Non-terminal antibody discovery methods and single cell assays

Patent metadata

Field	Value
Publication number	US-2024103009-A1
Application number	US-202218275855-A
Country	US
Kind code	A1
Filing date	Feb 4, 2022
Priority date	Feb 5, 2021
Publication date	Mar 28, 2024
Grant date	—

Abstract

Provided herein are methods of monitoring for the production of select antibodies in a non-human animal, comprising (a) immunizing a non-human animal with an immunogen; (b) obtaining a blood sample comprising antibody secreting cells (ASCs) from said non-human animal; and (c) individually assaying ASCs present in the blood sample, or a fraction thereof, for the production of select antibodies. Methods of guiding antibody production in a non-human animal for the production of select antibodies are also provided. In exemplary embodiments, the method comprises performing a cycle of (a) to (c), as above, and repeating the cycle when the percentage of ASCs producing select antibodies is below a threshold. In various aspects, the cycle is repeated until the percentage of ASCs producing select antibodies is at or above a threshold. Single cell assays are further provided herein.

First claim

What is claimed is:

1 . A method of monitoring for the production of select antibodies in a non-human animal, said method comprising
a. immunizing a non-human animal with an immunogen; b. obtaining a blood sample comprising antibody secreting cells (ASCs) from said non-human animal; and c. individually assaying ASCs present in the blood sample, or a fraction thereof, for the production of select antibodies.

2 . A method of guiding antibody production in a non-human animal for the production of select antibodies, said method comprising:
a. performing an initial immunization on a non-human animal with an immunogen; b. obtaining a blood sample comprising antibody secreting cells (ASCs) from said non-human animal; c. individually assaying ASCs present in the blood sample, or a fraction thereof, for the production of select antibodies; and d. performing a cycle of steps when the percentage of ASCs producing select antibodies is below a threshold, wherein the cycle comprises:
i. performing a subsequent immunization on the non-human animal with an immunogen when the percentage of ASCs producing select antibodies is below a threshold,
ii. obtaining a blood sample comprising ASCs from said non-human animal,
iii. individually assaying ASCs present in the blood sample, or a fraction thereof, for the production of select antibodies.

3 . The method of claim 1 or 2 , wherein the assaying comprises a single-cell, live-cell assay.

4 . The method of claim 3 , wherein multiple ASCs are simultaneously assayed.

5 . The method of any one of the preceding claims, comprising applying the blood sample, or a fraction thereof, to a matrix and assigning a unique address of the matrix to each ASC.

6 . The method of claim 5 , wherein a result of the assaying is the identification of each ASC producing select antibodies.

7 . The method of claim 6 , wherein the result comprises the identification of the unique address of each ASC producing select antibodies.

8 . The method of any one of claims 2 - 7 , wherein the cycle is carried out at least one time.

9 . The method of claim 8 , wherein the cycle is repeated until the number of ASCs producing select antibodies, as assayed in (iii), is at or above the threshold.

10 . The method of claim 9 , wherein the cycle is repeated at least two times.

11 . The method of any one of claims 2 - 10 , wherein the immunogen of the subsequent immunization is different from the immunogen of the initial immunization.

12 . The method of any one of claims 2 - 11 , wherein each subsequent immunization differs from a prior immunization in that (A) a different immunogen, adjuvant, and/or immunomodulatory agent is administered to the non-human animal, (B) a different dose of the immunogen is administered to the non-human animal, (C) the time between each administration of the immunogen, adjuvant, immunomodulatory agent is different, and/or (D) the route of administration for each administration of immunogen, adjuvant, immunomodulatory agent is different.

13 . The method of any one of claims 2 - 12 , wherein a different immunogen is used each time the non-human animal is immunized.

14 . A method of producing select antibodies in a non-human animal, comprising
a. performing an initial immunization on a non-human animal with an immunogen; b. obtaining a blood sample comprising antibody secreting cells (ASCs) from said non-human animal; c. individually assaying ASCs present in the blood sample, or a fraction thereof, for the production of select antibodies; d. performing a cycle of steps when the percentage of ASCs producing select antibodies is below a threshold, wherein the cycle comprises:
i. performing a subsequent immunization on the non-human animal with an immunogen when the percentage of ASCs producing select antibodies is below a threshold,
ii. obtaining a blood sample comprising ASCs from said non-human animal,
iii. individually assaying ASCs present in the blood sample, or a fraction thereof, for the production of select antibodies, and
e. isolating the select antibodies and/or an ASC producing the select antibodies.

15 . The method of claim 14 , comprising determining the nucleotide sequence encoding the heavy chain variable region of the select antibodies produced by an ASC and the nucleotide sequence encoding the light chain variable region of the select antibodies produced by the ASC, introducing into a host cell a first vector comprising the nucleotide sequence encoding the heavy chain variable region of the select antibodies and a second vector comprising the nucleotide sequence encoding the light chain variable region of the select antibodies, and isolating the antibodies produced by the host cell.

16 . The method of any one of the preceding claims, wherein the assaying comprises:
a. combining the ASCs within the matrix with (i) a capture reagent which binds to the select antibodies and comprises a solid support, (ii) a detection reagent which binds to the select antibodies and comprises a first detectable label, and (iii) a labeled target to which the select antibodies bind, wherein the labeled target comprises a second detectable label distinct from the first detectable label; b. assaying for the first detectable label and for the second detectable label; and; c. identifying the positions within the matrix at which both the first detectable label and the second detectable label are detected, wherein each identified position locates an individual ASC producing select antibodies.

17 . The method of claim 16 , wherein the capture agent comprises an antibody that binds to an antibody Fc domain attached to a solid support.

18 . The method of claim 16 or 17 , wherein the detection agent comprises an antibody that binds to an antibody Fc domain attached to a first detectable label.

19 . The method of claim 18 , wherein the antibody that binds to an antibody Fc domain of the capture agent is the same antibody of the detection agent.

20 . The method of any one of claims 16 - 19 , wherein the combining takes place in a well and the capture agent forms a monolayer in the well, optionally, wherein the ASCs are first exposed to the capture reagent, detection reagent, and/or labeled target in the well or immediately prior to being added to the well.

21 . The method of claim 20 , wherein the method comprises identifying the positions within the well at which both the first detectable label and the second detectable label are detected, wherein each identified position locates an individual ASC producing select antibodies.

22 . The method of any one of claims 16 - 19 , wherein the combining takes place in a microfluidic or nanofluidic chamber, a microwell or nanowell device, a microcapillary or nanocapillary tube, or a nanopen of a nanofluidic chip.

23 . The method of claim 22 , wherein the combining takes place in a nanopen of a nanofluidic chip.

24 . The method of claim 23 , wherein the method comprises identifying the position of each pen within the nanofluidic chip at which both the first detectable label and the second detectable label are detected, wherein each identified position locates an individual ASC producing select antibodies.

25 . The method of claim 23 or 24 , wherein a single ASC of the blood sample is moved into a pen of the nanofluidic chip through optoelectro positioning (OEP).

26 . The method of any one of t

Assignees

• Amgen Inc

Inventors

• Mcfadden Karyn E
• Chan Brian M
• Murawsky Christopher
• Winters Aaron George
• Santos Daniel

Classifications

• C07K16/28

  against receptors, cell surface antigens or cell surface determinants · CPC title

• C12N5/0087

  Purging against subsets of blood cells, e.g. purging alloreactive T cells · CPC title

• C12N5/0694

  Cells of blood, e.g. leukemia cells, myeloma cells · CPC title

• G01N33/541

  Double or second antibody {, i.e. precipitating antibody} · CPC title

• G01N33/56966

  Animal cells · CPC title