In the proposed research, GlycoT will employ its proprietary chemoenzymatic glycan-remodeling platform to establish a facile, robust, and scalable site-specific antibody conjugation technology. Conjugation of various functional molecules to antibodies are frequently used for a wide variety of applications within the life science sectors, such as fluorescent labeled antibodies for the detection and imaging, antibody-drug conjugate (ADC) for cancer therapy, antibody-antibiotic conjugate for infectious disease treatment, LYTAC for targeted degradation. The most exemplified application of antibody conjugation is the development of ADC therapeutics. Over the past 10 years, ADC has been developed as one of powerful and successful avenues for the treatment of cancer. For FDA-approved 11 ADCs and others in clinical trials, the payloads have been mainly conjugated to antibody by non-specific random linkage to either cysteine or arginine, resulting in heterogenous ADC regioisomers, with varied antigen affinity, aggregation potential, serum half-life, and other limitation. As a result, site-specific ADCs with improved pharmacokinetics, and enhanced therapeutic index have been developed. Among different approached to generate site-specific ADCs, remodeling of Fc-glycan on the conserved Asn-297 position to generate Fc-glycan specific ADC is particularly attractive. The use of the galactosyltransferase (GalT) mutants capable of accommodating modified UDP-Gal derivatives as the donor substrates has enabled the incorporation of a selected tag at the Fc glycans for subsequent site-specific conjugation with modified cytotoxic agents. This technology route has been adopted by several clinical stage companies. However, as the GalT mutant can only transfer azide or keto based small Gal-GDP derivative, the drug per antibody provided by this method is just 2. In contrast, another endoglycosidase-based transglycosylation method has overcome such limitation. This convergent approach consists of two key enzymatic steps: deglycosylation of the antibody by an endoglycosidase, and subsequent attachment of a tagged N-glycan to the deglycosylated antibody by an endoglycosidase mutant, which serves as the loading points for the functional molecules. The core enzyme of this platform, endoglycosidase mutant, can transfer either disaccharide or large glycan substrate with extended linker. Such flexibility of substrate size combability is unparallel by GalT, or other enzyme-based platform. In this project, GlycoT will further optimize and streamline this site-specific antibody conjugation technology. We will expand the glycan functionalization routes that can provide well-defined drug/ligand to antibody ratios. We will also establish an optimized cleavable peptide that is compatible with different functional molecules. To accomplish the research goals, we propose to pursue the following four specific aims in the Phase I study. Aim 1 is the functionalization of glycan to obtain broad and defined conjugation loading points. Aim 2 is the development of novel cleavable peptide linkers for the exatecan payload. Aim 3 is scalable synthesis of ADC with MMAE or exatecan payload. Aim 4 is the in vivo toxicity and anti-tumor efficacy tests.
Public Health Relevance Statement: Project Narrative Conjugation of various functional molecules to antibodies are frequently used for a wide variety of applications within the life science sectors. In the proposed research, GlycoT will employ its proprietary chemoenzymatic glycan-remodeling platform to establish a facile, robust, and scalable site-specific antibody conjugation technology.
Project Terms: Antibiotics; Antibiotic Agents; Antibiotic Drugs; Miscellaneous Antibiotic; Antibodies; Antigens; immunogen; Arginine; L-Arginine; Azides; Biological Sciences; Biologic Sciences; Bioscience; Life Sciences; Biotechnology; Biotech; Blood Circulation; Bloodstream; Circulation; Cells; Cell Body; Clinical Trials; Cysteine; Half-Cystine; L-Cysteine; Disaccharides; Pharmaceutical Preparations; Drugs; Medication; Pharmaceutic Preparations; drug/agent; Elements; Endoglycosidases; Enzymes; Enzyme Gene; Freedom; Liberty; Galactosyltransferases; Glycine; Aminoacetic Acid; Goals; Half-Life; Industrialization; Ligands; Methods; Esteroproteases; Peptidases; Protease Gene; Proteases; Proteinases; Proteolytic Enzymes; Peptide Hydrolases; Peptides; Pharmacokinetics; Drug Kinetics; Glycans; Polysaccharides; Research; Investigators; Researchers; Research Personnel; N-Acetylneuraminic Acids; Sialic Acids; sound; Technology; Uridine Diphosphate Galactose; UDP Galactose; UDPGal; Uridine Diphosphogalactose; Uridine Pyrophosphogalactose; Washington; Custom; base; dosage; Label; improved; Site; Clinical; Evaluation; Blood Serum; Serum; Toxicity Testing; Toxicity Tests; Therapeutic; Cytotoxic drug; Cytotoxic agent; Diagnostic; Adopted; Route; System; Heterograft; Heterologous Transplantation; Xenograft; Xenotransplantation; xeno-transplant; xeno-transplantation; Xenograft procedure; Services; mutant; antibody conjugate; Toxicities; Toxic effect; Therapeutic Index; novel; Basic Research; Basic Science; Position; Positioning Attribute; Modeling; cancer therapy; Cancer Treatment; Malignant Neoplasm Therapy; Malignant Neoplasm Treatment; anti-cancer therapy; anticancer therapy; cancer-directed therapy; Exatecan; Length; Affinity; Breast tumor model; mammary cancer model; mammary tumor model; Breast Cancer Model; in vivo; infectious disease treatment; Development; developmental; Image; imaging; mouse model; murine model; tumor; therapeutic development; therapeutic agent development; FDA approved; in vitro testing; efficacy testing; flexibility; flexible; phase 1 study; Phase I Study; operation; screening; Antibody-drug conjugates; efficacy study; antibody detection; antibody based detection; detect antibodies
