A TROP2/Claudin Program Mediates Immune Exclusion to Impede Checkpoint Blockade in Breast Cancer

Abstract

Immune exclusion inhibits anti-tumor immunity and response to immunotherapy, but its mechanisms remain poorly defined. Here, we demonstrate that Trophoblast Cell-Surface Antigen 2 (TROP2), a key target of emerging anti-cancer Antibody Drug Conjugates (ADCs), controls barrier-mediated immune exclusion in Triple-Negative Breast Cancer (TNBC) through Claudin 7 association and tight junction regulation. TROP2 expression is inversely correlated with T cell infiltration and strongly associated with outcomes in TNBC. Loss-of-function and reconstitution experiments demonstrate TROP2 is sufficient to drive tumor progression in vivo in a CD8 T cell-dependent manner, while its loss deregulates expression and localization of multiple tight junction proteins, enabling T cell infiltration. Employing a humanized TROP2 syngeneic TNBC model, we show that TROP2 targeting via hRS7, the antibody component of Sacituzumab govitecan (SG), enhances the anti-PD1 response associated with improved T cell accessibility and effector function. Correspondingly, TROP2 expression is highly associated with lack of response to anti-PD1 therapy in human breast cancer. Thus, TROP2 controls an immune exclusion program that can be targeted to enhance immunotherapy response.

Keywords: Antibody-Drug Conjugate; PD-1; TROP2; antitumor immunity; immune checkpoint blockade; immune exclusion; sacituzumab govitecan; tight junction; triple negative breast cancer; tumor immune microenvironment.

Figure 1.. TROP2 promotes mammary tumor growth and immune exclusion in immunocompetent hosts.

A, Top 20 pathways positively (blue) or negatively (green) associated with TROP2 expression in human breast cancer identified by Gene Set Variation Analysis (GSVA) using METABRIC dataset. Top and bottom quartiles of TROP2 expression were used to compute the association. B, Correlation between TROP2 and immune cell markers in multiple cancer types by TIMER analysis in the TCGA database. Red dashed box indicates breast cancers. C, TROP2 predicts poor overall survival in basal subtype of breast cancer. Generated by Kaplan-Meier Plotter database (https://kmplot.com/analysis/). D, Western blot for TROP2 in wildtype (WT) and Trop2 knockout (KO) 4T1 cells. E-G, WT and KO 4T1 tumor cell growth in cell culture (E); in immunocompromised (nude) hosts (n = 10 tumors per group) (F), and in syngeneic immunocompetent BALB/C hosts (WT, n = 12 tumors; KO, n = 10 tumors) (G).

Figure 2.. TROP2 promotes mammary tumor immune exclusion.

A and B, Representative images (A) and quantification (B) of CD3⁺ T cell IHC staining in WT and KO tumor margins and cores. C and D, Representative flow cytometry contour plots (C) and quantification (D) of CD44⁺ CD62L⁻ effector memory CD8⁺ T cells in WT and KO tumors. E and F, Representative flow cytometry contour plots (E) and quantification (F) of PD1⁺ activated CD8⁺ T cells in WT and KO tumors. G and H, Representative flow cytometry histogram (G) and quantification (H) of Granzyme B mean fluorescence intensity (MFI) in CD8⁺ T cells in WT and KO tumors. I, Percentages of CD8⁺ cells among CD3⁺ T cells in splenocytes harvested from mice treated with control IgG or anti-CD8⁺ depleting antibodies. J, Trop2-WT and -KO 4T1 tumor growth curves in BALB/C mice treated with control IgG or anti-CD8 depleting antibodies (WT, n = 10 tumors per group; KO/IgG, n = 8 tumors; KO/anti-CD8: n = 6 tumors). K, Kaplan-Meier survival curves of WT and KO tumor-bearing BALB/C mice treated with control IgG or anti-CD8 depleting antibodies (WT, n = 10 mice per group; KO/IgG, n = 9 mice; KO/anti-CD8: n = 8 mice).

Figure 3.. TROP2 promotes a tight junction-mediated barrier and inhibits a pro-inflammatory tumor immune microenvironment.

A, Enriched pathways from Gene set enrichment analysis (GSEA) on bulk RNA-seq of Trop2-WT and KO 4T1 tumors harvested from BALB/C hosts. B, Western blot analysis showing co-immunoprecipitation (co-IP) of Claudin7 and TROP2 using IgG or anti-TROP2 in Trop2-WT and KO 4T1 (left panel), MDA-MB468 (middle panel), and HCC1806 (right panel) TNBC cells. C, Representative tumor tissue immunofluorescence images of TROP2, tight junction molecule Claudin7, and DAPI (nuclear stain), showing TROP2/Claudin 7 co-localization, and loss of Claudin7 expression and membrane localization with TROP2 knockout. D, Quantification of proportion of membrane Claudin7-expressing cells in WT and KO 4T1 tumors for panel C. E, Representative tumor tissue immunofluorescence images of Occludin and DAPI (nuclear stain) showing loss of Occludin expression with TROP2 knockout. F, Quantification of Occludin intensity in WT and KO 4T1 tumors for panel E.

Figure 4.. TROP2 intracellular domain is dispensable for its tight junction and immune barrier function.

A, Histogram showing flow cytometry analysis of cell-surface TROP2 in Trop2-KO 4T1 reconstituted with control vector (Stuff), Full-Length TROP2, or C-terminal truncated ECDTM mutant. B, In vitro proliferation of Trop2-KO 4T1 reconstituted with control vector, Full-Length TROP2 or ECDTM truncated mutant. C, Tumor growth curves of Trop2-KO 4T1 reconstituted with control vector (Stuff), Full-Length TROP2, or C-terminal truncated ECDTM TROP2 mutant in BALB/C immunocompetent hosts (n = 10 tumors per group). D, Representative tumor tissue immunofluorescence images of Claudin7 in tumors harvested from panel C. E, Quantification of Claudin7 for panel D. F and G, Representative images (F) and quantification (G) of CD3⁺ T cell IHC staining in tumors harvested from panel C.

Figure 5.. TROP2 targeting via the naked anti-TROP2 antibody of SG promotes anti-PD1 efficacy.

A, Western blot of Trop2-KO 4T1 cells reconstituted with empty vector (EV) or human TROP2 (hTROP2). B, Western blot showing co-IP of hTROP2 and Claudin 7 in Trop2-KO 4T1 cells reconstituted with hTROP2. C, Tumor growth curves for KO+hTROP2 (hRS7+anti-PD1, n = 10 tumors; n = 8 tumors per group for other three groups) in BALB/C mice treated with Vehicle, hRS7, anti-PD1, or hRS7+anti-PD1 combination. Treatment started when tumors reached 100 mm³. Arrows indicate treatment days. D, Representative tumor tissue immunofluorescence images of Claudin7 in tumors harvested from panel C. E, Quantification of Claudin7 for panel D. F and G, Representative images (F) and quantification (G) of CD3⁺ T cell IHC staining in tumors harvested from panel C. H and I, Representative flow cytometry contour plots (F) and quantification (G) of TIM3⁻ early activated CD8⁺ PD1⁺ T cells in tumors harvested from panel C. *p<0.05, **p<0.01.

Figure 6.. High TROP2 associates with non-responses to immune checkpoint inhibitors in human breast cancers.

A, Odds ratios for associations between gene expression (PDCD1 (PD1), TACSTD2 (TROP2) and EPMCAM) and response to anti-PD1 therapy. Cohort 1: Response (evaluated radiologically using RECIST criteria) to combination Pembrolizumab, Carboplatin, and Nab-paclitaxel among patients with advanced TNBC; NR: non-responsive, R: responsive. Cohort 2: Response assessed by T cell receptor clonotype expansion following pre-operative single-agent Pembrolizumab for breast cancer, NE: no clonotype expansion, E: yes clonotype expansion. B, Violin plots derived from single-cell RNAseq data from the cohort in (A), showing subset signature scores among CD8⁺ T cells in tumors expressing low vs. high epithelial TROP2. CD8 signatures are derived from PMID: 37248301. C, Proposed model for TROP2-mediated immune exclusion in breast cancer. Left, TROP2 enables a claudin/tight junction-mediated mechanical barrier to exclude antitumor immune infiltration. Right, genetic deletion or pharmacological blockade of TROP2 deregulates the tight junction-mediated mechanical barrier and allows antitumor immune infiltration.