Recombinant fusion proteins and libraries from immune cell repertoires

• US 9,926,555 B2
• Filed: 05/18/2017
• Issued: 03/27/2018
• Est. Priority Date: 06/09/2015
• Status: Active Grant

• Current Assignee: GigaMune, Inc.
• Original Assignee: GigaMune, Inc.
• Inventors: Johnson, David Scott, Adler, Adam, Mizrahi, Rena
• Primary Examiner(s): Boesen, Christian C
• Application Number:
  US15/598,314
• Publication Number:
  US 20170247684A1
• Time in Patent Office: 313 Days
• Field of Search: None
• CPC Class Codes: C07K 14/7051 T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 2317/14 Specific host cells or culture conditions, e.g. components, pH or temperature
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12N 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms

Abstract

Disclosed herein are methods and compositions for generating a repertoire of recombinant fusion polypeptides from immune cells, and uses thereof.

Claims

1. A method for preparing a plurality of recombinant T cell receptor expression constructs, comprising:

providing a plurality of recombinant fusion polynucleotides each comprising a first polynucleotide encoding a first variable domain, a second polynucleotide encoding a second variable domain, and a linker polynucleotide linking the first and second polynucleotides;

circularizing one or more of said plurality of recombinant fusion polynucleotides;

inserting a third polynucleotide comprising a sequence encoding a promoter, a sequence encoding an internal ribosome entry site, or a sequence encoding a constant region between said first and second polynucleotide in each of said circularized recombinant fusion polynucleotides, thereby generating said plurality of recombinant T cell receptor expression constructs;

expressing said plurality of recombinant T cell receptor expression constructs to display a plurality of recombinant fusion polypeptides on a plurality of cell surfaces; and

enriching a subpopulation of said plurality of recombinant fusion polynucleotides encoding recombinant fusion polypeptides that can bind to an antigen by exposing said plurality of cell surfaces to said antigen and selecting based on binding between said antigen and one or more of said plurality of recombinant fusion polypeptides.

2. The method of claim 1, wherein said first and second variable domains are from a single cell from a mammalian donor.

3. The method of claim 1, wherein said first variable domain is from a T cell receptor beta chain, and said second variable domain is from a T cell receptor alpha chain.

4. The method of claim 1, wherein said plurality of recombinant T cell receptor expression constructs comprise at least 1,000, 10,000, or 100,000 unique cognate pairs of beta chain and alpha chain encoding sequences.