SBIR-STTR Award

While many HIV vaccine regimens have elicited robust viral-specific T cell responses, they have been unable to prevent infection. To date, passive immunotherapy with a small number of broadly neutralizing monoclonal antibodies (MAbs) given in combination have afforded the best protection against SHIV challenge in monkeys. However, in a global context, the large multiple doses required will create excessive demands on existing expensive microbial fermentation or cell-based production systems. Plant expression will provide an inexpensive, safe, and rapidly scalable alternative. Genetically engineered plants have been successfully used to produce many complex proteins, as diverse as IgG, sIgA, vaccine antigens, enzymes and growth factors. The aim of this Phase I study is to produce highly expressing MAbs in plants using Agrobacterium infiltration/ injection and assess their ability to broadly neutralize HIV-1 isolates in vitro. Several well characterized envelope-specific MAbs will be produced in tobacco; 2G12, 2F5, 4E10, IgG1b12 as well as a MAb against a CD4-inducible gp120 epitope. In order to examine, the effect of N-glycosylation on neutralization by these MAbs, both the ER- retained "high mannose" and the secreted "complex glycan" forms will be produced and compared to CHO-derived proteins. Concomitant with these studies, the slower process of generating stable transgenic tobacco plants will be initiated. This will be aided by coexpression of the DsRed fluorescent marker to identify antibody expressing lines and trace transgenic offspring. In the current environment, the use of a non-food, non-feed crop to produce important therapeutic proteins offers many advantages. In Phase II, those MAbs that are both expressed at high levels and have strong neutralizing activity will be produced by either transgenic plants or by large scale transient expression of tobacco plants and tested for their ability to prevent infection of monkeys challenged with an SHIV isolate.

Public Health Relevance:
Unlike HIV vaccines, the passive transfer of large amounts of monoclonal antibodies that broadly neutralize HIV virus has shown dramatic protection against a lethal challenge with a SHIV isolate given one day later. However, the cost of producing these recombinant proteins in traditonal expression systems would be exorbitant and a more inexpensive system is a high priority. In this proposal, these MAbs will be produced in tobacco plants and their ability to neutralize HIV in vitro and to protect monkeys in vivo will be compared to currently used and expensive CHO-derived MAbs.

Public Health Relevance:


Project narrative:
Unlike HIV vaccines, the passive transfer of large amounts of monoclonal antibodies that broadly neutralize HIV virus has shown dramatic protection against a lethal challenge with a SHIV isolate given one day later. However, the cost of producing these recombinant proteins in traditonal expression systems would be exorbitant and a more inexpensive system is a high priority. In this proposal, these MAbs will be produced in tobacco plants and their ability to neutralize HIV in vitro and to protect monkeys in vivo will be compared to currently used and expensive CHO-derived MAbs.

Thesaurus Terms:
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In the absence of specific intervention and with extended breast-feeding mother-to- child-transmission of HIV reaches a rate of ~35 % and infection of ~700,000 babies worldwide. Passive immunotherapy with a small number of broadly neutralizing monoclonal antibodies (MAbs) in SHIV challenged macaques bodes well for the ability of infusion of neutralizing MAb to prevent such transmission in humans. However the amount of MAb required would overwhelm current fermenter-based production systems and an alternate system for MAb production is a high priority. Due to recent innovations, transient plant (Nicotiana bentamiama) expression platforms now represent some of the most rapid, cost effective and productive in existence;capable of producing grams of recombinant MAbs in weeks. In the Phase I feasibility study, high mannose and complex glycoforms of 2G12, 2F5, 4E10 b12, m43 and m9 MAbs were transiently produced at 80-100 mg/kg leaves (except for the scFv m9) and each exhibited similar, and, in the case of b12, better neutralizing titers than their CHO-derived counterparts although differences were observed in antibody dependent cellular cytoxicity activity. In Phase II, high mannose, complex and 11,3-fucose/21,2-xylose-negative/low glycoforms of three new broad and potent MAbs (PG9, PG16, VRC01), in addition to b12 and 2G12 from Phase I, will be produced in a p19 enhanced plant expression system (200- 500mg/kg) and assessed for in vitro for both their neutralizing and F3-mediated antibody effector activity (ADCC and ADCVI). Six hundred mg of those forms with the best neutralizing activity will be produced for pharmacokinetics, immunogenicity and protection studies in macaques. Single MAbs and a cocktail of MAbs will be injected i.v. or s.c. weekly and vaginally challenged with a low dose of SHIV bi-weekly to more closely mimic doses encountered in human heterosexual exposure. These results will provide the preclinical data for transition into human trials.

Public Health Relevance:
With an ever increasing number of mother-to-baby-transmissions of the HIV virus in the developing world, the production of and immunotherapy with monoclonal antibodies that strongly neutralizing activity a high priority. However the facilities to manufacture such large amounts has not been available. In Phase I we showed that MAbs can be rapidly produced in a new transient plant expression systems in high levels and have similar (or better) neutralizing activity as the conventional CHO-derived forms. In Phase II we shall used an enhanced transient expression system to produce large amounts of new potent MAbs and assess their ability to protect macaques against SHIV virus infection.

Thesaurus Terms:
Adcc;Adcc Assay;Aids Virus;Acquired Immune Deficiency Syndrome Virus;Acquired Immunodeficiency Syndrome Virus;Address;Antibodies;Antibody -Dependent Cell Cytotoxicity;Antibody-Dependent Cellular Cytotoxicity;Assay;Bioassay;Biologic Assays;Biological Assay;Breast Feeding;Breastfeeding;Cell Cytoxicity, Antibody-Dependent;Clinic;Complex;D-Mannose;D-Xylose;Data;Deoxygalactose;Disease Progression;Dose;Drug Kinetics;Economics;Exhibits;Feasibility Studies;Fucose;Grant;Hiv;Hiv Infections;Hiv-1;Hiv-I;Hiv1;Htlv-Iii;Htlv-Iii Infections;Htlv-Iii-Lav Infections;Heterosexuals;Human;Human Immunodeficiency Viruses;Human T-Cell Leukemia Virus Type Iii;Human T-Cell Lymphotropic Virus Type Iii;Human T-Lymphotropic Virus Type Iii;Human Immunodeficiency Virus 1;Human, General;Itx;Immunodeficiency Virus Type 1, Human;Immunologically Directed Therapy;Immunotherapy;In Vitro;Infection;Infusion;Infusion Procedures;Intervention;Intervention Strategies;Lav-Htlv-Iii;Lymphadenopathy-Associated Virus;Macaca;Macaque;Man (Taxonomy);Man, Modern;Mannopyranose;Mannopyranoside;Mannose;Mediating;Metabolic Glycosylation;Moab, Clinical Treatment;Modeling;Monoclonal Antibodies;Mothers;Niaid;National Institute Of Allergy And Infectious Disease;Nicotiana;Passive Immunotherapy;Pharmacokinetics;Phase;Plants;Plants, General;Preclinical Testing;Prevention Measures;Production;Prophylactic Treatment;Prophylaxis;Recombinants;Sbir;Sbirs (R43/44);Shiv;Safety;Small Business Innovation Research;Small Business Innovation Research Grant;System;System, Loinc Axis 4;T-Lymphotropic Virus Type Iii Infections, Human;Testing;Tobacco;Transmission;Universities;Vertical Disease Transmission;Vertical Transmission;Viral Diseases;Virus Diseases;Virus-Hiv;Xylose;Alpha-Fucose;Antibody;Antibody Dependent Cell Mediated Cytotoxicity;Base;Cost Effective;Glycosylation;Human T Cell Leukemia Virus Iii;Human T Lymphotropic Virus Iii;Human Immunodeficiency Virus;Immune Therapy;Immunogenicity;In Vivo;Innovate;Innovation;Innovative;Interventional Strategy;Large Scale Production;Model;Monoclonal Antibody;Monoclonal Antibody Production;Mother To Child Transmission;Neutralizing Antibody;Neutralizing Mab;Neutralizing Monoclonal Antibodies;Passive Prophylaxis;Pre-Clinical;Preclinical;Prevent;Preventing;Response;Simian Hiv;Simian Human Immunodeficiency Virus;Transmission Process;University;Vector;Viral Infection;Virus Infection