Drug-to-antibody Ratio Research Articles

Abstract CT248: First in human trial of DB1305 in patients with advanced malignant solid tumors

Abstract Background: Trophoblast cell-surface antigen 2 (Trop-2) is known as tumor-associated calcium signal transducer 2 (TACSTD2) and plays a role in tumor progression, given its active interplay with several key molecular pathways associated with cancer development and progression. DB-1305 is a Trop-2 antibody-drug conjugate (ADC) composed of a humanized anti-Trop-2 immunoglobulin G1 (IgG1) monoclonal antibody, covalently linked to a proprietary DNA topoisomerase I inhibitor P1021 via a cleavable linker containing maleimide tetrapeptide, with a drug-to-antibody ratio (DAR) of approximately 4. Preclinical in vivo models show that DB-1305 demonstrates robust anti-tumor activity in Trop-2-high triple-negative breast cancer (TNBC), Trop-2-low small cell lung cancer (SCLC) and Trop-2-medium colon cancer but not Trop-2-negative SCLC, demonstrating the dependence of Trop-2 expression for the tumor-suppression activity of DB-1305 and appropriate safety profile supports the intended clinical use. This study aims to evaluate DB-1305 in terms of tolerability and preliminary anti-tumor activity in patients with advanced solid tumors. Methods: This is an open-label, multicenter, multiple-dose, Phase 1/2a study (NCT05438329), including dose escalation Phase 1 and dose-expansion Phase 2a in patients with pretreated advanced solid tumors. Patients should have histologically documented progressed/refractory disease on or after standard systemic anticancer treatments with proven benefits for their disease or without available standard treatments; adequate performance score (ECOG 0-1); adequate organ function and measurable disease as per RECIST v1.1 while without uncontrolled metastatic central nervous system (CNS) involvement and history of interstitial lung diseases. The dose escalation part will evaluate approximately five ascending dose levels of DB-1305 with accelerated titration for the first dose level followed by a “3+3” design for subsequent dose levels to identify the optimal dose, which will enroll up to 70 patients from the United States and China. Upon determining the optimal dose, seven tumor cohorts of 10-40 patients will be included in the dose expansion part, including non-small cell lung cancer, SCLC, hormone receptor-positive breast cancer, and TNBC. DB-1305 will be dosed in both parts until disease progression, clinical deterioration, withdrawal of consent, or unacceptable toxicity. The primary objectives are evaluating safety tolerability and identifying the optimal dose from Phase 1; assessing safety, tolerability, and objective response rate per RECIST v1.1 from Phase 2a. Secondary objectives include the assessment of the pharmacokinetics and immunogenicity of DB-1305. Exploratory objectives include the assessment of pharmacodynamic biomarkers and Exposure-Response correlation. As of 19 Dec 2022, 20 patients have been enrolled in the dose escalation part. Citation Format: Naoto T. Ueno, Jie Gao, Liming Liu, Rong Shi, Shengxue Liu, Lili Tang, Peizhen Miao, Yang Qiu, Wei Gu, Cheng Ying. First in human trial of DB1305 in patients with advanced malignant solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT248.

Abstract LB030: SHR-A1921, a novel TROP-2 ADC with an optimized design and well-balanced profile between efficacy and safety

Abstract Trop-2 is a promising target for ADC therapy due to its high expression in many solid tumors. The approval of Trodelvy, a Trop-2 directed ADC, for the treatment of refractory or drug-resistant triple negative breast cancer (TNBC) demonstrated the therapeutic value of Trop-2-targeted ADC. However, a Boxed Warning for severe or life-threatening neutropenia and severe diarrhea suggests the safety of Trodelvy needs to be improved. Here, we presented a novel Trop2-directed ADC, SHR-A1921, consisting of a topoisomerase I inhibitor (Proprietary payload, SHR9265) conjugated to a proprietary IgG1 mAb via cleavable linkers. SHR-A1921 demonstrated several advantages over other Trop-2 directed ADCs in the field. SHR9265 is a novel exatecan derivative designed by Hengrui with a better liposolubility and cellular permeability. SHR-A1921 had a drug-to-antibody ratio (DAR) of 4. Compared with other Trop-2-targeted ADCs in the field, such as Trodelvy, TINA-SHR79711 (a molecule synthesized using the published structure of DS-1062), and SKB264, SHR-A1921 has considerable advantages as follows: (1) Stronger binding affinity to both human and rhesus macaque TROP-2 than TINA-SHR79711; (2) Improved plasma stability in plasma of different species presumably due to the proper steric hindrance which was purposely designed on the payload for reducing non-intended cleavage; (3) Stronger bystander cell killing effect presumably due to the increased lipophilicity of the payload vs. that of the payload in TINA-SHR79711; (4) Superior in vivo efficacy in a PSCC2 CDX Model (FaDu) with high Trop-2 expression (TGI 101% vs 53% [TINA-SHR79711] @ 1 mpk) and in an ovarian cancer CDX Model (SK-OV-3) with moderate Trop-2 expression (TGI 63% vs. 23% [TINA-SHR79711] @ 3 mpk; 87% vs. 16% [TINA-SHR79711] @ 10 mpk); (5) ≥ 2X longer half-life in patients# vs. SKB264 vs. IMMU-132, supporting more flexible dosing frequency; (6) Lower free toxin/ADC ratio# regarding PK exposure in patients compared with SKB264 (< 1% vs. 5-6%); (7) approximately linear pharmacokinetics profile in patients with T1/2 ranging from 2.5 to 4.5 days. In summary, SHR-A1921 is a novel anti-TROP2-targeted ADC with a high permeable payload and optimized DAR demonstrating great stability and high potency in both in vitro and in vivo studies. SHR-A1921 also showed compelling efficacy and good safety profile from 50+ subjects of Phase I clinical trial in China (NCT05154604). Pivotal phase III trial for NSCLC is planned in China. (Notes: 1. TINA-SHR7971 is a molecule that Hengrui synthesized using the published structure of DS-1062. 2. PSCC: pharyngeal squamous cell carcinoma. #. non-head-to-head comparison.) Citation Format: Ning He, Chunpeng Yang, Yang Yang, Zhendong Xue, Jianyan Xu, Linda Zhao, Jun Feng, Xin Ye, Zhe Zhang, Feng He. SHR-A1921, a novel TROP-2 ADC with an optimized design and well-balanced profile between efficacy and safety [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB030.

Abstract LB219: Inducing significant and efficient tumor growth inhibition vs trastuzumab deruxtecan with low drug-load topoisomerase 1 inhibitor ADC using novel peptide linkers for payload conjugation

Abstract The Araris’ site-specific and one-step linker conjugation technology aims at generating stable, safe and highly potent ADCs without the need for antibody engineering prior to payload conjugation. Here, we generated an anti-HER2 ADC using a Topoisomerase 1 (Topo1) inhibitor as payload with highly favorable biophysical properties and superior anti-tumor efficacy compared to Trastuzumab deruxtecan in head-to-head in vitro and in vivo studies. Based on trastuzumab as the targeting antibody and a Topoisimerase 1 inhibitor as payload, we generated highly homogeneous and pure ADCs with a drug-to antibody-ratio (DAR) of 2. In in-vitro assays on target positive cell-lines, the Araris Topo 1 ADC demonstrated potent cell-cytotoxicity in the low nM-range similar to the approved Trastuzumab deruxtecan which has a DAR of 8. Moreover, the ADC showed excellent stability in mouse, cynomolgus and human sera exemplified by the absence of payload deconjugation or linker cleavage while Trastuzumab deruxtecan showed significant payload loss during the 14d incubation period. Interestingly, despite the improved stability, the kinetics for payload release was highly efficient in human Cathepsin B or human liver-lysosome (HLL) enzyme cleavage assays. Most importantly, the ADC was extremely stable in circulation as shown in pharmacokinetic studies in rodents, demonstrating an exposure profile similar to the unmodified trastuzumab parent antibody. In efficacy studies using an established NCI-N87 colon cancer model (therapeutic setting), a single injection of the Araris Topo 1 ADC at DAR2 at a dose of 52ug/kg (adjusted payload dose) induced superior anti-tumor activity compared to Trastuzumab deruxtecan at DAR of 8, injected at the same payload dose. Complete tumor regression of all tumors (7/7) was obtained at 104ug/kg payload dose and lasted throughout the whole study duration (total 80 days) and was very well tolerated. The data show that Araris Topo 1 ADCs assembled using novel peptide linkers, even at a DAR of as low as 2 have a very efficient anti-tumor activity suggesting optimal drug exposure, targeting and release of the payload. In summary, we show that the Araris Topo1 linker-payloads result in highly potent ADCs with very favorable biophysical properties and extremely efficient payload release as well as an antibody-like exposure profile making them ideal linker-payloads for solid tumor targeting. We anticipate the low-drug load to be favorable in avoiding excessive toxicities in non-targeted tissues. Finally, the Araris bioconjugation technology allows for the generation of tailor-made ADC candidates with improved therapeutic indices. Citation Format: Isabella Attinger-Toller, Rachael Fay, Romain Bertrand, Philipp Probst, Ramona Stark, Roger Santimaria, Dragan Grabulovski, Bernd Schlereth, Philipp Rene Spycher. Inducing significant and efficient tumor growth inhibition vs trastuzumab deruxtecan with low drug-load topoisomerase 1 inhibitor ADC using novel peptide linkers for payload conjugation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB219.

Abstract LB221: Novel peptide linker-based nectin-4 targeting ADC shows improved tolerability with long-lasting anti-tumor efficacy at low doses

Abstract The Araris site-specific and one-step peptide linker conjugation technology generates stable, safe and highly potent ADCs without the need for antibody engineering prior to payload conjugation. We generated an anti-Nectin-4 ADC that shows superior anti-tumor activity and tolerability compared to enfortumab-vedotin (EV) in head-to-head in vitro and in vivo studies. The Araris ADC is based on enfortumab as the targeting antibody and monomethyl auristatin E (MMAE) as payload. Using a peptide linker and site-specific enzymatic conjugation approach, we generated a pure ADC with a drug-to-antibody-ratio (DAR) of approximately 2 and above 98 percent monomeric content. The Araris ADC demonstrated potent cell cytotoxicity similar to the approved enfortumab-vedotin which has a DAR of 4, excellent stability in mouse, cynomolgus and human sera exemplified by the absence of payload deconjugation or linker cleavage while EV showed significant payload deconjugation. Despite high stability, the Araris ADC releases the free active MMAE metabolite at comparable rate to EV in human Cathepsin B or human liver-lysosome (HLL) enzyme cleavage assays. The ADC was also shown to be extremely stable in circulation in pharmacokinetic studies in rodents, leading to an intact ADC exposure profile comparable to the unmodified enfortumab parent antibody. No free payload was detectable in circulation during the 3 week study by LCMS-MRM. In efficacy studies using a SUM-190PT established breast cancer model, a single injection at a dose of 10 ug/kg normalized by payload induced a complete tumor regression lasting for more than 100 days (i.e. a very durable response or tumor eradication). EV administered at the same payload dose showed only a short and transient (until day 20 only) tumor regression with no animal (0/6) reaching a complete response. Despite the higher in vivo exposure and extremely efficient anti-tumor response at low payload doses, there was no increased toxicity but in contrast, overall tolerability was improved, i.e., less neutropenia, skin involvement and signs of toxicity - the skin toxicity being the dose-limiting toxicity of Enfortumab vedotin in humans and rats. Overall, the highest non-severely toxic dose (HNSTD) in 4-week repeat dose rat toxicity studies for the Araris ADC (25 mg/kg) was 5-fold higher compared to the HNSTD (5mg/kg) reported for Enfortumab vedotin. Our data impressively show that the Araris ADC has superior efficacy and durable anti-tumor response even at 3-fold lower payload dose compared to EV. The improved efficacy in mice and tolerability in rates resulted in a 8-fold better TI for the Araris ADC and offers the opportunity to develop a highly efficacious ADC having potentially lower dose-limiting toxicities such as peripheral neuropathy, rashes or neutropenia. Citation Format: Isabella Attinger-Toller, Philipp Probst, Romain Bertrand, Emma Renard, Ramona Stark, Roger Santimaria, Dragan Grabulovski, Bernd Schlereth, Philipp Rene Spycher. Novel peptide linker-based nectin-4 targeting ADC shows improved tolerability with long-lasting anti-tumor efficacy at low doses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB221.

One-Pot Assembly of Dual-Site-Specific Antibody-Drug Conjugates via Glycan Remodeling and Affinity-Directed Traceless Conjugation.

The drug-to-antibody ratio (DAR) value and dual-drug combination greatly influence the therapeutic index of antibody-drug conjugates (ADCs). The reported approaches usually require multifunctional branched linkers, a combination of complicated technologies, or protein-protein ligation, which may incorporate multihydrophobic fragments or result in low coupling efficiency. Herein, we developed a facile and efficient one-pot method to assemble dual-site-specific ADCs with defined DARs at both the N-glycosylation site and K248 site, either with the same payloads or with two types of payloads. The constructed dual-site ADCs showed acceptable homogeneity, excellent buffer stability, and enhanced in vitro and in vivo efficiency.

Abstract 2012: A novel antibody-drug conjugate platform enabling high drug-to-antibody ratios and greater payload flexibility

Abstract Historically, increasing the drug-to-antibody ratio (DAR) in an antibody-drug conjugate (ADC) significantly raises aggregation propensity as a result of the hydrophobicity of the payload. Consequently, DARs are commonly limited to 4, forcing developers to incorporate very high potency payloads to achieve the required efficacy. This, combined with chemical instability and off-target uptake, often results in dose-limiting toxicities and narrow therapeutic indexes. In this study, we used a novel and flexible multivalent linker technology to construct two ADCs targeting HER-2 with the monoclonal antibody, trastuzumab and interchain cysteine bioconjugation. One ADC incorporated monomethyl auristatin E (MMAE) as the drug payload at average DAR 16 whilst the second incorporated the camptothecin analog, SN38, as the drug payload also at average DAR 16. The ADCs were characterized by liquid chromatography-mass spectrometry (LC-MS), size-exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC) and SDS-PAGE and were then tested for efficacy in a range of HER-2 positive cell lines in vitro, and tumor growth inhibition studies using NCI-N87 cells in a mouse xenograft. Both ADCs remained > 95% monomeric (no aggregation) and demonstrated greater chemical stability in ex-vivo serum stability studies compared to comparator ADC constructs utilizing a clinically validated linker and the same payload release chemistry. In in vitro cell killing studies, the ADCs demonstrated target-specific cell killing with the SN38 average DAR 16 ADC achieving a disproportionate 30-fold increase in IC50 (by ADC concentration) compared to a DAR 8 comparator. Dose dependent tumor growth inhibition was observed in an HER-2 positive NCI-N87 mouse xenograft model with no change in mouse body weight, and good systemic exposure. These data demonstrate our capability, using a novel multivalent linker technology, to construct ADCs with DARs considerably greater than DAR 8 which are chemically stable, shield payloads from hydrophobic interactions (no aggregation), demonstrate good in vivo exposure profiles and which are highly efficacious in vitro and in vivo. Higher DARs will permit the incorporation of lower potency and targeted small molecule payloads into ADCs which will result in targeted cancer therapeutics with wider therapeutic indexes through the delivery of greater concentrations of payload to the tumor microenvironment and lower, payload-associated, dose-limiting toxicity. Citation Format: Ludovic Juen, Adam J. Collier, Anthony W. Tolcher, Myriam M. Ouberai. A novel antibody-drug conjugate platform enabling high drug-to-antibody ratios and greater payload flexibility [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2012.

Abstract 6298: A novel topoisomerase I inhibitor based anti-ICAM-1 antibody drug conjugate for the treatment of hematologic malignancies and solid tumors

Abstract Intercellular Cell Adhesion Molecule 1 (ICAM-1, CD54) is a type I transmembrane protein and a member of the immunoglobulin superfamily. ICAM-1 is involved in many key processes such as cell-cell interactions, signal transduction and leukocytes trans-endothelial migration. ICAM-1 is constitutively present at low levels on healthy tissues but highly expressed in some lymphoma, myeloma and some types of solid tumors including melanoma, non-small cell lung cancers and liver cancers. Previous attempt to target ICAM-1 with a monoclonal antibody bersanlimab (BI505, BioInvent) showed that it was well-tolerated but with limited clinical efficacy. To improve the effectiveness of targeting ICAM-1, we developed a novel ICAM-1 antibody drug conjugate (ADC). The ICAM-1 ADC is comprised of a humanized anti-ICAM-1 hIgG1 antibody conjugated to a novel topoisomerase I inhibitor through a cleavable linker at a drug-to-antibody ratio (DAR) of about 8. The antibody moiety of the ADC targets a unique epitope on ICAM-1 which is far from its ligand binding domain. Potent in vitro cytotoxicity of ICAM-1 ADC, with nanomolar to sub-nanomolar EC50, was observed on a panel of hematologic and solid tumor cell lines. In preclinical studies, one or two doses of ICAM-1 ADC at 5-6 mg/kg were generally sufficient to induce tumor regression in multiple cell line-derived xenograft models, including those of lung and liver cancers. In all models tested so far, the novel topoisomerase I inhibitor conjugated ICAM-1 ADC is at least as efficacious as the same ICAM-1 antibody conjugated to the benchmark topoisomerase I inhibitor linker payload deruxtecan (ICAM-1-DXd). Furthermore, in a repeat dose exploratory non-human primate safety study, the benchmark conjugate ICAM-1-DXd (DAR 8) was well-tolerated at 41 mg/kg, the highest dose tested. In conclusion, ICAM-1 is an attractive target for topoisomerase I inhibitor based ADC and warrants further investigation. Citation Format: Oi Kwan Wong, David Jackson, Lei Shi, Qi Fei, Xiaocheng Chen, Leonard Post. A novel topoisomerase I inhibitor based anti-ICAM-1 antibody drug conjugate for the treatment of hematologic malignancies and solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6298.

Abstract 1884: DB-1310, a novel Her3 targeting antibody-drug conjugate, exhibits therapeutic efficacy for solid tumors

Abstract Background: HER3 (ErbB3), a member of the human epidermal growth factor receptor family, is frequently overexpressed in various cancers. Its high expression is associated with poor prognosis in several solid tumors. Many anti-Her3 antibodies have been developed for cancer therapy. To date, only patritumab deruxtecan (U3-1402) showed clinical efficacy in both NSCLC and breast cancer among all Her3-targeting therapies. DB-1310 is a HER3 ADC composed of a novel humanized anti-Her3 immunoglobulin G1 (IgG1) monoclonal antibody, covalently linked to a proprietary DNA topoisomerase I inhibitor payload via a maleimide tetrapeptide-based cleavable linker with a drug antibody ratio (DAR) of approximately eight. In the present study, we aim to evaluate the efficacy and safety of DB-1310 in preclinical models. Methods: The binding of DB-1310 to Her3 and other HER families was measured by ELISA and SPR. The competition of binding epitope for DB-1310 and patritumab was tested by FACS. The sensitivity of breast cancer and lung cancer cell lines to DB-1310 was evaluated by in vitro cell killing assay. In vivo growth inhibition study evaluated the sensitivity of DB-1310 to Her3-positive breast cancer and lung cancer xenograft models. In addition, the efficacy of DB-1310 was compared to that of patritumab deruxtecan in tumor xenograft models. The pharmacokinetics and safety profile were also measured in cynomolgus monkey. Results: In this study, DB-1310 showed high affinity to both Human and Cyno Her3 without cross reacting with Her2, Her4 or EGFR. DB-1310 binds Her3 using a different epitope from patritumab. DB-1310 has high internalization capability and translocates into lysosomes after internalization. In vitro, DB-1310 exhibited cytotoxicity in a panel of Her3- expression human breast cancer and lung cancer cell lines. It also showed dose-dependent antitumor killing activity in a HER3-positive breast cancer HCC1569 xenograft model and a lung cancer NCI-H441 xenograft model. Tumor regression was observed in a NSCLC patient-derived xenograft model with EGFR mutation under DB-1310 treatment. Moreover, DB-1310 showed stronger tumor growth inhibition than that of patritumab deruxtecan at the same dose. The systemic exposure AUC0-last and Cmax of DB-1310 and total antibody increased dose proportionally from 1 mg/kg to 10 mg/kg with low released payload in serum. The DB-1310 also showed good safety profile with highest non-severely toxic dose (HNSTD) above 30mg/kg. Conclusions: DB-1310 exhibited its efficacy in tumor models both in vitro and in vivo. These studies suggest that DB-1310 as a novel HER3-targeting ADC showed antitumor efficacy for HER3 positive tumors with good safety profile. Citation Format: Xi Li, Jun Yao, Chen Qu, Luo Lan, Bing Li, Yu Zhang, Rong Shi, Haiqing Hua, Yang Qiu. DB-1310, a novel Her3 targeting antibody-drug conjugate, exhibits therapeutic efficacy for solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1884.

Abstract 2648: In vivo efficacy and preclinical safety profile of NN3201, an anti-cKIT antibody-drug conjugate, for treating SCLC and AML

Abstract Overexpression and activating mutations of cKIT are associated with tumor aggressiveness and poor prognosis in various cancer patients such as small cell lung cancer (SCLC), gastrointestinal stromal tumor (GIST), acute myeloid leukemia (AML), and mastocytoma. Since small molecule inhibitors including imatinib frequently have a limited therapeutic application due to eventual drug resistance and severe adverse events, we developed the cKIT-directed antibody-drug conjugate (ADC), NN3201, for the treatment of wild-type and mutant cKIT-positive cancers. NN3201 is an ADC conjugate of the antimitotic payload MMAE to the fully human 2G4 antibody, directed against cKIT, via the cysteine re-bridging technology ThioBridge® at a drug-to-antibody ratio (DAR) of 4. NN3201 displayed preferable biophysical properties and broadly potent in vitro antitumor activities across various cKIT-positive tumor cell lines. NN3201 exhibited potent in vivo antitumor activities in cKIT-positive tumor cell line xenograft and patient-derived tumor xenograft models of SCLC and AML, representing robust and durable antitumor responses. Furthermore, in a single-dose toxicity study with cynomolgus monkey, no significant or severe clinical toxicity signs were observed in animals dosed at 1, 2, and 3 mg/kg. Even though a decrease in reticulocyte, red cell mass (hemoglobin and red blood cell), neutrophil, and monocyte counts, along with bone marrow recovery and reticulocyte compensatory mechanism, red blood cell and white blood cell recovery were observed from day 14 in the low and medium dose groups and day 21 in the high dose group, dose-dependently. These preclinical results suggest that NN3201 is a potential therapeutic agent for the treatment of cKIT-positive cancers and support the further clinical evaluation in SCLC and AML. Citation Format: Han-Jik Ko, Sung Tae Kim, Jin Gu Cho, Jin-Ock Kim, Jung Tae Lee, Sang Gyu Park. In vivo efficacy and preclinical safety profile of NN3201, an anti-cKIT antibody-drug conjugate, for treating SCLC and AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2648.

Abstract 6297: Rationale for the development of a differentiated Trop2 ADC

Abstract Background: Trophoblast antigen 2 (Trop2) or Tumor-Associated Calcium Signal Transducer 2 (TACSTD2) is a transmembrane glycoprotein that is overexpressed in several solid tumor cancers. Antibody drug conjugate (ADC) targeting of Trop2 has been clinically validated. The Trop2 ADC Trodelvy® (also known as Sacituzumab govitecan/IMMU-132) is approved for therapy of TNBC and urothelial cancer, and Datopotamab DXd (DS-1062) is currently being tested in NSCLC. Peak Bio’s Thailanstatin suite of immunomodulatory linker-toxins (L-Ts) is a set of 7 related molecules with distinct ADC features that have been extensively characterized in vitro and in vivo as Her2 ADCs and is being used as a platform to generate a pipeline of potentially differentiated ADCs (AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1832). Results: M2.8 is a fully humanized Peak Bio antibody (Ab) that targets human Trop2 (hTrop2) with high binding affinity [Kd (M)= 8.97-09]. M2.8 was optimized for binding affinity and humanization relative to its parent M2.1 [Kd (M)= 1.78-08], and, both M2.1 and M2.8, display species-specificity for the human and cynomolgus monkey Trop2 proteins. When bound to hTrop2-positive cell lines, M2.1 and M2.8 can rapidly internalize and deliver an ADC payload. To identify the optimal release for therapeutic efficacy, we conjugated our family of L-Ts with hRS7 at a comparable drug-to-antibody ratio (DAR) of 4. Maximal tumor growth inhibition (TGI) was associated with the Trop2 ADCs that were conjugated via amine coupling to lysine residues using non-cleavable L-T L22, later renamed PH1. To determine the effect of antibody on TGI efficacy, different doses, and DARs of M2.1 PH1 ADC were compared to PH1 ADCs made from other Trop2 Abs. At low dose levels, M2.1 PH1 ADCs exhibited comparable TGI with hRS7 PH1, TINA PH1, and T6-16 PH1 ADCs, but exhibited significantly greater TGI than hRS7 SN38 (IMMU-132), suggesting that payload potency, rather than epitope, was important in targeting Trop2. The results also suggested that a PH1 DAR of 4 was sufficient to obtain 90% TGI with multiple anti-Trop2 Abs. M2.1 PH1 ADC was safely tolerated by cynomolgus monkeys at 3 and 6 mg/kg Q3W x 3 doses with mild-to-moderate changes in liver enzymes and platelets that reversed to baseline within 7-10 days of administration of each dose. The optimized M2.8 PH1 DAR4 ADCs killed Trop2-positive cell lines with single-digit nanomolar potency, while exhibiting low off-target cytotoxicity vs normal human skin fibroblasts and Trop2-negative cell lines in vitro. M2.8 PH1 ADC regressed all 200mm3 sized NCI-N87 tumors and 50% of these tumors exhibited long-term regression for 5 months. Conclusion: The target validation studies suggest that Trop2 PH1 ADCs were sufficiently differentiated in areas of preclinical efficacy and well tolerated in a toxicologically relevant host. Citation Format: Satyajit K. Mitra, William Monteith, Mary Do, Greg Tuffy, Scott Savage, Jeffrey Kang, Mastewal Abuhay, Teodora Losic, Sanjeevani Ghone, William E. Haskins, Vasu Jammalamadaka, Sanjeev Satyal. Rationale for the development of a differentiated Trop2 ADC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6297.

Abstract 1544: MBK-103, a potent novel conjugation platform-based antibody-drug conjugate, changing therapeutic options in folate receptor alpha positive cancer patients

Abstract Folate receptor alpha (FOLR1) is a clinically validated target, that is overexpressed extracellularly in numerous solid malignancies with a high unmet medical need. Ovarian, non-small cell lung and breast adenocarcinomas are among indications with the highest frequency of FOLR1-positive patients with one out of two patients showing upregulated topoisomerase I expression. MBK-103, an antibody-drug conjugate targeting FOLR1, is based on Mablink’s novel proprietary hydrophilic drug-linker platform. It is comprised of: (i) an Fc-attenuated humanized IgG1 monoclonal antibody, that selectively binds to FOLR1; (ii) a polysarcosine hydrophobicity masking entity allowing for a high drug-to-antibody ratio (DAR) of 8, while improving the pharmacokinetics and tolerability of the drug; (iii) a proprietary dipeptide cleavable unit and iv) exatecan, a potent topoisomerase I inhibitor. MBK-103 showed excellent ex vivo stability in human plasma enabling a prolonged half-life, similar to that of the unconjugated monoclonal antibody. This led to an increased drug exposure, resulting in a potent antitumor efficacy observed in a collection of more than ten in vivo mouse tumor models with a diverse FOLR1 expression, already at doses as low as 1-3 mg/kg. In addition, the distinct mode of action of exatecan along with the unique characteristics of the proprietary drug-linker platform resulted in the in vivo antitumor potency obtained also in colorectal cancer models, in which tubulin-inhibiting agents exhibit only limited therapeutic effects. The compound was also very well tolerated in cynomolgus monkeys with HNSTD at 50 mg/kg and even when administered repeatedly. High conjugation yields along with 100% homogeneity are major drivers of the successful ongoing manufacturing process with planned IND submission in Q4/2023. Citation Format: Warren Viricel, Louise Conilh, Edouard Leroy, Jean-Guillaume Lafay, Frederique Brune, Lenka Kyrych Sadilkova, Charles Dumontet. MBK-103, a potent novel conjugation platform-based antibody-drug conjugate, changing therapeutic options in folate receptor alpha positive cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1544.

Abstract 6320: CUSP06/AMT-707, a new CDH6-targeting antibody-drug conjugate, demonstrates potent antitumor activity in preclinical models

Abstract Cadherin-6 (CDH6), also known as K-cadherin, is a type II classic cadherin molecule that plays an important role in the embryonic development of the kidney, but has very limited expression in adult tissues. It is overexpressed in several human malignancies, primarily in ovarian, renal, but also (to a lesser degree) gastric, thyroid, cholangiocarcinoma and other tumor types. The characteristics of limited expression in normal tissues, high expression in tumor tissues, and rapid internalization upon antibody binding makes CDH6 a well-suited Antibody-drug Conjugate (ADC) target. We developed a novel CDH6-targeting ADC, CUSP06, consisting of a proprietary humanized antibody selective for CDH6, a protease cleavable linker, and an exatecan payload, with a drug-to-antibody ratio (DAR) of 8. CUSP06 selectively bound to cell surface CDH6 and was efficiently internalized into CDH6 positive ovarian and renal cancer cells. CUSP06 exhibited strong antiproliferative activity against several CDH6 positive cancer cell lines in vitro. Furthermore, compared to a DXd-based ADC, a exatecan-based ADC demonstrated improved bystander effect. Treatment with CUSP06 resulted in tumor regression in several CDH6 positive cell line derived xenograft (CDX) models, including PA-1, OVCAR3, and 786-O. In addition, CUSP06 demonstrated potent antitumor activity with tumor regression observed in two CDH6 low and high expressing patient derived xenograft (PDX) models. The preclinical activity of CUSP06 against CDH6-expressing tumors provide compelling support for the clinical development of CUSP06 in CDH6-expressing human cancers. CUSP06 is currently in IND-enabling studies. CUSP06 showed an expected toxicity profile consistent with it's exatecan payload in the ongoing pilot toxicology studies. We plan to initiate a Phase 1 first-in-human clinical trial in the 2nd half of 2023. Citation Format: Wei Lu, Jing Shi, Shu-Hui Liu, Nicole Covino, Xun Meng, Eric D. Slosberg. CUSP06/AMT-707, a new CDH6-targeting antibody-drug conjugate, demonstrates potent antitumor activity in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6320.

Abstract 6300: Therapeutic potential of EO-3021/SYSA1801, a Claudin18.2 antibody-drug conjugate, for the treatment of CLDN18.2-expressing cancers

Abstract Background: Claudin18.2 (CLDN18.2), a tight junction protein normally expressed only on gastric mucosa, is overexpressed in gastric, pancreatic, esophageal, ovarian, lung and other solid tumors. Unlike in normal tissue, CLDN18.2 is exposed on epithelial surfaces in malignancy. There are no approved targeted therapies for CLDN18.2-expressing cancers. An antibody-drug conjugate (ADC) composed of a monoclonal antibody (mAb) targeting CLDN18.2 with a monomethyl auristatin E (MMAE) payload site-specifically conjugated via a cleavable linker at a drug-antibody ratio (DAR) of 2, EO-3021/SYSA1801, was developed to target CLDN18.2-expressing cancer cells, minimize toxicities and maximize therapeutic index. Methods: HEK293-CLDN18.2 cells were used to evaluate CLDN18.2-dependent EO-3021 endocytosis, MMAE payload release, inhibition of proliferation and antibody dependent cellular cytotoxicity (ADCC). Cell cycle distribution and caspase-3/7 activity were measured 24 hours after treating BxPC3-CLDN18.2 pancreatic cells with EO-3021 or the unconjugated antibody, EO-3021 mAb. Inhibition of proliferation by EO-3021 was measured on cell lines ectopically expressing medium to high CLDN18.2 and on cell lines with endogenous low to medium CLDN18.2 expression. Xenograft studies evaluating tumor growth inhibition by EO-3021, EO3021 mAb, and cisplatin or gemcitabine were done in gastric (NUGC4; NUGC4-CLDN18.2), pancreatic (Patu8988S; BxPC3-CLDN18.2), and lung (NCI-H460) cancer models. Results: EO-3021 binding to cancer cells, endocytosis, MMAE release, and inhibition of proliferation were dependent on CLDN18.2 expression. EO-3021 (EC50: 172 ng/ml) and EO-3021 mAb (EC50: 130 ng/ml) demonstrated similar levels of ADCC. EO-3021 but not EO-3021 mAb promoted G2/M cell cycle arrest and apoptosis and exhibited potent activity across cell lines with low, medium, and high CLDN18.2 expression (IC50: 7-456 ng/mL). EO-3021 induced tumor regressions with a single dose across low, medium, and high CLDN18.2-expressing in vivo models derived from pancreatic (2-10 mg/kg), gastric (0.5-10 mg/kg), and lung cancers (4 mg/kg), respectively. In contrast, standard of care (SOC) chemotherapies and EO-3021 mAb did not induce tumor regressions across in vivo models. Conclusions: EO-3021 demonstrated profound in vivo antitumor activity and outperformed SOC in gastric, pancreatic, and lung cancer models. Results from in vitro and in vivo studies highlight the promising therapeutic potential of EO-3021/SYSA1801 for patients with CLDN18.2-expressing cancers. A Phase 1 study is ongoing to evaluate the safety, tolerability, pharmacokinetics and preliminary anti-tumor activity of SYSA1801 in patients with CLDN18.2 positive advanced solid tumors (NCT05009966). Citation Format: Mo Dan, Xiwu Hui, Yancui Wang, Can Yuan, Thomas O'Hare, Valerie Malyvanh Jansen, Shawn M. Leland, Yang Zhang, David Dornan, Xiaoyan Wang. Therapeutic potential of EO-3021/SYSA1801, a Claudin18.2 antibody-drug conjugate, for the treatment of CLDN18.2-expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6300.

Abstract 1545: Targeting anticancer agents to the tumor microenvironment with cathepsin B cleavable drug-linker conjugates of phosphatidylserine-binding proteins

Abstract In healthy cells, the membrane lipid phosphatidylserine (PS) is confined to the inner leaflet of the plasma membrane but in cancer cells and in the tumor vasculature PS is prevalent in the outer leaflet where it is highly immunosuppressive. In this study engineered proteins, such as Bavituximab, a chimeric monoclonal antibody that binds to PS via the abundant serum protein β2-glycoprotein 1, and constructs containing the PS binding domain of β2-glycoprotein 1 (betabodies), are conjugated to anticancer drug-linkers designed to be cleavable by cathepsin B. Cathepsin B is a cysteine protease that is abundant in the lysosomes of normal cells but is upregulated and trafficked to the cell surface and is secreted into the extracellular parenchyma in solid tumors, including breast and pancreatic cancer. A series of drug-linker constructs were synthesized that incorporate unique small-molecule payloads with dual mechanism of action, functioning as both potent tubulin-binding antimitotic agents and vascular disrupting agents. The payloads include benzosuberene-based aniline KGP156 and its phenolic congener KGP18, dihydronaphthalene-based KGP05, and indole-based OXi8006. These drug-linker constructs were reacted with N-acetyl-L-cysteine (NAC) and evaluated for cathepsin B cleavage, and liberation of payload after spontaneous 1,6-fragmentation and decarboxylation. The drug linker construct, NAC-succinimidocaproyl-L-valine-L citrulline-p-aminobenzyl carbamate derivatized with aminobenzosuberene, KGP156, was cleaved by cathepsin B (260 nM) with release of payload at the rate of 12.7 μM/min and by cathepsin L (600 nM) at a rate of 11. 5 μM/min. Other drug-linker constructs in this series demonstrated slower release of payload upon cleavage with cathepsin B or cathepsin L. The drug-linker construct incorporating aminodihydronaphthalene-based KGP05 released KGP05 at a rate of 2.4 μM/min with a comparable rate upon exposure to cathepsin L. Payload release from drug-linker constructs was not observed, over 24 h in the absence of enzyme. Drug-linker constructs without the Val-Cit dipeptide were not cleaved by cathepsin B. The most efficient cathepsin B drug-linker substrate, Mc-Val-Cit-PABC-KGP156, was conjugated to Baxituximab to form the corresponding antibody-drug conjugate (ADC). Baxituximab was first reacted with 2-iminothiolane, a thiolating agent that reacts with accessible lysine residues. Reaction with Ellman’s reagent revealed an -SH/antibody ratio of 3.5. Conjugation of thiolated Bavituximab with Mc-Val-Cit-PABC-KGP156 gave a drug/antibody ratio (DAR) of 3.6/1 after cleavage by cathepsin B and determination of released KGP156 by LC-MS. These studies revealed 1) distinct differences in cleavage rates for drug-linkers, 2) cleavage by cathepsin L, and 3) efficient cathepsin B mediated release of payload from the ADC. Citation Format: Yuling Deng, Jacob W. Ford, Jenny M. VanNatta, Casey J. Maguire, Deboprosad Mondal, Natalie Z. Phinney, Rolf A. Brekken, Kevin G. Pinney, Mary Lynn Trawick. Targeting anticancer agents to the tumor microenvironment with cathepsin B cleavable drug-linker conjugates of phosphatidylserine-binding proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1545.

Abstract 556: Bystander effects of EGFR-targeting 40H3 antibody-drug conjugates in glioblastoma with heterogeneous EGFR expression

Abstract An important aspect to determine the efficacy of a targeted macromolecule in treating brain tumors is penetration and activity in tumor across a heterogeneously intact blood-brain barrier. In this study, novel epidermal growth factor receptor (EGFR)-targeted antibody drug conjugates (ADCs) were constructed with a focus on cytotoxicity in glioblastoma (GBM) with an EGFR heterogeneous cell population. Initially, the cytotoxicity of free payloads with multiple mechanisms of cell kill was assessed. These included tesirine (TS, induces DNA cross-links), deruxtecan (Dxd) and SN38 (inhibit topoisomerase I), and an auristatin (MMAE) and maytansinoid (DM1) (inhibit microtubule polymerization). Across four GBM PDXs with varied EGFR expression (GBM6, 39 and 108- EGFRviii amplified; GBM10 - non-amplified), free TS and MMAE were consistently potent with EC50 values ranging from 0.50-45.9 pM and 7.9-229.1 pM, respectively. In contrast, the other free toxins were less potent; EC50 values: Dxd, 0.21-13.1 nM; SN38, 0.87-5.6 nM; DM1, 2.1-19.9 nM. Based on these results, the 40H3 EGFR-specific IgG was used to construct ADCs with TS and MMAE. 40H3-TS had a drug:antibody ratio (DAR) of 2.5 and was potently cytotoxic in GBM6, GBM39 and GBM108, while minimal cytotoxicity was observed in GBM10 or normal astrocyte SVG-A cells. 40H3-MMAE had a DAR of 3 and was similarly effective in GBM6 and GBM39 but less potent in GBM108, GBM10, and SVG-A. Bystander cytotoxicity was evaluated U87 cells expressing eGFP/fLuc2 (U87eGFP/fluc2) or EGFRviii (U87EGFRviii). Using live-cell imaging, U87EGFRviii cells treated with 40H3-TS or 40H3-MMAE had significantly reduced cell confluence relative to control. The same drug treatments in U87eGFP/fLuc2 cells had no effect on growth/confluence. However, in a 1:1 mixed culture, overall confluence was reduced from 89% in control to 35% and 32% after 40H3-TS (p<0.0001) or 40H3-MMAE (p<0.0001) treatment, respectively. This bystander killing of U87eGFP/fLuc2 cells was indicated by a reduction in confluence of green-fluorescent cells from 50% in control versus 29% with 40H3-TS (p<0.0001) and 26% with 40H3-MMAE (p<0.0001) treatment. In GBM39 orthotopic tumors, a single infusion of 10 or 20 µg 40H3-TS via convection enhanced delivery (CED) reduced the bioluminescence signal 7 days post treatment by ~10-fold (p=0.03) as compared to 40H3 control. However, 50% and 80% mortality was observed within a week of infusing 10 and 20 µg 40H3-TS, respectively. Neurotoxicity was associated with neuron loss in treated hemisphere as determined by NeuN staining. In summary, these data highlight the potential for novel EGFR-targeted ADCs to provide potent direct and bystander cytotoxicity to GBM cells. However, further selection and optimization of the conjugated toxins will be required to balance potency and bystander killing with toxicity for EGFR-targeted ADCs. Citation Format: Sonia Jain, Eric Chun Ho, Kendra A. Porath, Antonella Antignani, David J. FitzGerald, Jann N. Sarkaria. Bystander effects of EGFR-targeting 40H3 antibody-drug conjugates in glioblastoma with heterogeneous EGFR expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 556.

Abstract 1549: Preclinical characterization of GQ1010, a next generation Trop2 ADC with the best-in-class potential against diverse Trop2+ solid tumors

Abstract Background: While Trop2-targeting antibody-drug conjugates (ADC) have demonstrated meaningful clinical response for the treatment of solid tumors, these ADCs still exhibit significant safety issues that limit the full potential of Trop2 ADCs. GQ1010 was developed based on the intelligent Ligase-Dependent Conjugation (iLDC) technology platform of GeneQuantum to enhance the therapeutic window of Trop2 ADC by utilizing a stable linker and a next generation camptothecin analogue. Here we report that GQ1010 demonstrates superior anti-tumor activities against benchmark Trop2 ADCs while maintaining excellent safety profiles, and a potential for a larger therapeutic window, supporting further clinical investigation of GQ1010 in patient with diverse tumors. Method: To characterize GQ1010 in vitro, assays for target binding affinity, internalization, cell cycle, apoptosis, bystander killing, and in vitro cytotoxicity were used. A series of xenograft (CDX) models were used for in vivo efficacy evaluation. Ex vivo plasma stability study was performed to determine the linker stability by monitoring drug-antibody ratio (DAR) change and the payload shedding. In addition, in vivo payload distribution study was performed using a xenograft model. Toxicity study was performed in monkey, and clinical observation, body weight, food consumption, clinical pathology (hematology, coagulation, serum chemistry), and pathology were evaluated. Results: GQ1010 demonstrated more potent in vitro cytotoxicity than DS1062 in diverse Trop2+ cancer cell lines tested. GQ1010 also exhibited superior bystander killing than DS1062. GQ1010 further demonstrated strong in vivo antitumor activities in different CDX models including TNBC, gastric, head and neck (H&N) and pancreatic cancer, and the efficacies were better than DS1062 and Trodelvy®, consistent with the in vitro data. Ex vivo plasma stability data confirmed GQ1010 contains highly stable linker with a minimal DAR reduction and payload shedding, indicating expanded therapeutic window than the benchmark ADCs. Free payload distribution analysis in NCI-N87 model showed high target-specific payload delivery and ~90X payload enrichment in tumor than in plasma, confirming minimal shedding of the payload from GQ1010 during circulation in vivo. In the monkey toxicity study, GQ1010 demonstrated excellent safety without signal of interstitial lung disease (ILD) up to 80 mg/kg dosing. Conclusion: GQ1010 consistently demonstrate superior linker stability, in vitro and in vivo efficacies than DS1062 and Trodelvy®, while maintaining excellent safety. These data suggest GQ1010 has the potential as the next generation Trop2 ADC that can address the critical unmet needs for diverse cancer patients. Citation Format: Yajun Sun, Mingyu Hu, Cao Lv, Yu Si, Xiao Liu, Xinju Gao, Dong Yang, Guangming Chen, Boyu Zhong, Tony Zhang, Lili Shi, Gang Qin, Paul H. Song. Preclinical characterization of GQ1010, a next generation Trop2 ADC with the best-in-class potential against diverse Trop2+ solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1549.

Abstract 2638: Preclinical development of ADCT-212, a PSMA-targeted antibody-drug conjugate employing the pyrrolobenzodiazepine dimer SG2000 for PSMA-expressing cancers

Abstract Prostate-specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase that is highly expressed in nearly all prostate cancers with the highest expression in metastatic castration-resistant prostate cancer. Moreover, PSMA is expressed in the neovasculature that supplies most non-prostatic solid tumors, including carcinomas of the lung, colon, breast, kidney, liver, and pancreas. ADCT-212 is an antibody-drug conjugate composed of the human IgG1 antibody 2A10 directed against human PSMA, site-specifically conjugated using GlycoConnectTM technology to PL1801, which contains HydraspaceTM, a valine-alanine cleavable linker and the pyrrolobenzodiazepine (PBD) dimer warhead SG2000 (drug-antibody ratio of ~1.8). The purpose of this study was to characterize the in vitro and in vivo anti-tumor activity of ADCT-212 in human cancer cell lines and xenograft models and to determine its tolerability and pharmacokinetic (PK) in the rat. In vitro, ADCT-212 demonstrated potent cytotoxicity in a panel of PSMA-positive prostate cancer cell lines, whereas its activity was greatly reduced in PSMA-negative cell lines. ADCT-212 was efficiently internalized by PSMA-expressing LNCaP cells. Trafficking to the lysosomes started as early as 30 minutes and peaked at 1-2 hours post treatment. In line with the mechanism of action of the PBD dimer, ADCT-212 produced DNA interstrand cross-links (ICLs) that peaked by 12 hours and persisted for up to 36 hours post-treatment. In contrast, the peak of DNA ICLs formation for SG2000, the PBD dimer warhead alone, was observed immediately after 2-hour incubation, while a non-targeted PBD-ADC did not yield any appreciable DNA ICLs. Moreover, ADCT-212 showed indirect bystander killing activity in PSMA-negative PC3 cells incubated with conditioned medium from ADCT-212-treated LNCaP cells. In vivo, ADCT-212 showed strong antitumor activity against CWR22Rv1 and LNCaP prostate cancer xenograft models. In the CWR22Rv1 model, a tumor with heterogeneous PSMA expression, ADCT-212 achieved dose-dependent antitumor activity when administered as single dose at 2, 4 or 6 mg/kg, which resulted in increased survival compared to the control animals. In the LNCaP model, a single dose of ADCT-212 at 5 or 10 mg/kg resulted in strong and specific antitumor activity. Conversely, in the PSMA-negative PC3 xenograft model, ADCT-212 did not show anti-tumor activity compared to the controls, highlighting its target-mediated antitumor activity. ADCT-212 was tolerated as a single 20 mg/kg dose in male rats, with exposure data being indicative of a linear PK profile with a half-life of approximately 12 days. In conclusion, ADCT-212 demonstrated potent and specific in vitro and in vivo anti-tumor activity while it was stable and well tolerated in the rat, warranting further development of ADCT-212 into the clinic. Citation Format: Francesca Zammarchi, Karin Havenith, Lolke de Haan, Ian Kirby, Narinder Janghra, Veronica Gil, Pedro Alves, Kristina Zaitseva, Meghann Kerr, Ben Leatherdale, Afroze Patel, Marie Thoelke, Shiran Huang, John A Hartley, Patrick H van Berkel. Preclinical development of ADCT-212, a PSMA-targeted antibody-drug conjugate employing the pyrrolobenzodiazepine dimer SG2000 for PSMA-expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2638.

Phenyldivinylsulfonamides for the construction of antibody–drug conjugates with controlled four payloads

Phenyldivinylsulfonamides emerged from a series of divinylsulfonamides, demonstrating their ability to effectively re-bridge disulfide bonds. This kind of linkers was attached to monomethyl auristatin E (MMAE) and further conjugated with a model antibody, trastuzumab. After optimization, the linker 20 can deliver stable and highly homogenous DAR (Drug-to-Antibody Ratio) four antibody–drug conjugates (ADCs). The method was also applicable for other IgG1 antibodies to obtain ADCs with controlled four payloads. Moreover, the MMAE-bearing ADC is potent, selective and efficacious against target cell lines.

Aminooxy Click Modification of a Periodate-Oxidized Immunoglobulin G: A General Approach to Antibody-Drug Conjugates with Dye-Mediated Expeditious Stoichiometry Control.

A universal approach to the construction of antibody-drug conjugates (ADCs) has been developed. It relies on periodate oxidation of naturally present glycans of immunoglobulin G, followed by oxime ligation and, optionally, copper(I)-catalyzed alkyne-azide cycloaddition for conjugation with a toxic payload. The introduction of highly absorbing cyanine dyes into the linker allows for facile determination of the drug-antibody ratio. We applied this methodology to the synthesis of cytotoxic conjugates of an antibody against the tumor-associated antigen PRAME with doxorubicin and monomethyl auristatin E (MMAE). The resultant conjugates retained their affinity to a large extent, yet their cytotoxicity in vitro varied dramatically: while the doxorubicin-based conjugate did not produce any effect on cells, the MMAE-based one demonstrated specific activity against PRAME-expressing cancer cell lines. Importantly, the latter conjugate constitutes the first reported example of a PRAME-targeting ADC.

Implementation of Systematic Bioanalysis of Antibody–Drug Conjugates for Preclinical Pharmacokinetic Study of Ado-Trastuzumab Emtansine (T-DM1) in Rats

Antibody-drug conjugates (ADCs) are composed of monoclonal antibodies covalently bound to cytotoxic drugs by a linker. They are designed to selectively bind target antigens and present a promising cancer treatment without the debilitating side effects of conventional chemotherapies. Ado-trastuzumab emtansine (T-DM1) is an ADC that received US FDA approval for the treatment of HER2-positive breast cancer. The purpose of this study was to optimize methods for the quantification of T-DM1 in rats. We optimized four analytical methods: (1) an enzyme-linked immunosorbent assay (ELISA) to quantify the total trastuzumab levels in all drug-to-antibody ratios (DARs), including DAR 0; (2) an ELISA to quantify the conjugated trastuzumab levels in all DARs except DAR 0; (3) an LC-MS/MS analysis to quantify the levels of released DM1; and (4) a bridging ELISA to quantify the level of anti-drug antibodies (ADAs) of T-DM1. We analyzed serum and plasma samples from rats injected intravenously with T-DM1 (20 mg/kg, single dose) using these optimized methods. Based on these applied analytical methods, we evaluated the quantification, pharmacokinetics, and immunogenicity of T-DM1. This study establishes the systematic bioanalysis of ADCs with validated assays, including drug stability in matrix and ADA assay, for future investigation on the efficacy and safety of ADC development.