# US 2014/0155277 A1 · Adaptive Immunity Profiling and Methods for Generation of Monoclonal Antibodies

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- Number: US 2014/0155277 A1
- Document ID: US20140155277A1
- Status: Published application
- Kind: A1
- Application number: 14/174,767
- Filed: 2014-02-06
- Published: 2014-06-05
- Earliest claimed: 2009-01-15
- Assignees: Imdaptive Inc.
- Applicants: Imdaptive Inc.
- Inventors: Steven R. Wiley
- Web page: https://patentgrep.com/patent/US20140155277A1

## CPC

- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C07K 16/205: {Plasmodium}
- C12N 15/1034: {Isolating an individual clone by screening libraries}
- C40B 50/06: Biochemical methods, e.g. using enzymes or whole viable microorganisms
- C12N 15/1072: {Differential gene expression library synthesis, e.g. subtracted libraries, differential screening}
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)

## Abstract

Methods are provided for producing monoclonal antibody candidates using adaptive immunity profiling. In some aspects, the method provides for the use of massively parallel signature sequencing.

## Claims

### Claim 1

A method of generating monoclonal antibodies, comprising the steps of:
a) immunizing a host subject with an antigen;
b) creating a cDNA sequence library, the creating comprising the steps of:
i) isolating lymphocytes from the host subject;
ii) isolating mRNA from the lymphocytes;
iii) reverse transcribing the mRNA to cDNA;
iv) amplifying target antibody cDNA sequences; and
v) sequencing the target antibody cDNA sequences; and
c) analyzing the frequency of target antibody cDNA sequences, wherein target antibody cDNA sequences having a relatively high frequency are identified as candidate antibody sequences.

### Claim 2 (depends on 1)

The method of claim 1, wherein the lymphocytes comprise B cells and T cells.

### Claim 3 (depends on 1)

The method of claim 1, wherein the lymphocytes comprise isolated B cells.

### Claim 4 (depends on 1, 2, 3)

The method of claim 1-3, wherein analyzing the frequency of target antibody cDNA sequences comprises comparing the relative frequencies of target antibody cDNA sequences before and after exposure to the antigen, wherein target antibody cDNA sequences having a significantly higher frequency after exposure to the antigen are identified as candidate antibody sequences.

### Claim 5 (depends on 1, 2, 3, 4)

The method of any one of claims 1-4, further comprising re-immunizing the host subject with the antigen, creating a cDNA sequence library after the re-immunization, and comparing the relative frequencies of the target antibody sequences before and after the re-immunization, wherein antibody sequences present at a significantly higher frequency after the re-immunization are identified as mature candidate antibody sequences.

### Claim 6 (depends on 1, 2, 3)

The method of any one of claims 1-3, wherein analyzing the frequency of target antibody cDNA sequences comprises comparing the relative frequencies of target antibody cDNA sequences at two or more times after exposure to the antigen, wherein target antibody cDNA sequences having a significantly lower frequency in later times are identified as candidate antibody sequences.

### Claim 7 (depends on 1, 2, 3, 4, 5, 6)

The method of any one of claims 1-6, wherein the target antibody sequences comprise immunoglobulin (Ig) heavy chain variable domain sequences.

### Claim 8 (depends on 1, 2, 3, 4, 5, 6, 7)

The method of any one of claims 1-7, wherein the target antibody sequences comprise sequences of one or more complementarity determining regions (CDRs) of an immunoglobulin (Ig) heavy chain variable domain.

### Claim 9 (depends on 1, 2, 3, 4, 5, 6, 7, 8)

The method of any one of claims 1-8, further comprising subcloning the candidate antibody sequences with a light chain variable domain sequence and an Ig framework to produce a set of candidate monoclonal antibodies.

### Claim 10 (depends on 9)

The method of claim 9, wherein the Ig framework is a human Ig framework.

### Claim 11 (depends on 9, 10)

The method of claim 9 or 10, further comprising assaying the candidate monoclonal antibodies for affinity to the antigen.

### Claim 12 (depends on 9, 10, 11)

The method of any one of claims 9-11, further comprising subjecting the candidate monoclonal antibodies to positive selection to identify high affinity monoclonal antibodies.

### Claim 13 (depends on 12)

The method of claim 12, wherein subjecting the candidate monoclonal antibodies to positive selection comprises contacting the candidate monoclonal antibodies to the antigen bound to a substrate and isolating bound antibodies from unbound antibodies.

### Claim 14 (depends on 12, 13)

The method of claim 12 or 13, wherein the candidate monoclonal antibodies bind to an antigen with an affinity of at least 1×1010 liter/mole, measured as an association constant (Kaff).

### Claim 15 (depends on 12, 13, 14)

The method of any one of claims 12-14, further comprising sequencing the high affinity monoclonal antibodies.

### Claim 16 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

The method of any one of claims 1-15, wherein the relative frequencies of target antibody sequences are analyzed using a bioinformatics software algorithm.

### Claim 17 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)

The method of any one of claims 1-16, wherein the amplified target cDNA sequences are sequenced by massively parallel signature sequencing (MPSS).

### Claim 18 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)

The method of any one of claims 1-17, wherein the host subject is an experimental animal.

### Claim 19 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)

The method of any one of claims 1-18, wherein the host subject is a transgenic animal expressing human antibodies.

### Claim 20 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)

The method of any one of claims 1-19, wherein the host subject is a human patient that has been exposed to an antigen or a pathogen or is suffering from a disease which alters the adaptive immune response.

### Claim 21 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)

The method of any one of claims 1-20, wherein the host subject is immunized with multiple antigens.

### Claim 22 (depends on 21)

The method of claim 21, wherein the monoclonal antibody is reactive to multiple antigens.

### Claim 23 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)

The method of any one of claims 1-22, further comprising administering a background antigen and selecting against antibodies that are reactive to the background antigen.

### Claim 24 (depends on 23)

The method of claim 23, wherein the background antigen is administered after a first antigen.