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Supplementary Figures S1 to S11. Supplementary Figure S1: Schematic illustration of the approach used to create trastuzumab-maytansinoid ADC resistant cell lines. Supplementary Figure S2: Structures of payloads and ADCs. Supplementary Figure S3: 361-TM-resistant cells show differential responses to ADCs with modified linkers, payloads, and antibodies. Supplementary Figure S4: Ectoptic expression of Her2 in JIMT1-TM cells rescues sensitivity to TM-ADC. Supplementary Figure S5: Cell surface proteomic profiling of 361-TM resistant cells, with evidence of increased ABCC1. Supplementary Figure S6: Knockdown of ABCC1 (MRP1) in 361-TM cells sensitizes to ADCs. Supplementary Figure S7: Endosomal and lysosomal proteins altered in JIMT1-TM resistant cells as determined by proteomic profiling. Supplementary Figure S8: Immunoblot verification of elevated surface-associated proteins in JIMT1-TM resistant cells. Supplementary Figure S9: Co-localization analyses of ADC and lysosome via live-cell imaging. Supplementary Figure S10: Altered trafficking in JIMT1-TM cells are specific to TM-ADC and not transferrin receptor antibody. Supplementary Figure S11: LC-MRM analyses of released products from ADCs incubated with parental and resistant cell lines.

Supplementary Methods, Table S1 S2, and Figure Legends. Supplementary Table S1: Properties of cell lines made resistant to TM-conjugate. Supplementary Table S2: Relative resistance of selected free payloads and Cys-capped-released species in H69 (no ABCC1) and H69AR (high ABCC1 expressing) cell lines.

Bispecific antibodies which bind a tumor antigen and the CD3 heterodimer of the T cell receptor to form a synthetic immunological synapse represent a class of T cell engagers that has been clinically validated with the approvals of blinatumomab (Blinctyo®) and mosunetuzumab (Lunsumio®), targeting CD19 and CD20 respectively, for the treatment of B cell acute lymphoblastic leukemia and follicular lymphoma. Despite being a potent class of therapeutics, not all patients respond to therapy. Recent studies into state of blinatumomab-treated T cells highlight exhaustion and the lack of co-stimulation as potential factors in resistance. In solid tumors, the immunosuppressive tumor microenvironment is also a major factor while cytokine release is a safety consideration for all bispecifics that bind CD3 with strong affinity. To address the challenges of CD3 bispecifics, we have developed the EVOLVE™ platform, a trispecific antibody that consists of tumor targeting and attenuated CD3 binding affinity coupled with a CD2 agonist. We show that CD2 costimulation is superior to other pathways in its ability to sustain T cell activation and expansion and cytokine production over repeated cycles of stimulation. By integrating a CD2 agonist with an attenuated CD3 affinity bispecific, we can restore cytolytic potential without concomitant increase in cytokine release as compared to a strong CD3 affinity bispecific. The EVOLVE platform is also capable of inducing tumor cell killing by in vitro exhausted T cells whereas the bispecific did not. In terms of safety, the EVOLVE platform does not induce peripheral T cell activation and cytokine release in PBMCs in the absence of target as compared to CD3 and CD28 agonist antibodies. The superiority of the EVOLVE platform compared to a bispecific was also demonstrated in vivo where we observed 8/8 complete responses in EVOLVE-treated animals whereas the strong CD3 affinity bispecific showed no difference compared to isotype control using a CORL105 lung cancer xenograft model. We believe these experiments recapitulate the continual clinical challenges of using strong CD3 affinity bispecifics while demonstrating the advantages of the EVOLVE platform. Finally, we demonstrate the modular nature of the EVOLVE platform across diverse tumor antigens including B7H4, PSMA, CD20, and a novel squamous tumor antigen ULBP2. Together our data highlight the broad applications of the EVOLVE platform to improve T cell-mediated anti-tumor immunity and suggest its potential as an emerging, first-in-category immunotherapeutic strategy to address unmet medical needs in oncology. Citation Format: Mohosin Sarkar, Eric M. Tam, Guixian Jin, Shu Shien Chin, Abudukadier Abulizi, Oksana A. Sergeeva, Zengzu Lai, Evelyn Teran, Hayden Karp, Danielle Klaskin, Nana Adjoa Pels, Xingyue An, Jennifer Ziegler, Changqing Yuan, Maria Hackettt, Sonali Dhindwal, Amber Fearnley, Julio Rodriguez, Louis Matis, Jay S. Fine, Jeremy S. Myers. EVOLVETM: A novel T cell engager platform with integrated CD2 costimulation engineered for the treatment of immune suppressive tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2971.

CD3-bispecifics are antibody-based therapies that can simultaneously bind to a tumor cell surface antigen and T cells to establish a synapse between the tumor and T cell and activate T cell to induce specific killing of the tumor cell. CD3-bispecifics have demonstrated clinical success in B cell acute lymphoblastic leukemia and follicular lymphoma with approvals of that blinatumomab and mosunetuzumab that target B cell lineage antigens CD19 and CD20, respectively. However, clinical progress in deploying CD3-bispecifics for positive patient outcomes in solid tumors has been slow, due to tumor microenvironmental factors such as induction of T cell exhaustion, as well as the potential of CD3-bispecifics to mediate T cell anergy and dysfunction in the absence of adequate co-stimulation. Here we describe the development and preclinical validation of the EVOLVE platform, a tumor-targeted biologic that induces the formation of a synthetic synapse that simultaneously activates the T cell receptor complex and the CD2 receptor to optimize CD8 T cell effector phenotype and improve tumor cell killing ex vivo and in vivo, compared to matched CD3-bispecifics. We demonstrate that CD2 co-stimulation is superior to other forms of T cell co-stimulation in its ability to promote cytolytic co-stimulation, T cell cytokine production and T cell expansion. Furthermore, CD2 receptor expression is markedly elevated in tumor infiltrating lymphocytes across a broad set of tumor types, relative to the CD28 and 4-1BB costimulatory receptors. EVOLVE-mediated T cell activation is conditionally dependent on tumor antigen binding and can be tuned to promote optimal co-stimulation without increasing cytokine release relative to matched CD3-bispecifics. We also demonstrate the modular nature of the EVOLVE platform across diverse solid tumor antigens including B7H4 (VTCN1), and a novel squamous tumor antigen ULBP2. Our data highlight the broad applications of the EVOLVE platform to improve CD8 T cell-mediated anti-tumor immunity and suggest its potential as an emerging, first-in-category immunotherapeutic strategy to address unmet medical needs in oncology. Citation Format: Mandy Shu Shien Chin, Mohosin Sarkar, Eric Tam, Abudukadier Abulizi, Guixian Jin, Xingyue An, Evelyn Teran, Danielle M Klaskin, Nana Adjoa Pels, Maria Hackett, Oksana A Sergeeva, Hayden Karp, Julio Rodriguez, Sonali Dhindwal, Changqing Yuan, Zengzu Lai, Jennifer Zeiger, Amber Fearnley, Louis Matis, Jay Fine, Jeremy S Myers. EVOLVE: A novel costimulatory T cell engager platform engineered for the treatment of solid tumors [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B44.

Pharmacologic agents that interfere with nucleotide metabolism constitute an important class of anticancer agents. Recent studies have demonstrated that mTOR complex 1 (mTORC1) inhibitors suppress de novo biosynthesis of pyrimidine and purine nucleotides. Here, we demonstrate that mTORC1 itself is suppressed by drugs that reduce intracellular purine nucleotide pools. Cellular treatment with AG2037, an inhibitor of the purine biosynthetic enzyme GARFT, profoundly inhibits mTORC1 activity via a reduction in the level of GTP-bound Rheb, an obligate upstream activator of mTORC1, because of a reduction in intracellular guanine nucleotides. AG2037 treatment provokes both mTORC1 inhibition and robust tumor growth suppression in mice bearing non-small-cell lung cancer (NSCLC) xenografts. These results indicate that alterations in purine nucleotide availability affect mTORC1 activity and suggest that inhibition of mTORC1 contributes to the therapeutic effects of purine biosynthesis inhibitors.

Pharmacologic agents that interfere with nucleotide metabolism constitute an important class of anticancer agents. Recent studies demonstrated that mTOR complex 1 (mTORC1) inhibitors suppress de novo biosynthesis of pyrimidine and purine nucleotides. We have previously demonstrated that mTORC1 itself is suppressed by drugs that reduce intracellular purine nucleotide pools. Cellular treatment with AG2037, an inhibitor of the purine biosynthetic enzyme, GARFT, profoundly inhibits mTORC1 activity via a reduction in the level of GTP-bound Rheb, an obligate upstream activator of mTORC1, in part due to a reduction in intracellular guanine nucleotides. Purines are synthesized intracellularly in dynamic metabolic structures known as the purinosome. Our emerging data suggest that Rheb associates with the purinosome and treatment with AG2037 disrupts this interaction. We hypothesize that Rheb senses the availability of purines at the site of the purinosome and communicates purine sufficiency to activate mTORC1. The results of this project will increase our understanding of the regulation of purine biosynthesis and mTORC1 activation, two key processes in cell proliferation and cancer. Citation Format: Natasha Emmanuel, Elyssa Bernfeld, Shoba Ragunathan, Fang Wang, Qin Shan, Andreas Giannakou, Nanni Huser, Guixian Jin, Jeremy Myers, Keziban Unsal-Kacmaz, Robert T. Abraham. Exploring the interplay between the purinosome, a multienzyme, purine biosynthetic machine, and the Rheb-mTORC1 signaling axis [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A10.

Potent and selective TACE and MMP inhibitors utilizing the diazepine and thiazepine ring systems were synthesized and evaluated for biological activity in in vitro and in vivo models of TNF-α release. Oral activity in the mouse LPS model of TNF-α release was seen. Efficacy in the mouse collagen induced arthritis model was achieved with diazepine 20.

TNF-alpha converting enzyme (TACE) is a validated therapeutic target for the development of oral tumor necrosis factor-alpha (TNF-alpha) inhibitors. Here we report the pre-clinical results and characterization of a selective and potent TACE inhibitor, (2R, 3S)-2-([[4-(2-butynyloxy)phenyl]sulfonyl]amino)-N,3-dihydroxybutanamide (TMI-2), in various in vitro and in vivo assays. TMI-2 is a potent TACE inhibitor in an enzymatic FRET assay (IC50=2 nM). It is more than 250-fold selective over MMP-1, -7, -9, -14, and ADAM-10 in vitro. In cell-based assays and human whole blood, TMI-2 inhibits lipopolysaccharide (LPS)-induced TNF secretion with IC50s1 uM. Importantly, TMI-2 inhibits the spontaneous release of TNF-alpha in human synovium tissue explants of rheumatoid arthritis patients with an IC50 of 0.8 microM. In vivo, TMI-2 potently inhibits LPS-induced TNF-alpha production in mice (ED50=3 mg/kg). In the adjuvant-induced arthritis (AIA) model in rats, treatment with TMI-2 at 30 mg/kg and 100 mg/kg p.o. b.i.d. was highly effective in reducing joint arthritis scores. In a semi-therapeutic collagen-induced arthritis (CIA) model in mice, TMI-2 is highly effective in reducing disease severity scores after oral treatment at 100 mg/kg twice per day. In summary, TMI-2 is a potent and selective TACE inhibitor that inhibits TNF-alpha production and reduces the arthritis scores in pre-clinical models. TMI-2 represents a novel class of TACE inhibitors that may be effective and beneficial in the treatment of rheumatoid arthritis as well as other TNF-mediated inflammatory autoimmune diseases.

A series of benzodiazepine MMP/TACE inhibitors bearing polar moieties has been synthesized in an effort to optimize inhibitory activity against LPS-stimulated TNF production in human monocytes and oral activity in a murine LPS model.

Tumour necrosis factor α (TNFα)-converting enzyme (TACE/ADAM-17, where ADAM stands for a disintegrin and metalloproteinase) releases from the cell surface the extracellular domains of TNF and several other proteins. Previous studies have found that, while purified TACE preferentially cleaves peptides representing the processing sites in TNF and transforming growth factor α, the cellular enzyme nonetheless also sheds proteins with divergent cleavage sites very efficiently. More recent work, identifying the cleavage site in the p75 TNF receptor, quantifying the susceptibility of additional peptides to cleavage by TACE and identifying additional protein substrates, underlines the complexity of TACE-substrate interactions. In addition to substrate specificity, the mechanism underlying the increased rate of shedding caused by agents that activate cells remains poorly understood. Recent work in this area, utilizing a peptide substrate as a probe for cellular TACE activity, indicates that the intrinsic activity of the enzyme is somehow increased.

The mechanistic target of rapamycin complex 1 (mTORC1) regulates protein synthesis through effects on mRNA translation and ribosome biogenesis. Emerging evidence suggests that active mTORC1 also stimulates the de novo biosynthesis of purine and pyrimidine nucleotides. Here, using pharmacologic and genetic tools, we demonstrate that mTORC1 signaling is modulated by cellular purine nucleotide pools arising from the metabolic processes that generate ATP and GTP. Inhibition of GARFT, the enzyme that catalyzes the first folate-dependent step in purine synthesis, by the specific inhibitor AG2037, dramatically inhibits mTORC1 signaling via an AMPK-independent mechanism. GARFT inhibition suppresses the level of the activated form of the Rheb GTPase, a requisite upstream activator of mTORC1, through a global reduction in intracellular guanine-based nucleotides, and subsequently reduces Rheb protein farnesylation. Moreover, we demonstrate that mTORC1 blockade resulting from AG2037-mediated inhibition of GARFT impacts both translation initiation and pyrimidine biosynthesis and results in robust tumor growth inhibition of non-small cell lung cancer (NSCLC) xenografts. Our findings indicate that the regulatory relationship between mTORC1 activity and purine nucleotide pool is bidirectional, and suggest that mTORC1 inhibition contributes to the clinically established antiproliferative effects of purine biosynthesis inhibitors in cancer and inflammatory diseases. Citation Format: Kezi Unsal-Kacmaz, Natasha Emmanuel, Shoba Ragunathan, Qin Shan, Fang Wang, Andreas Giannakou, Nanni Huser, Guixian Jin, Jeremy Myers, Robert T. Abraham. Purine biosynthesis regulates mTORC1 by modulating Rheb GTPase activity [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B025.

Antibody–drug conjugates (ADC) are emerging as clinically effective therapy. We hypothesized that cancers treated with ADCs would acquire resistance mechanisms unique to immunoconjugate therapy and that changing ADC components may overcome resistance. Breast cancer cell lines were exposed to multiple cycles of anti-Her2 trastuzumab–maytansinoid ADC (TM-ADC) at IC80 concentrations followed by recovery. The resistant cells, 361-TM and JIMT1-TM, were characterized by cytotoxicity, proteomic, transcriptional, and other profiling. Approximately 250-fold resistance to TM-ADC developed in 361-TM cells, and cross-resistance was observed to other non–cleavable-linked ADCs. Strikingly, these 361-TM cells retained sensitivity to ADCs containing cleavable mcValCitPABC-linked auristatins. In JIMT1-TM cells, 16-fold resistance to TM-ADC developed, with cross-resistance to other trastuzumab-ADCs. Both 361-TM and JIMT1-TM cells showed minimal resistance to unconjugated mertansine (DM1) and other chemotherapeutics. Proteomics and immunoblots detected increased ABCC1 (MRP1) drug efflux protein in 361-TM cells, and decreased Her2 (ErbB2) in JIMT1-TM cells. Proteomics also showed alterations in various pathways upon chronic exposure to the drug in both cell models. Tumors derived from 361-TM cells grew in mice and were refractory to TM-ADC compared with parental cells. Hence, acquired resistance to trastuzumab–maytansinoid ADC was generated in cultured cancer cells by chronic drug treatment, and either increased ABCC1 protein or reduced Her2 antigen were primary mediators of resistance. These ADC-resistant cell models retain sensitivity to other ADCs or standard-of-care chemotherapeutics, suggesting that alternate therapies may overcome acquired ADC resistance. Mol Cancer Ther; 14(4); 952–63. ©2015 AACR.

Heteroaryl and cycloalkyl sulfonamide-hydroxamic acid MMP inhibitors were investigated. Of these, the pyridyl analogue 2 is the most potent and selective inhibitor of MMP-9 and MMP-13 in vitro.

Three new sulfated sterol dimers, fibrosterol sulfates A-C (1-3), have been isolated from the sponge Lissodendoryx (Acanthodoryx) fibrosa, collected in the Philippines. The structures were assigned on the basis of extensive 1D and 2D NMR studies as well as analysis by HRESIMS. Compounds 1 and 2 inhibited PKCzeta with IC(50) values of 16.4 and 5.6 microM, respectively.

Proteolytic cleavage of amyloid precursor protein by beta-secretase (BACE-1) and gamma-secretase leads to formation of beta-amyloid (A beta) a key component of amyloid plaques, which are considered the hallmark of Alzheimer's disease. Small molecule inhibitors of BACE-1 may reduce levels of A beta and thus have therapeutic potential for treating Alzheimer's disease. We recently reported the identification of a novel small molecule BACE-1 inhibitor N-[2-(2,5-diphenyl-pyrrol-1-yl)-acetyl]guanidine (3.a.1). We report here the initial hit-to-lead optimization of this hit and the SAR around the aryl groups occupying the S(1) and S(2') pockets leading to submicromolar BACE-1 inhibitors.

The proteolytic enzyme beta-secretase (BACE-1) produces amyloid beta (Abeta) peptide, the primary constituent of neurofibrillary plaques, implicated in Alzheimer's disease, by cleavage of the amyloid precursor protein. A small molecule inhibitor of BACE-1, (diaminomethylene)-2,5-diphenyl-1H-pyrrole-1-acetamide (1, BACE-1 IC(50)=3.7 microM), was recently described, representing a new small molecule lead. Initial SAR investigation demonstrated the potential of accessing the nearby S(3) and S(1)(') substrate binding pockets of the BACE-1 enzyme by building substituents off one of the phenyl substituents and guanidinyl functional group. We report here the optimization of guanidinyl functional group substituents on 1, leading to potent submicromolar BACE-1 inhibitors.

NIMA (never in mitosis arrest)-related kinase 2 (Nek2) is a serine/threonine kinase required for centrosome splitting and bipolar spindle formation during mitosis. Currently, two in vitro kinase assays are commercially available: (i) a radioactive assay from Upstate Biotechnology and (ii) a nonradioactive fluorescence resonance energy transfer (FRET) assay from Invitrogen. However, due to several limitations such as radioactive waste management and lower sensitivity, a need for more robust nonradioactive assays would be ideal. Accordingly, we have developed four quantitative and sensitive nonradioactive Nek2 in vitro kinase assays: (i) a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) using peptides identified from a physiologically relevant protein substrate, (ii) DELFIA using Nek2 itself, (iii) a homogeneous time-resolved FRET assay termed LANCE, and (iv) A method of detecting phosphorylated products by HPLC. The DELFIA and LANCE assays are robust in that they generated more than 10-fold and 20-fold increases in signal-to-noise ratios, respectively, and are amenable to robotic high-throughput screening platforms. Validation of all four assays was confirmed by identifying a panel of small molecule ATP competitive inhibitors from an internal corporate library. The most potent compounds consistently demonstrated less than 100 nM activity regardless of the assay format and therefore were complementary. In summary, the Nek2 in vitro time-resolved FRET kinase assays reported are sensitive, quantitative, reproducible and amenable to high-throughput screening with improved waste management over radioactive assays.

β-Secretase 1 (BACE1) is an aspartic protease believed to play a critical role in Alzheimer's disease. Inhibitors of this enzyme have been designed by incorporating the non-cleavable hydroxyethylene and statine isosteres into peptides corresponding to BACE1 substrate sequences. We sought to develop new methods to quickly characterize and optimize inhibitors based on the statine core. Minimal sequence requirements for binding were first established using both crystallography and peptide spot synthesis. These shortened peptide inhibitors were then optimized by using spot synthesis to perform iterative cycles of substitution and deletion. The present study resulted in the identification of novel “bis-statine” inhibitors shown by crystallography to have a unique binding mode. Our results demonstrate the application of peptide spot synthesis as an effective method for enhancing peptidomimetic drug discovery.

The fluorescence-based thermal shift assay is a general method for identification of inhibitors of target proteins from compound libraries. Using an environmentally sensitive fluorescent dye to monitor protein thermal unfolding, the ligand-binding affinity can be assessed from the shift of the unfolding temperature (ΔTm) obtained in the presence of ligands relative to that obtained in the absence of ligands. In this article, we report that the thermal shift assay can be conducted in an inexpensive, commercially available device for temperature control and fluorescence detection. The binding affinities obtained from thermal shift assays are compared with the binding affinities measured by isothermal titration calorimetry and with the IC50 values from enzymatic assays. The potential pitfalls in the data analysis of thermal shift assays are also discussed.

A series of benzodiazepine inhibitors of the MMPs and TACE has been developed. These compounds display an interesting selectivity profile and should be useful tools for exploring the biological relevance of such selectivity.

The structure-based design of potent sulfonamide hydroxamate TACE inhibitors bearing novel acetylenic P1' groups has led to compounds with excellent in vitro potency against TACE and selectivity over MMP-1.

Fluorogenic peptide substrates with fluorophore/quencher-capped ends have found extensive use in monitoring protease activity in the screening of small-molecule libraries for protease inhibitors. We report here the identification and characterization of a fluorogenic substrate for tumor necrosis factor-α converting enzyme (TACE). This substrate is a 10-amino-acid peptide (LAQAVRSSSR) capped with an o-aminobenzoyl group on the N-terminal end and with a 3-(2,4-dinitrophenyl)- l -2,3-diaminopropionic amide group on the C-terminal end. Exhaustive enzymatic conversion of the substrate to products resulted in a fluorescence enhancement of ∼11-fold. A single cleavage occurred at the A-V scissile bond of the peptide. The validity of this fluorimetric assay for TACE was corroborated by an independent HPLC method. Interestingly, the hydrolysis of the substrate displayed positive cooperativity with a Hill coefficient of 1.5, while the hydrolysis of the corresponding uncapped peptide displayed Michaelis–Menten kinetics. A kcat value of 21.6 s−1 and an S0.5 value of 342 μM were obtained for the fluorogenic substrate. The addition of the two capping groups on the two ends of the peptide enhanced the kcat value by 64-fold. Nine additional decapeptides that contained the same capping groups on the two ends and substitutions at the P1 and P1′ sites were also tested. TACE appears to slightly prefer the A-V scissile bond. The enzyme also cleaves scissile bonds such as F-V, A-I, and A-L efficiently.

A novel series of anthranilic acid-based inhibitors of MMP-1, MMP-9, and MMP-13 was prepared and evaluated both in vitro and in vivo. The most potent compound, 6e, has in vivo activity in a rat sponge-wrapped cartilage model.

Antibody-drug conjugates (ADCs) enable targeted delivery of therapeutics to cancer cells and offer potential for more selective therapy. Several ADCs are demonstrating promising clinical efficacy, however due to the complexity of human cancer, tumors become refractory to most drug treatments. We hypothesized that cultured tumor cells chronically treated with an ADC would acquire mechanisms of resistance unique to ADC-based therapy. Human breast cancer cell lines were exposed to multiple cycles of an anti-Her2 trastuzumab-maytansinoid conjugate (TM) at IC80 concentrations for 3 days followed by ∼1 week without treatment to simulate a maximally tolerated dose followed by recovery. After ∼2 months, significant resistance developed in JIMT1 and MDA-MB-361 cell lines. The potency of TM conjugate on drug-selected cell lines was reduced to the activity observed on Her2-negative cells (>20 & >270X in JIMT1 & 361 cell models, respectively). Flow cytometry revealed 58% and 25% decreases in Her2 receptor number in JIMT-TM & 361-TM, respectively. Proteomic profiling of surface proteins in JIMT-TM cells demonstrated significant increases in proteins involved in post-translational modification (e.g., ubiquitinating enzymes, kinases, and phosphatases), as well as elevated levels of endosomal and vesicle proteins (e.g., RAB family members), and proteins mediating microtubule and actin dynamics. Notably, ABC drug transporters were not altered in JIMT-TM cells. In 361-TM cells, an increase in ABCC1 (MRP1) was observed, but no changes in ABCB1 (MDR1) which typically effluxes tubulin inhibitors. These data suggest the acquisition of complex resistance mechanisms upon ADC treatment. The cross-resistance profile of these ADC refractory models was evaluated. Minimal or no resistance (1 - 6X) was observed to free drugs, including maytansine or other standard-of-care tubulin or DNA targeted therapeutics. In JIMT-TM cells, cross-resistance was observed to other trastuzumab-based ADCs, including those containing either non-cleavable or cleavable linkers, and delivering payloads with various biological mechanisms of action. In contrast, 361-TM cells (which were made resistant to an ADC delivering a tubulin inhibitor via a non-cleavable linker) retained significant sensitivity to ADCs containing cleavable linkers, even those containing other tubulin inhibitor-based payloads. Moreover, both 361-TM and JIMT-TM resistant cell lines retained sensitivity to ADCs delivering payloads with alternative (non-tubulin) mechanisms of action. Therefore, despite the reduction in antigen levels observed in both cell lines, modification of the linker and/or the payload was able to overcome resistance mediated by the initial ADC therapy. These data offer the potential to treat refractory tumors with ADCs containing the same antibody vehicle, but delivering alternative linkers or payloads. Citation Format: Xingzhi Tan, Guixian Jin, Jeremy Myers, Veronica Diesl, Max Follettie, My-Hanh Lam, Sylvia Musto, Kiran Khandke, Manoj Charati, Edmund Graziani, Andreas Maderna, Chakrapani Subramanyam, Frank Koehn, Russell Dushin, Kim Arndt, Christopher J. O'Donnell, Hans-Peter Gerber, Frank Loganzo. Tumor cells selected for resistance to an antibody-drug conjugate retain sensitivity to ADCs with modified linkers and payloads. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4629. doi:10.1158/1538-7445.AM2013-4629

Within the family of protein kinase C (PKC) molecules only the novel isoform member PRKCE (PKC∈) acts as a bona fide oncogene in in-vitro and in-vivo models of tumorigenesis. Previous studies have reported cancer-specific misexpression of PKC∈ at levels well above that of normal adjacent tissue in breast, prostate and lung tumors. We find that oncogenic Ras signaling promotes PKC∈ expression and results in hyperphosphorylation of CTNND1/p120-catenin (p120ctn). In this context, loss of PKC∈ by genetic or pharmacological means results in normalization of morphology and signaling responses. In a KRasD13-dependent breast cancer model loss of PKC∈ function results in growth inhibition in 2-dimensional (2D) and 3-dimensional (3D) culture systems as well as in orthotopic xenografts concomitant with the normalization of a subset of Ras-induced signaling responses. Using phospho-proteomic profiling analysis we observe that CTNND1 (p120ctn) phosphorylation at serine 268 (S268) occurs in a strictly PKC∈ dependent manner. Treatment with a specific PKC∈ inhibitor, PF-5263555, recapitulates the genetic loss of function phenotype and interferes with breast cancer cell growth in-vitro and in-vivo. We also show that PKC∈-mediated phosphorylation at S268 further stabilizes additional p120ctn phosphorylation sites and total protein levels of β-catenin. We demonstrate that p120ctn phosphorylation at S268 represents a specific readout for PKC∈ activity and as such can serve as a suitable biomarker for PKC∈ dysregulation in human cancer and for monitoring therapeutic response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1235. doi:1538-7445.AM2012-1235

Antibody-drug conjugates (ADCs) enable targeted delivery of therapeutics to cancer cells and offer potential for more selective therapy. Several new ADCs are demonstrating promising clinical efficacy, however due to the complexity of human cancer, tumors become refractory to most drug treatments. We hypothesized that cultured tumor cells chronically treated with an ADC would acquire mechanisms of resistance unique to ADC-based therapy. An anti-Her2 trastuzumab - maytansinoid conjugate (TM) was used. Human breast cancer cell lines sensitive (BT474) or insensitive (JIMT1 & MDA-MB-361) to unconjugated trastuzumab were used as models; all cells were highly responsive to TM conjugate. Cells were exposed to multiple cycles of TM at IC80 concentrations for 3 days followed by ∼1 week without treatment to simulate a maximally tolerated dose followed by recovery. After ∼2 months, minimal resistance was observed in BT474 cells, however significant resistance developed in JIMT and 361. The potency of TM conjugate on drug-selected cell lines was reduced to the activity observed on Her2-negative cells (>20× & >400× in JIMT & 361, respectively). In JIMT-TM cells, cross-resistance was observed to other trastuzumab-ADCs, including T-mcMMAD and T-vcMMAD (non-cleavable & cleavable linkers with dolastatin, respectively). In contrast, 361-TM cells retained partial sensitivity to ADCs containing vc-linked dolastatin and auristatins, including ADCs to other antigens. JIMT-TM & 361-TM cells showed low level resistance to free maytansine payload (∼ 4 − 6×). JIMT-TM was partially cross-resistant to free auristatins (∼4 − 5×), while 361-TM retained sensitivity to auristatins. Neither cell line showed marked resistance (1 − 3×) to other standard-of-care therapeutics such as paclitaxel, docetaxel, vinblastine, camptothecin, oxaliplatin, etoposide, doxorubicin, or 5FU. Interestingly, increased sensitivity was observed to Her2 kinase inhibitor neratinib in 361-TM cells. Flow cytometry revealed 58% and 25% decreases in Her2 receptor number in JIMT-TM & 361-TM, respectively, however partial resistance to free payloads suggests a complex resistance profile independent of Her2 levels. Proteomic profiling was conducted on surface proteins and organelle-enriched fractions. In JIMT-TM cells, significant increases were observed in proteins involved in post-translational modification, including ubiquitinating enzymes, kinases, and phosphatases. Interestingly, elevated levels of endosomal and vesicle proteins were noted, including RAB family members. Proteins mediating microtubule and actin dynamics were also increased in JIMT-TM resistant cells. Notably, ABC drug transporters were not altered in JIMT-TM cells. In 361-TM cells, an increase in ABCC1 (MRP1) was observed, but no changes in ABCB1 (MDR1) which typically effluxes tubulin inhibitors. Alterations in pathways involved in vesicle transport, cytoskeletal function, and phospho-signaling suggest unique ways by which cells may evade chronic ADC-based therapy. The observed sensitivity of 361-TM cells to other vc-linked-auristatin ADCs offers the potential for alternative conjugate-based therapy for cancers which fail TM treatment. Hence, these new ADC-refractory cell models will be valuable tools to interrogate molecular mechanisms underlying resistance to immunoconjugate therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C161.