SBIR-STTR Award

A research plan is proposed to engineer goats that produce fully human immunoglobulin. These animals will be evaluated as a candidate platform system for production of both targeted therapeutic immunoglobulin as well as reagent antibodies for standards and controls of serological assays of human clinical samples. Previous studies have demonstrated that ungulates such as cattle can produce large amounts of human immunoglobulins and show extremely high titers and neutralizing antibody activities against various antigens, including infectious diseases, toxins and other targets following multiple immunizations. In this study, SAB Capra LLC (SAB) and Utah State University intend to expand and optimize this technology by producing transchromosomic (Tc) goats containing a human artificial chromosome vector (HAC) which contains the entire germline repertoire of the human antibody genes. In addition, the participants will produce goat fibroblast cell lines where the endogenous goat antibody genes will be silenced using current gene editing technologies. There are three specific aims in this project: (1) develop the first human immunoglobulin producing Tc goats by transferring the HAC containing the germ line human antibody heavy and light chain genes to KO fetal fibroblast cell lines; (2) evaluate and characterize human immunoglobulin produced in the Tc goats from Aim 1 and determine the stability and antibody production of the Tc goats and compare the immunoglobulin response to Tc cattle as well as humans; (3) produce high titer targeted immunoglobulin to pandemic flu strains (H7N9 and/or H5N1) for target specific evaluation. Data generated from this study will enable further work to be conducted to determine the usefulness of small ungulates producing human antibodies used as production animals to produce targeted human immunoglobulin therapeutic candidates and diagnostic standards and controls to emerging diseases. If this project is completed successfully, it will also have a broad implication that SAB’s platform technology can be used in both a large (cattle) and small (goat) ungulate species providing greater flexibility in producing product candidates. The addition of a small ungulate species will reduce costs associated with generating antibodies for small volume products as well as targeted human immunoglobulins used as diagnostic standards and controls for testing human clinical samples.

Public Health Relevance Statement:
Project narrative Goats that produce fully human immunoglobulin will be produced and evaluated as a candidate system for production of both therapeutic immunoglobulin as well as reagent antibodies for standards and controls of serological assays of human clinical samples. Upon successfully completing this project, these animals will be used to produce hyperimmune targeted immunoglobulin against infectious disease, toxins, oncology targets, auto-immune targets and inflammation targets. This will provide an additional platform technology that can be used to address global unmet medical needs in a rapid and easily scalable production system leading to therapeutics to a wide variety of diseases.

Project Terms:
Address; Advanced Development; Animals; anti-influenza; Antibodies; Antibody Formation; Antigens; Autoimmune Process; Autoimmunity; base; Binding; Biological Assay; Biological Products; Biological Response Modifier Therapy; Cattle; Cell Line; Cells; Chromosomes, Artificial, Human; Clinical; Collaborations; Communicable Diseases; cost; Data; design; Development; Diagnostic; Diagnostic Reagent; Disease; Engineering; Enzyme-Linked Immunosorbent Assay; Evaluation; fetal; Fibroblasts; flexibility; Gene Cluster; Genes; Genetic Engineering; Genus Capra; Germ Lines; Goals; Goat; Human; Immunization; Immunoglobulin Genes; Immunoglobulin M; Immunoglobulins; Immunotherapeutic agent; Inflammation; Influenza A Virus, H5N1 Subtype; Influenza A Virus, H7N9 Subtype; Influenza vaccination; Knock-out; knockout gene; Light; Medical; Molecular; neutralizing antibody; oncology; Outcome; pandemic influenza; Participant; Production; Reagent; reagent standard; Recombinant Immune Globulin; Reporting; Research; Resort; response; Risk; Sampling; Serologic tests; Serological; somatic cell nuclear transfer; ST14 gene; System; targeted treatment; Technology; Testing; Therapeutic; therapeutic candidate; Toxin; Ungulate; Universities; Utah; Vaccination; vector; Work

The ultimate goal of this phase II STTR proposal is to expand the capabilities of SAB's diversitAb™ platform by continuing advanced development of Transchromosomic goats (TcGs). This will be accomplished by producing male and female goat endogenous immunoglobulin gene knockout cell lines that contain SAB Capra's human artificial chromosome (HAC) which encodes the entire repertoire of the germline human antibody genes thereby greatly improving the production efficiency of fully human antibodies in TcGs. To further advance the TcG platform towards commercial production of therapeutic and diagnostic antibody products, optimization of the antibody purification process, development of quality control assays, and further development of a purified TcG- derived pandemic influenza antibody product targeting H7N9 by completing in-vitro and in-vivo evaluation is also proposed. To accomplish these goals, endogenous immunoglobulin gene knockouts will be facilitated utilizing the CRISPR/Cas9 system in domestic Nubian/Boar goat fetal fibroblast (GFF) cells, and transfer of the SAB- designed HAC into the knockout cells will be accomplished by microcell mediated chromosome transfer (MMCT). HAC-containing knockout GFF cells will be used to produce embryos by somatic cell nuclear transfer (SCNT). After gestation and birth of multiple TcGs, molecular characterization will be completed, presence of the knockouts and HAC will be confirmed, and human IgG production will be confirmed and evaluated. Two of these TcGs will then be hyperimmunized with a plasmid DNA vaccine targeting H7N9. Plasma will be collected from the hyperimmunized TcGs, and anti-H7N9 fully human IgG will be purified from the TcG plasma. The in-vitro potency and in-vivo efficacy of the purified TcG-derived anti-H7N9 human IgG product will then be evaluated. Optimization of established purification procedures will be undertaken to ensure efficient and effective purification of TcG-derived human IgG from plasma, and development of quality control assays to evaluate product identity, purity, and potency will be completed. SAB's diversitAb™ platform for human antibody production is currently utilized in transchromosomic bovines against a wide range of antigens including viruses, bacteria, oncology targets, recombinant proteins, and DNA vaccines. This proven technology has the advantages of scalability, simplicity, and broad applicability. The addition of TcGs to the platform allows for simpler and cheaper rapid-response production of small volume targeted products as well as diagnostic reagents for serological testing of emerging infectious diseases.

Public Health Relevance Statement:
Project Narrative The research proposed in this application seeks to expand the capabilities of SAB's diversitAb™ platform by continuing advanced development of Transchromosomic goats (TcGs) that express human polyclonal antibodies for use as targeted immunotherapies and/or diagnostic reagents for serological testing. Immunotherapy products derived from TcGs will combine the beneficial effects of current animal derived antibody products with improved efficacy and safety while eliminating the risk of anaphylaxis and serum sickness associated with xenobiotic IgG administration. A TcG-derived anti-H7N9 fully human IgG product will also be developed through this proposal, potentially addressing the global need for highly effective therapeutics against avian lineage influenza virus infections in humans.

NIH Spending Category:
Biotechnology; Emerging Infectious Diseases; Genetics; Immunization; Immunotherapy; Infectious Diseases; Influenza; Pneumonia & Influenza; Vaccine Related

Project Terms:
Address; Adjuvant; Advanced Development; Affinity Chromatography; Anaphylaxis; Animals; Antibodies; Antibody Formation; Antigens; assay development; Autoimmunity; Bacteria; base; Biological Assay; Biological Products; Biological Response Modifier Therapy; Birds; Cattle; Cell Line; Cells; Chromosome Transfer; Chromosomes, Artificial, Human; Clinical; Collaborations; Collection; Communicable Diseases; CRISPR/Cas technology; design; Development; Diagnostic; Diagnostic Reagent; DNA Vaccines; efficacy study; Embryo; Embryo Transfer; Emerging Communicable Diseases; Ensure; Female; fetal; Fibroblasts; Filtration; Fractionation; Gene Cluster; Gene Expression; Genes; Genetic; Genetic Engineering; Genus Capra; Goals; Goat; Hemagglutinin; Human; hyperimmunization; Immunization; Immunoglobulin G; Immunoglobulin Genes; Immunoglobulin M; Immunoglobulins; Immunotherapeutic agent; Immunotherapy; improved; In Vitro; in vivo; in vivo evaluation; Inbred BALB C Mice; Infection; Inflammation; Influenza A virus; Influenza A Virus, H7N9 Subtype; influenzavirus; Knock-out; knockout gene; Light; Live Birth; male; Mediating; Molecular; mouse model; Multiple Birth Offspring; oncology; pandemic influenza; Peripheral; Phase; Plasma; Plasma Proteins; plasmid DNA; polyclonal human antibody; preclinical evaluation; Pregnancy; Preparation; Procedures; Process; Production; protein expression; Quality Control; reagent standard; Recombinant Proteins; Recombinants; Research; response; Risk; Safety; Sampling; Serologic tests; Serum; Serum Sickness; Small Business Technology Transfer Research; somatic cell nuclear transfer; System; Technology; Therapeutic; Therapeutic antibodies; Treatment Efficacy; Universities; Utah; vector; Virus; Virus Diseases; Work; Xenobiotics