Recombinant fusion proteins and libraries from immune cell repertoires

• US 9,422,547 B1
• Filed: 06/09/2015
• Issued: 08/23/2016
• Est. Priority Date: 06/09/2015
• Status: Active Grant

• Current Assignee: GigaGen Inc. (Grifols SA)
• Original Assignee: GigaGen Inc. (Grifols SA)
• Inventors: Adler, Adam, Mizrahi, Rena, Johnson, David Scott
• Primary Examiner(s): BOESEN, CHRISTIAN C
• Application Number:
  US14/734,953
• Time in Patent Office: 441 Days
• Field of Search: None
• US Class Current: 1/1
• 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 immunoglobulin 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 heavy chain variable domain and a second polynucleotide encoding a light chain variable domain, wherein said heavy chain variable domain and light 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 and 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 immunoglobulin expression constructs, thereby generating a recombinant immunoglobulin library.

2. The method of claim 1, further comprising:

inserting said at least 1,000 recombinant immunoglobulin expression constructs into a plurality of host cells; and

expressing said at least 1,000 recombinant immunoglobulin expression constructs in said plurality of host cells, thereby generating a recombinant immunoglobulin library comprising at least 1,000 unique recombinant immunoglobulins, wherein each of said at least 1,000 unique recombinant immunoglobulins comprises a linked heavy chain and light chain variable domain cognate pair from a single isolated cell, thereby generating a recombinant immunoglobulin library.

3. The method of claim 2, wherein said recombinant immunoglobulin library encodes at least 10,000, 100,000, or 1,000,000 unique recombinant immunoglobulin 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 4, wherein said medical criterion comprises a heightened immune response to hepatitis B, rabies, tetanus toxin, varicella-zoster, cytomegalovirus, or pneumococcus.

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

7. The method of claim 6, wherein said antigen of interest is a tumor antigen selected from the group consisting of:

CD19, CD20, CD22, CD52, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tumor necrosis factor receptor superfamily, member 10a (TRAILR1), receptor activator of nuclear factor kappa-B ligand (RANKL), insulin-like growth factor 1 receptor (IGF1R), epithelial cell adhesion molecule (EpCAM), carcinoembryonic antigen (CEA), and mucin 5AC.

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

thrombin, nicotinic acetylcholine receptor, thyroglobulin, TSH receptor, glutamate decarboxylase, phospholipase-A2-receptor (PLA2R), and muscle specific kinase (MUSK).

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

10. The method of claim 1, wherein said at least one mammalian donor has an autoimmune disease or cancer.

11. The method of claim 1, wherein said at least one mammalian donor has idiopathic (immune) thrombocytopenic purpura (ITP), Kawasaki'"'"'s vasculitis, B cell chronic lymphocytic leukemia (CLL), or primary immunodeficiencies.

12. The method of claim 1, wherein said recombinant immunoglobulin expression construct is an expression vector.

13. The method of claim 5, wherein said medical criterion comprises a heightened immune response to pneumococcus or hepatitis B.

14. The method of claim 7, wherein said antigen of interest is a tumor antigen selected from the group consisting of:

epithelial cell adhesion molecule (EpCAM) and epidermal growth factor receptor (EGFR).

15. The method of claim 8, wherein said antigen of interest is an autoimmune antigen selected from the group consisting of:

TSH receptor and thyroglobulin.

16. The method of claim 11, wherein said at least one mammalian donor has primary immunodeficiencies or B cell chronic lymphocytic leukemia (CLL).