Trophoblast Cell Surface Antigen 2 Expression Research Articles

Trophoblast cell-surface antigen 2 is a biomarker and potential therapeutic target in human colon cancer.

e15514 Background: Trophoblast cell-surface antigen 2 (TROP2), a transmembrane glycoprotein in epithelial cells, is implicated in malignant progression, notably in breast cancer therapy. However, the clinicopathologic aspects of TROP2 in colorectal cancer (CRC) are unclear. This study aims to explore the association between TROP2 expression and clinicopathological features in CRC and investigate the inhibitory effects of the anti-TROP2 antibody-SN-38 conjugate (ADC-TROP2) on colon cancer cells. Methods: A retrospective analysis of 1296 CRC patients who underwent colorectomy at Renji Hospital, Shanghai Jiaotong University School of Medicine, was conducted. Immunohistochemistry determined the expressions of TROP2, HER2, and PD-L1 in colon cancer tissues. Statistical analyses, including the chi-square test and Cox proportional hazards model, assessed associations. Sacituzumab Govitecan (ADC-TROP2 drug) cytotoxicity on human cancer cells was determined using the MTT method. Results: TROP2 was predominantly expressed at the cell membrane of cancer tissues, with 50% of cases TROP2-positive. The overexpression of TROP2 (staining extension 2+ or 3+) were found in 356 (27.5%) cases. The clinicopathological features of all 1296 patients based on the TROP2 expression in their cancer cells were summarized in Table 1. Compared to TROP2 negativity expression, the high TROP2 expression was significantly associated with the advantaged tumor stage (III/IV, P = 0.022), high tumor invasion depth (T3/T4, P = 0.002), lymph node metastasis (N1/N2, p = 0.026), HER-2 expression (2+/3+, p = 0.013) ,PD-L1 CPS (CPS≤5,P = 0.007). Sacituzumab Govitecan exhibited stronger inhibition in TROP2-positive human CRC cells in TROP2-negative CRC cells compared to TROP2-negative cells and the control SN-38 agent. Conclusions: TROP2 expression positive in 50% of CRC cases is associated with with advantaged tumor stage, high tumor invasion depth, lymph node metastasis. Sacituzumab Govitecan demonstrates potent targeting of TROP2-positive CRC cells, suggesting TROP2 as a potential biomarker for clinical prognosis and therapeutic targeting in colon cancer. [Table: see text]

Sacituzumab tirumotecan (SKB264/MK-2870) in patients (pts) with previously treated locally recurrent or metastatic triple-negative breast cancer (TNBC): Results from the phase III OptiTROP-Breast01 study.

104 Background: TROP2 (trophoblast cell surface antigen 2) is highly expressed in TNBC and is associated with worse survival. Sacituzumab tirumotecan (SKB264/MK-2870) is a TROP2 ADC developed with a novel linker to conjugate the payload, a belotecan-derivative topoisomerase I inhibitor with a drug-to-antibody-ratio (DAR) of 7.4. The hydrolytically linker permits both extracellular pH-sensitive cleavage and intracellular enzymatic cleavage to release the membrane permeable payload enabling the “bystander effect”. Here, we report the results from a phase Ⅲ study of sacituzumab tirumotecan in pts with advanced TNBC (OptiTROP-Breast01, NCT05347134). Methods: In this randomized phase Ⅲ trial, SKB264 was compared with physician's choice of chemotherapy (eribulin, vinorelbine, capecitabine, or gemcitabine) in pts with locally recurrent or metastatic TNBC who had received two or more prior therapies including at least one for metastatic setting. The primary endpoint was progression-free survival (PFS) by blinded independent central review (BICR). The TROP2 expression was determined by immunohistochemistry (IHC) using the semi-quantitative H-score method. Results: Pts were randomly assigned to receive SKB264 (n = 130) or chemotherapy (n = 133). The median age was 51 years; 87% had visceral metastases; 26% received prior PD-1/PD-L1 inhibitors; 48% received three or more prior lines of chemotherapy for advanced disease. The primary endpoint of PFS was met based on interim analysis (data cutoff: Jun 21, 2023) with a 69% reduction in risk of progression or death (HR 0.31; 95% CI, 0.22 to 0.45; P <0.00001). The median PFS assessed by BICR was 5.7 months (95% CI, 4.3 to 7.2) with SKB264 and 2.3 months (95% CI, 1.6 to 2.7) with chemotherapy; PFS at 6 months was 43.4% vs 11.1%. In the subset of pts with TROP2 H-score > 200, the median PFS was 5.8 months with SKB264 and 1.9 months with chemotherapy (HR 0.28; 95% CI, 0.17 to 0.48). At the first planned interim analysis for overall survival (OS) (data cutoff: Nov 30, 2023) with median follow-up of 10.4 months, OS was statistically significant in favor of SKB264 (HR 0.53; 95% CI, 0.36 to 0.78; P =0.0005); the median OS was not reached (95% CI, 11.2 to NE) with SKB264 and 9.4 months (95% CI, 8.5 to 11.7) with chemotherapy. The objective response rate assessed by BICR was 43.8% with SKB264 and 12.8% with chemotherapy. Most common grade ≥ 3 treatment-related adverse events (SKB264 vs chemotherapy) were neutrophil count decreased (32.3% vs 47.0%), anemia (27.7% vs 6.1%) and white blood cell count decreased (25.4% vs 36.4%). Conclusions: Sacituzumab tirumotecan monotherapy demonstrated statistically significant and clinically meaningful PFS and OS benefit over chemotherapy, with a manageable safety profile in pts with heavily pretreated advanced TNBC and limited treatment options. Clinical trial information: NCT05347134 .

Sacituzumab govitecan in patients with advanced or metastatic radioactive-iodine refractory thyroid carcinoma: The phase 2 SETHY, GETNE-T2318 trial.

TPS6130 Background: Current treatment options for advanced or metastatic differentiated thyroid carcinoma (DTC) with vascular endothelial growth factor inhibitors provide clinical benefit, despite the fact that most patients will acquire treatment resistance, whereas, advanced or metastatic anaplastic thyroid carcinoma (ATC) still lacks effective treatment options. Trophoblast cell surface antigen 2 (TROP-2) is highly expressed at the membrane of DTC and ATC, while it is rarely expressed in normal tissues. Moreover, TROP-2 is associated with tumor aggressiveness and poor prognosis. Targeting TROP-2 with sacituzumab govitecan, an antibody-drug conjugate with a SN-38 payload, showed efficacy in other cancer types such as triple negative or HER positive breast cancer or urothelial cancer and may be an effective treatment for thyroid carcinoma. SETHY is the first clinical trial with antibody-drug conjugates in thyroid cancer. Methods: The SETHY trial is a single-arm, multicohort, prospective, phase 2 trial of sacituzumab govitecan in patients with advanced or metastatic radioactive-iodine refractory thyroid cancer being recruited in 10 hospitals in Spain. Patients are ≥ 18 years, ECOG 0-1, should have recovered from any prior toxicity and have an adequate organ function. Patients will be included in two cohorts: DTC after progression (PD) to 1-3 prior systemic therapies (cohort 1) or ATC in 1st-line treatment or after failure of any systemic therapy (Cohort 2). Prior topoisomerase 1 inhibitors are not permitted. All patients will receive sacituzumab govitecan (10 mg/kg intravenously) on Days 1 and 8 of every 21-days cycle, until PD, death, study withdrawal, or unacceptable toxicity. Computed tomography (CT) or magnetic resonance imaging (MRI) scans and blood monitoring of tumor markers are performed every 12 weeks (Q12W) until PD. The primary endpoint is objective response rate (ORR) according to RECIST v1.1. Secondary endpoints include disease control rate, duration of response, progression-free survival, overall survival, safety, and quality of life (assessed through EORTC QLQ-C30 at baseline and Q12W until PD). Archival tumor samples will be collected at screening for retrospective central evaluation of TROP-2 expression levels and ancillary studies. The study uses a Simmon-II design considering a ORR of 5% as null hypothesis and an alternative ORR of 20% (α=0.1 one sided, β=0.2). In total, the study requires a total of 21 patients per cohort; 12 in the 1st stage and, if at least one response is reported, 9 additional in the 2nd stage. The study is approved and open to patient selection in Spain. Clinical trial identification: EU CT: 2023-504898-20-00 / NCT06235216 Clinical trial information: NCT06235216 .

Assessment of TROP2, CEACAM5 and DLL3 in metastatic prostate cancer: Expression landscape and molecular correlates

Therapeutic approaches targeting proteins on the surface of cancer cells have emerged as an important strategy for precision oncology. To capitalize on the potential impact of drugs targeting surface proteins, detailed knowledge about the expression patterns of the target proteins in tumor tissues is required. In castration-resistant prostate cancer (CRPC), agents targeting prostate-specific membrane antigen (PSMA) have demonstrated clinical activity. However, PSMA expression is lost in a significant number of CRPC tumors. The identification of additional cell surface targets is necessary to develop new therapeutic approaches. Here, we performed a comprehensive analysis of the expression heterogeneity and co-expression patterns of trophoblast cell-surface antigen 2 (TROP2), delta-like ligand 3 (DLL3), and carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) in CRPC samples from a rapid autopsy cohort. We show that DLL3 and CEACAM5 exhibit the highest expression in neuroendocrine prostate cancer (NEPC), while TROP2 is expressed across different CRPC molecular subtypes, except for NEPC. We further demonstrated that AR alterations were associated with higher expression of PSMA and TROP2. Conversely, PSMA and TROP2 expression was lower in RB1-altered tumors. In addition to genomic alterations, we show a tight correlation between epigenetic states, particularly histone H3 lysine 27 methylation (H3K27me3) at the transcriptional start site and gene body of TACSTD2 (encoding TROP2), DLL3, and CEACAM5, and their respective protein expression in CRPC patient-derived xenografts. Collectively, these findings provide insights into patterns and determinants of expression of TROP2, DLL3, and CEACAM5 with implications for the clinical development of cell surface targeting agents in CRPC.

Abstract PO4-15-03: Quantitative Measurement of TROP2 via Chromogenic Immunohistochemistry in Breast Cancer

Abstract Sacituzumab govitecan (SG) is an antibody-drug conjugate that targets human trophoblast cell-surface antigen 2 (TROP2), which is expressed in over 90% of breast cancer cases (Zaman et al, 2019). By delivering the cytotoxic SN-38 (topoisomerase I inhibitor) to TROP2 expressing breast cancer cells, SG showed promising antitumor activities in clinical trials and is now an approved treatment for triple-negative breast cancer (TNBC) and hormone receptor (HR)-positive HER2-negative breast cancer in the metastatic setting. Despite the prevalent expression of TROP2 in breast cancer, the objective response rates reported from clinical trials were about 30%, indicating the need for a diagnostic test that can identify which patients are likely to benefit from therapy. Although the drug is approved without a companion diagnostic assay, recent data from the TROPiCS-02 trial has shown that TROP2-low (H-score < 100) showed a non-significant hazard ratio for benefit from SG. In the same study, patients treated with SG with H-score >100 showed a significant hazard ratio for benefit compared to physician’s choice (Tolaney et al, ASCO 2023). This suggests that TROP2 expression level is associated with SG response and that a threshold for TROP2 expression level may aid patient selection. Although over 90% of breast cancer patients were considered TROP2-positive by IHC, the expression level of TROP2 was never quantitatively assessed in large breast cancer cohorts. Here we describe a quantitative chromogenic immunohistochemistry (IHC) assay with a cell line standard. Mass spectrometry was used to measure TROP2 peptide concentrations in the six standard cell lines, and the correlation between the mass spectrometry and IHC data for each cell line was then used to convert chromogenic signals to concentrations of TROP2 protein (fmol/mm2). The antibody used in this assay was rigorously validated, and the antibody concentration was optimized for the best signal-to-noise ratio. The optical density (OD) of chromogenic staining was measured using QuPath 0.4.3 (Qymia extension) by identifying the tumor area via object classifiers and manual editing and then calculating the area-normalized sum of OD. Collectively, this assay can measure up to 29.1 fmol/mm2 of TROP2. By applying this assay to two serial retrospective primary breast cancer cohorts from Yale University, we quantitatively measured TROP2 expression levels in 332 clinical cases. Not surprisingly, over 90% of cases showed some chromogenic signal, with a median TROP2 concentration of 2.1 fmol/mm2 and a maximum of 10.5 fmol/mm2. TROP2 expression levels showed no significant association with clinicopathologic characteristics including race, stage, BRCA mutation status, molecular subtype, HER2 IHC levels, and outcome. Further work is underway to optimize the assay toward the goal of determination of whether there is a quantitatively definable threshold for TROP2 expression below which patients are unlikely to benefit from SG or other TROP2-targeted therapies. Citation Format: Mengni He, Matthew Liu, Thazin Aung, Sneha Burela, Katherine Bates, Charles Robbins, David Rimm. Quantitative Measurement of TROP2 via Chromogenic Immunohistochemistry in Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-15-03.

Abstract CT283: Phase 3 MK-2870-007 study: MK-2870 (SKB264) plus pembrolizumab vs pembrolizumab alone as first-line treatment for metastatic non-small-cell lung cancer with PD-L1 tumor proportion score (TPS) ≥50%

Abstract Background: Pembrolizumab (anti-PD-1) monotherapy is a standard of care first-line treatment for advanced or metastatic non-small-cell lung cancer (NSCLC) with PD-L1 TPS ≥50% without actionable genetic alterations; however, some patients do not respond to therapy or experience disease progression. Trophoblast cell-surface antigen 2 (TROP2) is another candidate for anticancer treatment and overexpression of TROP2 is associated with poor prognosis in patients with NSCLC. MK-2870 (SKB264) is an antibody-drug conjugate (ADC) composed of the humanized TROP2 monoclonal antibody, a hydrolytically cleavable linker, and the cytotoxic drug KL610023. In patients with relapsed or refractory locally advanced or metastatic NSCLC, MK-2870 monotherapy showed encouraging antitumor activity with a manageable safety profile. The complementary mechanisms of action of MK-2870 and pembrolizumab may provide more potent antitumor activity when given in combination compared with each therapy alone. To investigate this further, the MK-2870-007 study will evaluate the efficacy and safety of the addition of MK-2870 to pembrolizumab vs pembrolizumab alone in patients with previously untreated metastatic NSCLC with PD-L1 TPS ≥50%. Trial Design: Approximately 614 eligible patients aged ≥18 y with previously untreated histologically or cytologically confirmed stage IV (per American Joint Committee on Cancer v8.0) NSCLC, PD-L1 TPS ≥50% and no EGFR, ALK, or ROS1 alterations (nonsquamous only); ECOG PS 0 or 1; and measurable disease per RECIST v1.1 will be enrolled. Patients with well-controlled HIV are permitted. Patients will be randomized 1:1 to receive either pembrolizumab 400 mg Q6W for up to 18 cycles plus MK-2870 4 mg/kg Q2W or pembrolizumab 400 mg IV Q6W alone until progressive disease, intercurrent illness, patient withdrawal, unacceptable toxicity, prolonged interruption of study drug, or until maximum number of cycles (18 cycles for pembrolizumab). Randomization stratification factors include ECOG PS (0 vs 1), histology (squamous vs nonsquamous), TROP2 expression (low vs medium vs high), and geographic region (East Asia vs North America/Western Europe/Australia vs Rest of World). The primary endpoint is OS. Secondary endpoints include PFS, ORR, and duration of response per RECIST v1.1 by blinded independent central review, patient-reported outcomes, and safety. Tumor imaging will occur at baseline, Q6W until wk 48, and Q12W thereafter until PD, start of new anticancer treatment, or withdrawal of consent. PD-L1 TPS will be assessed centrally using PD-L1 IHC 22C3 pharmDx (Agilent Technologies, Carpinteria, CA). AEs will be graded according to National Cancer Institute Common Terminology Criteria for Adverse Events v5.0. Enrollment began in December 2023 and is currently ongoing. Citation Format: Nikolaj Frost, Razi Ghori, Ananya Roy, Ana Tablante Nunes, James Chih-Hsin Yang. Phase 3 MK-2870-007 study: MK-2870 (SKB264) plus pembrolizumab vs pembrolizumab alone as first-line treatment for metastatic non-small-cell lung cancer with PD-L1 tumor proportion score (TPS) ≥50% [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT283.

Abstract CT282: MK-2870-004: A phase 3 study of MK-2870 (SKB264) versus chemotherapy for previously treated advanced or metastatic nonsquamous non-small-cell lung cancer (NSCLC) with EGFR mutations or other genomic alterations

Abstract Background: Trophoblast cell-surface antigen 2 (TROP2), a cell surface glycoprotein involved in the regulation of tumor growth and invasion, is broadly expressed and upregulated in several cancers, including NSCLC. MK-2870 (SKB264) is an antibody-drug conjugate (ADC) composed of the humanized anti-TROP2 monoclonal antibody, a hydrolytically cleavable linker, and the cytotoxic drug KL610023. MK-2870 has demonstrated encouraging antitumor activity and manageable toxicity in phase 1 and 2 studies of patients with solid tumors, including heavily pretreated EGFR-mutant NSCLC. We describe the design of the ongoing phase 3 MK-2870-004 study (NCT06074588) that will evaluate the efficacy and safety of MK-2870 vs chemotherapy for previously treated advanced or metastatic nonsquamous NSCLC with EGFR mutations or other genomic alterations. Trial design: The global, randomized, open-label, phase 3 MK-2870-004 study will enroll ~556 patients, including 456 with exon 19del or L858R EGFR mutations, and 100 with other genomic alterations. Eligible patients are aged ≥18 years with histologically or cytologically documented advanced (stage III not eligible for resection or curative radiation) or metastatic nonsquamous NSCLC with exon 19del or exon 21 L858R EGFR mutations or other genomic alterations in ALK, ROS1, BRAF V600E, NTRK, MET exon 14 skipping, or RET and those with less common EGFR mutations (exon 20 S768I, exon 21 L861Q, and/or exon 18 G719X). Patients must have progressive disease on or after 1 or 2 lines of prior tyrosine kinase inhibitor (TKI) therapy and a platinum-based therapy (may contain anti-PD-[L]1), measurable disease per RECIST v1.1, an available tumor sample, and adequate organ function. Prior EGFR TKI therapy must include a third-generation TKI for patients with T790M EGFR-mutant NSCLC. Patients with active central nervous system metastases or carcinomatous meningitis are ineligible, although those with previously treated and untreated stable brain metastasis are eligible for enrollment. Patients will be randomized (1:1) to MK-2870 4 mg/kg Q2W on Days 1, 15, and 29 of every 6-week cycle, or chemotherapy (docetaxel 75 mg/m2 Q3W or pemetrexed 500 mg/m2 Q3W) on Days 1 and 22 of every 6-week cycle. Randomization will be stratified by EGFR mutations with prior third generation TKI vs EGFR mutations with no prior third generation TKI vs other genomic alterations, brain metastasis (yes vs no), and TROP2 expression (low vs medium vs high). Dual primary endpoints are PFS (per RECIST v1.1 by blinded independent central review) and OS in patients with EGFR-mutant NSCLC. Key secondary endpoints include PFS and OS in all patients with NSCLC, ORR, DOR, patient-reported outcomes, and safety. Enrollment started in November 2023 and is currently ongoing. Citation Format: Federico Cappuzzo, Edward B. Garon, Myung-Ju Ahn, Shun Lu, Terufumi Kato, Enriqueta Felip, Parneet Cheema, Nicolas Girard, Mark Shamoun, Guoqing Wang, Bin Zhao, Rina Hui. MK-2870-004: A phase 3 study of MK-2870 (SKB264) versus chemotherapy for previously treated advanced or metastatic nonsquamous non-small-cell lung cancer (NSCLC) with EGFR mutations or other genomic alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT282.

Abstract 5760: Intra-tissue quantitative analysis using LC/MS will determine the mechanisms of interstitial pneumonia induced by antibody-drug conjugates

Abstract Background: Antibody-drug conjugates (ADCs) are a new class of therapeutics that combine a monoclonal antibody with a cytotoxic agent (known as their payloads) via a synthetic linker. While ADCs are one of game changer and are the fastest growing classes with novel clinical benefits recently, interstitial pneumonia (ILD) is a clinical problem as a fatal adverse event. However, the mechanism of ADC-induced ILD is under investigation. Purpose: We clarify ADC-ILD mechanism by analyzing released payload distribution in the lung using a novel Trophoblast cell surface antigen 2 (TROP2)-targeting ADC. Material and Methods: TROP2-eribulin is a novel TROP2-targeting ADC composed of the TROP2 antibody and cytotoxic payload eribulin conjugated by a cleavable linker. Four human cancer cell lines (A549, HeLa, NCI-H2110 and SKOV3) and one human- leukemia monocytic cell line (THP-1) were used in this study. Cell surface TROP2 antigen expression was evaluated by flow cytometry. We investigated the concentration and distribution of the TROP2-eribulin and the payload released from the TROP2-eribulin in tumor and lung tissue, using TROP2 low and high expression human cancer xenograft model that were treated with various doses of TROP2-eribulin. The concentration of erbulin was analyzed by liquid chromatograph mass spectrometry, and tissue distribution of ADCs was assessed by immunofluorescence (IF) assay. Results: In vitro analysis showed that the concentration of intracellularly released eribulin was significantly higher in NCI-H2110 with high TROP2 expression compared to A549 with low TROP2 expression (P < 0.05). In vivo analysis, the concentration of released eribulin in tumor tissue was approximately 10-fold higher in the NIC-H2110 xenograft model than the A549 xenograft model, while analysis of lung tissue showed that TROP2-eribulin was distributed in lung tissue in a dose-dependent manner of TROP2-eribulin regardless of TROP2 expression, with significantly more eribulin released in the high-dose group of TROP2-eribulin than in the other dose groups (P < 0.05). IF assay showed that TROP2-eribuilin localized to alveolar macrophages. In the analysis using THP-1 cell, which mimic macrophage function, the concentration of eribulin released from TROP2-eribuilin was significantly reduced by the use of an Fc receptor inhibitor (P < 0.05). Conclusions: Nonspecific uptake of ADC by alveolar macrophages via Fcγ receptors releases cytotoxic payload into lung tissue, regardless of tumor antigen expression levels. This understanding can help elucidate the pathogenesis of ADC-induced ILD. Citation Format: Shigehiro Koganemaru, Hirobumi Fuchigami, Chihiro Morizono, Hiroko Shinohara, Yasutoshi Kuboki, Keiji Furuuchi, Toshimitsu Uenaka, Toshihiko Doi, Masahiro Yasunaga. Intra-tissue quantitative analysis using LC/MS will determine the mechanisms of interstitial pneumonia induced by antibody-drug conjugates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5760.

Preclinical evaluation of the theranostic potential of 89Zr/177Lu-labeled anti-TROP-2 antibody in triple-negative breast cancer model

BackgroundTriple-negative breast cancer (TNBC) is one of the most lethal malignant tumors among women, characterized by high invasiveness, high heterogeneity, and lack of specific therapeutic targets such as estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Trophoblast cell-surface antigen-2 (TROP-2) is a transmembrane glycoprotein over-expressed in 80% of TNBC patients and is associated with the occurrence, progress, and poor prognosis of TNBC. The TROP-2 targeted immunoPET imaging allows non-invasive quantification of the TROP-2 expression levels of tumors, which could help to screen beneficiaries most likely to respond to SG and predict the response. This study aimed to develop a 89Zr/177Lu-radiolabeled anti-TROP-2 antibody (NY003) for immunoPET and SPECT imaging, as well as radioimmunotherapy (RIT) in TROP-2 (+)TNBC tumor-bearing model. Based on the camelid antibody, we developed a TROP-2 targeted recombinant antibody NY003. NY003 was conjugated with DFO and DTPA for 89Zr and 177Lu radiolabelling, respectively. The theranostic potential of [89Zr]Zr-DFO-NY003/[177Lu]Lu-DTPA-NY003 was evaluated through immunoPET, SPECT imaging, and RIT studies in the subcutaneous TROP-2 positive TNBC xenograft mice model.ResultsThe high binding affinity of NY003 to TROP-2 was verified through ELISA. The radiochemical purity of [89Zr]Zr-DFO-NY003/[177Lu]Lu-DTPA-NY003 exceeded 95% and remained stable within 144h p.i. in vitro. ImmunoPET and SPECT imaging showed the specific accumulation of [89Zr]Zr-DFO-NY003/[177Lu]Lu-DTPA-NY003 in MDA-MB-231 tumors and gradually increased with the time tested, significantly higher than that in control groups (P < 0.05). The strongest anti-tumor efficacy was observed in the high-dose of [177Lu]Lu-DTPA-NY003 group, followed by the low-dose group, the tumor growth was significantly suppressed by [177Lu]Lu-DTPA-NY003, the tumor volumes of both high- and low-dose groups were smaller than the control groups (P < 0.05). Ex vivo biodistribution and histological staining verified the results of in vivo imaging and RIT studies.ConclusionAs a drug platform for radiotheranostics, 89Zr/177Lu-radiolabeled anti-TROP-2 antibody NY003 could not only non-invasively screen the potential beneficiaries for optimizing SG ADC treatment but also suppressed the growth of TROP-2 positive TNBC tumors, strongly supporting the theranostic potential of [89Zr]Zr-DFO-NY003/[177Lu]Lu-DTPA-NY003.

Abstract C001: Retrospective analysis of TROP2 expression in colorectal cancer (CRC) primary tumors and liver metastases (LM) and its correlation with clinical factors

Abstract Background: Trophoblast cell surface antigen 2 (TROP2) is a transmembrane glycoprotein which is upregulated in CRC, is associated with tumorigenesis, and may serve as a drug target. We aimed to measure TROP2 expression by immunohistochemistry in CRC LM and primary tumors, and to determine how TROP2 expression correlates with prognosis and baseline clinical factors. Methods: TROP2 immunohistochemistry was retrospectively performed by a pathologist (ab214488; Abcam, Cambridge, UK) in paraffin-embedded CRC LM and primary tumor specimens (biopsy or resection, stored at the University of Colorado Surgical Pathology Archive) from adult patients between January 2006 and June 2022. Baseline clinical and survival data was collected. Differences in TROP2 frequency (range: 0-4), intensity (range: 0-3), and frequency x intensity combined scores (range: 0-12) for paired CRC primary and LM specimens were analyzed using a paired t-test. Additional statistical analyses included nonparametric Spearman’s correlation test (correlation of scores between LM and primary tumor), Mann Whitney U test (effect of prior systemic cancer treatment on TROP2 expression), Kaplan-Meier method (survival rates based on TROP2 expression), and log-rank test (differences in survival). Results: TROP2 was expressed in 100% (60/60) of CRC primary and 93% (52/56) of LM specimens, including 50 CRC primary-LM pairs and additional unpaired specimens (10 primary and 6 LM). Among all specimens, primary and LM specimens both had a median intensity score of 2, whereas CRC specimens had a slightly higher frequency score than LM specimens (2 vs. 1, respectively). In paired samples, the TROP2 frequency, intensity, and combined score distribution varied slightly between primary and LM specimens. The median intensity was higher in primary vs LM specimens (3 vs 2, respectively), mean difference was 0.26 (p=0.08), and correlation was weak (Spearman r=0.29, p=0.04). Similarly, the median frequency was higher in primary vs LM specimens (2 vs 1, respectively), mean difference was 0.20 (p=0.21), with moderate correlation (Spearman r=0.55, p&amp;lt;0.0001). The median combined score was higher in primary vs LM specimens (4 vs 3, respectively), mean difference was 0.98 (p=0.04), with moderate correlation (Spearman r=0.51, p=0.0001). Prior systemic cancer therapy did not significantly affect TROP2 intensity, frequency, or combined score in primary or LM specimens (data to be presented). Overall survival was not significantly different between low (&amp;lt;5) or high (≥5) TROP2 combined score (stratified by the median value) for primary (log-rank p=0.67) or LM specimens (log-rank p=0.66). Similarly, there was no difference in overall survival stratified by TROP2 frequency or intensity in CRC primary or LM (data to be presented). Conclusions: TROP2 expression was observed in both primary CRC and LM specimens, and the frequency x intensity combined score was significantly higher in primary CRC specimens compared to paired LM. Higher TROP2 expression in either primary tumors or LM did not correlate with overall survival. Citation Format: Robert W Lentz, Miriam Barnett, Michele Gerber, Jeffrey Kaplan, Adrie van Bokhoven, Kathleen C Torkko. Retrospective analysis of TROP2 expression in colorectal cancer (CRC) primary tumors and liver metastases (LM) and its correlation with clinical factors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C001.

Assessment of PD-L1, TROP2, and nectin-4 expression in penile squamous cell carcinoma

ObjectivesThere is an unmet need for therapeutically relevant biomarkers for advanced penile squamous cell carcinoma (pSCC). Proposed immunohistochemistry (IHC)-based biomarkers include programmed death-ligand 1 (PD-L1), trophoblast cell-surface antigen 2 (TROP2), and nectin-4; however, there is a paucity of data pertaining to these biomarkers. Herein, we investigated the expression of PD-L1, TROP2, and nectin-4 in a well-annotated cohort of pSCCs. MethodsA single-institution pathology archive was queried for patients who had a partial or total penectomy for pSCC between January 2000 and December 2022. Whole-slide sections were stained with antibodies against PD-L1 (22C3), TROP2, and nectin-4. Expression in tumor cells was quantified using H-scores (0–300). Associations between IHC expression, human papilloma virus (HPV) status, clinicopathologic findings, and outcome parameters were evaluated. ResultsThis study included 121 patients. For PD-L1, the median combined positive and H-scores were 1 and 0, respectively; 32.7 % of the cases had an H-score>0. Compared to PD-L1–negative tumors, PD-L1–positive tumors had higher pT stage and grade. The median TROP2 and nectin-4 H-scores were 230 and 140, respectively, with high TROP2 and nectin-4, defined by an H-score>200, noted in 80.7 % and 10.9 % of cases, respectively. High-risk HPV–positive cases had higher TROP2 and nectin-4 scores compared to HPV–negative cases. Patients with high TROP2 expression had significantly more disease progression, and patients with high nectin-4 expression had significantly fewer deaths due to disease. ConclusionsHigh expression of TROP2 and nectin-4 in pSCC support evaluation of these markers as therapeutic targets pending validation of our findings.

Comparative expression of trophoblast cell-surface antigen 2 (TROP2) in the different molecular subtypes of invasive breast carcinoma: An immunohistochemical study of 94 therapy-naive primary breast tumors

BackgroundSacituzumab govitecan, targeting trophoblast cell-surface antigen 2 (TROP2), is approved for the treatment of triple-negative and hormone receptor-positive/HER2-negative breast cancers. However, detailed studies comparing TROP2 protein expression in the different molecular subtypes of breast cancer are limited, and definitive evidence supporting the use of TROP2 as a biomarker for predicting response to this agent in patients with breast cancer is currently lacking. ObjectiveTo compare the expression of TROP2 in the different molecular subtypes of breast cancer. MethodsImmunohistochemical staining for TROP2 was performed on 94 therapy-naive primary invasive breast carcinomas, including 25 luminal A-like, 25 luminal B-like, 19 HER2-like, and 25 triple-negative tumors. ResultsIntermediate to high levels of TROP2 expression were observed in the majority of carcinomas of each molecular subtype, with a wide range of expression in each subtype. Occasional tumors with low or absent TROP2 expression were encountered, including two metaplastic carcinomas which were completely negative for TROP2. ConclusionsOur observations support the continued investigation of the efficacy of sacituzumab govitecan in all molecular subtypes of breast carcinoma. Furthermore, the observed wide range of expression of TROP2 suggests that TROP2 may have potential utility as a biomarker for predicting responsiveness to sacituzumab govitecan. If this proves to be the case, then immunohistochemical staining for TROP2 would be critical for identifying those patients whose tumors are completely negative for TROP2, since these patients may be least likely or unlikely to respond to this agent, and alternative therapies may be more appropriate in such instances.

Decoding TROP2 in breast cancer: significance, clinical implications, and therapeutic advancements.

Breast cancer is a heterogeneous disease characterized by distinct molecular subtypes, varied prognoses, and differential treatment responses. Understanding the molecular landscape and identifying therapeutic targets, such as trophoblast cell-surface antigen 2 (TROP2), is vital. TROP2 is notably overexpressed in breast cancer, playing a significant role in tumor growth, invasion, metastasis, and treatment resistance. While significant progress has been made in targeting TROP2 in breast cancer, several challenges and knowledge gaps remain. These challenges include the heterogeneity of TROP2 expression within breast cancer subtypes, resistance to its targeted therapies, potential off-target effects, limited therapeutic agents, and identifying optimal combination treatments. Integrating findings from clinical trials into clinical practice further complicates the landscape. This review article delves deep into TROP2 in breast cancer, highlighting its expression patterns, clinical implications, and therapeutic advancements. By understanding the role of TROP2, we can pave the way for personalized treatments, and transform the landscape of breast cancer care.

Selenium modulates AR/IGF-1R/EGFR and TROP2 signaling pathways and improves anticancer efficacy in murine mammary carcinoma 4T1

The micronutrient selenium (Se) has been shown to exert potential anticancer properties. This study aimed to evaluate the effects of Se (in Se yeast form) on the selenoproteins (SELENO), AR/IGF-1R/EGFR, PI3K/Akt/mTOR and Ras/Raf/ERK cascades, and immune checkpoint blockade in TNBC murine 4T1 cells. We also assessed the effects of combination treatment with chemotherapeutic doxorubicin and Se on trophoblast cell surface antigen 2 (TROP2) levels. Compared with the control groups, cells incubated with Se (0.25, 0.5, 0.75, 1.0, 1.5 µg Se/mL) have lower viability, raised intracellular Se concentrations and SELENO expression, and higher malondialdehyde products in a dose-dependent manner. Se induced the inactivation of AR/IGF-1R/EGFR and downregulation of the PI3K/Akt/mTOR and Ras/Raf/ERK signaling molecules. Se-treated cells also exhibited decreased mitochondrial membrane potential, reduced levels of the cell cycle regulatory protein cyclin D1, cancer stemness, metastatic and EMT-related markers, and increased apoptosis. Subsequently, Se treatment significantly suppressed PD-1/PD-L1 and CTLA-4 mRNA levels and proteins. Doxorubicin decreased 4T1 cell viability and TROP2 expression levels, but the addition of Se to doxorubicin contributed to further reductions. Similar responses to Se treatment were also observed in the human MDA-MB-231 and MCF-7 breast cancer cells. These results show that Se upregulates SELENO and anti-AR/IGF-1R/EGFR signaling in TNBC cells, thus inducing oxidative stress-dependent apoptosis and cell cycle arrest, stemness, EMT, and metastasis, as well as blocking the immune checkpoint molecules. TROP2 down-regulation with Se is also a potential anti-TNBC therapeutic target.

SKB264 (TROP2-ADC) for the treatment of patients with advanced NSCLC: Efficacy and safety data from a phase 2 study.

9114 Background: TROP2 (trophoblast cell surface antigen 2) is commonly overexpressed in non-small cell lung cancer (NSCLC) and associated with poor prognosis. SKB264 is a novel anti-TROP2 ADC developed using sulfonyl pyrimidine-CL2A-carbonate linker to conjugate its payload, a belotecan-derivative topoisomerase I inhibitor, to achieve an average Drug-to-antibody Ratio (DAR) of 7.4. The design was to achieve a more effective balance between stability in circulation and release of the ADC payload in tumor cells. Here we report clinical efficacy and safety results of SKB264 in the treatment of patients (pts) with NSCLC from a Phase 2 expansion cohort. TROP2 expression level by immunohistochemistry was assessed retrospectively. Correlation analyses between response and TROP2 level will be provided. Methods: This is a Phase 1/2, multicenter dose-escalation/expansion study in pts with relapsed or refractory locally advanced/metastatic NSCLC and other tumor types (NCT04152499). All NSCLC pts received SKB264 at 5 mg/kg IV Q2W. Tumor assessments based on RECIST 1.1 were performed every 8 weeks by investigators. Results: As of February 9th, 2023, 43 pts (63% male, 88% ECOG PS 1, median age 58 yrs [44-74]) were enrolled. Median follow-up was 11.5 months (mo; 95% CI, 10.4-12.2). Median treatment duration was 5.7 mo (range, 0.5-14.1). Among 39 response-evaluable pts, the ORR was 44% (17/39, 15 confirmed and 2 pending confirmation), median DoR was 9.3 mo (range, 1.3+ to 11.2+), 6-month DoR rate was 77%. For EGFR wild type subgroup (previously received median 2 lines of therapy including anti-PD-1/L1), the ORR was 26% (5/19), DCR was 89% (17/19), median PFS was 5.3 mo, and 9-month OS rate was 80.4%. For subgroup with TKI resistant EGFR mutant NSCLC (50% also failed at least one line of chemotherapy), the ORR was 60% (12/20), DCR was 100% (20/20), median PFS was 11.1 mo, and 9-month PFS rate was 66.7%. 67.4% (29/43) of pts had Grade ≥ 3 treatment-related adverse events (TRAEs). The most common Grade ≥3 TRAEs (occurred in ≥5% of pts) were neutrophil count decreased (32.6%), anemia (30.2%), white blood cell count (WBC) decreased (23.3%), stomatitis (9.3%), rash (7.0%), and lymphocyte count decreased (7.0%). Grade 4 TRAEs occurred only for neutropenia and WBC decreased. Most of the hematology toxicity occurred within the first two months of treatment and resolved after treatment with granulocyte colony stimulating factor or erythropoietin without blood transfusions. 23.3% (10/43) of the pts experienced dose reduction due to TRAEs. No neuropathy or drug-related ILD/pneumonitis was reported. No TRAEs led to treatment discontinuation or death. Conclusions: SKB264 at 5 mg/kg Q2W demonstrated encouraging anti-tumor activity and manageable safety profile in pts with relapsed or refractory locally advanced/metastatic NSCLC. TRAEs were mainly hematologic. Phase 3 studies of SKB264 in pts with advanced NSCLC have been planned. Clinical trial information: NCT04152499 .

TROP-2 is widely expressed in vulvar squamous cell carcinoma and represents a potential new therapeutic target.

Vulvar squamous cell carcinoma (VSCC) is a rare malignancy of the female genital tract with increasing incidence rates. Etiologically, HPV-dependent and HPV-independent VSCC are distinguished. Surgical treatment and/or radiotherapy represent the therapeutic mainstay for localized disease. For recurrent or metastatic VSCC, treatment options are limited. Research has identified trophoblast cell surface antigen 2 (TROP-2) to be broadly expressed across different tumor entities. The aim of the present study was to systematically investigate the expression of TROP-2 in VSCC. TROP-2 protein expression was investigated by immunohistochemistry in a cohort comprising n = 103 patients with primary VSCC. A four-tier scoring system (0: no staining, 1 + : low staining, 2 + : moderate staining, 3 + : high staining) was applied for quantification of protein expression. For further analyses, two groups (low TROP-2 expression: 0/1 + ; high TROP-2 expression: 2 + /3 +) were generated. The entire study cohort, as well as HPV-dependent and HPV-independent VSCC were considered separately. In the entire VSCC study cohort, TROP-2 expression was present in 97.1% of all cases (n = 100) with 74.8% displaying high TROP-2 expression (2 + /3 +). Only 2.9% of tumors showed absent TROP-2 expression. Of note, all HPV-dependent VSCC (n = 18) demonstrated high TROP-2 expression (2 + /3 +). In the subgroup of HPV-independent VSCC (n = 70), high TROP-2 expression was associated with favorable clinical outcomes based on log rank test and univariate cox analysis. TROP-2 protein expression is of prognostic value in HPV-independent VSCC. The broad expression of TROP-2 in VSCC indicates the TROP-2 directed ADC Sacituzumab govitecan as a potential new therapeutic strategy for VSCC patients.

Abstract LB027: In vivo delivery of novel CD89 fusion receptor to myeloid cells by mRNA activates anti-tumor immunity

Abstract Despite the revolution provided by immunotherapy, sustained clinical benefit has yet to be achieved for most of patients with advanced solid tumors. Myeloid cells readily accumulate in tumors, in some cases contributing up to 75% of the tumor mass. While supporting tumor growth and being associated with unfavorable prognosis, myeloid cells can mediate phagocytosis of cancer cells and cytotoxic tumor killing and promote adaptive immune responses through antigen presentation and co-stimulation, therefore representing promising targets in cancer therapy. The ability to engineer circulating and tumor associated myeloid cells to activate their ability to elicit anti-tumor adaptive immunity is an attractive approach to harness systemic anti-tumor immunity. However, it remains challenging to specifically target and activate myeloid cells in vivo. To overcome this hurdle, we have developed a novel in vivo myeloid cell engineering platform in which a chimeric antigen receptor (CAR) is generated by fusing a tumor recognition scFv with the alpha chain of human Fc receptors (CD89). The stable expression and function of these receptors requires the endogenously expressed common Fc receptor gamma chain (FcRγ), which expression is mostly restricted to myeloid cells. For in vivo engineering, the construct is encapsulated and delivered in lipid nanoparticles (LNP). Trophoblast cell surface antigen 2 (TROP2) is overexpressed in most human solid epithelial cancers, as compared to low expression in corresponding normal tissue. Increased TROP2 expression has been linked to increased tumor growth and has been implicated as a prognostic marker in these cancers, supporting the development of therapies targeting TROP2. Intravenous infusion of LNP encapsulating mRNA encoding the anti-TROP2-CD89 fusion protein results in the uptake of the LNPs and expression of the chimeric receptor fusion protein in myeloid cells. In immunodeficient xenograft models of hepatocellular carcinoma and triple negative breast cancer, delivery of LNP mRNA encoding GPC3-CD89 or TROP2-CD89 fusion proteins resulted in anti-tumor efficacy, confirming the ability of this approach to program myeloid cells. Repeat dosing studies showed significant anti-tumor efficacy following bi-weekly administration of TROP2-CD89. Furthermore, in the B16/10 syngeneic melanoma model, treatment with the melanoma antigen gp75-CD89 fusion protein was also associated with the initiation of broad systemic immune responses, characterized by tumor infiltration by activated CD8+ T cells, reduced tumor-associated Tregs and activation of antigen presenting cells in spleen. When infused in cynomolgus monkeys, TROP2-CD89 LNP led to cell surface expression of anti-TROP2 CAR in myeloid cells. Together these studies highlight the potential of in vivo delivery of CD89 fusion proteins to program myeloid cells to recognize and kill cancer. Citation Format: Thomas E. Prod'homme, Shannon Argueta, Hongyun Zhao, Michael Gorgievski, Neha Diwangi, Edward Cochran, Bruce McCreedy, Yuxiao Wang, Daniel Getts. In vivo delivery of novel CD89 fusion receptor to myeloid cells by mRNA activates anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB027.

Abstract 4396: TROP2 expression in non-small cell lung cancer

Abstract Introduction: Non-small cell lung cancer (NSCLC) is a major cause of cancer-related deaths globally with high unmet need. A variety of novel therapeutic strategies are being explored to improve patient outcomes including the use of antibody drug conjugates (ADCs) to deliver highly potent, cytotoxic payloads to tumors. Trophoblast cell-surface antigen 2 (TROP2), also known as tumor-associated calcium signal transducer 2 (TACSTD2), has emerged as an attractive target for ADCs. Sacituzumab govitecan-hziy is a novel ADC composed of a TROP2 antibody coupled to SN-38 and is being evaluated in NSCLC. High TROP2 mRNA expression is observed in many tumor types, however, TROP2 protein expression in NSCLC is not well-established. Here we characterized TROP2 expression across three independent datasets to explore the TROP2 relationship to baseline characteristics, molecular features of interest, and its prognostic value in NSCLC. Methods: We analyzed three independent datasets: (1) The Cancer Genome Atlas (TCGA) NSCLC adenocarcinoma (LUAD, N=660) and squamous cell carcinoma (LUSC, N=484) mRNA expression data, (2) a NSCLC adenocarcinoma FFPE tumor sample set (“Translational data set”; N=107), (3) an independent clinical NSCLC adenocarcinoma (N=103) and squamous cell carcinoma (N=37) data set. TROP2 IHC was performed on tumor sample sets (2) and (3) using the SP295 antibody (Robust Prototype Assay, Ventana) with H-Score and assessment of the percentage of membrane-positive tumor cells as readouts. Wilcoxon rank-sum and Kruskal-Wallis tests (continuous variables), Kaplan-Meier method (survival probability curves) and log-rank test (time to event outcome) were used (GraphPad Prism 8.1.2 and R version 4.0.5). Results: TROP2 mRNA was highly expressed in NSCLC and expression was similar in adenocarcinoma and squamous cell carcinoma in the TCGA data set. TROP2 expression did not vary with patient’s age, gender, race or tumor stage. Furthermore, TROP2 expression was not associated with TP53, KRAS or driver mutation status (including EGFR, ALK, ROS, RET, MET). TROP2 expression in the Translational sample set was consistent with the findings from the TCGA data analysis: TROP2 mRNA and protein expression were high and independent of patient baseline demographics, tumor stage and TP53 and KRAS mutation status. Finally, in an independent clinical NSCLC dataset, TROP2 protein was highly expressed and there was no relationship between TROP2 and baseline demographics including gender and age. TROP2 membrane H-scores and percentage of membrane-positive tumor cells also highly correlated in the two independent datasets. Conclusions: Three large independent datasets confirmed high expression of TROP2 in NSCLC both, as mRNA and protein. TROP2 expression in tumors did not differ between histological subtypes, baseline characteristics or clinically relevant driver alterations. Based on the TCGA data set, TROP2 expression was not prognostic for survival. Citation Format: Peiwen Kuo, Emon Elboudwarej, Lauri Diehl, Jilpa Patel, Sabeen Mekan, Juliane M. Jürgensmeier. TROP2 expression in non-small cell lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4396.

An assembly of TROP2-mediated signaling events.

Trophoblast cell surface antigen 2 (TROP2) is a calcium-transducing transmembrane protein mainly involved in embryo development. The aberrant expression of TROP2 is observed in numerous cancers, including triple-negative breast cancer, gastric, colorectal, pancreatic, squamous cell carcinoma of the oral cavity, and prostate cancers. The main signaling pathways mediated by TROP2 are calcium signaling, PI3K/AKT, JAK/STAT, MAPKs, and β-catenin signaling. However, collective information about the TROP2-mediated signaling pathway is not availablefor visualization or analysis. In this study, we constructed a TROP2 signaling map with respect to its role in different cancers. The data curation was done manually by following the NetPath annotation criteria. The described map consists of different molecular events, including 8 activation/inhibition, 16 enzyme catalysis, 19 gene regulations, 12 molecular associations, 39 induced-protein expressions, and 2 protein translocation. The data of the TROP2 pathway map is made freely accessible through the WikiPathways Database ( https://www.wikipathways.org/index.php/Pathway:WP5300 ). Development of TROP2 signaling pathway map.

Abstract P4-07-12: Development of Triple-negative breast cancer (TNBC) syngeneic models and TROP2-directed antibody-drug conjugate (ADC) surrogate to model therapeutic combinations

Abstract Background: Sacituzumab govitecan (SG, Trodelvy®) is a human trophoblast cell surface antigen 2 (TROP-2) directed antibody drug conjugate (ADC) coupled to an active form of irinotecan (SN-38) via our novel hydrolyzable linker (CL2A). SG is the only FDA-approved ADC treatment for TNBC patients in the second-line setting. TROP-2 is a transmembrane protein encoded by the tumor-associated calcium signal transducer 2 (TACSTD2) gene and highly expressed in TNBC, an aggressive type of cancer accounting for approximately 15% of all breast cancers. TROP-2 overexpression is associated with poor survival and relapse, but its biological function in TNBC remains poorly understood. Hypothesis/rationale: To better understand TROP-2 and TROP-2-directed ADC biology, we developed and characterized TROP2high vs TROP2low TNBC syngeneic tumors and an SG surrogate directed to murine TROP-2. Experimental design: We established 2 syngeneic TNBC models with differential TROP-2 expression: 4T1 cells were flow sorted into high (&amp;gt;95%) vs low (&amp;lt; 7%) TROP-2 expressors and EMT6 cells were transduced with a murine TACSTD2-encoding lentivirus. Balb/c mice were subcutaneously implanted with 0.5 × 10 E6 TROP-2high, TROP-2low, or parental tumor cells (4T1 or EMT6). Tumor immunophenotyping and transcriptomic analyses were performed 15 and 24 days after implantation. An SG mouse surrogate was engineered to mimic SG, using an anti-TROP-2 antibody (Rab64) that cross-reacts with human and murine TROP-2 covalently attached to SN-38 by the CL2A linker. SG surrogate activity was characterized in vitro and in 4T1 syngeneic models. Results: SG surrogate demonstrated high affinity for human and mouse TROP-2 (KD=1.1 and 1.4 nM, respectively) with SN-38 release rates and PK similar to that of SG. Flow cytometry analysis after bulk cell sorting of 4T1 or lentivirus transduction of EMT6 confirmed high TROP2 expression after at least 3 in vitro passages. Fifteen days after subcutaneous implantation, flow cytometry analysis of tumor single-cell suspensions revealed significant differences in immune infiltrates between 4T1-derived tumor groups (n=5/group; mean percentages in TROP2high vs TROP2low 4T1-derived tumors of cells expressing CD45: 65% vs 10%, P &amp;lt; 0.0001; CD8: 5.5% vs 1%, P = 0.0033; CD4: 22% vs 4%, P = 0.0055; macrophages: 12.5% vs 2.5%, P = 0.0002; myeloid cells: 52% vs 75%, P = 0.0066). In addition, TROP2high 4T1-derived tumors were smaller and had significantly less necrosis than TROP2low and unsorted 4T1-derived tumors 25 days after implantation. Finally, transcriptomics analyses of TROP2high vs TROP2low 4T1-derived tumors demonstrated the association of TACSTD2 expression levels with regulation of distinct molecular pathways. Conclusion: Syngeneic tumors derived from 4T1 cells with differential TROP2 expression levels are associated with differential cellular states and tumor microenvironment composition. In contrast, no significant phenotypic changes were observed in tumors derived from TACSTD2-transduced compared with mock-transduced EMT6 cells. Taken together, these results suggest that expression of the TACSTD2 gene is associated with, but not causative of, different tumor phenotypic states. Additional studies to investigate TROP-2 expression as a correlative marker of patient prognosis and the antitumor immune response are warranted. The effects of in vivo treatment with an SG surrogate on 4T1 tumor growth and immune phenotype will be discussed at the time of the presentation. Citation Format: Chih-Chien Chou, Jordan Kardos, Becky Yang, Jessica Orf, Rutwij Dave, Yurong Lai, Chingwei V. Lee, Giuseppe A. Papalia, Kelli Boyd, Lauri Diehl, Nathalie Scholler. Development of Triple-negative breast cancer (TNBC) syngeneic models and TROP2-directed antibody-drug conjugate (ADC) surrogate to model therapeutic combinations [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-07-12.