# US 2019/0169298 A1 · Recombinant monovalent antibodies and methods for production thereof

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- Number: US 2019/0169298 A1
- Document ID: US20190169298A1
- Status: Published application
- Kind: A1
- Application number: 16/183,300
- Filed: 2018-11-07
- Published: 2019-06-06
- Earliest claimed: 2005-11-28
- Assignees: Genmab A/S
- Applicants: Genmab A/S
- Inventors: Paul PARREN; Janine SCHUURMAN; Tom VINK; Willem Karel BLEEKER; Jan VAN DE WINKEL; Patrick VAN BERKEL; Frank BEURSKENS
- Web page: https://patentgrep.com/patent/US20190169298A1

## CPC

- C07K 16/2863: {against receptors for growth factors, growth regulators}
- A61P 1/00: Drugs for disorders of the alimentary tract or the digestive system
- A61P 1/04: for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
- A61P 1/16: for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
- A61P 1/18: for pancreatic disorders, e.g. pancreatic enzymes
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- A61P 3/14: for calcium homeostasis (vitamin D A61P3/02; parathyroid hormones A61P5/18; calcitonin A61P5/22; osteoporosis A61P19/10; bone metastasis A61P35/04)
- A61P 5/14: of the thyroid hormones, e.g. T3, T4
- A61P 7/06: Antianaemics
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- A61P 11/00: Drugs for disorders of the respiratory system
- A61P 11/06: Antiasthmatics
- A61P 13/12: of the kidneys
- A61P 17/00: Drugs for dermatological disorders
- A61P 17/06: Antipsoriatics
- A61P 19/02: for joint disorders, e.g. arthritis, arthrosis
- A61P 21/00: Drugs for disorders of the muscular or neuromuscular system
- A61P 21/04: for myasthenia gravis
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 25/02: for peripheral neuropathies
- A61P 25/28: for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
- A61P 27/02: Ophthalmic agents
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 31/18: for HIV
- A61P 35/00: Antineoplastic agents
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 16/2812: {against CD4}
- C07K 16/283: {against Fc-receptors, e.g. CD16, CD32, CD64 (CD23 C07K16/2851)}
- C07K 16/2887: {against CD20}
- C07K 2317/21: from primates, e.g. man
- C07K 2317/53: Hinge
- C07K 2317/54: F(ab')2
- C07K 2317/55: Fab or Fab'
- C07K 2317/56: variable (Fv) region, i.e. VH and/or VL
- C07K 2317/732: Antibody-dependent cellular cytotoxicity [ADCC]
- C07K 2317/734: Complement-dependent cytotoxicity [CDC]
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- C07K 2317/77: Internalization into the cell
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

The present invention provides monovalent antibodies with a long half-life when administered in vivo, methods of making such monovalent antibodies, pharmaceutical compositions comprising such antibodies, and uses of the monovalent antibodies.

## Claims

### Claim at position 1

1-228. (canceled)

### Claim 229

A method for producing a monovalent antibody, said method comprising
a) providing a nucleic acid construct encoding the light chain of said monovalent antibody, said construct comprising a nucleotide sequence encoding the VL region of a selected antigen specific antibody and a nucleotide sequence encoding the constant CL region of an Ig, wherein said nucleotide sequence encoding the VL region of a selected antigen specific antibody and said nucleotide sequence encoding the CL region of an Ig are operably linked together, and wherein, in case of an IgG1 subtype, the nucleotide sequence encoding the CL region has been modified such that the CL region does not contain any amino acids capable of forming disulfide bonds or covalent bonds with other peptides comprising an identical amino acid sequence of the CL region in the presence of polyclonal human IgG or when administered to a human being;
b) providing a nucleic acid construct encoding the heavy chain of said monovalent antibody, said construct comprising a nucleotide sequence encoding the VH region of a selected antigen specific antibody and a nucleotide sequence encoding a constant CH region of a human Ig, wherein the nucleotide sequence encoding the CH region has been modified such that the region corresponding to the hinge region and, as required by the Ig subtype, other regions of the CH region, such as the CH3 region, does not comprise any amino acid residues which participate in the formation of disulphide bonds or covalent or stable non-covalent inter-heavy chain bonds with other peptides comprising an identical amino acid sequence of the CH region of the human Ig in the presence of polyclonal human IgG or when administered to a human being, wherein said nucleotide sequence encoding the VH region of a selected antigen specific antibody and said nucleotide sequence encoding the CH region of said Ig are operably linked together;
c) providing a cell expression system for producing said monovalent antibody; and
d) producing said monovalent antibody by co-expressing the nucleic acid constructs of (i) and (ii) in cells of the cell expression system of (iii).

### Claim 230

A nucleic acid construct for expression of a monovalent antibody for pharmaceutical use, comprising i) a nucleic acid sequence encoding the CH region of an IgG4, wherein the nucleic acid sequence encoding the CH region has been modified such that the region corresponding to the hinge region in said CH region does not comprise any amino acid residues capable of participating in the formation of stable disulphide bonds with peptides comprising an amino acid sequence identical to the amino acid sequence of said CH region in the presence of polyclonal human IgG or when administered to a human being or (ii) a nucleotide sequence encoding a constant CH region of a human Ig, wherein the nucleotide sequence encoding the CH region has been modified such that the region corresponding to the hinge region and, as required by the Ig subtype, other regions of the CH region, such as the CH3 region, does not comprise any amino acid residues which participate in the formation of disulphide bonds or covalent or stable non-covalent inter-heavy chain bonds with other peptides comprising an identical amino acid sequence of the CH region of the human Ig in the presence of polyclonal human IgG or when administered to a human being or (iii) a sequence complementary thereto.

### Claim 231 (depends on 230)

A nucleic acid construct according to claim 230, wherein the nucleic acid sequence encoding the CH region has been modified such that the region corresponding to the hinge region does not comprise any cysteine residues.

### Claim 232 (depends on 230)

A nucleic acid construct according to claim 230, wherein said construct comprises a nucleic acid sequence encoding the VH region of an antigen specific antibody, or a sequence complementary thereto, wherein the nucleic acid sequence encoding the VH region is operably linked to the nucleic acid sequence encoding the CH region, or a sequence complementary thereto.

### Claim 233 (depends on 230)

A host cell comprising a nucleic acid construct according to claim 230.

### Claim 234 (depends on 231)

A method of treating a disease or disorder, wherein said method comprises administering to a subject in need of treatment a therapeutically effective amount of a monovalent antibody comprising a light chain and a heavy chain, wherein
a) said light chain comprises the amino acid sequence of the variable (VL) region of a selected antigen specific antibody and the amino acid sequence of the constant (CL) region of an Ig, and wherein, in case of an IgG1 subtype, the amino sequence of the constant (CL) region has been modified so that it does not contain any amino acids capable of participating in the formation of disulfide bonds or covalent bonds with other peptides comprising an identical amino acid sequence of the constant (CL) region of the Ig in the presence of polyclonal human IgG or when administered to a human being, and
b) said heavy chain comprises the amino acid sequence of the variable (VH) region of said selected antigen specific antibody and the amino acid sequence of the constant (CH) region of human Ig, wherein the amino acid sequence of the constant (CH) region has been modified so that the hinge region and, as required by the Ig subtype, other regions of the CH region, such as the CH3 region, does not contain any amino acid residues which participate in the formation of disulphide bonds or covalent or stable non-covalent inter-heavy chain bonds with other peptides comprising an identical amino acid sequence of the constant (CH) region of the human Ig in the presence of polyclonal human IgG or when administered to a human being,
a pharmaceutical composition comprising said antibody, an immunoconjugate comprising said antibody, or a nucleic acid construct according to claim 231.

### Claim 235 (depends on 234)

The method of claim 234, wherein the antibody comprises a human IgG4 CH region.

### Claim 236 (depends on 234)

The method of claim 234, wherein the heavy chain comprises the amino acid sequence of the heavy chain variable (VH) region of said selected antigen specific antibody and the amino acid sequence of SEQ ID NO: 16.

### Claim 237 (depends on 236)

The method of claim 236, wherein the CL region is the constant region of the kappa light chain of a human IgG.

### Claim 238 (depends on 232)

The method of claim 232, wherein the CL region comprises the amino acid sequence of SEQ ID NO: 2.

### Claim 239 (depends on 236)

The method of claim 236, wherein the CL region is the constant region of the lambda light chain of a human IgG.

### Claim 240 (depends on 239)

The method of claim 239, wherein the CL region comprises the amino acid sequence of SEQ ID NO: 4.

### Claim 241 (depends on 236)

The method of claim 236, wherein the light chain and the heavy chain are connected to each other via one or more disulphide bonds or via an amide bond.

### Claim 242 (depends on 234)

The method of claim 234, wherein the heavy chain comprises the amino acid sequence of the heavy chain variable (VH) region of the selected antigen specific antibody and the amino acid sequence of SEQ ID NO: 22.

### Claim 243 (depends on 234)

The method of claim 234, wherein the heavy chain comprises the amino acid sequence of the heavy chain variable (VH) region of said selected antigen specific antibody and the amino acid sequence of the heavy chain constant (CH) region of human IgG1, wherein the CH region comprises the amino acid sequence set forth in SEQ ID NO: 19, wherein Lys (K) in position 292 of SEQ ID NO: 19 has been replaced by Arg (R); and wherein the hinge region of the heavy chain lacks cysteine residues.

### Claim 244 (depends on 243)

The method of claim 243, wherein the CL region is a kappa light chain CL region comprising the amino acid sequence set forth in SEQ ID NO: 18, wherein the terminal cysteine residue in position 106 of SEQ ID NO: 18 has been replaced with a different amino acid residue or has been deleted.

### Claim 245 (depends on 243)

The method of claim 243, wherein the CL region is a lambda light chain CL region having the amino acid sequence set forth in SEQ ID NO: 17, wherein the cysteine residue in position 104 of SEQ ID NO: 17 has been replaced with a different amino acid residue or has been deleted.

### Claim 246 (depends on 234)

The method of claim 234, wherein the heavy chain comprises the amino acid sequence of the heavy chain variable (VH) region of said selected antigen specific antibody and the amino acid sequence of the heavy chain constant (CH) region of human IgG1, wherein the CH region comprises the amino acid sequence set forth in SEQ ID NO: 19, wherein Lys (K) in position 292 of SEQ ID NO: 19 has been replaced by Arg (R), and Ser (S) in position 14 of SEQ ID NO: 19 has been replaced by Cys (C); and wherein the hinge region of the heavy chain lacks cysteine residues.

### Claim 247 (depends on 234)

The method of claim 234, wherein the heavy chain comprises the amino acid sequence of the heavy chain variable (VH) region of said selected antigen specific antibody and the amino acid sequence of the heavy chain constant (CH) region of IgG2, wherein the CH region comprises the amino acid sequence set forth in SEQ ID NO: 20, wherein the following amino acid substitutions have been made: Arg (R) in position 234 of SEQ ID NO: 20 has been replaced by Gln (Q), Met (M) in position 276 of SEQ ID NO: 20 has been replaced by Val (V), Lys (K) in position 288 of SEQ ID NO: 20 has been replaced by Arg (R), Gln (Q) in position 298 of SEQ ID NO: 20 has been replaced by Glu (E), and Pro (P) in position 324 of SEQ ID NO: 20 has been replaced by Leu (L); and wherein the hinge region lacks cysteine residues.

### Claim 248 (depends on 234)

The method of claim 234, wherein the heavy chain comprises the amino acid sequence of the heavy chain variable (VH) region of said selected antigen specific antibody and the amino acid sequence of the heavy chain constant (CH) region of IgG3, wherein the CH region comprises the amino acid sequence set forth in SEQ ID NO: 21, wherein one or more of the following amino acid substitutions have been made: Arg (R) in position 285 of SEQ ID NO: 21 has been replaced by Gln (Q), Ser (S) in position 314 of SEQ ID NO: 21 has been replaced by Asn (N), Asn (N) in position 322 of SEQ ID NO: 21 has been replaced by Lys (K), Met (M) in position 327 of SEQ ID NO: 21 has been replaced by Val (V), Lys (K) in position 339 of SEQ ID NO: 21 has been replaced by Arg (R), Gln (Q) in position 349 of SEQ ID NO: 21 has been replaced by Glu (E), Be (I) in position 352 of SEQ ID NO: 21 has been replaced by Val (V), Arg (R) in position 365 of SEQ ID NO: 21 has been replaced by His (H), Phe (F) in position 366 of SEQ ID NO: 21 has been replaced by Tyr (Y), and Pro (P) in position 375 of SEQ ID NO: 21 has been replaced by Leu (L); and wherein the hinge region of the heavy chain lacks cysteine residues.