Dangshe Ma

A general approach to site-specific antibody drug conjugates

Significance Here we demonstrate the ability to genetically incorporate nonnative amino acids into proteins in mammalian cells using both transient and stable platform expression systems that provide yields and fidelities compatible with commercial applications. To illustrate the utility of this methodology we have generated chemically homogeneous antibody drug conjugates (NDCs) with precise control over the site and stoichiometry of drug conjugation. In rodent xenograft models these NDCs display improved properties, including half-life, efficacy and safety, relative to conventional heterogeneous ADCs. These advances allow the generation of therapeutic antibody drug conjugates with medicinal chemistry like control over structure, which should greatly facilitate the optimization of their pharmacological activities. Using an expanded genetic code, antibodies with site-specifically incorporated nonnative amino acids were produced in stable cell lines derived from a CHO cell line with titers over 1 g/L. Using anti-5T4 and anti-Her2 antibodies as model systems, site-specific antibody drug conjugates (NDCs) were produced, via oxime bond formation between ketones on the side chain of the incorporated nonnative amino acid and hydroxylamine functionalized monomethyl auristatin D with either protease-cleavable or noncleavable linkers. When noncleavable linkers were used, these conjugates were highly stable and displayed improved in vitro efficacy as well as in vivo efficacy and pharmacokinetic stability in rodent models relative to conventional antibody drug conjugates conjugated through either engineered surface-exposed or reduced interchain disulfide bond cysteine residues. The advantages of the oxime-bonded, site-specific NDCs were even more apparent when low–antigen-expressing (2+) target cell lines were used in the comparative studies. NDCs generated with protease-cleavable linkers demonstrated that the site of conjugation had a significant impact on the stability of these rationally designed prodrug linkers. In a single-dose rat toxicology study, a site-specific anti-Her2 NDC was well tolerated at dose levels up to 90 mg/kg. These experiments support the notion that chemically defined antibody conjugates can be synthesized in commercially relevant yields and can lead to antibody drug conjugates with improved properties relative to the heterogeneous conjugates formed by nonspecific chemical modification.

Assessment of near-infrared fluorophores to study the biodistribution and tumor targeting of an IL13 receptor α2 antibody by fluorescence molecular tomography

Non-invasive imaging using radiolabels is a common technique used to study the biodistribution of biologics. Due to the limited shelf-life of radiolabels and the requirements of specialized labs, non-invasive optical imaging is an attractive alternative for preclinical studies. Previously, we demonstrated the utility of fluorescence molecular tomography (FMT) an optical imaging modality in evaluating the biodistribution of antibody-drug conjugates. As FMT is a relatively new technology, few fluorophores have been validated for in vivo imaging. The goal of this study was to characterize and determine the utility of near-infrared (NIR) fluorophores for biodistribution studies using interleukin-13 receptor subunit alpha-2 antibody (IL13Rα2-Ab). Eight fluorophores (ex/em: 630/800 nm) with an N-hydroxysuccinimide (NHS) linker were evaluated for Ab conjugation. The resulting antibody-fluorophore (Ab-F) conjugates were evaluated in vitro for degree of conjugation, stability and target-binding, followed by in vivo/ex vivo FMT imaging to determine biodistribution in a xenograft model. The Ab-F conjugates (except Ab-DyLight800) showed good in vitro stability and antigen binding. All Ab-F conjugates (except for Ab-BOD630) resulted in a quantifiable signal in vivo and had similar biodistribution profiles, with peak tumor accumulation between 6 and 24 h post-injection. In vivo/ex vivo FMT imaging showed 17–34% ID/g Ab uptake by the tumor at 96 h. Overall, this is the first study to characterize the biodistribution of an Ab using eight NIR fluorophores. Our results show that 3-dimensional optical imaging is a valuable technology to understand biodistribution and targeting, but a careful selection of the fluorophore for each Ab is warranted.

Abstract 2092: Characterizing NIR dye-IL13RA2 antibody conjugates for biodistribution studies in xenograft tumor models by fluorescence molecular tomography (FMT)

Background: The antibody based therapies are promising anti-cancer therapeutic modalities with minimal toxicity and maximum efficacy. The efficacy of these agents is regulated by their biodistribution and targeting. The contemporary methods of testing the biodistriution of large molecule drugs are expensive and tedious. The development of simple and rapid methodology, such as optical imaging, can enable effective screening of a larger number of compounds. Here we have used Fluorescence Molecular Tomography (FMT), an optical imaging technique to study biodistribution and tumor targeting of IL3RA2 antibody (Ab). Methods: Different near infrared (NIR) fluorophores (λmax:650-800nm) were conjugated to the Ab. The fluorophore conjugation protocol was optimized to achieve a degree of labeling (DOL) of 1-3 for all the conjugates. The properties of Ab-fluorophore conjugate (Ab-F) were compared to unlabeled Ab using SEC-HPLC and cell binding assays in three cell lines with varying expression of IL13RA2: A375(+++), U87MG(+) and H460(-). For in vivo evaluation, Ab-F conjugates were administered intravenously at a dose of 2 nmol fluorophore to nu/nu mice bearing A375. Similar studies were also conducted in U87MG and H460 xenografts with selected Ab-F. The mice were imaged longitudinally (6 time-points) for up to 96 hrs using FMT4000 and the data were analyzed using TrueQuant software. Results: The SEC-HPLC and flow cytometry studies demonstrated that conjugation of Ab with most of the fluorophores did not change its stability or functionality. The in vivo fluorescence data from all the NIR dyes showed a peak tumor accumulation of the Ab-F at 6h and was maintained until 96 hrs. Quantitation of various Ab-F conjugated revealed that 2-6% of the injected dose was accumulated in the A375 tumors. In contrast to tumor profile, there was a steep decline in heart signal (a surrogate for blood/ plasma concentration), suggesting fast clearance from blood. The in vivo and ex vivo data suggested that there was 15-80pmol of Ab-F conjugate accumulated in the tumors at 96h. In addition, Alexa Flour® (AF)680 and AF750 showed minimal non-specific accumulation in other organs, whereas VivoTag® (VT)680 and BODIPY®630 showed a significantly higher non-specific accumulation in liver. Conclusions: These results show that the biological properties of Ab were not changed by conjugation with various NIR fluorophores at DOL Citation Format: Parul Gupta, Dangshe Ma, Rachel Roach, Mary Spilker, Mauricio Leal, Cedo Bagi, Anand Giddabasappa. Characterizing NIR dye-IL13RA2 antibody conjugates for biodistribution studies in xenograft tumor models by fluorescence molecular tomography (FMT). [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 2092.

Abstract 868: Creating a superior, site-specific anti-HER2 antibody-drug conjugate (NG-HER2 ADC) for treatment of solid tumors

Antibody-drug conjugates (ADCs) have emerged as an important class of cancer therapeutics. The FDA approval of Kadcyla (T-DM1), a single agent for treatment of HER2-positive advanced metastatic breast cancer, was a significant milestone in the field of targeted therapy, as the first and only ADC for treatment of solid tumors. Despite the 3-month improvement over standard of care in the median survival, almost all the patients eventually became refractory to T-DM1. We have identified several possible areas for improvement: 1) The potency of T-DM1 as confirmed by the Phase III clinical data is restricted to high HER2 tumors which leaves moderate or low HER2 expressing patients without access to T-DM1 treatment; 2) The 48% overall response rate is indicative of intrinsic resistance to T-DM1 and all T-DM1 treated patients eventually relapse. 3) The randomized lysine conjugation in T-DM1 generates heterogeneity of the product. We have developed a novel, site-specific anti-HER2 ADC (NG-HER2 ADC) and evaluated it in comparative preclinical studies with T-DM1. The results show that the NG-HER2 ADC is ∼ 10 fold more potent than T-DM1 in HER2 3+ xenograft models of breast and gastric cancers. Our proprietary cleavable and permeable linker-payload can mediate bystander effect and this enables potent anti-tumor activity in non-HER2 amplified breast cancer and heterogeneous low HER2 NSCLC PDX models, where T-DM1 is ineffective. Our ADC can overcome T-DM1 resistance in in vitro and in vivo models.. Our site-specific ADC at HNSTD of 9 mg/kg in cynomolgus monkeys showed high AUC, long half-life and had normal clinical observations with no marked neutropenia. On the contrary, conventional conjugates with cleavable linker payloads typically have severe bone marrow toxicity as DLT above 5 mg/kg. The therapeutic index for NG-HER2 ADC is significantly greater than T-DM1 in all models tested. NG-HER2 ADC has a projected clinical efficacious dose of ∼1 mg/kg, compared to 3-5 mg/kg for T-DM1, based on PK/PD modeling. In addition, the activity of the NG-HER2 ADC shows increased infiltration of CD8 positive effector cells, an essential component for immuno-oncology (IO) efficacy, in a syngeneic HER2 overexpressing model. This property potentially allows the combination of the ADC with IO drugs to improve the long-term, overall survival. Our data provides preclinical proof of concept for NG-HER2 ADC with best-in-class potential and is currently being tested in preparation for clinical trials for treatment of HER2 solid tumors. Citation Format: Dangshe Ma, Bitha Narayanan, Kim Marquette, Edmund Graziani, Frank Loganzo, Manoj Charati, Nadira Prashad, Nathan Tumey, Jon Golas, Christine Hosselet, George Hu, Frank Barletta, Alison Betts, Judy Lucas, Chris O’Donnell, Lioudmila Tchistiakova, Hans-Peter Gerber, Puja Sapra. Creating a superior, site-specific anti-HER2 antibody-drug conjugate (NG-HER2 ADC) for treatment of solid tumors. [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 868.

Abstract 644: Impact of conjugation site on pharmacokinetics and off-target toxicity of site-specific antibody drug conjugates

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Site-specific conjugation of toxic payload to antibody yields homogeneous ADCs resulting in improved therapeutic index over conventional conjugation. To understand the role of conjugation site on pharmacokinetics, off-target toxicity, cysteine(cys) was engineered into a humanized anti-IL13Rα2 antibody, hAB08, at various sites within the IgG1/k constant domains. Single cys mutants L443C and Q347C with DAR 2, and double cys mutants K392C+L443C, L443C/kK183C and Q347C/kK183C with DAR 4 were conjugated using auristatin 0101 via maleimidocapronic - valine-citruline-p-aminobenzyloxycarbonyl (vc, cleavable linker). All cys mutants and their conjugates maintained their binding properties compared to parental hAb08. Cysteine substitution sites were selected based on conjugation efficiency and in vitro stability. In vivo efficacy was evaluated in mouse xenograph tumor models. Pharmacokinetics (PK) and off target toxicity (TOX) were assessed in both mouse and rat in vivo studies. Our data demonstrate that that all site specific conjugates show improved in vitro stability, comparable or better in vivo efficacy in mice xenograph models and in vivo stability (ratio of ADC-AUC/Ab-AUC) compared to conventional conjugates. However PK and off-target toxicity of antibody drug conjugates had been significantly impacted by conjugation site and animal species. L443 mutants show higher ADC-AUC, lower clearance and longer half life than Q347 mutants in mice while a reverse TK behavior was observed in rat. All site specific conjugates show higher dose tolerance and less off-target toxicity relative to conventional conjugates in rat. This off-target toxicity improvement depends on sites of conjugation. The L443 mutants exhibited a better off-target TOX profile relative to Q347 mutants. Taken together, site-specific conjugation improved in vivo efficacy and off-target toxicity relative to conventionally conjugated ADC. PK differences and off-target toxicity improvements depend on conjugation sites and animal species. Note: This abstract was not presented at the meeting. Citation Format: Dangshe Ma, Fang Jin, Frank Barletta, George Hu, Nathan Tumey, Haige Zhang, Tao He, Eric Sousa, Manoj Charti, Kiran Khadke, Judy Lucas, Darren Ferguson, Christoper Brown, Weijun Ma, Scott Gatto, William Brady, Edmund Graziani, Hans-Peter Gerber, Puja Sapra, Lioudmila Tchistikova. Impact of conjugation site on pharmacokinetics and off-target toxicity of site-specific antibody drug conjugates. [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 644. doi:10.1158/1538-7445.AM2015-644

Abstract 1241: Targeting the IL-13 receptor alpha 2 with novel antibody-drug conjugates for the treatment of cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The IL-13 receptor alpha 2 (IL-13Rα2) is a transmembrane protein that is highly expressed on several cancers including glioblastoma, ovarian, pancreatic cancers. It can internalize after binding to its ligand IL-13 or in response to ligation with an antibody. In addition, we have discovered that IL-13Rα2 is up-regulated in tumor cell lines that are resistant to chemotherapeutic drugs. These properties make IL-13Rα2 an attractive target for antibody-drug conjugate (ADC), an emerging modality of molecularly targeted cancer therapies. A humanized monoclonal antibody, hAB08 with an affinity of 0.17 nM to the soluble recombined hIL-13Rα2, was conjugated with two novel microtubule inhibitors (MTIs), MTI1 and MTI2, developed by Pfizer. In vitro cytotoxicity of the conjugates was tested against IL-13Rα2 positive and negative cell lines. These conjugates were effective against the IL-13Rα2 positive cell lines (PC3MM2 and A375), having an IC50 ranging from 1.1 to 3.8 ng Ab/mL. However, both of the ADCs were not active against the IL-13Rα2 negative H460 cell line. When treating mice with established human PC3MM2 and A375 tumors, these conjugates were efficacious at 3 mg/kg in reducing tumor growth. In the PC3MM2 model, treatment with hAB08-MTI1 and hAB08-MTI2 resulted in 5 out of 8 or 3 out of 8 animals without measurable tumors at Day 76, respectively. In contrast, the vehicle control group and control ADC (hIgG8.8-MTI1 and hIgG8.8-MTI2) groups were terminated at Day 15, 15, 19 due to the large size of tumors, respectively. These in vitro and in vivo results demonstrate potent antitumor activities of hAB08-MTI1 and hAB08-MTI2 against IL-13Rα2 positive tumors. The findings support the development of novel hAB08-MTI1 or hAB08-MTI2 ADCs as a molecularly targeted therapy for cancer patients that are resistant to standard chemotherapy. Citation Format: Dangshe Ma, Haige Zhang, Fang Jin, Manoj Charati, Kiran Khandke, Judy Lucas, Max Follettie, L. Nathan Tumey, Hans-Peter Gerber, Lioudmila Tchistiakova, Puja Sapra. Targeting the IL-13 receptor alpha 2 with novel antibody-drug conjugates for the treatment of cancer. [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 1241. doi:10.1158/1538-7445.AM2013-1241