Resistant Models Research Articles

Abstract 537: Preclinical PD-1 resistance models: Crucial tools for unveiling PD-1 resistance mechanisms and advancing novel treatment strategies

Abstract In the cancer-immunity cycle, the immune checkpoint PD-1 and its ligand PD-L1 collaborate to facilitate tumor immune evasion and promote tumor progression by thwarting immunity-induced apoptosis. Immune checkpoint inhibitors disrupt this alliance by preventing PD-1 from binding to PD-L1, thereby reactivating the patient's immune system. While anti-PD-1/PD-L1 therapy has demonstrated remarkable efficacy in treating solid tumors, durable responses are observed in only a minority of patients. The majority of patients do not experience the full benefits of anti-PD-1/PD-L1 therapy, and a significant proportion of initial responders eventually develop acquired resistance. Thus, the establishment of reliable preclinical PD-1 resistance models is of paramount importance for uncovering resistance mechanisms and devising novel treatment strategies. Common mechanisms contributing to PD-1/PD-L1 resistance include the absence of appropriate tumor antigens, inactivation of tumor surface MHC molecules, aberrant IFNγ signaling pathways, and the presence of an immunosuppressive tumor microenvironment. We have successfully generated multiple drug resistance models at both cellular and animal levels, offering valuable tools for the investigation of diverse drug resistance mechanisms. For instance, our animal model of drug resistance, induced by long-term treatment with the PD-1 antibody (Keytruda), simulates the clinical scenario of acquired resistance due to prolonged medication. Through gene editing techniques, we have created a drug-resistant cell line (CT26-B2M KO) by disrupting the B2M gene in the CT26 cell line, thereby mimicking a tumor model with B2M functional deficiency in mice. Additionally, we have compiled efficacy data from various naturally occurring resistant models, which serve as valuable resources for studying primary resistance or intricate resistance mechanisms. In summary, preclinical PD-1 resistance models serve as crucial tools for researchers to dissect the intricacies of PD-1 resistance mechanisms and offer essential guidance for the development of innovative treatment strategies. Citation Format: Yini Zhu, Yuan Fang, Huiyi Wang, Jun Xing, Lu Yang, Jing Zhao, Xiang Gao, Cunxiang Ju. Preclinical PD-1 resistance models: Crucial tools for unveiling PD-1 resistance mechanisms and advancing novel treatment strategies [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 537.

Abstract 4737: The combination of the BET inhibitor JQ1 and gemcitabine induces regressions in gemcitabine resistant patient-derived xenograft models of pancreatic cancer

Abstract Efforts to develop targeted therapies for drug resistant tumors have been relatively unsuccessful. The 5-year survival for patients with advanced pancreatic cancer is ~3%. Gemcitabine is usually combined with nab-paclitaxel as a standard of care for treatment of locally advanced or metastatic pancreatic ductal adenocarcinoma (PDAC). However, no chemotherapeutic regimens thus far evaluated have been curative. We developed the first PDAC patient-derived xenograft (PDX) models in which resistance against gemcitabine was acquired in vivo. We used RNA-seq and ingenuity pathway analysis to compare expression profiles of paired parent vs gemcitabine resistant PDX models. This comparison suggested a previously unreported mechanism for gemcitabine resistance, involving aberrations of cholesterol biosynthesis and lipid metabolism pathways. We also made the unexpected observation that gemcitabine-resistant PDAC models derived from primary tumors have high levels of 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), an enzyme essential to cholesterol biosynthesis and rate limiting in ketogenesis. Notably, the BET bromodomain inhibitor JQ1 decreases HMGCS2 levels; and BET inhibitors + gemcitabine exert synergistic cytotoxicity in gemcitabine-resistant PDAC cells. We determined that the BET bromodomain inhibitor JQ1 + gemcitabine induces regressions of two in vivo gemcitabine-resistant tumor models which we developed directly from primary human tumor tissue. These mechanism-based studies suggest that inhibiting HMGCS2 represents a novel approach to therapy for PDAC. We propose that combining gemcitabine with agents that decrease levels of HMGCS2 comprises effective treatment for patients with drug resistant PDAC. Citation Format: Aubrey L. Miller, Samuel C. Fehling, Eric Josh Brown, Eric O. Heard, Tracy L. Gamblin, Rebecca B. Vance, Karina J. Yoon. The combination of the BET inhibitor JQ1 and gemcitabine induces regressions in gemcitabine resistant patient-derived xenograft models of pancreatic cancer [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 4737.

Abstract 538: Establishment of PARP inhibitor-induced resistant patient-derived ovarian cancer xenograft models

Abstract Introduction: Poly (ADP-ribose) polymerases (PARPs) can recruit DNA repair effectors and cope with DNA single-strand breaks. For DNA double-strand breaks (DSB), there are two main repair mechanisms: non-homologous end joining repair (NHEJ) which is prone to error due to infidelity events, and BRCA1/2 mediated homologous recombination repair (HR) restoring the original sequence. Therefore, inhibition of PARPs may cause both single stranded break repair deficiency and HR deficiency in BRCA1/2 deficient patients, leading to cell death. The concept of “synthetic lethality” has led to the development of PARP inhibitors, including Olaparib, Niraparib and Rucaparib in clinical practice. Despite encouraging efficacy of these PARP inhibitors, acquired resistance, such as HR repair restoration, drug efflux pump upregulation and replication fork protection, has limited their clinical benefits. Therefore, to investigate novel therapeutic strategies which can overcome the acquired resistance, we established patient-derived xenograft (PDX) ovarian cancer models with PARP inhibitor-induced resistance. Methods: OV9402, an ovarian cancer PDX model with BRCA 1 c.3155del, was implanted subcutaneously and treated with Niraparib at 35 mg/kg, daily, for 129 days. Relapse tumors (tumor #1 and tumor #2) were isolated and inoculated into naïve mice. Then the 2nd round of treatment (Niraparib, 35 mg/kg, daily) was given when the mean tumor volume reached 140 mm3. The tumors showing no significant response to Niraparib were collected on either day 32 (#1 derived tumor model) and day 22 (#2 derived tumor model) after randomization and their gene changes were analyzed by RNA sequencing (RNA-seq). Results: Niraparib significantly inhibited OV9402 tumor growth, with complete regression observed in nine out of ten mice on day 54 after the 1st round of treatment. However, tumor regrowth was observed in two out of the nine mice from day 68 onwards. The relapsed tumors (tumor #1 and tumor #2) from these two mice were collected and re-inoculated into 5 naïve mice to establish tumor models for the 2nd round of Niraparib treatment, separately. For #1 derived tumor model, all the tumors presented complete resistance to the Niraparib treatment. RNA-seq data showed that the gene expression of ABCB1, which encodes for efflux pumps, was upregulated. In parallel, complete resistance to Niraparib was observed in all the tumors derived from 2# tumor bearing mice. The RNA-seq data demonstrated that an acquired secondary genetic deletion on BRCA1 genes, which restored the functional protein expression loss in the parental line and restored HR repair, was found in the tumors. Conclusion: Ovarian cancer PDX models that have effectively developed resistance to PARP inhibitors have the potential to serve as valuable preclinical tools to evaluate potential next-generation PARP inhibitors and innovative combination strategies. Citation Format: Li Hua, Shuang Li, Chenpan Nie, Xiaobo Chen, Guo Sheng, Ludovic Bourre, Jingjing Wang. Establishment of PARP inhibitor-induced resistant patient-derived ovarian cancer xenograft models [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 538.

Abstract 945: In vivo anti-tumor activity of onvansertib, a PLK1 inhibitor, combined with gemcitabine or carboplatin in platinum- resistant ovarian carcinoma patient-derived xenograft models

Abstract Background: The standard treatment for high grade ovarian carcinoma (HGOC) is cytoreductive surgery followed by a platinum based therapy. Despite an initial high rate of complete remission, most of the patients relapse with a platinum resistant disease. Patients with resistant tumors have few options, including monotherapy with gemcitabine, and new effective therapeutic alternatives are needed. The Polo-like kinase 1 (PLK1) is a master regulator of mitosis and recent evidences suggest its role in interfering with several DNA repair mechanisms. We investigated the effect of onvansertib (ONVA), a highly selective ATP-competitor PLK1 inhibitor, in combination with carboplatin (CARBO) and gemcitabine (GEM) in platinum-resistant HGOC patient-derived xenografts (PDXs). Methods: The combination of ONVA with either CARBO or GEM was investigated in two platinum-resistant HGOC PDXs, one intrinsically resistant to platinum (MNHOC#315), the other made resistant after repeated in vivo treatments with cisplatin (MNHOC#266R). Tumors were subcutaneously (s.c.; MNHOC#315) or orthotopically (i.p.; MNHOC#266R) transplanted in nude mice, mice were treated for 4 weeks with vehicle, single agents or drug combinations and followed for tumor growth (s.c. model) and survival. Results: CARBO- and GEM-ONVA combinations were well tolerated, with a maximum body weight loss of 6.6%. Both combinations showed strong anti-tumor activities in the two tested models. In MNHOC#315 PDX, CARBO- and GEM-ONVA combinations caused a tumor stabilization that extended beyond the dosing period and resulted in a higher tumor growth inhibition as compared to vehicle and single agents treated mice. In i.p. MNHOC#266R PDX, single agents were inactive, while a statistically significant increase in survival was observed in CARBO/ONVA and in GEM/ONVA treated mice as compared to vehicle treated mice, with a 6.6- and 7-fold increase in mean survival time, respectively. Pharmacodynamic studies are ongoing to decipher the mechanisms underlying the synergic effects of both combinations. Conclusions: The combinations of ONVA with either CARBO or GEM showed potent anti-tumor activities in two platinum-resistant HGOC PDXs. Additional studies in other HGOC platinum-resistant models, both in vivo and in vitro, as well pharmacodynamic studies are ongoing to validate these results and to get insights in the mechanisms underlying their efficacy. Citation Format: Michela Chiappa, Federica Guffanti, Ilaria Mengoli, Maya Ridinger, Giovanna Damia. In vivo anti-tumor activity of onvansertib, a PLK1 inhibitor, combined with gemcitabine or carboplatin in platinum- resistant ovarian carcinoma patient-derived xenograft models [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 945.

Abstract 4061: Preclinical characterization of MBS309, a conditionally active PD-1-dependent IL-2 mutation with significantly superior anti-tumor efficacy with safety

Abstract Although PD-1/PD-L1 therapies have shown significant benefit among a range of tumor types, the majority of malignancy patients do not respond or develop resistance after initial tumor regression. Development of new modality to overcome primary and acquired resistance to anti-PD-1/PD-L1 therapy is a huge unmet medical need, and IL-2 is believed a potentially powerful approach for that. However, the current clinical application of IL-2 in cancer immunotherapy has been limited by its severe toxicity. Herein, we developed MBS309 (PD1/IL2m), a novel PD-1-targeted IL-2 mutation with significantly enhanced therapeutic window. MBS309 has the similar high affinity and blockade efficacy with monoclonal antibody to PD-1, and its IL-2 mutation has reduced binding to IL-2Rβγ and abolished binding to IL-2Rα. Our in vitro assays, including HEK-blue assay and CD3+ T cell assay, both indicated that the IL-2 receptor agonist activity of MBS309 was conditionally induced only the antibody cis-binding to both PD-1 and IL-2 Rβγ. And the maximal agonist activities of MBS309 in HEK-blue assay and CD3+ T cell assay were much lower than wild-type IL-2 and RG6279 analog enabling its high tolerated drug doses in vivo. In our in vivo tumor murine models, 1mg/kg MBS309 showed much better suppression effect in MC38/hPD-L1 tumor than 1mg/kg RG6279 analog and Pembrolizumab (1mg/kg) combination with wild type IL-2 (0.1mg/kg) ; And MBS309 at the dosage of 5mg/kg can significantly inhibit tumor growth in PD-1 resistant Pan-02 xenograft models without any side-effects observed, while RG6279 analog at the dosage of 5mg/kg induced severe body reduction of the mice and resulted in 80% (4/5) mice death after two dosages. MBS309 showed similar PK profile with anti-PD-1 monoclonal antibody and much better than RG6279 analog in mouse model. 20mg/kg of MBS309 was well tolerated in mouse, while 10mg/kg of RG6279 analog or PF-07209960 analog show severe toxicity and resulted in death of all the mice. Pharmacodynamic studies in murine models showed that MBS309 prioritized active tumor infiltrated PD-1+ CD8 T cells rather than other organs. Taken together, MBS309 effectively expands tumor infiltrated T-cells, and avoids system immune cytokine side effects compared to other PD-1-targeted 2nd generation IL-2Rβγ agonist modalities currently in clinical development. And it shows much higher anti-tumor effect compared to anti-PD-1/-L1 antibodies alone in both PD-1 sensitive and resistant tumor models. MBS309 has demonstrated a favorable safety profile and highly anti-tumor activities in vivo, which has the potential to supporting its clinical development for the treatment of cancer. Citation Format: Jiangmei Li, Guangzhong Lin, Lunfeng Zhang, Feng Li. Preclinical characterization of MBS309, a conditionally active PD-1-dependent IL-2 mutation with significantly superior anti-tumor efficacy with safety [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 4061.

Abstract 2819: Novel drug resistant CDX models for anti-cancer drugs discovery

Abstract Oncogene BRAF is located on chromosome 7q34, consisting of 766 amino acids and 22 exons, encoding a serine/threonine protein kinase belonging to the RAF family and plays a critical role in the MAPK signaling pathway. The mutation of BRAF was documented in nearly 8% of all human cancers including melanoma (60%), thyroid (60%), and lung adenocarcinoma (10%). The most common mutation of BRAF is V600E (Class I), which was found in more than 70% in these cancers. Despite the clinical success of approved small molecule inhibitors of BRAF V600E (vemurafenib, dabrafenib and encorafenib), this remains an area of unmet medical need because of primary or acquired drug resistance. The construction of BRAF drug-resistant cell lines plays a pivotal role in cancer research. It serves as a crucial tool for discovery of the mechanisms of drug resistance. In this study, we have developed a systematic approach for constructing drug-resistant cell lines, inducing resistance through the gradual escalation of drug concentrations. The validation of drug resistance involves morphological recordings of parent and drug-resistant cell lines, drug resistance index in vitro through IC50 measurements and in vivo through the inhibition of tumor growth (TGI) which compare to those in the parental cell line. We successfully established Vemurafenib and Dabrafenib resistant A375 cell lines (A375 Vemurafenib R, A375 Dabrafenib R) with 21 and 31.5 folds of resistance index, respectively. The in vivo drug resistance efficacy was also tested by implanting Vemurafenib resistant cells subcutaneously into NOD-SCID mice and showed that A375 Vemurafenib R exhibited robust tolerance to 50 mg/kg Vemurafenib treatment (P.O.21 days, 50mpk, TGI=17.4%), whereas the parental cells were nearly completely inhibited (P.O.21 days, 50mpk, TGI=92.3%). In addition, single-cell sequencing of A375 Vemurafenib R revealed significant gene expression differences in the MAPK and EGFR signaling pathways, aiding in the identification of new targets, and signaling pathways associated with drug resistance, thereby providing potential targets for new drug development. Citation Format: Hai-Ting Dai, Tie-Jun Bing, Zhi-Da An, Jie Yang, Tian-Yu Pang, Jia-Wei Gao, Yun-Yi Bo, Wen-Jen Yu. Novel drug resistant CDX models for anti-cancer drugs discovery [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 2819.

Abstract 1930: Combination of KRASG12C and FGFR1 inhibitors as a resistance-overcoming therapeutic strategy for maximizing therapeutic impact of KRASG12C inhibitors

Abstract INTRODUCTION: Lung cancer is the leading cause of cancer-related deaths worldwide. KRAS is the most frequent mutated driver gen in lung adenocarcinoma (LUAD). KRASG12C inhibitors (G12Ci) have revealed promising results in the clinic, having being approved two G12Ci for the treatment of LUAD patients, which marks the first approved targeted therapy for KRAS-mutant tumors. Nevertheless, these therapies face the same limitation as other targeted therapies, the therapeutic potential of these inhibitors can be impaired by resistance mechanisms. Deciphering resistance mechanisms to G12Ci is of prime relevance to predict which patients may benefit from these therapies and to propose resistance-overcoming therapeutic strategies for maximizing therapeutic impact of these inhibitors. MATERIAL AND METHODS: We generated 10 acquired resistant LUAD cell lines to G12Ci (Sotorasib and Adagrasib), exposing sensitive cells to increasing concentrations of drugs. All the acquired resistant models were characterized at the proteomic (phospho-Array and Western Blot) and transcriptomic (WTS) levels to find signatures that define the resistance.The efficacy of G12Ci was tested in vitro in a panel of 8 cell lines and 7 PDX-derived organoids (PDXDO) models and in vivo in 7 patient-derived xenografts (PDX) models to classify them as resistant, sensitive or partially sensitive. The efficacy of the combination of FGFR1 and G12Ci was tested in vitro in the panel of 10 acquired resistant cell lines but also in the parental cell lines and PDXDOs (sensitive and intrinsic resistant) and in vivo in the intrinsic resistant PDXs. RESULTS AND DISCUSSION: The characterization of 10 acquired resistant cell lines at the proteomic and transcriptomic levels showed differences in the expression and/or activation of Fibroblast Growth Factor Receptors (FGFRs) in more than 50% of them. The combination of KRASG12C and FGFR1 inhibitors in all acquired resistant cell lines showed efficacy, regardless of whether acquired resistance was mediated or not by FGFR1 overexpression/overactivation.In addition, the combination of KRASG12C and FGFR1 inhibitors showed more efficacy than the monotherapies in most intrinsically resistant cell lines, PDXDOs and PDXs models. Finally, we tested the combination with FGFR1i in sensitive or partially responsive models, observing an improvement in efficacy compared to monotherapy with G12Ci. CONCLUSIONS: The activation or overexpression of FGFR1 acts as a mechanism of acquired resistance to KRASG12C inhibitors.-The combination of FGFR and KRASG12C inhibitors is effective as an acquired/intrinsic resistance-overcoming therapeutic strategy in cell lines, PDXDOs and PDXs models.-The combination of FGFR and KRASG12C inhibitors is also synergistic for sensitive models, which could maximize therapeutic impact of KRASG12C inhibitors. Citation Format: Alba Santos, Patricia Plaza, Marta Jimenez, David Gómez-Sánchez, Luis Paz-Ares, Irene Ferrer. Combination of KRASG12C and FGFR1 inhibitors as a resistance-overcoming therapeutic strategy for maximizing therapeutic impact of KRASG12C inhibitors [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 1930.

Abstract 3357: Identifying mechanisms of acquired resistance to lorlatinib utilizing a genome-wide CRISPR-Cas9 screen

Abstract Survival rates for neuroblastoma (NB), a predominantly pediatric solid tumor arising from the sympathoadrenal neural crest, have improved incrementally from 25% to 50% over three decades with dramatic escalation in therapy intensity. Gain-of-function mutations in anaplastic lymphoma kinase (ALK), of which 85% occur at residues F1174, F1245, and R1275, confer inferior survival compared to patients with wild-type ALK. Lorlatinib, a third generation ALK inhibitor, has completed Phase 1 testing in the NANT Consortium and has moved to frontline therapy in the Children’s Oncology Group. The robust and sustained activity observed with minimal toxicity is transient in relapse patients harboring MYCN amplification and ALK activation. We aim to elucidate and target mechanisms of acquired resistance that emerge from lorlatinib treatment. We utilized the Brunello library, a genome-wide CRISPR-CAS9 loss of function screen targeting 19,114 genes (4 sgRNA per gene), which allows for a high confidence of detection of hits. We screened four clinically representative cell lines: Kelly (ALKF1174L, MYCN-amplified, p53P117T), SMS-SAN (ALKF1174L, MYCN-amplified), LAN5 (ALKR1275Q, MYCN-amplified), and SY5Y (ALKF1174L, MYCN-nonamplified). sgRNAs were ranked according to the degree of depletion or enrichment. We validated top depleted targets through cell-based assays and immunoblotting. We identified several actionable targets across multiple NB cell lines that, when depleted, increase the efficacy of lorlatinib, including FGFR2, BCL2L1, SOX11, SMARCA2, and SMARCD2. Genetic knockout of FGFR2 led to a 40% reduction (p<0.05) in KO cells upon lorlatinib treatment compared to control. PROTAC degradation of the anti-apoptotic BCL-xL (BCL2L1) revealed the combination of lorlatinib and the PROTAC led to a decrease in IC50 in vitro. Additionally, we identified conserved core components of the SWI/SNF complex, a subfamily of ATP-dependent chromatin remodelers, as well as SOX11, a regulator of the SWI/SNF complex and transcription factor of the core regulatory circuitry in adrenergic high-risk NB. In conclusion, we identified actionable targets across four NB cell lines that, when depleted, increase the efficacy of lorlatinib. Depletion of FGFR2 led to increased sensitivity to lorlatinib, however enzymatic inhibition showed minimal effect. Future investigation into dual targeting of ALK and FGFR2 through novel alternative methods is warranted. Furthermore, we observed a synergistic relationship between lorlatinib and a BCL-xL PROTAC in vitro. Ongoing in vivo studies will test the combinatorial efficacy in lorlatinib-naïve and resistant models. Finally, we are investigating epigenetic remodeling by the SWI/SNF complex and its regulators in NB cells during lorlatinib treatment through targeted genetic manipulation of SOX11 to understand its role as a master transcriptional regulator of MYCN and FGFR2. Citation Format: Joshua Renn Kalna, Smita Matkar, Emily O'Drisoll, Skye Balyasny, Matteo Calafatti, Mark Gerelus, Dave Groff, Tina Acholla, Colleen E. Casey, Paul Kamitsuka, Grant Li, Steven J. Pastor, Gabriela Witek, Kateryna Krytska, Jarrett Lindsay, Ophir Shalem, Yael P. Mossé. Identifying mechanisms of acquired resistance to lorlatinib utilizing a genome-wide CRISPR-Cas9 screen [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 3357.

Abstract 6909: KRASG12C inhibitor resistance in patient-derived non-small cell lung cancer models

Abstract Introduction: One quarter of lung adenocarcinomas (LUAD) harbor KRAS-mutations, with KRASG12C accounting for the majority (40%). GDP-KRASG12C inhibitors (G12Ci) have been developed for this patient cohort, and two of these are approved for clinical use in the US. While response rates to G12Ci tested in clinical trials have been between 35-50%, studies have identified mechanisms of adaptive resistance to these novel agents, limiting their use as monotherapies. However, primary (intrinsic) resistance to these compounds has not been explored extensively. We leveraged our patient-derived xenograft (PDX) and xenograft-derived organoid (XDO) development programs to study primary resistance to the novel G12Ci, AZD4625. Methods: Twelve KRASG12C PDX models were established which recapitulated patient tumor histology, and genomic, transcriptomic, and methylome profiles. From these 12 PDX, six long-term (passage >10) XDO were generated. PDX were treated with AZD4625 chronically for four weeks as well as in an acute dosing pharmacodynamic study, where tumors were harvested for further analysis. Results: The AZD4625 drug screen in our PDX models reproduced the G12Ci response rate observed in clinical trials. Four of twelve tumor models reduced in size on treatment, while the remainder were considered resistant as these tumors increased in size on treatment. Sensitive tumors became necrotic while those resistant remained proliferative. XDO models recapitulated their originating PDX genomic alterations, histology, as well as responses to AZD4625. During the acute pharmacodynamic screen in our PDX models, we observed a decrease in pERK1/2 and pS6 protein expression in sensitive but not resistant models, despite acute DUSP6 gene expression decreases in every model. Global proteomic analysis highlighted differences between each model. Conclusion: PDX and XDO are useful models to study resistance to this novel class of inhibitor, with the KRASG12C NSCLC PDX models exhibiting a treatment response rate that is similar to those observed in clinical trials. Global proteomics suggests uniqueness of each model in our cohort, hinting that a universal combination of specific targeted agents to treat KRASG12C LUAD tumors may be insufficient. Citation Format: Joshua C. Rosen, Nhu-An Pham, Quan Li, Pinjiang Cao, Katrina Hueniken, Takamasa Koga, Nikolina Radulovich, Alex Koers, Michael Niedbala, Sarah Ross, Adrian Sacher, Ming-Sound Tsao. KRASG12C inhibitor resistance in patient-derived non-small cell lung cancer models [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 6909.

Abstract 2722: CTN001, a first Twin Fc-immune cell engager, with a superior potency against Her2+ and Her2-low cancers by engaging multiple Fc-mediated mechanisms

Abstract Many therapeutic monoclonal antibodies mediate the immune effector functions through the engagement of their fragment crystallizable (Fc) domain and Fcγ receptors on the immune cells. However, these functions are limited due to the low affinity of their interactions and the requirement of abundancy of antigens for the avidity effects. Recently, mutation-based Fc engineered antibodies showed their enhanced immune effector functions mostly through the NK cell-mediated ADCC. To overcome the limited improvement of current Fc engineering, we developed an innovated platform, Twin Fc-Immune Cell Engager (Twin Fc-ICE) composed of a single Fab domain and two Fc domain with drug-like properties. Here we demonstrated the superior efficacy of CTN001, a first Twin Fc-ICE, in in vitro and in vivo Her2+ tumor models. CTN001 bound to various types of Fcγ receptor-expressing immune cells with stronger affinity, supported by its better in vitro affinity to all types of Fcγ receptors than wild type and Fc-engineered antibodies. To evaluate the pharmacological efficacies, CTN001 was tested for its functions of in vitro antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) and in vivo efficacy with Her2+ xenograft models. In Her2+ models, both CTN001 and trastuzumab induced ADCC-mediated effects, but CTN001 showed better anti-tumor cytotoxicity through ADCP and CDC than trastuzumab. Importantly, CTN001 also showed cytotoxic effects on Her2-low tumor cells. Consistently, CTN001 showed better anti-tumor efficacy than trastuzumab or its combination with pertuzumab in Her2+ tumor xenograft model and, surprisingly, even in Her2-low or resistant models. These results suggest that CTN001 can provide new therapeutic options for treating Her2-low patients differentiated from antibody-drug conjugates (ADCs), and that the Twin Fc-ICE can be an innovative platform to overcome limitations of existing therapeutics. Citation Format: Deokjae Lee, Sujin Yoon, Jiseon Bae, Minju Kim, Ki-June Lee, Eunmi Kim, Hyunkyu Park, Eun S. Choi, Gi-Hyeok Yang. CTN001, a first Twin Fc-immune cell engager, with a superior potency against Her2+ and Her2-low cancers by engaging multiple Fc-mediated mechanisms [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 2722.

Abstract 1224: 3D models of chemotherapy- and PARPi-resistant ovarian cancer indicate essential roles for JAK/STAT signaling in mediating drug resistance

Abstract Ovarian cancer is the deadliest gynecological cancer with a low 5-year overall survival rate that affects over 300,000 women globally each year. Given lack of routine screening procedures, ovarian cancer patients are frequently diagnosed with late-stage disease that rapidly progress despite initial responses to front line therapy. PARPi and VEGFi targeted therapies have improved progression free survival in small subsets of patients but have largely failed to improve overall survival. Since the robust uptake of PARPis in the front line setting, resistance to this class of drugs is a growing problem. The literature is filled with many novel treatment approaches, but the vast majority fail to gain traction in pre-clinical models and early phase trials. A contributing factor to these disappointments relates to the prevalent use of 2D monoculture model systems that do not recapitulate the tumor microenvironment. To address this issue, we utilized the Rastrum 3D cell printer to grow ovarian cancer cells and PDX models in bio-functional matrices which provide optimal stiffness and extracellular matrix components specific to the ovarian tumor microenvironment. We found that ovarian cancer cells grown under these conditions exhibit dramatic up-regulation of the JAK/STAT signaling pathway, including autocrine/paracrine signaling factors, which correlated with increased sensitivity to JAK/STAT inhibitors. This affect was lost when STAT1 and STAT3 knockout cell lines were grown in this 3D environment. Ovarian tumors are known to be rich in cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). We incorporated primary CAFs into this 3D culture system and found they could promote ovarian cancer cell growth only in cancer cells with intact JAK/STAT signaling further indicating the essential role of this pathway in mediating tumor progression. Co-culture with M2 macrophages was also found to induce ovarian cancer cell growth, particularly in resistant models. Conversely, co-culture with M1 macrophages inhibited treatment naïve ovarian cancer cell growth but not resistant cell line growth. Single-cell RNAseq of the complex 3D cultures elucidated profound differences in the cross-talk between these cell types that is dependent on cancer cell sensitivity to standard-of-care agents. Finally, the addition of JAK/STAT inhibitors was found to substantially alter this cross-talk between cell types and inhibit cancer cell viability. These data further implicate the importance of JAK/STAT signaling, specifically in clinically relevant 3D models, in supporting ovarian cancer progression and response to chemotherapy and PARPi treatment. Ongoing studies are aimed at confirming these findings in pre-clinical mouse models and to elucidate the most effective JAK/STAT inhibitors in vivo with the goal of informing the design of future clinical trials. Citation Format: Esther Rodman, Xiyin Wang, Hannah Smith, Xiaonan Hou, Scott Kaufmann, John Weroha, John Hawse. 3D models of chemotherapy- and PARPi-resistant ovarian cancer indicate essential roles for JAK/STAT signaling in mediating drug resistance [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 1224.

Abstract 6912: Pretreatment multiomics in conjunction with preclinical trials on patient-derived xenograft models of cholangiocarcinoma can identify chemotherapy response signatures

Abstract Background Cholangiocarcinoma (CCA) is a heterogeneous malignancy. Platinum-based chemotherapy (Gemcitabine and Cisplatin) has been the standard treatment for over a decade and is the base for the current immunotherapy combinations. Response rates are typically between 20-30%. Herein, we present outcomes of preclinical efficacy trials on CCA PDX models in correlation with baseline multiomics profiling of CCA patient-derived xenograft (PDX) models to identify molecular features associated with response. Methods Tumor samples from twenty-eight CCA PDX were used to isolate RNA and protein to be included in multiomics analysis (Phosphoproteomics, Proteomics, and Transcriptomics). A subset of PDX models were treated with various doses of Gemcitabine and Cisplatin. The Median Efficacy Index (MEI) was calculated as the ratio of the difference in median of delta in tumor volume at the last day of the study between the treatment and the control arms divided by the median of delta in tumor volume of the control at the last day of the treatment (positive= growth, negative=inhibition, and 0=no efficacy). A Hierarchical All-Against-All algorithm was utilized to compare multiomics features to the MEI and pathway analysis completed for identified molecular features. Results After conducting preclinical efficacy trials, the five tested CCA PDX models were classified into sensitive, moderately sensitive, and resistant models based on the MEI on each treatment arm. The top up-regulated genes were identified for Gemcitabine response (SRPK2, GLK2, and RIOK2) and Cisplatin response (NTRK1, PRPF4B, and SRPK2). The top up-regulated genes related to Gemcitabine resistance were NTRK1, PIP5K1C, and CSNK2A2, while those related to Cisplatin resistance were SRPK1, PRPF4B, and CSNK2A1. Conclusion Cholangiocarcinoma multiomics signatures can predict therapeutic response to standard-of-care chemotherapy in preclinical models. Novel mechanisms of Gemcitabine and Cisplatin resistance were identified related to NTRK1, SRPK1 and SRPK2. Resistant signatures will be challenged with the appropriate targeted therapies on the resistant CCA PDX models. Citation Format: Amro M. Abdelrahman, Danielle M. Carlson, Erik Jessen, Dong-Gi Mun, Isaac Lynch, Alessandro Fogliati, Stella Konadu Adjei Antwi, Aushinie Abeynayake, Akhilesh Pandey, Mark J. Truty, Gregory J. Gores, Rory L. Smoot. Pretreatment multiomics in conjunction with preclinical trials on patient-derived xenograft models of cholangiocarcinoma can identify chemotherapy response signatures [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 6912.

Abstract 6068: Discovery of BTX-9341, a bifunctional degrader of CDK4 and CDK6 for HR+/HER2- breast cancer

Abstract CDK4/6 inhibitors (CDK4/6i) such as palbociclib, abemaciclib and ribociclib are used to treat HR+/HER2- breast cancer, but patients can develop resistance via many mechanisms, several of which converge on the upregulation of the cyclin D-CDK4/6 signaling node. This has been shown to limit the effectiveness of CDK4/6i in ER+ breast cancer with up to 20% patients exhibiting innate resistance and up to 70% patients developing acquired resistance after 3 years on therapy (Scheidemann, 2021). To address acquired resistance, we sought a degrader approach. We utilized our PRODEGY platform of Cereblon (CRBN) binders to synthesize CRBN mediated CDK4/6 bifunctional degraders and identified BTX-9341 as a development candidate. Breast cancer cell lines treated with BTX-9341 showed up to 85% degradation of CDK4 and CDK6 with DC50s <1nM. CDK4/6 phosphorylates the protein RB which releases the transcription factor E2F, inducing the expression of genes which promote cell cycle progression. We examined RB phosphorylation by in-cell western, E2F target gene expression by qPCR and cell cycle progression by propidium iodide staining followed by flow cytometry. BTX-9341 was potent in all downstream assays, with phospho-RB IC50s <30nM, E2F target gene downregulation and G0/G1 cell cycle arrest at concentrations as low as 10nM. These downstream effects were sustained throughout 72 hours with BTX-9341 treatment but recovered more rapidly with Palbociclib treatment. We used a 2D colony formation assay (CFA) to assess inhibition of proliferation by cell cycle arrest. BTX-9341 potently inhibited cell proliferation with CFA IC50s of 20-50nM while CDK4/6i had CFA IC50s of 50-1000nM. This increased activity was due to CRBN mediated target degradation, as demonstrated by a shift in CFA IC50 values in a CRBN knockout cell line towards the values seen with the inhibitors. In palbociclib-resistant HR+/HER2- cell line models BTX-9341 maintained a low CFA IC50 (<150nM) while CDK4/6i displayed micromolar CFA IC50s. BTX-9341 displays excellent pharmacokinetic properties which allowed for oral dosing in xenograft studies. In several breast cancer xenograft models, BTX-9341 showed dose-dependent tumor growth inhibition, tumor regression at higher dose levels, and was effective with multiple alternate dosing regimens. These results show that BTX-9341 displays excellent single agent activity in vitro and in vivo and that this activity is maintained in CDK4/6i resistant models. This indicates that a degrader approach to targeting this pathway may be more effective than current therapies, and that using this modality in a post CDK4/6i setting may be more effective than switching CDK4/6 inhibitors. Reference: Scheidemann, Erin R, and Ayesha N Shajahan-Haq. “Resistance to CDK4/6 Inhibitors in Estrogen Receptor-Positive Breast Cancer.” International journal of molecular sciences vol. 22,22 12292. 14 Nov. 2021, doi:10.3390/ijms222212292 Citation Format: Hannah Majeski, Akinori Okano, Kirti Chahal, Angela Pasis, Casey Carlson, Arvind Shakya, Qiao Liu, Shenlin Huang, Aparajita Hoskote Chourasia, Leah Fung. Discovery of BTX-9341, a bifunctional degrader of CDK4 and CDK6 for HR+/HER2- breast cancer [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 6068.

Abstract 1420: Mechanism of NPRL2 gene therapy induced antitumor immunity in KRAS/STK11mt aPD1 resistant metastatic NSCLC

Abstract NPRL2/TUSC4 is a tumor suppressor gene whose expression is reduced in many cancers including NSCLC. Restoration of NPRL2 induces cell cycle arrest and apoptosis. We investigated the antitumor immune responses to NPRL2 gene therapy in aPD1 resistant KRAS/STK11mt NSCLC in a humanized mouse model. Humanized mice were generated by transplanting human cord blood-derived CD34 stem cells into NSG mice. Mice harboring > 25% human CD45 cells were considered humanized. Lung metastases were developed in humanized mice by injecting KRAS/STK11mt/aPD1R A549 cells, which were treated with i.v. injection of NPRL2 nanoparticles (DOTAP-NPRL2) with or without pembrolizumab (aPD1). NPRL2 treatment reduced lung metastases, whereas pembrolizumab was ineffective. The antitumor effect was greater in humanized than non-humanized mice suggesting the role of immune cells. The antitumor effect was associated with increased infiltration of human cytotoxic immune cells and decreased numbers of Treg in tumors. NPRL2+ pembrolizumab was not synergistic in this resistant model but was synergistic in KRASwt/aPD1S H1299 tumors. Cytotoxic immune cells in tumors were associated with the antitumor effect. Consistent with A549, NPRL2 showed a significantly strong antitumor effect on other KRASmt/aPD1R syngeneic LLC2 tumors whereas aPD1 was not effective. The antitumor effect of NPRL2 was correlated with HLA-DR+ DC, CD11c DC, TILs and NK cells in TME. The antitumor effect of NPRL2 was abolished upon in-vivo depletion of CD4, CD8 T, and MΦ in the LLC2 tumor model. However, no effect was found on in-vivo depletion of NK cells. Nanostring analysis on lung metastasis resulted in a distinct pattern of human gene expression by NPRL2. T cell functional genes, including IFNγ, CD8b, CD7, TNFSF18, ITGA1, GATA3 and TBX21 were significantly increased. Conversely, the negative regulatory genes, including FOXP3, TGFB1, TGFB2, and IL-10RA were inhibited. NPRL2 also downregulated T cell co-inhibitory molecules, including CTLA4, ICOS, LAG3, PDCD1, CD274, IDO1, PDCD1LG2, CD47, and KLRB1. Stably expressing NPRL2 clones were established, and tumors in humanized mice with these clones exhibited significantly slower growth compared to controls. TME analysis showed an upregulation of human CD45, CD3, CD8 T, HLA-DR+ DC and a downregulation of Tregs, CD3+PD1+T, MDSC, and CD163+ TAM in tumors expressing NPRL2. The stable cells showed a substantial increase in both colony formation inhibition and apoptosis. Stable clones showed heightened sensitivity to carboplatin in colony formation, apoptosis, and PARP cleavage assays. Stable expression of NPRL2 resulted in the downregulation of both AKT-mTOR and MAPK pathways by inhibition of pAKT, pmTOR, pPRAS40, p4E-BP1, pS6, and pERK1/2. Taken together, NPRL2 gene therapy induces antitumor activity on KRAS/STK11mt/aPD1R tumors through DC-mediated antigen presentation and cytotoxic immune cell activation Citation Format: Ismail M. Meraz, Mourad Majidi, Renduo Song, Feng Meng, Lihui Gao, Qi Wang, Jing Wang, Elizabeth Shpall, Jack A. Roth. Mechanism of NPRL2 gene therapy induced antitumor immunity in KRAS/STK11mt aPD1 resistant metastatic NSCLC [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 1420.

Abstract 1934: The PLK1 inhibitor, onvansertib, is active as monotherapy and in combination with cetuximab in RAS wild-type colorectal cancer patient-derived xenografts

Abstract Background: Cetuximab and panitumumab are monoclonal antibodies targeting the epidermal growth factor receptor (EGFR), that provide clinical benefit to metastatic colorectal cancer (mCRC) patients with RAS wild-type (RASWT) tumors. Unfortunately, intrinsic or acquired resistance to these therapies limits their clinical effectiveness, necessitating the development of more efficient therapeutic strategies. Onvansertib is an oral, small molecule, selective inhibitor of the PLK1 kinase, currently in clinical development for KRAS-mutant mCRC. The aim of this study was to assess the anti-tumor activity of onvansertib monotherapy and in combination with cetuximab in RASWT CRC patient-derived xenograft (PDX) models. Methods: Twenty RASWT CRC PDXs were selected and engrafted in nude mice. Once tumors reached 200-350-mm3, mice were treated with vehicle, onvansertib (60mg/kg, QD), cetuximab (20mg/kg, BIW) or the combination for 18 days. PDX models were chosen based on their sensitivity to cetuximab, resulting in a selection of 7 cetuximab-sensitive (CetuxS) and 13 cetuximab-resistant (CetuxR) PDXs, including 7 with intrinsic resistance and 6 with acquired-resistance. Tumor volume change from baseline (TVC) was calculated as 100%x(Vt-Vo)/Vo and tumor growth inhibition (TGI) as 100%x(TVCcontrol-TVCtreated)/TVCcontrol. Tumor regression was defined as TVCD18<0 and tumor stasis 0≤TVCD18<100. Results: Cetuximab sensitivity was confirmed in the selected models, cetuximab induced tumor regression in all the CetuxS PDXs, while no or limited activity was observed in the resistant models (median TGI=29%, IQR 16-61). Onvansertib exhibited potent anti-tumor activity in 17 (85%) PDXs, resulting in tumor regression (n=11) or tumor stasis (n=6). Onvansertib TGI at Day 18 was not significantly different in CetuxS PDXs (median TGI 102, IQR 76-103) compared to CetuxR PDXs (median TGI 108, IQR 74-124), supporting that onvansertib anti-tumor activity is independent of the sensitivity/resistance to cetuximab. The combination of onvansertib and cetuximab induced tumor regression in 18 (90%) PDXs. The combination showed significantly improved efficacy compared to individual therapies in some of the models. Conclusions: Onvansertib monotherapy displayed potent anti-tumor activity in RASWT CRC PDX models, independently of their sensitivity to cetuximab. Additionally, onvansertib combined with cetuximab exhibited either comparable or superior anti-tumor activity than the monotherapies. Collectively, this data supports the clinical development of the PLK1 inhibitor onvansertib for RASWT mCRC. Citation Format: Maya Ridinger, Preeti Kanikarla Marie, Fengqin Gao, Zhensheng Liu, Giulia Maddalena, David Menter, Alexey Sorokin, Tod Smeal, Scott Kopetz. The PLK1 inhibitor, onvansertib, is active as monotherapy and in combination with cetuximab in RAS wild-type colorectal cancer patient-derived xenografts [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 1934.

Abstract 5871: Unveiling mechanisms of CDK4/6 inhibitor resistance in ER+ breast cancer models with acquired resistance

Abstract Introduction: Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors, including palbociclib, ribociclib, and abemaciclib, have revolutionized the treatment of estrogen receptor-positive (ER+) breast cancer. However, the development of resistance to these inhibitors poses a formidable challenge in the clinical management of ER+ breast cancer. This study aims to explore the common resistance mechanisms in three CDK4/6 inhibitor-resistant models induced by chronic exposure to these agents. Methods: We developed models of resistance to palbociclib, ribociclib, and abemaciclib by repeatedly exposing murine xenografts to these CDK4/6 inhibitors. Resistance was confirmed through cell viability assays and in vivo tumorigenesis studies. The tumors were sequenced using NGS analyses, and genes of interest were validated through Western blotting and qPCR. Results: In all resistant models, we identified a common resistant mechanism via NGS analyses, which was characterized by the overexpression of the FAT1, CCNE1, and AR genes. The upregulation of these genes at mRNA and protein levels was confirmed by qPCR analyses and western blotting, respectively. Our data indicate that upregulation of FAT1, CCNE1, and AR genes may play a critical role in mediating resistance to CDK4/6 inhibitors, namely palbociclib, ribociclib, and abemaciclib. Conclusion: Our investigation uncovered a shared mechanism, characterized by the overexpression of FAT1, CCNE1, and AR genes, that underlay CDK4/6 inhibitors-induced drug resistance in ER+ breast cancer. These findings shed light on the shared pathways that drive CDK4/6 inhibitor resistance and provide insights into potential therapeutic targets for overcoming this resistance in breast cancer patients. Understanding these mechanisms is crucial for the development of more effective treatment strategies for CDK4/6 inhibitor-resistant cancers. Citation Format: Bin Li, Gaoxiang Liu, Ting Ni, Wenting Shi, Qingyang Gu. Unveiling mechanisms of CDK4/6 inhibitor resistance in ER+ breast cancer models with acquired resistance [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 5871.

Abstract 2726: A novel trispecific antibody HC010 targeting PD-1/CTLA-4/VEGF for the potential treatment in anti-PD-1 antibody resistant NSCLC patients

Abstract Background and Aim: Immune checkpoint inhibitors (anti-PD-1/CTLA-4 antibody) have been approved and widely used for the first line treatment of non-small cell lung cancer (NSCLC). However, most patients are experiencing resistant to PD-1 blockade. In recent clinical trials, anti-VEGF antibody has shown to improve the clinical outcomes of anti-PD-1 antibodies and overcome resistance to checkpoint blockade. We developed a PD-1/CTLA-4/VEGF trispecific antibody (HC010) by site-specifically fusing anti-PD-1 scFv and anti-CTLA-4 nanobody to the defined site of anti-VEGF antibody. The aim of this study was to evaluate the potential use of HC010 for treatment of anti-PD-1 antibody resistant NSCLC patients. Method: Humanized mice were established in NCG mice by reconstituting human PBMC before inoculating the PDX tissue, which was derived from a NSCLC patient treated with two cycles of an anti-PD-1 antibody sintilimab. When tumors were approx. 170mm3, mice were dosed with HC010 twice weekly for 3 weeks. Pembrolizumab, sintilimab and a Standard of Care (SoC) agent docetaxel were included as reference. For IHC analysis, tumor samples were harvested 24h post final dose and fixed with 10% NFB overnight and subsequently stained with CD31 and CD8 to explore the change in tumor microenvironment after HC010 treatment. For preclinical toxicity assessment, HC010 was intravenously administered once weekly for four weeks in naïve cynomolgus monkeys and immunogenicity/local tolerance/clinical pathology were evaluated during the dosing and recovery phase. Results: HC010 showed a significant tumor inhibition (TGI=81%) when compared with the vehicle control. Pembrolizumab and sintilimab alone exhibited limited anti-tumor effect. Docetaxel only showed a partial response during the dosing phase. By analyzing tumor samples, there was a significant increase in CD8+ positive T cells in HC010 treated tumors in contrast to the vehicle group and pembrolizumab/sintilimab treated groups, suggesting that HC010 can significantly improve CD8+ T cell infiltration. Expression of CD31 as an angiogenic and vasculogenic marker, was significantly reduced in HC010 treated tumors. In the toxicity study, HC010 at selected doses showed no obvious toxicity in cynomolgus monkeys. Conclusion: Our trispecific antibody HC010 targeting PD-1/CTLA-4/VEGF showed a superior anti-tumor effect in a sintilimab resistant PDX animal model when compared with pembrolizumab and sintilimab as well as SoC. By analyzing tumor microenvironment, HC010 overcame acquired resistance by modulating tumor microenvironment such as increase in intra-tumoral cytotoxic T cell infiltration and normalization of blood vessels. Together with preclinical efficacy and safety data, HC010 showed potential benefits in treatment of anti-PD-1 antibody resistant NSCLC patients. Citation Format: Diandong Jiang, Yue Xi, Chen Xu, Xingding Wang, Qiaoshan Yin, Zhaohui Li, Teddy Yang. A novel trispecific antibody HC010 targeting PD-1/CTLA-4/VEGF for the potential treatment in anti-PD-1 antibody resistant NSCLC patients [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 2726.

Abstract 2621: Preclinical Efficacy of DM002, a bispecific HER3 × MUC1 antibody-drug conjugate with a novel DNA topoisomerase I inhibitor, in solid tumor models

Abstract MUC1 is a known tumor-associated antigen (TAA), but previous anti-MUC1 agents have shown limited clinical efficacy due to neutralization by the soluble N-terminal autoproteolytic product of MUC1. More recently, HER3-targeted ADCs, such as Patritumab deruxtecan, have shown encouraging early clinical activity in a variety of tumors. However, the majority of patients did not exhibit response to paritumab deruxtecan treatment. Therefore, improved therapies targeting these TAAs are needed. As HER3 and MUC1 are co-expressed in a wide range of cancer types, we hypothesized that simultaneous targeting of HER3 and MUC1 with a bispecific antibody-drug conjugate (bsADC) could potentially lead to increased efficacy, reduced resistance and improved safety in the clinical setting. We generated an anti-HER3 × MUC1 bispecific antibody using the fully human common light chain antibody transgenic mice (RenLite®). The HER3 × MUC1 bsAb showed improved cell binding and internalization in vitro compared to the parental mAb, suggesting synergy between the two arms. We then conjugated the HER3xMUC1 bsAb with vcMMAE for proof-of-concept studies, or with our novel DNA topoisomerase I inhibitor linker/payload conjugates (BLD1102) to make DM002-BLD1102 bsADC. Both bsADCs potently inhibited the growth of HER3+MUC1+ PDX tumors. DM002-vcMMAE also showed greater in vivo efficacy than its parental mAb ADCs, consistent with their in vitro internalization activity. DM002-BLD1102 also showed strong anti-tumor activity in DM002-vcMMAE resistant PDX models, suggesting the superiority of this new DNA topoisomerase I inhibitor linker/payload over the classical vcMMAE linker/payload. In conclusion, the HER3 × MUC1 bsAb is a promising agent for the treatment of HER3 and MUC1 positive tumors. Citation Format: Yifu Zhang, Chengzhang Shang, Nannan Wang, Gao An, Chaoshe Guo, W. Frank An, Yi Yang. Preclinical Efficacy of DM002, a bispecific HER3 × MUC1 antibody-drug conjugate with a novel DNA topoisomerase I inhibitor, in solid tumor models [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 2621.

Abstract 1083: Cell membrane-anchored and tumor-targeted IL12 T-cell therapy destroys cancer-associated fibroblasts and disrupts extracellular matrix in heterogenous osteosarcoma xenograft models

Abstract Background: The extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs) play major roles in tumor progression, metastasis, and the poor response of many solid tumors to immunotherapy. CAF-targeted CAR-T cell therapy cannot infiltrate ECM-rich tumors such as osteosarcoma. Method: In this study, we used RNA sequencing to assess whether the recently invented membrane-anchored and tumor-targeted IL12-armed (attIL12) T cells, which bind cell-surface vimentin (CSV) on tumor cells, could destroy CAFs to disrupt the ECM. We established an in vitro model of the interaction between osteosarcoma CAFs and attIL12-T cells to uncover the underlying mechanism by which attIL12-T cells penetrate stroma-enriched osteosarcoma tumors. Results: RNA sequencing demonstrated that attIL12-T cell treatment altered ECM-related gene expression. Immunohistochemistry staining revealed disruption or elimination of high-density CAFs and ECM in osteosarcoma xenograft tumors following attIL12-T cell treatment, and CAF/ECM density was inversely correlated with T-cell infiltration. Other IL12-armed T cells, such as wild-type IL12- or tumor-targeted IL12-T cells, did not disrupt the ECM because this effect depended on the engagement between CSV on the tumor cell and its ligand on the attIL12-T cells. Mechanistic studies found that attIL12-T cell treatment elevated IFNγ production upon interacting with CSV+ tumor cells, suppressing TGFβ secretion and in turn upregulating FAS-mediated CAF apoptosis. CAF destruction reshaped the tumor stroma to favor T-cell infiltration and tumor inhibition. Conclusions: This study unveiled a novel therapy—attIL12-T cells—for targeting CAFs/ECM. These findings are highly relevant to humans because CAFs are abundant in human osteosarcoma. What is already known on this topic: As the major source of ECM factors in tumors, the FAP+αSMA+ subtype of CAFs within tumors induce production of ECM factors, preventing antitumor T cells from accessing the core regions of tumors. What this study adds: Our group demonstrated that attIL12-T cell transfer (US Patent: 11421010) induced T-cell infiltration into and eliminated large osteosarcoma PDX tumors. Comparison of gene expression in sensitive or resistant PDX models to attIL12-T cell therapy elucidated the ECM-targeting mechanism of attIL12-T cells. Our in vitro system further determined that the CSV-attIL12 interaction mediated IFNγ upregulation, which triggered FAS induction in CAFs to induce CAF apoptosis. In sum, attIL12-T cell treatment shifts tumor stroma from a TGFβ-dependent CAF-promoting phenotype to an IFNγ-induced immune-inflamed phenotype. How this study might affect research, practice or policy: attIL12-T cell transfer is a novel anti-ECM strategy that renders ECM-rich tumors more susceptible to T cell-mediated tumor killing. Citation Format: Jiemiao Hu, Alexander Lazar, Wei-Lien Wang, Wendong Zhang, Zhiliang Jia, Dristhi Ragoonanan, Jian Wang, Xueqing Xia, Kris Mahadeo, Richard Gorlick, Shulin Li. Cell membrane-anchored and tumor-targeted IL12 T-cell therapy destroys cancer-associated fibroblasts and disrupts extracellular matrix in heterogenous osteosarcoma xenograft models [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 1083.

Abstract 4993: CJRB-101 induces immune responses through the GUT-TME axis in immune cell-driven mechanism in lung cancer models

Abstract Background Live biotherapeutic products (LBPs) are known to enhance immune responses through the GUT-TME axis. Here, we investigate the GUT-TME-related immune cell profiling and signals associated with the anti-cancer effects of CJRB-101. Methods Tumors from NSCLC patients (anti-PD-1 refractory) were transplanted in Hu-CD34-NSG to establish humanized patient-derived xenograft (PDX) models. CJRB-101 was administered at 1x109 CFU (p.o., BID) or combination with anti-PD-1 (10 mpk, i.p., BIW). TME was analyzed using multiplex IHC, flow cytometry and scRNA sequencing. Samples were collected from C3PQ syngeneic mice at multiple timepoints for GUT-TME immune cell profiling. For depletion assay, immune cells were individually depleted during the combination treatment. TLR4-mediated mechanism was evaluated using ex vivo and in vivo assay treated with CJRB-101 or cell membrane of CJRB-101. Results CJRB-101 combined with pembrolizumab effectively suppressed tumor growth in anti-PD-1 resistant PDX models. Further analysis revealed a correlation between the activity of NK cells and angiogenesis inhibition. Abundance of NK/NKT was higher in the CJRB-101 treated group compared to the vehicle group in multiple PDX models. The expression of GZMB and IFNG in NK/NKT cells was significantly higher in the CJRB-101 treated group compared to the vehicle group in YHIM2014 (p<0.001). CJRB-101 treated group showed significantly higher expression of antiangiogenic IL1B (p<0.001) while the expression of pro-angiogenic VEGFA (p=0.002) was lower compared to the vehicle group in YHIM2014 cancer cells. Macrophages in the intestine were increased at Day 3 in the CJRB-101 group compared to anti-PD1, while NK cells, granulocytes, CD3+, CD4+, CD8+ T cells increased at Day 10 in the syngeneic model. Depletion assay confirmed that macrophages, CD8+ T cells and neutrophils were pivotal in the anti-cancer effects of CJRB-101. We observed that TAK242 and MD2 reduced the IL-6 secretion of Raw264.7 in a dose-dependent manner. The cell membrane played a key role in increasing BMDM M1 polarization and repolarization of M2 to M1, and that inhibition of TLR4 resulted in a decrease in BMDM repolarization. TLR inhibition also demonstrated that TGI decreased from 34% to 20% when treated with cell membrane + TAK242 compared to cell membrane monotherapy. Conclusions This study showed that macrophages were the dominant immune population in the early stages then T cells (CD3, CD4, CD8), NK cells and granulocytes became more active in the latter stages of the GUT-TME-axis immune response. In vivo results indicated that anti-cancer efficacy of CJRB-101 is immune-cell driven by TLR4-dependent stimulation of key immune cell populations (macrophages, CD8+ T cells, neutrophils) modulated by the cell membrane of CJRB-101. CJRB-101 is currently undergoing clinical investigation for treatment of patients with advanced NSCLC. Citation Format: Arim Min, Bo-eun Kwon, Seong-san Kang, Sujeong Baek, Junwon Yang, Hyunkyung Park, Jieun Im, Hyunjeong Kim, Jaemin Kim, Jieun Kwon, Dong Kwon Kim, Jii Bum Lee, Hyeonseok Oh, Seung Min Yang, Yu Jin Han, Mi hyun Kim, Heekyung Han, Kwangmin Na, Young Taek Kim, Mi Ran Yun, Jae Hwan Kim, Youngseon Byeon, Young Seob Kim, Min Hee Hong, Sun Min Lim, Kyoung-Ho Pyo, Byoung Chul Cho. CJRB-101 induces immune responses through the GUT-TME axis in immune cell-driven mechanism in lung cancer models [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 4993.