# US 2018/0022829 A1 · Multispecific antigens binding fragments and multispecific antibodies derived therefrom comprising mutant ch1 and cl-kappa domains

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2018/0022829 A1
- Document ID: US20180022829A1
- Status: Published application
- Kind: A1
- Application number: 15/463,498
- Filed: 2017-03-20
- Published: 2018-01-25
- Earliest claimed: 2011-07-07
- Assignees: Centre National De La Recherche Scientifique; Azienda Ospedaliera Papa Giovanni XXIII; Universite de Lausanne
- Applicants: Centre National de la Recherche Scientifique; Azienda Ospedaliera Papa Giovanni XXIII; Universite de Lausanne
- Inventors: Jean Kadouche; Jean-Pierre Mach; Olivier Michielin; Vincent Zoete; Justyna Iwaszkiewicz; Martine Cerutti; Sylvie Choblet; Josee Golay
- Web page: https://patentgrep.com/patent/US20180022829A1

## CPC

- C07K 16/468: {Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies}
- C07K 16/2833: {against MHC-molecules, e.g. HLA-molecules}
- C07K 16/2896: {against molecules with a "CD"-designation, not provided for elsewhere}
- C07K 2317/31: multispecific
- C07K 2317/522: CH1 domain
- C07K 2317/55: Fab or Fab'
- C07K 2317/732: Antibody-dependent cellular cytotoxicity [ADCC]
- 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 relates to multispecific antibody constructs comprising Fab fragments having mutations at the interface of the CH1 and CL domains, said mutations preventing heavy chain/light chain mispairing.

## Claims

### Claim at position 1

-12. (canceled)

### Claim 13

A multispecific antigen-binding fragment comprising at least two Fab fragments with different CH1 and CL domains, wherein said Fab fragments comprise:
a. a mutated Fab fragment consisting of:
i. the VH and VL domains of an antibody recognizing an epitope of interest;
ii. a CH1 domain of a human immunoglobulin comprising substitution of the leucine residue at position 143 of said CHI domain with a glutamine residue and substitution of the serine residue at position 188 of said CH1 domain with a valine residue; and
iii. a CL-kappa domain of a human immunoglobulin comprising substitution of the valine residue at position 133 of said CL domain with a threonine residue and substitution of the serine residue at position 176 of said CL domain with an valine residue; and
b. at least another Fab fragment which is selected from the group consisting of
b1) a mutated Fab fragment consisting of:
i. the VH and VL domains of an antibody recognizing an epitope of interest;
ii. a CH1 domain of a human immunoglobulin comprising substitution of the threonine residue at position 192 of said CH1 domain with a glutamic acid residue; and
iii. a CL-kappa domain of a human immunoglobulin by substitution of the asparagine residue at position 137 of said CL domain with a lysine residue and substitution of the serine residue at position 114 of said CL domain with an alanine residue;
b2) a mutated Fab fragment consisting of:
i. the VH and VL domains of an antibody recognizing an epitope of interest;
ii. a CH1 domain of a human immunoglobulin comprising substitution of the leucine residue at position 124 of said CH1 domain with an alanine residue and substitution of the leucine residue at position 143 of said CH1 domain with a glutamic acid residue; and
iii. a CL-kappa domain of a human immunoglobulin comprising substitution of the valine residue at position 133 of said CL domain with a tryptophane residue;
b3) a mutated Fab fragment consisting of:
i. the VH and VL domains of an antibody recognizing an epitope of interest;
ii. a CH1 domain of a human immunoglobulin comprising substitution of the valine residue at position 190 of said CHI domain with an alanine residue; and
iii. a CL-kappa domain of a human immunoglobulin comprising substitution of the leucine residue at position 135 of said CL domain with a tryptophane residue, and substitution of the asparagine residue at position 137 of said CL domain with an alanine residue; and
b4) a wild-type Fab fragment consisting of wild consisting of wild-type CH1 and wild-type CL domains of an immunoglobulin, and the VH and VL domains of an antibody recognizing an epitope of interest;
wherein the position numbers used for the CH1 and CL domains refer to Kabat numbering and each Fab fragment recognizes a different epitope of interest, and said Fab fragments are tandemly arranged in any order, the C-terminal end of the CH1 domain of the first Fab fragment being linked to the N-terminal end of the VH domain of the following Fab fragment through a polypeptide linker.

### Claim 14 (depends on 13)

The multispecific antigen-binding fragment of claim 13, wherein said Fab fragments comprise:
a. a mutated Fab fragment consisting of:
i. the VH and VL domains of an antibody recognizing an epitope of interest;
ii. a CH1 domain of a human immunoglobulin comprising substitution of the leucine residue at position 143 of said CHI domain with a glutamine residue and substitution of the serine residue at position 188 of said CH1 domain with a valine residue; and
iii. a CL domain of a human immunoglobulin comprising substitution of the valine residue at position 133 of said CL domain with a threonine residue and substitution of the serine residue at position 176 of said CL domain with a valine residue; and
b. a Fab fragment consisting of:
i. the VH and VL domains of an antibody recognizing an epitope of interest;
ii. a CH1 domain of a human immunoglobulin comprising substitution of the threonine residue at position 192 of said CHI domain with a glutamic acid residue; and
iii. a CL-kappa domain of a human immunoglobulin by substitution of the asparagine residue at position 137 of said CL domain with a lysine residue and substitution of the serine residue at position 114 of said CL domain with an alanine residue.

### Claim 15 (depends on 13)

The multispecific antigen-binding fragment of claim 13, wherein the polypeptide linker has a length of at least 20 amino-acids.

### Claim 16 (depends on 13)

The multispecific antigen-binding fragment of claim 13, wherein the polypeptide linker comprises all or part of the sequence of the hinge region of one or more immunoglobulin(s) selected among IgA, IgG, and IgD.

### Claim 17 (depends on 13)

The multispecific antigen-binding fragment of claim 13, wherein the polypeptide linker has the sequence of SEQ ID NO:9.

### Claim 18 (depends on 13)

A multispecific antibody having two identical antigens-binding arms, each consisting of an antigens-binding fragment of claim 13.

### Claim 19 (depends on 13, 14, 15, 16, 17)

A multispecific antibody having two identical antigens-binding arms and an immunoglobulin-like structure, wherein the multispecific antibody comprises:
a. two identical antigens-binding arms, with each consisting of the antigen-binding fragment of any of claims 13 to 17,
b. the dimerized CH2 and CH3 domains of an immunoglobulin;
c. the hinge region of an IgA, IgG, or IgD, linking the C-terminal ends of CH1 domains of the antigens-binding arms to the N-terminal ends of the CH2 domains.

### Claim 20 (depends on 13)

A polynucleotide comprising a sequence encoding the heavy chain of an antigen-binding fragment of claim 13.

### Claim 21 (depends on 18)

A polynucleotide comprising a sequence encoding the heavy chain of the multispecific antibody of claim 18.

### Claim 22 (depends on 13)

A host-cell transformed with a polynucleotide encoding a heavy chain of an antigen-binding fragment of claim 13 and at least two polynucleotides encoding two different light chains, wherein the first light chain pairs specifically with a first VH/CH1 region of said heavy chain and the second light chain pairs specifically with a second VH/CH1 region of said heavy chain.

### Claim 23 (depends on 22)

The host cell of claim 22, wherein at least one of said light chains is the light chain of a mutated Fab fragment.

### Claim 24 (depends on 17)

A host-cell transformed with a polynucleotide encoding the multispecific antibody of claim 17 and at least two polynucleotides encoding two different light chains, wherein the first light chain pairs specifically with a first VH/CH1 region of said heavy chain and the second light chain pairs specifically with a second VH/CH1 region of said heavy chain.

### Claim 25 (depends on 24)

The host cell of claim 24, wherein at least one of said light chains is the light chain of a mutated Fab fragment.

### Claim 26 (depends on 22, 24)

A host cell of claim 22 or 24, which is additionally transformed with a third polynucleotide encoding a third light chain which is different from the first and second light chains, and which pairs specifically with a third VH/CH1 region of said heavy chain.

### Claim 27 (depends on 22)

A method of preparing an antigen-binding fragment wherein the method comprises culturing the host-cell of claim 22 and recovering said antigen-binding fragment from the culture.

### Claim 28 (depends on 24)

A method of preparing a multispecific antibody, wherein the method comprises culturing the host cell of claim 24 and recovering the antibody from the culture.

### Claim 29 (depends on 13)

A therapeutic composition comprising the multispecific antigen-binding fragment of claim 13 and a pharmaceutically acceptable vehicle.

### Claim 30 (depends on 18)

A therapeutic composition comprising a multispecific antibody of claim 18 and a pharmaceutically acceptable vehicle.