Stable-isotope labeled glycopeptides
Antibody Drug Conjugates
Intravenous immunoglobulin (IVIG) is pooled immunoglobulins collected and purified from thousands of healthy donors. It has been widely used for the treatment of autoimmune disease and chronic inflammation such as immune thrombocytopenia, Guillain-Barre syndrome, Kawasaki disease, and rheumatoid arthritis (RA). Global sale of IVIG is expected to increase at an annual rate of 6.8% and is projected to reach $11.6 billion by 2020. Despite its wide applications for anti-inflammation treatment, current IVIG preparation has distinct limitations, including long time infusion (4-6 h) associated with high volume of IVIG treatment, variable efficacy, shortage of supplies, inconsistence from batch to batch, potential contaminations, and side effects. As a result, alternatives for IVIG with more potent therapeutic efficacy (thus much lower doses), more consistent in components, and minimal side effects are highly desired. Recent studies in animal models and human patients have suggested that the sialylated Fc glycoform of IVIG, which is a minor component consisting of ca. 10% of IVIG, could be the major active form that confers the anti-inflammatory activities. GlycoT is applying our innovative and proprietary platform to establish an efficient, scalable chemoenzymatic process for producing homogeneous and optimal glycoforms of IVIG (hyersialylated IVIG) with improved anti-inflammatory activities.
So far, GlycoT scientists have established an efficient, scalable chemoenzymatic process for producing fully sialylated IVIG (hsIVIG) (over 95% sialylation) on gram-scale. Preliminary animal studies indicated that the glycan remodeled human IVIG (hsIVIG) exhibited over 10-fold higher anti-inflammatory activity than the commercial human IVIG a mouse model of rheumatoid arthritis. With this promising preliminary result, GlycoT plans to further scale up the production of hsIVIG, to carry out efficacy study in multiple animal model of inflammations, and to perform initial preclinical studies such as PK/PD, and toxicity. A rigorous quality control system with extensive biological and physiochemical characterization of the quality and stability of the cGMP grade hsIVIG will be established aiming at human phase I clinical trials. The R&D work of hsIVIG is currently supported by a small business innovation research (SBIR) grant awarded by NIH (R43GM123823)
Stable-isotope labeled Fc-glycopeptide standards
GlycoT is constructing a library of stable isotope labeled Fc glycopeptides as standards for mass-spec based absolute quantitation of Fc glycosylations of antibodies. Immunoglobulin G (IgG) is an important class of glycoproteins that have been involved in many immunological functions. All IgGs contain an N-glycan at conserved Asn297 position in its Fc domain. Aberrant change of Fc glycosylation in Asn297 has been observed in many diseases, such as rheumatoid arthritis, tubuclouses infection, dengue virus infection in patients, and many types of cancers. Taken together, site-specific Fc glycan analysis and absolute quantitation is of great importance to IgG therapeutics, biomarker discovery, and monitoring and diagnosis of diseases and disease progression. The use of mass spectrometry in combination with isotopically enriched and quantified internal standards for quantitative analysis has become routine in metabolomics and proteomics. Isotope-labeled glycans are also emerging as tools for the absolute quantification of individual glycan levels in complex mixtures. However, comprehensive stable isotope-labeled glycopeptide standards are lacking.
GlycoT is developing a C13-labeled IgG-Fc glycopeptide library using our technology platform to fulfill this unmet market need. The Fc glycopeptide library can be used as standards for biomarker discovery, for diagnosis and monitoring of diseases and disease progression, and for quality control in manufacturing antibody-based biopharmaceutical products. The R&D work at GlycoT in this area is being supported by a Phase I SBIR grant awarded by NIGMS/NIH (R43GM128547).
Antibody-drug conjugate (ADC)
Antibody–drug conjugates (ADCs) are emerging as a very promising strategy for selective delivery of highly toxic drugs to target cells, which takes advantage of the specificity of antibodies. Despite the rapid progresses in this area, however, site-specific conjugation to produce homogeneous ADCs remains a challenging task. Several approaches have been developed to obtain structurally defined homogeneous ADCs. One approach is to introduce additional cysteine, unnatural amino acid, or other tags to serve as a handle for chemo-selective ligation with a drug. This approach will require the expression of the new recombinant antibody with the engineered tags and there may be an issue of potential immunogenicity. Another approach is to introduce a tag such as an azide by modifying the Fc glycans of antibodies. These usually include desiaylation, galactosylation, and resialylation with an azide-tagged sialic acid residues for click conjugation with a drug. Alternatively, sequential desialylation, degalactosylation, and regalactosylation with an azide-tagged galactose substrate can be performed, with final click conjugation of a drug. The advantage of this approach is that the glycan modification will not change the protein portions and will not affect Fab binding to antigens. However, there are a few potential weaknesses with the current approach: 1) there are antibody glycoforms such as high-mannose type that cannot be modified by this approach to introduce tags, thus the resulting ADCs can be still heterogeneous; 2) if the antibody has both Fc and Fab glycans, then there would be no selection on the glycans; and 3) the approach involves several enzymatic transformations on an intact antibody, which can be tedious and it may be difficult to push each step to completion to obtain a homogeneous product.
GlycoT is developing a novel chemoenzymatic method for antibody-drug conjugation through site-specific attachment of drugs to the Fc glycans. This platform technology would be generally applicable to any existing monoclonal antibodies or recombinant antibodies produced in various host systems regardless the heterogeneous glycoforms. In addition, the drug/antibody ratio (DAR) can be precisely controlled to give a homogeneous product by this technology.