Recombinant fusion proteins and libraries from immune cell repertoires

• US 10,689,641 B2
• Filed: 04/30/2019
• Issued: 06/23/2020
• Est. Priority Date: 06/09/2015
• Status: Active Grant

• Current Assignee: GigaGen Inc.
• Original Assignee: GigaGen Inc.
• Inventors: Johnson, David Scott, Adler, Adam, Mizrahi, Rena
• Primary Examiner(s): Boesen, Christian C
• Application Number:
  US16/399,759
• Publication Number:
  US 20190256841A1
• Time in Patent Office: 420 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 kit for producing a library of recombinant immunoglobulin expression constructs, the kit comprising:

a. a plurality of at least 10,000 unique, recombinant fusion polynucleotides, each comprising a first polynucleotide and a second polynucleotide and a linker polynucleotide,

the first polynucleotide encoding a heavy chain variable domain from a cognate pair from a single isolated mammalian B cell and an IgG heavy chain constant domain fragment; and

the second polynucleotide encoding a light chain variable domain from the cognate pair from the single isolated mammalian B cell and an IgK light chain constant domain; and

the linker polynucleotide linking the first and second polynucleotides and comprising a restriction site; and

b. a third recombinant polynucleotide, not operationally linked to the recombinant fusion polynucleotides, comprising a first AOX1 promoter sequence and a sequence encoding a portion of the IgG heavy chain constant domain, wherein the third recombinant polynucleotide comprises a first end sequence and a second end sequence, each overlapping with a first or second end sequence of each recombinant fusion polynucleotide; and

c. a fourth recombinant polynucleotide, not operationally linked to the recombinant fusion polynucleotides or the third recombinant polynucleotide, comprising a second AOX1 promoter sequence.

2. The kit of claim 1, wherein each single isolated mammalian cell is isolated from a human donor immunized with an antigen.

3. The kit of claim 1, wherein the plurality of recombinant fusion polynucleotide comprises at least 100,000 unique linear polynucleotides.