# US 11,999,800 B2 · Bispecific and tetravalent CD137 and FAP molecules for the treatment of cancer

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- Number: US 11,999,800 B2
- Document ID: US11999800B2
- Status: Granted
- Kind: B2
- Application number: 17/323,844
- Filed: 2021-05-18
- Published: 2021-11-25
- Granted: 2024-06-04
- Earliest claimed: 2020-05-19
- Assignees: Boehringer Ingelheim International GmbH
- Applicants: Boehringer Ingelheim International GmbH
- Inventors: Eric Borges; Pankaj Gupta; Daniel Christopher Rowe; Justin M. Scheer; Abdallah Souabni; Inigo Tirapu; Joseph Ronald Tumang
- Web page: https://patentgrep.com/patent/US11999800B2

## CPC

- C07K 16/40: against enzymes
- A61P 35/00: Antineoplastic agents
- C07K 16/2878: {against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95}
- A61K 2039/507: {Comprising a combination of two or more separate antibodies}
- C07K 2317/31: multispecific
- C07K 2317/565: Complementarity determining region [CDR]
- C07K 2317/622: Single chain antibody (scFv)
- 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
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

This invention relates to binding molecules that bind specifically to CD137 and FAP and their use in medicine, pharmaceutical compositions containing the same, and methods of using the same as agents for treatment and/or prevention of cancer.

## Claims

### Claim 1

A bispecific and tetravalent binding molecule having two antigen binding sites that bind specifically to CD137 (4-1BB Ligand Receptor, 4-1BB) and two antigen binding sites that bind specifically to Fibroblast Activation Protein (FAP) wherein each antigen binding site that binds specifically to CD137 (4-1BB Ligand Receptor) is part of an immunoglobulin molecule comprising a heavy chain variable region comprising heavy chain complementarity determining regions (CDRs) comprising the amino acid sequences of SEQ ID NO:295 (CDR1), SEQ ID NO:18 (CDR2) and SEQ ID NO: 9 (CDR3) and a light chain variable region comprising light chain CDRs comprising the amino acid sequences of SEQ ID NO:22 (CDR1), SEQ ID NO:23 (CDR2) and SEQ ID NO:14 (CDR3); and
the antigen binding sites that bind specifically to Fibroblast Activation Protein (FAP) each comprise single chain variable fragments (scFvs) comprising heavy chain CDRs comprising the amino acid sequences of SEQ ID NO:333 (CDR1), SEQ ID NO:144 (CDR2) and SEQ ID NO:145 (CDR3) and light chain CDRs comprising the amino acid sequences of SEQ ID NO:138 (CDR1), SEQ ID NO:139 (CDR2) and SEQ ID NO:140 (CDR3).

### Claim 2 (depends on 1)

The bispecific and tetravalent binding molecule of claim 1, wherein each of said scFvs has a VL-VH orientation from N-to C-terminus.

### Claim 3 (depends on 1)

The bispecific and tetravalent binding molecule of claim 1, wherein each of said scFvs is fused to the C-terminus of a heavy chain of the immunoglobulin molecule.

### Claim 4 (depends on 1)

The bispecific and tetravalent binding molecule of claim 1, wherein each of said immunoglobulin molecules is an IgG.

### Claim 5 (depends on 1)

The bispecific and tetravalent binding molecule of claim 1, wherein each of said scFvs is fused to the immunoglobulin molecule by a peptide linker having a length of between 4 to 20 amino acids.

### Claim 6 (depends on 1)

The bispecific and tetravalent binding molecule of claim 1 wherein each of said immunoglobulin molecules comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO.:20 and a variable light chain comprising the amino acid sequence of SEQ ID NO.:25.

### Claim 7 (depends on 1)

The bispecific and tetravalent binding molecule of claim 1, wherein each of said scFvs comprises
a variable heavy chain comprising the amino acid sequence of SEQ ID NO.:142 and a variable light chain comprising the amino acid sequence of SEQ ID NO.:146.

### Claim 8

An immunoglobulin-like binding molecule comprising
an immunoglobulin heavy chain fused to a scFv at its C-terminus comprising the amino acid sequence of SEQ ID NO:159, and a light chain comprising the amino acid sequence of SEQ ID NO:160.

### Claim 9 (depends on 1)

A nucleic acid molecule comprising a polynucleotide sequence encoding at least one polypeptide of a bispecific and tetravalent binding molecule of claim 1, or an expression vector or expression vectors containing such a nucleic acid molecule.

### Claim 10 (depends on 9)

A host cell containing a nucleic acid molecule of claim 9.

### Claim 11 (depends on 1)

A method of production of a bispecific and tetravalent binding molecule of claim 1, comprising:
(i) cultivating the host cell containing a nucleic acid molecule encoding the binding molecule of claim 1 or multiple nucleic acid molecules or an expression vector or expression vectors containing such a nucleic acid molecule or nucleic acid molecules under conditions allowing expression of the bispecific and tetravalent binding molecule; and,
(ii) recovering the bispecific and tetravalent binding molecule.

### Claim 12 (depends on 1)

A pharmaceutical composition, comprising a binding molecule of claim 1 together with a pharmaceutically acceptable carrier and optionally one or more further active ingredients.

### Claim 13 (depends on 1)

A method of treatment of cancer comprising administering an effective amount of the bispecific and tetravalent binding molecule of claim 1 to a patient in need thereof.

### Claim 14 (depends on 13)

The method of claim 13, wherein said method further comprises administering an effective amount of an anti-PD-1 antibody to said patient in need thereof.

### Claim 15

A bispecific and tetravalent binding molecule having two antigen binding sites that bind specifically to CD137 (4-1BB Ligand Receptor, 4-1BB) and two antigen binding sites that bind specifically to Fibroblast Activation Protein (FAP), comprising two identical immunoglobulin heavy chains each fused to a single chain variable fragment (scFv) at its C-terminus, each comprising the amino acid sequence of SEQ ID NO:159, and two identical light chains, each comprising the amino acid sequence of SEQ ID NO:160.

### Claim 16 (depends on 15)

A pharmaceutical composition comprising the bispecific and tetravalent binding molecule of claim 15 and a pharmaceutically acceptable carrier.

### Claim 17 (depends on 15)

A nucleic acid molecule the comprising a polynucleotide sequence encoding at least one polypeptide of the bispecific and tetravalent binding molecule of claim 15 or an expression vector or expression vectors containing such a nucleic acid molecule.

### Claim 18 (depends on 17)

A host cell containing the nucleic acid molecule of claim 17.

### Claim 19 (depends on 15)

A method of production of the bispecific and tetravalent binding molecule of claim 15, comprising:
(i) cultivating the host cell containing a nucleic acid molecule encoding the immunoglobulin-like binding molecule of claim 15 or multiple nucleic acid molecules or an expression vector or expression vectors containing such a nucleic acid molecule or nucleic acid molecules under conditions allowing expression of the bispecific and tetravalent binding molecule; and
(ii) recovering the bispecific and tetravalent binding molecule.

### Claim 20 (depends on 16)

A method of treatment of cancer comprising administering an effective amount of the pharmaceutical composition of claim 16 to a patient in need thereof.

### Claim 21 (depends on 2)

The bispecific and tetravalent binding molecule of claim 2, wherein each of the two scFvs is fused to the C-terminus of a heavy chain of the immunoglobulin molecule.

### Claim 22 (depends on 8)

A host cell comprising a nucleic acid molecule or multiple nucleic acid molecules comprising polynucleotide sequences encoding the immunoglobulin-like binding molecule of claim 8.

### Claim 23 (depends on 15)

A host cell comprising a nucleic acid molecule or multiple nucleic acid molecules comprising polynucleotide sequences encoding the bispecific and tetravalent binding molecule of claim 15.

### Claim 24

An immunoglobulin-like binding molecule comprising an immunoglobulin heavy chain fused to a scFv at its C-terminus having the amino acid sequence consisting of SEQ ID NO:159, and a light chain having the amino acid sequence consisting of SEQ ID NO:160.

### Claim 25 (depends on 8)

A pharmaceutical composition, comprising the immunoglobulin-like binding molecule of claim 8 and a pharmaceutically acceptable carrier.

### Claim 26 (depends on 24)

A pharmaceutical composition, comprising the immunoglobulin-like binding molecule of claim 24 and a pharmaceutically acceptable carrier.

### Claim 27 (depends on 16)

The pharmaceutical composition of claim 16, wherein said pharmaceutical composition comprises one or more further active ingredients.

### Claim 28 (depends on 25)

The pharmaceutical composition of claim 25, wherein said pharmaceutical composition comprises one or more further active ingredients.

### Claim 29 (depends on 26)

The pharmaceutical composition of claim 26, wherein said pharmaceutical composition comprises one or more further active ingredients.

### Claim 30 (depends on 12)

The pharmaceutical composition of claim 12, wherein said pharmaceutical composition is lyophilized.

### Claim 31 (depends on 16)

The pharmaceutical composition of claim 16, wherein said pharmaceutical composition is lyophilized.

### Claim 32 (depends on 25)

The pharmaceutical composition of claim 25, wherein said pharmaceutical composition is lyophilized.

### Claim 33 (depends on 26)

The pharmaceutical composition of claim 26, wherein said pharmaceutical composition is lyophilized.

### Claim 34 (depends on 24)

A nucleic acid molecule comprising a polynucleotide sequence encoding at least one polypeptide of the immunoglobulin-like binding molecule of claim 24 or an expression vector or expression vectors containing such a nucleic acid molecule.

### Claim 35 (depends on 24)

A host cell comprising a nucleic acid molecule or multiple nucleic acid molecules comprising polynucleotide sequences encoding the immunoglobulin-like binding molecule of claim 24.

### Claim 36 (depends on 8)

A method of production of the immunoglobulin-like binding molecule of claim 8, comprising:
(i) cultivating the host cell containing a nucleic acid molecule encoding the immunoglobulin-like binding molecule of claim 8 or multiple nucleic acid molecules or an expression vector or expression vectors containing such a nucleic acid molecule or nucleic acid molecules under conditions allowing expression of the immunoglobulin-like binding molecule; and
(ii) recovering the immunoglobulin-like binding molecule.

### Claim 37 (depends on 24)

A method of production of the immunoglobulin-like binding molecule of claim 24, comprising:
(i) cultivating the host cell containing a nucleic acid molecule encoding the immunoglobulin-like binding molecule of claim 24 or multiple nucleic acid molecules or an expression vector or expression vectors containing such a nucleic acid molecule or nucleic acid molecules under conditions allowing expression of the immunoglobulin-like binding molecule; and
(ii) recovering the immunoglobulin-like binding molecule.

### Claim 38

A bispecific and tetravalent binding molecule having two antigen binding sites that bind specifically to CD137 (4-1BB Ligand Receptor, 4-1BB) and two antigen binding sites that bind specifically to Fibroblast Activation Protein (FAP), comprising two identical immunoglobulin heavy chains each fused to a single chain variable fragment (scFv) at its C-terminus, each consisting of the amino acid sequence of SEQ ID NO:159, and two identical light chains, each consisting of the amino acid sequence of SEQ ID NO:160.

### Claim 39 (depends on 38)

A pharmaceutical composition, comprising the bispecific and tetravalent binding molecule of claim 38 and a pharmaceutically acceptable carrier.

### Claim 40 (depends on 39)

The pharmaceutical composition of claim 39, wherein said pharmaceutical composition comprises one or more further active ingredients.

### Claim 41 (depends on 39)

The pharmaceutical composition of claim 39, wherein said pharmaceutical composition is lyophilized.

### Claim 42 (depends on 38)

A method of production of the bispecific and tetravalent binding molecule of claim 38, comprising:
(i) cultivating a host cell containing a nucleic acid molecule encoding said bispecific and tetravalent binding molecule, or containing multiple nucleic acid molecules encoding said bispecific and tetravalent binding molecule, or containing an expression vector or expression vectors containing such a nucleic acid molecule or nucleic acid molecules, under conditions allowing expression of the bispecific and tetravalent binding molecule; and
(ii) recovering the bispecific and tetravalent binding molecule.

### Claim 43 (depends on 15)

A method of treatment of cancer comprising administering an effective amount of the bispecific and tetravalent binding molecule of claim 15 to a patient in need thereof.

### Claim 44 (depends on 43)

The method of claim 43, wherein said method further comprises administering an effective amount of an anti-PD-1 antibody to said patient in need thereof.

### Claim 45 (depends on 24)

A method of treatment of cancer comprising administering an effective amount of the immunoglobulin-like binding molecule of claim 24 to a patient in need thereof.

### Claim 46 (depends on 45)

The method of claim 45, wherein said method further comprises administering an effective amount of an anti-PD-1 antibody to said patient in need thereof.

### Claim 47 (depends on 38)

A method of treatment of cancer comprising administering an effective amount of the bispecific and tetravalent binding molecule of claim 38 to a patient in need thereof.

### Claim 48 (depends on 47)

The method of claim 47, wherein said method further comprises administering an effective amount of an anti-PD-1 antibody to said patient in need thereof.