# US 7,608,429 B2 · Methods and compositions for increasing antibody production

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- Number: US 7,608,429 B2
- Document ID: US7608429B2
- Status: Granted
- Kind: B2
- Application number: 10/697,995
- Filed: 2003-10-30
- Published: 2005-03-03
- Granted: 2009-10-27
- Earliest claimed: 2002-10-31
- Assignees: Genentech, Inc.
- Inventors: Dorothea Reilly; Daniel Yansura
- Web page: https://patentgrep.com/patent/US7608429B2

## CPC

- C07K 16/2896: {against molecules with a "CD"-designation, not provided for elsewhere}
- C07K 16/22: against growth factors {; against growth regulators}
- C07K 16/4291: {against IgE}
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/53: Hinge
- C07K 2319/00: Fusion polypeptide
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- 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)

## Abstract

The invention provides methods and compositions for improved expression and production of recombinant antibodies in host cell expression systems. In particular, prokaryotic expression and production of antibodies with modified hinge cysteine residues are provided. The invention further provides compositions, kits and articles of manufacture for practicing methods of the present invention.

## Claims

### Claim 1

A method of producing increased yields of an intact heavy and light chain-comprising antibody, wherein the intact antibody comprises a heavy chain variant of a reference antibody heavy chain, where the variant heavy chain lacks at least one inter-heavy chain hinge region disulfide bond as compared to the reference heavy chain, comprising
(a) expressing in a host cell an antibody heavy chain-encoding polynucleotide, and a polynucleotide encoding antibody light chains capable of functionally pairing with the antibody heavy chains encoded by the polynucleotide,
wherein the polynucleotide encoding the variant heavy chain polypeptide is made by a method comprising:
(i) providing a nucleic acid comprising sequence encoding the reference antibody heavy chain polypeptide having at least one inter-heavy chain hinge region disulfide bond;
(ii) modifying the nucleic acid to a variant nucleic acid encoding an antibody heavy chain polypeptide which cannot form at least one of the inter-heavy chain hinge region disulfide bond or bonds formed in the reference heavy chain, wherein the variant hinge region cannot form at least one inter-heavy chain hinge region disulfide bond because it is modified to lack a cysteine residue present in a corresponding non-variant heavy chain polypeptide by either deletion of the cysteine residue or substitution of the cysteine residue with an amino acid residue not capable of forming an inter-heavy chain disulfide bond;
(b) culturing the host cell under conditions permissive for expression of the heavy and light chain antibody polypeptides and pairing of the heavy and light chain antibody polypeptides,
wherein the amount of intact antibody produced in the host cell is at least 10% greater than the amount of an antibody comprising the corresponding non-variant heavy chain polypeptide expressed under similar culture conditions; and
(c) recovering said intact antibody from the host cell.

### Claim 2 (depends on 1)

The method of claim 1, wherein the method provides a nucleic acid comprising sequence encoding an antibody heavy chain polypeptide having at least two inter-heavy chain hinge region disulfide bonds, and the at least two inter-heavy chain disulfide bonds of the antibody variant heavy chain are eliminated by deletion, or by substitution of the antibody heavy chain sequence with an amino acid residue not capable of forming an inter-heavy chain disulfide linkage.

### Claim 3 (depends on 2)

The method of claim 2, wherein all inter-heavy chain disulfide bonds of the antibody variant heavy chain are eliminated by deletion, or by substitution of the antibody heavy chain sequence with an amino acid residue not capable of forming an inter-heavy chain disulfide bond.

### Claim 4 (depends on 2)

The method of claim 2, wherein the method provides a nucleic acid comprising sequence encoding an antibody heavy chain polypeptide having at least two cysteines involved in inter-heavy chain hinge region disulfide bonds, and the at least two cysteines are substituted in a hinge region of the antibody heavy chain.

### Claim 5 (depends on 4)

The method of claim 4, wherein all of the cysteine residues in said variant hinge region are modified or deleted.

### Claim 6 (depends on 1)

The method of claim 1, wherein a cysteine of a hinge region is deleted.

### Claim 7 (depends on 1)

The method of claim 1, wherein said cysteine residue is modified to be a serine residue.

### Claim 8 (depends on 1)

The method of claim 1, wherein said antibody heavy chain-encoding polynucleotide encodes a full-length antibody heavy chain polypeptide.

### Claim 9 (depends on 1)

The method of claim 1, wherein said antibody heavy chain-encoding polynucleotide encodes a humanized heavy chain polypeptide.

### Claim 10 (depends on 1)

The method of claim 1, wherein said antibody heavy chain-encoding polynucleotide encodes a human heavy chain polypeptide.

### Claim 11 (depends on 1)

The method of claim 1, wherein said antibody comprises a human heavy chain constant domain and a human light chain constant domain.

### Claim 12 (depends on 1)

The method of claim 1, wherein said antibody heavy chain is selected from the group consisting of IgG, IgA and IgD.

### Claim 13 (depends on 1)

The method of claim 1, wherein said antibody heavy chain is selected from the group consisting of IgG, IgA, IgE, IgM and IgD.

### Claim 14 (depends on 12)

The method of claim 12, wherein the antibody is IgG.

### Claim 15 (depends on 14)

The method of claim 14, where said antibody is IgG1 or IgG2.

### Claim 16 (depends on 1)

The method of claim 1, wherein said antibody is a therapeutic, an agonist, an antagonist, a diagnostic, a blocking or a neutralizing antibody.

### Claim 17 (depends on 1)

The method of claim 1, wherein the heavy chain-encoding polynucleotide and the light chain-encoding polynucleotide comprise a single polynucleotide.

### Claim 18 (depends on 1)

The method of claim 1, wherein the heavy chain-encoding polynucleotide and the light chain-encoding polynucleotide are separate polynucleotides.

### Claim 19 (depends on 1)

The method of claim 1, further comprising determining that the intact antibody having the variant heavy chain polypeptide is biologically active or retains binding activity to the same antigen as the antibody having the non-variant heavy chain polypeptide.

### Claim 20 (depends on 1)

The method of claim 1, wherein the amount of intact antibody produced in the host cell is at least 25% greater than the amount of an antibody comprising the corresponding non-variant heavy chain polypeptide expressed under similar culture conditions.

### Claim 21 (depends on 20)

The method of claim 20, wherein the amount of intact antibody produced in the host cell is at least 50% greater than the amount of an antibody comprising the corresponding non-variant heavy chain polypeptide expressed under similar culture conditions.

### Claim 22 (depends on 21)

The method of claim 21, wherein the amount of intact antibody produced in the host cell is at least 75% greater than the amount of an antibody comprising the corresponding non-variant heavy chain polypeptide expressed under similar culture conditions.

### Claim 23 (depends on 1)

The method of claim 1, wherein the intact antibody having the variant heavy chain polypeptide and the corresponding antibody having the non-variant heavy chain polypeptide have substantially similar antigen binding capabilities.

### Claim 24 (depends on 1)

The method of claim 1, wherein the intact antibody having the variant heavy chain polypeptide and the corresponding antibody having the non-variant heavy chain polypeptide have substantially similar FcR binding capabilities.

### Claim 25 (depends on 1)

The method of claim 1, wherein the intact antibody having the variant heavy chain polypeptide and the corresponding antibody having the non-variant heavy chain polypeptide have substantially similar pharmacokinetic values.

### Claim 26 (depends on 1)

The method of claim 1, wherein said host cell is prokaryotic.

### Claim 27 (depends on 26)

The method of claim 26, wherein said host cell is a gram-negative bacterial cell.

### Claim 28 (depends on 27)

The method of claim 27, wherein said host cell is E. coli.

### Claim 29 (depends on 26)

The method of claim 26, further comprising expressing in the host cell a polynucleotide encoding at least one prokaryotic polypeptide selected from the group consisting of disulfide bond A (DsbA), disulfide bond C (DsbC), disulfide bond G (DsbG) and FkpA.

### Claim 30 (depends on 29)

The method of claim 29, wherein the polynucleotide encodes both DsbA and DsbC.

### Claim 31 (depends on 28)

The method of claim 28, wherein the E. coli is of a strain deficient in endogenous protease activities.

### Claim 32 (depends on 1)

The method of claim 1, wherein said intact antibody having the variant heavy chain polypeptide is recovered from a cell lysate of the host cell.

### Claim 33 (depends on 1)

The method of claim 1, wherein said intact antibody having the variant heavy chain polypeptide is recovered from a culture medium or a periplasm of the host cell.

### Claim 34 (depends on 1)

The method of claim 1, wherein the polynucleotide encoding the variant heavy chain polypeptide further encodes a secretion signal sequence operably linked to the polynucleotide.

### Claim 35 (depends on 34)

The method of claim 34, wherein the secretion signal sequence comprises a prokaryotic secretion signal sequence.

### Claim 36 (depends on 35)

The method of claim 35, wherein the prokaryotic secretion signal sequence is endogenous to a prokaryotic host cell.

### Claim 37 (depends on 1)

The method of claim 1, wherein the host cell is a prokaryotic cell, an Archaebacteria cell, a Eubacteria cell, a gram-negative cell or a gram-positive cell.

### Claim 38 (depends on 37)

The method of claim 37, wherein the host cell is an Escherichia cell, an E. coli cell, a Bacilli cell, a B. subtilis cell, an Enterobacteria cell, a Pseudomonas species cell, a P. aeruginosa cell, a Salmonella sp. cell or an S. typhimurium cell, a Serratia marcescans cell, a Klebsiella sp. cell, a Proteus sp. cell, a Shigella sp. cell, a Rhizobia sp. cell, Vitreoscilla sp. cell, or a Paracoccus sp. cell.