Payloads For Antibody-drug Conjugates Research Articles

Evaluation of Quantitative Platforms for Single Target Mass Spectrometry Imaging.

(1) Imaging of pharmaceutical compounds in tissue is an increasingly important subsection of Mass Spectrometry Imaging (MSI). Identifying proper target engagement requires MS platforms with high sensitivity and spatial resolution. Three prominent categories of drugs are small molecule drugs, antibody-drug conjugate payloads, and protein degraders. (2) We tested six common MSI platforms for their limit of detection (LoD) on a representative compound for each category: a Matrix-Assisted Laser Desorption/Ionization (MALDI) Fourier Transform Ion Cyclotron, a MALDI-2 Time-of-Flight (ToF), a MALDI-2 Trapped Ion Mobility Spectrometry ToF, a Desorption Electrospray Ionization Orbitrap, and 2 Atmospheric Pressure-MALDI Triple Quadrupoles. Samples were homogenized tissue mimetic models of rat liver spiked with known concentrations of analytes. (3) We found that the AP-MALDI-QQQ platform outperformed all 4 competing platforms by a minimum of 2- to 52-fold increase in LoD for representative compounds from each category of pharmaceutical. (4) AP-MALDI-QQQ platforms are effective, cost-efficient mass spectrometers for the identification of targeted analytes of interest.

DNA sequence-selective G-A cross-linking ADC payloads for use in solid tumour therapies

Antibody-Drug Conjugates (ADCs) are growing in importance for the treatment of both solid and haematological malignancies. There is a demand for new payloads with novel mechanisms of action that may offer enhanced therapeutic efficacy, especially in patients who develop resistance. We report here a class of Cyclopropabenzindole-Pyridinobenzodiazepine (CBI-PDD) DNA cross-linking payloads that simultaneously alkylate guanine (G) and adenine (A) bases in the DNA minor groove with a defined sequence selectivity. The lead payload, FGX8-46 (6), produces sequence-selective G-A cross-links and affords cytotoxicity in the low picomolar region across a panel of 11 human tumour cell lines. When conjugated to the antibody cetuximab at an average Drug-Antibody Ratio (DAR) of 2, an ADC is produced with significant antitumour activity at 1 mg/kg in a target-relevant human tumour xenograft mouse model with an unexpectedly high tolerability (i.e., no weight loss observed at doses as high as 45 mg/kg i.v., single dose).

Discovery of novel polyamide-pyrrolobenzodiazepine hybrids for antibody-drug conjugates

Pyrrolobenzodiazepine (PBD) dimers are well-known highly potent antibody drug conjugate (ADC) payloads. The corresponding PBD monomers, in contrast, have received much less attention from the ADC community. We prepared several novel polyamide-linked PBD monomers and evaluated their utility as ADC payloads. The unconjugated polyamide-PBD hybrids exhibited potent antiproliferative activity (IC50 range: 10−11–10−8 M) against a variety of HER2-expressing cancer cell lines. Several peptide-linked variants of the lead compound were prepared and conjugated to trastuzumab to afford ADCs with drug-to-antibody (DAR) ratios ranging from 3 to 5. The ADCs exhibited antigen-dependent cytotoxicity in vitro and potently suppressed tumor xenograft growth in vivo in a target-dependent manner. Moreover, the ADCs were well-tolerated in both mouse and rat. This work demonstrates for the first time that PBD polyamide hybrids can serve as effective ADC payloads.

Abstract 1749: A new class of DNA sequence-selective G-A cross-linking antibody-drug conjugate (ADC) payloads

Abstract Antibody-Drug Conjugates (ADCs) are rapidly growing in importance as a targeted cancer therapy, with over ten approved, and many currently undergoing clinical trials. ADCs comprise of a cytotoxic agent (i.e., the "payload") conjugated via a chemical linker to a tumor-targeting antibody. There is a demand for novel ADC payloads with unique mechanisms of action, enhanced tolerability profiles and improved physicochemical properties. Such payloads could lead to ADCs with therapeutic efficacy in patients resistant to other therapies, and payloads with reduced hydrophobicity should lead to improved conjugation, minimal aggregation, and higher Drug-Antibody Ratios (DARs). We report here, studies on a novel class of Cyclopropabenzindole-Pyridinobenzodiazepine (CBI-PDD) payloads, designed through molecular modeling, which form cross-links between Guanine and Adenine bases within the DNA minor groove with defined sequence specificity. Eleven novel analogs will be described which have been structurally modified with various functional groups to allow tunable hydrophobicity, and new vectors through which antibodies may be attached. Most of these analogs are highly cytotoxic, with some exhibiting IC50 values in tumor cells down to the femtomolar range (e.g., FGX37-140, 0.9 pM in both RAJI and Jurkat cells, 72 h incubation). The DNA interactivity of these analogs has been studied using a range of methods such as gel-based DNA footprinting and cross-linking, FRET melting and Transcription Factor (TF) Array assays, the results of which will be described. Overall, the analogs appear to favor the formation of intrastrand rather than interstrand DNA cross-links and can inhibit the DNA binding of several key cancer-related transcription factors such as NFκB. To explore the potential of these analogs as ADC payloads, one member, FGX8-46, was conjugated to the EGFR-targeting antibody Cetuximab in stochastic fashion (DAR2) to produce Cetuximab-(FGX16-11). This ADC had an unexpectedly high Maximum Tolerated Dose (MTD) in a standard mouse model of at least 45 mg/kg, perhaps reflecting the novel mechanism of action of the payload. It was then evaluated in a Human Tumor Xenograft study based on BALB/c mice transplanted with the EGFR-expressing human colon cancer cell line SW-48 using single doses ranging from 1 mg/kg to 40 mg/kg. Cetuximab-(FGX16-11) was active at all dose levels including 1 mg/kg which provided tumor suppression out to approximately three weeks. Complete tumor suppression out to 50 days was seen with 20 mg/kg. Overall, the unique mechanism of action, potent cytotoxicity, and excellent in vivo tolerability and significant efficacy in ADC format, make the CBI-PDDs a promising new class of ADC payloads suitable for further development. Citation Format: George Procopiou, Paul J. Jackson, Daniella M. di Mascio, Jennifer L. Auer, Paolo Andriollo, Ilona Pysz, Khondaker M. Rahman, Keith R. Fox, David E. Thurston. A new class of DNA sequence-selective G-A cross-linking antibody-drug conjugate (ADC) payloads [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1749.

Abstract 5293: Rationally developing antibody drug conjugates targeting genomically stable gastric cancer

Abstract Background: Gastric cancer (GC) ranks the fifth most common cancer and the fourth most frequent cause of cancer-related deaths. Genomically stable gastric cancers (GSGCs) represent 20% of all GCs and GSGC patients have the worst prognosis among all GC subtypes. Although two HER2-targeted antibody-drug conjugates (ADCs), RC48 and DS8201, have shown promising clinical efficacies, most GSGC patients do not benefit from these ADCs due to the infrequent and heterogeneous HER2 expression. Therefore, there remains a significant and unmet medical need for developing novel ADCs targeting GSGC tumors. Methods: In this presented study, a quantitative and unbiased approach was first used to identify potential targets for GSGC in a panel of human GSGC cell lines in reference with two normal human gastric epithelial cells. Cell membrane localizations of potential targets on GSGC cell lines were determined by immunofluorescent (IF) staining. To correlate it with clinically-relevant GSGC tumor samples, immunohistochemical (IHC) staining of potential targets were performed in human diffuse GC tumor tissues along with normal gastric tissues. Next, GSGC cellular internalization capability of antibodies against potential targets was evaluated. Then, an optimized target was selected for developing GSGC-targeted ADCs. A series of ADC formulations with different linkers and payloads were constructed and characterized. Their anti-tumor activities against GSGC cell lines were subsequently screened in order to identify the optimized ADC formulation. Eventually, the anti-tumor activity and biosafety profile of the optimized ADC formulation were determined in suppressing tumor growth, progression and metastasis by using established orthotopic and peritoneal metastasis models of GSGC. Results: We first identified CD54 as a potential molecular target for human GSGCs using comparative flow cytometric analysis. We next evaluated its potential as an ADC target for GSGC-targeted therapy by determining its overexpression levels, cell membrane localizations, and cell internalization activity in GSGC cells. Furthermore, we constructed and characterized a panel of CD54-targeted ADCs and identified an optimized ADC formulation by quantitatively determining their half maximum inhibitory concentrations (IC50s) and validated its anti-tumor efficacy against GSGC tumors. Meanwhile, there was no evidence to indicate histopathological damages to normal organs in the optimized ADC treated group. Conclusions: In this study, we identified a molecular target for GSGC using an unbiased and quantitative screening assay and explored its potential application in developing GSGC-targeted ADCs. In order to maximize its efficacy, we optimized the ADC formulations against GSGC cells using different ADC linkers and payloads. Our study provides a promising targeted therapeutic candidate for the clinical treatment of GSGC. Citation Format: Rui Xu, Li Yuan, Xiao-Qing Guan, Shi-Li Yao, Bing Zhu, Tong Yang, Chun Liu, Peng Guo, Jiang-Jiang Qin, Xiang-Dong Cheng. Rationally developing antibody drug conjugates targeting genomically stable gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5293.

A Novel NAMPT Inhibitor-Based Antibody-Drug Conjugate Payload Class for Cancer Therapy.

Inhibition of intracellular nicotinamide phosphoribosyltransferase (NAMPT) represents a new mode of action for cancer-targeting antibody–drug conjugates (ADCs) with activity also in slowly proliferating cells. To extend the repertoire of available effector chemistries, we have developed a novel structural class of NAMPT inhibitors as ADC payloads. A structure–activity relationship-driven approach supported by protein structural information was pursued to identify a suitable attachment point for the linker to connect the NAMPT inhibitor with the antibody. Optimization of scaffolds and linker structures led to highly potent effector chemistries which were conjugated to antibodies targeting C4.4a (LYPD3), HER2 (c-erbB2), or B7H3 (CD276) and tested on antigen-positive and -negative cancer cell lines. Pharmacokinetic studies, including metabolite profiling, were performed to optimize the stability and selectivity of the ADCs and to evaluate potential bystander effects. Optimized NAMPTi-ADCs demonstrated potent in vivo antitumor efficacy in target antigen-expressing xenograft mouse models. This led to the development of highly potent NAMPT inhibitor ADCs with a very good selectivity profile compared with the corresponding isotype control ADCs. Moreover, we demonstrate—to our knowledge for the first time—the generation of NAMPTi payload metabolites from the NAMPTi-ADCs in vitro and in vivo. In conclusion, NAMPTi-ADCs represent an attractive new payload class designed for use in ADCs for the treatment of solid and hematological cancers.

Characterization and Potential Mitigation of Corneal Effects in Nonclinical Toxicology Studies in Animals Administered Depatuxizumab Mafodotin.

Purpose: To characterize the ocular toxicity of an antibody-drug conjugate (ADC), depatuxizumab mafodotin (Depatux-m), in nonclinical species and to evaluate the effects of drug-antibody ratios (DARs), variations of the ADC construct, and potential methods for mitigation of the corneal toxicity. Depatux-m contains the potent cytotoxic agent monomethyl auristatin F as the ADC payload. Methods: Depatux-m was administered intravenously to cynomolgus monkeys at doses up to 30 mg/kg and to mice up to 100 mg/kg. Ocular toxicity was evaluated by clinical ophthalmic examinations and histopathology. Potential mitigation was tested through agents to block target engagement and multiple topical ophthalmic treatments (antioxidant, vasoconstrictor, tear stimulant). Results: Effects primarily involved corneal epithelium and were dose-dependent with respect to onset, severity, and time to reversal in both monkeys and mice. On slit lamp biomicroscopy, the initial effect in monkeys was superficial multifocal punctate opacities (granularity), which migrated axially and were followed by pigmentation and multifocal punctate fluorescein staining. Microscopically, findings were characterized by single-cell necrosis, pigmentation, disordered basilar layer, and thinning of the corneal epithelium. Increased toxicity was associated with a higher DAR or more stably attached linker. Treatment with agents to block target engagement did not affect toxicity, and none of the topical treatments was successful. Conclusions: The corneal findings observed were similar to the effects described in clinical trials with Depatux-m and other ADCs. Collectively, these studies and available literature support the hypothesis that ADC-mediated toxicity is driven primarily by mechanism of action of the payload.

Abstract P5-13-04: NF1 mutations render HER2+ breast cancer highly sensitive to T-DM1 by altering microtubule dynamics

Abstract Background: Despite major technological and conceptual advancements, treatment decisions in HER2+ metastatic breast cancer (mBC) remain largely based on clinical evidence, with no established predictive biomarkers to direct treatment for individual patients. The tumour suppressor Neurofibromatosis 1 (NF1) has been implicated in endocrine resistance but its role remains incompletely characterized in mBC. NF1 is best known as a GTPase-activating protein (GAP) that attenuates RAS signalling. However, the GAP function is likely not limited to RAS, and NF1 has been involved in other GTP-dependent processes including cytoskeletal dynamics. Data mining and analysis of public mutational registries revealed NF1 mutations as particularly enriched in HER2+ mBC compared to other molecular subtypes. Methods: To investigate the biological consequences of NF1 loss, we generated NF1 KO HER2+ mBC cell lines (BT474 and SKBR3) by CRISPR-Cas9 and both 2D and 3D proliferations assays were used for drug sensitivity profiling; live-cell imaging, high-resolution confocal microscopy and an ad-hoc computational algorithm were employed to study cell fate and microtubule conformational changes. Patient data were obtained from the Northwestern University through a prospective observational study in mBC patients. Results: Screening of several compounds approved for HER2+ mBC showed that response was generally equal or reduced in NF1 KO vs WT cells. However, response to trastuzumab-emtansine (T-DM1) was significantly increased in NF1 KO cells (IC50 ~0,3 vs 1,6 μg/mL in NF1WT). This sensitization was not observed with other antibody drug conjugates (ADCs) like DS-8201 and was reproducible with maytansine alone, suggesting a pharmacologically relevant NF1 activity on microtubules. Using the FUCCI(Ca) reporter, which tracks cell cycle progression at single-cell level, we saw a more prominent G2/M phase arrest and cell death upon T-DM1 treatment in NF1 KO compared to WT cells. Notably, NF1 KO cells exhibited a higher frequency of aberrant mitotic figures (chromosome alignment defects and multipolar spindle formation) and stronger β-galactosidase activity, an established marker of senescence. Collectively, these results suggest that NF1 KO cells become particularly subject to T-DM1-triggered mitotic catastrophe. Dephosphorylation of GTP-bound tubulin is required for appropriate microtubular dynamics; so-called “GTP islands” within the inner microtubule region are prone to rapid repolymerization and are normally kept at low levels. We hypothesize that expanded GTP-tubulin islands generated by the loss of NF1 GAP activity is a major cause of microtubular instability in NF1 KO cells. Preliminary evidence in support of this model was obtained by quantification of GTP-tubulin with a specific antibody. Finally, we assessed the predictive role of NF1 as a biomarker for T-DM1 response in a cohort of 300 mBC patients with mutational data in circulating tumour DNA (Guardant 360); we identified 13 heavily pretreated patients (>4 prior lines) who received T-DM1, of which 3 had loss-of-function NF1 mutations and 10 were NF1 WT. Median progression-free survival was higher in NF1-mutated than WT patients (334 vs 80 days); given the small sample size, these results cannot yet be considered significant (p=0.14). Conclusions: These results provide preliminary mechanistic and clinical evidence supporting the use of NF1 loss to guide treatment in HER2+ mBC. As novel HER2-specific agents are being rapidly added to the therapeutic arsenal, we propose biology-driven criteria to identify patients that may benefit specifically from T-DM1. In addition, NF1 dependence for correct microtubular dynamics may be exploited by other inhibitors of microtubular polymerization in use as ADC payloads, further extending the potential usefulness of NF1 determination. Citation Format: Bruno A Duso, Elena Gavilán Dorronzoro, Giulia Tini, Maria R de Filippo, Emanuele Bonetti, Maria R Ippolito, Chiara Soriani, Paolo D'Amico, Simona Rodighiero, Giuseppe Curigliano, Stefano Santaguida, Massimo Cristofanilli, Pier Giuseppe Pelicci, Luca Mazzarella. NF1 mutations render HER2+ breast cancer highly sensitive to T-DM1 by altering microtubule dynamics [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-04.

Design and Development of Glucocorticoid Receptor Modulators as Immunology Antibody-Drug Conjugate Payloads.

Glucocorticoid receptor modulators (GRM) are the first-line treatment for many immune diseases, but unwanted side effects restrict chronic dosing. However, targeted delivery of a GRM payload via an immunology antibody-drug conjugate (iADC) may deliver significant efficacy at doses that do not lead to unwanted side effects. We initiated our α-TNF-GRM ADC project focusing on identifying the optimal payload and a linker that afforded stable attachment to both the payload and antibody, resulting in the identification of the synthetically accessible maleimide-Gly-Ala-Ala linker. DAR 4 purified ADCs were shown to be more efficacious in a mouse contact hypersensitivity model than the parent α-TNF antibody. Analysis of P1NP and corticosterone biomarkers showed there was a sufficient therapeutic window between efficacy and unwanted effects. In a chronic mouse arthritis model, α-TNF-GRM ADCs were more efficacious than both the parent α-TNF mAb and an isotype control bearing the same GRM payload.

Mechanistic insight into the repair of C8-linked pyrrolobenzodiazepine monomer-mediated DNA damage.

Pyrrolobenzodiazepines (PBDs) are naturally occurring DNA binding compounds that possess anti-tumor and anti-bacterial activity. Chemical modifications of PBDs can result in improved DNA binding, sequence specificity and enhanced efficacy. More recently, synthetic PBD monomers have shown promise as payloads for antibody drug conjugates and anti-bacterial agents. The precise mechanism of action of these PBD monomers and their role in causing DNA damage remains to be elucidated. Here we characterized the damage-inducing potential of two C8-linked PBD bi-aryl monomers in Caulobacter crescentus and investigated the strategies employed by cells to repair the same. We show that these compounds cause DNA damage and efficiently kill bacteria, in a manner comparable to the extensively used DNA cross-linking agent mitomycin-C (MMC). However, in stark contrast to MMC which employs a mutagenic lesion tolerance pathway, we implicate essential functions for error-free mechanisms in repairing PBD monomer-mediated damage. We find that survival is severely compromised in cells lacking nucleotide excision repair and to a lesser extent, in cells with impaired recombination-based repair. Loss of nucleotide excision repair leads to significant increase in double-strand breaks, underscoring the critical role of this pathway in mediating repair of PBD-induced DNA lesions. Together, our study provides comprehensive insights into how mono-alkylating DNA-targeting therapeutic compounds like PBD monomers challenge cell growth, and identifies the specific mechanisms employed by the cell to counter the same.

Abstract LBA018: Discovery of novel functional TROP2 antibodies for treatment of epithelial cancers

Abstract Trophoblast cell surface antigen-2 (TROP2) is a membrane-bound protein with an extracellular domain highly expressed in a wide range of late-stage epithelial cancers. TROP2 upregulation in solid cancers is associated with increased tumor aggressiveness, metastasis, and an overall decreased survival in large groups of difficult-to-treat cancers, making it an attractive target for cancer therapy. TROP2 positive cancers have been successfully targeted by antibody-drug conjugates (ADC) but not naked functional antibodies. ADC’s have associated drug toxicity in healthy tissues, off-target effects, and payload delivery issues. Given ADC dose-limiting side effects, narrow therapeutic windows, and efficacy limitations, there is a pressing need to improve anti-TROP2 therapeutics for patients who are poorly served by current therapies, if at all. Understanding these needs, we applied our proprietary heavy chain-only antibody (HCAb) transgenic mouse platform to the creation of therapeutic anti-TROP2 antibodies. After immunization, anti-TROP2 antibodies were selected using VHH (variable region of HCAbs)-phage-display. Due to their size and shape, VHH antibodies target a broader spectrum of epitopes compared to full size VH/VL antibodies. Further, VHH antibodies are associated with high affinity, stability, and robustness. In this abstract, we describe five novel anti-TROP2 HCAbs without ADC payloads that induce in vivo tumor regression in murine xenograft models of human cancers. When tested in an in vitro viability assay with the MDA-MB-453 triple negative breast cancer (TNBC) cell line, a novel anti-TROP2 HCAb significantly reduced cancer cell numbers compared to other anti-TROP2 antibodies in the absence of complement or effector cells. In the in vivo MDA-MB-453 TNBC established xenograft model, tumor regression was achieved with five different TROP2 HCAbs at 8mg/kg administered once per week, and in the in vivo established model of MDA-MB-231 strong anti-tumor efficacy was also achieved. In binding cross-reactivity experiments, anti-TROP2 HCAbs showed similar binding to human and cynomolgus TROP2 and low binding to the mouse or rat orthologs. Anti-TROP2 HCAbs bind to a panel of TROP2-expressing human cancer cell lines, including a wide range of human epithelial tumours, such as breast, colon, lung and pancreatic cancers. Epitope binding by competitive ELISA showed that binding to TROP2 by these HCAbs could not be competed by hRS7 or other anti-TROP2 antibodies demonstrating that they target epitopes different from known TROP2 antibodies. Our preclinical data demonstrates the potent anti-tumor activity on TROP2-positive cancers by novel anti-TROP2 HCAbs without the need for cytotoxic payloads. These results and additional data highlight the feasibility of developing functional anti-TROP2 antibodies for clinical use which avoid the challenges of ADCs. Citation Format: Israel Matos, Hiba Zahreddine, Yahya Ashraf, Aditya Pandey, Emily Chen, Liying Gong, Alex Zhou, Claire Bonfils, Aniel Moya, Yun Cui, Xiaowei Wang, Elijus Undzyz, Shugang Yao, Jacynthe Toulouse, Dominic Hou, Gordon Ngan, Luis da Cruz, David Young. Discovery of novel functional TROP2 antibodies for treatment of epithelial cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA018.

Abstract P196: Novel hydrophilic drug linkers enable exatecan-based antibody-drug conjugates with promising physiochemical properties and in vivo activity

Abstract The physiochemical properties of an antibody-drug conjugate (ADC) can impact its stability and pharmacokinetics/pharmacodynamics and are one of the key design attributes. Typically, ADCs with better hydrophilicity are less prone to aggregation and have lower systemic clearance and greater anti-tumor activities. However, the need of incorporating lipophilic payloads with enhanced bystander activity has posed significant challenges to ADC and linker design, especially at higher drug-antibody ratios (DAR). Here we present novel hydrophilic linkers that can greatly improve the hydrophilicity of ADCs conjugated to lipophilic payloads such as exatecan. Exatecan is camptothecin analogue that has failed as a small molecule drug in clinical trials due to lack of therapeutic window but retains considerable promise as an ADC payload because of its high potency and resistance to Pgp efflux. By introducing highly polar PEG, polyhydroxyl and/or polycarboxyl groups, we generated hydrophilic linkers that enable site specific, highly homogeneous conjugation of exatecan to multiple prototypical monoclonal antibodies at DAR8. The linkers may also allow for other DARs such as DAR4 and DAR16. These exatecan-based ADCs with novel hydrophilic linkers were evaluated for their binding affinity and stability and compared with corresponding naked antibodies and deruxtecan-based and conventional vedotin-based ADCs for hydrophilicity, pharmacokinetics, in vitro and in vivo anti-tumor activities. Safety assessments in cynomolgus monkeys have also been planned. ADCs conjugated with the novel hydrophilic linkers were stable at 37oC for 15 days, after 5 cycles of freeze-thaw, and at concentrations as high as 100 mg/mL, as assessed by visual inspection, hydrophobic interaction chromatography, and size-exclusion chromatography. Binding affinity to target-positive cell lines was similar to corresponding naked antibodies. At DAR8, these ADCs were more hydrophilic than deruxtecan-based ADCs and DAR2 species of conventional vedotin-based ADCs. These ADCs also demonstrated potent in vitro cell growth inhibition and induced comparable or stronger tumor regression with single or multiple dosing in multiple mouse xenograft models. The pharmacokinetics profiles of these ADCs are similar to those of the naked antibodies. In summary, our novel hydrophilic linkers can enable conjugation of exatecan-based ADCs at high DARs with favorable physiochemical properties, which result in robust stability, pharmacokinetics, potency, and the potential for a meaningful therapeutic window. Citation Format: Haidong Liu, Lei Wang, Julia Gavrilyuk, Tae Han, Baiteng Zhao, Xiao Shang. Novel hydrophilic drug linkers enable exatecan-based antibody-drug conjugates with promising physiochemical properties and in vivo activity [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P196.

Abstract 1873: GlycoConnect™ ADCs based on topoisomerase 1 inhibitor exatecan (SYNtecan E™) show excellent in vivo efficacy and tolerability

Abstract Camptothecins form a powerful class of chemotherapy drugs (e.g. irinotecan, topotecan, belotecan), based on their ability to inhibit topoisomerase I. Some camptothecins, such as DXd and SN-38, have also proven powerful payloads for antibody-drug conjugates (ADCs), as exemplified by the recent market approval of Enhertu® and Trodelvy®, respectively. We have reported earlier that the native glycan of monoclonal antibodies is a priviliged conjugation site for ADCs (GlycoConnect™)1, while the highly polar spacer technology (HydraSpace™)2 enables the conjugation of any cytotoxic payload, providing ADCs with significantly expanded therapeutic index (TI) versus mainstream ADC technologies. We here show that exatecan (DX-8951f), a clinically validated and potent campothecin, is readily combined with HydraSpace™ technology to provide linker-payload (SYNtecan E™). Homogeneous and stable GlycoConnect™ADCs were generated from trastuzumab and SYNtecan E™ and compared head-to-head with DXd or SN-38payload, as well as deruxtecan, the linker-payload applied in Enhertu®. Complete tumor regression was observed in a mouse xenograft study (BT-474) after a single dose, while safety studies in mouse corroborated the high tolerability of SYNtecan E™ ADCs. Taken together, these studies demonstrate the potential of SYNtecan E™ for application in stable and site-specific ADCs with excellent therapeutic index. 1 van Geel et al. Chemoenzymatic Conjugation of Toxic Payloads to the Globally Conserved N-Glycan of NativemAbs Provides Homogeneous and Highly Efficacious Antibody−Drug Conjugates. Bioconj. Chem. 2015, 26, 2233-2242. 2 Verkade et al. A Polar Sulfamide Spacer Significantly Enhances the Manufacturability, Stability, and Therapeutic Index of Antibody-Drug Conjugates. Antibodies 2018, 7, 12, doi:10.3390/antib7010012. Citation Format: Floris van Delft. GlycoConnect™ ADCs based on topoisomerase 1 inhibitor exatecan (SYNtecan E™) show excellent in vivo efficacy and tolerability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1873.

Abstract 1832: Development of a splicing modulator-based ADC payload class with immune stimulatory properties for cancer therapy

Abstract Background: Thailanstatins are naturally occurring anti-proliferative compounds that target spliceosomes and modulate pre-mRNA splicing. Alterations in splicing machinery and mRNA splicing is common in cancer and represents a potential susceptibility that can be exploited by targeted delivery of a splicing modulator to tumors with antibody drug conjugates (ADCs). Results: PH1 payload is a novel derivative of Thailanstatin optimized for metabolic stability and anti-tumor activity. PH1 is an efficient modulator of splicing in vitro and in vivo and a poor substrate for the MDR1 transporter. Transcriptome analysis of PH1- treated NCI-N87 cells revealed multiple classes of mis-splicing events including elevated expression of transcripts with skipped exons (3364 genes/4x change) and retained introns (332 genes/2.5x change). Translation of transcripts with retained introns and novel exon-exon junctions results in generation of neoepitopes. Upon conjugation to Trastuzumab, Tras PH1 ADC exhibited nanomolar potency specific to HER2-expressing cancer cells, with durable anti-tumor response in vivo and favorable pharmacokinetic exposure in mice. Due to the potential for generation of neoepitopes, combination studies of Tras PH1 with a checkpoint inhibitor antibody was performed in a syngeneic MC38-HuHer2 model. Single agent and combination treatments induced tumor recruitment of CD11b+ positive myeloid cell subsets. While the combination treatment eradicated tumors, the treated animals also developed tumor-specific immunity. A toxicology study performed in cynomolgus monkeys confirmed favorable PK profile and a wide safety margin for Tras PH1 ADC. Conclusions: We present the development of a splicing modulator-based payload class with the ability to target tumor mRNA splicing, induce tumor neoepitopes, recruit myeloid cells and generate anti-tumor immunity. These findings support the development of ADCs using this novel class of immunostimulatory payload. Citation Format: Satyajit K. Mitra, Vasu Jammalamadaka, Jeffrey Kang, Teodora Losic, Greg Tuffy, Tony W. Liang, Kim Tipton, Adriana Lopez, Scott Savage, William Monteith, William E. Haskins, Melissa S. Jurica, Sanjeev Satyal, Mary Do. Development of a splicing modulator-based ADC payload class with immune stimulatory properties for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1832.

Abstract 1319: Rapid accumulation of cytotoxic payload in tumor tissue drives BT5528 activity in tumor models

Abstract BT5528 was developed as a Bicycle® toxin conjugate to deliver monomethyl auristatin E (MMAE) -payload to EphA2 overexpressing tumors. It consists of a bicyclic peptide targeting the tumor antigen EphA2, linked to the cytotoxin MMAE via a molecular spacer and cleavable linker. Administration of BT5528 results in rapid uptake of payload into EphA2 overexpressing xenograft tumors associated with persistent toxin levels in tumor tissue and limited systemic exposure of both parent drug and payload (1). BT5528 showed a favorable preclinical profile supporting the initiation of a first-in-human Phase I/II study (NCT04180371) to investigate safety and efficacy of BT5528 in indications with evidence of EphA2 expression including non-small-cell lung cancer (NSCLC), ovarian cancer, triple-negative breast cancer (TNBC), gastric/upper gastrointestinal (GI), pancreatic and urothelial cancers (2). We have used EphA2 overexpressing tumor xenograft models in both mice and rats to elucidate the relative differences between toxin payload delivery and systemic exposure of BT5528. Comparison of payload-delivery and systemic exposure from EphA2 -targeted Bicycle toxin conjugate and antibody-drug conjugate (ADC) demonstrate the differentiation of these toxin-payload delivery platforms. ADC payload delivery depends upon a sustained plateau-like pharmacokinetic (PK) profile that exposes target and potentially nontarget organs to intact ADC for several days. In contrast, BT5528 achieves prolonged toxin delivery to tumors, following transient exposure in plasma of less than one hour. A Phase I/II study of BT5528 in patients with solid tumors is ongoing, which will investigate if this PK novel, differentiated pharmacokinetic profile, unique to Bicycle toxin conjugates, gives rise to a favorable balance of efficacy and safety.

Linker Design Impacts Antibody-Drug Conjugate Pharmacokinetics and Efficacy via Modulating the Stability and Payload Release Efficiency.

The development of antibody-drug conjugates (ADCs) has significantly been advanced in the past decade given the improvement of payloads, linkers and conjugation methods. In particular, linker design plays a critical role in modulating ADC stability in the systemic circulation and payload release efficiency in the tumors, which thus affects ADC pharmacokinetic (PK), efficacy and toxicity profiles. Previously, we have investigated key linker parameters such as conjugation chemistry (e.g., maleimide vs. disulfide), linker length and linker steric hindrance and their impacts on PK and efficacy profiles. Herein, we discuss our perspectives on development of integrated strategies for linker design to achieve a balance between ADC stability and payload release efficiency for desired efficacy in antigen-expressing xenograft models. The strategies have been successfully applied to the design of site-specific THIOMABTM antibody-drug conjugates (TDCs) with different payloads. We also propose to conduct dose fractionation studies to gain guidance for optimal dosing regimens of ADCs in pre-clinical models.

C3 ester side chain plays a pivotal role in the antitumor activity of Maytansinoids

Maytansinoids, the chemical derivatives of Maytansine, are commonly used as potent cytotoxic payloads in antibody-drug conjugates (ADC). Structure-activity-relationship studies had identified the C3 ester side chain as a critical element for antitumor activity of maytansinoids. The maytansinoids bearing the methyl group at C3 position with D configuration were about 100 to 400-fold less cytotoxic than their corresponding L-epimers toward various cell lines. The detailed mechanism of how chirality affects the anticancer activity remains elusive. In this study, we determined the high-resolution crystal structure of tubulin in complex with maytansinol, L-DM1-SMe and D-DM1-SMe. And we found the carbonyl oxygen atom of the ester moiety and the tail thiomethyl group at C3 side chain of L-DM1-SMe form strong intramolecular interaction with the hydroxyl at position 9 and the benzene ring, respectively, fixing the bioactive conformation and enhancing the binding affinity. Additionally, ligand-based and structure-based virtually screening methods were used to screen the commercially macrocyclic compounds library, and 15 macrocyclic structures were picketed out as putatively new maytansine-site inhibitors. Our study provides a possible strategy for the rational discovery of next-generation maytansine site inhibitors.

Axially Substituted Silicon Phthalocyanine Payloads for Antibody–Drug Conjugates

AbstractIR700, a silicon phthalocyanine (SiPc) photosensitizer, is an antibody–drug conjugate payload used clinically. It is, however, the sole SiPc payload to date, possibly due to the difficulty of its synthesis, resulting from its asymmetric phthalocyanine skeleton. Here, we report a new axially substituted SiPc payload that is more easily synthesized. Trastuzumab conjugated with the SiPc showed light- and antigen-dependent cytotoxicity in HER2-overexpressing cancer cell lines.

Quantitative Evaluation of the Effect of Antigen Expression Level on Antibody-Drug Conjugate Exposure in Solid Tumor.

Antibody-drug conjugates (ADCs) rely on high expression of target antigens on cancer cells to effectively enter the cell and release a cytotoxic payload. Previous studies have shown that ADC efficacy is not always tied to antigen expression. However, our recent in vitro study suggests a linear relationship between antigen expression and the intracellular levels of the ADC payload. In this study, we have explored the relationship between antigen expression and intratumoral ADC exposure in vivo. Using trastuzumab-vc-MMAE (T-vc-MMAE) and four cell lines with varying expression of human epithelial growth factor receptor 2 (HER2), the pharmacokinetics of total trastuzumab, released ("free") MMAE, and total MMAE were evaluated in a tumor xenograft model. Nude mice were implanted with tumors originating from BT-474, MDA-MB-453, MCF-7, and MDA-MB-468 cell lines and dosed with 10 mg/kg or 1 mg/kg of ADC. Observed data were mathematically characterized using a mechanism-based PK model. A strong positive correlation was observed between antigen expression levels and free/total MMAE exposure (R2 ≥ 0.91) (total MMAE being the sum of released and conjugated MMAE) within the tumor, but not for total trastuzumab exposure. The PK model was able to recapitulate plasma PK through simulation; however, the tumor PK was overpredicted or underpredicted in some cases potentially due to differences in tumor vasculature or extracellular matrix conditions. Our results indicate a linear relationship between antigen expression and tumor exposure of free/total ADC payload in vivo, validating our previous finding in vitro, while also revealing the need to understand complex physiology of the tumor to predict tumor PK of ADC and its components. Our findings also support the concept of antigen expression screening in patients for targeted therapies like ADCs to achieve the maximum therapeutic benefit of the treatment.

Amino DSA analogues as payloads for antibody-drug conjugates with multiple sites for conjugation. Initial studies and solid phase synthesis

Duocarmycins are highly potent and promising anticancer payloads for ADC applications. They tolerate a range of chemical modifications which allow the chemist to modulate both their biophysical and pharmacological properties. The possibility to synthesize these payloads on resin and orthogonally add linkers while immobilized on the solid phase, would allow a combinatorial design of payload analogues with linkers, potentially aided by automation. Working towards this goal, we report a concise and high yielding synthesis of an alkylating unit suitable for solid phase synthesis (10, 9 steps, 34% yield) and demonstrate its applicability to the synthesis of duocarmycin SA analogues (19, 20). An intermediate for traditional solution phase synthesis (8) is also described in 7 steps and 44% yield. A side reaction with potential application to the stereoselective synthesis of these derivatives has also been described.