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The mechanisms of action of cytotoxic monoclonal antibodies (mAbs) include FcγR-dependent mechanisms to induce antibody-dependent cell cytotoxicity (ADCC). In mice, ADCC relies on mFcγRIII expressed on NK cells, while human NK cells depend on hFcγRIIIA (hCD16A). However, human IgG1 display significant affinity differences between mouse and human FcγRs, which can impact their efficacy in preclinical models. Additionally, testing an affinity-enhanced Fc-engineered antibody in a mouse without the human FcγR counterparts provides limited predictive value. Therefore, preclinical mouse models expressing human FcγRs are essential to evaluate clinical Fc potent antibody. In this study, a hCD16A-CB17SCID humanized (HuGEMM) mouse model, featuring hCD16A knock-in on mouse NK cells, was used to evaluate the ADCC function of the CLDN18.2 mAb zolbetuximab. Using SNU-601 cell line, a high expression hCLDN18.2 model, the efficacy of zolbetuximab in hCD16A-CB17SCID mice was compared to CB17SCID WT mice in SNU-601 xenograft. Surface expression of human CD16 on NK cells from peripheral blood (PB) and spleen of hCD16A-CB17SCID HuGEMM mice was verified by flow cytometry. In vivo studies involved 1×107 SNU-601 cells subcutaneously inoculated into hCD16A-CB17SCID HuGEMM models or CB17SCID WT models. Tumor-bearing models were treated with zolbetuximab (10 mg/kg, i.p., BIW×13 doses) or hIgG1 (10 mg/kg, i.p., BIW×13 doses) as an isotype control. At the study endpoint, NK cell function related markers in PB, tumor tissues and hCLDN18.2+ tumor cell apoptosis from SNU-601 tumors were assessed by flow cytometry. hCD16A expression was confirmed on NK cells from PB and spleen in hCD16A-CB17SCID HuGEMM mice. SNU-601 tumor showed similar growth profile as was observed in hCD16A-CB17SCID HuGEMM and WT models. Significant tumor growth inhibition (TGI) of SNU-601 tumor was observedwith zolbetuximab compared to the isotype control, with hCD16A-CB17SCID HuGEMM models showing 55% TGI, higher than the 33% TGI observed in CB17SCID WT models. At the study endpoint, similar percentages of mNKp46+ mCD49b+ NK cells, mCD107a+ mNKp46+ mCD49b+ NK cells, and mKi67+ mNKp46+ mCD49b+ NK cells were observed between hCD16A-CB17SCID HuGEMM and CB17SCID WT models. Apoptosis analysis indicated that zolbetuximab induced apoptosis in hCLDN18.2+ tumor cells compared to control groups in both hCD16A-CB17SCID HuGEMM and CB17SCID WT models. This study indicates that hCD16A-CB17SCID HuGEMM models exhibit enhanced tumor growth inhibition in response to zolbetuximab treatment compared to CB17SCID WT models, demonstrating the potential of this model as a more effective preclinical model for evaluating ADCC-mediated therapies. Rong Wang, Xiaoze Wang, Kaixia Lian, Rongfei Lu, Haijuan Yu, Jie Lin, Ludovic Bourre, Jingjing Wang. Comparison of ADCC-mediated efficacy of zolbetuximab in hCD16A-CB17SCID humanized mice and CB17SCID WT mice bearing the SNU-601 tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7335.
For decades, KRAS was considered undruggable due to the lack of suitable binding sites. However, advancements in bioengineering and chemistry have enabled the approval of targeted therapies. Success was first seen with allele-specific targeting of KRASG12C in non-small cell lung cancer (NSCLC), leading to the approval of sotorasib (AMG510, LumakrasTM). Despite its clinical benefits, resistance emerged in some patients due to secondary KRAS mutations, which necessitates next-generation or combination therapy development. In this study, we outline the development of KRASG12C inhibitor-resistant models overcome this hurdle. Secondary KRAS mutations (Y96D/C/S, H95D/Q/R, R68S, Q61H, A59T/S, and Q99L) were introduced by CRISPR/Cas9 in MIA PaCa-2 with a homozygous KRASG12C mutation. Knock-in of point mutation was validated by Sanger sequencing. Cell viability was assessed by CellTiter-Glo (CTG) with AMG510 and MRTX849 (Adagrasib, KrazatiTM). RAS-MAPK pathway activity was evaluated by western blot. Xenograft models of MIA PaCa-2 cells with Y96D/C, H95D/Q/R, R68S, Q61H and A59T were established. Additionally, in vitro chronic dosing of AMG510 generated AMG510-resistant MIA PaCa-2 and NCI-H358 cell lines were validated by CellTiter-Glo and western blot. RNA-seq identified potential resistance mechanisms. Xenograft models were also established. A successful homozygous point mutation knock-in was confirmed by Sanger sequencing. Cells expressing double-mutant alleles KRAS G12C Y96D/C/S, A59T/S and R68S showed resistance to both AMG510 and MRTX849, while KRAS G12C H95D/Q/R was more resistant to MRTX849, and KRAS G12C Q61H, Q99L didn’t show significant resistance. Persistent phosphorylated ERK (pERK) and pRSK levels indicated sustained RAS-MAPK activity in cells expressing KRAS G12C Y96D, H95D, A59T/S, and R68S, even at high KRAS inhibitor concentrations. Furthermore, a KRAS G12C Y96D/C, A59T, Q61H, R68S and H95D/Q/R double mutant cell-derived xenograft was established in vivo. Additionally, MIA PaCa-2 AMG510-resistant and NCI-H358 AMG510-resistant cells showed resistance to AMG510 and MRTX849 in cell viability assays. RNA-seq data identified c-MET amplification in AMG510-resistant MIA PaCa-2 cell, while FGFR1/3/4 amplification was found in AMG510-resistant NCI-H358 cells. CRISPR/Cas9-engineered KRAS secondary mutations cell lines displayed differentially resistant profile to KRASG12C inhibitors, and drug-induced resistant cell models developed in vitro displayed KRAS-independent mechanisms of resistance. These novel cell models offer a valuable preclinical platform to evaluate therapeutic strategies to overcome resistance to KRAS-targeted therapies. Jian Feng, Dan Zhang, Aaron Hua, Chenpan Nie, Jessie(Jingjing) Wang, Ludovic Bourre, Jun Zhou, Peng Wang. Developing KRASG12C inhibitor-resistant tumor models for efficacy evaluation of next-generation anticancer therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5524.
Antibody-drug conjugates (ADCs) are monoclonal antibodies targeting tumor cells linked to cytotoxic drugs via chemical linkers. Trop-2 targeting ADCs like sacituzumab govitecan (SG), datopotamab deruxtecan (Dato-DXd), and SKB-264 have shown success in clinical settings. However, they face challenges such as complex pharmacokinetics, insufficient tumor targeting, suboptimal payload release, side effects and drug resistance. This study developed SG-resistant PDX models to replicate clinical resistance to support new therapy development and understanding of resistance mechanisms. Subcutaneous PDX models were established and characterized by histology and sequencing, with Trop-2 expression assessed by immunohistochemistry (IHC) and RNA sequencing (RNA-seq). For in vivo efficacy studies, models were treated 3-4 weeks after tumor inoculation with SG (3 or 5 mg/kg, i.v.) and Dato-DXd (5 mg/kg, i.v.) based on clinical SG treatment schedule. Tumor growth inhibition (TGI) was calculated as [1 - (mean treated tumor volume / mean control tumor volume)] * 100%. Homologous recombination deficiency (HRD) scores were calculated (using scarHRD and PureCN from whole exome sequencing, with a cutoff value of 38). A female TNBC patient underwent 8 rounds of chemotherapy and targeted treatment, including 2 cycles of SG in the sixth round. Tissues collected at different treatment stages following SG treatment were used to establish BR9690, BR9806 and BR9801 PDX models. High Trop-2 expression was detected at both gene and protein levels in all three models. To evaluate Trop-2 ADCs efficacy, SG and Dato-DXd were tested in the PDX models. Tumor progression was observed in BR9690 for both SG (3mg/kg TGI=9.40% and 5mg/kg TGI=16.77%) and Dato-DXd (TGI=32.15%). In BR9806, tumor progression was observed in SG with limited TGI (3mg/kg TGI=27.52% and 5mg/kg TGI=25.75%), while Dato-DXd showed a partial response (TGI=59.47%). For BR9801, both SG and Dato-DXd showed limited TGI (SG: 3mg/kg TGI=-2.60% and 5mg/kg TGI=0.21%, Dato-DXd: TGI=24.78%). These results indicate clinical consistency with SG resistance and resistance to other Trop2-ADCs as the disease progresses. Besides, HRD scores for all three PDX models were above the cutoff (BR9690: 42, BR9806: 39, BR9801: 50) indicating genomic instability. In this study, the limited response of the models despite HRD may suggest other factors affecting the drug’s efficacy targeting DNA repair mechanism, such as acquired resistance mechanisms or drug efflux activity, overriding the vulnerability provided by HRD. The study successfully established longitudinal breast cancer PDX models from a patient with demonstrated resistance to Trop2-ADCs. These models will aid in evaluating new treatments or combination therapies and understanding drug resistance mechanisms. Qingzhi Liu, Jinxi Wang, Xu Zhang, Wubin Qian, Likun Zhang, Ludovic Bourre, Jingjing Wang. The development of PDX models from ADC-resistant breast cancer patient tissue for next-generation therapy evaluation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1272.
Antibody-drug conjugates (ADCs) represent a breakthrough therapeutic class that provides tumor-targeted cytotoxicity while stimulating immune responses, offering promising potential when combined with immunotherapies and particularly with PD-1 inhibitors. Traditional xenograft tumor models developed in immunodeficient mice are employed to evaluate ADCs' anti-tumor efficacy. However, these models lack competent immune systems and human antigen expression in normal tissues, limiting their capacity to assess immune responses and toxicity. To address these limitations, we developed HuCell, mouse tumor cells engineered to express tumor associated antigen (hTAA) such as HER2, and HuGEMM, humanized for a specific drug target within mice with a functional immune system. In this study HuCell/HuGEMM humanized mouse models were developed to evaluate the efficacy of ADCs in combination with anti-PD-1 immunotherapy and toxicity. Surface expression of human HER2, Trop2, and Nectin-4 on MC-38 HuCell was verified using flow cytometry before and after inoculation on mice. 5×106 HuCells were subcutaneously inoculated into hHER2, hTrop2, hNectin4 and hHER2/hPD-1 models in C57BL/6 background or wild-type (WT) models. Tumor-bearing models were treated with monotherapy of trastuzumab-deruxtecan (T-Dxd), sacituzumab govitecan or enfortumab vedotin, or combination therapy with anti-PD-1 antibodies. For the toxicity study, 180 mg/kg T-Dxd and 100 mg/kg trastuzumab-emtansine (T-DM1) were injected into hHER2 mice weekly for two doses by intravenous injection. A highly HER2-expressing HuCell line (clone #2) successfully grew in hHER2 models but not in WT models, with tumors displaying nearly 100% of human Her2 positive cells with high surface expression (2×105 molecules per cell). In dose response study, T-Dxd significantly inhibited tumor growth at 5 mg/kg and 10 mg/kg in C57BL/6-hHER2. Combination treatment of T-Dxd (4 mg/kg+2 mg/kg, QW) with Keytruda (5 mg/kg, BIW×2) resulted in a complete response rate (8/8) with 87% TGI (p<0.01) compared to T-Dxd monotherapy (7/8) with 96% TGI (p<0.001) in hHER2/PD-1 double knock in mice. In a toxicity study, T-DM1 showed liver toxicity, whereas T-Dxd did not. Both T-DM1 and T-Dxd exhibited notable hematological toxicity, consistent with clinical observations. In the Trop2 and Nectin-4 HuCell/HuGEMM studies, similar results were observed. HuGEMM models demonstrated superior positive cell rate and single-cell antigen expression levels. Furthermore, a synergistic effect was observed when PD-1 therapy was combined with monotherapy. HuCell and HuGEMM models provide an advanced platform to evaluate ADC drug monotherapy and combination therapies with immune checkpoint inhibitors. Additionally, they offer a sophisticated solution to assess both on-target and off-target toxicity of ADCs. Xiaoze Wang, Rong Wang, Kaixia Lian, Rongfei Lu, Jian Feng, Jun Zhou, Xiaolong Tu, Xinhe Feng, Jie Lin, Fuping Xu, Ludovic Bourre, Jingjing Wang. Novel humanized cell and murine models expressing tumor-associated antigens for ADC toxicity and IO combination evaluation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7270.
