title: Automated pipeline for rapid production and screening of HIV-specific monoclonal antibodies using pichia pastoris.
abstract: Monoclonal antibodies (mAbs) that bind and neutralize human pathogens have great therapeutic potential. Advances in automated screening and liquid handling have resulted in the ability to discover antigen-specific antibodies either directly from human blood or from various combinatorial libraries (phage, bacteria, or yeast). There remain, however, bottlenecks in the cloning, expression and evaluation of such lead antibodies identified in primary screens that hinder high-throughput screening. As such, "hit-to-lead identification" remains both expensive and time-consuming. By combining the advantages of overlap extension PCR (OE-PCR) and a genetically stable yet easily manipulatable microbial expression host Pichia pastoris, we have developed an automated pipeline for the rapid production and screening of full-length antigen-specific mAbs. Here, we demonstrate the speed, feasibility and cost-effectiveness of our approach by generating several broadly neutralizing antibodies against human immunodeficiency virus (HIV).
authors.affiliation: Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts.
authors.forename: Kartik A
authors.initials: KA
authors.lastname: Shah
authors.affiliation: Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts.
authors.forename: John J
authors.initials: JJ
authors.lastname: Clark
authors.affiliation: Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts.
authors.forename: Brittany A
authors.initials: BA
authors.lastname: Goods
authors.affiliation: Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Politano
authors.affiliation: Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Mozdzierz
authors.affiliation: Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts.
authors.forename: Ross M
authors.initials: RM
authors.lastname: Zimnisky
authors.affiliation: Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts.
authors.forename: Rachel L
authors.initials: RL
authors.lastname: Leeson
authors.affiliation: Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts. clove@mit.edu.|MIT Center for Biomedical Innovation, Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts. clove@mit.edu.
authors.forename: J Christopher
authors.initials: JC
authors.lastname: Love
authors.affiliation: Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts. kerryluv@mit.edu.|MIT Center for Biomedical Innovation, Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts. kerryluv@mit.edu.
authors.forename: Kerry R
authors.initials: KR
authors.lastname: Love
mesh_terms: D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D057134:Antibodies, Neutralizing / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods; D006678:HIV / Q000276:immunology*; D015483:HIV Antibodies / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D013997:Time Factors
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D015483:HIV Antibodies; D011994:Recombinant Proteins
keywords: HIV; OE-PCR; automation; monoclonal antibody