Joshua D. Thomas

An engineered selenocysteine defines a unique class of antibody derivatives.

Selenocysteine is cotranslationally inserted into proteins by recoding the stop codon UGA from termination to selenocysteine insertion. The nucleophilic selenol group of selenocysteine endows this rare amino acid with unique chemical reactivity that allows regiospecific covalent conjugation in the presence of the other natural amino acids. Using a mammalian expression system, we generated an IgG1-derived Fc fragment with a C-terminal selenocysteine in yields comparable to conventional monoclonal antibodies and conjugated it to an electrophilic derivative of a peptidomimetic that binds with high affinity and specificity to integrin α4β1. Through this conjugation, both the biological and chemical components are endowed with pharmacological advantages. We demonstrate that whereas the Fc protein increases the circulatory half-life from minutes to days and mediates transcytosis through binding to the neonatal Fc receptor, the peptidomimetic introduces cross-species binding to cell surface integrin α4β1 and blocks its interaction with vascular cell adhesion molecule-1. Compared with conventional monoclonal antibodies, our technology benefits economically from combining a generic biological component with a variable chemical component.

A Polymer-Based Antibody-Vinca Drug Conjugate Platform: Characterization and Preclinical Efficacy

Antibody-drug conjugates (ADC) are an emerging drug class that uses antibodies to improve cytotoxic drug targeting for cancer treatment. ADCs in current clinical trials achieve a compromise between potency and physicochemical/pharmacokinetic properties by conjugating potent cytotoxins directly to an antibody at a 4:1 or less stoichiometric ratio. Herein, we report a novel, polyacetal polymer-based platform for creating ADC that use poly-1-hydroxymethylethylene hydroxymethyl-formal (PHF), also known as Fleximer. The high hydrophilicity and polyvalency properties of the Fleximer polymer can be used to produce ADC with high drug loading without compromising physicochemical and pharmacokinetic properties. Using trastuzumab and a vinca drug derivative to demonstrate the utility of this platform, a novel Fleximer-based ADC was prepared and characterized in vivo. The ADC prepared had a vinca-antibody ratio of 20:1. It exhibited a high antigen-binding affinity, an excellent pharmacokinetic profile and antigen-dependent efficacy, and tumor accumulation in multiple tumor xenograft models. Our findings illustrate the robust utility of the Fleximer platform as a highly differentiated alternative to the conjugation platforms used to create ADC currently in clinical development.

Chemically Programmed Bispecific Antibodies That Recruit and Activate T Cells

Background: Bispecific antibodies in cancer therapy operate through simultaneous binding of tumor cells and T cells. Results: Bispecific antibodies with chemically programmed tumor cell specificity through a C-terminal selenocysteine residue kill tumor cells potently and specifically by recruiting and activating T cells. Conclusion: Chemically programmed bispecific antibodies are active. Significance: Chemically programmed bispecific antibodies have a broad utility in cancer therapy. Bispecific antibodies (biAbs) that mediate cytotoxicity by recruiting and activating endogenous immune cells are an emerging class of next-generation antibody therapeutics. Of particular interest are biAbs of relatively small size (∼50 kDa) that can redirect cytotoxic T cells through simultaneous binding of tumor cells. Here we describe a conceptually unique class of biAbs in which the tumor cell specificity of a humanized antibody fragment that recognizes CD3 on T cells is chemically programmed through a C-terminal selenocysteine (Sec) residue. We demonstrate that through chemically programmed specificity for integrin α4β1 or folate receptor 1 (FOLR1), and common specificity for CD3, these hybrid molecules exert potent and specific in vitro and ex vivo cytotoxicity toward tumor cell lines and primary tumor cells in the presence of primary T cells. Importantly, the generic nature of chemical programming allows one to apply our approach to virtually any specificity, promising a broad utility of chemically programmed biAbs in cancer therapy.

Application of a trifunctional reactive linker for the construction of antibody-drug hybrid conjugates.

A flexible, trifunctional poly(ethylene glycol)-succinamide-Lysine-Lysine-maleimide (PEG-SU-Lys-Lys-mal) linker was employed to simultaneously allow biotin tagging and cell-surface targeting through an integrin alpha(4)beta(1)-binding peptidomimetic that was regiospecifically conjugated to an IgG1-derived Fc fragment with an engineered C-terminal selenocysteine residue. The resulting antibody derivative mediates Fc receptor binding by virtue of the Fc protein and selectively targets cancer cells expressing human integrin alpha(4)beta(1). The PEG-SU-Lys-Lys-mal linker may have general utility as an organic tether for the construction of antibody-drug conjugates.

Harnessing the fcμ receptor for potent and selective cytotoxic therapy of chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a B-cell malignancy in need of new, effective, and safe therapies. The recently identified IgM receptor FcμR is overexpressed on malignant B cells in CLL and mediates the rapid internalization and lysosomal shuttling of IgM via its Fc fragment (Fcμ). To exploit this internalization and trafficking pathway for targeted drug delivery, we engineered an IgM-derived protein scaffold (Fcμ) and linked it with the cytotoxic agent monomethylauristatin F. This Fcμ-drug conjugate was selectively toxic for FcμR-expressing cell lines in vitro and for CLL cells but not autologous normal T cells ex vivo. Notably, the cytotoxic activity of the Fcμ-drug conjugate was maintained in CLL cells carrying a 17p deletion, which predicts resistance to standard chemotherapy. Next, we tested the possible therapeutic application of the Fcμ-drug conjugate in immunodeficient NOD/SCID/IL-2Rγ(null) (NSG) mice engrafted with peripheral blood cells from patients with leukemia. Three intravenous injections of the Fcμ-drug conjugate over a 10-day period were well tolerated and selectively killed the human CLL cells but not the coengrafted autologous human T cells. In summary, we developed a novel strategy for targeted cytotoxic therapy of CLL based on the unique properties of FcμR. FcμR-targeted drug delivery showed potent and specific therapeutic activity in CLL, thus providing proof of concept for FcμR as a valuable therapeutic target in CLL and for IgM-based antibody-drug conjugates as a new targeting platform.

Abstract LB-231: A novel, highly potent HER2-targeted antibody-drug conjugate (ADC) for the treatment of low HER2-expressing tumors and combination with trastuzumab-based regimens in HER2-driven tumors

Antibody-drug conjugates are effective in the treatment of HER2-amplified breast cancer and Hodgkin9s lymphoma, but current ADC technologies have faced limitations expanding the addressable patient population and target space. Ado-trastuzumab emtansine (T-DM1) is an ADC with 3-4 cytotoxic drugs per antibody that was recently approved for HER2 IHC 3+ or HER2-amplified breast cancer. Even within this high HER2-expressing population, several studies have now shown greater T-DM1 benefit in patients with HER2 mRNA expression above the median. These data suggest the need for more potent anti-HER2 ADCs to maximize benefit for HER2 IHC 3+ or amplified patients, and to extend HER2 ADC therapy to low HER2-expressing patients (HER2 IHC 1+/2+). XMT-1522 is an anti-HER2 ADC that uses a novel, human anti-HER2 antibody optimized for cytotoxic payload delivery, and is non-competitive with trastuzumab or pertuzumab for HER2 binding. Each antibody is conjugated to ∼15 proprietary auristatin molecules using Fleximer, a biodegradable hydrophilic polymer. XMT-1522 shows nanomolar potency in cultured tumor cells with HER2 receptor densities as low as 10,000 per cell, and is typically 1-3 logs more potent than T-DM1 across a panel of 25 tumor cell lines. In mouse xenograft studies XMT-1522 has excellent pharmacokinetic properties and achieves complete tumor regressions at well-tolerated doses. In the high HER2-expressing N87 gastric cancer model (800,000 HER2 receptors/cell), complete regressions are achieved with a single 1 mg/kg dose of XMT-1522, while 10 mg/kg T-DM1 is required for comparable activity. In the same model, the XMT-1522/trastuzumab/pertuzumab triple combination results in tumor regressions where the same doses of XMT-1522 alone or the trastuzumab/pertuzumab doublet result in tumor stasis. In the low HER2-expressing JIMT-1 breast cancer (79,000 HER2/cell) and SNU5 gastric cancer (22,000 HER2/cell) models, complete regressions are achieved with single 1 mg/kg or 0.67 mg/kg doses of XMT-1522, respectively, while T-DM1 is inactive at doses ≥10 mg/kg. In non-human primates XMT-1522 demonstrates good stability of drug conjugate in plasma with t1/2 ∼5 days (comparable to antibody t1/2) and minimal exposure to free payload. Despite the high potency of XMT-1522 in low HER2 tumor models, there is no XMT-1522-related toxicity observed in critical HER2-expressing tissues including heart and lung. The preclinical data support testing XMT-1522 as a single agent in tumors with low HER2 expression where current HER2-directed therapies are not indicated. Furthermore, combination of XMT-1522 with trastuzumab and/or pertuzumab achieves efficient cytotoxic payload delivery while retaining the potential for full inhibition of HER2 signaling, which may be necessary to improve on current regimens in HER2-driven tumors. Citation Format: Donald A. Bergstrom, Natalya Bodyak, Alex Yurkovetskiy, Peter U. Park, Michael DeVit, Mao Yin, Laura Poling, Joshua D. Thomas, Dmitry Gumerov, Dongmei Xiao, Elena Ter-Ovanesyan, LiuLiang Qin, Alex Uttard, Alex Johnson, Timothy B. Lowinger. A novel, highly potent HER2-targeted antibody-drug conjugate (ADC) for the treatment of low HER2-expressing tumors and combination with trastuzumab-based regimens in HER2-driven tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-231. doi:10.1158/1538-7445.AM2015-LB-231

Abstract 596: Optimization of lead antibody selection for XMT-1522, a novel, highly potent HER2-targeted antibody-drug conjugate (ADC)

XMT-1522 is an anti-HER2 ADC that incorporates HT-19, a novel, human anti-HER2 antibody optimized for cytotoxic payload delivery. Several parameters such as cell binding, internalization rate, cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC) downstream signaling, affinity maturation, NHP cross-reactivity, normal human tissue cross-reactivity and in vivo efficacy were used to screen a wide range of antibodies to select a lead candidate optimized for use in ADC applications. In addition HT-19 was selected to be non-competitive for HER2 binding with existing therapies - trastuzumab or pertuzumab, to allow for potential combinability. In vivo efficacy as an ADC did not directly correlate with typical parameters used in antibody screening cascade such as in vitro cytotoxicity, internalization or binding affinity. The lead antibody underwent affinity maturation, and despite increases in affinity, affinity maturation significantly decreased the in vivo efficacy of the ADC in vivo xenograft models. This phenomenon was observed in all the antibody hits. HT-19 showed antibody-dependent cell-mediated cytotoxicity (ADCC) activity. When administered as a single agent in NCI-N87 gastric cancer xenograft model it had biological activity at 20 mg/kg as well as at 3 mg/kg. Consistent with the hypothesis that a non-competitive ADC is combinable with current anti-HER2 regimens, the combination of XMT-1522 with trastuzumab and/or pertuzumab showed more rapid internalization, more complete HER2 degradation, and significantly great anti-tumor activity in the NCI-N87 gastric cancer xenograft model relative to XMT-1522 alone or the combination of pertuzumab + trastuzumab. Citation Format: Natasha Bodyak, Alex Yurkovetskiy, Dmitry R. Gumerov, Dongmei Xiao, Joshua D. Thomas D. Thomas, Laura L. Poling, LiuLiang Qin, Mao Yin, Michael J. DeVit, Peter U. Park, Winnie Lee, Bianka Prinz, Donald A. Bergstrom, Timothy B. Lowinger. Optimization of lead antibody selection for XMT-1522, a novel, highly potent HER2-targeted antibody-drug conjugate (ADC). [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 596.

Abstract 641: Trastuzumab-dolaflexin, a highly potent Fleximer-based antibody-drug conjugate, demonstrates a favorable therapeutic index in exploratory toxicology studies in multiple species

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Antibody-drug conjugates (ADCs) represent a promising drug class that relies on monoclonal antibody recognition of specific cancer-associated antigens for targeted delivery of chemotherapeutic agents. Fleximer® ADCs utilize a polymer-based conjugation platform to enable high drug-antibody ratios (DAR) and significantly greater anti-tumor potency compared to ADCs with DAR 3-4. We previously showed that trastuzumab-dolaflexin (T-dolaflexin), a HER2-targeted ADC comprised of the antibody trastuzumab conjugated to ∼15 proprietary auristatin molecules via Fleximer, has excellent pharmacokinetics and efficacy in mouse models (AACR 2014 Abstract #2645). T-dolaflexin is efficacious at a single dose of 0.67 mg/kg in mouse xenograft models, and achieves prolonged tumor-free survival after a single 2 mg/kg dose in a low HER2-expressing model that is insensitive to ado-trastuzumab emtansine (T-DM1). We sought to test the tolerability of T-dolaflexin in exploratory mouse, rat and non-human primate toxicology studies. Cohorts of 6 mice were treated with a single dose of vehicle or T-dolaflexin at 20, 30 or 40 mg/kg and followed for 21 days. The 20 and 30 mg/kg doses were well-tolerated based on body weight loss and mortality and achieved a therapeutic index (TI) in mouse >40X. Cohorts of 4 female cynomolgus monkeys were treated with a single dose of vehicle or T-dolaflexin at 0.67, 1.34 or 2.68 mg/kg (payload doses 600, 1200 and 2400 μg/m2, respectively). Day 3 necropsy was performed on 2 animals per cohort, with recovery necropsy on Day 22 in the remaining 2 animals. All animals survived until scheduled necropsy with limited body weight loss. There were no test-article related findings on gross pathology. Most notable clinical pathology findings were transaminase elevations (primarily AST), and decreased platelet counts at Day 8. One high dose animal demonstrated laboratory findings consistent with hepatic injury with full recovery by Day 22 and no findings on gross or microscopic pathology at necropsy. There was no evidence of myelosuppression. Microscopic pathology findings were limited, with no test-article related findings in HER2-expressing organs including heart, lungs and GI tract. All doses were considered well-tolerated. Toxicokinetics demonstrated good stability of drug conjugate in plasma with t1/2 ∼5 days (comparable to antibody t1/2) and minimal exposure to free payload. Plasma exposure at the 2.68 mg/kg dose in cyno was several fold higher than mouse exposure at the minimally efficacious dose of 0.67 mg/kg, and was comparable to the mouse exposure associated with prolonged tumor free survival after a single 2mg/kg dose. Trastuzumab-dolaflexin, a highly potent Fleximer-based ADC, demonstrates a favorable TI in tumor models with low HER2 expression where current HER2-directed therapies are inactive. Citation Format: Natalya Bodyak, Alex Yurkovetskiy, Peter U. Park, Dmitry R. Gumerov, Michael DeVit, Mao Yin, Joshua D. Thomas, LiuLiang Qin, Timothy B. Lowinger, Donald A. Bergstrom. Trastuzumab-dolaflexin, a highly potent Fleximer-based antibody-drug conjugate, demonstrates a favorable therapeutic index in exploratory toxicology studies in multiple species. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 641. doi:10.1158/1538-7445.AM2015-641

Abstract 2645: Advantages of polyacetal polymer-based ADCs: Application to low expression targets

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The application of biodegradable polymers to antibody-drug conjugate (ADC) design can provide numerous advantages, including significantly higher drug-antibody ratios, the use of alternative payloads with potencies considered insufficient for direct conjugation, the improvement of ADC physico-chemical properties, especially for highly hydrophobic payloads, and the potential expansion of protein recognition scaffolds beyond the commonly used IgGs. The basis of the novel polymer-based conjugation system described herein is a hydrophilic, fully biodegradable polyacetal carrier, (poly(1-hydroxymethylethylene hydroxymethylformal) or PHF) modified with chemically orthogonal linkers. A bioconjugation linker is used for efficient covalent attachment of a targeting moiety to the PHF scaffold, while a second, chemically distinct linker is used to attach multiple copies of a drug payload to the polymer to control the mechanism and rate of drug release. Utilizing multiple copies of a proprietary dolastatin derivative chemically conjugated to PHF, we have developed a potent and effective drug conjugation platform for ADC application, which has been named Dolaflexin™. Here, we report the preparation and characterization of a novel trastuzumab DolaflexinTM ADC, employing a maleimide-based bioconjugation approach. The resulting ADC, with a drug-antibody ratio of 20, exhibits enhanced stability and improved pharmacokinetics, with a prolonged plasma half-life and tumor-specific accumulation. Active drug release and accumulation in tumor tissue was also confirmed by LC/MS/MS methods. The activity of this novel trastuzumab-dolaflexin ADC was evaluated in multiple tumor xenograft models with significant variations in target antigen expression levels. Models including BT474 breast cancer, NCI-N87 gastric cancer, and JIMT1 breast cancer models were utilized, and comparisons to a variety of controls and ADC reference standards were made. Significant advantages of the polyacetal polymer-based ADCs in comparison to conventional ADCs, particularly in models with low target antigen expression, were observed. Details of these studies and potential applications for the development of new ADC therapeutics based on this approach will be presented. Citation Format: Alex Yurkovetskiy, Natalya Bodyak, Mao Yin, Joshua D. Thomas, Patrick Conlon, Cheri A. Stevenson, Alex Uttard, LiuLiang Qin, Dmitry R. Gumerov, Elena Ter-Ovaneysan, Venu R. Gurijala, Dennis McGillicuddy, Roberta E. Glynn, Michael DeVit, Laura L. Poling, Peter U. Park, Timothy B. Lowinger. Advantages of polyacetal polymer-based ADCs: Application to low expression targets. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2645. doi:10.1158/1538-7445.AM2014-2645