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Antibody–drug conjugates (ADCs) that incorporate the exatecan derivative DXd in their payload are showing promising clinical results in solid tumor indications. The payload has an F-ring that also contains a second chiral center, both of which complicate its synthesis and derivatization. Here we report on new camptothecin-ADCs that do not have an F-ring in their payloads yet behave similarly to DXd-bearing conjugates in vitro and in vivo. This simplification allows easier derivatization of camptothecin A and B rings for structure–activity relationship studies and payload optimization. ADCs having different degrees of bystander killing and the ability to release hydroxyl or thiol-bearing metabolites following peptide linker cleavage were investigated.

Tumors with BRCA1/2 mutations and other homologous repair deficiencies (HRD) are vulnerable to agents that target the remaining DNA repair pathways, including platinum-containing chemotherapy agents and molecules targeting poly (ADP-ribose) polymerase-1 (PARP1). Despite the clinical benefit achieved with these drugs, many patients achieve incomplete disease control and resistance often emerges. With the goal of addressing this clinical need, we applied our proprietary CRISPRomics technology to identify novel targets in cancer indications characterized by defects in DNA repair pathways. One of the top ranked targets was the deubiquitinating enzyme USP1. USP1 has established roles in DNA damage repair processes including Translesion Synthesis and the Fanconi Anemia pathway. We developed KSQ-4279, a potent, highly selective inhibitor of USP1. KSQ-4279 was active in cells, leading to the accumulation of mono-ubiquitinated substrates of USP1 and inhibited the proliferation of cancer cell lines with BRCA mutations or other HRD alterations. Studies investigating the effect of combining KSQ-4279 with PARP inhibitors revealed clear evidence of synergy in cell lines with partial or no sensitivity to each agent alone. To investigate how the distinct mechanisms of action of KSQ-4279 and PARP inhibitors would be reflected in their resistance profiles, we used our CRISPRomics technology to perform functional genomic resistance screens. The top scoring resistance genes for KSQ-4279 were distinct from those identified for PARP inhibitors, which raised the possibility that combining PARP and USP1 inhibitors may provide more durable disease control by reducing the emergence of resistance. Evaluation of KSQ-4279 in patient-derived ovarian and triple-negative breast cancer xenograft models demonstrated dose-dependent tumor growth inhibition as a single agent and in combination with PARP inhibitors. In xenograft models that were insensitive or only partially sensitive to PARP inhibitors, the combination of KSQ-4279 and Olaparib led to tumor regressions and durable tumor control. This data supports the ongoing clinical trial of KSQ-4279 in patients with tumors harboring BRCA1/2 or other HRD mutations, both as a single agent and in combination with PARP inhibitors. Citation Format: Louise Cadzow, Erica Tobin, Pamela Sullivan, Sol Shenker, Sumeet Nayak, Janid Ali, Hugh Gannon, Anne Dodson, Paula Grasberger, Alyssa Carlson, Michael McGuire, Jehrod Brenneman, Hanlan Liu, Andrew Olaharski, Kerstin Sinkevicius, Jeff Hixon, Elsa Krall, Mike Schlabach, Matt Goulet, Jeremy Wilt, Patricia Harris, Frank Stegmeier, Andrew Wylie. KSQ-4279: A first-in-class USP1 inhibitor for the treatment of cancers with homologous recombination deficiencies [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 ND01.

Src homology 2 domain-containing phosphatase (SHP2), a ubiquitously expressed non-receptor tyrosine phosphatase, plays a critical role in the regulation of the MAPK signaling pathway and cellular proliferation. Activating mutations in SHP2 are associated with the development of multiple malignancies including leukemia, lung cancer and neuroblastoma. In addition, SHP2 promotes the conversion of oncogenic KRAS to its active GTP-bound state and it’s inhibition can enhance efficacy of GDP-KRASG12C inhibitors as well as other MAPK pathway inhibitors (RAF, MEK and ERK) which have suboptimal clinical efficacy as single agents. As a result, inhibition of SHP2 through genetic manipulation or pharmacological means has been shown to suppress tumor growth and presents an attractive potential avenue for the treatment of malignancies as monotherapy or in combination with other MAPK/PI3K inhibitors. Here we describe BBP-398, a potent, orally bioavailable allosteric small molecule inhibitor of SHP2. BBP-398 displays high selectivity against other phosphatases, kinases, GPCRs, transporters and hERG. Predicted human PK properties show good oral bioavailability with half-life of ~12-16 hours enabling continuous daily dosing and optimal therapeutic index in combination with other targeted therapeutics. In cellular assays, BBP-398 demonstrates potent pERK/DUSP6 inhibition and loss of viability across a panel of cell lines with active MAPK signaling, such as mutant EGFR and KRASG12C. In vivo, BBP-398 strongly suppresses RAS-ERK signaling in RTK- or RAS-driven xenografts. In the EGFR-dependent non-small cell lung cancer (NSCLC) HCC827 and esophageal squamous cell carcinoma KYSE-520 xenograft models, BBP-398 drives dose dependent efficacy consistent with the level of target inhibition. Detailed analysis of tumor response shows that efficacy is driven by maintaining better than 50% inhibition of pERK for most of the dosing interval. In addition to its strong single agent activity, BBP-398 also leads to enhanced efficacy in vitro and in vivo when used in combination with targeted therapeutics against driver MAPK genetic alterations, such as KRAS, EGFR or MET. Combination targeting, such as with the GDP-KRASG12C inhibitor sotorasib in the NSCLC NCI-H358 xenograft model, or with the mutant EGFR inhibitor osimertinib in the HCC827 erlotinib resistant (ER) xenograft model, drives strong suppression of MAPK activity and results in tumor regressions. Collectively, these findings highlight that SHP2 inhibition is a promising molecular therapeutic strategy in cancer which can potentially strongly suppress tumor growth as a single agent or in combination with other MAPK pathway inhibitors. Given its preclinical properties and projected favorable clinical pharmacokinetic profile, BBP-398 is currently being evaluated in a Phase 1/1b trial in patients with advanced solid tumors (NCT04528836). Citation Format: James P. Stice, Sofia Donovan, Yuting Sun, Nancy Kohl, Barbara Czako, Faika Mseeh, Paul Leonard, Anna Wade, Justin Lim, Phil Jones, Eli Wallace, Kerstin Sinkevicius, Pedro Beltran. BBP-398, a potent, small molecule inhibitor of SHP2, enhances the response of established NSCLC xenografts to KRASG12C and mutEGFR inhibitors [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 P207.

Genomic instability is a hallmark of most cancers that enables tumor cells to adapt and evolve, but also creates vulnerabilities that can be therapeutically exploited. Cancer therapies that target the DNA Damage Response (DDR) have proven to be efficacious across a broad range of cancer types, but often present a narrow therapeutic window and limited duration of response due to the development of resistance. Through mining our proprietary CRISPRomics® Oncology dataset we identified the ubiquitin protease, USP1, as an attractive target with activity in ovarian and triple negative breast cancers (TNBC). USP1 facilitates DNA repair via its role regulating the Fanconi anemia complex and translesion synthesis. We developed a series of potent, selective USP1 inhibitors to investigate the therapeutic potential of targeting USP1 in tumor settings dependent on those DNA repair processes. When profiled across a broad range of cancer cell line models, the USP1-inhibitor KSQ-4279 showed anti-proliferative effects in a subset of cell lines, often characterized by the presence of homologous recombination deficiencies (HRD), including mutations in BRCA1/2. To better understand the mechanism of action of KSQ-4279, we employed large scale functional genomic screens using KSQ-4279, olaparib, and cisplatin, in combination with a DNA-repair focused CRISPR library, to identify genetic determinants of sensitivity and anticipate modes of drug resistance. In addition to recovering genetic associations between KSQ-4279 and genes that modify USP1's direct substrates, our screens identified genetic interactions of KSQ-4279 with multiple DNA repair pathways, including genes involved in ubiquitin-mediated signaling events that regulate the response to DNA damage and replication stress. These studies revealed that the profile of resistance to KSQ-4279 is distinct from, and in some cases complementary to, other DNA damaging agents, and provide a mechanistic rationale for the use of KSQ-4279 in combination with other agents that target DNA repair. Consistent with the largely non-overlapping resistance profile of USP1 and PARP inhibitors, we found that the combination of KSQ-4279 with olaparib was able to induce strong and durable regressions across a number of ovarian and TNBC PDX models. Citation Format: Sol Shenker, Hugh Gannon, Alyssa Carlson, Paula Grasberger, Pamela Sullivan, Chris Middleton, Anne Dodson, Caroline Bullock, Michael McGuire, Erica Tobin, Kerstin Sinkevicius, Mike Schlabach, Frank Stegmeier, Louise Cadzow, Andrew Wylie. Functional genomic characterization of the USP1 inhibitor KSQ-4279 reveals a distinct mechanism of action and resistance profile relative to other DDR targeting drugs [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 1337.

Dysregulation of ADAM9, a member of the ADAM (a disintegrin and metalloproteinase) family of proteases, has been implicated in tumor progression and metastasis, as well as pathological neovascularization. ADAM9 overexpression correlates with poor prognosis in multiple cancers. We have shown that ADAM9 is overexpressed in multiple solid tumor indications and that anti-ADAM9 antibodies are efficiently internalized and degraded by tumor cell lines making ADAM9 an attractive target for antibody-drug conjugate (ADC) development. Here, we describe IMGC936, the first ADAM9-targeting ADC to enter preclinical development. IMGC936 is comprised of a high-affinity humanized antibody site-specifically conjugated to DM21, a next-generation linker-payload that combines a maytansinoid microtubule-disrupting payload with a stable peptide linker at a drug-antibody ratio of two. To maximize the potential for IMGC936 activity, the M252Y/S254T/T256E (YTE) mutation was introduced into the CH2 domain of the antibody to increase in vivo plasma half-life and exposure. In vitro studies demonstrated targeted cytotoxicity of IMGC936 across a panel of ADAM9-positve tumor cell lines with activity at least 2 logs greater than a non-targeting conjugate. Consistent with the in vitroactivity, an anti-ADAM9-DM21 conjugate displayed compelling anti-tumor activity in multiple xenograft models representing non-small cell lung, gastric and colorectal cancers. For example, in the EBC-1 non-small cell lung cancer subcutaneous xenograft model with only moderate ADAM9 expression (H-score of 130), anti-ADAM9-DM21 not only induced tumor growth delay but produced complete and durable remissions in 6/6 mice following a single intravenous dose of 8.6 mg Ab/kg (100 ug DM21/kg). IMGC936 demonstrated a favorable pharmacokinetic profile with good conjugate stability in non-human primates. Importantly, IMGC936 was well-tolerated following repeat dosing in cynomolgus monkeys with no ADAM9 target-related toxicities identified at doses exceeding the levels required for anti-tumor activity in murine xenograft models. Based on the totality of the preclinical data, IMGC936 represents a promising therapeutic candidate to target a wide range of ADAM9-expressing tumors. Citation Format: Stuart Hicks, Deryk Loo, Kerstin Sinkevicius, Juniper Scribner, Bhaswati Barat, Nicholas Yoder, Christopher Espelin, Marian Themeles, Francine Chen, Jacquelynn Lucas, Jennifer Brown, Bahar Matin, Megan Fuller, Jenny Lee, Paulin Salomon, Juliet Costoplus, Sadiqa Yancey, Gundo Diedrich, Sergey Gorlatov, Thomas Son, Michael Chiechi, Pam Li, Michael Spliedt, Valentina Ciccarone, Jeff Hooley, Nadia Gantt, James Tamura, Kerry Donahue, Paul Moore, Syd Johnson, Thomas Chittenden, Richard Gregory, Ezio Bonvini. IMGC936, a first-in-class ADAM9-targeting antibody-drug conjugate, demonstrates promising anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1533.

ImmunoGen’s newest antibody-drug conjugate (ADC) design uses the novel maytansinoid linker-payload, DM21-C that bears a peptidase/protease cleavable linker. These ADCs show good bystander killing of proximal antigen negative cells, suggesting the generation of cell-permeable catabolites. The goal of this study was to identify the catabolites generated upon incubation in antigen-positive cancer cells (both cell pellet and media), in mouse plasma, as well as in in vitro catabolic systems. Upon incubation with a cysteine-conjugated DM21-C ADC, the small molecule fraction was extracted with an organic solvent and analyzed by high resolution mass spectrometry after chromatographic separation. Through comparison to reference standards, mass spectral signal extraction of possible products, and database search of unknown peaks, the potential in vitro catabolite products of DM21-C conjugate were identified. Information on linker-payload stability in these model systems, including plasma stability, in vitro metabolism of the ADC, and catabolic products that were both retained and effluxed from cancer cells was obtained. As a result, we identified DM51 (the thiol- resulting from self-immolation of the cleaved linker-payload) as a major catabolite of the DM21-C ADC. Citation Format: Janet Lau, Paulin Salomon, Kerstin Sinkevicius, Juliet Costoplus, Megan Fuller, Raymond Xu, Stuart Hicks, Ravi Chari, Wayne Widdison, Nicholas Yoder, Thomas Keating. LC-MS based catabolite identification study of an ADC with DM21-C, a novel maytansinoid linker-payload [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 538.

DARPin® molecules are small engineered proteins, derived from natural ankyrin repeat proteins that are selected to bind to specific targets with high affinity. Individual DARPin® molecules can be linked together genetically in order to create multi-specific drug molecules. This versatility of DARPin® molecules makes them an attractive alternative to antibodies for the development of drug conjugates. We have developed DARPin® drug conjugates (DDCs) using a model EGFR multi-specific DARPin® molecule, consisting of four DARPin® domains linked together. DARPin® constructs were conjugated to the indolinobenzodiazepine mono-imine DGN549, a potent DNA alkylating payload. Biophysical characterization showed the DDCs to be well behaved in stability and solubility assays. The in vitro binding and cytotoxicity of these DDCs were evaluated. The DARPin® conjugates displayed in vitro potency in direct cytotoxicity assays across a panel of cell lines expressing EGFR. The impact of conjugation on PK parameters and in vivo efficacy is currently under evaluation. DARPin® drug conjugates combine the potency observed with antibody drug conjugates and the modular design of DARPin® molecules to create designer therapeutics. Citation Format: Laura A. Laviolette, Cynthia J. Guidi, Christian Reichen, Qifeng Qiu, Luke Harris, Patricia Schildknecht, Stefanie Fischer, Zita Arany, Tanja Hospodarsch, Anna Skaletskaya, Megan Fuller, Stephen Abbott, Rebecca McCarthy, Jenny Lee, Katherine Francisco, Kerstin Sinkevicius, Sharlene Adams, Christopher Espelin, Emily Reid, Wei Li, Carla Marashio, Kerry Donahue, Stuart Hicks, Dan Snell. Generation of site-specific DARPin® drug conjugates using EGFR as a model system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 215.