhibitory receptor expressed on a variety of immune cells including T cells, natural killer (NK) cells and different myeloid cells. In the tumor microenvironment, binding of class I MHC (in particular HLA-G) to ILT2 on immune cells mediates a strong inhibitory effect, which manifests in inhibition of antitumor cytotoxicity of T and NK cells, and prevention of phagocytosis of the tumor cells by macrophages.MethodsWe describe here the development and characteristics of BND-22, a novel, humanized monoclonal antibody that selectively binds to ILT2 and blocks its interaction with classical MHC I and HLA-G. BND-22 was evaluated for its binding and blocking characteristics as well as its ability to increase the antitumor activity of macrophages, T cells and NK cells in various in vitro, ex vivo and in vivo systems.ResultsCollectively, our data suggest that BND-22 enhances activity of both innate and adaptive immune cells, thus generating robust and comprehensive antitumor immunity. In humanized mice models, blocking ILT2 with BND-22 decreased the growth of human tumors, hindered metastatic spread to the lungs, and prolonged survival of the tumor-bearing mice. In addition, BND-22 improved the antitumor immune response of approved therapies such as anti-PD-1 or anti-EGFR antibodies.ConclusionsBND-22 is a first-in-human ILT2 blocking antibody which has demonstrated efficient antitumor activity in various preclinical models as well as a favorable safety profile. Clinical evaluation of BND-22 as a monotherapy or in combination with other therapeutics is under way in patients with cancer.Trial registration numberNCT04717375.
Background: Ig-like transcripts (ILTs) are a family of immuno-modulating receptors expressed on immune cells. ILT2 is an inhibitory receptor expressed on both innate and adaptive immune cells. It has been shown to bind to MHC class I molecules and binds with the highest affinity to HLA-G, an immunosuppressive MHC molecule expressed by multiple tumor types. ILT2-mediated inhibition can lead to impairment of immune cell proliferation, differentiation, phagocytosis, cytotoxicity and cytokine secretion, identifying the ILT2 signaling axis as a potential novel target for anti-cancer immunotherapy. Here we describe preclinical characterization of BND-22, a humanized IgG4, ILT2 antagonist antibody developed for the treatment of solid tumors. Methods: BND-22 binding to ILT2 was evaluated by flow cytometry, ELISA and surface plasmon resonance. We investigated the ability of BND-22 to reverse ILT2-mediated immune suppression of macrophages and lymphocytes. Macrophage and NK cell effector functions were studied in vitro and ex vivo in the presence of several cancer cell lines and patient tumor tissues using flow cytometry, time-lapse live-cell-microscopy, and functional cell-based assays. Xenograft subcutaneous melanoma and colorectal carcinoma and melanoma lung metastases models were used to evaluate the in vivo anti-tumor effect of BND-22. Results: BND-22 binds ILT2, but not other ILT-family receptors, with low nanomolar affinity and blocks its interaction with HLA-G in a concentration-dependent manner. ILT2 blockade with BND-22 increased macrophage-mediated phagocytosis of tumor cell lines and patient-excised tumors, enhanced the cytotoxicity of NK cells against multiple types of cancer cells, and increased NK cell production of Granzyme B and IFNγ. ILT2 and PD-1 were found to be expressed by different sub-populations of CD45+ cells. Co-blockade of ILT2 and PD-1 showed a synergistic effect in increasing pro-inflammatory cytokine production by patient-derived PBMC in response to autologous tumor cells. In subcutaneous melanoma and colorectal carcinoma xenograft mouse models, BND-22 enhanced the anti-tumor activity of macrophages, inhibited tumor growth and prolonged survival. BND-22 also potently increased the anti-tumor activity of lymphocytes and inhibited metastases spread in an in vivo melanoma lung lesion model. Conclusions: BND-22, a first-in-class, anti-ILT2 antagonist antibody induces a potent macrophage and lymphocyte-driven anti-tumor immune response in multiple in vitro, ex vivo and in vivo models. BND-22 offers a differentiated, multi immune cell-based mechanism of action. Safety, tolerability, and anti-tumor activity of BND-22 will be explored in a first-in-human clinical trial in cancer patients with tumor types known to express HLA-G. Citation Format: Ilana Mandel, Dana Haves, Ilana Goldshtein, Tsuri Peretz, Dror Alishekevitz, Yair Sapir, Sharon Hashmueli, Itay Friedman, Tehila Ben Moshe. BND-22, a first-in-class, anti-ILT2 monoclonal antibody inhibits the immunosuppressive effects of HLA-G and enhances anti-tumor activity of immune cells in preclinical in vitro, ex vivo, and in vivo models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3266.
2571 Background: Leukocyte Ig-like receptor B1 [LILRB1; ILT2] is an inhibitory receptor expressed on various immune cells. ILT2 binds to classical and nonclassical MHC class I molecules, with highest affinity to HLA-G. ILT2-mediated inhibition leads to impairment of immune cell proliferation, differentiation, phagocytosis, cytotoxicity and cytokine secretion. Antagonism of ILT2 signaling may serve as a novel target for anti-cancer immunotherapy. SAR444881 (BND-22) is a novel humanized IgG4 monoclonal antagonist antibody which selectively binds to ILT2 and blocks its interaction with MHC I molecules. SAR444881 induces robust macrophage and lymphocyte-driven anti-tumor activity in in vitro and in vivo models. To overcome the limitation that LILRB proteins are not expressed in rodents, we conducted a series of in vivo studies using humanized mouse models, cancer patient biopsies and ex vivo co-culture systems to interrogate the pharmacodynamic (PD) response of ILT2 antagonism as well as inform the combinatorial and patient enrichment strategies for SAR444881. Methods: SAR444881-induced modulation of PD biomarkers was evaluated in humanized xenograft models. Ex vivo co-culture system has been established using patient tumor tissues and isolated PBMC or other immune cells. Biomarker expression on immune cells and secreted soluble proteins were monitored by flow cytometry and ELISA, respectively. Procured tumor samples of patients with various advanced solid tumors, including Head and neck squamous cell carcinoma (HNSCC), Gastric Cancer (GC), Non-Small Cell Lung Cancer (NSCLC) and Colorectal Cancer (CRC) were utilized to evaluate protein or gene expression levels of potential predictive biomarkers (ILT2, HLA = G, PD-L1) using immunohistochemistry (IHC) and whole transcriptomic analysis (RNAseq). Results: 1) PD biomarkers: in murine models, SAR444881 modulates intra-tumoral sub-populations of CD8+ T cells, NK cells and Macrophage polarization; 2) Combination strategy: functional study in an ex vivo system showed that addition of an anti-PD-1 antibody induced an increased production of pro-inflammatory cytokines, including IFNγ and TNFα, compared to SAR444881 or anti-PD-1 alone. Concomitantly, combining SAR444881 with cetuximab resulted in increased phagocytosis compared to isotype control. 3) Predictive biomarkers: protein and gene profiling results suggest enriched expression of ILT2 in majority of biopsies from several tumor types including NSCLC, HNSCC and CRC. Conclusions: These data inform the PD response biomarkers, combination, and patient enrichment strategies for the clinical development of SAR444881 to maximize its benefits for cancer patients. An ongoing phase 1/2 trial of SAR444881 mono- and combination therapy in patients with advanced solid tumors is testing these concepts in the clinic (NCT04717375).
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