Trophoblast Cell Surface Antigen Research Articles

Assessment of immunohistochemical expression of trophoblast cell surface antigen 2 (Trop 2) in oral squamous cell carcinoma and its clinicopathological correlation

Trophoblast cell surface antigen 2 (Trop 2) is a transmembrane glycoprotein that is involved in a variety of oncogenic cell signaling pathways. Its heterogeneous expression in normal and cancerous tissues makes it a diagnostic and prognostic marker for various malignancies, including Oral Squamous Cell Carcinoma (OSCC). To assess the immunohistochemical expression of Trop 2 in OSCC and evaluate its clinicopathological correlation. Histopathologically diagnosed 40 cases of OSCC and their respective clinical records were retrieved and subsequent clinical staging and histopathological grading was done. Formalin fixed parrafin embedded sections were subjected to immunohistochemistry with anti Trop 2 antibody using standard DAB technique. The immunohistochemical expression of Trop 2 was evaluated using immunostaining intensity and mean integrated score followed by its comparison with age, gender, anatomical site, clinical stages and histopathological grades of OSCCTrop 2 expression was positive in 97.5% of cases. There was a statistically significant difference (p value<0.001) between immunostaining intensity and integrated score of Trop 2 among various histopathological grades of OSCC. As grade of OSCC increased, integrated score decreased. Trop 2 is one of the biomarkers that may serve as a useful tool to detect the changes undergoing at the molecular level which have prognostic or therapeutic significance.

Trophoblast Cell Surface Antigen 2 (Trop2) Is Expressed in Cases of EBV-Positive Diffuse Large B-Cell Lymphoma Emerging from Angioimmunoblastic T-Cell Lymphoma

Trophoblast Cell Surface Antigen 2 (Trop2) Is Expressed in Cases of EBV-Positive Diffuse Large B-Cell Lymphoma Emerging from Angioimmunoblastic T-Cell Lymphoma

Antibody Drug Conjugate Sacituzumab Govitecan Enables A Sequential TOP1/PARP Inhibitor Cancer Therapy Strategy in Breast Cancer Patients.

The Antibody-Drug Conjugate (ADC) Sacituzumab govitecan (SG) comprises the topoisomerase 1 (TOP1) inhibitor SN-38, coupled to a monoclonal antibody targeting trophoblast cell surface antigen 2 (TROP-2). Poly (ADP-ribose) polymerase (PARP) inhibition may synergize with TOP1 inhibitors and SG, but previous studies combining systemic PARP and TOP1 inhibitors failed due to dose-limiting myelosuppression. Here, we assess proof-of-mechanism and clinical feasibility for SG and talazoparib employing an innovative sequential dosing schedule. In vitro models tested pharmacodynamic endpoints, and in a phase 1b clinical trial (NCT04039230) 30 patients with metastatic Triple-Negative Breast Cancer (mTNBC) received SG and talazoparib using a concurrent (N=7) or sequential (N=23) schedule. Outcome measures included safety, tolerability, preliminary efficacy and establishment of a recommended phase 2 dose (RP2D). We hypothesized that tumor-selective delivery of TOP1i via SG would reduce non-tumor toxicity and create a temporal window, enabling sequential dosing of SG and PARP inhibition. In vitro, sequential SG followed by talazoparib delayed TOP1 cleavage complex clearance, increased DNA damage and promoted apoptosis. In the clinical trial, sequential SG/talazoparib successfully met primary objectives and demonstrated median PFS of 7.6 months without Dose-Limiting Toxicities (DLTs), while concurrent dosing yielded 2.3 months PFS and multiple DLTs including severe myelosuppression. While SG dosed concurrently with talazoparib is not tolerated clinically due to an insufficient therapeutic window, sequential dosing of SG then talazoparib proved a viable strategy. These findings support further clinical development of the combination and suggest that ADC-based therapy may facilitate novel, mechanism-based dosing strategies.

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 PO3-15-12: Expression and co-expression patterns of TROP2 and HER2 in breast cancer: implications for bispecific antibody-drug conjugate therapy

Abstract Introduction: Antibody-drug conjugates (ADCs) targeting trophoblast cell surface antigen 2 (TROP2), such as sacituzumab-govitecan and datopotamab-deruxtecan, have recently been developed. A preclinical study has reported the efficacy of anti-human epidermal growth factor receptor 2 (HER2) and TROP2 bispecific ADCs in treating HER2 and TROP2 co-expressing tumors, including HER2-low tumors in various solid tumors (Chengzhang Shang et al: AACR 2023). In this study, we aimed to evaluate the expression of TROP2 by immunohistochemistry (IHC) at different sites and investigate the changing status and co-expression of HER2 and TROP2 among breast cancer patients. Methods: We collected 244 archival paired samples of primary tumors and metastatic sites from 111 breast cancer patients treated in our hospital from 2000 to 2018. Estrogen receptor (ER), progesterone receptor (PgR), and HER2 status were determined from previous pathology reports. HER2 low was defined as HER2 IHC score of 1+ or 2+ with negative fluorescent in situ hybridization (ISH). In addition, we determined the expression of TROP2 using IHC assays and categorized the results based on the histochemical score (H-score). Results: Among the samples, ER+HER2- was 139, ER+HER2+ was nine, ER-HER2+ was eight, and triple-negative (TN) was 88. HER2 3+ or 2+ with ISH positive was 17, HER2 low was 112, HER2 negative was 115. The median TROP2 H-score was 10, with a mean of 37.5 (range: 0-265). Overall, 27% of the samples had H-score of 0, 73% had H-score of 1<, 47% had H-score of 10<, and 12% had H-score of 100< . No statistically significant association was observed between TROP2 expression and breast cancer subtypes (ER+HER2-; H-score of 0 24%, H-score of 1< 76%, H-score of 10< 44%, H-score of 100< 14%, TN; H-score of 0 30%, H-score of 1< 70%, H-score of 10< 54%, H-score of 100< 11%). On the other hand, samples from the metastatic site showed a higher rate of TROP-2 expression H-score of 1< compared to the primary site (primary site; H-score of 0 36%, H-score of 1< 64%, metastatic site; H-score of 0 18%, H-score of 1< 82%; p< 0.01). The rates of TROP2-positive expression defined as H-score of 1<, varied among the metastatic sites, with 100% in liver, 92% in brain, 89% in lymph nodes, 64% in lung, and 60% in bone. Regarding the co-expression of HER2 and TROP2, among HER2-positive samples, 35% had TROP2 H-score of 0, 24% had TROP2 H-score of 1<, and 41% had TROP2 H-score of 10<, while among HER2 low samples, 21% had TROP2 H-score of 0, 27% had TROP2 H-score of 1<, 39% had TROP2 H-score of 10<, and TROP2 H-score of 100< was found in 13% of patients. Based on the definition of co-expression such as TROP2 H-score of 1< and HER2 1+ or more, overall, 40% of the samples had co-expression of HER2 and TROP2, and the metastatic site had a higher rate of co-expression than the primary tumor (49%, 32% respectively, p=0.002). Conclusions: This study demonstrates that TROP-2 expression by IHC in breast cancer varies between primary and metastatic sites in breast cancer. Metastatic sites and specific organs show higher rate of TROP2 expression. Additionally, 40% of the samples had the co-expression of HER2 and TROP2, suggesting a potential target for future bispecific ADCs therapy. Citation Format: Mai Onishi, Tatsunori Shimoi, Yuki Kojima, Shu Yazaki, Taro Yamanaka, Rui Kitadai, Asuka Kawachi, Hitomi Okuma, Mai Hoshino, Ayumi Saito, Munehiro Ito, Aiko Maejima, Tadaaki Nishikawa, Kazuki Sudo, Emi Noguchi, Yasuhiro Fujiwara, Masayuki Yoshida, Kan Yonemori. Expression and co-expression patterns of TROP2 and HER2 in breast cancer: implications for bispecific antibody-drug conjugate therapy [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 PO3-15-12.

Abstract PO2-23-12: Innovative Metastatic Breast Cancer Therapy, CBT300, Reverses Drug and Immune Resistance

Abstract For many patients with metastatic breast cancer (MBC), their disease is treatable but incurable. Although the rate of death from MBC has significantly decreased over the last 10 years due to early detection and new treatment options, MBC still accounts for over 40,000 deaths each year in the US that has been consistent for the past 30 years. Recently, it is estimated that 90% of those deaths were due to drug resistant recurrent disease. Nearly 45% of MBC deaths can be attributed to a subpopulation of patients with metastatic triple negative breast cancer (mTNBC). Despite MBC’s initial sensitivity to chemotherapy, it often recurs at greater than 40% in stage I-III patients and greater than 80% in stage IV patients which have a dismal 5-year survival of 12%. African American women have a 40% greater incidence of death from MBC despite a 4% lower risk of diagnosis. Unfortunately, current therapies do little to reduce drug resistance and death for recurrent drug resistant MBC. Currently, the only therapies approved by the FDA for recurrent TNBC patients are Keytruda plus chemotherapy and an Antibody Drug Conjugate (ADC) called Sacituzumab Govitecan. Keytruda (anti-PD-L1) is an immune checkpoint inhibitor(ICI) given with chemotherapy (nab-paclitaxel, paclitaxel, or gemcitabine plus carboplatin). The second recently approved therapy, Sacituzumab Govitecan (SG), is composed of an antibody to human trophoblast cell-surface antigen 2 (Trpo-2) coupled to SN-38 (topoisomerase I inhibitor). Although these results are very encouraging, the 80% of TNBC patients that either do not have high positive PD-L1 tumors, or don’t respond to these therapies and become drug resistant suggests that most patients with TNBC will have recurrent disease with little or no hope for survival. For contrast, our novel MBC therapy, CBT300, targets cell surface GRP78 that has been found in over 95% of MBC and in 93% of TNBC tumors. We can now show that inhibition of cell surface GRP78 can a) induce apoptosis of drug resistant TNBC cells in vitro and in vivo, b) eliminate drug resistance showing synergistic effects with chemotherapy in vitro and in vivo, c) decrease amount of chemotherapy in combination with CBT300 and d) reduce immune suppression. Recent publications show that GRP78 is found on many types of tumor cell surfaces but not on normal cell surfaces. In fact, tumor cell surface GRP78 (csGRP78) is important for many aspects of MBC development, including cell survival, proliferation, chemoresistance, angiogenesis, metastasis formation, immune suppression, and stem cell formation. Recently, it has been shown that increased cell surface GRP78 expression in TNBC patients was significantly associated with later stage, increased distant metastasis, increased aggressiveness, shorter disease-free survival, and decreased overall survival. In studies to help understand how cell surface GRP78 causes MBC progression and drug resistance, we discovered a novel GRP78 binding transmembrane protein on TNBC cells called Receptor Tyrosine Kinase Orphan Receptor-1 (ROR1). Using the GRP78 binding domain from ROR1 and a human Fc IgG1 domain, we created a biologic fusion protein that is a potent and cell surface specific GRP78 inhibitor, CBT300. We now show that CBT300’s elimination of cell surface GRP78 destabilizes and removes oncofetal proteins ROR1, Cripto-1, and checkpoint protein PD-L1 from tumor cell surfaces resulting in reversal of chemoresistance, reduction in immune suppression, inhibition of stem cell phenotype and increased tumor cell apoptosis. Since ROR1, Cripto-1 and PD-L1 are three of the major redundant pathways for tumor drug resistance, immune suppression and stem cell formation, the ability to inhibit all these pathways with CBT300, a single safe and very efficacious therapy, is very innovative and could be a significant advance for the treatment of MBC. Citation Format: Elizabeth Stolarik, Jorryn Zelek, Mariah Thigpen, Anita Davidson, Donald Davidson. Innovative Metastatic Breast Cancer Therapy, CBT300, Reverses Drug and Immune Resistance [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 PO2-23-12.

Abstract PO3-13-11: Quantitative Multiplex Immunofluorescence Assay for TROP2 and HER2 Expression in Breast Cancer: Towards Guiding Patient Selection for Antibody Drug Conjugate Therapies

Abstract Emerging antibody drug conjugate (ADC) therapies targeting human trophoblast cell-surface antigen (TROP2) and human epidermal growth factor receptor 2 (HER2) are transforming the treatment landscape for breast cancer. Sacituzumab govitecan (SG) and trastuzumab deruxtecan (T-DXd) have gained approval for an overlapping set of "HER2-low" metastatic breast cancers, including hormone receptor (HR)-positive HER2 non-amplified and "triple-negative" subtypes. Nevertheless, the optimal selection of patients and treatment sequencing for these ADC therapies remains a clinical challenge. Clinical trial objective response rates to SG are approximately 30%, compared to 30-90% for T-DXd depending on HER2 expression levels. While both drugs are thought to be targeted therapies, the value of measuring the target and the best methods to do so are still not established. We believe that quantitative measurement of TROP2 and HER2 antigen expression levels could establish thresholds for responders, enabling more effective patient selection for ADC therapies. Here, we present a TROP2, high-sensitivity HER2, and cytokeratin (CK) quantitative immunofluorescence (QIF) multiplex assay. Using a ten-cell line standard array and proteomic mass spectrometry, we can convert tumor compartment QIF intensity to protein concentration in fmol/mm2 for tissue specimens. Anti-HER2, anti-TROP2 antibodies, and fluorescence detection systems were titrated and combined to maximize signal-to-noise ratio on our cell line standard array and breast cancer tissue microarrays (TMA). The multiplex assay was designed for automated slide stainers (Leica BOND Rx) and fluorescence slide scanning (Rarecyte CyteFinder II HT). We perform our analyses in QuPath using an image processing plugin developed for automated QIF/IHC analysis (Qymia). Reproducible TROP2 and HER2 QIF scoring (R² > 0.95) was achieved across multiple staining batches using serial sections of breast cancer TMAs and cell standard arrays. This assay has a TROP2 linear range between 0.63 - 9.17 fmol/mm2 (about 1 million TROP2 receptors/cell) and HER2 linear range between 0.09 - 0.565 fmol/mm2 (about 60,000 HER2 receptors/cell). We then applied this multiplex assay to two serial retrospective primary breast cancer cohorts from Yale University to quantitatively measure TROP2 and HER2 expression (338 clinical cases). We find a weak negative correlation between TROP2 and HER2 expression in our breast cancer cohorts (Pearson r = -0.14, p = 0.0097, n = 338). TROP2 expression levels were above the limit of detection (LOD) in 90.2% of cases, with 4.1% exceeding the limit of linearity (LOL), and a mean TROP2 expression of 4.05 fmol/mm2. For HER2, 67.2% of cases were above the LOD, with 7.1% exceeding the LOL, and a mean HER2 expression of 0.186 fmol/mm2. Both TROP2 and HER2 were below the LOD in 3.0% of cases, which we define as “negative”. We found 29.9% expressed TROP2 and were HER2-negative, and 6.8% expressed HER2 and were TROP2-negative. Our future studies will aim to quantitatively define expression thresholds for T-DXd and SG response with the goal to produce a clinical grade assay for ADC patient selection and determine the value of the assay to help select which ADC to give first. HER2 and TROP2 protein expression summary in Yale breast cancer cohort Table 1: Summary of HER2 and TROP2 protein expression levels in serial retrospective primary breast cancer cohort of 338 cases using our high-sensitivity HER2 and TROP2 multiplex immunofluorescent assay Citation Format: Charles Robbins, Mengni He, Revekka Khaimova, Katherine Bates, Nay Nwe Nyein Chan, Daniel Liebler, Regan Fulton, David Rimm. Quantitative Multiplex Immunofluorescence Assay for TROP2 and HER2 Expression in Breast Cancer: Towards Guiding Patient Selection for Antibody Drug Conjugate Therapies [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 PO3-13-11.

A new TROP2-targeting antibody-drug conjugate shows potent antitumor efficacy in breast and lung cancers

Trophoblast cell surface antigen 2 (Trop2) is considered to be an attractive therapeutic target in cancer treatments. We previously generated a new humanized anti-Trop2 antibody named hIMB1636, and designated it as an ideal targeting carrier for cancer therapy. Lidamycin (LDM) is a new antitumor antibiotic, containing an active enediyne chromophore (AE) and a noncovalently bound apoprotein (LDP). AE and LDP can be separated and reassembled, and the reassembled LDM possesses cytotoxicity similar to that of native LDM; this has made LDM attractive in the preparation of gene-engineering drugs. We herein firstly prepared a new fusion protein hIMB1636-LDP composed of hIMB1636 and LDP by genetic engineering. This construct showed potent binding activities to recombinant antigen with a KD value of 4.57 nM, exhibited binding to Trop2-positive cancer cells and internalization and transport to lysosomes, and demonstrated powerful tumor-targeting ability in vivo. We then obtained the antibody-drug conjugate (ADC) hIMB1636-LDP-AE by molecular reconstitution. In vitro, hIMB1636-LDP-AE inhibited the proliferation, migration, and tumorsphere formation of tumor cells with half-maximal inhibitory concentration (IC50) values at the sub-nanomolar level. Mechanistically, hIMB1636-LDP-AE induced apoptosis and cell-cycle arrest. In vivo, hIMB1636-LDP-AE also inhibited the growth of breast and lung cancers in xenograft models. Moreover, compared to sacituzumab govitecan, hIMB1636-LDP-AE showed more potent antitumor activity and significantly lower myelotoxicity in tumors with moderate Trop2 expression. This study fully revealed the potent antitumor efficacy of hIMB1636-LDP-AE, and also provided a new preparation method for LDM-based ADC, as well as a promising candidate for breast cancer and lung cancer therapeutics.

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 CT247: Updated efficacy and safety of anti-TROP2 ADC SKB264 (MK-2870) for previously treated advanced NSCLC in Phase 2 study

Abstract Background: TROP2 (trophoblast cell surface antigen 2) is commonly over-expressed in non-small cell lung cancer (NSCLC) and associated with poor prognosis. SKB264 (MK-2870) is a TROP2 ADC developed with a novel linker to conjugate the payload, a belotecan-derivative topoisomerase I inhibitor. The linker is affected by both extracellular pH-sensitive cleavage and intracellular enzymatic cleavage within tumor cells, which leads to efficiently release payload inside tumor cells as well as within tumor microenvironment to exert its anti-tumor effects. Here, we present the updated data from a Phase 2 expansion cohort for patients (pts) with advanced NSCLC. Methods: Pts with previously treated advanced NSCLC were enrolled to receive SKB264 at 5 mg/kg Q2W until disease progression or unacceptable toxicity (KL264-01, NCT04152499). Tumor assessment was performed every 8 weeks per RECIST v1.1 assessed by investigator. Results: As of Nov 22, 2023, 43 NSCLC pts had been enrolled. Median follow-up was 17.2 months (mo). 21 pts with EGFR wild type had received median prior 3 regimens of therapy including anti-PD-1/L1 inhibitors. 22 pts with EGFR mutant had progressed on or after TKI therapy, 50% of whom also failed at least one line of chemotherapy. Updated efficacy results are shown in the Table. Overall, 30 pts (69.8%) experienced Grade ≥3 treatment-related adverse events (TRAEs). The most common Grade ≥3 TRAEs were neutrophil count decreased (34.9%), anemia (30.2%), WBC count decreased (25.6%), stomatitis (9.3%), and rash (7.0%). No TRAEs leading to treatment discontinuation or deaths occurred. No drug-related ILD/pneumonitis was reported. Conclusions: The updated data continues to demonstrate that SKB264 monotherapy delivers promising clinical efficacy with manageable toxicity in pretreated advanced NSCLC pts. A Phase 3 global study of SKB264 in pts with 3L+ EGFR mutant NSCLC (NCT06074588) and a Phase 3 study of SKB264 in China in pts with 2L EGFR mutant NSCLC (NCT05870319) are ongoing. TABLE 1: NAND Table Clinical outcomes by EGFR mutation status Overall (N=43) EGFR mutant (N=22) EGFR wild type Total (N=21) Non-squamous (N=9) Squamous (N=12) ORR*, % 43.6% 60.0% 26.3% 22.2% 30.0% Median DoR, mo (95%CI) 9.3 (3.7, 10.3) 8.7 (3.7, 10.3) 9.6 (3.5, NE) / / Median PFS, mo (95% CI) 7.2 (5.4, 11.3) 11.5 (5.7, 12.9) 5.3 (3.5, 6.2) 5.8 (1.5, 12.1) 5.1 (1.9, 9.3) Median OS, mo (95% CI) 22.6 (13.1, NE) 22.7 (19.7, NE) 14.1 (10.7, NE) 16.2 (5.8, NE) 12.8 (3.5, NE) 12-mo OS rate, % (95% CI) 69.0% (52.7, 80.7) 81.0% (56.9, 92.4) 57.1% (33.8, 74.9) 66.7% (28.2, 87.8) 50.0% (20.8, 73.6) 18-mo OS rate, % (95% CI) 56.5% (40.1, 70.0) 76.2% (51.9, 89.3) 35.9% (16.0, 56.4) 44.4% (13.6, 71.9) 30.0% (7.7, 56.9) *Including confirmed or unconfirmed response. Based on response evaluable pts (≥1 on-study scans) with 4 pts (2 EGFR mutant pts with non-squamous histology and 2 EGFR wild type pts with squamous histology) excluded. Citation Format: Wenfeng Fang, Ying Cheng, Zhendong Chen, Wei Wang, Yongsheng Li, Yongmei Yin, Xingya Li, Huiting Xu, Guohua Yu, Yanjun Mi, Zev A. Wainberg, Jordi Rodon, Xiang Wang, Xian Wang, Xiaoqing Zhang, Xiaoping Jin, Lian Lu, Junyou Ge, Jin Li, Li Zhang. Updated efficacy and safety of anti-TROP2 ADC SKB264 (MK-2870) for previously treated advanced NSCLC in Phase 2 study [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 CT247.

Abstract CT038: Preliminary efficacy and safety results of anti-TROP2 ADC SKB264 (MK-2870) in patients (pts) with previously treated advanced gastric (G) or gastroesophageal junction (GEJ) cancer from a Phase 2 study

Abstract Background: Overexpression of TROP2 (trophoblast cell surface antigen 2) in advanced gastric cancer is known as a poor prognostic factor. SKB264 (MK-2870) is a TROP2 ADC developed with a novel linker to conjugate the payload, a belotecan-derivative topoisomerase I inhibitor. The linker is affected by both extracellular pH-sensitive cleavage and intracellular enzymatic cleavage within tumor cells, which leads to efficiently release payload inside tumor cells as well as within tumor microenvironment to exert its anti-tumor effects. Here, we report the preliminary results from a Phase 2 expansion cohort in pts with advanced G/GEJ cancer. Methods: Pts with previously treated inoperable advanced G/GEJ adenocarcinoma were enrolled to receive SKB264 monotherapy at 5 mg/kg Q2W until disease progression or unacceptable toxicity in Phase 2 expansion cohort of KL264-01 study (NCT04152499). Pts with heavily pre-treated G/GEJ cancer were enrolled first, and then the cohort was amended to enroll pts with only one prior therapy of chemotherapy and anti-PD-1/L1 therapy. Tumor assessment was performed every 8 weeks per RECIST v1.1 assessed by investigator. Results: At data cutoff date (Nov 22, 2023), a total of 48 pts were enrolled and followed up for at least 9 weeks. 24 pts (50.0%) had received one prior line of therapy (2L), while 24 pts (50.0%) had received ≥ 2 prior lines of therapy (3L+). 40 pts (83.3%) had received prior anti-PD-1/L1 inhibitors. Treatment-related adverse events (TRAEs) of ≥ Grade 3 were reported in 52.1% of pts. The most common ≥ Grade 3 TRAEs (occurred in ≥5% of pts) were anemia (20.8%), neutrophil count decreased (18.8%), WBC decreased (12.5%) and neutropenia (6.3%). TRAEs leading to dose reduction and dose delay occurred in 18.8% and 33.3% of pts, respectively. No TRAEs leading to treatment discontinuation or deaths occurred. No neuropathy or drug-related ILD/pneumonitis was reported. Of 41 response-evaluable pts (defined as ≥ 1 on-study scans), the ORR was 22.0% (9 PRs, 2 pending confirmation) and disease control rate (DCR) was 80.5%. The ORRs in the 2L and 3L+ setting were 27.3% (including 2 pending confirmation) and 15.8%, respectively. Median duration of response (DoR) was 7.5 months (mos). In the subset of 3L+ pts (n=24 including 54.2% of pts with ≥ 4 prior lines of therapy) with more mature follow-up (median follow up of 14.6 mos), median progression free survival (mPFS) was 3.7 mos (95% Cl: 2.6, 5.4); median overall survival (mOS) was 7.6 mos (95% Cl: 5.3, 15.5) with 12-mo OS rate of 32.6%. Conclusions: The preliminary data suggests that pts with heavily pre-treated advanced G/GEJ cancer could achieve durable response and potentially prolonged OS from SKB264 monotherapy, with a manageable safety profile. A Phase 3 global study of SKB264 monotherapy vs SOC in 3L+ G/GEJ adenocarcinoma is being planned. Citation Format: Jordi Rodon, Zev A. Wainberg, Mingjun Zhang, Tianshu Liu, Bo Liu, Guohua Yu, Yongmei Yin, Shuang Zhang, Yanjun Mi, Xingya Li, Xian Wang, Yunpeng Liu, Xiang Wang, Ying Cheng, Xiaoqing Zhang, Yalan Yang, Junyou Ge, Omobolaji O. Akala, Elliot Chartash, Jin Li. Preliminary efficacy and safety results of anti-TROP2 ADC SKB264 (MK-2870) in patients (pts) with previously treated advanced gastric (G) or gastroesophageal junction (GEJ) cancer from a Phase 2 study [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 CT038.

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.

ImmunoPET imaging of Trop2 in patients with solid tumours.

Accurately predicting and selecting patients who can benefit from targeted or immunotherapy is crucial for precision therapy. Trophoblast cell surface antigen 2 (Trop2) has been extensively investigated as a pan-cancer biomarker expressed in various tumours and plays a crucial role in tumorigenesis through multiple signalling pathways. Our laboratory successfully developed two 68Ga-labelled nanobody tracers that can rapidly and specifically target Trop2. Of the two tracers, [68Ga]Ga-NOTA-T4, demonstrated excellent pharmacokinetics in preclinical mouse models and a beagle dog. Moreover, [68Ga]Ga-NOTA-T4 immuno-positron emission tomography (immunoPET) allowed noninvasive visualisation of Trop2 heterogeneous and differential expression in preclinical solid tumour models and ten patients with solid tumours. [68Ga]Ga-NOTA-T4 immunoPET could facilitate clinical decision-making through patient stratification and response monitoring during Trop2-targeted therapies.

Abstract 3136: Towards development of a novel biparatopic trop2-ADC

Abstract Background: Trophoblast cell surface antigen 2 (TROP-2) is a member of Tumor-associated Calcium Signal Transducer family (TACSTD2), which is a 35kDa transmembrane protein with 4 N-linked glycosylation sites. The overexpression of TROP2 in various cancers is associated with poor prognosis and increased risk of metastasis. Anti-TROP2 antibody drug conjugates (ADCs) are emerging as an innovative therapeutic intervention for cancer treatment. While these efforts were rewarded by the FDA approval of Trodelvy (Sacituzumab govitecan), a Trop2 targeted ADC, in 2020 for the TNBC patients, tumor heterogeneity could present one of the limitations for the Trop2-targeting therapy, leading to drug resistance. Here, we report our effort in developing a novel biparatopic TROP2 ADC which demonstrate superior cell killing activity compared with the monospecific TROP2 ADC, including certain resistant cell lines for the approved therapy. Methods: Various biparatopic Abs were engineered using two different mAbs targeting the two non-overlapping epitopes of Trop2. The physical stability and biological activities were characterized. Binding competition assay was utilized for epitope binning using SPR. Cell binding affinity was determined using flow cytometry. Cellular internalization activity was measured using Incucyte system, and tumor cell killing efficacy was evaluated in multiple cancer cell lines. CDX mouse models were utilized to investigate the TROP2-ADC in vivo efficacy. Results: We identified a biparatopic TROP2 mAb using two monospecific TROP2 mAbs with A recognizing the same epitope as Trodelvy, and B binding to a non-overlapping epitope vs Sacituzumab’s. mAb-B demonstrates superior ability to recognize a few resistance cell lines to mAb-A. The biparatopic TROP2 mAb-A/B displays a KD of 0.81 nM protein binding compared with 6.42 nM and 0.26 nM, respectively for mAb-A and mAb-B. Importantly, the biparatopic mAb-A/B demonstrates significantly faster cellular internalization activity compared to those of the individual mAbs, signifying the biparatopic scaffold could drive receptor clustering and subsequently enhance the internalization, trafficking to the lysosome and degradation of the target. As expected, the ADC of the biparatopic mAb-A/B conjugated with a TOPi payload shows cancer cell killing efficacy in both in vitro and in vivo experiments. Furthermore, we tested mAb-A, -B and biparatopic mAb-A/B for their binding affinity to 14 missense mutations in TROP2 ECD expressed in HEK293 cells. Interestingly, mAb-A showed decreased binding in 5 mutant cell lines, while mAb-B and biparatopic mAb-A/B maintains activity. Conclusion: We have engineered a novel TROP2 biparatopic antibody with unique biological features as well as superior in vitro and in vivo activities. Such a biparatopic ADC presents its potential to be a best-in-class ADC therapy by overcoming the limitation of the monospecific ADC therapy such as drug resistance. Citation Format: Yinan Wu, Brittany Jiang, Julliet Rivera, Fang Wang, Jing Zhou, Lin Liu, Silvia Hudson, Will Draper, Jennifer Li, Xiaoshan Min, Hang Chen, Zhulun Wang. Towards development of a novel biparatopic trop2-ADC [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 3136.

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.

Abstract 6757: The anti-tumor efficacy of Th1-specific TROP2 vaccine (AST-07X) in a triple-negative breast cancer murine model

Abstract Background: The trophoblast cell-surface antigen 2 (TROP2) is associated with enhanced tumor proliferation and poor prognosis in breast cancer. TROP2 overexpresses on various epithelial cancer cell surfaces, and is commonly found in prostate, colon, pancreas, lung, and breast cancer. Therefore, anticancer strategy targeting TROP2 may be a potential treatment option for patients with metastatic triple-negative breast cancer (TNBC). Sacituzumab govitecan, a Trop-2-directed antibody drug conjugate (ADC), has shown significant benefits associated with progression-free and overall survival compared to standard chemotherapy for treating metastatic TNBC; however, its toxic effects were more frequent than that of chemotherapy (NCT02574455). In this study, we discovered TROP2 target peptides with T-helper 1 (Th1) polarization and anticancer efficacy. We then evaluated the anti-tumor efficacy of these epitopes and possibility of co-administration with ADC in a TNBC murine model. Materials and Methods: Th1 peptides were identified using the Th-Vac® platform and the predictive binding affinity ranking that indicates potential immunogenicity points of interest for HLA class II. Human peripheral blood mononuclear cell (PBMC) Th1-specific polarization-induced activity and in vivo immunogenicity (Module 2b) were evaluated using flow cytometry and ELISpot analysis. The anti-tumor efficacy of the selected Th1 peptide was evaluated against MDA-MB-23 inoculated athymic Balb/c mice. Each peptide was administered intradermally once weekly for a total of four times at a concentration of 100 μg/head. Sacituzumab govitecan (0.81 mg/kg) was administered intravenously once weekly, for a total of two times. Results: Ten peptides with high affinity for HLA class II were selected, and three epitopes that induce differentiation of T cells secreting TROP2-specific IFN-γ were subsequently selected. These epitopes (p1, p3 and p6) showed tumor growth inhibition efficacy of 52.4%, 38.5%, and 57.2%, respectively, compared to the control (vehicle) group in the TNBC tumor-bearing mice. The cocktail vaccine (AST-07X), which combined these epitopes, was the most dominant at 65.4%. The anti-tumor efficacy of the combination group of the cocktail vaccine and ADC was 77.4% compared with the control group, showing a potent synergistic effect compared to the 46.1% anti-tumor efficacy of the ADC-mono group. Conclusion: AST-07X that induce TROP2-specific Th1 anticancer immune responses, showed sufficient anti-tumor efficacy; these effects were evident under immunodeficient conditions, such as those of TNBC tumor-bearing athymic mice. Synergy effects were also induced via combined ADC administration. We are currently conducting further research on humanized mice to address the limitations of the immunodeficient murine model. Citation Format: Jinho Kang, Hyo-Hyun Park, Jin Kyeong Choi, Seong-Yong Jang, Min-Ah Kim, Myeong-Kyu Park, Ashley Youngmin Kim, Eunkyo Joung, Hun Jung. The anti-tumor efficacy of Th1-specific TROP2 vaccine (AST-07X) in a triple-negative breast cancer murine model [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 6757.

Abstract 1869: The AI-powered KisoSeek™ platform identifies functional anti-TROP2 heavy-chain antibodies with potent anti-cancer activity

Abstract Functional heavy-chain antibodies (HCAbs) are promising therapeutic agents for targeted cancer therapy. Since they do not have a light chain, HCAbs have a small antigen-binding domain that can unlock new functionalities by reaching epitopes that are inaccessible to classical antibodies. HCAbs can be obtained by immunizing camelids, but this approach is expensive, time-consuming, and results in a limited antibody repertoire. Previously, we engineered the KisoMouse™- a transgenic mouse with multi-camelid species VHH/VH genes and the complete alpaca D and J gene segments integrated into the mouse IgH locus, along with a functional loss of the heavy chain CH1 domain to eliminate light chain pairing. KisoMouse™ are healthy and generate a robust serum HCAb response against a diverse range of immunogens. Trophoblast cell surface antigen-2 (TROP2) is a membrane protein overexpressed in numerous solid tumor types such as breast, colon, and lung cancers. TROP2 is associated with tumor aggressiveness and decreased survival and has emerged as an attractive therapeutic target. An anti-TROP2 antibody-drug conjugate has been approved for breast and bladder cancer, but challenges related to payload toxicity and resistance highlight the need for alternative therapeutics. We had previously identified an anti-TROP2 HCAb with high potency in in vivo preclinical models of human breast and colorectal cancer. Conventional antibody discovery approaches are resource-intensive and can fail to identify rare antibodies. To overcome such limitations, we developed the KisoSeek™ platform, a proprietary AI-powered toolkit that categorizes antibodies in an immune response based on their structural and biophysical features. With KisoSeek™, we analyzed the entire antibody repertoire generated from KisoMouse™ immunized with TROP2 and rapidly identified HCAbs with similar features as the high potency reference antibody described above. We selected three additional antibodies based on their in vitro TROP2 affinity, competitiveness for the reference antibody’s epitope, and yield in CHO cells. These candidates showed comparable potent efficacy in inducing regression in human breast and colorectal xenograft models, akin to the original reference antibody. In summary, the KisoSeek™ platform identified novel anti-TROP2 HCAbs out of the KisoMouse™ antibody repertoire that were similar to our original reference antibody. This demonstrates how KisoMouse™ and KisoSeek™ are two powerful complementary platforms for novel clinical antibody discovery. By categorizing the entire antibody repertoire and identifying antibodies with similar characteristics as known functional antibodies, KisoSeek™ has the potential to leverage existing discovery efforts to uncover many more rare yet potent therapeutic antibodies - the proverbial needles in a haystack. Citation Format: Sophie E. Cousineau, Amit Subedi, Richard Wargachuk, Juliana The, Xiaowei Wang, Wenyang Hou, Amogh Nair, Wansu Qiu, Alex Zhou, Tiffany Cheng, Morteza Babaie, Benyamin Gojogh, Marinieve Montero, Hiba Zahreddine, Elijus Undzys, Lucy Lai, Gordon Ngan, Luis Da Cruz, David Young. The AI-powered KisoSeek™ platform identifies functional anti-TROP2 heavy-chain antibodies with potent anti-cancer activity [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 1869.

Abstract 5804: Preclinical evaluation of LCB84, a novel next-generation TROP2 directed ADC utilizing a cancer selective linker and an MMAE payload

Abstract LCB84 is an antibody-drug conjugate (ADC) that consists of an antibody targeting human trophoblast cell-surface antigen-2 (TROP2) linked to monomethyl auristatin E (MMAE) via a beta-glucuronidase cleavable linker with a drug-to-antibody ratio (DAR) of 4. LCB84 demonstrates tumor selective killing by multiple mechanisms, increasing both safety and efficacy in parallel. Firstly, the over-expression of TROP2 in many solid tumors allows for targeted delivery of MMAE by LCB84 to Trop2-positive tumor tissues. Secondly, the overexpression of beta-glucuronidase in the lysosome of most tumors facilitates the selective release of active payload within tumor cells compared to normal healthy tissues. Thirdly, the intrinsic selectivity of tubulin inhibitor payloads, such as MMAE, for proliferating cells provides additional protection for non-proliferating normal cells. MMAE is also cell-permeable in its free drug form and can lead to increased efficacy of LCB84 via a bystander effect, enabling activity in heterogenous tumors. LCB84 demonstrates superior efficacy in mouse tumor xenograft studies when compared to benchmark TROP2-targeting ADCs, including sacituzumab govitecan and datopotamab deruxtecan. LCB84 also shows an improved safety profile compared to these clinical benchmarks, as well as compared to traditional MMAE-bearing ADCs, in both in vitro and in vivo toxicity assessments. Additionally, LCB84 shows a stable preclinical pharmacokinetic profile with minimal loss of payload over time. The utilization of an ADC for target-mediated delivery, in combination with a stable beta-glucuronide trigger for selective payload release in tumors, allows for a superior preclinical therapeutic index which is expected to translate to a best-in-class ADC in the clinic. Citation Format: Stephen Slocum, Hyejung Kim, Chul-Woong Chung, Changsik Park. Preclinical evaluation of LCB84, a novel next-generation TROP2 directed ADC utilizing a cancer selective linker and an MMAE payload [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 5804.

Abstract 4086: ABN202 (anti-Trop-2-interferon-beta mutein): A potent antibody-cytokine fusion protein for the treatment of Trop-2 positive solid tumors

Abstract Trop-2, trophoblast cell-surface antigen 2, is a type 1 transmembrane glycoprotein overexpressed in many solid cancers, including breast, lung, pancreatic, bile duct and bladder cancer. Overexpression of Trop-2 in cancer patients is associated with disease progression and poor prognosis. The antibody-drug conjugate (ADC) targeting Trop-2, Sacituzumab-govitecan (Trodelvy®), is approved for patients with triple-negative breast cancer (TNBC) and bladder cancer. However, Trop-2 targeting ADCs exhibit a high frequency of grade 3/4 adverse events and a short duration of response. Therefore, there is an unmet medical need for new treatment strategies beyond ADCs. In a previous study, we designed ABN202 (αTrop-2) to comprise a Trop-2 targeting antibody (Sacituzumab) and IFN-β mutein (ABN102) for the Trop2-positive tumor-specific delivery of ABN102. We confirmed anti-cancer activities of ABN202 against Trop-2 positive bladder cancer. In this study, we evaluated in vitro and in vivo anti-cancer efficacy of ABN202 (αTrop-2) in Trop-2 positive TNBC, bile duct cancer and bladder cancer cell lines. To identify response biomarkers for ABN202 (αTrop-2), we analyzed cell lines exhibiting both response and non-response to ABN202 (αTrop-2). In conclusion, ABN202 (αTrop-2) demonstrated potent anti-cancer efficacy compared to Trodelvy®. Our results support ABN202 (αTrop-2) as a promising drug candidate against Trop2-positive solid tumors. Citation Format: Hee Geon Park, Hyun Kyung Lee, Myeung Ryun Seo, Daa Eun Kim, Sang Beom Bang, Ji Yang Lee, Sung Youl Hong, Kyoung Song, Chan Gyu Lee, Hae Min Jeong, Sae Hyung Lee, Na Young Kim, Jun Young Choi, Young Kee Shin. ABN202 (anti-Trop-2-interferon-beta mutein): A potent antibody-cytokine fusion protein for the treatment of Trop-2 positive solid tumors [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 4086.

Correlation between trophoblast cell-surface antigen-2 (Trop-2) expression and pathological complete response in patients with HER2-positive early breast cancer treated with neoadjuvant docetaxel, carboplatin, trastuzumab, and pertuzumab.

The prognostic and predictive role of trophoblast cell-surface antigen-2 (Trop-2) overexpression in human epidermal growth factor receptor 2-positive (HER2-positive) breast cancer is currently unknown. We retrospectively analyzed Trop-2 expression and its correlation with clinicopathologic features and pathological complete response (pCR) in HER2-positive early breast cancer (EBC) patients treated with neoadjuvant docetaxel, carboplatin, trastuzumab, and pertuzumab in the PHERGain study. Trop-2 expression at baseline was determined in formalin-fixed, paraffin-embedded primary tumor biopsies by immunohistochemistry and was first classified into expressing (Trop-2-positive) or not-expressing (Trop-2-negative) tumors. Then, it was classified by histochemical score (H-score) according to its intensity into low (0-9), intermediate (10-49), and high (≥ 50). The association between clinicopathologic features, pCR, and Trop-2 expression was performed with Fisher's exact test. Forty-one patients with tissue evaluable for Trop-2 expression were included, with 28 (68.3%) Trop-2-positive tumors. Overall, 17 (41.46%), 14 (34.15%), and 10 (24.40%) tumors were classified as low, intermediate, and high, respectively. Trop-2 expression was significantly associated with decreased pCR rates (50.0% vs. 92.3%; odds ratio [OR] 0.05; 95% CI, 0.002-0.360]; p adjusted = 0.01) but was not correlated with any clinicopathologic features (p ≥ 0.05). Tumors with the highest Trop-2H-score were less likely to obtain a pCR (OR 0.03; 95% CI, 0.001-0.290, p adjusted < 0.01). This association was confirmed in univariate and multivariate regression analyses. These findings suggest a potential role of Trop-2 expression as a biomarker of resistance to neoadjuvant chemotherapy plus dual HER2 blockade and may become a strategic target for future combinations in HER2-positive EBC patients.