Cleavable Linker Research Articles

Abstract LB057: Pre-clinical characterization of IBI343, a site-specifically conjugated anti-Claundin18.2 ADC, for treating solid tumors

Abstract Claudin18.2 is important for the formation and function of tight junctions between cells in epithelial tissues. Normally expressed only in gastric mucosa, Claudin18.2 is overexpressed in multiple types of solid tumors, including gastric, pancreatic, breast and colon cancers. Despite being a well-established tumor-associated antigen, the clinical value and feasibility of targeting Claudin18.2 by various modalities are yet-to-be fully demonstrated. IBI343 is a fully humanized anti-Claudin18.2 monoclonal antibody conjugated to Exatecan via site-specific glycol-conjugation with an average drug-to-antibody ratio (DAR) of 3.6. IBI343 showed robust antigen-specific in vitro cytotoxicity in cancer cell lines with varying levels of Claudin18.2 expression. In addition, due to the cleavable linker design and high hydrophilicity of the payload, IBI343 demonstrated potent bystander killing effects in vitro. The glycan-based conjugation technology leads to enhanced stability of the conjugated linker-payload and the entire ADC molecule, which results in superior pharmacokinetics. IBI343 showed potent in vivo anti-tumor efficacy with complete tumor regressions in multiple xenograft models. Furthermore, it demonstrated strong in vitro and in vivo synergistic effects with various anti-cancer agents, including immune checkpoint blockers and DNA damage repair pathway inhibitors. IBI343 displayed good safety profile in monkey GLP toxicology study (HNSTD = 30 mg/kg). These pre-clinical findings show that IBI343 is a promising anti-cancer agent with large therapeutic window, and support clinical exploration of IBI343 in patients with Claudin18.2-expressing tumors. A companion late-breaking abstract of clinical phase 1 study results (NCT05458219) will also be presented in AACR 2024. Citation Format: Shuaixiang Zhou, Xiong Yao, Jian Guan, Keke Fei, Jia Lu, Weiwei Wu, Yang Liu, Tianyu Zhu, Zhuangguang Liao, Shi Chen, Bingliang Chen, Kaijie He. Pre-clinical characterization of IBI343, a site-specifically conjugated anti-Claundin18.2 ADC, for treating solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB057.

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

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

Abstract CT168: A phase 1/2a, multicenter, open-label, non-randomized first-in-human study to assess the safety, tolerability, pharmacokinetics, and preliminary antitumor activity of DB-1310 (a HER3-targeting ADC) in patients with advanced/metastatic solid tumors

Abstract Background: Overexpression of epidermal growth factor receptor 3 (HER3 or ERBB3) has been reported in various cancers, including lung, prostate, head and neck, breast, and ovarian cancers, and is associated with disease progression and poor prognosis. DB-1310 is an antibody-drug conjugate (ADC) comprised of a humanized anti-HER3 IgG1 monoclonal antibody, covalently linked to a proprietary DNA topoisomerase I inhibitor (P1021) via a maleimide tetrapeptide-based cleavable linker, with a high drug-to-antibody ratio (~8). Preclinical studies of DB-1310 demonstrated promising antitumor activity in solid tumors and a tolerable safety profile, warranting further clinical development. Methods: This global, first-in-human, Phase 1/2a study includes dose escalation and expansion parts to assess the safety, tolerability, pharmacokinetics, and antitumor activity of DB-1310 in patients (pts) with pretreated advanced/metastatic solid tumors (NCT05785741). Eligible pts must have progressed on or after standard systemic anticancer treatments have ECOG PS ≤1, and evidence adequate organ function. Phase 1 part will evaluate 5 ascending dose levels of DB-1310 with a standard “3+3” design to identify the maximum tolerated dose (MTD) and/or recommended phase 2 dose (RP2D) in pts with advanced/metastatic solid tumors. Phase 2a part will initiate after MTD and/or RP2D are determined and enroll pts with non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) activating mutation, NSCLC without EGFR activating mutation, castration-resistant prostate cancer, head and neck squamous cell carcinoma, and HER2-positive (immunohistochemistry [IHC] 3+, or IHC 2+ and in situ hybridization-positive) breast cancer (BC). DB-1310 is administrated intravenously every 3 weeks as monotherapy, or plus trastuzumab in pts with HER2-positive BC only, until discontinuation criteria are met. The study plans to enroll approximately 95 pts in Phase 1 and 192 in Phase 2a from the United States and China. Citation Format: Aaron Lisberg, Shun Lu, Alexander Starodub, Harshad Amin, Julia Rotow, Lin Wu, Alexander Spira, Erika Hamilton, Weidi Shen, Liming Liu, Zhongyuan Zhu, Wei Gu, Yang Qiu, Rong Shi, Shengxue Liu. A phase 1/2a, multicenter, open-label, non-randomized first-in-human study to assess the safety, tolerability, pharmacokinetics, and preliminary antitumor activity of DB-1310 (a HER3-targeting ADC) in patients with advanced/metastatic solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT168.

Abstract CT291: A phase 1/2, first-in-human study of DS-3939a in patients with advanced solid tumors: A new DXd ADC targeting TA-MUC1

Abstract Background: Mucin 1 (MUC1), a single-transmembrane glycoprotein, is expressed on the apical membrane of the epithelial surface. In normal tissues, it is highly glycosylated and protects the underlying epithelia. In cancer cells, however, MUC1 is hypoglycosylated due to changes in the expression patterns of some sialyltransferases, and exposes new epitopes on MUC1 in tumors, referred to as tumor-associated MUC1 (TA-MUC1). TA-MUC1 loses cell polarity and is redistributed over the cell surface and within the cytoplasm. Overexpression of TA-MUC1 in several malignancies is associated with poor prognosis and development of metastasis, thus making it an attractive therapeutic target. DS-3939a is a novel antibody-drug conjugate in development for the treatment of malignant tumors composed of a humanized anti-TA-MUC1 antibody, a peptide-based cleavable linker, and a potent topoisomerase I inhibitor (deruxtecan [DXd]). This study will assess the safety, tolerability, and efficacy of DS-3939a in patients with advanced solid tumors. Trial design: This phase 1/2, first-in-human, open-label, multicenter, 2-part, dose-escalation, and dose-expansion study (NCT05875168) is active and plans to enroll a maximum of 430 adult patients. Part 1 (dose-escalation) is accruing patients with locally advanced, metastatic, or unresectable urothelial, non-small cell lung, breast, ovarian, biliary tract, or pancreatic cancers, and Part 2 (dose-expansion) will enroll patients with various advanced solid tumors. In Parts 1 and 2, DS-3939a will be administered intravenously on Day 1 of a 21-day cycle. The primary endpoints in Parts 1 and 2 include safety and tolerability of DS3939a as assessed by the number of patients with dose-limiting toxicities (Part 1 only), treatment-emergent adverse events, and other safety parameters. Part 2 will also evaluate efficacy by objective tumor response rate per RECIST version 1.1 as a primary endpoint. Secondary endpoints include the disease control rate, duration of response, time to response, progression-free survival, overall survival, TA-MUC1 expression (detected by immunohistochemistry), the pharmacokinetic profile of DS-3939a, and the number of patients with anti-drug antibodies against DS-3939a. Citation Format: Noboru Yamamoto, Toshihiko Doi, Manish R. Patel, Ignacio Garrido-Laguna, Sutun Wu, Satoshi Nishioka, Keiko Nakajima, Benedito A. Carneiro. A phase 1/2, first-in-human study of DS-3939a in patients with advanced solid tumors: A new DXd ADC targeting TA-MUC1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT291.

Anti-CD45 PBD-based antibody-drug conjugates are effective targeted conditioning agents for gene therapy and stem cell transplant

Stem cell gene therapy and hematopoietic stem cell transplantation (SCT) require conditioning to ablate the recipient's hematopoietic stem cells (HSCs) and create a niche for gene-corrected/donor HSCs. Conventional conditioning agents are non-specific, leading to off-target toxicities and resulting in significant morbidity and mortality. We developed tissue-specific anti-human CD45 antibody-drug conjugates (ADCs), using rat IgG2b anti-human CD45 antibody clones YTH24.5 and YTH54.12, conjugated to cytotoxic pyrrolobenzodiazepine (PBD) dimer payloads with cleavable (SG3249) or non-cleavable (SG3376) linkers. Invitro, these ADCs internalized to lysosomes for drug release, resulting in potent and specific killing of human CD45+ cells. In humanized NSG mice, the ADCs completely ablated human HSCs without toxicity to non-hematopoietic tissues, enabling successful engraftment of gene-modified autologous and allogeneic human HSCs. The ADCs also delayed leukemia onset and improved survival in CD45+ tumor models. These data provide proof of concept that conditioning with anti-human CD45-PBD ADCs allows engraftment of donor/gene-corrected HSCs with minimal toxicity to non-hematopoietic tissues. Our anti-CD45-PBDs or similar agents could potentially shift the paradigm in transplantation medicine that intensive chemo/radiotherapy is required for HSC engraftment after gene therapy and allogeneic SCT. Targeted conditioning both improve the safety and minimize late effects of these procedures, which would greatly increase their applicability.

Abstract 3134: SDP03923-000-9106, a novel and optimized LIV-1 ADC with superior anti-tumor efficacy in breast cancer xenograft models

Abstract Background: LIV-1, also known as SLC39A6 or ZIP6, is a transmembrane protein belonging to the zinc transporter family. LIV-1 is a promising target for antibody-drug conjugate (ADC) therapy due to its broad expression in tumors and limited normal tissue expression. Ladiratuzumab vedotin, a LIV-1 targeting ADC with payload of MMAE, is now in phase I clinical trial stage and is the only LIV-1 ADC under clinical development worldwide. The preliminary clinical results demonstrated promising efficacy in TNBC (ORR=32%) but no response in HR+/HER2- breast cancer (ORR=0%), and typical MMAE-related adverse events were observed. Herein, we presented a novel LIV-1 directed ADC, SDP03923-000-9106, consisting of a LIV-1 mAb developed in house conjugated to a proprietary topoisomerase I inhibitor via a cleavable linker with an optimized average drug-to-antibody ratio (DAR) of 6. Method: The in vitro binding affinity of SDP03923-000-9106 against LIV-1 protein was measured by Surface Plasmon Resonance (SPR) assays. The expression levels of LIV-1 in different cancer cell lines were detected by flow cytometry and IHC staining. In vitro activity against cancer cell lines was analyzed by CellTiter Glo assays following 10 days of treatment. The in vivo anti-tumor activities were investigated in both CDX and PDX models of breast cancer. Results: SDP03923-000-9106 is a novel anti-LIV-1 ADC comprised of a humanized LIV-1 monoclonal antibody conjugated to the potent topoisomerase I inhibitor (a novel exatecan derivative designed by Hengrui with a better liposolubility and cellular permeability) by a cleavable linker. SDP03923-000-9106 had an optimal drug-to-antibody ratio (DAR) of 6 and showed strong binding affinity to LIV-1 and favorable internalization. SDP03923-000-9106 exhibited robust anti-tumor activity in cell lines across breast cancer, esophageal squamous cancer, gastric cancer, prostate cancer and non-small cell lung cancer. In the LIV-1 highly expressed breast cancer cell line of MCF-7, SDP03923-000-9106 demonstrated a strong inhibitory effect with IC50 of 5.36 nM. Moreover, in a LIV-1 high-expressing patient-derived xenograft model of breast cancer, SDP03923-000-9106 showed pronounced tumor regression at both doses of 3 and 10 mg/kg and showed stronger anti-tumor activity (TGI: 109%) at the same dose of 3 mg/kg versus BMK-MMAE (TGI: 75%; BMK-MMAE is synthesized by Hengrui using the published structure of Ladiratuzumab vedotin). Conclusion: In summary, SDP03923-000-9106 is a novel LIV-1 targeted ADC with a highly permeable payload demonstrating great anti-tumor activity across multiple types of tumors in preclinical studies. The promising preclinical data support advancing SDP03923-000-9106 into clinical testing, and Investigational New Drug (IND) application to NMPA has been submitted. Citation Format: Yanling Gong, Xing Sun, Changyong Yang, Simeng Chen, Wei Zhang, Cheng Liao. SDP03923-000-9106, a novel and optimized LIV-1 ADC with superior anti-tumor efficacy in breast 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 3134.

Abstract 4700: Induced phenotype targeted ADC by using caspase enzymatic cleavable peptide linker to overcome tumor heterogeneity

Abstract Antibody-drug conjugates (ADCs) have ushered in a new era of precision cancer therapy, and the caspase-3-cleavable linker has demonstrated its potential as a game-changing addition to the ADC toolkit. The DEVD peptide, specifically cleaved by caspase and lysosomal enzymes, in combination with the KG peptide, provides the necessary flexibility to achieve targeted cancer cell apoptosis. This research explores the key attributes and actions of the linker, aiming to highlight its potential for revolutionizing ADC therapy. The caspase-3-cleavable linker operates through caspase-3 activation during apoptosis. After targeting cancer cells with specific antigens, the ADC is internalized into the cells, and the linker is cleaved by caspase-3, releasing the drug and initiating apoptosis. The specificity of caspase-3 extends the drug’s effect to neighboring cells, even those lacking the target antigen. This cascade effect ensures the continuous activation of ADCs until all cancer cells are eradicated. Firstly, it overcomes tumor heterogeneity by activating the drug outside of the cell. Traditional bystander killing effects rely on internalization followed by drug release and penetration of neighbor cancer cells. However, this linker's unique mechanism ensures that the drug is activated by caspase-3 outside the cell without the need for internalization, effectively targeting all cancer cells within the tumor tissue. This is achieved through the induced phenotype targeting effect (IPTE), which involves caspase-3 cleavage of the linker, inducing a phenotype in the tumor tissue that targets all cancer cells, regardless of their specific markers. Secondly, it maintains therapeutic efficacy even as the target antigen decreases. Current targeted therapies face a challenge as target antigens may diminish during treatment, leading to reduced drug effectiveness. However, the unique mechanism of the linker ensures that the drug continues to be activated as long as there is caspase-3 secretion, resulting in continuous activation of ADCs in the tumor, with a feedback amplification loop that ensures therapeutic efficacy until all cancer cells are eliminated. Lastly, the linker's superior physicochemical properties allow for a maximum drug-to-antibody ratio (DAR). Its hydrophilic nature enables independent drug conjugation, increasing therapeutic potential and mitigating aggregation issues. This innovation addresses the current limitations in ADC conjugation technologies. In summary, the caspase-3-cleavable linker represents a significant leap forward in the field of ADCs. Its unique attributes offer promising prospects for more effective and targeted cancer treatments. This linker is poised to revolutionize the landscape of targeted cancer therapy, bringing us one step closer to the dream of eradicating cancer cells with precision and efficiency. Citation Format: Ha Kyeong Lee, Byoungmo Kim, Hyo Won Chang, Sang Yoon Kim, Youngro Byun. Induced phenotype targeted ADC by using caspase enzymatic cleavable peptide linker to overcome tumor heterogeneity [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 4700.

Abstract 5907: Pre-clinical evaluation of a novel antibody drug conjugate (ADC) LM-317 targeting NaPi2b

Abstract Introduction: NaPi2b is a multi-transmembrane type II sodium-dependent phosphate transporters that regulates phosphate homeostasis in normal physiological condition [1]. It is highly expressed in ovarian cancer (OC), non-small-cell lung cancer (NSCLC), and papillary thyroid cancer, with limited expression in normal tissue, making it a promising target for antibody-drug conjugates (ADCs) [2,3]. LM-317 is a novel NaPi2b targeted humanized monoclonal antibody LM-117 conjugated to the next-generation topoisomerase I inhibitor LDX2 via a cleavable linker, with antibody-drug ratio of 8. Here, we present the preclinical in vivo and in vitro results of LM-317. Results: LM-317 is highly specific cross-reactive with rhesus monkeys, cynomolgus monkeys, mouse, and rat NaPi2b. Concentration-dependent cell binding and internalization effects of LM-317 were observed in human NaPi2b-expressing cells, and OC and NSCLC cells expressing NaPi2b, with EC-50 of 0.92 - 9.7 nM and 1.51 - 4.76 nM, respectively. The cytotoxicity effect of LM-317 in NaPi2b -positive tumor cells was superior to LM-117-DXD, with IC-50 of 0.002 - 0.52nM. The in vivo studies showed that 1 mg/kg of LM-317 almost completely eradicated tumors in the OVCAR-3 xenograft mice model of OC, and similar antitumor activity was observed in the NCI-H175-xenograft mice model of NSCLC at the dose of 3 mg/kg. Single dose of 6 mg/kg of LM-317 showed sustained tumor eradication in the NSCLC xenograft -mouse model. LM-317 also exhibited potent tumor regression in the PDX model of OC and NSCLC. The tumor growth inhibition (TGI) value was 80%-100% in OC and NSCLC PDX models with different expression degree of NaPi2b after treatment with LM-317. The antitumor activity of LM-317 in cell line- and PDX models of OC and NSLCL was comparable to those of LM-117-DXD. Conclusion: The novel ADC LM-317 targeting NaPi2b showed robust antitumor activity in vitro, and in the OC and NSCLC cell line derived models and PDX models. LM-317 could potentially provide therapeutic benefit to treat NaPi2b-positive tumors including OC and NSCLC in the future. Reference: 1.S. Banerjee et al. Cancer Treatment Reviews 112 (2023) 1024892.Finstad CL, Lloyd KO, Federici MG, et al. Clin. Cancer Res. 1997;3(8):1433-14423.Zhang et al. Tumor Biology July 2017: 1-7. Citation Format: Wentao Huang, Zhifang Liu, Yifan Li, Heng Pan, Xia Qin, Da Fei, Runsheng Li. Pre-clinical evaluation of a novel antibody drug conjugate (ADC) LM-317 targeting NaPi2b [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 5907.

Abstract 3195: SHR-4602, next generation of HER2-targeting ADC with potent anti-tumor efficacy overcoming the resistance of HER2 ADC with Topo I inhibitor payload in xenograft models

Abstract Background: Current clinical HER2-targeting ADCs (i.e. DS-8201) have shown promising clinical efficacy in HER2 expressing or mutant cancer patients. However, most of them will ultimately experience disease progression due to different mechanisms. Herein, we presented a novel HER2 ADC, SHR-4602, which demonstrated good in vitro and in vivo anti-tumor activities and could overcome the resistance caused by HER2 ADC including DS-8201 analogue (synthesized using DS-8201 structure) and SHR-A1811 (composed of trastuzumab, a cleavable linker, and a Topo I inhibitor payload, in phase III clinical trials) in xenograft models. Method: We used a stable enzyme-cleavable linker technology to conjugate the HER2 ADC using the amino acid sequence of pertuzumab and ER300 (eribulin derivative). The in vitro biological and anti-tumor activities were assessed by a series of assays. The in vivo anti-tumor efficacies were evaluated in the JIMT-1 (HER2 low expressing) as well as the SHR-A1811 and DS-8201 analogue induced JIMT-1 resistance (caused by treating with SHR-A1811 followed by DS-8201 analogue for a long-term) xenograft models in BALB/c nude mice. Results: Through the SPR assay, SHR-4602 exerted single nanomolar binding affinity to human and rhesus monkey HER2, but no binding activity against rat HER2. It demonstrated varying inhibitory activity on the proliferation of cancer cell lines with various HER2 expression levels, with the IC50 at the range of 0.007 nM to 8.21 nM. SHR-4602 showed highly efficient internalization in NCI-N87 HER2 expressing cells and could be translocated to the lysosome followed by releasing ER300. The cytotoxic activity of SHR-4602 was predominantly mediated by released ER300 through microtubule dynamics inhibition, G2/M arrest and apoptosis induction. Furthermore, SHR-4602 showed a strong in vitro bystander effect in the co-culture of HER2 positive/negative cells. SHR-4602 showed significant anti-tumor efficacy in JIMT-1, SHR-A1811 and DS-8201 analogue both-resistant JIMT-1 xenograft models. In SHR-A1811 and DS-8201 analogue both-resistant xenograft models, treatment with 10 mg/kg SHR-A1811 or DS-8201 analogue exerted negligible anti-tumor activity (TGI% < 17%), whereas SHR-4602 at 2.5, 5 and 10 mg/kg significantly inhibited the tumor growth (p < 0.001, vs. vehicle), with TGI% of 84.96%, 101.64% and 113.11% on Day 42, respectively, which indicated a superior anti-tumor efficacy against SHR-A1811 and DS-8201 analogue both-resistant xenograft models. Conclusion: SHR-4602 represents a new generation HER2-targeting ADC to deliver a potent cytotoxin to HER2 expressing tumor cells and may be more efficacious to the tumors in which current anti-HER2 therapies are ineffective. SHR-4602 is now in the phase I clinical development stage for advanced malignant solid tumors (NCT05819684). Citation Format: Yunan Tian, Xing Sun, Changyong Yang, Cheng Liao. SHR-4602, next generation of HER2-targeting ADC with potent anti-tumor efficacy overcoming the resistance of HER2 ADC with Topo I inhibitor payload in 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 3195.

Abstract 3123: Preclinical characterization of AMB302/GQ1011, a first-in-class FGFR3 antibody-drug conjugate (ADC) with topoisomerase 1 inhibitor against diverse FGFR3 overexpression or alteration

Abstract Background: Molecular alterations of oncogene FGFR3 in various cancers include FGFR3 gene fusions, activating mutations, and overexpression as a driver mutation. AMB302/GQ1011 is a novel FGFR3-targeting ADC conjugated with an innovative topoisomerase I inhibitor (TopoIx) using intelligent Ligase-Dependent Conjugation (iLDC) technologies developed by GeneQuantum (GQ), providing high homogeneity, excellent druggability, and superior linker stability. Based on preclinical characterization, AMB302/GQ1011 exhibits impressive anti-tumor activities against bladder cancer (BC), head and neck squamous cell carcinoma (HNSCC), and glioblastoma (GBM) with either FGFR3 overexpression or FGFR3 alterations including gene fusions, demonstrating potential as a first-in-class FGFR3 ADC against FGFR3 relevant solid tumor indications. Methods: For biomarker analysis, patient tissue microarrays and tissue blocks were subjected to IHC staining for FGFR3 to assess its real-world intensity. To characterize AMB302/GQ1011, in vitro assays and ex vivo plasma stability were evaluated to confirm the anti-tumor effect and linker stability. In vivo anti-tumor effects of AMB302/GQ1011 were assessed on various CDX and PDX models with FGFR3 overexpression or alterations. The potential toxicity profile of AMB302/GQ1011 was evaluated in NHP via repeated intravenous infusions. Results: AMB302/GQ1011 conjugates a novel FGFR3-targeting antibody (Aimedbio Inc.) with TopoIx (GQ) via a cleavable linker through iLDC platform. In IHC analysis, we detected high and widespread FGFR3 expression in various tumors. In in vitro cytotoxicity assay, AMB302/GQ1011 showed excellent antitumor activity against FGFR3-expressing cells. In various CDX and PDX models with FGFR3 expression or alterations, AMB302/GQ1011 demonstrated superior efficacy compared with benchmark ADCs and FGFR pan-inhibitors. AMB302/GQ1011 dramatically prolonged survival in GBM orthotopic PDX models with F3-T3 fusion by >150% in combination with TMZ. Particularly, in combination with an immune checkpoint inhibitor (ICI), AMB302/GQ1011 exhibited an excellent synergistic antitumor effect. In a monkey toxicity study, AMB302/GQ1011 demonstrated a high tolerability up to 60 mg/kg dosing, without severe adverse effects. Conclusion: AMB302/GQ1011 demonstrated a potent antitumor efficacy in vitro and in vivo models with FGFR3 overexpression and alterations across various solid tumors. Notably, AMB302/GQ1011 suggests the potential for combination with ICI treatment. Moreover, AMB302/GQ1011 exhibited excellent tolerability at high doses in NHP, indicating its safety profile. Our data suggest that AMB302/GQ1011 has potential to become a first-in-class FGFR3-targeting ADC for solid tumors with FGFR3 overexpression or alterations. Citation Format: Byeongkwi Min, Sunghyun Lee, Wonsik Jung, Lina Wang, Yajun Sun, Hyejin Kim, Nam-Gu Her, Paul H. Song, Gang Qin, Do-Hyun Nam. Preclinical characterization of AMB302/GQ1011, a first-in-class FGFR3 antibody-drug conjugate (ADC) with topoisomerase 1 inhibitor against diverse FGFR3 overexpression or alteration [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 3123.

Abstract 1862: ZW191 - a FRα-targeting antibody drug conjugate with strong preclinical activity across multiple FRα-expressing indications

Abstract Background: Folate Receptor alpha (FRα) is a validated cancer target that is prevalently expressed in multiple cancers with high unmet need, including ovarian cancer and other gynecological cancers, NSCLC, endometrial cancer and TNBC. Due to FRα’s favorable expression profile, multiple antibody-drug conjugates (ADCs) are being explored in this setting. Here we present the preclinical characterization of a new anti-FRα ADC, ZW191. ZW191 is a bystander active antibody drug conjugate (ADC) comprised of a humanized IgG1 antibody conjugated to a novel camptothecin-based topoisomerase 1 inhibitor (TOPO1i) payload, ZD06519, via a maleimidocaproyl (MC) anchor and a glycyl glycyl phenylalanyl glycyl (GGFG)-aminomethyl (AM) cleavable linker at a drug-to-antibody ratio (DAR) of 8. ZW191 is projected to undergo IND submission in 2024. Materials and Methods: The novel antibody and drug-linker components of ZW191 were generated, characterized, and optimally integrated. The apparent binding affinity and cellular internalization of the ZW191 antibody were determined in FRα-expressing cells. Tumor spheroid cancer cell cultures were utilized to determine ZW191’s degree of tissue penetration and its cytotoxicity. The bystander activity of ZW191 was assessed using antigen positive and negative co-culture experiments. The anti-tumor activity of ZW191 was evaluated in a panel of ovarian cancer, NSCLC, endometrial cancer and TNBC patient-derived xenografts (PDX) spanning a range of FRα expression. ZW191 was evaluated in toxicology and pharmacokinetic (PK) studies performed in non-human primates (NHP). Results: The antibody component of ZW191 features a favorable binding profile with strong binding to FRα, and drives superior tumor spheroid penetration, cellular internalization, and payload delivery compared to FRα targeted antibodies used in multiple other ADCs. ZW191 demonstrates potent activity in FRα expressing cell cultures and effective bystander activity. In a panel of PDX models representing a range of FRα expression, ZW191 demonstrates compelling anti-tumor activity across ovarian and endometrial cancers, NSCLC and TNBC. ZW191 demonstrates an encouraging tolerability profile in NHP, with favorable PK. The promising efficacy, tolerability, and PK support the potential of ZW191 as a novel therapeutic agent that may help address unmet need in patients with high and low FRα expressing cancers. Citation Format: Sam Lawn, Andrea Hernandez Rojas, Raffaele Colombo, Jodi Wong, Kaylee Wu, Vincent Fung, Manuel Lasalle, Mark E. Petersen, Lemlem Degefie, Araba Sagoe-Wagner, Dunja Urosev, Luying Yang, Ambroise Wu, Catrina Kim, Amos Chua, Kurt Stahl, Geoffrey C. Winters, John C. Fann, Jamie Rich, Stuart Barnscher. ZW191 - a FRα-targeting antibody drug conjugate with strong preclinical activity across multiple FRα-expressing indications [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 1862.

Abstract 3210: Raptamer-drug conjugates as molecularly targeted cancer therapeutics

Abstract The use of aptamers as a therapeutic targeting moiety holds the promise of building on the success of antibody-drug conjugates (ADCs). While ADCs are an attractive modality, they face limitations, including limited penetration into solid tumors and a complicated production process for large-scale manufacturing. Aptamers have several potential advantages over antibodies as a targeting moiety including: (i) a smaller size that enables better tumor penetration; (ii) chemical synthesis which allows rapid scale-up and ease of incorporating modifications; (iii) a superior safety profile from lack of immunogenicity and (iv) room temperature stability. Therefore, the use of aptamers in conjunction with drugs (similar to ADCs) holds promise for development of targeted therapeutics. We have identified a novel therapeutic target for certain cancers (COF-01) using proteomics data and immunohistochemistry from patient-derived tumors. Both the COF-01 protein and related mRNA expression was shown to be significantly increased in non-small cell lung cancer, head and neck cancer and pancreatic cancer. Here, we present a new therapeutic approach that targets COF-01-positive cancer cells using next generation aptamer conjugates. We performed a cell- and protein-based selection using our proprietary bead-based combinatorial libraries to generate next-generation base-modified ssDNA aptamers (Raptamers) against COF-01. Binding affinities of the COF-01 Raptamer candidates were determined by concentration curves using biolayer interferometry. The dissociation constants for the COF-01 Raptamers ranged from 80 nM - 200 nM. COF-01-specific Raptamer-drug conjugates (Rap-DCs) were developed by conjugating the Raptamers to monomethyl auristatin E via a cleavable linker. In vitro efficacy data showed the COF-01 Rap-DC can selectively induce cytotoxicity in different types of COF-01-positive human cancer cells (A431, CAL 27, BxPC-3). There was a significant decrease in cell viability of COF-01-positive cells when compared to COF-01-negative control cells (IC50 values ranged from 5.19 nM - 14.11 nM). In vivo efficacy testing in a COF-01-postive cell-derived xenograft mouse model showed a significant decrease in tumor volume in animals treated with the COF-01 Rap-DC when compared to saline- and erlotinib-treated controls. This provides proof-of concept for the use of high-affinity Raptamers against novel molecular targets, such as COF-01. This platform can be used to develop Rap-DCs against emergent neo-antigens on cancer cells that have escaped chemotherapy. Citation Format: Stephanie P. Vega, Nancy E. Ward, Uksha Saini, Leniher Castan-Chibas, Garima Kaushik, Daniel Ciznadija, Dev Chatterjee, Michael J. Heffernan, Atul Varadhachary, Michael Ritchie. Raptamer-drug conjugates as molecularly targeted cancer therapeutics [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 3210.

Abstract 6042: CBP-8008: A first-in-class targeted pan-Bet protein degradation therapy using bi-specific XDC (Bi-XDC) technology for TNBC and mCRPC

Abstract Bi-XDC is a breakthrough cellular targeting technology using multi-targeting ligands to effectively deliver the payload to and into specific cells. Several anticancer Bi-ligand drug conjugates including CBP-1008, CBP-1018, and CBP-1019 have been brought into various clinical stages in China and US. Targeted protein degradation molecules can degrade previously “undruggable” target proteins through their catalytic mechanism of action. The field target protein degradation has evolved since 2001, and more than 20 PROTACs are under clinical trials at different stages. To increase the efficacy vs. tolerability window, we combine Bi-XDC technology and the catalytic approach of targeted protein degradation, to form C-PROTAC, by taking advantage of our privileged Bi-XDC technology and PROTAC’s degradation property. Further medicinal chemistry optimization, spacer optimization, and evaluation of many linker payloads led to the identification of our preclinical candidate CBP-8008, a highly potent pan-Bet protein degrader conjugated to bi-ligand via a cleavable linker. A small panel of cell lines was screened to identify which tumors were sensitive to the pan-Bet protein degradation. All the tested breast cancer and prostate cell lines were the most sensitive to the payload. CBP-8008 had a high affinity to the Prostate Specific Membrane Antigen (PSMA) and the folate receptor alpha (FRα). Much more CBP-8008 were binding the 293T-FP which overexpress both FR-α and PSMA than the cell lines which only express one target (293T-F or 293T-P) or negative cell line (293T). CBP-8008 can quickly enter cells through endocytosis after treatment for 15 min, and degradation of BRD2, BRD3, and BRD4 was observed in MDA-MB-231 after 6 h, which was similar to its payload. The compound was very stable in mouse and human plasma in vitro, and limited free payload was detected which potentially avoids hematotoxicity in clinical. In the tumor-beaning mice DMPK study, fewer free payload was detected in 6 hours while much more free payload was detected in the tumor than in the blood and the time was extended to 72 hours after a single dose treatment. We evaluated CBP-8008 in TNBC and mCRPC xenograft model and robust efficacy was observed as lower as 3 MPK, significantly better than BMS-986158, a potent Bet inhibitor in clinical. In conclusion: CBP-8008 is currently in preclinical development as a potential first-in-class pan-Bet protein degradation therapy using Bi-XDC technology for TNBC and mCRPC Citation Format: Tao Wang, Zhiguang Zhang, Guitao Wang, Fan Pan, Robert Huang. CBP-8008: A first-in-class targeted pan-Bet protein degradation therapy using bi-specific XDC (Bi-XDC) technology for TNBC and mCRPC [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 6042.

Abstract 3133: A novel bispecific antibody-drug conjugate targeting HER2 and Trop-2 displays potent antitumor activity in a variety of tumor models with promising preclinical characteristics

Abstract Antibody-drug conjugates (ADCs) have exhibited considerable advancements in cancer therapy, though their efficacy is often confined to specific patient subsets or results in modest clinical responses due to low and heterogenous expression of drug targets in tumors. Targeting more than one tumor-associated antigens (TAAs) represents a new strategy to broaden treatment applicability and minimize on-target toxicity of ADC. Our analysis of co-expression of HER2 and Trop-2, two clinically validated ADC targets, in tumors of various histological malignancies manifests the great potential for an ADC simultaneously targeting these two antigens. We describe here a novel bispecific ADC (BIO-201) designed to co-target HER2 and Trop-2. This compound comprises a bispecific anti-HER2/Trop-2 antibody conjugated with a novel topoisomerase I inhibitor via a cleavable linker. BIO-201 demonstrates increased or comparable cell binding and internalization compared to parental antibodies. In in vitro cytotoxicity assays, BIO-201 effectively kills cancer cells with an IC50 in the sub nM range, irrespective of whether they co-express HER2 and Trop-2 or express only one antigen. Co-culture studies reveal that BIO-201 possesses a good bystander killing effect on drug target-negative tumor cells. In vivo studies further demonstrate the superior anti-tumor activity of BIO-201 in various tumor xenograft models, including those expressing different levels of HER2 and/or Trop-2, as well as models resistant to HER-2-targeting therapies. Moreover, BIO-201 exhibits favorable plasma stability, preclinical pharmacokinetics (PK), and toxicity profiles. In conclusion, these findings suggest that the anti-HER2/Trop-2 bispecific ADC, BIO-201, holds significant promise as an effective therapy for a diverse range of cancers, especially those expressing heterogenous levels of HER2 or Trop-2, and has the potential to benefit a broad patient population. Citation Format: Haihong (Helen) Zhong, Yan Xiong, Han Huang, Yuming Pan, Na Wang, Bin Sun, Shen Liu, Wei Yuan, Dingguo Liu, Jan Fang, Haifeng Bao. A novel bispecific antibody-drug conjugate targeting HER2 and Trop-2 displays potent antitumor activity in a variety of tumor models with promising preclinical characteristics [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 3133.

Abstract 5083: Discovery of an asymmetric IgG-like bispecific ADC targeting EGFR/HER3

Abstract Background: The epidermal growth factor receptor (EGFR) family, consisting of EGFR, HER2, HER3, and HER4, plays a critical role in tumorigenesis and tumor progression. Inhibition of ERBBs using either monoclonal antibodies (mAb) or small-molecule tyrosine kinase inhibitors have been approved for use for the treatment of various types of cancers. Despite relative success, many patients acquire resistance to long-term benefit due to alternative compensatory signaling pathways. Accordingly, activating mutations or overexpression of HER3 has been reported in many cancers, such as breast, ovarian, lung, colorectal, melanoma, head and neck, cervical and prostate cancers. Here, we report the discovery of a humanized anti-EGFR x HER3 IgG-like bispecific antibody-drug conjugate (ADC). The ADC takes a asymmetric 1+1 form through HC-HC and HC-LC charge-based heterodimerization, which is then conjugated to a topoisomerase 1 inhibitor (TOP1i) payload via a cleavable linker. The affinity of EGFR and HER3 was optimized to improve the therapeutical window, and has shown potent anti-tumor efficacy in various tumor models with limited toxicity supporting further development in clinic. Methods: An anti-EGFR x HER3 bispecific ADC (PM1300) was generated by introducing unique mutations to the CH1-CL domains of each binding arm to avoid HC-LC mispairing. KIH mutations were also introduced to each CH3 domain to support HC/HC heterodimerization and generate an IgG-like bispecific with one arm binding to EGFR and the other to HER3. TOP1i payload was conjugated to the IgG-like bispecific by the endogenous cysteine residues at DAR 8 via a clearable linker. Results: Through affinity optimization, PM300 was found to preferentially bind to EGFR/HER3 double-positive cells rather than EGFR single-positive cells. PM1300 showed significantly increased internalization and anti-proliferation activity against EGFR/HER3 double-positive cells, with limited activity towards single positive-cells. PM1300 used an optimized linker for better payload release and serum stability. PM1300 induced a potent anti-tumor efficacy in various CDX models representing different EGFR and HER3 expressing levels and mutations. Importantly, PM1300 exhibited a good safety profile in NHP consistent with the optimal affinity for EGFR and HER3. These results demonstrate that PM1300 has a promising efficacy/safety therapeutical window in preclinical models supporting further development in the clinic. Conclusion: A bispecific ADC targeting EGFR x HER3 was discovered, named PM1300, which has an asymmetric 1+1 IgG-like structure and optimized affinity. This allows for preferential binding to EGFR/HER3 double-positive cancer cells, rather than EGFR single-positive cells. This may significantly contribute to minimizing safety risk that is common for EGFR-targeting agents. Citation Format: Liandi Chen, Shaogang Peng, Chao Wang, Xiaoniu Miao, Yao Yan, Jun Xing, Weifeng Huang, Andy Tsun, Yingda Xu, Xiaolin Liu, Yi Luo. Discovery of an asymmetric IgG-like bispecific ADC targeting EGFR/HER3 [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 5083.

Abstract 6582: Preclinical characterization of IPH45, a novel topoisomerase I inhibitor ADC targeting Nectin-4 for the treatment of Nectin-4 expressing tumors

Abstract Background: Nectin-4 is a cell membrane adhesion protein overexpressed in several solid tumors, including urothelial, breast, lung, ovarian, and pancreatic cancers, with limited expression in normal tissues, making it an ideal target for antibody-drug conjugates (ADC). Enfortumab Vedotin (EV, PADCEV®) is an ADC targeting Nectin-4 with a monomethyl auristatin E (MMAE) payload approved for the treatment of patients with urothelial carcinoma (UC), but with limited clinical activity reported beyond UC. In addition, EV causes peripheral neuropathy and skin toxicity, leading to treatment discontinuation. To overcome these limitations and the mechanism of resistance to EV, IPH45, a novel topoisomerase I inhibitor ADC targeting Nectin-4 with an improved therapeutic index, was developed. Methods: In vitro studies were performed to investigate internalization, tumor cell killing and bystander effect, antibody-dependent cellular cytotoxicity (ADCC) and complement activation. In vivo efficacy studies were performed on cell line-derived xenografts (CDX) of triple negative breast cancer, patient-derived xenografts (PDX) of urothelial carcinoma and syngeneic tumor models expressing human Nectin-4. IPH45 efficacy was compared to EV. Toxicology studies to determine the dose range were conducted in rat and non-human primates (NHP). Results: IPH45 consists of a humanized IgG1 targeting a unique epitope in human Nectin-4, non-overlapping with EV, a cleavable linker, and a topoisomerase I inhibitor with a high drug-to-antibody ratio (DAR). In vitro, IPH45 is internalized upon binding to its target and induces direct killing of Nectin-4 expressing tumor cells as well as bystander killing of Nectin-4 non-expressing tumor cells in mixed culture. IPH45 mediates ADCC and complement-mediated killing. IPH45 demonstrates activity in CDX and PDX models with similar effective dose as EV. Moreover, IPH45 shows anti-tumor efficacy in vitro and in vivo in primary and secondary EV resistant models. In dose-range finding toxicology studies in rats and NHP, IPH45, unlike EV, does not elicit skin toxicity. IPH45 remains well tolerated up to the highest dose tested, suggesting a potentially larger therapeutic index than MMAE-based ADCs. Pharmacokinetic analyses in mice, rats, and NHP show minimal release of free toxin in the serum and half-life compatible with Q2W or less frequent dosing schedules in clinic. Conclusion: IPH45 is a topoisomerase I ADC targeting Nectin-4 with the potential for larger therapeutic index than EV, improved safety and dosing regimen, and ability to overcome primary and secondary resistance to EV or other Nectin-4 ADC in development. IPH45 is progressing to the clinic where the translatability of this unique profile into improved patient outcomes will be assessed across indications. Citation Format: Romain Remark, Cécile Bonnafous, Laura Chiossone, Caroline Soulas, Cyril Perrier, Guillaume Habif, Sivan Bokobza, Aurélie Maguer, Rachel Courtois, Julie Lopez, Grégory Fenaux, Olivier Benac, Barbara Carrette, Robin Letay-Drouet, Raja Bonifay, Séverine Augier, Léa Simon, Benjamin Rossi, Ariane Morel, Agnès Represa, Nicola Beltraminelli, Yannis Morel, Carine Paturel, Eric Vivier. Preclinical characterization of IPH45, a novel topoisomerase I inhibitor ADC targeting Nectin-4 for the treatment of Nectin-4 expressing tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6582.

Abstract 6573: Targeting DLK1, a Notch ligand, with an antibody-drug conjugate in adrenocortical carcinoma

Abstract Adrenocortical carcinoma (ACC) is an aggressive and rare endocrine malignancy with poor prognosis. First-line therapy (adrenolytic agent mitotane in combination with chemotherapy) for recurrent and metastatic ACC has limited efficacy and there are no approved second-line therapies. Antibody-drug conjugates (ADCs) are an emerging immunotherapeutic class in which a cytotoxic payload is directed to tumor cells via antibodies to cell surface proteins. As Notch ligands, such as DLL3, are ADC targets in neuroendocrine cancers, we screened for expression of Notch ligands in ACC. Among Notch ligands (DLL1, DLL3, DLK1, JAG1, JAG2), DLK1 (delta-like non-canonical Notch ligand 1) was the most highly expressed. Moreover, ACC had the near highest expression of DLK1 across all TCGA tumors likely because this ligand is of adrenal origin (with limited expression in other normal tissues apart from the pituitary gland and ovaries). By immunohistochemistry, we verified DLK1 to be expressed, at variable levels, in 97% of ACC tumors (n=30/31). We then tested a DLK1-directed antibody-drug conjugate (ADCT-701; DLK1-PBD), consisting of a humanized anti-DLK1 monoclonal antibody coupled to DNA damaging pyrrolobenzodiazepine (PBD) dimers (SG3199; drug-to-antibody ratio~1.8) via a cleavable linker in ACC pre-clinical models. ADCT-701 exhibited potent, nanomolar cytotoxicity in DLK1-expressing ACC cell lines and ACC patient-derived short-term organoid cultures. ADCT-701 cytotoxicity was dependent on DLK1 expression as DLK1 knockout and DLK1 overexpression abrogated and facilitated cytotoxicity, respectively. Mechanistically, ADCT-701 induced cytotoxicity through DLK1 dependent receptor-mediated internalization and DNA damage (γH2AX) as well as apoptosis (annexin V/PI positive cells, cleaved PARP, and cleaved caspase-3). In vivo studies showed that ADCT-701 was highly effective against DLK1-positive ACC xenografts and PDX models with durable anti-tumor activity. Our pre-clinical data demonstrate DLK1 as an important therapeutic target in ACC and support an upcoming phase I clinical trial of ADCT-701 in patients with neuroendocrine tumors including ACC (NCT06041516). Citation Format: Nai-Yun Sun, Suresh Kumar, Amber Weiner, Yoo Sun Kim, Arnulfo Mendoza, Rosa Nguyen, Reona Okada, Yves Pommier, Dan Martinez, Jennifer Pogoriler, Sharon Diskin, John Maris, Jaydira Del Rivero, Nitin Roper. Targeting DLK1, a Notch ligand, with an antibody-drug conjugate in adrenocortical carcinoma [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 6573.

Abstract 3140: BL-M05D1, a novel claudin 18.2-targeting ADC, demonstrates specific potent anti-tumor efficacy in preclinical evaluation

Abstract Claudin 18.2, is a member of the tight junction protein family, and an isoform of claudin18. It is therapeutically targeted in cancer due to its limited expression in normal tissue and prevalent expression in a variety of human carcinomas. To develop a promising therapeutic anti-tumor agent, we generated BL-M05D1, an anti-Claudin 18.2-Ed-04 ADC. It is comprised of a novel, specific monoclonal antibody against Claudin 18.2 (5103F3), having a wt Fc that can mediate ADCC and CDC in vitro. BL-M05D1 is composed of (5103F3), a cathepsin B cleavable linker, and a novel topoisomerase I inhibitor agent (Ed-04). The novel Ed-04 is a derivative of the alkaloid camptothecin and mediates cell cycle arrest at the S phase and subsequent apoptosis. BL-M05D1 achieves a high drug-to-antibody-ratio (DAR=8) with a highly stable linker and is readily internalized and trafficked to lysosomal cellular compartments. The antitumor efficacy of BL-M05D1 was evaluated in xenograft tumor models. BL-M05D1 exhibited strong tumor inhibition capacity toward the human gastric cancer cell MKN-45-Claudin18.2 (#C11D) xenograft model, and human pancreatic cancer cell Patu8988s-Claudin18.2 (#C7G) xenograft model. In summary, these studies suggest BL-M05D1, a novel Claudin 18.2-targeting ADC, is potentially efficacious in the treatment of Claudin 18.2-expressing carcinomas. The clinical phase I is being undertaken to evaluate safety, dosing and observations of preliminary signs of efficacy. Citation Format: Weili Wan, Kelcey Dinkel, Austin Sanford, Shuwen Zhao, Shi Zhuo, Yong Zhang, Lan Chen, Ruigang Li, Jahan Salar Khalili, Xiao Sa, Yongqi Yan, Xuejiao Shen, Yi Zhu. BL-M05D1, a novel claudin 18.2-targeting ADC, demonstrates specific potent anti-tumor efficacy in preclinical evaluation [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 3140.

Abstract 1812: Development of novel topoisomerase 1 inhibitor PBX-7 payload-based ADC including tandem cleavable linker system

Abstract Antibody-Drug Conjugates (ADC) employing camptothecin-based payloads are gaining high interests as a new modality to treat various cancers, especially solid tumors. Among them, novel ADCs containing synthetic camptothecin-based payloads specially optimized for ADC applications (e.g., DXd) are of special interest due to their successful clinical results. Despite the remarkable success of ADCs exploiting these payloads, such as Enhertu (DS-8201a), there are still clear unmet needs, including the improvement of safety profile (minimization of ILD and neutropenia) and the development of novel ADCs with multiple MoA payloads to combat cancer heterogeneity. To address these unmet needs, we have synthesized and evaluated a series of PBX-7 payloads, derived from novel camptothecin FL118, a potent, dual inhibitor of Top 1/anti-apoptotic pathway known for its favorable safety profile. These compounds showed strong in vitro cytotoxicity across various cancer cell lines and inhibition of Top 1 comparable to DXd or other related camptothecin compounds. They had excellent safety profile in the preliminary toxicity study in mice that can be attributed to their physicochemical properties. PBX-7 series payloads based ADC exhibited superior cellular cytotoxicity and in vivo tumor regression compared to Enhertu in Her2-positive cell line and CDX mouse model. Notably, PBX-7 based ADC demonstrated remarkable efficacy in reducing tumor volume more than Enhertu in the T-DM1-resistant JIMT-1 xenograft mouse model. Furthermore, PBX-7 based ADC have bystander effects similar to Enhertu, leading to reduced viability of antigen-negative cells when co-cultured with antigen-positive cell lines. In NHP toxicity study in Cynomolgus monkeys, no sign of toxicity was observed when dosed with Trastuzumab-PBX-7016 up to 30mg/kg. As another way of addressing unmet needs, we synthesized novel linker including tandem cleavage site to reduce payload mediated toxicity. Two distinct enzyme, beta-glucuronidase and cathepsin B cleave linker sequentially. This linker system has a higher stability during circulation in the body and higher specific payload release in the tumor tissue than single cleavable linker. In conclusion, our research shows the promising potential of novel camptothecin PBX-7 based ADCs and tandem cleavable linker system as potent and safe ADC candidates for cancer therapy. These advancements hold great promise in addressing the current challenges in ADC development with their enhanced safety profiles and targeted efficacy. Citation Format: Younghwa Chun, Areum Go, Hyun Yong Cho, Byeong Sung Lee, Seunggun Shin, JaeHyeon Lee, Sohui Kweon, Hyemin Han, Suhyun Park, Doo Young Jung. Development of novel topoisomerase 1 inhibitor PBX-7 payload-based ADC including tandem cleavable linker system [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 1812.

Abstract 4695: Effect of fully human anti-PTGFRN antibody drug conjugate on head neck cancer cells and patient derived xenografts

Abstract Prostaglandin F2 Receptor Negative regulator, or PTGFRN, also known as FPRP is a 175 kDa transmembrane protein associated with metastatic characteristics of certain cancer types and a member of the tetraspanin family. PTGFRN expression is undetectable to low in normal tissues whereas it is elevated in several cancers such as head and neck, mesothelioma and medulloblastoma. PTGFRN has also been reported to be upregulated in many different types of cancer, increasingly so in metastatic cancer cells, compared to their non-metastatic counterparts PTGFRN expression has been reported to be a poor prognostic factor for glioblastoma making it a potential novel cancer therapeutic target. We have previously shown that PTGFRN is an internalizable cell surface protein making it suitable for targeting by antibody drug conjugate (ADC). We have developed fully human monoclonal antibodies to PTGFRN by immunizing humanized transgenic TC mice with human PTGFRN. Within 60 days from the start of immunization we were able to select several fully human anti-PTGFRN monoclonal antibodies that were examined by several functional assays that included flow binding, internalization, affinity determination and cell killing as antibody drug conjugates. Several monoclonal antibodies were selected and evaluated for their ability to inhibit tumor formation in several cancers. Selected antibodies were then conjugated to duocarmycin by a cleavable linker. The present study proposed to examine the effect of one such anti-PTGFRN ADC on head and neck cancer cell lines and patient derived xenografts that had been analyzed for their PTGFRN expression by western blot analysis using anti-PTGFRN antibody. Cells lines and PDX that were positive for PTGFRN expression were used to examine the effect of anti-PTGFRN ADC on their in vitro and in vivo proliferation. The data show that anti-PTGFRN ADC inhibits tumor formation in a dose-dependent fashion. These data also establish PTGFRN as cancer druggable target and the selected fully human anti-PTGFRN antibody as an attractive antibody for the development of new antibody drug conjugate in Oncology. These data also emphasize the potential of humanized transgenic TC mice for the development of fully human monoclonal antibodies, thus bypassing the need for humanization and affinity maturation required by other humanized mice or phase display. Citation Format: Jorge Marquez, Jianping Dong, Mitsuo Oshimura, Jun Hayashi, Ginette Serrero. Effect of fully human anti-PTGFRN antibody drug conjugate on head neck cancer cells and 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 4695.