Human Cell Surface Antigens Research Articles

Sacituzumab Govitecan Demonstrates Efficacy across Tumor Trop-2 Expression Levels in Patients with Advanced Urothelial Cancer.

Human trophoblast cell surface antigen 2 (Trop-2) is a protein highly expressed in urothelial cancer (UC). Sacituzumab govitecan (SG) is a Trop-2-directed antibody drug conjugate with a hydrolysable linker and a potent SN-38 payload. This study explored Trop-2 expression in tumors treated with SG in cohorts 1 to 3 (C1-3) from the TROPHY-U-01 study and evaluated whether efficacy was associated with Trop-2 expression. TROPHY-U-01 (NCT03547973) is an open-label phase II study that assessed the efficacy and safety of SG (alone or in combinations) in patients with unresectable locally advanced or metastatic UC (mUC). Archival tumor samples collected at enrollment for C1-3 were analyzed for Trop-2 membrane expression by considering histological scores (H-scores; scale 0-300) and the percentage of membrane positive tumor cells at low magnification (4×). The association of Trop-2 with clinical endpoints [objective response rate (ORR), progression-free survival (PFS), and overall survival (OS)] was evaluated. In C1-3, tissue was collected from 158 (82%) of 192 treated patients, and 146 (76%) had evaluable Trop-2 data. Trop-2 was highly expressed in tumor samples. The median [interquartile range (IQR)] Trop-2 H-score was 215 (180-246), and the median (IQR) percentage of membrane positive tumor cells was 91% (80-98). Trop-2 expression at any level was observed in 98% of patients. Furthermore, ORR, PFS, and OS benefits were observed across all Trop-2 expression levels. Trop-2 protein is highly expressed in UC, as confirmed by examining tumors from patients enrolled in the TROPHY-U-01 trial. The results indicate that SG demonstrates efficacy in mUC across Trop-2 expression levels.

Preliminary exploration of Trop2 ADC in combination with immune checkpoint inhibitors in the different molecular subtypes of invasive breast carcinoma.

e12558 Background: Human trophoblastic cell surface antigen 2 (Trop2) is a transmembrane protein overexpressed in breast cancer tissues. However, it is present at minimal levels in adult somatic tissues, making it an ideal target for treatment with antibody-drug conjugates (ADCs). Sacituzumab govitecan, an anti-Trop2 ADC, has been approved for the treatment of triple-negative breast cancer and urothelial cancer. Recent studies have demonstrated that human cytotoxic T lymphocytes can recognize Trop2. Accordingly, we aimed to assess the role of Trop2 in the tumor immune activity of different subtypes of breast cancer, and laying the foundation for further studies on the efficacy of the combination of Trop2 ADC and immune checkpoint inhibitors in breast cancer. Methods: We retrospectively retrieved tissues from 152 invasive breast carcinoma of no special type and 111 benign breast tumor tissues and undergoing surgical resection at our institution from 2018 to 2023. Immunohistochemistry was used to detect Trop2 and PD-L1 expression and lymphatic invasion in patients, and their correlations with clinicopathological factors were analyzed. Trop2 expression was evaluated using the H-score. The median as the cut-off value, a score of 0–240 indicated low expression, and 240–300 indicated high expression. Cases with CPS ≥ 1% were considered positive for PD-L1. Results: The expression was Trop2 in breast cancer tissues than in benign tumor tissues (P<0.0001). Immune infiltration analysis suggested that Trop2 expression was correlated with immune cell infiltration and the abundance (P<0.001). Kaplan–Meier plotter analysis demonstrated that high Trop2 expression was related to poor prognosis in patients with triple-negative breast cancer (overall survival: HR=1.53, P=0.029; relapse-free survival: HR=1.26, P=0.045). In contrast, Trop2 expression was not significantly associated with prognosis in other breast cancer subtypes. However, there was no statistically significant correlation found between PD-L1 expression and Trop-2 expression in breast cancer. Conclusions: The findings suggest that Trop2 correlates with poor prognosis and immune cell infiltration in breast cancer. This indicates the role of Trop2 as a potential prognostic biomarker, laying the foundation for further research focusing on the immune regulatory role of Trop2. [Table: see text]

Functional Characterization and Optimization of Switchable Allogeneic Chimeric Antigen Receptor T Cells for Targeting CD19 and CD20 in B Cell Malignancies

Chimeric antigen receptor (CAR) T cell therapy has demonstrated significant efficacy in B cell malignancies. However, despite clinically manageable on-target/off-tumor toxicity when targeting antigens like CD19 and BCMA, severe cytokine release and neurotoxicity remain a clinical challenge. Additionally, patients may require immunoglobulin replacement therapy, and antigen-loss is recognized as a cause of relapse (PMID: 30275569, 31798590, 36263838, 37051246 and 37122700). To overcome these limitations, we developed a novel switchable CAR-T cell therapy that simultaneously targets both CD19 and CD20 in B cell malignancies. This innovative therapy is based on our reverse universal chimeric antigen receptor platform (RevCAR), a 2-component CAR-T technology (Figure 1; PMID: 34638268 and 32923149). The first component is a universal CAR-T cell, incorporating a CAR based on a short, non-immunogenic peptide motif derived from the human nuclear La/SSB autoantigen. The CAR itself does not recognize any human cell surface antigen. However, it is specifically targeted by an antibody-based binder in the second component, a soluble adaptor called targeting module (TM). This TM confers specificity against the desired cancer antigens of choice. Consequently, the availability of the TM effectively controls the activity of RevCAR-T cells. We have recently reported preclinical development of an allogeneic RevCAR-T cell therapy for CD123-positive hematologic malignancies (AVC-201; Ehninger et al. 2023, AACR Abstract 4095; NCT05949125). To target CD123, which is also expressed on normal hematopoietic progenitor cells, a TM with a short half-life was selected (R-TM123), enabling a rapid switch-off of the RevCAR system by TM withdrawal to avoid acute toxicity and continued aplasia. As CD19 and CD20 have a substantially more favorable safety profile and autologous conventional CAR-T cells and bispecific T cell engagers with respective target-specificities are approved in certain B cell malignancies, a half-life extended TM was developed to allow for dosing every 1-2 weeks. After an extensive screen for an optimized TM targeting CD19 and CD20, we screened multiple RevCAR variants differing in hinge, transmembrane and costimulatory domains for optimal anti-tumor potency using both preclinical in vitro and in vivo models. Profound efficacy and complete tumor control could be observed for optimized RevCAR constructs (Figure 2). While short-term in vitro cytotoxicity assays already gave a small hint towards in vivo efficacy, surprisingly, long-term in vitro rechallenge assay results did not correlate with in vivo efficacy. A summary of functional characterization data will be presented at the meeting with a focus on the lack of predictability of certain in vitro models and underlying hypotheses. Insights from these studies are now flowing into preclinical development of a novel switchable allogeneic CAR-T cell product candidate targeting CD19 and CD20 in B cell malignancies engineered to fully overcome graft-versus-host disease as well as graft rejection by host T and NK cells.

Abstract 4095: Preclinical characterization of switchable allogeneic chimeric antigen receptor T cells to support first in human clinical study in CD123-positive hematologic and lymphatic malignancies

Abstract Approximately 80% of AML patients express CD123 on their leukemic blasts and CD123 is frequently expressed on other hematologic and lymphatic malignancies. Chimeric antigen receptor (CAR) T-cell therapy has demonstrated significant efficacy in B cell malignancies. Breakthrough of conventional CAR-T technology in AML has been hampered by expression of suitable target antigens on normal hematopoietic progenitor cells posing a risk for continued aplasia. Thus, innovative approaches putting the power of CAR-T technology under the control of reliable and fast-acting on/off switches to avoid and/or abrogate acute and long-term side effects are required. We have recently reported clinical proof-of-concept for autologous switchable CAR-T cell therapy in AML (Wermke et al. Blood 2021). However, manufacturing of autologous products is very costly and rrAML patients are often in urgent need for a product and multiple prior lines of treatment may be detrimental for product quality. While current clinical allogeneic approaches successfully prevent graft-versus-host disease, observed persistence has been limited due to rejection of the grafted T cells by host immune cells. Here, we report preclinical development of a donor-derived allogeneic switchable CAR-T therapy for CD123-positive hematologic and lymphatic malignancies (AVC-201). The reverse universal chimeric antigen receptor platform (RevCAR) is a 2-component CAR-T platform. The first component is a universal CAR-T cell. Its binding domain is a short, non-immunogenic peptide that by itself does not recognize any human cell surface antigen but is specifically bound by an scFv included in the second component, a soluble adaptor called targeting module (TM). To target CD123, a TM with a short half-life was selected (R-TM123), enabling a rapid switch-off of the RevCAR system by TM withdrawal to avoid acute and long-term toxicity usually associated with continuous activation of CAR-T cells. The allogeneic cells are generated by three edits with CRISPR-Cas9, which is meant to fully overcome graft-versus-host disease as well as graft rejection by host T and NK cells. Therefore, we expect a persistent T cell product that can be re-expanded with additional TM cycles. A summary of preclinical characterization of AVC-201 will be presented at the meeting including in vitro and in vivo pharmacology and toxicology experiments. Activation of Allo-RevCAR-T cells is strictly dependent on the presence of CD123-positive target cells and R-TM123. Allo-RevCAR-T cells redirected by R-TM123 efficiently lyse CD123-positive AML cells in vitro and in vivo, and in vitro EC50 values are in the low picomolar range of R-TM123 concentrations. In conclusion, preclinical data support the clinical exploration of AVC-201 in a first in human study. Citation Format: Armin Ehninger, Johannes Spehr, Simon Loff, Anika Langer, Julia Riewaldt, Julia Reinhardt, Jan-Erik Meyer, Josephine Dietrich, Gabriel Jurado, Reynald Lescarbeau, Marc Cartellieri. Preclinical characterization of switchable allogeneic chimeric antigen receptor T cells to support first in human clinical study in CD123-positive hematologic and lymphatic malignancies. [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 4095.

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 (>95%) vs low (< 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 < 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.

Hiroshi Shiku, MD, PhD: In Memoriam (1943–2022)

Hiroshi Shiku, MD, PhD: In Memoriam (1943–2022)

TROP2: a potential marker in diagnosis of thyroid neoplasms.

The human trophoblast cell surface antigen (TROP-2), a transmembrane glycoprotein, has recently been investigated as a valuable marker of thyroid epithelial neoplasms. In this study, we aimed to demonstrate the diagnostic utility of TROP2 in thyroid neoplasms. A total of 308 cases, including 170 cases of different PTC variants, 50 cases of benign and non-neoplastic lesions, and 86 cases of other neoplasms (NIFT-P, poorly differentiated thyroid carcinoma, anaplastic thyroid carcinoma, follicular thyroid carcinoma, Hürthle cell carcinoma, and medullary thyroid carcinoma), were included in this study. Only membranous staining with TROP2 was considered positive. In PTC, classical (n = 35), tall cell (n = 41), follicular (n = 39), solid (n = 19), hobnail (n = 10), Warthin-like (n = l0), columnar (n = 7), and oncocytic variant (n = 9) were 100%, 97.6%, 5.1%, 42.1%, 90%, 90%, 28.6%, and 33.3% positive, respectively. A negative reaction was observed in all 50 cases of benign and non-neoplastic lesions and also in surrounding normal thyroid tissue in all cases. Negative reactions were observed in 83 (n = 86) of other thyroid neoplasms including NIFT-P (n = 20), poorly differentiated thyroid carcinoma (n = 10), anaplastic thyroid carcinoma (n = 10), follicular thyroid carcinoma (n = 18), Hürthle cell carcinoma (n = 18), and medullary thyroid carcinoma (n = 10). Only focal positivity was observed in three Hürthle cell carcinoma cases. TROP2 was a helpful marker for differentiating PTC, especially in the classic, tall cell, hobnail, and Warthin-like variants, benign and non-neoplastic lesions, and other neoplasms of the thyroid.

Immunomodulatory Function of Polyvinylpyrrolidone (PVP)-Functionalized Gold Nanoparticles in Vibrio-Stimulated Sea Urchin Immune Cells.

We investigated the role of the gold nanoparticles functionalized with polyvinylpyrrolidone (PVP–AuNPs) on the innate immune response against an acute infection caused by Vibrio anguillarum in an in vitro immunological nonmammalian next-generation model, the sea urchin Paracentrotus lividus. To profile the immunomodulatory function of PVP–AuNPs (0.1 μg mL−1) in sea urchin immune cells stimulated by Vibrio (10 μg mL−1) for 3 h, we focused on the baseline immunological state of the donor, and we analysed the topography, cellular metabolism, and expression of human cell surface antigens of the exposed cells, as well as the signalling leading the interaction between PVP–AuNPs and the Vibrio-stimulated cells. PVP–AuNPs are not able to silence the inflammatory signalling (TLR4/p38MAPK/NF-κB signalling) that involves the whole population of P. lividus immune cells exposed to Vibrio. However, our findings emphasise the ability of PVP–AuNPs to stimulate a subset of rare cells (defined here as Group 3) that express CD45 and CD14 antigens on their surface, which are known to be involved in immune cell maturation and macrophage activation in humans. Our evidence on how PVP–AuNPs may stimulate sea urchin immune cells represents an important starting point for planning new research work on the topic.

Structural insights into the cis and trans assembly of human trophoblast cell surface antigen 2

SummaryHuman trophoblast cell surface antigen 2 (TROP-2) is an important target of tumor therapy, and antibody-drug conjugates with sacituzumab targeting TROP-2 have been approved for the treatment of triple-negative breast cancer. Here, we report the crystal structures of TROP-2-ECD, which can be either cis- or trans-dimers depending on which distinct but overlapping interfaces is used to engage with monomers. The cis- or trans-tetrameric forms of TROP-2 can also be assembled with a non-overlapping interface with either cis- or trans-dimerization, suggesting that cis- and trans-dimers cluster on the cell surface. The binding site of sacituzumab on TROP-2 is mapped to be located on a stretched polypeptide in CPD (Q237-Q252), which is not involved in either cis- or trans-interactions. The present findings will improve understanding of the molecular assembly of TROP-2 on tumor cells and shed light on future design of biologics for tumor therapy.

Abstract 1827: Efficacy and characterization of a specific and potent monoclonal antibody, LYT-210, against immunosuppressive gd1 T cell in cancer

Abstract Background: Targeting and engineering γδ T cells has emerged as an orthogonal therapeutic approach in oncology with capacity to modulate both innate and adaptive immunity. Solid tumors (e.g. pancreatic ductal adenocarcinoma (PDAC), melanoma, glioblastoma, ovarian-, breast- cancer etc.), can be enriched for γδ1T cells, where they mediate a robust, multi-faceted immunosuppression. We verified the phenotypic and functional features, and the immunosuppressive potential of cancer patient γδ1T cells and have developed a therapeutic antibody (mAb), LYT-210, for selective depletion of these immune evasion culprits. LYT-210 is highly specific, fully human, species cross-reactive IgG1 antibody, aimed for IND enabling studies and clinical development. Methods: γδ1T cells were profiled and studied from blood of healthy and cancer donors, and from surgically excised primary or metastatic tumors. A proprietary, synthetic Fab-phage library was used to generate LYT-210, a fully human IgG1 mAb. Functional assays (ADCC/ADCP) confirmed an expected and desired, Fcγ-dependent LYT-210 mode of action. We used healthy and patient PBMCs to assess the dynamics, dose dependence and specificity of γδ1 T cell killing with LYT-210. Patient tumor organoids were used for ex vivo efficacy studies. Purified proteins and engineered cell lines served to determine specificity and affinity of LYT-210. Retrogenix protein array (>5500 cell surface and secreted proteins) was used to identify possible off-target binding of LYT-210. Results: Anti-δ1antibody was used to treat patient tumor organoids (n = 29) and showed potent intra-tumoral T cell activation across tumor types (e.g. colorectal and liver cancer). Additionally, LYT-210 exerted potent ADCP and ADCC on: i) stable cell lines expressing γδ1TCRs and ii) γδ1T cells from patient PBMCs and did so by sparing αβ T cells and γδ2 T cells. Furthermore, LYT-210 induced ADCP and ADCC were Fcγ receptor dependent, confirming LYT-210-specific and effector cell dependent mode of action. LYT-210 induced γδ1 T cell-specific death rapidly, i.e. within 30 minutes of treatment with an EC50 of 26 pM and with the effect sustained over 70 hours of culture. Cell surface proteomics microarray analysis showed very weak cross reaction of LYT-210 with CEACAM5 (a known human cancer cell surface antigen), but in confirmatory assays had minimal binding to CEACAM5 expressed onto HEK293 cells (~200 nM) and no binding at all to purified CEACAM5. The Retrogenix array and additional assays therefore confirm the profound specificity of LYT-210 to γδ1T cells. Conclusions: We have developed a novel, therapeutic immuno-oncology strategy specifically targeting imunosuppressive γδ1cells. Our data support that this therapeutic approach may have the potential to be transformative for the treatment of cancers where γδ1T cells drive a pro-tumorigenic, immunosuppressive environment. Citation Format: Tatyana Panchenko, Wei Wang, Eric Denbaum, Akiko Koide, Takamitsu Hattori, Aleksandra Filipovic, George Miller, Shohei Koide. Efficacy and characterization of a specific and potent monoclonal antibody, LYT-210, against immunosuppressive gd1 T cell in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1827.

The role and mechanism of human trophoblastic cell surface antigen 2 in the invasion and metastasis of ovarian cancer

Objective: To explore the role and molecular mechanism of trophoblastic cell surface antigen 2 (Trop2) in the invasion and migration of ovarian cancer. Methods: Through the data mining of Cancer Cell Line Encyclopedia and TCGA database, the clinical significance of Trop2 expression was analyzed. Western blot was used to detect Trop2 protein expression in ovarian cancer cell lines including A3O, A1780 and SKOV3. SKOV3 cells were used to construct Trop2-short hairpin RNA (shRNA) cell model. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was used to detect the SKOV3 mRNA expression in SKOV3-shRNA and SKOV3-NC cells. Cell counting kit-8 (CCK8) was used to detect the proliferation of SKOV3-shRNA cells and SKOV3-NC cells. Flow cytometry was used to detect cell cycle and apoptosis in two groups of cells. Transwell array was used to detecte the invasion and migration of SKOV3-shRNA cells and SKOV3-NC cells. Western blot was used to detect the protein expressions of AKT, p-AKT, β-catenin, caspase3, bcl-2, E-cadherin and vimentin. Results: Trop2 mRNA highly expressed in ovarian cancer, and was related to the tumor stage and patient survival. Compared with A3O cells, Trop2 overexpressed in A1780 and SKOV3 cells (P<0.05). The relative expression levels of Trop2 mRNA in SKOV3-NC group and SKOV3-shRNA group were 1.18±0.24 and 0.42±0.08, with statistically significant difference (P<0.05). The results of CCK-8 array showed that the cell viability of SKOV3-NC group was significantly higher than that of SKOV3-shRNA group (P<0.05). The proportion of G(0)/G(1) cells in SKOV3-NC and SKOV3-shRNA groups were (38.67±4.22)% and (60.24±8.17)%, respectively. G(0)/G(1) arrest was observed in SKOV3-shRNA cells (P<0.05). The apoptosis rate of SKOV3-shRNA group was (26.32±1.81)%, significantly higher than (6.54±1.32)% of SKOV3-NC group (P<0.05). The number of migrating SKOV3 cells in the SKOV3-shRNA and SkOV3-NC groups were 1 255.83±108.44 and 1 679.71±213.92, while the number of invading cells were 242.49±52.09 and 473.54±73.11, respectively. Compared with the SKOV3-NC group, the number of migrating and invading SKOV3-shRNA group was significantly reduced (all P<0.05). The expressions of p-AKT2, Bcl-2, vimentin and β-catenin were down-regulated, and the expressions of caspase 3 and E-cadherin were up-regulated in SKOV3-shRNA cells. There was no significant change in the total protein level of AKT. Conclusions: Trop2 expression is related to ovarian cancer stage and postoperative survival. Trop2 can promote ovarian cancer cell proliferation and metastasis by activating the AKT/β-catenin signaling pathway and knockdown of Trop2 inhibits the progression of ovarian cancer.

Abstract 6465: Clinicopathological features of TROP-2 and pTROP-2 in gastric cancer

Abstract Introduction: The human trophoblast cell-surface antigen TROP-2 is a cell surface glycoprotein receptor involved in tight junctions at the epithelial barrier. Although TROP-2 is reportedly overexpressed in various epithelial tumors and its expression correlates with aggressive tumor behavior, there is no report of the clinicopathological features of gastric cancer (GC) with TROP-2 and phosphorylated TROP-2 (pTROP-2) expression. We evaluated the clinicopathologic significance of TROP-2 and pTROP-2 expression in GC patients. Materials and methods: We retrospectively analyzed the cases of 720 GC patients who underwent resection for GC. The expressions of TROP-2 and pTROP-2 in each tumor were evaluated by immunohistochemistry. We then divided the patients into the TROP-2 high group, TROP-2 low group, pTROP-2 high group, and pTROP-2 low group. The association between TROP-2 or pTROP-2 expression and clinicopathological variables were assessed by the chi-square test. Overall survival was analyzed by Kaplan-Meier method, and compared by log-rank test. The Cox proportional hazards model was used for multivariate analysis. Results: TROP-2 high group was found to be overexpressed in 330 (46.7%) tumor samples. Significantly higher expression of TROP-2 was significantly correlated with intestinal type, tumor invasion depth, lymph node metastasis, lymphatic invasion, and venous invasion. pTROP-2 high group was found to be overexpressed in 306 (43.4%) tumor samples. Significantly higher expression of pTROP-2 was correlated with intestinal type, low tumor invasion depth, no lymph node metastasis, and no lymphatic invasion. TROP2 overexpression was predictive for poor overall survival of patients with GC (p = 0.0002, log rank test), while multivariate analysis to overall survival revealed that TROP-2 overexpression was not an independent prognostic marker (p=0.06). In contrast, pTROP-2 overexpression was predictive for good overall survival of patients with GC (p = 0.004, log rank test). Conclusion: TROP-2 might be associated with metastatic ability of gastric cancer cells, resulting poor prognosis of patients with GC. Citation Format: Syuhei Kushiyama, Masakazu Yashiro, Sadaaki Nishimura, Shingo Togano, Kanji Kuroda, Atsushi Sugimoto, Tomohiro Sera, Yurie Yamamoto, Tomohisa Okuno, Yuichiro Miki, Hiroshi Nakada, Masaichi Ohira. Clinicopathological features of TROP-2 and pTROP-2 in gastric cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6465.

Affinity maturation, humanization, and co-crystallization of a rabbit anti-human ROR2 monoclonal antibody for therapeutic applications

Antibodies are widely used as cancer therapeutics, but their current use is limited by the low number of antigens restricted to cancer cells. A receptor tyrosine kinase, receptor tyrosine kinase-like orphan receptor 2 (ROR2), is normally expressed only during embryogenesis and is tightly down-regulated in postnatal healthy tissues. However, it is up-regulated in a diverse set of hematologic and solid malignancies, thus ROR2 represents a candidate antigen for antibody-based cancer therapy. Here we describe the affinity maturation and humanization of a rabbit mAb that binds human and mouse ROR2 but not human ROR1 or other human cell-surface antigens. Co-crystallization of the parental rabbit mAb in complex with the human ROR2 kringle domain (hROR2-Kr) guided affinity maturation by heavy-chain complementarity-determining region 3 (HCDR3)-focused mutagenesis and selection. The affinity-matured rabbit mAb was then humanized by complementarity-determining region (CDR) grafting and framework fine tuning and again co-crystallized with hROR2-Kr. We show that the affinity-matured and humanized mAb retains strong affinity and specificity to ROR2 and, following conversion to a T cell-engaging bispecific antibody, has potent cytotoxicity toward ROR2-expressing cells. We anticipate that this humanized affinity-matured mAb will find application for antibody-based cancer therapy of ROR2-expressing neoplasms.

Knockdown of Trop2 inhibits proliferation and migration and induces apoptosis of endometrial cancer cells via AKT/β-catenin pathway.

Endometrial cancer (EC) is the most common gynaecologic malignancy in western countries and has been reported to account for about 7% of female malignant tumours and 20% to 30% of female genital system malignant tumours. Accumulating evidence showed the expression of human trophoblast cell surface antigen 2 (Trop2) was abnormal in many cancers; however, the expression and role of Trop2 in EC are not clear. The Trop-2 protein expression was detected by western blot in EC cells. Cell proliferation, apoptosis, and migration were measured by CCK-8, flow cytometry, and Transwell assay, respectively. The epithelial mesenchymal transition (EMT) and AKT/β-catenin signalling pathway-related proteins in EC cell lines were detected by western blot assay following Trop2 gene silencing. The present study revealed that the Trop2 protein was highly expressed in EC cell lines compared with human endometrial epithelial cells. The Trop2 mRNA and protein were obviously decreased following transfection with Trop2-siRNA sequence in KLE and Ishikawa cells. Meanwhile, Trop2 gene silencing in KLE and Ishikawa cells strongly inhibited cell proliferation and migration and increased cell apoptosis. Investigation into the molecular mechanism indicated that the Trop2 gene silencing suppressed EMT and AKT/β-catenin signalling pathway activation. SIGNIFICANCE OF THE STUDY: These findings suggested that Trop2 silencing inhibited EC cell proliferation and migration and promoted cell apoptosis. The mechanism might be related to the inhibition of the AKT/β-catenin signalling pathway in EC cells. Therefore, Trop2 may be a potential therapeutic target for the treatment of EC.

Silencing of the TROP2 gene suppresses proliferation and invasion of hepatocellular carcinoma HepG2 cells.

ObjectivesOverexpression of human trophoblast cell surface antigen 2 (Trop2) has been observed in many cancers; however, its roles in proliferation, apoptosis, migration, and invasion of hepatocellular carcinoma (HCC) remain unclear. Thus, this study aimed to characterize the function of Trop2 in HCC.MethodsTrop2 protein expression was detected by immunohistochemistry in HCC tissues. Cell proliferation, apoptosis, and invasion were respectively measured by CCK-8, flow cytometry, Transwell, and wound healing assays. Expression levels of epithelial–mesenchymal transition-related proteins and Trop2 protein in HCC cell lines were detected by western blotting after silencing of the TROP2 gene.ResultsTrop2 protein was highly expressed in HCC tissues and HCC cell lines. Trop2 mRNA and protein expression levels decreased in HepG2 and HCCLM3 cells after transfection with Trop2 siRNA. Silencing of the TROP2 gene in HepG2 and HCCLM3 cells strongly inhibited cell proliferation and migration, while enhancing cell apoptosis. Investigation of the molecular mechanism revealed that silencing of the TROP2 gene suppressed epithelial–mesenchymal transition of HepG2 and HCCLM3 cells.ConclusionsThe results of the present study may improve understanding of the role of Trop2 in regulation of cell proliferation and invasion, and may aid in development of novel therapy for HCC.

Siglec-6 on Chronic Lymphocytic Leukemia Cells Is a Target for Post-Allogeneic Hematopoietic Stem Cell Transplantation Antibodies.

Although the 5-year survival rate of chronic lymphocytic leukemia (CLL) patients has risen to >80%, the only potentially curative treatment is allogeneic hematopoietic stem cell transplantation (alloHSCT). To identify possible new monoclonal antibody (mAb) drugs and targets for CLL, we previously developed a phage display-based human mAb platform to mine the antibody repertoire of patients who responded to alloHSCT. We had selected a group of highly homologous post-alloHSCT mAbs that bound to an unknown CLL cell surface antigen. Here, we show through next-generation sequencing of cDNAs encoding variable heavy-chain domains that these mAbs had a relative abundance of ∼0.1% in the post-alloHSCT antibody repertoire and were enriched ∼1,000-fold after three rounds of selection on primary CLL cells. Based on differential RNA-seq and a cell microarray screening technology for discovering human cell surface antigens, we now identify their antigen as Siglec-6. We verified this finding by flow cytometry, ELISA, siRNA knockdown, and surface plasmon resonance. Siglec-6 was broadly expressed in CLL and could be a potential target for antibody-based therapeutic interventions. Our study reaffirms the utility of post-alloHSCT antibody drug and target discovery. Cancer Immunol Res; 6(9); 1008-13. ©2018 AACR.

Abstract 3964: Mesothelin targeting immunotoxin LMB-100 alters the profile of tumor-secreted proteins

Abstract Recombinant immunotoxins are antibody-based therapies that carry a bacterial toxin payload. LMB-100 is a next generation immunotoxin currently being tested in clinical trial. It targets the human cell surface antigen mesothelin (MSLN). MSLN is made by many solid tumors but has no expression in the parenchyma of vital organs. LMB-100 carries a Pseudomonas exotoxin payload that works by irreversibly modifying elongation factor-2 (EF-2), inactivating it and halting protein synthesis. To determine whether non-lethal doses of LMB-100 might inhibit tumor cell secretion of cytokines and growth factors that maintain the pancreatic cancer microenvironment, human pancreatic cancer cells were treated with LMB-100 and conditioned medium was assayed for tumor-secreted factors by ELISA and Luminex assays. Decreased levels of multiple secreted factors were detected including VEGF, PDGF, MMP-1, SPARC, and OPN. Conversely, levels of MIF, a stored cytokine released under stress conditions, increased dramatically upon LMB-100 treatment. To determine whether this also occurs in vivo, tumor-bearing athymic nude mice were treated with LMB-100 and levels of tumor-secreted factors in intratumoral fluid were measured. A statistically significant decrease in the same factors was found in treated mice compared to control mice. To determine whether these decreases in tumor-secreted factors could change the tumor microenvironment and alter the immune milieu, a mouse syngeneic model of pancreas cancer sensitive to LMB-100 was developed. LMB-100 treatment of this mouse cell model caused a statistically significant decrease in the angiogenesis factors VEGF and Angiopoietin-2 in conditioned medium. To determine if this also occurs in vivo, immune-competent mice bearing intraperitoneal or orthotopic tumors were treated with immunotoxin or vehicle then intratumoral fluid was assayed for secreted proteins contributed by both tumor and stromal cells. No detectable change in intratumoral fluid concentration was seen for VEGF, Antiopoietin-2 or a panel of 11 other secreted factors. In conclusion, LMB-100 does inhibit tumor cell production of secreted proteins, but this change is insufficient to alter the total concentration of secreted factors in the tumor microenvironment. Citation Format: Salma El-Behaedi, Rebekah Landsman, Michael Rudloff, Emily Kolyvas, Rakan AlBalawy, Christine Alewine. Mesothelin targeting immunotoxin LMB-100 alters the profile of tumor-secreted proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3964.

Curcumin inhibits cell proliferation and motility via suppression of TROP2 in bladder cancer cells.

Bladder cancer (BC) has become a serious health problem and represents the second most commonly diagnosed urological tumor. Curcumin is a principal active natural component of turmeric and has long been used in Asia as a traditional herbal medicine. Curcumin suppresses cell growth in various types of cancer, including BC, by regulating numerous molecular signaling pathways. The human trophoblast cell surface antigen 2 (Trop2) belongs to the tumor-associated calcium signal transducer gene family. Trop2 has been described as a cancer driver and is deregulated in various types of cancer. However, whether Trop2 is involved in curcumin-induced BC cell inhibition remains to be elucidated. The present study hypothesized that Trop2 may be a promising target of curcumin in BC cells. It was found that Trop2 was closely involved in curcumin-induced cell proliferation suppression, mobility inhibition, apoptosis, and cell cycle arrest in BC cells. Curcumin decreased the expression of Trop2 and its downstream target cyclin E1, and increased the level of p27. The overexpression of Trop2 enhanced the oncogenic activity of BC cells, whereas downregulation of the expression of Trop2 suppressed cell proliferation and mobility, increased apoptosis, and sensitized BC cells to curcumin treatment. Therefore, Trop2 may be a promising target of curcumin in BC cells and the inhibition of Trop2 may be an important method for the therapeutic management of patients with BC.

TROP2 promotes cell proliferation and migration in osteosarcoma through PI3K/AKT signaling.

Human trophoblast cell surface antigen 2 (TROP2) has been noted to serve an important role in the proliferation and migration of various types of human cancers. However, the potential role and the molecular mechanisms of TROP2 in osteosarcoma (OS) remain largely unclear. In the present study, high expression of TROP2 in human OS tissues and cell lines was observed. Overexpression of TROP2 promoted the proliferation and migration of OS cell lines, while TROP2 knockdown markedly decreased cell growth and migration. Furthermore, it was revealed that TROP2 overexpression significantly activated the phosphoinositide 3‑kinase/protein kinase B (PI3K/AKT) signaling pathway. Collectively, these results suggested that TROP2 may promote OS cell proliferation and migration via PI3K/AKT signaling and may serve as a novel treatment target for OS.

The trophoblast cell surface antigen 2 and miR-125b axis in urothelial bladder cancer.

Human trophoblast cell surface antigen 2 (Trop-2) is a 40-kDa transmembrane glycoprotein that was first identified as a marker of human trophoblast cells. Trop-2 acts on cell proliferation, adhesion, and migration by activating a number of intracellular signalling pathways. Elevated Trop-2 expression has been demonstrated in several types of cancer and correlated with aggressiveness and poor prognosis. Since no data are available on Trop-2 in bladder cancer (BC), the purpose of the study was to determine its levels in tissue specimens from normal individuals and patients with BC at different stages. Moreover, since according to recent evidence Trop-2 is a miR-125b target, miR-125b expression was also assessed in tissue specimens. Finally, the effect of the Trop-2/miR-125b axis on the proliferation and migration of BC cells was evaluated in vitro.The Trop-2/miR-125b axis was seen to be differentially expressed in normal urothelium, non-invasive BC and invasive BC tissue. Significant miR-125b down-regulation was associated with a significant increase in Trop-2 protein levels in BC tissue and correlated with disease severity. In vitro analysis confirmed the role of miR-125b in down-modulation of Trop-2 protein levels and showed that Trop-2/miR-125b axis affects cellular proliferation in bladder tissue.In conclusion, our findings highlight a role for the Trop-2/miR-125b axis in BC progression and suggest Trop-2 and miR-125b as diagnostic/prognostic marker candidates as well as druggable targets for innovative therapeutic approaches.