Recombinant fusion proteins and libraries from immune cell repertoires

• US 9,738,699 B2
• Filed: 05/19/2016
• Issued: 08/22/2017
• Est. Priority Date: 06/09/2015
• Status: Active Grant

• Current Assignee: GigaMune, Inc.
• Original Assignee: GigaGen Inc.
• Inventors: Johnson, David Scott, Adler, Adam, Mizrahi, Rena
• Primary Examiner(s): BOESEN, CHRISTIAN C
• Application Number:
  US15/159,705
• Publication Number:
  US 20160362470A1
• Time in Patent Office: 460 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 recombinant T cell receptor library, comprising:

providing primary immune cells from at least one mammalian donor;

isolating in a plurality of monodisperse droplets single immune cells from said primary immune cells;

generating a plurality of recombinant fusion polynucleotides each comprising a first polynucleotide encoding a beta chain variable domain and a second polynucleotide encoding an alpha chain variable domain, wherein said beta chain variable domain and alpha chain variable domain on each of said plurality of recombinant fusion polynucleotides are a cognate pair from one of said isolated primary immune cells, wherein each of said plurality of recombinant fusion polynucleotides further comprise a linker polynucleotide linking said first and second polynucleotides;

circularizing each of said plurality of linear recombinant fusion polynucleotides; and

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 at least 1,000 unique recombinant T cell receptor expression constructs, thereby generating said recombinant T cell receptor library.

2. The method of claim 1, further comprising:

inserting said at least 1,000 unique recombinant T cell receptor expression constructs into a plurality of host cells; and

expressing said at least 1,000 unique recombinant T cell receptor expression constructs in said plurality of host cells, thereby generating the recombinant T cell receptor library comprising at least 1,000 unique recombinant T cell receptors, wherein each of said at least 1,000 unique recombinant T cell receptors comprises a linked beta chain and alpha chain variable domain cognate pair from a single isolated cell, thereby generating the recombinant T cell receptor library.

3. The method of claim 2, wherein said recombinant T cell receptor library encodes at least 10,000, 100,000, or 1,000,000 unique recombinant T cell receptor proteins.

4. The method of claim 1, wherein said at least one mammalian donor is selected based on a medical criterion.

5. The method of claim 1, wherein said at least one mammalian donor was exposed to an antigen of interest or a pathogen.

6. The method of claim 5, wherein said antigen of interest is an antigen selected from the group consisting of:

testis specific antigen, regulatory T cell specific antigen, FOXP3, FOXO3, WT1, MART-1, MAGE-A3, gp100, and NY-ESO1.

7. The method of claim 5, wherein said antigen of interest is an antigen selected from a pathogenic virus consisting of:

CMV, EBV, BK virus, herpes virus, adenovirus, and HIV.

8. The method of claim 1, wherein said at least one mammalian donor was immunized against a pathogen.

9. The method of claim 1, wherein said recombinant T cell receptor expression constructs are expression vectors.

10. The method of claim 3, wherein said recombinant T cell receptor library encodes at least 100,000 unique recombinant T cell receptor proteins.

11. The method of claim 6, wherein said antigen of interest is MART-1 or MAGE-A3.

12. The method of claim 7, wherein said antigen interest is an antigen of CMV.