SBIR-STTR Award

There has been growing interest in the use of antibody drug conjugates (ADCs) for the treatment of cancer as mounting data suggests an increase in anti-tumor effectiveness and reduced toxicity, compared with the administration of unlabeled antibodies in combination with chemotherapy.1-4 The two ADCs that are currently approved by the FDA, brentuximab vedotin (Adcetris) and ado-trastuzumab emtansine (T-DM1; Kadcyla) are both heterogeneous mixtures in terms of both the location of the drug and the number of drugs per IgG. Recent evidence has shown that differentially labeled antibodies, i.e. labeled at different locations and with different numbers, can have distinct therapeutic and pharmacokinetic properties5 and some subpopulations can show little, if any, therapeutic activity yet account for most of the toxicity.6-8 Therefore, there has been a movement towards the development of site-specific ADCs, which are precisely labeled with drugs at pre-defined locations. Currently, there are four general approaches for the generation of site-specific ADCs. These include the introduction of cysteine-tags into the genetic code of IgG,6, 9 the use of unnatural amino acids,10 the addition of peptide tags that are recognized and modified with enzymes (e.g. formylglycine generating enzyme or transglutaminase),8, 11 and glycan modifications (e.g. via glycotransferase).12 While each approach has its own unique advantages, shortcomings can include low product yields, inefficient drug conjugation, unstable and/or hydrophobic antibody-drug linker chemistry, or incompatibility with glycosylated antibodies. We propose to further develop a new site-specific bioconjugation approach, Proximity-Based Sortase Ligation (PBSL), that can produce ADCs in high yields, allows different drugs to be site-specifically added to the heavy and/or light chains, is compatible with glycosylated IgG, and offers unlimited flexibility in antibody-drug linker chemistry. Therefore, we believe that this technology will provide a new, favorable approach for the production of site- specific ADCs that will be of interest to the pharmaceutical industry. The specific aims for this proposal are: Aim 1: Use PBSL to produce site-specific anti-prostate specific membrane antigen (PSMA)-MMAE ADCs and characterize their properties; Aim 2: Evaluate the binding and efficacy of site-specific anti-PSMA-MMAE ADCs in vitro

Public Health Relevance Statement:
The overall goal of this proposal is to develop a new site-specific bioconjugation technology that enables the highly efficient production of antibody-drug conjugates in high yields.

Project Terms:
Antibodies; Antibody Avidity; Antibody Formation; Antibody Specificity; Antibody-drug conjugates; antitumor drug; base; Binding; Biological Assay; cancer therapy; Chemistry; chemotherapy; Cysteine; cytotoxic; cytotoxicity; Data; Development; Drug Industry; Drug Kinetics; drug production; Drug Stability; Effectiveness; Enzymes; Extracellular Domain; FDA approved; flexibility; FOLH1 gene; formylglycine; Generations; Genetic Code; glycosylated IgG; Goals; Human; Hydrophobicity; Immunoglobulin G; Immunoglobulins; In Vitro; interest; J591 Monoclonal Antibody; Label; Length; Ligation; Light; Location; Modification; Movement; neoplastic cell; Peptides; Pharmaceutical Preparations; Polysaccharides; Preparation; Production; Property; Site; Solubility; sortase; System; Technology; Therapeutic; Toxic effect; Transglutaminases; Trastuzumab; tumor; unnatural amino acids; Variant

There has been growing interest in the use of antibody drug conjugates (ADCs) for the treatment of cancer asmounting data suggests an increase in anti-tumor effectiveness and reduced toxicity, compared with theadministration of unlabeled antibodies in combination with chemotherapy. Recent evidence has shown thatdifferentially labeled antibodies, i.e. labeled at different locations and with different numbers, can have distincttherapeutic and pharmacokinetic properties and some subpopulations can show little, if any, therapeutic activityyet account for most of the toxicity. Therefore, there has been a movement towards the development of site-specific ADCs, which are precisely labeled with drugs at pre-defined locations.We have recently developed two new approaches for the preparation of highly uniform ADCs, one site-specificbioconjugation approach, Proximity-Based Sortase-mediated protein Ligation (PBS-PL), whereby sortase isused to ligate drugs to a peptide tag that has been introduced into the antibody backbone, and one region-specific bioconjugation approach, Proximity-based Sortase Isopeptide Ligation (PBS-IL), which allows for thelabeling of native antibodies with reduced variability compared with current lysine/cysteine residue labelingapproaches. Both methods produce ADCs in high yields, are compatible with glycosylated IgG, and offerunlimited flexibility in antibody-drug linker chemistry. Therefore, we believe that these technologies will providenew, favorable approaches for the production of ADCs that will be of interest to the pharmaceutical industry.In this proposal, we have partnered with Integral Molecular and the University of Pennsylvania to developADCs against claudin-18 (CLDN 18.2). CLD18.2 is ectopically expressed in several cancers includingpancreatic cancer, which is the focus of this proposal. Outcomes for pancreatic cancer patients are poor with a5-year survival of <8% due to a lack of effective treatment modalities. Therefore, the development of newtherapies are a clinical necessity. The highly selective expression of CLDN18.2 in cancer, with no detectableexpression on any healthy adult tissues that are accessible to antibodies, make it an attractive option fortargeted therapy. We will prepare various ADCs using PBS-PL and PBS-IL and will identify the conjugationapproach and ADC formulation that is expected to be most favorable for clinical translation, based on serumstability, pharmacokinetics, and efficacy. Pre-clinical testing will be performed in an orthotopic pancreatic tumormodel in syngeneic mice. The specific aims for the proposal are: Aim 1: Produce and characterize anti-CLDN18.2-vcMMAE ADCs using PBS-PL and PBS-IL; Aim 2: Evaluate the binding and efficacy of anti-CLDN18.2-vcMMAE ADCs in vitro; Aim 3. Determine the pharmacokinetics and efficacy of anti-CLDN18.2ADCs in a murine tumor model

Public Health Relevance Statement:
The overall goal of this proposal is to utilize two unique bioconjugation technologies to produce highly homogeneous anti-CLDN18.2 antibody drug conjugates (ADC) for the treatment of pancreatic cancer and to identify the optimal approach and lead ADC for continued development into a clinical therapeutic.

Project Terms:
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