Highly parallel assays for simultaneous identification of antibody sequences and binding partners

Patent metadata

Field	Value
Publication number	US-2020149031-A1
Application number	US-201616063394-A
Country	US
Kind code	A1
Filing date	Dec 21, 2016
Priority date	Dec 21, 2015
Publication date	May 14, 2020
Grant date	—

Abstract

The present invention relates to methods for isolating the sequences of an antibody that reacts with a disease related antigen, e.g., an autoantigen, without knowing the identity of the antigen (sequence or structural epitope) a priori. The methods can also be used to identify an antigen that mediates a disease state, e.g., an autoantigen implicated in an autoimmune disorder or a tumor response.

First claim

1 . A method for determining an antibody sequence, or fragment thereof that interacts with one or more display particles, the method comprising
optionally, subjecting a sample comprising one or more antibody secreting cells (ASCs) or ASC precursor cells to conditions suitable for expansion of the one or more ASCs or ASC precursor cells, to form an expanded sample comprising ASCs; distributing the sample into a plurality of containers to provide a plurality of subsamples; optionally, subjecting one or more subsamples to conditions suitable for expansion of the one or more ASCs or ASC precursor cells in the one or more subsamples to form one or more expanded subsamples comprising ASCs; capturing antibodies, present in one or more of the subsamples, on a solid substrate in one or more of the containers to provide captured antibodies; exposing the captured antibodies in one or more of the containers to an antigen display library comprising a plurality of display particles for a time sufficient to allow for interaction of the captured antibodies with the one of more of the display particles; capturing one or more display particles that interact with one or more of the antibodies; optionally washing the solid substrate to remove unbound display particles; and sequencing an antibody nucleic acid or antigen binding fragment thereof that interacts with one or more of the display particles.

2 . The method of claim 1 , further comprising,
performing a functional assay on one or more of the subsamples or expanded subsamples to identify one or more functional subsamples, wherein the functional assay measures a property of an antibody secreted by an ASC in the one or more subsamples or expanded subsamples; identifying one or more functional subsamples based on the results of the functional assay; and wherein the capturing antibodies comprises capturing antibodies from one or more functional subsamples on a solid substrate in one or more containers to provide captured antibodies.

3 . The method of claim 1 , further comprising determining the containers that contain the captured display particles prior to the sequencing step.

4 . The method of claim 3 , wherein determining the containers that contain the captured display particles comprises
imaging containers to detect a signal from a detection reagent to identify positive containers that include captured display particles.

5 - 8 . (canceled)

9 . The method of claim 2 , wherein the functional assay is an antibody affinity or specificity assay.

10 . (canceled)

11 . The method of claim 2 , wherein the functional assay is an antibody ELISPOT assay, a cytokine neutralization assay, a virus neutralization assay or an enzyme neutralization assay.

12 . The method of claim 2 , wherein the antibodies from the one or more functional subsamples are captured in the same container as the container used for the functional assay.

13 . The method of claim 2 , wherein the antibodies from the one or more functional subsamples are captured in a different container from the container used for the functional assay.

14 . The method of claim 1 , comprising subjecting the sample comprising one or more antibody secreting cells (ASCs) or ASC precursor cells to conditions suitable for expansion of the one or more of the ASCs or ASC precursor cells, to form an expanded sample comprising ASCs.

15 . The method of claim 1 , wherein one or more subsamples are subjected to conditions suitable for expansion of one or more antibody secreting cells (ASCs) or ASC precursor cells in the one or more subsamples to form one or more expanded subsamples comprising ASCs.

16 . The method of claim 1 , wherein the conditions suitable for expansion comprise conditions suitable for polyclonal activation or conditions suitable for antigen-specific activation.

17 . (canceled)

18 . The method of claim 1 , wherein the conditions suitable for expansion comprise the use of an EL-4-B5 cell line as a feeder layer.

19 . The method of claim 1 , wherein the conditions suitable for expansion comprise the use of CD40 ligand.

20 . The method of claim 1 , wherein the conditions suitable for expansion comprise treatment with Epstein Barr virus, CD40L, one or more toll like receptor agonists, phorbol 12-myristate 13-acetate (PMA) in combination with ionomycin or phytohaemagglutinin (PHA) activation, irradiated allogeneic peripheral blood mononuclear cells (PBMC) in combination with soluble anti-CD3 mAB, or a combination thereof.

21 - 39 . (canceled)

40 . The method of claim 1 , wherein the antigen display library is a phage display library, yeast display library, ribosome display library, mRNA display library, cDNA display library, or a combination thereof.

41 - 44 . (canceled)

45 . A method for determining an antibody nucleic acid of an antibody that interacts with one or more targets of interest, the method comprising,
enriching an antibody display library for binding to one or more targets to provide an enriched antibody display library; transferring the enriched antibody display library into a mammalian, yeast or bacterial cell line engineered to express the antibodies present in the enriched antibody display library to produce a cell line comprising antibody secreting cells (ASCs); distributing the ASCs into a plurality of containers to provide ASC subsamples; interacting one or more antibodies in one or more ASC subsamples with particles that express the one or more targets; and determining the nucleic acid sequence(s) of an antibody that interacts with the one or more targets of interest.

46 . (canceled)

47 . The method of claim 45 , wherein the one or more targets comprises an integral membrane protein, a G-protein coupled receptor (GCPR) or an ion channel.

48 - 49 . (canceled)

50 . The method of claim 45 , wherein enriching comprises panning the antibody display library against a cell line that does not express the one or more targets of interest to deplete the library of irrelevant library members.

51 . The method of claim 45 , wherein the enriched antibody display library is transferred to a mammalian cell line, and the mammalian cell line expresses the antibodies as Fab fragments.

52 . The method of claim 45 , wherein the enriched antibody display library is transferred to a yeast cell line, and the yeast cell line expresses the antibodies as Fab fragments.

53 - 58 . (canceled)

Assignees

• Univ British Columbia

Inventors

• Falconer Ester
• Hansen Carl Lars Genghis
• Lecault Veronique
• Lisaingo Kathleen
• Heyries Kevin Albert

Classifications

• C12N15/1034

  Isolating an individual clone by screening libraries · CPC title

• C40B50/06

  Biochemical methods, e.g. using enzymes or whole viable microorganisms · CPC title

• C40B40/02 Primary

  Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors · CPC title

• C12N15/1037 Primary

  Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display · CPC title

• G01N33/6845

  Methods of identifying protein-protein interactions in protein mixtures · CPC title