Dorin Toader

A Biparatopic HER2-Targeting Antibody-Drug Conjugate Induces Tumor Regression in Primary Models Refractory to or Ineligible for HER2-Targeted Therapy.

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.

Preclinical Evaluation of MEDI0641, a Pyrrolobenzodiazepine-Conjugated Antibody–Drug Conjugate Targeting 5T4

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.

Abstract 2970: MEDI4276, a HER2-targeting antibody tubulysin conjugate, displays potent in vitro and in vivo activity in preclinical studies

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.

Abstract B170: Discovery of tubulysin payloads for antibody drug conjugates with potent in vitro activity and in vivo efficacy in solid tumor models

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.

Abstract 3601: Antibody-drug conjugates (ADCs) with tubulysin and PBD warheads, maintain potent in vitro cytotoxicity against multidrug-resistant tumor cells expressing P-glycoprotein (P-gp)

Development of resistance to initially-effective therapies remains a major challenge in the treatment of cancer. Resistance to antibody-drug conjugates (ADCs) can arise due to many factors including down-regulation of the target, modified internalization or trafficking of the internalized ADC/antigen complexes, decreased sensitivity to the warhead conjugated to the ADC, or combinations of these. One of the common mechanisms of resistance to ADCs is increased expression of efflux drug pumps such as P-glycoprotein (P-gp) which can then transport small molecule warheads out of the cell leading to multi-drug resistance (MDR). In fact, the warheads used in the two clinically-approved ADCs, Adcetris® and Kadcyla™, have been reported to be P-gp substrates and P-gp overexpression can lead to acquired resistance against these ADCs. Studies were conducted to determine if pyrrolobenzodiazepine (PBD) dimer or tubulysin warheads, and/or ADCs conjugated with these warheads were susceptible to P-gp-mediated resistance. The P-gp susceptibility of these warheads and associated ADCs was evaluated by comparing the relative cytotoxicity of warheads/ADCs in parental tumor cell lines compared to the same cell lines that have been manipulated to overexpress P-gp. Verapamil, an inhibitor of P-gp-mediated efflux, was used to confirm whether decreased sensitivity was due to P-gp activity. Unlike known P-gp substrates MMAE, paclitaxel, and vinblastine, the majority of PBD and tubulysin warheads tested were not significant P-gp substrates. These warheads and ADC9s conjugated with these warheads potently induced cytotoxicity of the parental cancer cell lines, and maintained this potency in the same cell lines overexpressing P-gp. Interestingly, certain tubulysins had differential susceptibility to P-gp depending on whether they were conjugated or depending on the linker that was used. For example, one tubulysin warhead showed moderate susceptibility to P-gp-mediated efflux as a naked warhead, but ADCs conjugated with this warhead had equivocal efficacy in parental and P-gp-overexpressing cell lines. An alternative tubulysin warhead was equipotent as an unconjugated small molecule in either parental or P-gp-overexpressing cell lines, however an ADC conjugated with this warhead using a cleavable linker had no activity in the resistant cells; an effect that was reversible with verapamil treatment, confirming the role of P-gp. These data suggest that ADCs conjugated with either PBD or tubulysin warheads may be active in MDR settings where resistance is mediated by P-gp expression, however activity against MDR cancers may be dependent on the particular warhead that was used and/or the particular linker that is used to conjugate the warhead to the antibody. Citation Format: Shenlan Mao, Ryan Fleming, Binyam Bezabeh, Nazzareno Dimasi, Dorin Toader, Thais Cailleau, Philip Howard, Changshou Gao, Bob Hollingsworth, Adeela Kamal, Jay Harper. Antibody-drug conjugates (ADCs) with tubulysin and PBD warheads, maintain potent in vitro cytotoxicity against multidrug-resistant tumor cells expressing P-glycoprotein (P-gp). [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 3601. doi:10.1158/1538-7445.AM2015-3601

Abstract 2659: The MedImmune ADC platform: Building highly potent and specific cancer drugs

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA MedImmune has made a major commitment to developing antibody-drug conjugates for cancer. Our ADC initiative builds on our previous experience with this area and with other armed antibody technologies, which have generated several drugs currently undergoing clinical development. We have leveraged our expertise in antibody engineering to develop new antibody constructs for ADC development, including variants that facilitate site-specific conjugation of the payload to the antibody. This technology circumvents problems associated with random payload conjugation, resulting in a more homogeneous drug product as well as improving the stability and potency of the ADC. We have developed new, potent payloads, teaming up with Spirogen to apply their expertise in the pyrrolobenzodiazapine (PBD) dimer payload technology. The PBD payloads are versatile and potent, allowing use of multiple types of linkers and adjustment of potency to very high levels (picomolar IC50 range). The mechanism of action of the PBDs is different from other commonly used ADC payloads, inducing DNA damage that may evade DNA repair mechanisms and killing both bulk cancer cells and cancer stem cells. We have also developed other, novel ADC payloads. Our ADC target discovery approach permits rapid identification and validation of targets specifically suited for this technology. This includes the early generation and use of tool ADCs for target evaluation. We are applying this state-of-the-art ADC platform to advancing multiple projects as a major component of our oncology drug development strategy. Citation Format: Robert E. Hollingsworth, Adeela Kamal, Philip W. Howard, John A. Hartley, David Tice, Changshou Gao, Nazzareno Dimasi, Haihong Zhong, Jay Harper, Zhan Xiao, Dorin Toader, Chris Martin, Herren Wu, Norman Greenberg, Bahija Jallal. The MedImmune ADC platform: Building highly potent and specific cancer drugs. [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 2659. doi:10.1158/1538-7445.AM2014-2659

Abstract 2459: Discovery of small molecule TAK1 inhibitors with antiproliferative activity in cell lines derived from hematological malignancies.

Transforming growth factor β-activated kinase 1 (TAK1), a member of the MAPKKK family, is a key mediator of proinflammatory and stress signaling. It was shown previously that inhibition of TAK1 via the use of siRNA or small-molecule kinase inhibitors can inactivate NF-κB, down-regulate p38, and activate the intrinsic caspase pathway, resulting in profound induction of apoptosis. Herein we report the discovery of 5-fluoro-4-(imidazo[1,2-b]pyridazin-3-yl)-N-phenylpyrimidin-2-amines as TAK1 ATP competitive small molecule inhibitors. These compounds inhibit the kinase activity of TAK1 in vitro with low nanomolar potency. Evidence is presented that supports a mechanism of action consistent with inhibition of TAK1 kinase activity within the NF-κB pathway. Optimization of potency and selectivity for this series from hit to lead will be discussed, including the structure-based design and crystallographic determination of the binding mode. The outcome of efforts aimed at improving the in vivo pharmacokinetics of compounds is described. Results of kinase selectivity profiling for both an in vitro probe compound - AZ TAK1 - and an in vivo probe compound will be presented. Furthermore, we report low nanomolar antiproliferative activities in cell lines derived from Haematological Malignancies for these compounds. This cellular activity profile is suggesting that TAK1 inhibition presents therapeutic potential in Haematological Cancers. Citation Format: Dorin Toader, Jamal C. Saeh, Nin Guan, Francoise Powell, Raymond Chen, Corinne Reimer, Kate Byth. Discovery of small molecule TAK1 inhibitors with antiproliferative activity in cell lines derived from hematological malignancies. [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 2459. doi:10.1158/1538-7445.AM2013-2459