Drug Conjugate Targeting Research Articles

Detection of SEZ6, a therapeutic target, in medullary thyroid carcinoma.

Seizure-related 6 homolog (SEZ6) is a cDNA that strongly associated with neuroendocrine differentiation. Recently, SEZ6 expression was found in a subset of small cell lung carcinoma (SCLC). Furthermore, ABBV-011, a novel antibody drug conjugate targeting SEZ6 has been developed and is currently in clinical trial in treating SCLC and neuroendocrine neoplasms, including medullary thyroid carcinoma (MTC). We herein presented the first evidence that SEZ6 was highly expressed in MTC. SEZ6 immunoexpression was studied in 78 MTCs and correlated with clinicopathologic characteristics, outcome, and molecular profile. SEZ6 was highly expressed in primary tumors, regional recurrence, and distant metastasis. Using two different SEZ6 antibody clones SC17.14 and 14E5, SEZ6 immunopositivity was seen in 91% to 93% of primary MTCs, 100% of regional recurrence, and 75% to 83% of distant metastasis. High level of SEZ6 immunoexpression determined using H score was associated with male sex, advance stage, and extrathyroidal thyroidal extension. There was no correlation between SEZ6 expression and outcome or RET/RAS mutation status in MTC. The frequency of SEZ6 positivity in MTC without RET/RAS mutations were 83%. SEZ6 may serve as a novel biomarker for MTCs. Although SEZ6 lacks any prognostic values in MTC, its positivity in 91% to 93% of MTCs, including MTCs without RET and RAS mutations, renders SEZ6-targetted antibody-drug conjugate therapy a promising targeted therapy for MTCs.

437TiP An open-label, multicenter, phase II study to evaluate the safety and efficacy of BB-1701, a novel antibody drug conjugate (ADC) targeting HER2, in previously treated patients (pts) with HER2+ or HER2-low unresectable or metastatic (M) breast cancer (BC)

437TiP An open-label, multicenter, phase II study to evaluate the safety and efficacy of BB-1701, a novel antibody drug conjugate (ADC) targeting HER2, in previously treated patients (pts) with HER2+ or HER2-low unresectable or metastatic (M) breast cancer (BC)

DB-2304, a Duality Immune Modulating Antibody‒Drug Conjugate (DIMAC) targeting BDCA2, displays strong potency in the suppression of pDC functions.

Abstract Study Objective: BDCA2 is specifically expressed on plasmacytoid dendritic cells (pDCs), whose overproduction of type I IFN is crucial in SLE pathogenesis. Combining anti-BDCA2 mAB with glucocorticoids (GCs) has shown clinical efficacy. Previous studies revealed superior anti-inflammatory effects of anti-BDCA2 antibody‒drug conjugate (ADC), motivating the development of DB-2304, an anti-BDCA2 ADC using a novel glucocorticoid receptor agonist as a payload. This study assessed the preclinical efficacy and safety of DB-2304. Methods: A novel BDCA2 mAB, Hu033-03, was developed and conjugated with the DIMAC payload to generate DB-2304. The inhibitory potency of DB-2304 was evaluated in human PBMCs in vitro. The PK and safety profile and pDC function suppression were characterized in cynomolgus monkeys. Results: Hu033-03 exhibited high affinity for BDCA2, inducing rapid internalization upon binding. DB-2304 showed enhanced suppression of IFNa production and a broader spectrum of proinflammatory cytokines in human PBMCs without cytotoxicity. Reduction in pDC cytokine production capability and surface BDCA2 level occurred in an ADC serum concentration-dependent manner. DB-2304 exhibited a favorable safety profile, with an HNSTD of 125 mg/kg in cynomolgus monkeys. Conclusions: DB-2304 has demonstrated potent in vitro and in vivo anti-inflammatory effects on type I IFN genes and proinflammatory cytokines in pDC. These findings strongly support future clinical investigations of DB-2304.

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 2615: Preclinical development of a next generation antibody drug conjugate (ADC) targeting cMET for treatment of solid tumors

Abstract cMET (mesenchymal-epithelial transition factor), which belongs to the MET family, is a type of receptor tyrosine kinase that is expressed on the surfaces of various epithelial cells. The elevation of cMET can promote the development and progression of multiple cancers. cMET- ADCs have shown promising clinical activity with highest cMET levels, indicating tumor cMET levels may be limited for efficacy. YL211, a novel cMET-ADC was developed by leveraging on MediLink’s tumor microenvironment activable linker-payload platform (TMALIN platform), which could release the payload both in the tumor cell and in the tumor microenvironment. YL211 is comprised of an anti-cMET humanized monoclonal antibody conjugated to a novel topoisomerase 1 inhibitor via a protease-cleavable linker. The novel linker-payload and site-specific conjugation of YL211 results in a homogeneous product with a DAR (Drug-to-Antibody Ratio) of 8. Furthermore, the advancement in linker-payload design has facilitated the development of more hydrophilic ADCs, which exhibit minimal impact on the antibody's characteristics while being less prone to aggregation and showcasing lower systematic clearance. Along with release of the payload in the tumor microenvironment extracellularly, upon binding to cMET on tumor cell surfaces, the antigen-ADC complexes are internalized to lysosomal vesicles and subsequently release the toxic payload. The cytotoxic features drive cell cycle arrest, apoptosis, elevation of PARP/caspase 7 expression, as well as cell death and bystander effect. These findings translated in vivo where YL211 showed superior efficacy in causing tumor regressions and good tolerability in both cell lines and patient-derived xenograft (CDX and PDX) mouse models with both low to high cMET expression levels (H358, CR5088, ect.). The in vivo pharmacokinetics in monkeys showed that YL211 is highly stable with less than 0.1% (molar ratio) payload released in circulation. GLP safety evaluations demonstrated good safety profiles and no off-target toxicity in monkeys. These non-clinical data suggest the stability of the linker in circulation as well as efficient release of the payload in tumors with improved therapeutic window. Taken together, preclinical data suggest that YL211 could be a promising treatment strategy for cMET positive cancer patients. Citation Format: Liang Xiao, Wei Lian, Shuai Song, Qigang Liu, Qing Zong, Sasha Stann, Jiaqiang Cai, Tongtong Xue. Preclinical development of a next generation antibody drug conjugate (ADC) targeting cMET for treatment of solid 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 2615.

Abstract 3141: LCB02A an antibody drug conjugate (ADC) targeting CLDN18.2 expressing solid tumors

Abstract Claudin18.2 (CLDN18.2) is an isoform of CLDN18 with very limited expression in gastric epithelial cells in normal tissues, and aberrant overexpression in gastric, pancreatic, esophageal, ovarian, lung, and other solid tumors. This expression pattern presents a significant hurdle in developing an ADC targeting CLDN18.2 due to potential on-target toxicity to the gastrointestinal (GI) tract. LCB02A is an antibody-drug conjugate (ADC) that targets CLDN18.2, composed of a topoisomerase 1 inhibitor (TOPO1i) as payload (DAR4) linked to the CLDN18.2 specific binding PR301839 hClaudin18.2 (CLDN18.2) is an isoform of CLDN18 with very limited expression in gastric epithelial cells in normal tissues, and aberrant overexpression in gastric, pancreatic, esophageal, ovarian, lung, and other solid tumors. This expression pattern presents a significant hurdle in developing an ADC targeting CLDN18.2 due to potential on-target toxicity to the gastrointestinal (GI) tract. LCB02A is an antibody-drug conjugate (ADC) that targets CLDN18.2, composed of a topoisomerase 1 inhibitor (TOPO1i) as payload (DAR4) linked to the CLDN18.2 specific binding PR301839 humanized IgG1 antibody utilizing LegoChem Biosciences’ ConjuAllTM technology. LCB02A showed potent in vitro cytotoxicity in CLDN18.2 expressing gastric and pancreatic cancer cell lines. LCB02A also showed excellent anti-tumor efficacy in various gastric and pancreatic cell line-derived xenograft (CDX) models and patient-derived xenograft (PDX) models. Preliminary toxicity assessment in cynomolgus monkeys (CLDN18.2 cross-reactive species) demonstrated that LCB02A was well tolerated, with repeat doses of up to 60 mg/kg. In conclusion, LCB02A is a promising ADC for the treatment of CLDN18.2 expressing solid tumors including gastric cancer (GC) and pancreatic ductal adenocarcinoma (PDAC). umanized IgG1 antibody utilizing LegoChem Biosciences’ ConjuAllTM technology. LCB02A showed potent in vitro cytotoxicity in CLDN18.2 expressing gastric and pancreatic cancer cell lines. LCB02A also showed excellent anti-tumor efficacy in various gastric and pancreatic cell line-derived xenograft (CDX) models and patient-derived xenograft (PDX) models. Preliminary toxicity assessment in cynomolgus monkeys (CLDN18.2 cross-reactive species) demonstrated that LCB02A was well tolerated, with repeat doses of up to 60 mg/kg. In conclusion, LCB02A is a promising ADC for the treatment of CLDN18.2 expressing solid tumors including gastric cancer (GC) and pancreatic ductal adenocarcinoma (PDAC). Citation Format: Eun ji Baek, Chul-Woong Chung, Changsik Park, Taeik Jang, Yeojin Jeong, Sang-Ah Lee. LCB02A an antibody drug conjugate (ADC) targeting CLDN18.2 expressing solid 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 3141.

Abstract 1916: Discovery of a novel Nectin4 IPSC-derived cell therapy for the treatment of solid tumors

Abstract Century Therapeutics is developing a Nectin4-targeting IPSC-derived cell therapy for the treatment of Nectin4 expressing solid tumors. The Nectin proteins (Nectin1, Nectin2, Nectin3 and Nectin4) are members of the calcium independent immunoglobulin superfamily of cell adhesion molecules (CAMs) involved in adhesive processes that help in the growth and development of tissues. Nectin 1, 2 and 3 are normally found in adult tissue whereas Nectin4 is mainly found in normal placental and embryonic tissue. Nectin4 is also expressed in different types of cancer including urothelial, ovarian, lung and head/neck cancers. Nectin4 is established as a biomarker for worse prognosis in cancers and is linked with carcinogenesis and disease severity. These features establish Nectin4 as a promising target for anti-tumor drug development. Enfortumab vedotin, an antibody drug conjugate targeting Nectin4 positive tumors, has demonstrated benefit in clinical trials and is FDA approved for treatment of advanced bladder cancer. We have identified novel single-domain antibodies (VHH) that bind to multiple epitopes on Nectin4 extracellular domain. The VHH antibodies were engineered into chimeric antigen receptor (CAR) formats and characterized for expression, cell activation through antigen engagement, and cytotoxicity activity in primary T-cells. Selected binders demonstrated efficacy in multiple CAR formats in primary T-cells in a mouse xenograft model using OVCar-3 tumor cells. The lead CARs engineered into primary T-cells demonstrated tumor inhibition similar to a reference CAR using the ASG-5ME antibody (Enfortumab) as the Nectin4 binder. The CARs were engineered into our IPSC-derived iNK and iT cells and demonstrated cytotoxicity activity against a panel of cell lines with a range of cell surface expression of Nectin4. Based on these studies, Century Therapeutics is advancing the lead Nectin4 binder for development of an IPSC-derived cell therapy to treat Nectin4 positive solid tumors. Citation Format: Jill M. Carton, Chris Dower, Michael Miller, John Wheeler, Sean Heron, Hillary J. Millar, David Walker, Daniel J. Perry, Andriana Lebid, Luis Cocka, Dae Hwan Kim, Barry Morse, Buddha Gurung, Maisha Harris, Annalise Jethon, Michael Naso, Hy Levitsky. Discovery of a novel Nectin4 IPSC-derived cell therapy for the treatment of solid 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 1916.

Safety and efficacy of enfortumab vedotin in patients with metastatic/locally advanced urothelial cancer: Real-world evidence from a European database.

553 Background: Enfortumab vedotin (EV) is an antibody drug conjugate targeting Nectin-4. It was approved by EMA/FDA in patients (pts) with metastatic/ locally advanced urothelial cancer post platinum and immune check point inhibitors following the results of the EV-301 trial. We report updated efficacy and safety data of EV in a large European cohort of real-world pts (GUARDIANS consortium) treated in hospitals and private practices. Methods: Retrospective data were collected from 25 German and Swiss hospitals and private practices for pts who received EV. Adverse events (AEs) were reported according to Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 criteria. Objective responses were evaluated by local investigators according to Response Evaluation Criteria in Solid Tumors version 1.1. Progression-free survival (PFS) and overall survival (OS) were estimated using the Kaplan-Meier method. Results: Weidentified 188 pts (32.4% female) with a median age of 66 yrs (range 31-89; 22.3% >/= 75 yrs). Eastern Cooperative Oncology Group performance status (ECOG PS) was 0/1/2/3-4/unknown in 36/39/11/3/11%. EV was administered in the fourth or later line in 43% of pts. Overall response rate (ORR) was 46.3% (partial remission: 42.0%, complete response 4.3%), disease control rate was 58.0%. Median OS (mOS) was 12.0 months (mo) (95% confidence interval 9.65-14.35) and median PFS (mPFS) was 7.0 mo (95% confidence interval 5.43-8.57). Any-grade AEs were observed in 71% and CTCAE grade ≥3 AEs in 32% of pts. Most common AEs were peripheral sensory neuropathy (33.5% any grade), skin toxicity (24.5%) and fatigue (22.9%). In pts >/= 75 yrs, females, and pts treated in >/=4th line mOS, mPFS, ORR, and toxicities were comparable to younger and male counterparts and pts treated in <4th line, respectively. Limitations are retrospective design and short follow-up. Conclusions: Anti-tumor activity of EV in real-world pts including difficult-to-treat subgroups is comparable to the results of the pivotal EV-301 trial. No new safety signals were observed.

Metabolite characterisation of the peptide–drug conjugate LN005 in liver S9s by UHPLC-Orbitrap-HRMS

LN005 is a peptide–drug conjugate (PDC) targeting glucose-regulated protein 78 (GRP78) to treat several types of cancer, such as breast, colon, and prostate cancer. As a new drug modality, understanding its metabolism and elimination pathways will help us to have a whole picture of it. Currently, there are no metabolic studies on LN005; therefore, this study aimed to investigate the metabolism of LN005, clarify its metabolic profile in the liver S9s of different species, and identify the major metabolic pathways and differences between species. The incubation samples were measured by ultra-high performance liquid chromatography combined with orbitrap tandem mass spectrometry (UHPLC-Orbitrap-HRMS). The results showed that LN005 was metabolised by liver S9s, and four metabolites were identified. The main metabolic pathway of LN005 in liver S9s was oxidative deamination to ketone or hydrolysis. Similar metabolic profiles were observed in mouse, rat, dog, monkey, and human liver S9s, indicating no differences between these four animal species and humans. This study provides information for the structural modification and optimisation of LN005 and affords a reference for subsequent animal experiments and human metabolism of other PDCs.

Mirvetuximab Soravtansine in FRα-Positive, Platinum-Resistant Ovarian Cancer

BackgroundMirvetuximab soravtansine-gynx (MIRV), a first-in-class antibody–drug conjugate targeting folate receptor α (FRα), is approved for the treatment of platinum-resistant ovarian cancer in the United States.MethodsWe conducted a phase 3, global, confirmatory, open-label, randomized, controlled trial to compare the efficacy and safety of MIRV with the investigator’s choice of chemotherapy in the treatment of platinum-resistant, high-grade serous ovarian cancer. Participants who had previously received one to three lines of therapy and had high FRα tumor expression (≥75% of cells with ≥2+ staining intensity) were randomly assigned in a 1:1 ratio to receive MIRV (6 mg per kilogram of adjusted ideal body weight every 3 weeks) or chemotherapy (paclitaxel, pegylated liposomal doxorubicin, or topotecan). The primary end point was investigator-assessed progression-free survival; key secondary analytic end points included objective response, overall survival, and participant-reported outcomes.ResultsA total of 453 participants underwent randomization; 227 were assigned to the MIRV group and 226 to the chemotherapy group. The median progression-free survival was 5.62 months (95% confidence interval [CI], 4.34 to 5.95) with MIRV and 3.98 months (95% CI, 2.86 to 4.47) with chemotherapy (P<0.001). An objective response occurred in 42.3% of the participants in the MIRV group and in 15.9% of those in the chemotherapy group (odds ratio, 3.81; 95% CI, 2.44 to 5.94; P<0.001). Overall survival was significantly longer with MIRV than with chemotherapy (median, 16.46 months vs. 12.75 months; hazard ratio for death, 0.67; 95% CI, 0.50 to 0.89; P=0.005). During the treatment period, fewer adverse events of grade 3 or higher occurred with MIRV than with chemotherapy (41.7% vs. 54.1%), as did serious adverse events of any grade (23.9% vs. 32.9%) and events leading to discontinuation (9.2% vs. 15.9%).ConclusionsAmong participants with platinum-resistant, FRα-positive ovarian cancer, treatment with MIRV showed a significant benefit over chemotherapy with respect to progression-free and overall survival and objective response. (Funded by ImmunoGen; MIRASOL ClinicalTrials.gov number, NCT04209855.)

Abstract B128: Preclinical characterization of ETx-22, a next-generation antibody drug conjugate (ADC) targeting nectin-4

Abstract Nectin-4 is overexpressed in a variety of cancers including urothelial, breast, cervix, lung and ovarian, with low level of expression in normal tissues. Enfortumab vedotin (EV), an anti-Nectin-4 ADC with a monomethyl auristatin (MMAE) payload, has been a significant advancement in the treatment of urothelial cancer. Yet, it is associated with important skin toxicity owing to the expression of nectin-4 in the skin, and peripheral neuropathy secondary to its toxin. We have developed a next-generation anti-nectin-4 ADC, ETx-22, which consists of a humanized IgG1, Fcg-silent antibody that is bound via maleimide-ß-glucuronide poly-sarcosine linkers to 8 molecules of the topoisomerase-I inhibitor, exatecan. Here, we describe the key pharmacological experiments, in-vivo efficacy and results from the good laboratory practice (GLP) toxicology studies. In-vitro studies have shown that the ETx-22 antibody exhibits a high affinity for tumor-expressed Nectin-4, but 10-fold lower affinity for nectin-4 expressed on skin, with significantly higher internalization in tumor cells. In-vivo efficacy was evaluated in various patient-derived xenograft models. ETx-22 was shown to be consistently superior to EV as well as chemotherapy, in a broad array of tumors, including urothelial, ovarian, cervical and triple negative breast cancer irrespective of the degree of nectin-4 expression. Antitumor activity was also observed in MMAE-resistant models. GLP studies evaluated repeated doses of ETx-22 in cynomolgus monkeys at two doses, 10 and 20 mg/kg. The treatment was administered as 30-minute intravenous infusions once every 2 weeks for a total of three cycles. The ETx-22 antibody alone was also evaluated at 20 mg/kg. Both ETx-22 doses were well tolerated and all animals survived up to scheduled euthanasia. The common observation at both doses was mild multifocal skin hyperpigmentation, which was reversible. No skin rash or pruritus was reported. Mild transient decrease in red blood cells was observed in between cycles. No gastrointestinal toxicities were observed. No signs of interstitial lung disease or eye toxicity were detected. Based on these findings, the highly non- severely toxic dose (HNSTD) is at least 20 mg/kg. The half-life of ETx-22 ranged from 3.36 – 3.6 days. In conclusion, preclinical and pharmacological shows that ETx-22 has better tolerance, longer half-life, and higher efficacy to EV. No skin rash was reported in toxicology studies. A first in human study (EXCEED) is planned to start in 2023. Citation Format: Hatem Azim, Florence Lhospice, Xavier Preville, Joanna Fares, Marc Lopez, Emerence Crompot, Emmanuelle Josselin, Remy Castellano, Maria Wehbeh, Yves Collette, Jack Elands, Daniel Olive. Preclinical characterization of ETx-22, a next-generation antibody drug conjugate (ADC) targeting nectin-4 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B128.

Abstract B133: Anti-tumor efficacy of GMF-1A3-MMAE, an antibody drug conjugate targeting cell surface cleaved Amphiregulin in endocrine-resistant breast cancer

Abstract The Epidermal Growth Factor Receptor ligand, Amphiregulin, is a key proliferative effector of estrogen receptor signaling in breast cancer and also plays a role in other malignancies. Amphiregulin is a single-pass transmembrane protein proteolytically processed by TACE/ADAM17 to release the soluble EGFR ligand, leaving a residual transmembrane stalk that is subsequently internalized. Here, we describe the development of an antibody drug conjugate, GMF-1A3-MMAE, targeting an AREG neo-epitope revealed following ADAM17-mediated cleavage. The antibody does not interact with uncleaved Amphiregulin, providing a novel means of targeting cells with high rates of Amphiregulin shedding. Using fluorescent dye conjugation, we demonstrated that the antibody is internalized by cancer cells in a manner dependent on the presence of cell surface cleaved Amphiregulin. Antibodies conjugated with monomethyl auristatin E (MMAE) were cytotoxic in vitro and induced rapid regression of established breast tumor xenografts in immunocompromised mice. We further demonstrate that these antibodies recognize the Amphiregulin neo-epitope in formalin fixed paraffin embedded tumor tissue, suggesting their utility as a companion diagnostic for patient selection. A pan-cancer tissue microarray analysis indicates that the target is commonly detected at high levels (&amp;gt; 50% of cases) in breast, prostate, liver and lung cancer. In normal tissue microarrays, cleaved Amphiregulin was generally detected at moderate levels in several tissues with only 7 of 93 tissue cores having levels comparable to the high levels commonly found in tumors. These data suggest therapeutic opportunities for GMF-1A3-based ADCs in multiple malignancies, however the broad off-tumor detection of cleaved Amphiregulin suggest linker/payload optimization may be necessary for an adequate therapeutic window. Citation Format: Kristopher A Lofgren, Paraic A Kenny. Anti-tumor efficacy of GMF-1A3-MMAE, an antibody drug conjugate targeting cell surface cleaved Amphiregulin in endocrine-resistant breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B133.

Debio 1562M, a Next Generation Antibody Drug Conjugate (ADC) Targeting CD37 for AML and MDS Treatment

CD37 is a trans-membrane protein specifically expressed on normal hematopoietic tissues such as B cells, neutrophils and macrophages, and has demonstrated roles in adhesion, cell migration (de Winde et al, 2018; Wee et al, 2015), survival and apoptotic signals (Lapalombella et al, 2012). Increased expression of CD37 has been observed in various hematological cancers such as lymphomas, Chronic Lymphocytic Leukemia (CLL) and Acute Myeloid Leukemia (AML) (Beckwith et al, 2014; Deckert et al, 2013; Pereira et al, 2015; Yan et al, 2021). Higher expression of CD37 was also associated with poor patient outcome in AML (Zhang et al, 2020; Larkin et al, ASH 2018). Debio 1562M is a second-generation novel ADC directed against CD37 and including Debiopharm's proprietary Multilink cleavable linker-payload technology. Multilink improves manufacturing to achieve homogenous high drug antibody ratio while retaining excellent solubility as well as favorable biologic characteristics with preserved internalization kinetics on target cells. Debio 1562M is conjugated to 8 molecules of a tubulin-binding agent and despite cleavable linker that ensure effective intracellular delivery of toxic payload, we show sustained plasma stability in vitro and in vivo with no premature extracellular release. In this work, we report Debio 1562M specificity and potent antitumor activity in multiple AML models both in vitro and in vivo. First, in our in vitro cytotoxicity assay, Debio 1562M displayed nano- to picomolar anti-proliferative activity in both AML cell lines and primary samples. Out of 30 primary samples tested, 20 were sensitive to Debio 1562M with IC50 ranging from 1 to 10nM, this regardless of their mutation status, subtype or medical history. Interestingly, we observed that AML cells have high internalization capability and therefore relatively low levels of surface CD37 is sufficient for cytotoxic activity. The specificity of Debio 1562M was demonstrated by using 3 different CD37 knock out AML cell lines which were generated by CRISPR-Cas9 technology. Parental and CD37 KO cells were treated for 72h with a dose range of Debio 1562M and viability was measured (Figure 1). While Debio 1562M has an IC50 below 1nM in all 3 parental cell lines, its activity is reduced by more than 2 log in the KO cell lines. In vivo, in 3 different AML cell line xenograft mouse models, a single injection of Debio 1562M at 5mg/kg induced complete tumor regression and all mice remained alive at study termination, more than 60 days after treatment, except one mouse in one model. In a patient derived xenograft (PDX) model, the same dose of Debio 1562M significantly reduced tumor burden in blood and bone marrow with sustained activity 42 days after treatment (37% and 98% of blasts in blood and bone marrow respectively in control animals versus 0% and 3% in treated mice, pvalue&amp;lt;0.001). Furthermore, we evaluated the activity of Debio 1562M over venetoclax and azacytidine combination in the MV4-11 Luciferase xenografted mouse model. NCG mice were inoculated with 1x10 7 cells in the tail vein and randomization in groups of 8 mice was performed 14 days later. Mice received either one administration of Debio 1562M at 5mg/kg or a combination of venetoclax (QDx14 100mg/kg) and azacitidine (QDx5 3,5mg/kg). Complete regression of the tumor was observed in all mice after Debio 1562M administration and all mice survived at study termination, 75 days after treatment while none survived after 56 days when treated with venetoclax and azacitidine combination (Figure 2). The toxicology profile of Debio 1562M in mice showed expected clinical signs related to the payload's off target toxicity but with a significant safety margin. Interestingly, no peripheral neuropathy symptoms were detected, as commonly observed for this class of payload. Additionally, Multilink TM-payload construct alone was not associated with any signs of toxicity. On-target toxicity profile couldn't be evaluated as Debio 1562M isn't cross reactive with mouse CD37, however 1 st generation CD37 ADC has already demonstrated safety and tolerability in Phase 1 and 2 clinical trials.Overall, our data confirm the relevance of CD37 targeting for AML treatment. The excellent antitumor activity and tolerability of Debio 1562M in several pre-clinical models now pave the way for upcoming clinical development.

CD46-ADC Reduces the Engraftment of Multiple Myeloma Patient-Derived Xenografts.

An antibody-drug conjugate (ADC) targeting CD46 conjugated to monomethyl auristatin has a potent anti-myeloma effect in cell lines in vitro and in vivo, and patient samples treated ex vivo. Here, we tested if CD46-ADC may have the potential to target MM-initiating cells (MM-ICs). CD46 expression was measured on primary MM cells with a stem-like phenotype. A patient-derived xenograft (PDX) model was implemented utilizing implanted fetal bone fragments to provide a humanized microenvironment. Engraftment was monitored via serum human light chain ELISA, and at sacrifice via bone marrow and bone fragment flow cytometry. We then tested MM regeneration in PDX by treating mice with CD46-ADC or the nonbinding control-ADC. MM progenitor cells from patients that exhibit high aldehyde dehydrogenase activity also have a high expression of CD46. In PDX, newly diagnosed MM patient samples engrafted significantly more compared to relapsed/refractory samples. In mice transplanted with newly diagnosed samples, CD46-ADC treatment showed significantly decreased engraftment compared to control-ADC treatment. Our data further support the targeting of CD46 in MM. To our knowledge, this is the first study to show preclinical drug efficacy in a PDX model of MM. This is an important area for future study, as patient samples but not cell lines accurately represent intratumoral heterogeneity.

Generation of a Novel SORT1×HER2 Bispecific Antibody-Drug Conjugate Targeting HER2-Low-Expression Tumor.

Human epidermal growth factor receptor 2 (HER2) is considered an ideal antibody-drug conjugate (ADC) target because the gene is overexpressed in many tumors compared to normal tissues. Multiple anti-HER2 ADCs conjugated with different toxic payloads bring benefits to patients with high HER2 expression. However, HER2-targeted ADC technology needs further optimization to improve its effect for the treatment of patients with low HER2 expression. We hypothesized that bispecific antibody-drug conjugate (bsADC) targeting HER2 and Sortilin-1 (SORT1) would overcome this limitation. SORT1 is a suitable target for pairing with HER2 to generate a bispecific antibody (BsAb) since the gene is co-expressed with HER2 in tumors and possesses rapid internalization. We developed a BsAb (bsSORT1×HER2) that exhibited strong binding and internalization activity on HER2-low-expression tumor cells and facilitated higher HER2 degradation. The bsSORT1×HER2 was further conjugated with DXd to generate a bsADC (bsSORT1×HER2-DXd) that showed strong cytotoxicity on HER2-low-expression tumor cells and antitumor efficacy in an MDA-MB-231 xenograft mice model. These results demonstrated that employment of a SORT1×HER2-targeted bsADC may be promising to improve the antitumor efficacy of HER2-targeted ADC for the treatment of tumors with low HER2 expression.

A Novel Fc-Optimized Antibody Drug Conjugate Targeting CD7 As a Therapeutic Strategy in T-Cell Acute Lymphoblastic Leukemia

Despite progress in improving treatment regimens for patients with T-cell acute lymphoblastic leukemia (T-ALL), the therapeutic options are still limited, and especially antibody-based immunotherapy is not established. The CD7 antigen represents a promising target structure in T-ALL since it is strongly expressed in different T-ALL subtypes including early T-cell precursor (ETP)-ALL. Therefore, different approaches are currently pursued for targeting CD7, including CAR-T cell therapy. Due to its high internalization capacity CD7 also represents an ideal target structure for antibody drug conjugates (ADC). Here, a novel antibody engineering approach for CD7-targeting was evaluated in vitro and in xenograft mouse models of T-ALL. A CD7 antibody was optimized for its ability to trigger antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) by introducing two amino acid substitutions (S239D/I332E; DE-variant) in the Fc-domain. In addition, the Fc-engineered antibody was conjugated to the microtubule-disrupting agent monomethyl auristatin E (MMAE) via an enzymatic-cleavable linker (mc-vc-PABC). The Fc-optimized ADC, CD7-DE-vcMMAE, as well as the unconjugated antibody, CD7-DE, showed improved binding to activating Fcγ receptors (FcγRIIa, FcγRIIIa) compared to a CD7 antibody lacking the DE-modification. This resulted in potent ADCC- and ADCP-activity against different T-ALL cell lines mediated by NK-cells or macrophages as effector cells (EC 50 ADCC, 1-7 pM). In contrast to the CD7-DE antibody, CD7-DE-vcMMAE showed strong cytotoxic effects independently of immune effector cell engagement by releasing its cytotoxic compound into T-ALL cells in an antigen-restricted manner, thereby inducing G2/M cell cycle arrest and apoptosis. CD7-DE-vcMMAE was active at subnanomolar concentrations demonstrating dose-dependent cytotoxic effects in six T-ALL cell lines (IC 50 0.2 - 1 nM). The extent of maximum growth inhibition ranged between 54-98 % and correlated with CD7 antigen density. Yet, although CD7-DE-vcMMAE did not kill CD7-negative cells directly, the linker design facilitated bystander killing activity. Thus, in co-culture experiments using CEM cells and CEM-CD7-knockout cells mimicking CD7-antigen escape, the ADC demonstrated significant killing of neighbouring CD7-negative cells, thereby extending its mode of action. The antitumor activity of the CD7-ADC was further investigated in xenograft mouse models of T-ALL. In a first model, CEM cells were injected subcutaneously into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice and CD7-DE-vcMMAE treatment was evaluated in comparison to the unconjugated antibody CD7-DE. Treatment with CD7-DE-vcMMAE led to a significantly reduced tumor growth in comparison to CD7-DE or untreated animals (p&amp;lt;0.05; see panel A). A preclinical phase II-like patient-derived xenograft (PDX) study employing eight randomly selected T-ALL-PDX samples from pediatric and adult patients was conducted. PDX-cells were injected intravenously into NSG mice and treatment was started when the leukemia load reached 1 % human blasts in the peripheral blood, reflecting an overt leukemia situation. Animals receiving therapy with CD7-DE-vcMMAE showed significant prolongation of median survival in comparison to animals treated with a similarly designed control ADC targeting an irrelevant antigen (control-DE-vcMMAE) or which were left untreated (median survival time; CD7-DE-vc-MMAE: 82 days; untreated/control-DE-vcMMAE: 60 days; p&amp;lt;0.05; see panel B). Importantly, no leukemic blasts were found in the peripheral blood, spleen or bone marrow in animals treated with CD7-DE-vcMMAE and surviving the experimental period of 150 days. Together, the novel ADC CD7-DE-vcMMAE showed a unique set of Fc effector functions, potent direct growth inhibitory effects, bystander killing activity and efficacy in xenograft models of T-ALL. These results exhibit CD7-DE-vcMMAE as a promising therapeutic strategy and form the basis for new approaches in the treatment of T-ALL.

A Potential Antibody–Drug Conjugate Targeting Human LIV1 for the Treatment of Triple-Negative Breast Cancer

AbstractTriple-negative breast cancer (TNBC), which accounts for 15 to 20% of incidents of breast cancer, is the only breast cancer subtype that lacks targeted treatments. It was reported in the literature that LIV1 was highly expressed in TNBC and other solid tumors. This makes LIV1 a potential target for the treatment of TNBC. This study aimed to develop an anti-LIV1 antibody for the treatment of TNBC. In this study, a novel anti-LIV1 antibody Ab1120 was developed and conjugated with monomethyl auristatin E (MMAE) to obtain the antibody–drug conjugate, Ab1120-vcMMAE. The Cell Counting Kit-8 method was used to assess the killing effect of the antibody–drug conjugate on cell lines MDA-MB−231 (high LIV1 expression of breast cancer cell line), MDA-MB-468 (low LIV1 expression of breast cell line), and 293C18 (LIV1-negative human embryonic kidney cell). The antitumor effect of Ab1120-vcMMAE on an MDA-MB-231 xenograft model was determined by evaluating the tumor volume and body weight after its treatment. In vitro analysis showed that Ab1120-vcMMAE is a potent inhibitor against the proliferation of a LIV1 overexpression cell line. The in vivo results demonstrated its antitumor activity in the cell-derived xenograft breast tumor mouse model. The results of this study suggest that Ab1120-vcMMAE may be used as a new therapeutic drug for patients with LIV1 high-expression breast cancer.

Preclinical radiolabeling, in vivo biodistribution and positron emission tomography of a novel pyrrolobenzodiazepine (PBD)-based antibody drug conjugate targeting ASCT2

IntroductionAnti-ASCT2 antibody drug conjugate (ADC) MEDI7247 has been under development as a potential anti-cancer therapy for patients with selected relapsed/refractory hematological malignancies and advanced solid tumors by MedImmune. Although promising efficacy was observed in the clinic, pharmacokinetic (PK) analyses observed low exposure of MEDI7247 in phase I hematological patients. To investigate the biodistribution properties of MEDI7247, MEDI7247 and control antibodies were radiolabeled with zirconium-89 and in vitro and in vivo properties characterized. MethodsMEDI7247 (human anti-ASCT2 antibody conjugated with pyrrolobenzodiazepine (PBD)) and MEDI7519 (MEDI7247 without PBD drug conjugate) and an isotype control antibody drug conjugate construct were conjugated with p-isothiocyanatobenzyl-deferoxamine (Df) and radiolabeled with zirconium-89. In vitro studies included determining the radiochemical purity, protein integrity, immunoreactivity (Lindmo analysis), apparent antigen binding affinity for ASCT2-positive cells by Scatchard analysis and serum stability of the radiolabeled immunoconjugates. In vivo studies included biodistribution and PET/MRI imaging studies of the radiolabeled immunoconjugates in an ASCT2-positive tumor model, HT-29 colorectal carcinoma xenografts. ResultsConditions for the Df-conjugation and radiolabeling of antibody constructs were determined to produce active radioimmunoconjugates. In vivo biodistribution and whole body PET/MRI imaging studies of [89Zr]Zr-Df-MEDI7519 and [89Zr]Zr-Df-MEDI7247 radioimmunoconjugates in HT-29 colon carcinoma xenografts in BALB/c nude mice demonstrated specific tumor localization. However, more rapid blood clearance and non-specific localization in liver was observed for [89Zr]Zr-Df-MEDI7247 and [89Zr]Zr-Df-MEDI7519 compared to isotype control ADC. Except for liver and bone, other normal tissues demonstrated clearance reflecting the blood clearance for all three constructs and no other abnormal tissue uptake. Conclusions and advances in knowledgePreclinical biodistribution analyses of [89Zr]Zr-Df-MEDI7247 and [89Zr]Zr-Df-MEDI7519 showed the biodistribution pattern of anti-ASCT2 ADC MEDI7247 was similar to parental MEDI7519, and both antibodies showed specific tumor uptake compared to an isotype control ADC. This study highlights an important role nuclear medicine imaging techniques can play in early preclinical assessment of drug specificity as part of the drug development pipeline.

Efficacy and safety of trastuzumab deruxtecan (T-DXd) in patients (pts) with HER2-expressing solid tumors: DESTINY-PanTumor02 (DP-02) interim results.

LBA3000 Background: T-DXd is an antibody drug conjugate targeting HER2 and is approved in HER2-expressing breast (BC) and gastric (GC) cancers. HER2 expression is prevalent in other solid tumors. The efficacy of current treatments (Tx) in these populations, including studies with HER2-directed Tx, is modest, revealing a significant unmet medical need. Clinically meaningful activity of T-DXd was seen in HER2-expressing tumors in a phase 1 study (NCT02564900). Methods: DP-02 (NCT04482309) is an open-label phase 2 study of T-DXd 5.4 mg/kg q3w in pts with HER2-expressing (immunohistochemistry [IHC] 3+ or IHC 2+ by local or central testing) locally advanced or metastatic disease that progressed after ≥1 systemic Tx or that has no Tx options. Cohorts with biliary tract (BTC), bladder (URO), cervical (CC), endometrial (EC), ovarian (OC), pancreatic (PC), or other tumors (excluding BC, GC, colorectal cancer, and non-small cell lung cancer) were enrolled. Efficacy and safety were analyzed in all pts who received ≥1 dose of T-DXd. The primary endpoint was investigator-assessed confirmed objective response rate (ORR). Secondary endpoints included duration of response (DOR), disease control rate, progression-free and overall survival, and safety. Results: At data cutoff (16 Nov 2022; median follow-up, 9.7 mo), 267 pts had been treated (median, 2 prior lines of Tx [range, 0-13]); 75 pts were IHC 3+ and 125 were IHC 2+ by central testing. In all 267 pts, the ORR was 37.1% and median DOR (mDOR) was 11.8 mo; in pts with IHC 3+ expression, the ORR was 61.3% and mDOR was 22.1 mo. ORR per cohort is shown in all pts and those with centrally confirmed HER2 IHC 3+ or IHC 2+ expression. Grade (G) ≥3 adverse events (AEs) occurred in 58.4% of pts; 11.6% discontinued Tx due to AEs. Adjudicated drug-related interstitial lung disease/pneumonitis occurred in 18 pts (6.7% [G1, n=6; G2, n=11; G5, n=1]). Conclusions: This is the first tumor-agnostic global study of T-DXd in a broad range of HER2-expressing solid tumors. T-DXd showed encouraging ORR, particularly in pts with IHC 3+ expression, durable clinical benefit, and a manageable safety profile in this heavily pretreated population. These interim results show T-DXd to be a potential new Tx option for pts with HER2-expressing solid tumors. Clinical trial information: NCT04482309 . [Table: see text]