Inhibiting CXCR4 reduces immunosuppressive effects of myeloid cells in breast cancer immunotherapy

Patients with triple negative breast cancer (TNBC) show only modest response rates to immune checkpoint inhibitor immunotherapy, motivating ongoing efforts to identify approaches to boost efficacy. Using an immunocompetent mouse model of TNBC, we investigated combination therapy with an anti-PD-1 immunotherapy antibody plus balixafortide, a cyclic peptide inhibitor of CXCR4. Cell-based assays demonstrated that balixafortide functions as an inverse agonist, establishing a mode of action distinct from most compounds targeting CXCR4. Combination anti-PD-1 plus balixafortide significantly reduced growth of orthotopic tumors and extended overall survival relative to single agent therapy or vehicle. Adding balixafortide to anti-PD-1 increased numbers of tertiary lymphoid structures, a marker of local tumor immune responses associated with favorable response to immunotherapy in TNBC. Single cell RNA sequencing revealed that combination anti-PD-1 plus balixafortide reduced T cell exhaustion and increased markers of effector T cell activity. Combination therapy also reduced signatures of immunosuppressive myeloid derived suppressor cells (MDSCs) in tumors. MDSCs isolated from mice treated with anti-PD-1 plus balixafortide showed reduced inhibition of T cell proliferation following ex vivo stimulation. These studies demonstrate that combining inhibition of CXCR4 with anti-PD-1 to enhances responses to checkpoint inhibitor immunotherapy in TNBC, supporting future clinical trials.