Bispecific Antibody-drug Conjugates Research Articles

Novel bispecific antibody-drug conjugate targeting PD-L1 and B7-H3 enhances antitumor efficacy and promotes immune-mediated antitumor responses

BackgroundAntibody-drug conjugates (ADCs) offer a promising approach, combining monoclonal antibodies with chemotherapeutic drugs to target cancer cells effectively while minimizing toxicity.MethodsThis study examined the therapeutic efficacy and potential mechanisms of...

1426P BL-B01D1, an EGFR x her3 bispecific antibody-drug conjugate (ADC), in patients with locally advanced or metastatic esophageal squamous cell carcinoma (ESCC)

1426P BL-B01D1, an EGFR x her3 bispecific antibody-drug conjugate (ADC), in patients with locally advanced or metastatic esophageal squamous cell carcinoma (ESCC)

54P BL-B01D1, an EGFR x HER3 bispecific antibody-drug conjugate (ADC), in patients with locally advanced or metastatic biliary tract carcinoma (BTC)

54P BL-B01D1, an EGFR x HER3 bispecific antibody-drug conjugate (ADC), in patients with locally advanced or metastatic biliary tract carcinoma (BTC)

1959O BL-B01D1, an EGFR x HER3 bispecific antibody-drug conjugate (ADC), in patients with locally advanced or metastatic urothelial carcinoma (UC)

1959O BL-B01D1, an EGFR x HER3 bispecific antibody-drug conjugate (ADC), in patients with locally advanced or metastatic urothelial carcinoma (UC)

Developing ‘Next-Gen’ Therapeutics: a First-in-Class B7H3/PTK7 Topo-I-Payload Bispecific ADC

Bispecific ADCs (bsADCs) are positioned as an emerging, next-generation, class of therapeutics, combining the dual-targeting capabilities of bispecific antibodies (bsAbs) with the potent cytotoxicity of antibody-drug conjugates (ADCs). The resulting bsADC offers improved efficacy and safety for the treatment of heterogeneous solid tumors.

BL-B01D1, a first-in-class EGFR–HER3 bispecific antibody–drug conjugate, in patients with locally advanced or metastatic solid tumours: a first-in-human, open-label, multicentre, phase 1 study

Antibody-drug conjugates have promising clinical activity in the treatment of solid tumours. BL-B01D1 is a first-in-class EGFR-HER3 bispecific antibody-drug conjugate. We aimed to assess the safety and preliminary antitumour activity of BL-B01D1 in patients with locally advanced or metastatic solid tumours. This first-in-human, open-label, multicentre, dose-escalation and dose-expansion phase 1 trial was conducted in seven hospitals in China, enrolling patients aged 18-75 years (dose escalation; phase 1a) or older than 18 years (dose expansion; phase 1b), with a life expectancy of at least 3 months, an Eastern Cooperative Oncology Group performance status of 0-1, and histologically or cytologically confirmed locally advanced or metastatic solid tumours that had progressed on current standard treatment. In the phase 1a i3+3 design, patients received intravenous BL-B01D1 at three different schedules: 0·27 mg/kg, 1·5 mg/kg, and 3·0 mg/kg weekly; 2·5 mg/kg, 3·0 mg/kg, and 3·5 mg/kg on days 1 and 8 of each cycle every 3 weeks; or 5·0 mg/kg and 6·0 mg/kg on day 1 of each cycle every 3 weeks. The primary objectives of phase 1a were to identify the safety, maximum tolerated dose, and dose-limiting toxicity. In phase 1b, patients were treated in two schedules: 2·5 and 3·0 mg/kg on days 1 and 8 every 3 weeks, or 4·5, 5·0, and 6·0 mg/kg on day 1 every 3 weeks. The primary objectives of phase 1b were to assess the safety and recommended phase 2 dose of BL-B01D1, and objective response rate was a key secondary endpoint. Safety was analysed in all patients with safety records who received at least one dose of BL-B01D1. Antitumour activity was assessed in the activity analysis set which included all patients who received at least one dose of BL-B01D1 every 3 weeks. This trial is registered with China Drug Trials, CTR20212923, and ClinicalTrials.gov, NCT05194982, and recruitment is ongoing. Between Dec 8, 2021, and March 13, 2023, 195 patients (133 [65%] men and 62 [32%] women; 25 in phase 1a and 170 in phase 1b) were consecutively enrolled, including 113 with non-small-cell lung cancer, 42 with nasopharyngeal carcinomas, 13 with small-cell lung cancer, 25 with head and neck squamous cell carcinoma, one with thymic squamous cell carcinoma, and one with submandibular lymphoepithelioma-like carcinoma. In phase 1a, four dose-limiting toxicities were observed (two at 3·0 mg/kg weekly and two at 3·5 mg/kg on days 1 and 8 every 3 weeks; all were febrile neutropenia), thus the maximum tolerated dose was reached at 3·0 mg/kg on days 1 and 8 every 3 weeks and 6·0 mg/kg on day 1 every 3 weeks. Grade 3 or worse treatment-related adverse events occurred in 139 (71%) of 195 patients; the most common of which were neutropenia (91 [47%]), anaemia (76 [39%]), leukopenia (76 [39%]), and thrombocytopenia (63 [32%]). 52 (27%) patients had a dose reduction and five (3%) patients discontinued treatment due to treatment-related adverse events. One patient was reported as having interstitial lung disease. Treatment-related deaths occurred in three (2%) patients (one due to pneumonia, one due to septic shock, and one due to myelosuppression). In 174 patients evaluated for activity, median follow-up was 6·9 months (IQR 4·5-8·9) and 60 (34%; 95% CI 27-42) patients had an objective response. Our results suggest that BL-B01D1 has preliminary antitumour activity in extensively and heavily treated advanced solid tumours with an acceptable safety profile. Based on the safety and antitumour activity data from both phase 1a and 1b, 2·5 mg/kg on days 1 and 8 every 3 weeks was selected as the recommended phase 2 dose in Chinese patients. Sichuan Baili Pharmaceutical. For the Chinese translation of the abstract see Supplementary Materials section.

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

Abstract Introduction: Antibody-drug conjugates (ADCs) targeting trophoblast cell surface antigen 2 (TROP2), such as sacituzumab-govitecan and datopotamab-deruxtecan, have recently been developed. A preclinical study has reported the efficacy of anti-human epidermal growth factor receptor 2 (HER2) and TROP2 bispecific ADCs in treating HER2 and TROP2 co-expressing tumors, including HER2-low tumors in various solid tumors (Chengzhang Shang et al: AACR 2023). In this study, we aimed to evaluate the expression of TROP2 by immunohistochemistry (IHC) at different sites and investigate the changing status and co-expression of HER2 and TROP2 among breast cancer patients. Methods: We collected 244 archival paired samples of primary tumors and metastatic sites from 111 breast cancer patients treated in our hospital from 2000 to 2018. Estrogen receptor (ER), progesterone receptor (PgR), and HER2 status were determined from previous pathology reports. HER2 low was defined as HER2 IHC score of 1+ or 2+ with negative fluorescent in situ hybridization (ISH). In addition, we determined the expression of TROP2 using IHC assays and categorized the results based on the histochemical score (H-score). Results: Among the samples, ER+HER2- was 139, ER+HER2+ was nine, ER-HER2+ was eight, and triple-negative (TN) was 88. HER2 3+ or 2+ with ISH positive was 17, HER2 low was 112, HER2 negative was 115. The median TROP2 H-score was 10, with a mean of 37.5 (range: 0-265). Overall, 27% of the samples had H-score of 0, 73% had H-score of 1<, 47% had H-score of 10<, and 12% had H-score of 100< . No statistically significant association was observed between TROP2 expression and breast cancer subtypes (ER+HER2-; H-score of 0 24%, H-score of 1< 76%, H-score of 10< 44%, H-score of 100< 14%, TN; H-score of 0 30%, H-score of 1< 70%, H-score of 10< 54%, H-score of 100< 11%). On the other hand, samples from the metastatic site showed a higher rate of TROP-2 expression H-score of 1< compared to the primary site (primary site; H-score of 0 36%, H-score of 1< 64%, metastatic site; H-score of 0 18%, H-score of 1< 82%; p< 0.01). The rates of TROP2-positive expression defined as H-score of 1<, varied among the metastatic sites, with 100% in liver, 92% in brain, 89% in lymph nodes, 64% in lung, and 60% in bone. Regarding the co-expression of HER2 and TROP2, among HER2-positive samples, 35% had TROP2 H-score of 0, 24% had TROP2 H-score of 1<, and 41% had TROP2 H-score of 10<, while among HER2 low samples, 21% had TROP2 H-score of 0, 27% had TROP2 H-score of 1<, 39% had TROP2 H-score of 10<, and TROP2 H-score of 100< was found in 13% of patients. Based on the definition of co-expression such as TROP2 H-score of 1< and HER2 1+ or more, overall, 40% of the samples had co-expression of HER2 and TROP2, and the metastatic site had a higher rate of co-expression than the primary tumor (49%, 32% respectively, p=0.002). Conclusions: This study demonstrates that TROP-2 expression by IHC in breast cancer varies between primary and metastatic sites in breast cancer. Metastatic sites and specific organs show higher rate of TROP2 expression. Additionally, 40% of the samples had the co-expression of HER2 and TROP2, suggesting a potential target for future bispecific ADCs therapy. Citation Format: Mai Onishi, Tatsunori Shimoi, Yuki Kojima, Shu Yazaki, Taro Yamanaka, Rui Kitadai, Asuka Kawachi, Hitomi Okuma, Mai Hoshino, Ayumi Saito, Munehiro Ito, Aiko Maejima, Tadaaki Nishikawa, Kazuki Sudo, Emi Noguchi, Yasuhiro Fujiwara, Masayuki Yoshida, Kan Yonemori. Expression and co-expression patterns of TROP2 and HER2 in breast cancer: implications for bispecific antibody-drug conjugate therapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-15-12.

Abstract PS08-07: BL-B01D1, a first-in-class EGFRxHER3 bispecific antibody-drug conjugate, in patients with Locally Advanced or Metastatic Breast Cancer and other Solid Tumor: Results from a phase 1 study

Abstract Background: BL-B01D1 is a first-in-class novel antibody drug conjugate (ADC) consisting of an EGFRxHER3 bispecific antibody bounded to a novel TOP-I inhibitor payload via a cleavable linker. We now present safety/efficacy data from a phase I study of BL-B01D1 in breast cancer. Methods: This study included patients (pts) with locally advanced or metastatic breast cancer (BC) and other solid tumors. BL-B01D1 was administered intravenously at doses of 2.5mg/kg Day 1 & Day 8 every 3 weeks (D1D8Q3W) or 5.0mg/kg Day 1 every 3 weeks (D1Q3W) during dose escalation (D-ESC, i3+3) based on the information obtained during the first-in-human study in solid tumors. A subset of pts will be enrolled in the dose-expansion (D-EXP) phase. Results: As of June 26, 2023, 42 pts were enrolled and received at least one dose (D-ESC, n=8; D-EXP, n=34) of BL-B01D1. Only one DLT of febrile neutropenia was observed at 5.0mg/kg D1 Q3W, maximum tolerated dose (MTD) has not been reached. D-EXP was conducted at 2.5mg/kg D1D8 Q3W. Forty-one pts with BC and 1 pt with non-small cell lung cancer (NSCLC) were enrolled in this study. The most common TRAEs ( >10%, all grade/≥ G3) were leukopenia (67%/24%), neutropenia (55%/33%), anemia (55%/26%), thrombocytopenia (60%/24%), nausea (38%/0%), vomiting (38%/0%), stomatitis (31%/2%), asthenia (29%/0%), hypokalemia (21%/5%), aspartate aminotransferase increased (19%/0), alanine aminotransferase increased (19%/0%), decreased appetite (19%/0%), hypertriglyceridemia (19%/0%), hyperglycemia (19%/0%), hyperglycemia (17%/0%), weight decreased (14%/0%), diarrhea (12%/0%), epistaxis (12%/0%), hypercholesterolemia (12%/0%). No ILD was observed. Twenty-four pts. were evaluable for efficacy (at least 1 tumor assessment). Updated information will be provided during the meeting. Conclusions: BL-B01D1 demonstrated encouraging efficacy in metastatic/locally advanced breast cancer that have failed standard of care, especially in pts with TNBC. The safety profile showed adequate safety and tolerability. Clinical trial information: NCT05470348. Efficacy in Patients with Breast Cancer 1 Including pts whose PRs were not yet confirmed but still under treatment. Citation Format: Jiong Wu, Jian Zhang, Yiqun Du, Wen Zou, Muran Ding, Hui Yang, Sa Xiao, Hongwei Wang, Hai Zhu, Martin Olivo, Yi Zhu. BL-B01D1, a first-in-class EGFRxHER3 bispecific antibody-drug conjugate, in patients with Locally Advanced or Metastatic Breast Cancer and other Solid Tumor: Results from a phase 1 study [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS08-07.

A novel bispecific antibody drug conjugate targeting HER2 and HER3 with potent therapeutic efficacy against breast cancer.

Antibody drug conjugate (ADC) therapy has become one of the most promising approaches in cancer immunotherapy. Bispecific targeting could enhance the efficacy and safety of ADC by improving its specificity, affinity and internalization. In this study we constructed a HER2/HER3-targeting bispecific ADC (BsADC) and characterized its physiochemical properties, target specificity and internalization in vitro, and assessed its anti-tumor activities in breast cancer cell lines and in animal models. The HER2/HER3-targeting BsADC had a drug to antibody ratio (DAR) of 2.89, displayed a high selectivity against the target JIMT-1 breast cancer cells in vitro, as well as a slightly higher level of internalization than HER2- or HER3-monospecific ADCs. More importantly, the bispecific ADC potently inhibited the viability of MCF7, JIMT-1, BT474, BxPC-3 and SKOV-3 cancer cells in vitro. In JIMT-1 breast cancer xenograft mice, a single injection of bispecific ADC (3 mg/kg, i.v.) significantly inhibited the tumor growth with an efficacy comparable to that caused by combined injection of HER2 and HER3-monospecific ADCs (3 mg/kg for each). Our study demonstrates that the bispecific ADC concept can be applied to development of more potent new cancer therapeutics than the monospecific ADCs.

Abstract LB043: VBC101-F11: An innovative EGFR/cMet bispecific antibody drug conjugate (ADC) targeting key oncogenic drivers in solid tumors

Abstract EGFR and cMet are two classic growth factor receptors frequently co-expressed in solid tumors at significantly high levels, while only limited to moderate expression in normal tissues. Beyond their co-expression patten, the aberrant or over expression in their pathway are implicated in oncogenesis. Notably, MET pathway activation either through increased cMet expression or its ligand, is often associated with resistance to EGFR tyrosine kinase inhibitors (TKI). Therefore, the interplay between EGFR and cMet, coupled with their differential expression in tumors versus normal tissue substantiates the rationale for a bispecific approach that targets both receptors to enhance tumor specificity. VBC101-F11 is a bispecific anti-EGFR/cMet ADC engineered to deliver clinically validated cytotoxic agents to tumor cells with a drug-to antibody ratio (DAR) of 4. This innovative design distinguishes VBC101-F11 from other EGFR/cMet-targeting agents on the market or in clinical trials (ABBV399 or AZD9592), optimizing both efficacy and safety. 1. VBC101-F11 features a low-affinity EGFR arm coupled with a high-affinity cMet arm. Incorporating the low-affinity EGFR arm enhances binding avidity and internalization, resulting in a superior cytotoxicity with a 5-fold improvement in IC50 compared to ABBV-399 (targeting cMet only). This nuanced EGFR arm design ensures a reduction in EGFR-related off-target toxicity in normal tissues. 2. The biparatopic design in cMet arm further underscores our strategic rationale by demonstrating a synergistic effect in terms of binding, internalization, and functional blocking with a 10-fold improvement in EC50 compared to the monospecific parental antibodies that target a single epitope. This ensures VBC101-F11’s maximized efficacy in tumor cells. 3. VBC101-F11’s innovative nanobody-based format sets our molecule apart. Living imaging data in mouse model reveals that VBC101-F11 (90kD) achieves superior tumor penetration and accumulation from 1 hour to over 90 hours surpassing the mAb ABBV399 and bispecific antibody AZD9592, both at 150kD. 4. VBC101-F11 has demonstrated outstanding molecular developability in stress tests, particularly in human and non-human primate (NHP) plasma, where both the antibody component and the entire ADC molecule exhibit remarkable stability, positioning VBC101-F11 as a viable candidate for future Chemistry, Manufacturing, and Controls (CMC) development. 5. In in vivo CDX models using various lung cancer cell lines, VBC101-F11 equipped with MMAE warhead displayed a marked superiority in tumor growth inhibition (TGI of 60% vs. 17%) at a single dose of 3mg/kg compared to MMAE conjugating ADC ABBV-399. Further payload exploration, such as DNA replication inhibitor, has also yielded promising results, with VBC101-F11-new payload showing comparable efficacy to the same class ADC AZD9592 in models with low EGFR and high cMet expression (100% TGI) and in those with moderate EGFR/cMet expression (~ 60% TGI). In summary, VBC101-F11, with unique design and distinguished attributes, has demonstrated its remarkable potential in the landscape of anti-EGFR/cMet therapies. The encouraging efficacy observed in preclinical models supports the molecule’s advancement into clinical development, positioning it as a best-in-class bispecific ADC for treating tumors in patients. Citation Format: Wei (Vivian) Wang, Jing Li, Lin Ma, Man Xu, Qun Yin. VBC101-F11: An innovative EGFR/cMet bispecific antibody drug conjugate (ADC) targeting key oncogenic drivers in 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 LB043.

Abstract LB448: VBC103: An innovative Trop2/Nectin4 targeted bispecific antibody drug conjugate (ADC) in bladder urothelial carcinoma (UC), triple-negative breast cancer (TNBC) and beyond

Abstract Trop2 and Nectin4 are transmembrane proteins involved in cancer pathogenesis. The therapeutic potential of targeting Trop2 or Nectin4 has been demonstrated through the approval of Trodelvy™ (Sacituzumab govitecan; SG) and Padcev™ (Enfortumab Vedotin; EV). However, the clinical use of these agents is accompanied by safety concerns. Trodelvy carries Boxed Warnings for severe or life-threatening neutropenia and severe diarrhea, while Padcev is associated with serious skin reactions. These safety issues underscore the importance of improving the drugs' safety profiles. It should be noted that the co-expression of Trop2 and Nectin4 is high in solid tumors while limited or moderate in normal tissues, presents an opportunity for more tumor-specific targeting and supports the bispecific design that could potentially address these safety issues. VBC103 is a bispecific antibody-drug conjugate (ADC) that selectively targets both Trop2 and Nectin4, which are highly co-expressed in UC, TNBC and other tumors. It delivers a TOPOi payload with a strong bystander effect to tumor cells. Our unique design maximizes efficacy while minimizing safety concerns. VBC103 has the following features to differentiate itself from other drugs targeting Trop2 or Nectin4 separately in clinical development and on the market. 1. Modulated affinity and valency. VBC103 incorporates a moderate affinity arm that targets Trop2 and a moderate affinity but high avidity arm that targets Nectin4, allowing for enhanced binding avidity, internalization, and cytotoxicity compared to its parental molecules (anti-Nectin4 parental antibody) or approved drug like EV. Additionally, the moderate affinity of both Trop2 and Nectin4 in VBC103 helps to reduce target-driven toxicities in normal tissues. 2. Innovative format of VHH-Fc fusion protein (MW 90kDa) demonstrated the superior tumor penetration, accumulation (MFI ratio in tumor: 280 VS 174) and distribution (MFI ratio in liver: 10 VS 18) compared to the IgG1 mAb- enfortumab (MW 150kDa). 3. Stronger bystander cell killing effect of the TOPOi payload vs. that of the payload used in EV. 4. Superior in vivo efficacy. In the UC CDX model, TGI 95% [VBC103, 5 mpk] vs 77% [EV, 8 mpk] vs 70% [EV+SG, 4+4 mpk] (Q3W X 3; ADC molecular equivalence) at Day 42 post injection. In the TNBC CDX model, VBC103 demonstrated continued superior efficacy compared to EV (maximum tolerated dose) and SG when administered as a single dose treatment at Day 21 post injection. 5. Wide therapeutic widow (TW). Preliminary toxicity studies in cynomolgus monkeys demonstrated good tolerability of repeated doses (18 and 36 mpk) of VBC103, which is molecular equivalent to 30 and 60 mpk of IgG1 ADC. These findings could establish a TW of >10 (HNSTD/MED). 6. Good developability. VBC103 also exhibited excellent developability based on plasma stability and other stress test data, which suggests it a good candidate in future CMC development. In summary, VBC103, with its unique bispecific design and differentiated features in affinity, valency, linker-payload, has shown promising potential as a first-in-class ADC candidate. Discovery studies have revealed favorable efficacy and toxicity profiles, supporting advancing VBC103 into clinical trials. Citation Format: Wei Wang, Jing Li, Lingyu Guan, Man Xu, Qun Yin. VBC103: An innovative Trop2/Nectin4 targeted bispecific antibody drug conjugate (ADC) in bladder urothelial carcinoma (UC), triple-negative breast cancer (TNBC) and beyond [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 LB448.

Abstract 1870: LCB36, a bi-specific antibody drug conjugate (BsADC) utilizing ConjuAll conjugation technology and proprietary prodrug payload selectively activated in cancer cells for treating B-cell blood cancers expressing CD20 and/or CD22

Abstract Although frontline chemoimmunotherapy elicits responses in many B-Cell malignancy patients, treatment options remain limited, and clinical outcomes are suboptimal for those with relapsed or refractory diseases. Minimal residual disease (MRD) can lead to disease relapse despite achieving complete remission through targeted therapy, and tumor heterogeneity is responsible for MRD and compromises the efficacy of targeted therapy, especially in B-cell malignancies. Antibody-drug conjugates (ADCs) play a crucial role in oncology indications, and bispecific antibodies (BsAbs) represent one of the fastest-growing classes of next generation antibody therapeutics for cancer therapy. Several advantages of targeting dual tumor-associated antigens (TAAs) with BsAbs and bi-specific antibody drug conjugates (BsADCs) have been proposed, including improved efficacy, heightened tumor cell specificity, and reduced side effects in normal tissues. Notably, the ability of BsADCs to bind to cancer cells expressing even just one antigen has prompted further investigation into their efficacy in heterogeneous cancer models. LCB36 is a BsADC composed of a BsAb targeting both clinically validated CD20 and CD22, which are expressed in B-Cell malignancy, and a proprietary DNA cross-linking PBD prodrug that is site-specifically conjugated to the BsAb using ConjuAll technology. We assessed the expression levels of CD20 and CD22 in the cancer cell lines. While most cells expressed both antigens, some populations showed a low expression level of CD20 and a high expression level of CD22, while others exhibited the opposite pattern, suggesting heterogeneous expression of the two antigens within the same cell line. To confirm the heterogeneity of the two antigens in blood cancer patients, we analyzed single-cell sequencing data [GSE132509]. Each patient had CD20 and CD22 double-positive cells along with CD20 or CD22 single-positive cells, suggesting the possibility of relapse due to MRD following CD20 or CD22 single-targeted therapy. LCB36 demonstrated superior efficacy in CD20 and CD22 double-positive cell lines and cell-line derived xenografts (CDXs) models compared to ADCs targeting either CD20 or CD22, while showing comparable efficacy to single-target ADCs in CD20 or CD22 single-positive cancer cell lines and CDXs. Pharmacokinetic and toxicokinetic studies of LCB36 were analyzed in rats and Cynomolgus monkeys, respectively, using LC-MS/MS bioanalysis. Preliminary toxicity studies in rats and Cynomolgus monkeys demonstrated that LCB36 is well tolerated. In conclusion, LCB36, composed of a BsAb that binds to two clinically validated targets, showed efficacy at well-tolerated doses and has the potential to increase event-free survival periods by reducing MRD. Citation Format: Wihak Kim, Namjeong Choi, Cul-Woong Chung, Hwanhee Oh. LCB36, a bi-specific antibody drug conjugate (BsADC) utilizing ConjuAll conjugation technology and proprietary prodrug payload selectively activated in cancer cells for treating B-cell blood cancers expressing CD20 and/or CD22 [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 1870.

Abstract 1871: Discovery and characterization of NXV01c, an EGFR × cMET bispecific nanobody drug conjugate with potent anti-tumor activity

Abstract EGFR and cMET are proven cancer targets co-expressed in diverse tumor types. EGFR × cMET bispecific antibody has been approved for the treatment of NSCLC, supporting a simultaneous targeting strategy. In addition to this dual targeting benefit, bispecific antibody drug conjugates (ADCs) targeting EGFR and cMET have also been developed to further improve anti-tumor activity and tissue selectivity. Sufficient target affinity, good cellular internalization and high tumor infiltration are critical for an ADC to mediate therapeutic activity. To this end, we developed the first EGFR × cMET bispecific nanobody conjugated with monomethyl auristatin E (MMAE) as payload (NXV01c). Lead nanobodies against EGFR and cMET were respectively selected from immune libraries by phage display, followed by highly efficient humanization and optimization by neoX’s computation platform. The bispecific nanobody with Fc (NXV01) was constructed by “knobs-into-holes” heterodimerization and then homogeneously conjugated with MMAE via a lysosomal cleavable valine-citrulline dipeptide linker. The resulting bispecific nanobody drug conjugate (NDC), NXV01c, was evaluated in multiple tumor cell lines and tumor xenograft models. NXV01, the pre-conjugate bispecific nanobody, bound EGFR and cMET with nanomolar potency (BLI) and inhibited the phosphorylation of cellular EGFR and cMET with nanomolar IC50 (ELISA). Moreover, it did not activate cellular cMET. Importantly, the NDC NXV01c is highly homogeneous: it has an average DAR of 3.87 and >95% of NXV01c has a DAR of 4. To examine stability of conjugation, NXV01C was incubated in human plasma (37°C) for 14 days, the maximum free drug release rate (by LC/MS/MS) is 0.68%, which is much lower than that of DS8201. In vitro, NXV01c was rapidly internalized into H1975 cells which co-expressed EGFR and cMET. It inhibited the growth of H1975 (lung) and SNU5 (gastric) cancer cells with picomolar activity but spared normal keratinocytes. NXV01c also inhibited the proliferation of additional cell lines derived from lung, gastric, esophageal, and liver cancer, and the level of inhibition positively associated with the expression density of both targets. In an H1975 cell line-derived xenograft model, NXV01C exhibited potent and dose-dependent anti-tumor activity. Treatments at 3 and 10 mg/kg once per week for 2 weeks led to shrinkage and complete regression of tumor, respectively. In patient-derived xenograft models of NSCLC and esophageal cancer, NXV01C led to tumor regression and elimination without noticeable toxic effects. EGFR × cMET bispecific nanobody drug conjugate NXV01c has favorable drug-like properties and demonstrated superior anti-tumor effect in vitro and in vivo. The results suggest NXV01C can be an effective solution for EGFR/cMET bearing tumors commonly found in diverse malignancies. Citation Format: Ran Wu, Puwei Yuan, Yang Xie, Jianxiu Guo, Fei Zhang, Fan Liu, Taylor B. Guo. Discovery and characterization of NXV01c, an EGFR × cMET bispecific nanobody drug conjugate with potent anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1871.

Abstract 3753: Co-expression proteomic analysis of antibody drug conjugate receptor and payload biomarkers in breast cancer

Abstract The use of antibody drug conjugates (ADC) targeting HER2, and Trop 2 has made a great difference in the treatment of breast cancer. Such agents consist of an antibody targeting the receptor which is conjugated to a payload chemotherapy agent. Chemotherapy involves the use of anti-tubulin agents (e.g. docetaxel), topoisomerase inhibitors etc. We examined 507 breast cancer samples using targeted proteomics to quantitate biomarkers for ADC targets such as HER2, HER3, EGFR and Trop2 in our clinical proteomics (CLIA) laboratory. We also measured biomarkers that are associated with response and biomarkers that are associated with resistance to chemotherapeutic agents. Resistance markers include TUBB3 (taxanes), and response markers include TOPO1 (irinotecan, topotecan). Methods: Formalin fixed, paraffin embedded (FFPE) tissues blocks of breast cancer were sectioned, marked for tumor, laser capture microdissected, digested and analyzed by LC-selected reaction monitoring mass spectrometry. Seventy-two proteins were quantitated. Discussion:In our previous work, we have determined that >740 amol/µg HER2 corresponds to IHC 3+/2+ and FISH +. Based on the HER2 quantitation for each sample, the samples were segregated into three groups. Negative (Group A, <150 amol/µg), High expressors (Group C, >740 amol/µg), and Detected (Group B, 151-739 amol/µg). Groups A, B, and C were 39%, 46%, and 43% of the cases respectively. In Group A (HER2 negative), HER3 was detected in 14% of cases with a 3x range (181-602 amol/µg), EGFR was detected in 53% of samples with 139x range (108-14986 amol/µg), Trop2 was detected in 59% of cases ranging from 256 to 11900 amol/µg, Topo1 was detected in 86% of cases with a 12x range (402-4870 amol/µg) and TUBB3 was detected in 63% of cases at levels ranging from 755 to 11750 amol/µg. In Group B (HER2 detected), HER3 was detected in 38% with a 3x range (178-565 amol/µg), EGFR was detected in 34% at levels ranging from 104-7040 amol/µg, Trop2 was detected in 63% ranging from 157 to 12808 amol/µg, Topo1 was detected in 94% with a 12x range (413-5100 amol/µg), and TUBB3 was detected in 51% of cases at levels ranging from 757 to 9311 amol/µg.In Group 3 (HER2 highly expressed), HER3 was detected in 43% with a 5x range (189- 890 amol/µg), EGFR was detected in 30% at levels ranging from 101 to 565 amol/µg, Trop2 was detected in 68% ranging from 310 to14650 amol/µg, Topo1 was detected in 95% with a 6x range (487- 2885 amol/µg), and TUBB3 was detected in 64% of samples at levels ranging from 829 to 2300 amol/µg. Conclusion:The capacity to multiplex 72 protein biomarkers from 2-3 FFPE sections will help in design of ADCs bispecific antibodies and/or bispecific ADCs. Citation Format: Negin Ghafourian, Sheeno P. Thyparambil, Richard Mortensen, James Chou, Robert Heaton, Xuefeng B. Ling. Co-expression proteomic analysis of antibody drug conjugate receptor and payload biomarkers in 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 3753.

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 2616: BCG033, a novel bispecific antibody-drug conjugate targeting PTK7 and TROP2, demonstrates preclinical efficacy against triple-negative breast cancer and other solid tumor xenografts

Abstract Metastatic triple-negative breast cancer (TNBC) patients have poor overall survival, highlighting the need for novel treatments. Although the TROP2-targeting ADC sacituzumab govitecan recently received accelerated approval from the FDA for the treatment of patients with metastatic TNBC, the on-target toxicity of this single-target agent has limited clinical efficacy. We sought to improve the specificity and efficacy of future TNBC-targeted therapies by generating a bispecific antibody-drug conjugate (bsADC) targeting TROP2 and PTK7, another tumor-associated antigen (TAA) that is highly expressed in TNBC and correlates with poor prognosis and metastatic disease. We previously generated fully human antibodies targeting PTK7 and TROP2 from Biocytogen’s fully human, common light chain antibody transgenic RenLite® mice, which were selected such that their monovalent antibodies exhibited reduced internalization profiles for improved tumor selectivity. These antibodies were then constructed into a bispecific antibody targeting PTK7 × TROP2, which demonstrated reactivity to human and cynomolgus monkey antigens and binding to multiple cancer cell lines, including TNBC, with high affinity. PTK7 × TROP2 bsAb also showed enhanced internalization in vitro compared with parental monovalent antibodies. Next, the PTK7 x TROP2 bsAb was conjugated to vcMMAE with a drug-to-antibody ratio (DAR) of 4 to generate a bsADC (BCG033). BCG033-vcMMAE demonstrated superior activity to benchmark and parental ADCs in a cell line-derived xenograft (CDX) TNBC model with low PTK7 expression. Here, we demonstrate that BCG033-vcMMAE ADCs also exhibited superior efficacy to benchmark ADCs in PDX models, including TNBC xenografts and NSCLC xenografts. We next tested the efficacy of the PTK7 x TROP2 bsAb conjugated to BLD1102, Biocytogen’s novel, proprietary linker/payload composed of a DNA Topo I inhibitor payload and a highly hydrophilic protease-cleavable linker, with a DAR of 8. BCG033-BLD1102 showed potent activity in a colorectal cancer PDX model in which benchmark ADCs were inactive, even at high doses. In summary, BCG033 conjugates demonstrate promising preclinical efficacy in vivo, which ultimately could provide a new treatment option for TNBC and other solid tumors expressing PTK7 and TROP2. Citation Format: Sufei Yao, Chengzhang Shang, Gao An, Chaoshe Guo, W. Frk an, Yi Yang. BCG033, a novel bispecific antibody-drug conjugate targeting PTK7 and TROP2, demonstrates preclinical efficacy against triple-negative breast cancer and other solid tumor 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 2616.

Abstract 2619: A novel EGFR × HER3-targeting bispecific antibody drug-conjugate, BCG019, demonstrates robust anti-tumor efficacy in preclinical evaluation

Abstract EGFR and HER3 are receptor tyrosine kinases that are highly expressed in multiple epithelial tumors. EGFR point mutations and small insertions within the kinase domain are common in lung cancer. HER3 is overexpressed in patients who are resistant to EGFR-TKI therapy, and is one of the common resistance mechanisms of EGFR and HER2-targeted therapy. We hypothesize that simultaneous targeting of EGFR and HER3 with a bispecific ADC will have the potential to overcome the resistance caused by HER3 after EGFR-targeted therapy and obtain better efficacy than the combination of EGFR monotherapy and HER3 monotherapy. We first generated fully human bispecific antibodies (bsAbs) targeting EGFR and HER3 using RenLite® mice, which contain the full human heavy chain variable domain with a common human kappa light chain to facilitate bispecific antibody assembly. The anti-EGFR × HER3 bsAb demonstrated reactivity to human and cynomolgus monkey antigens and binding to multiple cancer cell lines, including NSCLC, gastric, pancreatic, colorectal, and breast cancer cell lines. The anti-EGFR × HER3 bsAb also showed high internalization activity in cell lines expressing EGFR and HER3. The anti-EGFR × HER3 bsAb was then conjugated with vc-MMAE (valine-citrulline-monomethyl auristatin E) for proof-of-concept studies, as well as BLD1102, Biocytogen’s novel, proprietary linker/payload system composed of a DNA Topo I inhibitor payload (BCPT02) and highly hydrophilic protease-cleavable linker, to generate EGFR- and HER3-targeting bispecific ADC candidates BCG019. In vivo efficacy of the bsADC candidates were evaluated in cell-derived NSCLC and gastric cancer xenografts and in patient-derived gastric and colorectal xenograft models. Both bsADCs showed superior anti-tumor activity when compared to benchmark ADCs. Collectively, these results suggest that our novel anti-EGFR × HER3 bsADC has therapeutic potential for EGFR and HER3 co-expressing tumors. Citation Format: Zhuolin Li, Chengzhang Shang, Gao An, Chaoshe Guo, W. Frank An, Yi Yang. A novel EGFR × HER3-targeting bispecific antibody drug-conjugate, BCG019, demonstrates robust 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 2619.

Abstract 3126: A novel pegylated bispecific antibody-drug conjugate (P-BsADC) JY207b targeting cancers co-expressing PD-L1 and CD47

Abstract ADCs have showed improved efficacy and target selectivity comparing to the non-specific small molecule cytotoxicity drugs. However, conventional IgG based ADC has been hindered in solid tumor therapies by the poor tumor penetration, low internalization efficiency, unexpected efflux from tumor cells, and narrow therapeutic window due to the on target/off tumor toxicity, etc. To address these issues, we have developed the pegylated bispecific ADC platform and proved the concept with JY207, which targets PD-L1 and CD47 with a 30k PEG-MMAE linker-payload. JY207 has demonstrated advantages in tumor penetration, internalization efficiency, without efflux post internalization, lacking Fc related toxicity, and better tumor inhibition than DS-8201a in a model positive for Her2, PD-L1 and CD47. In this study, we further improved the compound by site-specific conjugating the fusion protein of two scFvs that target PD-L1 and CD47, with a 40K PEG-MMAE linker-payload. As expected, the new compound JY207b retained all the advantages of JY207. Additionally, JY207b did not bind to human red blood cells at all even though an ultra-sensitive detection method was employed, while CD47 antibodies with reported low affinity bound >90% red blood cells under our ultra-sensitive detection method. Meanwhile, JY207b could still preferentially bind to CD47/PD-L1 double positive tumor cells, thus reducing the possibility of on-target toxicities. In the in vitro cytotoxicity assays, the compound JY207b demonstrated strong potencies in CD47/PD-L1 double positive tumor cells, with almost no killing effect to CD47 or PD-L1 single positive tumor cells. In CDX models and a PDX model of transplanted tumor tissues from lung cancer patients, the compound JY207b demonstrated strong tumor inhibition efficacies at low doses. More interestingly, although the elimination half-life (T1/2) of JY207b in mice is 59.17±3.824 hours, the elimination time of JY207b in tumors is beyond 1 week by the IVIS imaging examination, which allows for weekly dosing in PK unfavorable mouse animals for pegylated protein drugs. Moreover, preliminary repeated-dosing toxicological study has found the maximum tolerated dose of JY207 in CD47/PD-L1 double transgenic mice to be 50mg/kg. These studies suggested favorable anti-tumor effect of JY207 both in vitro and in vivo, and its mechanism of action has been well described. These findings provide evidence for the efficacy of JY207 as a clinical treatment for patients with CD47/PD-L1 double positive cancers. Citation Format: Yu (Yvonne) Wen, Shumin Liu, Weidong Lyu, Yang Lei, Shuangyu Tan, Zibin Wu, Shuqiang Yin, Qiudong Zhuo, Dechun Wu. A novel pegylated bispecific antibody-drug conjugate (P-BsADC) JY207b targeting cancers co-expressing PD-L1 and CD47 [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 3126.

Abstract 2617: BCG022, a novel HER3 × MET bispecific antibody-drug conjugate (bsADC), demonstrates promising anti-tumor efficacy

Abstract HER3 and MET expression act as bypass resistance mechanisms, conferring resistance to multiple therapeutic agents, such as EGFR TKI treatment. HER3 and MET are also co-expressed at high prevalence in multiple tumor types, including gastric, colorectal, breast, and non-small-cell lung cancer (NSCLC). In addition, HER3 and MET are frequently overexpressed in liver metastases from patients with colorectal cancer, indicating that targeting both targets may provide clinical benefit. We previously reported generation of a fully human bispecific antibody (bsAb) targeting HER3 and MET using RenLite® mice, which contain the full human heavy chain variable domain with a common human kappa light chain to facilitate bispecific antibody assembly. This bsAb demonstrated reactivity to human and cynomolgus monkey antigens and binding to multiple cancer cell lines, including NSCLC, gastric, colorectal, and hepatocellular cancer cell lines. The bsAb also showed enhanced internalization activity compared to parental monovalent antibodies in the NUGC-4 cell line expressing HER3 and MET. Here, we evaluated the efficacy of the bsAb when conjugated to two distinct payloads: vcMMAE and BLD1102, Biocytogen’s novel, proprietary linker/payload system composed of a DNA Topo I inhibitor payload (BCPT02) and a highly hydrophilic protease-cleavable linker. The in vivo efficacy of BCG022-vcMMAE and BCG022-BLD1102 was evaluated in cell-derived NSCLC and gastric cancer xenografts, and in patient-derived gastric and pancreatic xenograft models. Both demonstrated strong anti-tumor activity, with efficacy greater than or comparable to benchmark ADCs. Taken together, these promising preclinical results suggest that the BCG022 bsADC could be a novel treatment for HER3 and MET co-expressing tumors. Citation Format: Zhuolin Li, Chengzhang Shang, Zhenyan Han, Gao An, Chaoshe Guo, W. Frank An, Yi Yang. BCG022, a novel HER3 × MET bispecific antibody-drug conjugate (bsADC), demonstrates promising anti-tumor efficacy [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 2617.

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.