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Supplementary Figure S1. Comparable internalization rates for 5T4_0108 and the 5T4-Tub ADC;Supplementary Figure S2. Affinity optimization improves in vitro cytotoxicity of a 5T4-ADC;Supplementary Figure S3. Induction of apoptosis by 5T4-PBD and 5T4-Tub;Supplementary Figure S4. 5T4 is expressed on CSCs of multiple cancer cell lines;Supplementary Figure S5. Only PBD is capable of inducing cytotoxicity of CSC populations in vitro;Supplementary Table S1. Improved cytotoxic potency following affinity optimization of 5T4 antibody; Supplementary Table S2. Quantification of cell surface-associated 5T4 in various cancer cell lines;Supplementary Table S3. Tumorigenicity of CSC populations derived from NCI- N87 and MDA-MB-361 xenografts; Supplementary Materials & Methods

STING pathway agonism has emerged as a potential therapeutic strategy to stimulate anti-tumor immune responses. We have previously shown that tumor cell-targeted antibody-drug conjugates (ADCs) carrying a novel STING agonist induce anti-tumor activity without causing substantial elevations in systemic cytokine levels, thus suggesting a therapeutic advantage of STING agonist ADCs relative to unconjugated agonists. ADCs constitute a proven therapeutic modality that is ideally suited to enable systemic administration and delivery of the conjugated drug to desired cell types within the tumor microenvironment. In addition to delivering STING agonist into the antigen-expressing tumor cells, antigen-bound ADCs deliver STING agonist to tumor-resident myeloid cells through Fcγ receptor (FcγR)-mediated internalization. In this study we investigated the mechanism of FcγR-mediated internalization of the tumor cell-targeted STING-agonist ADCs into myeloid cells and the nature of the subsequent STING pathway activation. We developed flow cytometry-based assays to determine the changes in FcγRI, FcγRII, and FcγRIII cell surface detection levels in the presence of ADCs specifically designed to be either proficient or deficient in FcγR-binding. Combined with functional assays based on co-culture of cancer cells and FcγRI knock out myeloid cells, we identified FcγRI as the major Fcγ receptor that mediates target-bound ADC internalization into myeloid cells in vitro. Even though FcγRI is expressed only by a subset of CD11b+ myeloid cells, tumor cell-targeted ADCs induce greater production of interferons and other cytokines and more potent cancer cell killing than CD11b-targeted-ADCs, which deliver STING agonist into FcγRI- (non-productive) as well as FcγRI+ (productive) myeloid cells. Finally, we demonstrate that myeloid cells within dissociated primary human tumors from multiple donors express FcγRI and are capable of tumor cell killing in response to tumor cell-targeted STING agonist ADCs in vitro. In summary, our data indicate that the ADC-mediated delivery of a STING agonist specifically into FcγRI-expressing myeloid cells efficiently activates innate immune responses in the most relevant immune cell types within the tumor microenvironment. Citation Format: Naniye Malli Cetinbas, Kalli C. Catcott, Travis Monnell, Jahna Soomer-James, Keith Bentley, Susan Clardy, Bingfan Du, Eoin Kelleher, Marina Protopopova, Cheri Stevenson, Joshua D. Thomas, Alex Uttard, Dorin Toader, Jeremy Duvall, Raghida Bukhalid, Marc Damelin, Timothy B. Lowinger. Tumor cell-targeted STING-agonist antibody-drug conjugates achieve potent anti-tumor activity by delivering STING agonist specifically to tumor cells andFcγRI-expressing subset of myeloid cells [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 2114.

We present here a novel therapeutic agent, XMT-2056, that results in robust anti-tumor activity mediated by an immune response through targeted delivery of a STING agonist to the tumor microenvironment. By leveraging an antibody-drug conjugate (ADC) strategy, systemic administration of a STING agonist with tumor-targeted delivery can be achieved, potentially overcoming limitations of either intratumoral or intravenous administrations of unconjugated, small molecule STING agonists. XMT-2056 was generated through conjugation of Immunosynthen, a platform that employs a novel STING agonist payload specifically designed for ADCs, to HT-19, a HER2-targeting antibody which binds to a novel epitope and does not compete for binding with either trastuzumab or pertuzumab. Initial results showed XMT-2056 has target-dependent anti-tumor activity in vivo and is well tolerated in non-human primates at significantly higher exposure levels than those required for anti-tumor activity. To evaluate the impact of HER2 expression level on the activity of XMT-2056, in vivo studies in gastric and breast cancer models with varying HER2 expression levels were conducted, and XMT-2056 showed potent anti-tumor activity in a dose dependent and target dependent manner including in models with very low expression of HER2. Because the antibody employed in XMT-2056 does not compete for binding with trastuzumab or pertuzumab, we hypothesized that there could be benefit in combining with such approved HER2-targeted therapies. This advantage was demonstrated in vivo as the combination of XMT-2056 and trastuzumab or pertuzumab showed greater anti-tumor activity compared to the administration of either agent alone. Further efforts to elucidate the mechanism(s) of the observed benefit of these combinations will be discussed. Given the innate immune activation by XMT-2056, there is also a strong rationale for combination with immune checkpoint inhibitors. To this end, administration of an XMT-2056 surrogate ADC in combination with an anti-PD1 agent improved anti-tumor activity in a ratHER2-engineered EMT-6 syngeneic mouse model. Together these data support the potential of XMT-2056 both as a monotherapy and in combination with other HER2 targeted agents as well as checkpoint inhibitors. Citation Format: Jeremy R. Duvall, Raghida A. Bukhalid, Naniye M. Cetinbas, Kalli C. Catcott, Kelly Lancaster, Keith W. Bentley, Suzanna Clark, Susan Clardy, Scott D. Collins, Anouk Dirksen, Elizabeth Ditty, Bingfan Du, Eugene W. Kelleher, Travis Monnell, Marina Protopopova, Caitlin Routhier, Cheri Stevenson, Elena Ter-Ovanesyan, Joshua D. Thomas, Alex Uttard, Jason Wang, Phonphimon Wongthida, Ling Xu, Annika Yau, Jeffrey Zurita, Dorin Toader, Marc Damelin, Timothy B. Lowinger. XMT-2056, a HER2-targeted Immunosynthen STING-agonist antibody-drug conjugate, binds a novel epitope of HER2 and shows increased anti-tumor activity in combination with trastuzumab and pertuzumab [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 3503.

Background STING pathway agonism has emerged as a potential therapeutic mechanism to stimulate an innate anti-tumor immune response. While in principle systemic administration of a STING agonist would have many therapeutic benefits, including the delivery of STING to all tumor lesions, such an approach may be limited by toxicity. Antibody-drug conjugates (ADCs) constitute a proven therapeutic modality that is ideally suited to allow systemic administration while stimulating the innate immunity in a targeted manner. We have previously demonstrated that targeted delivery of a STING agonist with an ADC induces robust anti-tumor immune responses. Methods Herein we investigated the mechanism of action of tumor cell-targeted STING agonist ADCs. We evaluated STING pathway activation and anti-tumor activity elicited by ADCs harboring either wild type (wt) or mutant Fc deficient in Fcγ receptor (FcγR) binding in wt or STING knockout (ko) cancer cell mono-cultures, immune cell co-cultures, and in in vivo tumor models. Results Consistent with previous reports, the majority of cancer cell lines tested failed to induce STING pathway following STING agonist payload treatment in mono-cultures. In cancer cell:THP1 monocytic cell co-cultures, tumor-targeted STING agonist ADCs with wt Fc exhibited robust STING activation, whereas Fc-mutant ADCs or non-targeted control ADCs had minimal activity. Similar results were obtained when THP1 cells were treated in plates coated with target antigen without cancer cells, demonstrating STING activation in THP1 cells following FcγR-mediated uptake of antigen-bound ADCs. Tumor-targeted Fc-wt ADCs led to marked induction of STING pathway and cancer cell-killing in cancer cell:PBMC or primary monocyte co-cultures, and complete tumor regressions in in vivo tumors. Surprisingly, while at reduced levels relative to the Fc-wt ADCs, Fc-mutant ADCs exhibited significant activity in these in vitro and in vivo models, suggesting that tumor cell-intrinsic STING pathway may be activated in the presence of cues from immune cells. Consistently, STING agonist payload treatment in the presence of conditioned media from PBMC and primary monocyte but not from THP1 cultures, led to STING activation in cancer cell mono-cultures. Moreover, Fc-mutant ADCs had diminished activity in STING ko cancer cell:PBMC or primary monocyte co-cultures, demonstrating the contribution of tumor cell-intrinsic STING activation to the anti-tumor activity elicited by tumor cell-targeted STING agonist ADCs. Conclusions In conclusion, we demonstrated that tumor cell-targeted STING agonist ADCs induce robust anti-tumor activity through mechanisms involving both FcγR and tumor antigen-mediated ADC internalization and subsequent induction of STING pathway in immune cells and tumor cells.

Janus kinases (JAKs) have been demonstrated to be critical in cytokine signaling and have thus been implicated in both cancer and inflammatory diseases. The JAK family consists of four highly homologous members: JAK1-3 and TYK2. The development of small-molecule inhibitors that are selective for a specific family member would represent highly desirable tools for deconvoluting the intricacies of JAK family biology. Herein, we report the discovery of a potent JAK1 inhibitor, 24, which displays ∼1000-fold selectivity over the other highly homologous JAK family members (determined by biochemical assays), while also possessing good selectivity over other kinases (determined by panel screening). Moreover, this compound was demonstrated to be orally bioavailable and possesses acceptable pharmacokinetic parameters. In an in vivo study, the compound was observed to dose dependently modulate the phosphorylation of STAT3 (a downstream marker of JAK1 inhibition).

Checkpoint kinase 1 (CHK1) inhibitors are potential cancer therapeutics that can be utilized for enhancing the efficacy of DNA damaging agents. Multiple small molecule CHK1 inhibitors from different chemical scaffolds have been developed and evaluated in clinical trials in combination with chemotherapeutics and radiation treatment. Scaffold morphing of thiophene carboxamide ureas (TCUs), such as AZD7762 (1) and a related series of triazoloquinolines (TZQs), led to the identification of fused-ring bicyclic CHK1 inhibitors, 7-carboxamide thienopyridines (7-CTPs), and 7-carboxamide indoles. X-ray crystal structures reveal a key intramolecular noncovalent sulfur-oxygen interaction in aligning the hinge-binding carboxamide group to the thienopyridine core in a coplanar fashion. An intramolecular hydrogen bond to an indole NH was also effective in locking the carboxamide in the preferred bound conformation to CHK1. Optimization on the 7-CTP series resulted in the identification of lead compound 44, which displayed respectable drug-like properties and good in vitro and in vivo potency.

Key defining attributes of an antibody-drug conjugate (ADC) include the choice of targeting antibody, linker, and the drug-to-antibody ratio (DAR). The choice of DAR, within the constraints of acceptable physicochemical properties for the given platform, is a function of balancing delivery of sufficient payload to targeted cells with the ability to achieve sustained in vivo exposures. Previous reports have described lower DAR mc-VC-MMAE conjugates, DAR = 1-2, that demonstrated higher in vivo exposure and lower clearance when compared to higher DAR (e.g. 4-8) counterparts. In theory, high DAR conjugates may be especially desirable when targeting low antigen expressing tumors or when lower potency payloads are used, as each binding and internalization event results in greater payload delivery. Here we report a systematic exploration of DAR across a much wider range than has been previously reported, by combining THIOMAB® protein engineering technology with the Dolasynthen platform. Homogeneous, site-specific ADCs spanning a discrete range of DARs – 2, 4, 6, 12, and 18 – were made by conjugation of Trastuzumab IgG1 THIOMAB constructs with 1, 2, or 3 engineered cysteines to monomeric or trimeric Dolasynthen. The cytotoxicity of the resulting well-defined ADCs was assessed in vitro in cell lines with high or low expression of HER2 antigen. Pharmacokinetic data for all test articles in mice were generated in tumor bearing mice. In high HER2 expressing cell lines, in vitro cytotoxicity by payload was comparable across DARs. In a lower HER2 expressing system, the higher DAR ADCs performed better. In vivo, our data demonstrated comparable pharmacokinetics for the Dolasynthen conjugates across all DARs. These results illustrate the utility of a DAR ranging platform, such as Dolasynthen when evaluating ADCs as it enables the interrogation of a range of antibody and payload dosing regimens. Citation Format: Kalli C. Catcott, Susan Clardy, Jack Sadowsky, Rebecca K. Rowntree, Naniye Malli Centibas, Ling Xu, Andy Polson, Kenneth Avocetien, Tyler Carter, Mark Nazzaro, Dokyong "DK" Kim, Thomas H. Pillow, Neelie Zacharias, Cong Wu, Jeffrey Zurita, Elizabeth Ditty, Stephen Bradley, Alex Uttard, Bingfan Du, William S. Sawyer, Doug Leipold, Gail Lewis Phillips, LiuLiang Qin, Kelly Slocum, Geoffrey Del Rosario, Ginny Li, Shang-Fan Yu, David Lee, Radha Iyengar, Marc Damelin, Dorin Toader, Timothy B. Lowinger. Site-specific Dolasynthen ADCs demonstrate consistent exposure across a wide range of drug-to-antibody ratios [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 P167.

BackgroundThe tumor microenvironment is a complex, multicellular system, composed not only of malignant cancer cells but also of a diversity of stromal cells including vascular cells, immune cells, and fibroblasts that support tumorigenesis. Antigens expressed on these cells tend to be widely expressed across a range of malignancies, presenting unique opportunities for development of anti-cancer therapies.MethodsWe have previously demonstrated that STING-agonist antibody-drug conjugates (Immunosynthen ADCs) targeting tumor cell antigens induce target-dependent anti-tumor immune responses in vitro and in vivo. To that effect, we hypothesized that Immunosynthen ADCs targeting tumor-associated antigens would coordinate immune-mediated killing of cancer cells not expressing the tumor-associated antigens (antigen-negative cancer cells) and induce anti-tumor activity.ResultsHerein, we demonstrate that targeting tumor-associated antigens with STING-agonist ADCs activate the STING pathway in immune cells via Fcγ receptor-mediated uptake. In addition, due to the intrinsic ability of certain tumor-associated cells to activate the STING pathway, STING-agonist ADCs targeting those cells can induce STING signaling in both the targeted cells and the immune cells, which constitutes a therapeutic advantage of ADCs that activate the STING pathway. In triple co-cultures of antigen-positive tumor-associated cells, antigen-negative cancer cells, and immune cells, the STING-agonist ADC specifically induced potent cell killing of the antigen-negative cancer cells with minimal impact on the immune and tumor-associated cells, thus representing a non-traditional, yet highly effective mechanism of ADC targeting. In vivo efficacy studies showed that STING-agonist ADCs developed for two tumor-associated antigens induced complete, sustained tumor regressions in syngeneic tumor models and exhibited immunological memory after rechallenge. CD8+ T cells contributed to the anti-tumor activity of the STING-agonist ADCs.ConclusionsIn summary, Immunosynthen STING-agonist ADCs targeting tumor-associated antigens represent a novel approach for ADC-mediated cancer immunotherapy and enable the multifaceted activation of the STING pathway in a tumor-targeted manner beyond tumor antigens.

STING pathway plays a critical role in inducing anti-tumor immunity by upregulating Type 1 Interferon (IFN) and IFN-stimulated genes within the tumor microenvironment in response to cytosolic nucleic acid ligands. Therefore, the STING pathway agonism has emerged as a potential therapeutic mechanism to stimulate an anti-tumor innate immune response. Intratumorally injected free STING-agonists that are currently being evaluated in the clinic by others have shown limited effects in non-injected lesions. Antibody-drug conjugates (ADCs) constitute a proven therapeutic modality that enables tumor-targeted drug delivery with systemic administration. We have previously demonstrated that the tumor cell-intrinsic STING pathway is activated in the presence of cues from immune cells and contributes to the anti-tumor activity of tumor cell-targeted Immunosynthen STING-agonist ADCs, in which a STING-agonist payload is conjugated to a tumor cell-targeting antibody. Here we investigated the nature of the STING pathway activation in tumor cells and its contribution to the anti-tumor activity elicited by STING agonism. Leveraging ADCs with a wild type (wt) or mutant Fc (deficient in Fcγ receptor -FcγR- binding), we delivered a STING-agonist simultaneously to tumor-resident immune and cancer cells or only to cancer cells through FcγR-mediated and/or tumor antigen-mediated ADC internalization. We utilized these ADCs in in vivo human tumor xenograft models and STING wt or knock out (ko) cancer cell:immune cell co-cultures and evaluated gene expression, cytokine production, and anti-tumor activities induced by STING-agonist ADCs. Surprisingly, Nanostring analysis of the human tumor xenografts from mice treated with tumor cell-targeted STING-agonist ADCs revealed human tumor cell-specific activation of Type III IFNs. In human cancer cell:immune cell co-cultures, treatment with tumor cell-targeted STING-agonist ADCs also led to marked upregulation of Type III IFNs, which was significantly reduced in STING ko cancer cell:immune cell co-cultures, suggesting that the cancer cells may contribute majority of the Type III IFNs downstream of STING pathway activation. Blocking Type III IFNs with neutralizing antibodies in cancer cell:immune cell co-cultures inhibited the production of key cytokines, including Type I IFN, and nearly abolished tumor cell-killing in response to STING-agonist ADC treatment, indicating that the Type III IFNs play an important role in the anti-tumor activity induced by STING activation. These studies reveal a previously underappreciated mechanism of STING agonist anti-tumor activity. The ability of tumor cell-targeted STING-agonist ADCs to activate STING in both tumor cells and in tumor-resident immune cells may represent a significant therapeutic advantage of an Immunosynthen ADC approach to STING agonism. Citation Format: Naniye Malli Cetinbas, Travis Monnell, Kalli Catcott, Winnie Lee, Pamela Shaw, Kelly Slocum, Kenneth Avocetien, Keith Bentley, Susan Clardy, Brian Jones, Eoin Kelleher, Rebecca Mosher, Joshua D. Thomas, Dorin Toader, Jeremy Duvall, Raghida A. Bukhalid, Marc Damelin, Timothy B. Lowinger. Tumor cell-intrinsic STING pathway activation leads to robust induction of Type III Interferons and contributes to the anti-tumor activity elicited by STING agonism [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 1773.

STING pathway agonism has emerged as a potential therapeutic mechanism to stimulate an innate anti-tumor immune response. However, the systemic administration of a free STING agonist may be limited by toxicity, and broad biodistribution may not be ideal. Antibody-drug conjugates (ADCs) constitute a proven therapeutic modality that enables tumor-targeted delivery and thus is ideally suited to systemic administration with reduced toxicity. To develop an optimized STING-agonist ADC platform, we designed a novel STING-agonist specifically tailored for use in an ADC. Determination of the co-crystal structure confirmed that the agonist binds to the closed, or ‘active', conformation of the STING homodimer. The resulting Immunosynthen platform, which was developed specifically for the selected STING agonist payload, was used to generate XMT-2056, a tumor antigen-targeted STING-agonist ADC with excellent drug-like properties and >100-fold increased potency as compared to the free STING-agonist payload. In mice, XMT-2056 induced robust anti-tumor immune activity, with only minimal increases in systemic cytokine levels, and exhibited significant benefit over the benchmark free STING-agonist payload in both regards. Additionally, in vitro and in vivo studies demonstrate that XMT-2056 is able to activate the STING pathway in both tumor-resident immune cells and tumor cells, offering a potential advantage over other innate immune activating pathways. XMT-2056 was well-tolerated in non-human primates at significantly higher exposure levels than those required for anti-tumor activity, and the ADC exhibited favorable pharmacokinetics after repeat doses. Together these data support the clinical development of XMT-2056. Citation Format: Jeremy R. Duvall, Raghida A. Bukhalid, Naniye M. Cetinbas, Kalli C. Catcott, Kelly Slocum, Kenneth Avocetien, Keith W. Bentley, Stephen Bradley, Susan Clardy, Scott D. Collins, Elizabeth Ditty, Timothy Eitas, Brian D. Jones, Eugene W. Kelleher, Winnie Lee, Travis Monnell, Rebecca Mosher, Marina Protopopova, LiuLiang Qin, Pamela Shaw, Elena Ter-Ovanesyan, Joshua D. Thomas, Phonphimon Wongthida, Ling Xu, Liping Yang, Jeffrey Zurita, Dorin Toader, Marc Damelin, Timothy B. Lowinger. XMT-2056, a well-tolerated, Immunosynthen-based STING-agonist antibody-drug conjugate which induces anti-tumor immune activity [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 1738.

XMT-1660 is a novel Dolasynthen-based antibody drug conjugate carrying a DolaLock payload with controlled bystander effect and targeting B7-H4, a tumor antigen that is broadly expressed on the cell surface in breast, ovarian and endometrial cancers. B7-H4 (VTCN1) exerts immunosuppressive effects by suppression of T cell proliferation and is expressed on tumor-associated macrophages (TAMs) as well as epithelial tumor cells. XMT-1660 is comprised of an anti-B7-H4 antibody site-specifically conjugated to Dolasynthen, with a total of 6 DolaLock Auristatin F-HPA (AF-HPA) anti-tubulin payloads per antibody (DAR-6). To select the optimal ADC, three ADCs using the same antibody and DolaLock payload were compared: site-specific Dolasynthen-based DAR-2 and DAR-6 ADCs, and a stochastically conjugated Dolaflexin-based DAR-12 ADC. In vitro, no significant differences were observed among the 3 ADCs: all exhibited specific recognition of B7-H4 and elicited potent cytotoxicity against B7-H4-expressing cancer cells. In vivo, XMT-1660 consistently exhibited more anti-tumor activity than the other ADCs in TNBC models and ER+/HER2- models after single, equivalent doses based on payload. XMT-1660 demonstrated dose-dependent anti-tumor activity and induced sustained tumor regressions after a single administration. XMT-1660 and the Dolasynthen DAR-2 ADC both exhibited improved pharmacokinetics in mouse relative to the Dolaflexin DAR 12 ADC. These data indicate that XMT-1660 exhibited a superior preclinical profile to the other ADCs and more generally demonstrate the importance of DAR-ranging studies to identify the optimal antibody-drug conjugate for a given target. These results, as well as results from exploratory toxicology studies in non-human primates, strongly support the clinical development of XMT-1660. Citation Format: Shawn P. Fessler, Jason Wang, Scott D. Collins, LiuLiang Qin, Kenneth Avocetien, Ling Xu, Ronald Eydelloth, Steven Vonderfecht, Chen-Ni Chin, Steven Bradley, Susan Clardy, Anouk Dirksen, Elizabeth Ditty, Bingfan Du, Dokyong Kim, Rebecca Mosher, Elena Ter-Ovanesyen, Kelly Slocum, Alex Uttard, Phonphimon Wongthida, Jeffrey Zurita, Dorin Toader, Marc Damelin, Timothy B. Lowinger. XMT-1660, a B7-H4-targeted Dolasynthen antibody-drug conjugate for the treatment of breast cancer [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 907.

Significant development and scale-up work was completed on the synthesis of an antibody drug conjugate payload based on the tubulysin natural products. This work included the development of new routes to the tubuvaline and tubuphenylaniline portions of the molecules, as well as extensive optimization of the solid phase peptide synthesis used to assemble the molecule. The initial route (21 steps longest linear sequence, 0.01% overall yield) was improved to a new, more robust route (19 steps longest linear sequence, 2.4% overall yield) affording a 240-fold increase in overall yield and allowing delivery of over 86 g of the required molecule.

Herein we report structure–cytotoxicity relationships for analogues of N14-desacetoxytubulyisn H 1. A novel synthetic approach toward 1 enabled the discovery of compounds with a range of activity. Calculated basicity of the N-terminus of tubulysins was shown to be a good predictor of cytotoxicity. The impact of structural modifications at the C-terminus of 1 upon cytotoxicity is also described. These findings will facilitate the development of new tubulysin analogues for the treatment of cancer.

We have identified a class of azabenzimidazoles as potent and selective JAK1 inhibitors. Investigations into the SAR are presented along with the structural features required to achieve selectivity for JAK1 versus other JAK family members. An example from the series demonstrated highly selective inhibition of JAK1 versus JAK2 and JAK3, along with inhibition of pSTAT3 in vivo, enabling it to serve as a JAK1 selective tool compound to further probe the biology of JAK1 selective inhibitors.

The Dolasynthen platform incorporates the highly potent anti-mitotic agent auristatin F-HPA (AF-HPA), with its associated DolaLock mechanism of controlled bystander effect, and enables the synthesis of antibody-drug conjugates (ADCs) with precise control of the drug-to-antibody ratio (DAR) and site-specific bioconjugation. XMT-1592 is a novel ADC comprised of an anti-NaPi2b antibody and Dolasynthen, conjugated in a site-specific manner to yield DAR 6. NaPi2b, also known as SLC34A2, is a transmembrane sodium-phosphate transporter that is broadly expressed on tumor cells in ovarian carcinoma, NSCLC lung adenocarcinoma and other tumor types. Recent studies have shown that NaPi2b expression is enriched in the EGFR and KRAS mutant subtypes of lung adenocarcinoma. Binding studies showed a specific, high-affinity interaction of XMT-1592 with NaPi2b that was not affected by conjugated Dolasynthen. XMT-1592 elicited potent and specific in vitro cytotoxicity against NaPi2b-expressing ovarian carcinoma cells. XMT-1592 exhibited potent and specific in vivo activity in NaPi2b-expressing tumor xenografts derived from ovarian carcinoma or lung adenocarcinoma. Consistent with the targeted delivery benefits of the ADC approach, XMT-1592 yielded high and sustained concentrations of AF-HPA to tumors but not normal tissues. To evaluate the benefits of site-specific bioconjugation of Dolasynthen, we conducted in vitro and in vivo comparisons of XMT-1592 to a stochastically conjugated version of the ADC. XMT-1592 had improved in vivo activity, pharmacokinetics, and clinical pathology relative to its stochastic counterpart. Taken together, these results support XMT-1592 as a development candidate for the treatment of NaPi2b-expressing tumors. Citation Format: Shawn Fessler, Anouk Dirksen, Scott D. Collins, Ling Xu, Winnie Lee, Jason Wang, Ron Eydelloth, Elena Ter-Ovanesyen, Jeffrey Zurita, Elizabeth Ditty, Barrett Nehilla, Susan Clardy, Susan Clardy, Tyler Carter, Kenneth Avocetien, Mark Nazzaro, Nam Le, Kalli C. Catcott, Alex Uttard, Bingfan Du, Chen-Ni Chin, Rebecca Mosher, Kelly Slocum, Liuliang Qin, David Lee, Dorin Toader, Marc Damelin, Timothy B. Lowinger. XMT-1592, a site-specific Dolasynthen-based NaPi2b-targeted antibody-drug conjugate for the treatment of ovarian cancer and lung adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2894.

STING pathway agonism has emerged as a potential therapeutic mechanism to stimulate an innate anti-tumor immune response. While in principle systemic administration of a STING agonist would have many therapeutic benefits, including the delivery of STING to all tumor lesions, such an approach may be limited by toxicity. Antibody-drug conjugates (ADCs) constitute a proven therapeutic modality that is ideally suited to enable systemic administration without associated toxicity concerns via a targeted delivery strategy. Herein, we demonstrate that systemically administered STING agonist ADCs have greater anti-tumor activity as well as greatly improved tolerability compared to an intravenously (IV) administered, unconjugated (free) agonist. We generated novel STING agonist ADCs by leveraging our Immunosynthen platform, in which the chemical scaffold for bioconjugation is optimized for the STING agonist, resulting in an ADC that has desirable physicochemical and drug-like properties. We have studied the in vitro activity and mechanism of action of STING agonist ADCs in monoculture and co-culture systems. STING agonist ADCs were at least 100-fold more potent in inducing interferon and cytokines as well as tumor cell-killing relative to free agonist. STING agonist ADCs against several targets (antigens) have been evaluated for anti-tumor activity and pharmacodynamic and pharmacokinetic properties in multiple xenograft and syngeneic models. A single administration of STING agonist ADC resulted in target-dependent, durable, and complete regressions. Importantly, the STING agonist ADC led to an increase in tumor-localized inflammatory cytokines and significant immune cell infiltration, while levels of systemic cytokines remained low. In contrast, IV administered free agonist induced up to 100-fold higher levels of systemic cytokines with concomitant body weight loss but only modest tumor growth delay. In summary, Immunosynthen represents a novel STING agonist ADC platform. We have demonstrated target-dependent anti-tumor immune responses in vitro and in vivo for multiple targets, tumor models, and mouse strains. In each case the STING agonist ADC was more active and better tolerated than the IV administered free agonist. Citation Format: Raghida A. Bukhalid, Jeremy R. Duvall, Naniye Malli Cetinbas, Kalli C. Catcott, Kenneth Avocetien, Keith W. Bentley, Stephen Bradley, Tyler Carter, Chen-Ni Chin, Susan Clardy, Scott D. Collins, Timothy Eitas, Brian D. Jones, Eugene W. Kelleher, Rebecca Mosher, Mark Nazzaro, Marina Protopopova, Pamela Shaw, Kelly Slocum, Elena Ter-Ovanesyan, LiuLiang Qin, Joshua D. Thomas, Ling Xu, Liping Yang, Jeffrey Zurita, Dorin Toader, Marc Damelin, Timothy B. Lowinger. Systemic administration of STING agonist antibody-drug conjugates elicit potent anti-tumor immune responses with minimal induction of circulating cytokines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6706.

Antibody-drug conjugate (ADC) which delivers cytotoxic drugs specifically into targeted cells through internalization and lysosomal trafficking has emerged as an effective cancer therapy. We show that a bivalent biparatopic antibody targeting two non-overlapping epitopes on HER2 can induce HER2 receptor clustering, which in turn promotes robust internalization, lysosomal trafficking, and degradation. When conjugated with a tubulysin-based microtubule inhibitor, the biparatopic ADC demonstrates superior anti-tumor activity over ado-trastuzumab emtansine (T-DM1) in tumor models representing various patient subpopulations, including T-DM1 eligible, T-DM1 ineligible, and T-DM1 relapsed/refractory. Our findings indicate that this biparatopic ADC has promising potential as an effective therapy for metastatic breast cancer and a broader patient population may benefit from this unique HER2-targeting ADC.

Antibody-drug conjugates (ADCs) are a therapeutic modality that enables the targeted delivery of highly potent cytotoxic payloads to tumors. This chapter describes the components of the ADCs and discusses the medicinal chemistry principles that guide the design of this class of therapeutics. A description of main classes of drugs and the linkers used to attach to monoclonal antibodies with an emphasis on the design, historical development, and linking strategies is presented. Clinical use of the approved ADCs for the treatment of cancer is briefly described.

Antibody–drug conjugates (ADC) are used to selectively deliver cytotoxic agents to tumors and have the potential for increased clinical benefit to cancer patients. 5T4 is an oncofetal antigen overexpressed on the cell surface in many carcinomas on both bulk tumor cells as well as cancer stem cells (CSC), has very limited normal tissue expression, and can internalize when bound by an antibody. An anti-5T4 antibody was identified and optimized for efficient binding and internalization in a target-specific manner, and engineered cysteines were incorporated into the molecule for site-specific conjugation. ADCs targeting 5T4 were constructed by site-specifically conjugating the antibody with payloads that possess different mechanisms of action, either a DNA cross-linking pyrrolobenzodiazepine (PBD) dimer or a microtubule-destabilizing tubulysin, so that each ADC had a drug:antibody ratio of 2. The resulting ADCs demonstrated significant target-dependent activity in vitro and in vivo; however, the ADC conjugated with a PBD payload (5T4-PBD) elicited more durable antitumor responses in vivo than the tubulysin conjugate in xenograft models. Likewise, the 5T4-PBD more potently inhibited the growth of 5T4-positive CSCs in vivo, which likely contributed to its superior antitumor activity. Given that the 5T4-PBD possessed both potent antitumor activity as well as anti-CSC activity, and thus could potentially target bulk tumor cells and CSCs in target-positive indications, it was further evaluated in non-GLP rat toxicology studies that demonstrated excellent in vivo stability with an acceptable safety profile. Taken together, these preclinical data support further development of 5T4-PBD, also known as MEDI0641, against 5T4+ cancer indications. Mol Cancer Ther; 16(8); 1576–87. ©2017 AACR.

Herein we report structure-cytotoxicity relationships for analogues of N

Antibody-drug conjugates (ADCs) consist of a tumor-targeted antibody, a drug (payload) with specified mechanism of action, and the chemical framework for attaching them to each other. By selective delivery of the payload to the tumor and not to normal tissues, ADCs can provide greater efficacy and tolerability than systemic chemotherapies, which can translate to longer duration of treatment and response, as well as more options for combination therapies without the concern of overlapping toxicity. To preempt potential resistance to therapy, we have engineered a dual-payload ADC (DP-ADC) that delivers two mechanistically distinct payloads to a single target cell. To build a precision dual payload-ADC (DP-ADC), we leveraged our Synthemer platform, which enables us to chemically attach the payloads to a synthetic scaffold in a defined manner, and then to chemically attach the loaded scaffold to the antibody. This approach allows for unambiguous control of architecture and ratio of the payloads: the payloads are incorporated in a fixed ratio into the scaffold and subsequently into the ADC. Moreover, the synthetic design also allows for selection of solubilizing groups and charge compensation for each payload, allowing for optimal properties of the ADC. Our prototype DP-ADC combines the microtubule inhibitor auristatin F hydroxypropyl amide (AF-HPA) with the DNA monoalkylator I-BiP (related to pyrrolobenzodiazepine), at a payload ratio of 3:1. The drug-to-antibody ratio (DAR) was 12 AF-HPA and 4 I-BiP (16 total), achieved by conjugating 4 of the loaded dual-payload scaffolds to the antibody. The DP-ADC exhibited comparable antigen binding to the unconjugated antibody, and cytotoxic potency comparable to both single-payload ADCs alone. Using cell-based assays that specifically probe the mechanism of action of each payload, we demonstrated that the DP-ADC exerted both expected mechanisms of action on the tumor cell in a target-dependent manner. Considerations for the selection of payload combinations will also be presented. While synergistic action is typically assumed to be preferred if not required, a recent study demonstrated that the observed clinical benefit of most combinations can be explained by the drugs’ independent modes of action, not synergy. Indeed, AF-HPA and I-BiP did not exhibit synergy in cytotoxicity studies in cancer cell lines, yet their combination in a DP-ADC is still expected to confer potential clinical benefit over either single-payload ADC across a patient population. Citation Format: Jeremy R. Duvall, Marc Damelin, Mariya V. Kozytska, Barrett J. Nehilla, Marina Protopopova, Patrick R. Conlon, LiuLiang Qin, Mark Nazzaro, Josh D. Thomas, Qingxiu Zhang, Dorin Toader, Timothy B. Lowinger. An antibody-drug conjugate carrying a microtubule inhibitor and a DNA alkylator exerts both mechanisms of action on tumor cells [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 232.

Dolasynthen is a novel, fully synthetic, structurally homogeneous platform that enables the construction of ADCs with tunable drug-to-antibody ratios (DAR), from a low of 2 to a high of 24. The resulting ADCs exhibit excellent physicochemical properties and fully homogeneous conjugates can be created through a variety of bioconjugation chemistries. Analogous to our first platform, Dolaflexin®, Dolasynthen is loaded with the proprietary payload Auristatin F hydroxypropylamide (AF-HPA) with precisely defined numbers of the cytotoxin per Dolasynthen scaffold.Studies that evaluate the tolerability and efficacy of Dolasynthen in preclinical models are described herein. ADCs containing a range of DAR values were generated following conjugation of Dolasynthen to two different monoclonal antibodies. The DAR of the ADCs was achieved by controlled reduction of native disulfide bonds in IgG1 antibodies, chromatographic fractionation, or through use of site-specific conjugation technologies. ADCs with both DAR6 and DAR12 were evaluated in vitro and also in vivo in the mouse, rat and monkey, for efficacy, tolerability and PK. Dolasynthen conjugates had excellent physicochemical properties and displayed the expected cell binding and in vitro cytotoxicities. In vivo pharmacology of Dolasynthen ADCs in in vivo xenograft models showed dose-dependent tumor growth inhibition at low mg/kg mAb doses. Tolerability in the rat at multiple doses was determined, including histopathological evaluation. Dolasynthen ADCs showed excellent PK characteristics in mouse, rat and NHP. Overall, the Dolasynthen platform appears to offer significant potential for clinical application. Citation Format: Dorin Toader, Marc Damelin, Anouk Dirksen, Shawn P. Fesler, Scott D. Collins, Barrett J. Nehilla, Jian Xu, Ling Xu, Kalli C. Cattcott, Alex Uttard, Winnie Lee, Susan Clardy, Cheri A. Stevenson, LiuLiang Qin, Patrick R. Conlon, Mariya V. Kozytska, Chen-Ni Chin, David H. Lee, Timothy B. Lowinger. Dolasynthen–a novel, homogeneous Auristatin F hydroxypropyl amide antibody-drug conjugate platform [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 2687.

Antibody–drug conjugates (ADCs) have become a powerful platform to deliver cytotoxic agents selectively to cancer cells. ADCs have traditionally been prepared by stochastic conjugation of a cytotoxic drug using an antibody’s native cysteine or lysine residues. Through strategic selection of the mammalian expression host, we were able to introduce azide-functionalized glycans onto a homogeneously glycosylated anti-EphA2 monoclonal antibody in one step. Conjugation with an alkyne-bearing pyrrolobenzodiazepine dimer payload (SG3364) using copper-catalyzed click chemistry yielded a site-specific ADC with a drug-to-antibody ratio (DAR) of four. This ADC was compared with a glycoengineered DAR two site-specific ADC, and both were found to be highly potent against EphA2-positive human prostate cancer cells in both an in vitro cytotoxicity assay and a murine tumor xenograft model.

TGF-β–activated kinase 1 (TAK1), a member of the MAPK kinase family, plays a key role in B-cell growth and development. In the present study, we examined the potential role of TAK1 as a therapeutic target for lymphoma. Here, we show that the active phosphorylated form of TAK1 is abundantly expressed in a panel of lymphoma cell lines, including mantle cell, anaplastic large cell, and Hodgkin lymphoma cell lines. Silencing TAK1 expression via the use of siRNA inhibited the activation of NF-κB and p38 and induced apoptosis in lymphoma cell lines. Moreover, submicromolar concentrations of AZ-TAK1, a novel ATP-competitive small molecule inhibitor of TAK1, dephosphorylated TAK1, p38, and IκB-α in lymphoma cell lines. These molecular events were associated with the release of cytochrome c into the cytosol, down-regulation of X-linked inhibitor of apoptosis, activation of caspase 9, and induction of apoptosis. We also demonstrate that primary lymphoma cells express TAK1 and pTAK1 and were sensitive to AZ-TAK1–mediated cell death. Collectively, our data demonstrate an essential role for TAK1 in regulating critical survival mechanisms in lymphoma and suggest that it may serve as a therapeutic target.

Checkpoint Kinase-1 (Chk1, CHK1, CHEK1) is a Ser/Thr protein kinase that mediates cellular responses to DNA-damage. A novel class of Chk1 inhibitors, triazoloquinolones/triazolones (TZ's) was identified by high throughput screening. The optimization of these hits to provide a lead series is described.

Checkpoint kinase-1 (Chk1, CHEK1) is a Ser/Thr protein kinase that mediates the cellular response to DNA-damage. A novel class of 2-ureido thiophene carboxamide urea (TCU) Chk1 inhibitors is described. Inhibitors in this chemotype were optimized for cellular potency and selectivity over Cdk1.

Using structure-based design, a new class of inhibitors of protein tyrosine phosphatase-1B (PTP1B) has been identified, which incorporate the 1,2,5-thiadiazolidin-3-one-1,1-dioxide template.

Resistance to antibody-drug conjugates (ADC) has been observed in both preclinical models and in the clinic. It has been previously reported that an ADC targeting CD33 with a DNA cross-linking pyrrolobenzodiazepine (PBD) dimer was active in models of multidrug-resistant (MDR) AML, suggesting that PBDs, delivered as part of a molecular-targeted cancer therapy may be effective in MDR settings. However it could still be possible to develop resistance to PBD-conjugated ADCs though other mechanisms that remain unknown. Studies were conducted to determine if tumor cells have innate resistance to PBDs or an anti-5T4 ADC delivering PBD warhead. It was found that both the ADC and its PBD warhead, SG3199, completely eradicated MDA-MB-361 breast cancer cells and NCI-N87 gastric cancer cells in culture, suggesting a lack of intrinsic resistance in these cell lines. The potential emergence of acquired resistance was investigated by culturing MDA-MB-361 cells in gradually-increasing concentrations of either the ADC or SG3199 for over a year and the variants resistant to either the ADC (361-ADCr) or the PBD warhead (361-PBDr) were established as determined by decreased sensitivity in cytotoxicity assays. Transcriptional profiling characterization of these two cell models demonstrated that neither of them had significant upregulation of P-glycoprotein (P-gp) or other drug efflux transporter genes. Significant 5T4 antigen down-regulation was observed in the 361-ADCr cells but not the 361-PBDr cells and these cells also remained sensitive to alternate treatments including ADCs targeting a different surface antigen with the same PBD warhead, suggesting that down-regulation of target was likely the main driver of resistance in 361-ADCr cells. Further transcriptional profiling analyses conducted on 361-PBDr cells suggest a multi-factorial process may be needed to develop resistance to PBD warheads as differential expression of genes associated with DNA damage and repair, drug metabolism and cell cycle was observed. Similar gene expression profiles were observed in both cultured cells and in tumors derived from these cells. Studies to validate these differentially expressed genes, including proteomic and phosphoproteomic profiling are currently being conducted. Collectively these data could identify how resistance to ADCs conjugated with PBD payloads may develop in the clinic and more importantly, provide insight into developing strategies to overcome such resistance. Citation Format: Shenlan Mao, Andrew Garcia, Tracy Chen, Raghothama Chaerkady, Allison Marreno, Helen Zhong, Dorin Toader, Nazzareno Dimasi, Maureen Kennedy, Philip Howard, Changshou Gao, Herren Wu, Ronald Herbst, David Tice, Jay Harper. Tumor cells treated with a PBD-based antibody-drug conjugate targeting 5T4 develop acquired resistance due to 5T4 down-regulation and response to alternate therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2064. doi:10.1158/1538-7445.AM2017-2064

2-Arylpropenals H2CdC(Ar)CHO are of considerable synthetic value for the preparation of tryptophans,1 vinylic peroxyketals,2 2,2′-bipyridines,3 and functionalized cycloheptenes.4 Accordingly, a variety of methods have been developed for the preparation of 2-arylpropenals based on transformations of various styrenes as follows: (i) reaction of Grignards derived from R-bromo styrenes with ethyl orthoformate;5 (ii) oxidation with selenium dioxide,6 or bromination followed by hydrolysis,7 of R-methylstyrenes; and (iii) three-step cyclopropanation, ring opening, and hydrolysis of styrenes.8 Further routes use 2-arylethanols9 and R-chloroacetals as starting materials.10 However, the application of 2-arylpropenals in synthesis is accompanied by several problems. The compounds are highly toxic.11 The double bond in H2CdC(Ar)CHO, activated by both the aryl and the carbonyl group, is highly reactive; consequently, these molecules are claimed to be unstable above -6 °C,8 although 2-(4-methoxycarbonylphenyl)propenal was isolated apparently at room temperature12 and although 2-phenylpropenal can be distilled at 79-82 °C at reduced pressure.6 At room temperature, atropaldehyde (2-phenylpropenal) undergoes a spontaneous hetero Diels-Alder dimerization to give the corresponding dihydropyran.6 Furthermore, while conjugate additions occur readily, to our best knowledge, the only 1,2-additions of carbon nucleophiles to 2-arylpropenals are allylmagnesium chloride, propynyl bromide in the presence of aluminum amalgam to 2-phenylpropenal,13 and 1-bromo-2-phenylpropene in the presence of zinc and ammonium chloride to 2-(4-methoxycarbonylphenyl)propenal.14 Hence, the development of a route to masked 2-arylpropenals is considerably attractive. We now describe such substrates in which the double bond is masked, allowing for clean 1,2-addition to the aldehyde moiety. Deprotection of the double bond regenerates the unsaturation and renders a formal 1,2addition to 2-arylpropenals.

Reactions of α-aryl-, α-heteroaryl-, and α-heteroatom-substituted masked alkenyllithiums with aldehydes and ketones provide a general method for the synthesis of allylic alcohols substituted with an aryl or heteroaryl in the β position and aryl, heteroaryl or alkyl substituents in the α position via a [1,4]-C→O silicon rearrangement. In the case of reactions with enolizable aldehydes and ketones, anhydrous CeCl3 was used as an additive. High diastereoselectivities are observed for allyl alcohols produced from α-substituted aldehydes.

The title compounds 1a−e readily undergo deprotonation and subsequent reactions with the appropriate electrophiles to form intermediates of types 4 and 7 which, upon treatment with Lewis acids, cyc...

Vicinal elimination of trimethylsilyl and benzotriazolyl groups from 2-benzotriazolyl-2-aryl-3-ketopropylsilanes forms, along with the expected 1,1-disubstituted ethylenes, significant amounts of the corresponding chalcones. A study of this transformation by carbon labeling suggests the intermediate formation of cyclopropanes. Stabilizing/destabilizing (electron-donor/acceptor) para-substituents on the aryl group affect the product distribution of the elimination in a manner consistent with the proposed mechanism.

A general regiospecific method for the synthesis of (p-nitroaryl)diarylmethanes has been developed starting from diarylmethanols and 2- and/or 3-substituted nitrobenzenes. This utilizes the quantitative condensation between benzotriazole and diarylmethanols under acidic catalysis and in the presence of perfluorocarbon fluids, followed by vicarious nucleophilic substitution of the resulting diarylmethylbenzotriazoles upon nitrobenzenes in moderate to high yield. Oxidative nucleophilic substitution of hydrogen is observed as a side process. These vicarious nucleophilic substitutions complement Friedel−Crafts reactions for the synthesis of triarylmethanes.

Transforming growth factor-β activated kinase-1 (TAK1) is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family that regulates several signaling pathways including NF-κB signal transduction and p38 activation. TAK1 deregulation has been implicated in human diseases including cancer and inflammation. Here, we show that, in addition to its kinase activity, TAK1 has intrinsic ATPase activity, that (5Z)-7-Oxozeaenol irreversibly inhibits TAK1, and that sensitivity to (5Z)-7-Oxozeaenol inhibition in hematological cancer cell lines is NRAS mutation status and TAK1 pathway dependent. X-ray crystallographic and mass spectrometric studies showed that (5Z)-7-Oxozeaenol forms a covalent complex with TAK1. Detailed biochemical characterization revealed that (5Z)-7-Oxozeaenol inhibited both the kinase and the ATPase activity of TAK1 following a bi-phase kinetics, consistent with the irreversible inhibition mechanism. In DoHH2 cells, (5Z)-7-Oxozeaenol potently inhibited the p38 phosphorylation driven by TAK1, and the inhibition lasted over 6 h after withdrawal of (5Z)-7-Oxozeaenol. Profiling (5Z)-7-Oxozeaenol in a panel of hematological cancer cells showed that sensitive cell lines tended to carry NRAS mutations and that genes in TAK1 regulated pathways were enriched in sensitive cell lines. Taken together, we have elucidated the molecular mechanism of a TAK1 irreversible inhibitor and laid the foundation for designing next generation TAK1 irreversible inhibitors. The NRAS-TAK1-Wnt signaling network discerned in our study may prove to be useful in patient selection for TAK1 targeted agents in hematological cancers.

Anions formed from the lithiation of a variety of 1-(arylmethyl)- and 1-(heteroarylmethyl)benzotriazoles 1 with n-BuLi underwent addition to aliphatic and aromatic aldehydes and cyclic and acyclic ketones. Subsequent in situ thermal rearrangements of the intermediates in the presence of zinc bromide provided one-carbon chain-extended or ring-expanded alpha-aryl- and alpha-heteroaryl-substituted ketones 2 in moderate to excellent yields in simple one-pot operations with excellent regioselectivity in most cases. Substituent effects on the relative migration rates were investigated in the insertion reactions of 1-(4-methoxybenzyl)benzotriazole (1e) with XC(6)H(4)COPh. The small and negative Hammett rho(+) value (-0.92) suggested that the rearrangements proceed via early, reagent-like, electron deficient transition states.

Antibody drug conjugates (ADCs) combine the specificity of antibodies with the potent cytotoxicity of small molecule drugs and have shown to provide therapeutic options for various cancers. We report herein the discovery of a HER2-targeting ADC MEDI4276 that showed potent cell killing activity in vitro in cancer cell lines that express the HER2 receptor. The observed in vitro activity translated into in vivo tumor growth inhibition in various xenograft mouse models. MEDI4276 is a homogeneous molecule with precise control of drug loading following site specific conjugation of a cytotoxic drug. The drug in MEDI4276 is MMETA, a fully synthetic analog of the tubulysin family that showed pM potency in a panel of cancer cell lines. MMETA was conjugated to the antibody via engineered cysteines with a maleimide-bearing mc-Lys protease cleavable linker. The antibody in MEDI4276 is a bivalent biparatopic antibody targeting two distinct non-overlapping epitopes on HER2 that leads to antibody-receptor clustering following binding and thus promoting internalization, lysosomal trafficking and degradation. The combination of enhanced internalization and potent cytotoxic drug allows for this ADC to kill tumor cell populations with a broader range of HER2 expression. Preclinical studies showed that MEDI4276 induced tumor regression in HER2-positive tumor models that had developed acquired resistance to T-DM1 and in a number of models with lower HER2 expression that are refractory to T-DM1 treatment. Overall, our findings underscore the potential application of MEDI4276 to treat a large patient population that is ineligible for or relapsed/refractory to current HER2-targeted therapies. MEDI4276 is currently being investigated in a Phase I clinical trial. Citation Format: John Li, Dorin Toader, Samuel R. Perry, Vanessa Muniz-Medina, Leslie Wetzel, Marlon C. Rebelatto, Mary Jane Masson Hinrichs, Ryan Fleming, Binyam Bezabeh, Pamela Thompson, Nazzareno Dimasi, Brandon Lam, Xian-Qing Yu, Changshou Gao, Rakesh Dixit, Steven Coats, Jane Osbourn, Herren Wu. MEDI4276, a HER2-targeting antibody tubulysin conjugate, displays potent in vitro and in vivo activity in preclinical studies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2970.

Checkpoint kinases CHK1 and CHK2 are activated in response to DNA damage that results in cell cycle arrest, allowing sufficient time for DNA repair. Agents that lead to abrogation of such checkpoints have potential to increase the efficacy of such compounds as chemo- and radiotherapies. Thiophenecarboxamide ureas (TCUs) were identified as inhibitors of CHK1 by high throughput screening. A structure-based approach is described using crystal structures of JNK1 and CHK1 in complex with 1 and 2 and of the CHK1-3b complex. The ribose binding pocket of CHK1 was targeted to generate inhibitors with excellent cellular potency and selectivity over CDK1and IKKβ, key features lacking from the initial compounds. Optimization of 3b resulted in the identification of a regioisomeric 3-TCU lead 12a. Optimization of 12a led to the discovery of the clinical candidate 4 (AZD7762), which strongly potentiates the efficacy of a variety of DNA-damaging agents in preclinical models.

One of the grand challenges in kinase drug discovery is the design of small-molecule inhibitors with selectivity profiles that will ultimately be efficacious in the clinic. Current medicinal chemistry strategies make heavy use of structural, biophysical and computational approaches to achieve this multi-faceted goal. Here we review structure-based approaches underlying the development of several molecules that are currently in clinical trials, including the cMet inhibitor ARQ197 and the Bcr–Abl inhibitor ponatinib. We highlight the challenge posed by the emergence of resistance mutants and discuss promising lead generation strategies to obtain selective inhibitors of protein and lipid kinases such as targeting of specific sites, the use of fragment-based approaches and new chemical probes based on metal complexes.

Antibody drug conjugates (ADCs) combine the specificity of antibodies with the potency of small molecule cytotoxic drugs and have the potential to provide significant efficacy as a treatment for cancer. The objective of this work was to identify potent new cytotoxic ADC payloads that can be used to target diverse tumor types. Here we report for the first time the discovery of fully synthetic tubulysin payloads which belong to a class of highly cytotoxic natural products that disrupt the cellular microtubule network leading to apoptosis of tumor cells. Our fully synthetic tubulysin payloads are comprised of: (i) a tubulysin warhead that displays pM potency, (ii) a protease cleavable amino-acid sequence and (iii) a tether bearing a reactive maleimide group. Tubulysin-based ADCs were generated via site-specific conjugation of these payloads to cysteines engineered into antibodies against cancer antigen target oncofetal protein 5T4. The resulting ADCs showed potent in vitro cell killing and in vivo efficacy in multiple solid tumor xenograft models including prostate cancer, non-small cell lung adenocarcinoma, breast cancer and gastric carcinoma. Furthermore, specific structural features of the tubulysin warhead, linker design and antibody engineering were shown to impact the overall in vitro and in vivo properties of the ADCs. Thus, these synthetic tubulysin payloads represent novel microtubule network disrupting compounds that display potent preclinical anti-tumor activity as an ADC that could be advanced to the clinic. Citation Format: Dorin Toader, Jay Harper, Chris Lloyd, Rose Marwood, David Bannister, Shenlan Mao, Cui (Tracy) Chen, Haihon (Helen) Zhong, Vahe Bedian, Fengjiang Wang, Lakshmaiah Gingipalli, Melisa Vasbinder, Pamela Thompson, Ryan Fleming, Byniam Bezabeh, Nazzareno Dimasi, Changshou Gao, Adeela Kamal. Discovery of tubulysin payloads for antibody drug conjugates with potent in vitro activity and in vivo efficacy in solid tumor models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B170.

Checkpoint kinase 1 (Chk1, CHEK1) is a Ser/Thr protein kinase that plays a key role in mediating the cellular response to DNA-damage. Synthesis and evaluation of a previously described class of Chk1 inhibitors, triazoloquinolones/triazolones (TZs) is further described herein. Our investigation of structure-activity relationships led to the identification of potent inhibitors 14c, 14h and 16e. Key challenges included modulation of physicochemical properties and pharmacokinetic (PK) parameters to enable compound testing in a Chk1 specific hollow fiber pharmacodynamic model. In this model, 16e was shown to abrogate topotecan-induced cell cycle arrest in a dose dependent manner. The demonstrated activity of TZs in this model in combination with a chemotherapeutic agent as well as radiotherapy validates this series of Chk1 inhibitors. X-ray crystal structures (PDB code: 2YEX and 2YER) for an initial lead and an optimized analog are also presented.

A general regiospecific method for the synthesis of (p-nitroaryl)diarylmethanes has been developed starting from diarylmethanols and 2- and/or 3-substituted nitrobenzenes. This utilizes the quantitative condensation between benzotriazole and diarylmethanols under acidic catalysis and in the presence of perfluorocarbon fluids, followed by vicarious nucleophilic substitution of the resulting diarylmethylbenzotriazoles upon nitrobenzenes in moderate to high yield. Oxidative nucleophilic substitution of hydrogen is observed as a side process. These vicarious nucleophilic substitutions complement Friedel−Crafts reactions for the synthesis of triarylmethanes.