SBIR-STTR Award

The goal of this proposal is to establish a robust and scalable process for production of specially engineered glycoforms of intravenous immunoglobulin (IVIG) with markedly enhanced anti-inflammatory activity. We propose to achieve this goal by exploiting a chemoenzymatic glycosylation remodeling technology. IVIG is a mixture of antibodies prepared from the pooled sera of thousands of healthy donors. It has been widely used for the treatment of autoimmune disease and chronic inflammation such as rheumatoid arthritis (RA), with multi-billion-dollar annual sales globally. Although IVIG treatment is efficacious in a number of indications, current IVIG preparations have distinct limitations, including variable efficacy, the need of unusually high doses, shortage of supplies, 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 have suggested that the sialylated Fc glycoform of IVIG could be the major active form that confers the anti-inflammatory activities. It has been demonstrated that the enrichment of the Fc sialylation glycoforms could achieve full activity of IVIG in several mouse models of autoimmune disease with less than 1/10 of the doses of commercial IVIG. In addition, other studies demonstrate that the glycosylation sialylation of Fab glycan may also play a role in anti-inflammatory activities. Therefore, modification of IVIG with full sialylation at both Fc and Fab domain points to a very promising approach to improving the therapeutic efficacy with more homogeneous components. Attempts to perform sequential galactosylation and sialylation of IVIG to obtain fully sialylated IVIG resulted in only partial success, due to the tremendous heterogeneity of the original glycoforms of IVIG, the relatively low efficiency of enzymatic sialylation, and side reactions. In addition, the global sequential addition of monosaccharides could not achieve selective glycan transformation at either Fc or Fab domains. On the other hand, recombinant antibodies from mammalian cell lines has been hitherto difficult to produce fully sialylated Fc glycoforms. To meet with this challenge, Prof. Lai-Xi Wang’s laboratory has developed a chemoenzymatic platform technology that permits specific glycoengineering of heterogeneous antibodies to provide structurally well-defined, homogeneous glycoforms. In particular, making use of the substrate specificity of a handful glycosynthases, the technology was capable of distinguishably engineering the Fab and Fc glycans, as demonstrated by the glycosylation remodeling of cetuximab. This technology development has resulted in 8 US patent applications: three have been issued and five are pending. To further develop and apply this platform technology, GlycoT Therapeutics (a startup) was recently founded focusing on exploring the chemoenzymatic technology for improving protein therapeutics. In this SBIR grant application, we propose to apply the chemoenzymatic technology for glycan remodeling of IVIG aiming to improve its anti-inflammatory efficacy. We will achieve the goal by performing three specific aims. Aim 1: Establish a scalable chemoenzymatic process for production of fully sialylated intravenous immunoglobulin (IVIG) on a relatively large scale. Aim 2: Perform site-specific glycoengineering of IVIG at the Fc and Fab domains for evaluating the contributions of Fab and Fc glycosylations to anti-inflammatory activity. Aim 3: Evaluate anti-inflammatory activity of various glycoforms of IVIG in mouse inflammation models This Phase I studies will pave a way to produce sufficient amount of well-defined Fc and Fab glycoforms of IVIG for expected Phase II evaluation for extensive preclinical studiesls.

Public Health Relevance Statement:
Project Narrative The goal of this proposal is to establish a robust and scalable process for production of specially engineered glycoforms of intravenous immunoglobulin (IVIG) with markedly enhanced anti-inflammatory activity.

Project Terms:
Adverse effects; Animal Model; Animals; Anti-inflammatory; Anti-Inflammatory Agents; Antibodies; Applications Grants; Arthritis; Autoimmune Diseases; Cell Line; Cetuximab; Chronic; Collaborations; Dose; Engineering; Evaluation; Fab domain; Fc domain; General Hospitals; glycosylation; Goals; Heterogeneity; improved; Inflammation; Intravenous Immunoglobulins; K/BxN model; Laboratories; Legal patent; Mammalian Cell; Maryland; Massachusetts; medical schools; Methods; Minor; Modeling; Modification; Molecular; Monosaccharides; mouse model; Mus; Phase; phase 1 study; Play; Polysaccharides; pre-clinical; Preparation; Process; Production; Reaction; Recombinant Antibody; Rheumatoid Arthritis; Role; Sales; sialylation; Side; Site; Small Business Innovation Research Grant; Substrate Specificity; success; Technology; technology development; Therapeutic; therapeutic protein; Treatment Efficacy; Universities

The goal of the proposed studies is to continue and expand GlycoT’s research in a Phase I SBIR study to establish a robust and scalable process for production of hypersialylated glycoforms of intravenous immunoglobulin (IVIG) with markedly enhanced anti-inflammatory activity. We will achieve this goal by exploiting an innovative chemoenzymatic glycosylation remodeling technology to produce the glycoengineered IVIG and to test their efficacy in animal models. IVIG is a mixture of antibodies prepared from the pooled sera of thousands of healthy donors. High dosage of IVIG (1-2 g/kg) has been widely applied to treat autoimmune disease and chronic inflammation, such as rheumatoid arthritis (RA), with multi-billion-dollar annual sales globally. Despite its wide applications for treatment, current IVIG preparation has distinct limitations, including long time infusion (4- 6 h), variable efficacy, shortage of supplies, and side effects. As a result, alternatives for IVIG with more potent therapeutic efficacy (thus much lower doses) and minimal side effects are highly desired. While the precise molecular mechanisms for the anti-inflammatory activities of IVIG is still not well-understood, recent studies in animal models 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. It has been demonstrated that the enrichment of the Fc sialylation glycoforms could achieve full activity of IVIG in several mouse models of autoimmune disease with less than 10% of the doses of commercial IVIG. Therefore, modification of IVIG with full sialylation points to a very promising approach to improving the therapeutic efficacy. Attempt to increase Fc sialylaton of IVIG by sequential galactosylation and sialylation of IVIG has been pursued, but met with only partial success due to the tremendous heterogeneity of the original glycoforms of IVIG, the relatively low efficiency of enzymatic sialylation, and side reactions. In this application, we apply a chemoenzymatic platform technology originally developed at University of Maryland to achieve fully Fc sialylated glycoform of IVIG. In the phase I study, GlcyoT has successfully established an efficient, scalable chemoenzymatic process for producing gram-scale of hypersialylated glycoforms of IVIG. The Fc glycan of engineered IVIG is more than 95% tetra-sialylated and exhibited 10-fold higher anti-inflammatory activity than the commercial IVIG in a mouse model of rheumatoid arthritis. The proposed Phase II study will further scale up the IVIG glycan-remodeling technology, establish a rigorous quality control system, and carry out intensive efficacy, toxicity, and pharmacodynamics study, paving the way for further preclinical studies and future human clinical trials.

Public Health Relevance Statement:
Project Narrative The goal of this proposal is to enhance the anti-inflammatory activity of intravenous immunoglobulin (IVIG) through glycoengineering.

Project Terms:
acute toxicity; Animal Model; animal safety; Animals; Anti-inflammatory; Antibodies; antigen binding; Autoimmune Diseases; Biological; Biological Sciences; Chronic; Clinical Trials; Cyclic GMP; dosage; Dose; Drug Kinetics; Endoglycosidases; Endotoxins; Engineering; Ensure; Excision; Exhibits; Future; glycosylation; Goals; Heterogeneity; Human; Immobilization; improved; Inflammation; Infusion procedures; innovation; Intravenous Immunoglobulins; Kilogram; Maryland; Minor; Modeling; Modification; Molecular; mouse model; novel; Oryctolagus cuniculus; Pharmacodynamics; Phase; phase 1 study; phase 2 study; Polysaccharides; preclinical study; Preparation; Procedures; Process; Production; Proteins; Protocols documentation; Quality Control; Rattus; Reaction; Research; Rheumatoid Arthritis; Sales; scale up; sialylation; Side; side effect; Small Business Innovation Research Grant; Specificity; success; System; Technology; Testing; Time; Toxic effect; Treatment Efficacy; Universities; Work