Adeela Kamal

Rational design, biophysical and biological characterization of site-specific antibody-tubulysin conjugates with improved stability, efficacy and pharmacokinetics

Antibody-drug conjugates (ADCs) are among the most promising empowered biologics for cancer treatment. ADCs are commonly prepared by chemical conjugation of small molecule cytotoxic anti-cancer drugs to antibodies through either lysine side chains or cysteine thiols generated by the reduction of interchain disulfide bonds. Both methods yield heterogeneous conjugates with complex biophysical properties and suboptimal serum stability, efficacy, and pharmacokinetics. To limit the complexity of cysteine-based ADCs, we have engineered and characterized in vitro and in vivo antibody cysteine variants that allow precise control of both site of conjugation and drug load per antibody molecule. We demonstrate that the chemically-defined cysteine-engineered antibody-tubulysin conjugates have improved ex vivo and in vivo stability, efficacy, and pharmacokinetics when compared to conventional cysteine-based ADCs with similar drug-to-antibody ratios. In addition, to limit the non-target FcγRs mediated uptake of the ADCs by cells of the innate immune system, which may result in off-target toxicities, the ADCs have been engineered to lack Fc-receptor binding. The strategies described herein are broadly applicable to any full-length IgG or Fc-based ADC and have been incorporated into an ADC that is in phase I clinical development.

Selecting an Optimal Antibody for Antibody–Drug Conjugate Therapy: Internalization and Intracellular Localization

Antibody-drug conjugates (ADCs) combine the selectivity of a monoclonal antibody with the killing potency of a cytotoxic drug. For an antibody to function as a successful component of an ADC, it needs to bind to the target antigen on the surface of tumor cells and then be internalized by the cell. Following internalization, the ADC has to be transported to the lysosome where subsequent intracellular processing of the ADC will release the biologically active drug to exert its cytotoxic effects on tumor cells. This chapter describes some of the techniques that are currently used to determine internalization and proper intracellular trafficking of antibodies in order to select an optimal antibody for ADC therapeutics.

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.

Novel Role for NFAT3 in ERK-Mediated Regulation of CXCR4

The G-protein coupled chemokine (C-X-C motif) receptor CXCR4 is linked to cancer, HIV, and WHIM (Warts, Hypogammaglobulinemia, Infections, and Myelokathexis) syndrome. While CXCR4 is reported to be overexpressed in multiple human cancer types and many hematological cancer cell lines, we have observed poor in vitro cell surface expression of CXCR4 in many solid tumor cell lines. We explore further the possible factors and pathways involved in regulating CXCR4 expression. Here, we showed that MEK-ERK signaling pathway and NFAT3 transcriptional factor plays a novel role in regulating CXCR4 expression. When cultured as 3D spheroids, HeyA8 ovarian tumor cells showed a dramatic increase in surface CXCR4 protein levels as well as mRNA transcripts. Furthermore, HeyA8 3D spheroids showed a decrease in phospho-ERK levels when compared to adherent cells. The treatment of adherent HeyA8 cells with an inhibitor of the MEK-ERK pathway, U0126, resulted in a significant increase in surface CXCR4 expression. Additional investigation using the PCR array assay comparing adherent to 3D spheroid showed a wide range of transcription factors being up-regulated, most notably a> 20 fold increase in NFAT3 transcription factor mRNA. Finally, chromatin immunoprecipitation (ChIP) analysis showed that direct binding of NFAT3 on the CXCR4 promoter corresponds to increased CXCR4 expression in HeyA8 ovarian cell line. Taken together, our results suggest that high phospho-ERK levels and NFAT3 expression plays a novel role in regulating CXCR4 expression.

Abstract A49: Anti-DLL4 antibodies inhibit cancer stem cells in small cell lung cancer.

Small cell lung cancer (SCLC) is a very aggressive lung cancer with features suggesting enrichment in cancer stem cells (CSCs). Delta-like ligand 4 (DLL4) is a membrane bound ligand for Notch receptors critical for functional angiogenesis. Blocking DLL4 signaling increases the density of nonfunctional blood vessels and hypoxia of tumors, and thereby inhibits growth of tumor xenografts in mice. In addition, growing evidence implicates DLL4 Notch signaling pathway in the maintenance of CSCs. Here we investigated the anti-CSC activity of anti-DLL4 mAbs using in vitro and in vivo models of SCLC. Biochemical and flow cytometry analyses revealed that multiple SCLC cell lines express DLL4, and notably, SCLC spheres cultured under CSC-enriching conditions express higher levels of DLL4. Prior to determining the ability of anti-DLL4 mAb in inhibiting CSC in vivo, we first characterized the phenotype of CSCs in the SCLC cell line NCI-H69. NCI-H69 cells enriched for high CD133 expression were more tumorigenic and expressed higher levels of Nanog, Oct 3/4, and EZH2, which are genes crucial for maintenance of CSCs, than cells with low or negative levels of CD133. Moreover, RNA level of DLL4 was found to be three fold higher in CD133 high cells compared to CD133 low cells, suggesting that expression of DLL4 correlates with the CSC phenotype. In vivo, an anti-DLL4 antibody leads to inhibition of NCI-H69 tumor growth when the antibody is administered as a single agent, or in combination with cisplatin+etoposide or topotecan, chemotherapy agents commonly used in treatment of SCLC. To determine if blockade of DLL4 inhibits CSCs in vivo, NCI-H69 tumor xenografts treated with anti-DLL4 mAb were analyzed by flow cytometry. A subset of dissociated tumor cells expressed CD133 and DLL4, and a reduction of these populations of cells was observed in the anti-DLL4 antibody treated groups. In summary, these studies highlight that, in addition to vascular expression, (1) DLL4 is frequently expressed in SCLC cells, (2) DLL4 expression correlates with a CSC phenotype, and (3) that DLL4 blockade using an anti-DLL4 mAb results in inhibition of CSCs. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A49. Citation Format: Patrick Strout, Martin Korade, Ching Ching Leow, Ivan Inigo, Suneetha Thomas, Elaine Hurt, Jon Chesebrough, Adeela Kamal, Song Cho. Anti-DLL4 antibodies inhibit cancer stem cells in small cell lung cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A49.

Abstract 5462: MEDI3185, a potent anti-CXCR4 antibody, inhibits tumor cell migration, signaling and tumor growth in preclinical models.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The chemokine receptor CXCR4 is a seven-transmembrane G-protein coupled receptor that mediates chemotaxis and cell migration upon stimulation via its ligand, stromal-derived factor 1 (SDF-1), also called CXCL12. CXCR4 is normally expressed on bone marrow stem and progenitor cells, various circulating lymphocytes, endothelial precursor cells, tissue macrophages and fibroblasts but the aberrant overexpression of CXCR4 is linked to various hematological malignancies, solid tumors and metastatic neoplasms. Moreover, CXCR4 overexpression is correlated with poor prognosis in many types of cancer, including breast, ovarian, colon, pancreatic, AML and glioblastomas. CXCR4 inhibition using siRNA, small-molecule and peptide inhibitors has demonstrated that it can inhibit tumor growth by blocking tumor cell survival/proliferation, metastasis, angiogenesis and tumor immune infiltrates. Here we describe a novel, fully human, antagonistic antibody to CXCR4, MEDI3185, which blocks SDF-1 binding to CXCR4. MEDI3185 has picomolar binding affinity to human CXCR4 and exhibits no significant binding to other chemokine receptors such as CCR4 or CXCR3. In vitro studies demonstrated that MEDI3185 inhibited tumor cell migration, blocked SDF-1 induced tumor cell signaling and induced apoptosis of tumor cells. In preclinical human tumor xenograft models in mouse, MEDI3185 showed single-agent tumor growth inhibition in multiple myeloma and B-cell Burkitts lymphoma models and had combination activity in ovarian models. In addition, MEDI3185 extended survival as combination therapy in mouse models of CLL and also blocked lung tumor burden in a disseminated ovarian model. Combined, these data suggest that MEDI3185 is a potent CXCR4 antibody for the treatment of both hematological and solid tumors because it has pleiotropic effects on tumor biology that may enhance the efficacy of the current standard of care. Citation Format: Adeela Kamal, Youzhen Wang, Philipp Steiner, Anne-Marie Mazzola, Leslie Wetzel, Melissa Passino, Brenda McDermott, Keven Huang, Vahe Bedian, Norman Greenberg. MEDI3185, a potent anti-CXCR4 antibody, inhibits tumor cell migration, signaling and tumor growth in preclinical models. [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 5462. doi:10.1158/1538-7445.AM2013-5462

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 1801: ERK-mediated regulation of NFAT3 enhances CXCR4 expression in HeyA8 ovarian cell line.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The G-protein coupled chemokine (C-X-C motif) receptor CXCR4 is linked to cancer, HIV, and WHIM (Warts, Hypogammaglobulinemia, Infections, and Myelokathexis) syndrome. While CXCR4 is widely studied and characterized extensively to be over-expressed in multiple human tumor tissues, we have seen poor surface CXCR4 expression in many solid tumor cell lines in vitro. To better understand how CXCR4 is regulated, cells grown under 3-D spheroid conditions were compared to normal adherent culturing conditions. When cultured as 3D spheroids, HeyA8 cells showed a dramatic increase in surface CXCR4 protein levels as well as mRNA transcripts. Furthermore, HeyA8 3D spheroids showed a decrease in pERK levels when compared to adherent cells. Using an inhibitor of the MEK-ERK pathway, the treatment of adherent HeyA8 cells with U0126 resulted in a significant increase in surface CXCR4 expression similar to 3D spheroids. Additional investigation using PCR array showed a wide range of transcription factors being up-regulated but notably a > 20 fold increase in NFAT3 transcription factor. Using a calcineurin inhibitor, cyclosporin A diminished the U0126 treated up-regulation of CXCR4 surface expression. Finally, Chromatin Immunoprecipitation analysis showed direct binding of NFAT3 on the CXCR4 promoter to increase CXCR4 expression in HeyA8 ovarian cell line. Taken together, our results suggest that phospho-ERK levels regulate CXCR4 expression through the NFAT3 signaling pathway. Citation Format: Keven Huang, Christine Kiefer, Adeela Kamal. ERK-mediated regulation of NFAT3 enhances CXCR4 expression in HeyA8 ovarian cell line. [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 1801. doi:10.1158/1538-7445.AM2013-1801 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.