# US 12,180,295 B2 · Humanized anti-SIRPα antibodies

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- Number: US 12,180,295 B2
- Document ID: US12180295B2
- Status: Granted
- Kind: B2
- Application number: 17/292,182
- Filed: 2019-11-15
- Published: 2021-12-16
- Granted: 2024-12-31
- Earliest claimed: 2018-11-15
- Assignees: Byondis B.V.
- Applicants: Byondis B.V.
- Inventors: Gijsbertus Franciscus Maria Verheijden
- Web page: https://patentgrep.com/patent/US12180295B2

## CPC

- C07K 16/2803: {against the immunoglobulin superfamily}
- C07K 16/2896: {against molecules with a "CD"-designation, not provided for elsewhere}
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61K 39/395: Antibodies (agglutinins A61K38/36 {; as drug carriers A61K47/50}); Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K 2039/505: {comprising antibodies}
- A61P 35/00: Antineoplastic agents
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/33: Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
- C07K 2317/72: Increased effector function due to an Fc-modification
- C07K 2317/732: Antibody-dependent cellular cytotoxicity [ADCC]
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- C12N 5/10: Cells modified by introduction of foreign genetic material
- 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)
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention relates to humanized antibodies against SIRPα that are suitable for use in anti-cancer therapy. The invention further relates to the use of the humanized anti-SIRPα antibodies in the treatment of human solid tumours and haematological malignancies, optionally in combination with further anti-cancer therapeutics.

## Claims

### Claim 1

A humanized anti-SIRPα antibody or an antigen-binding fragment thereof, comprising heavy chain complementarity determining regions (HCDR) and light chain complementarity determining regions (LCDR) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the antibody or the antigen-binding fragment thereof comprises:
a. HCDR1 comprising SEQ ID NO:36;
b. HCDR2 comprising SEQ ID NO:44;
c. HCDR3 comprising SEQ ID NO:45;
d. LCDR1 comprising SEQ ID NO:39;
e. LCDR2 comprising SEQ ID NO:40; and
f. LCDR3 comprising SEQ ID NO:41.

### Claim 2 (depends on 1)

The humanized anti-SIRPα antibody or the antigen-binding fragment thereof according to claim 1, wherein the anti-SIRPα antibody or the antigen-binding fragment thereof has one or more properties from the group consisting of:
a. the anti-SIRPα antibody or the antigen-binding fragment thereof binds human SIRPα1 with a binding affinity of at least 10−10 M, as analysed by surface plasmon resonance at 25° C. using human SIRPα1 extracellular domain as shown in SEQ ID NO:51;
b. the anti-SIRPα antibody or the antigen-binding fragment thereof binds human SIRPαBIT with a binding affinity of at least 10−10 M, as analysed by surface plasmon resonance at 25° C. using human SIRPαBIT extracellular domain as shown in SEQ ID NO:52;
c. the anti-SIRPα antibody or the antigen-binding fragment thereof binds cynomolgus monkey SIRPα with a binding affinity of at least 10−8 M, as analysed by surface plasmon resonance at 25° C. using cynomolgus SIRPα extracellular domain as shown in SEQ ID NO:56;
d. the anti-SIRPα antibody or the antigen-binding fragment thereof does not bind human SIRPγ as measured by T-cell binding using flow cytometry;
e. the anti-SIRPα antibody or the antigen-binding fragment thereof does not bind human SIRPγ as analysed by surface plasmon resonance at 25° C. using human SIRPγ extracellular domain as shown in SEQ ID NO:55; and
f. the anti-SIRPα antibody or the antigen-binding fragment thereof is not immunogenic as determined by IL-2 enzyme-linked immunosorbent spot (ELISpot) and/or T-cell proliferation assay.

### Claim 3 (depends on 1)

The humanized anti-SIRPα antibody or the antigen-binding fragment thereof according to claim 1, wherein:
a. the anti-SIRPα antibody or the antigen-binding fragment thereof binds human SIRPα1 with a binding affinity of at least 10−10 M, as analysed by surface plasmon resonance at 25° C. using human SIRPα1 extracellular domain as shown in SEQ ID NO:51;
b. the anti-SIRPα antibody or the antigen-binding fragment thereof binds human SIRPαBIT with a binding affinity of at least 10−10 M, as analysed by surface plasmon resonance at 25° C. using human SIRPαBIT extracellular domain as shown in SEQ ID NO:52;
c. blocks CD47 binding to SIRPα1 and SIRPαBIT as analysed by dissociation from captured CD47 by surface plasmon resonance; and
d. the anti-SIRPα antibody or the antigen-binding fragment thereof does not bind human SIRPγ as measured by T-cell flow cytometry staining.

### Claim 4 (depends on 3)

The humanized anti-SIRPα antibody or the antigen-binding fragment thereof according to claim 3, wherein:
a. the anti-SIRPα antibody or the antigen-binding fragment thereof binds human SIRPα1 with a binding affinity of at least 10−11 M, as analysed by surface plasmon resonance at 25° C. using human SIRPα1 extracellular domain as shown in SEQ ID NO:51;
b. the anti-SIRPα antibody or the antigen-binding fragment thereof binds human SIRPαBIT with a binding affinity of at least 10−11 M, as analysed by surface plasmon resonance at 25° C. using human SIRPαBIT extracellular domain as shown in SEQ ID NO:52;
c. blocks CD47 binding to SIRPα1 and SIRPαBIT as analysed by dissociation from captured CD47 by surface plasmon resonance; and
d. the anti-SIRPα antibody or the antigen-binding fragment thereof does not bind human SIRPγ as measured by T-cell flow cytometry staining.

### Claim 5 (depends on 1)

The humanized anti-SIRPα antibody or the antigen-binding fragment thereof according to claim 1, comprising:
a. a heavy chain variable domain which comprises 4 heavy chain framework regions, HFR1 to HFR4, and the 3 complementarity determining regions HCDR1 to HCDR3 that are operably linked in the order HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4, wherein each of the heavy chain framework regions has at least 90% amino acid sequence identity with the corresponding framework amino acid sequence of SEQ ID NO:8, or wherein HFR1 to HFR4 differ from SEQ ID NO:8 in one or more of the amino acid substitutions as defined in SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, and SEQ ID NO:80, respectively; and
b. a light chain variable domain which comprises 4 light chain framework regions, LFR1 to LFR4, and the 3 complementarity determining regions LCDR1 to LCDR3 that are operably linked in the order LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, wherein each of the light chain framework regions has at least 90% amino acid sequence identity with the corresponding framework amino acid sequence of SEQ ID NO: 9, or wherein LFR1, LFR2 and/or LFR4 differ from SEQ ID NO:9 in one or more acid substitutions as defined in SEQ ID NO:81, SEQ ID NO: 82, and SEQ ID NO:83, respectively.

### Claim 6 (depends on 5)

The humanized anti-SIRPα antibody according to claim 5, comprising a modified human IgG1 Fc region, comprising an amino acid substitution at one or more positions of a wild-type human IgG1 Fc region selected from the group consisting of L234, L235, G237, D265, D270, N297, A327, P328, and P329 according to Eu numbering.

### Claim 7 (depends on 1)

The humanized anti-SIRPα antibody or the antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the antibody or the antigen-binding fragment thereof comprises the HCVR amino acid sequence of SEQ ID NO:8 and the LCVR amino acid sequence of SEQ ID NO:9.

### Claim 8 (depends on 7)

The humanized anti-SIRPα antibody according to claim 7, comprising a modified human IgG1 Fc region, comprising an amino acid substitution at one or more positions of a wild-type human IgG1 Fc region selected from the group consisting of L234, L235, G237, D265, D270, N297, A327, P328, and P329 according to Eu numbering.

### Claim 9 (depends on 8)

The humanized anti-SIRPα antibody according to claim 8, comprising the amino acid substitutions L234A and L235A; L234E and L235A; L234A, L235A and P329A; or L234A, L235A and P329G.

### Claim 10 (depends on 1)

The humanized anti-SIRPα antibody according to claim 1, comprising a modified Fc region that exhibits reduced binding to a human Fcα or Fcγ receptor compared to the same anti-SIRPα antibody comprising a wild-type Fc region.

### Claim 11 (depends on 1)

The humanized anti-SIRPα antibody according to claim 1, comprising a modified human IgG1 Fc region, comprising an amino acid substitution at one or more positions of a wild-type human IgG1 Fc region selected from the group consisting of L234, L235, G237, D265, D270, N297, A327, P328, and P329 according to Eu numbering.

### Claim 12 (depends on 11)

The humanized anti-SIRPα antibody according to claim 11 comprising the amino acid substitutions L234A and L235A; L234E and L235A; L234A, L235A and P329A; or L234A, L235A and P329G.

### Claim 13 (depends on 1)

A pharmaceutical composition comprising the humanized anti-SIRPα antibody or antigen-binding fragment thereof according to claim 1 and a pharmaceutically acceptable excipient.

### Claim 14 (depends on 1)

A method of treating cancer, which comprises administering to a patient in need of such treatment an effective amount of (i) the humanized anti-SIRPα antibody or antigen-binding fragment thereof according to claim 1, and (ii) a therapeutic antibody, wherein the cancer is a human solid tumour or a hematological malignancy.

### Claim 15 (depends on 14)

The method according to claim 14, wherein said cancer comprises tumour cells; and said therapeutic antibody (i) is directed against a membrane-bound target on the surface of the tumour cells and (ii) comprises a human Fc region that binds to activating Fc receptors present on human immune effector cells.

### Claim 16 (depends on 15)

The method according to claim 15, wherein the cancer is selected from the group consisting of breast cancer, colon carcinoma, neuroblastoma, melanoma, osteosarcoma, B-cell lymphomas, lymphoma, acute myeloid leukaemia, chronic myeloid leukaemia, chronic lymphatic leukaemia, acute lymphoblastic leukaemia, non-Hodgkin's lymphoma, including follicular lymphoma and diffuse large B-cell lymphoma, hepatocellular carcinoma, multiple myeloma, bladder cancer, gastric cancer, ovarian cancer, head and neck cancer, pancreatic cancer, renal carcinoma, prostate cancer, hepatocellular carcinoma and lung cancer.

### Claim 17 (depends on 16)

The method according to claim 16, which further comprises administering a further anti-cancer therapeutic compound.

### Claim 18 (depends on 17)

The method according to claim 17, wherein the further anti-cancer therapeutic compound is a targeted therapeutic agent.

### Claim 19 (depends on 14)

The method according to claim 14, wherein said (i) anti-SIRPα antibody or the antigen-binding fragment thereof and (ii) therapeutic antibody are administered sequentially.

### Claim 20 (depends on 14)

The method according to claim 14, wherein said anti-SIRPα antibody or the antigen-binding fragment thereof, has a partly or completely disrupted Fc effector function.

### Claim 21 (depends on 1)

A nucleic acid molecule comprising a nucleotide sequence encoding the humanized anti-SIRPα antibody according to claim 1.

### Claim 22 (depends on 21)

A host cell comprising the nucleic acid molecule according to claim 21.