Longitudinal tissue analysis and correlation of microenvironmental changes with combined immunotherapy and targeted therapy response in metastatic breast cancer.

1022 Background: The ability to interrogate changes within the tumor microenvironment before, during and following therapeutic intervention could yield important understanding of treatment response and causes for disease progression.Here we conducted a multicenter phase II clinical trial (NCT04521179) examining the effect of a novel CTLA-4/PD-L1 bispecific (KN046) antibody in combination with a a novel anti-HER2 bispecific (KN026) antibody in treatment resistant metastatic breast cancer. Our on-going trial demonstrated that in advanced HER2-positive breast cancer (HER2+ BC) patients, who have progressed after prior anti-HER2 combinational therapies, the objective response rate (ORR) of this chemo-free therapy of KN026 in combination of KN046 was about 47.2% ( 95% CI: 30.4-64.5). To explore the underlying mechanism of this regimen, we collected tumor specimens from patients before and after receiving this combinational treatment for emerging multi-modal molecular analyses ("Multi-omics") to provide an in-depth description of the tumor immune microenvironment and its correlation with treatment response. Methods: We performed matched pre- and on-treatment investigative biopsies on index tumors and performed single-cell RNA sequencing (scRNA-seq) and single-cell T cell receptor sequencing (scTCR-seq) analysis. Results: We performed comprehensive scRNA-seq on tumor biopsies obtained from a total of 17 patients that evaluable for overall response. Among them, 13 patients had two biopsies taken, and four patients had one biopsy collected either before or during treatment. Single-cell RNA and T cell receptor sequencing from 334,183 cells from site-matched tumors reveal significant temporal shift of various immune cell populations and phenotypes within the tumor microenvironment associated with treatment responses. In-depth analysis of subpopulations revealed that CD8 + T cells are activated in responsive patients during treatment, and T REG cells, one of CD4 + T cell subtypes, are activated in non-responsive patients after therapy. Moreover, we also found that combined-therapy activates cDCs and induces an inflammation shift in Mϕs of responsive patients. Conclusions: We identified that regulatory T cells are activated while effector T cells, natural killer cells, and dendritic cells were significantly depleted in non-responding tumors. The immune response in responsive patients was effectively enhanced, whereas in nonresponsive patients, it was significantly diminished. And higher baseline levels of Mϕs were associated with therapeutic resistance. These results support that longitudinal analysis of tumor microenvironment to generate multi-omics data that can lead to rich insight disease process and to provide clinical value in evaluating treatment responses. Clinical trial information: NCT04521179 .