# US 9,775,332 B2 · Human artificial chromosome vector

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- Number: US 9,775,332 B2
- Document ID: US9775332B2
- Status: Granted
- Kind: B2
- Application number: 15/070,842
- Filed: 2016-03-15
- Published: 2016-08-18
- Granted: 2017-10-03
- Earliest claimed: 2009-11-17
- Assignees: Sab, LLC
- Applicants: Sab, LLC
- Inventors: Yoshimi Kuroiwa; Hiroaki Matsushita; Akiko Kanai
- Web page: https://patentgrep.com/patent/US9775332B2

## CPC

- A01K 67/0278: Knock-in vertebrates, e.g. humanised vertebrates
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 16/1278: Bacillus (G)
- C12N 15/85: for animal cells
- C12N 15/8509: {for producing genetically modified animals, e.g. transgenic}
- A01K 2207/15: Humanized animals
- A01K 2217/052: inducing gain of function
- A01K 2217/072: maintaining or altering function, i.e. knock in
- A01K 2227/101: Bovine
- A01K 2267/01: Animal expressing industrially exogenous proteins
- C12N 2800/208: of mammalian origin, e.g. minichromosome
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- 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)

## Abstract

A human artificial chromosome vector comprising a gene encoding a human antibody heavy chain, a gene encoding a human antibody light chain, and a gene encoding an IgM heavy chain constant region derived from a nonhuman animal.

## Claims

### Claim 1

A human artificial chromosome (HAC) vector, wherein the HAC comprises:
a) a gene encoding a human antibody heavy chain, wherein the gene encoding the human antibody heavy chain encodes at least a human IgG (γ) heavy chain, a human IgA (α) heavy chain or both human IgG and IgA heavy chains,
b) a gene encoding a human antibody light chain, wherein the gene encoding the human antibody light chain encodes either kappa (κ) human antibody light chain or lambda (λ) human antibody light chain, or both κ and λ human antibody light chains, and
c) a gene encoding a chimeric IgM (μ) heavy chain constant region, wherein the gene encoding the chimeric IgM heavy chain constant region comprises a human IgM heavy chain constant region gene substituted with DNA encoding a bovine or a goat IgM heavy chain constant region gene; wherein the gene encoding the chimeric IgM heavy chain constant region encodes constant region (CH) domains CH1, CH2, CH3 and CH4, and transmembrane domains (TM) TM1 and TM2; and wherein the encoded human CH4, TM1 and TM2 domains are completely substituted with the encoded CH4, TM1 and TM2 domains of the bovine or the goat IgM heavy chain constant region gene.

### Claim 2 (depends on 1)

The human artificial chromosome vector according to claim 1, wherein the gene encoding the human antibody heavy chain encodes human IgA (α), human IgD (δ), human IgE (ε), human IgG (γ) and human IgM (μ) heavy chains.

### Claim 3 (depends on 1)

The human artificial chromosome vector according to claim 1, wherein the gene encoding the human antibody heavy chain comprises a human chromosome 14 fragment comprising the 14q32 region.

### Claim 4 (depends on 1)

The human artificial chromosome vector according to claim 1, wherein the gene encoding the bovine or the goat IgM heavy chain constant region is bovine IgM heavy chain constant region.

### Claim 5 (depends on 1)

A goat having the human artificial chromosome vector according to claim 1.

### Claim 6 (depends on 1)

A bovine having the human artificial chromosome vector according to claim 1.

### Claim 7 (depends on 1)

The HAC vector of claim 1, wherein the gene encoding the human antibody light chain comprises a human chromosome 2 fragment comprising the 2p11.2-12 region, a human chromosome 22 fragment comprising the 22p11.2 region, or both a human chromosome 2 fragment comprising the 2p11.2-12 region and a human chromosome 22 fragment comprising the 22p11.2 region.

### Claim 8 (depends on 1)

The HAC vector of claim 1, wherein the HAC vector further comprises a gene encoding a human antibody surrogate light chain selected from the group consisting of VpreB1, VpreB3, and lamda-5.

### Claim 9 (depends on 1)

The HAC vector of claim 1, wherein the encoded human CH3 domain is completely substituted with the encoded CH3 domain of the bovine or the goat IgM heavy chain constant region.

### Claim 10 (depends on 9)

The HAC vector of claim 9, wherein the encoded CH2 domain is a chimeric bovine-human CH2 domain or a chimeric goat-human CH2 domain.

### Claim 11 (depends on 9)

The HAC vector of claim 9, wherein the encoded human CH2 domain is completely substituted with the encoded CH2 domain of the bovine or the goat IgM heavy chain constant region gene.

### Claim 12 (depends on 11)

The HAC vector of claim 11, wherein the encoded CH1 domain is a chimeric bovine-human CH1 domain or a chimeric goat-human CH1 domain.

### Claim 13 (depends on 11)

The HAC vector of claim 11, wherein the encoded human CH1 domain is completely substituted with the encoded CH1 domain of the bovine or the goat IgM heavy chain constant region gene.

### Claim 14 (depends on 1)

A method for producing a human antibody, comprising:
a) administering a target antigen into a transgenic goat having the HAC vector of claim 1,
b) accumulating the human antibody specific to the antigen in serum of the transgenic goat, and
c) recovering the human antibody specific to the antigen from the serum of the transgenic goat.

### Claim 15 (depends on 14)

The method of claim 14, wherein the transgenic goat produces a serum concentration of at least 1,000 ug/mL of human IgG.

### Claim 16 (depends on 14)

The method of claim 14, wherein the endogenous goat immunoglobulin M (μ) heavy-chains of the transgenic goat are inactivated.

### Claim 17 (depends on 1)

A method for producing a human antibody, comprising:
a) administering a target antigen into a transgenic bovine having the HAC vector of claim 1,
b) accumulating the human antibody specific to the antigen in serum of the transgenic bovine, and
c) recovering the human antibody specific to the antigen from the serum of the transgenic bovine.

### Claim 18 (depends on 17)

The method of claim 17, wherein the transgenic bovine produces a serum concentration of at least 1,000 ug/mL of human IgG.

### Claim 19 (depends on 17)

The method of claim 17, wherein the endogenous bovine immunoglobulin M (μ) heavy-chains of the transgenic bovine are inactivated.