Recombinant fusion proteins and libraries from immune cell repertoires

• US 9,926,554 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:
  US15/598,313
• Publication Number:
  US 20170247683A1
• 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 of generating a T cell receptor library, comprising:

providing a plurality of circularized polynucleotide constructs, each comprising a first polynucleotide, a second polynucleotide, and a linker polynucleotide linking the first and second polynucleotides, wherein said first polynucleotide comprises a region encoding a first variable domain from a single isolated cell, wherein said second polynucleotide comprises a region encoding a second variable domain from said single isolated cell, and wherein each of said plurality of circularized polynucleotide constructs comprises a cognate pair of linked first and second variable domains from a single isolated cell;

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

inserting said plurality of recombinant T cell receptor expression constructs into a plurality of host cells; and

expressing said plurality of recombinant T cell receptor expression construct in said plurality of host cells, thereby generating a recombinant T cell receptor library comprising a plurality of recombinant T cell receptors, wherein each of said plurality of recombinant T cell receptors comprises a linked beta chain variable domain and alpha chain variable domain cognate pair from a single isolated cell.

2. The method of claim 1, wherein said insertion of said third polynucleotide is performed in parallel for said plurality of circularized polynucleotide constructs.

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 are expression vectors.

5. The method of claim 1, wherein said insertion of said third polynucleotide comprises a method selected from the group consisting of:

Gibson assembly, site-specific digestion and ligation, and targeted recombination.