Antagonistic and agonist activities of CX3CR1 monoclonal antibody == We tested whether the CX3CR1 antibody had agonist or antagonistic activity when bound to CX3CR1 on the cell surface using CHO cells transfected to express mouse CX3CR1 and a -gal reporter

Antagonistic and agonist activities of CX3CR1 monoclonal antibody == We tested whether the CX3CR1 antibody had agonist or antagonistic activity when bound to CX3CR1 on the cell surface using CHO cells transfected to express mouse CX3CR1 and a -gal reporter. mediators to attract myeloid populations, and 4) generation of tumor-inflammasome. The CX3CR1 monoclonal antibody reduces migration of tumor cells and decreases secretion of immune suppressive soluble mediators by tumor cells. In combination with anti-PD-1 immunotherapy, this CX3CR1 monoclonal antibody enhances survival in an immunocompetent mouse colon carcinoma model through a decrease in tumor-promoting myeloid populations. Thus, this axis is involved in the mechanisms of resistance to anti-PD-1 immunotherapy and the combination therapy can overcome a portion of the resistance mechanisms to anti-PD-1. Keywords:CX3CR1, CX3CL1, PD-1, tumor immune evasion, cancer immunotherapy == 1. Introduction == Immunotherapy with anti-PD-1, PD-L1 and CTLA-4 antibodies has revolutionized cancer treatment (1,2). Anti-PD-1 immunotherapy is FDA approved in multiple cancer types; however, resistance mechanisms result in only a moderate percentage of clinical responses (3). The abundance of immune suppressive myeloid cells is a major resistance mechanism to anti-PD-1 in multiple tumor types (4,5). Aberrant myelopoiesis is a hallmark event in cancer where myeloid cells with immune suppressive properties infiltrate the tumor microenvironment (6). Thus, combination therapies that block the generation and maintenance of immune suppressive myeloid populations are promising approaches to enhance clinical responses to anti-PD-1 therapy (5,7,8). Non-responders to anti-PD-1 in non-small cell lung cancer showed an increase in the plasma concentrations of the CX3CR1 ligand, CX3CL1 (9). An immune suppressive myeloid population defined as CX3CR1+CD206+ may strongly influence the outcome of the response to anti-PD-1 therapy since tumor-CX3CR1+CD206+ myeloid cells were reduced after response to anti-PD-1 in a T3 sarcoma mouse model (10). In summary, the failure to reduce CX3CR1+ myeloid populations may result in non-response and resistance to anti-PD-1 immunotherapy. CX3CR1 binds to its ligand, CX3CL1 (also known as fractalkine or neurotactin), which has membrane-bound and shed forms. The CX3CL1-CX3CR1 axis promotes chemotaxis of CX3CR1+ cells towards soluble CX3CL1 as well as adhesion of CX3CR1+ cells to membrane-bound CX3CL1 (11,12). CX3CR1 activation induces signaling events (13). Following interaction with CX3CL1, CX3CR1 signaling supports tumorigenesis through several mechanisms: 1. CX3CL1 in Rabbit Polyclonal to H-NUC the tumor milieu promotes an influx of CX3CR1+ myeloid cells; a hallmark event of aberrant myelopoiesis, 2. CX3CL1 in the tumor milieu promotes migration of multiple CX3CR1+ tumor types such as breast cancer, prostate cancer, CLL, neuroblastoma, glioblastoma, pancreatic ductal carcinoma (PDAC), colon carcinoma, gastric cancer, skin cancer, lung cancer, osteosarcoma, melanoma, multiple myeloma, and bladder cancer (1418), and 3. CX3CL1 in the tumor milieu promotes activation of several oncogenic pathways following interaction with CX3CR1 in tumors (1936). CRISPR deletion of CX3CR1 or its blockade in human tumor lines of PDAC, breast cancer, prostate cancer, bladder cancer and glioblastoma results in a decrease in the ability of tumor cells to migrate and metastasize (34,37). CX3CR1 deficient mice show a reduction in tumor infiltrating macrophages in SL4 colon carcinoma and skin cancer model (15,38). A small molecule antagonist to CX3CR1 has shown efficacy in preclinical models of breast cancer (34); however, a challenge of small molecule CX3CR1 antagonists is that they can target several related GPCRs (G Protein-coupled receptors) and are not unique LY278584 to CX3CR1. An LY278584 antibody approach might have advantages due to its specificity for CX3CR1, and better receptor engagement based on avidity wherein a higher receptor expression will be required for effective cell depletion by ADCC (39). Together, this led us to investigate whether blockade of the CX3CL1-CX3CR1 axis using an antibody approach would augment the response to anti-PD-1 LY278584 therapy and increase the number of responders in preclinical models whilst offering clinical translatability. We generated a novel monoclonal antibody that binds to CX3CR1 with high affinity, blocks its interaction with CX3CL1 and antagonizes the immune-suppressive signals of this axis. We observe an improvement in response.