SIRP-ALPHA IMMUNOGLOBULIN FUSION PROTEINS

• US 20160177276A1
• Filed: 08/14/2015
• Published: 06/23/2016
• Est. Priority Date: 08/15/2014
• Status: Active Grant

• Current Assignee: Merck Patent GmbH (Merck & Co., Inc.)
• Original Assignee: Merck Patent GmbH (Merck & Co., Inc.)
• Inventors: Zizlsperger, Nora, Sircar, Aroop, Lo, Kin-Ming
• Granted Patent: US 11,021,694 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: C07K 14/70596 Molecules with a "CD"-designation not provided for elsewhere
• CPC Class Codes: C07K 16/2803 against the immunoglobulin superfamily
• CPC Class Codes: C07K 16/2827 against B7 molecules, e.g. CD80, CD86
• CPC Class Codes: C07K 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 16/2887 against CD20
• CPC Class Codes: C07K 16/30 from tumour cells
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 2317/31 multispecific
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C07K 2319/33 fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
• CPC Class Codes: C07K 2319/74 containing a fusion for binding to a cell surface receptor
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12Y 301/03048 Protein-tyrosine-phosphatase (3.1.3.48)

Abstract

The invention discloses immunoglobulin fusion proteins designed to bind both CD47 and a tumor cell antigen. The immunoglobulin fusion proteins include a SIRPα moiety that binds CD47 and an antigen binding site for a tumor cell antigen.

Claims

1. A SIRPα

immunoglobulin fusion protein comprising;

an IgV extracellular domain of SIRPα

or a SIRPα

variant comprising an amino acid sequence at least 80% identical to residues 3-115 of SEQ ID NO;

6 or to residues 3-114 of SEQ ID NO;

8; and

an immunoglobulin molecule or portion thereof that binds to a surface antigen on a disease promoting cell.

2. The SIRPα

immunoglobulin fusion protein of claim 1, wherein the disease promoting cell is a tumor cell and the surface antigen is a tumor antigen.

3. The immunoglobulin fusion protein of claim 1, wherein the SIRPα

variant comprises an amino acid sequence at least 85%, at least 90%, or at least 95% identical to residues 3-115 of SEQ ID NO;

6 or to residues 3-114 of SEQ ID NO;

8.

4. The immunoglobulin fusion protein of claim 1, wherein the SIRPα

variant comprises an amino acid sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to residues 1-115 of SEQ ID NO;

6 or to residues 1-114 of SEQ ID NO;

8.

5. The immunoglobulin fusion protein of claim 1, wherein the SIRPα

variant comprises an amino acid sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to a sequence selected from the group consisting of;

residues 1-114 of SEQ ID NO;

193, residues 1-115 of SEQ ID NO;

194, residues 1-115 of SEQ ID NO;

195, residues 1-115 of SEQ ID NO;

196, residues 1-114 of SEQ ID NO;

197, residues 1-114 of SEQ ID NO;

198, residues 1-115 of SEQ ID NO;

199, residues 1-114 of SEQ ID NO;

200, and residues 1-115 of SEQ ID NO;

190.

6. The immunoglobulin fusion protein of claim 1, wherein the IgV extracellular domain comprises a sequence selected from the group consisting of residues 1-115 of SEQ ID NO:

6, residues 1-114 of SEQ ID NO;

8, 1-114 of SEQ ID NO;

193, residues 1-115 of SEQ ID NO;

194, residues 1-115 of SEQ ID NO;

195, residues 1-115 of SEQ ID NO;

196, residues 1-114 of SEQ ID NO;

197, residues 1-114 of SEQ ID NO;

198, residues 1-115 of SEQ ID NO;

199, residues 1-114 of SEQ ID NO;

200, and residues 1-115 of SEQ ID NO;

190.

7. The immunoglobulin fusion protein of claim 1, wherein the SIRPα

variant comprises an amino acid sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to residues 1-343 of SEQ ID NO;

6.

8. The immunoglobulin fusion protein of claim 1, wherein the SIRPα

variant comprises a modification to an amino acid at one or more positions corresponding to positions 6, 27, 31, 37, 54, 56, 66, or 72 of SEQ ID NO;

6 or SEQ ID NO;

8.

9. The immunoglobulin fusion protein of claim 8, wherein the modification is a substitution selected from the group consisting of:

V6I;

V27I;

A27I;

I31R;

I31T;

Q37W;

Q37H;

E54P;

H56P;

S66Q;

L66Q; and

M72R.

20. The immunoglobulin fusion protein of claim 1, wherein the SIRPα

variant comprises a modification to an amino acid at one or more positions corresponding to positions 4, 6, 27, 31, 35, 37, 47, 52, 53, 54, 56, 66, 67, 68, 72, 92, or 94 of SEQ ID NO;

6 or SEQ ID NO;

8.

21. The immunoglobulin fusion protein of claim 20, wherein the modification is a substitution, deletion, or insertion of an amino acid.

23. The immunoglobulin fusion protein of claim 1, wherein the substitution is one or more selected from:

a substitution at a position corresponding to position 4 wherein the substitution is L4V;

a substitution at a position corresponding to position 6 of SEQ ID NO;

6 or SEQ ID NO;

8 selected from V6A, V6C, V6D, V6E, V6G, V6I, V6L, V6M, V6N, V6Q, V6S, or V6T;

a substitution at a position corresponding to position 27 of SEQ ID NO;

6 selected from A27C, A27D, A27G, A27H, A27I, A27L, A27N, A27K, A27Q, A27R, A27S, A27T, A27V, or corresponding to position 27 of SEQ ID NO;

8 selected from V27A, V27C, V27D, V27G, V27H, V27I, V27K, V27L, V27N, V27Q, V27R, V27S, V27T;

a substitution at a position corresponding to position 31 of SEQ ID NO;

6 or SEQ ID NO;

8 selected from I31A, I31C, I31E, I31K, I31Q, I31R, I31T, or I31V;

a substitution at a position corresponding to position 35 of SEQ ID NO;

6 or SEQ ID NO;

8 selected from P35A, P35C, P35E, P35G, P35N, P35Q, or P35S;

a substitution at a position corresponding to position 37 of SEQ ID NO;

6 or SEQ ID NO;

8 selected from Q37A, Q37C, Q37E, Q37G, Q37H, Q37K, Q37L, Q37M, Q37N, Q37R, Q37S, Q37T, or Q37W;

a substitution at a position corresponding to position 47 of SEQ ID NO;

6 or SEQ ID NO;

8 selected from E47A, E47C, E47D, E47F, E47G, E47H, E47I, E47K, E47L, E47M, E47N, E47Q, E47R, E47S, E47T, E47V, E47W, or E47Y;

a substitution at a position corresponding to position 52 of SEQ ID NO;

6 or SEQ ID NO;

8 selected from Q52A, Q52C, Q52E, Q52H, or Q52M;

a substitution at a position corresponding to position 53 wherein the substitution is K53R;

a substitution at a position corresponding to position 54 of SEQ ID NO;

6 or SEQ ID NO;

8 selected from E54D or E54P;

a substitution at a position corresponding to position 56 of SEQ ID NO;

6 or SEQ ID NO;

8 selected from H56A, H56C, H56D, H56E, H56F, H56G, H56I, H56K, H56L, H56M, H56N, H56P, H56Q, H56R, H56S, H56T, H56V, H56W, or H56Y;

a substitution at a position corresponding to position 66 of SEQ ID NO;

6 selected from L66A, L66C, L66D, L66E, L66F, L66G, L66H, L66I, L66K L66M, L66N, L66P, L66Q, L66S, L66T, L66V, L66W, L66Y;

or corresponding to position 66 of SEQ ID NO;

8 selected from S66A, S66C, S66D, S66E, S66F, S66G, S66H, S66I, S66I, S66K, S66L, S66M, S66N, S66P, S66Q, S66R, S66V, S66W, S66Y;

a substitution at a position corresponding to position 67 of SEQ ID NO;

6 or SEQ ID NO;

8 selected from T67A, T67C, T67D, T67E, T67F, T67G, T67H, T67I, T67L, T67M, T67N, T67Q, T67R, T67S, T67V, T67W, or T67Y;

a substitution at a position corresponding to position 68 wherein the substitution is K68R;

a substitution at a position corresponding to position 72 of SEQ ID NO;

6 or SEQ ID NO;

8 selected from M72A, M72C, M72D, M72E, M72F, M72G, M72H M72I, M72K, M72L, M72N M72Q, M72R, M72S, M72T, M72V, M72W, or M72Y;

a substitution at a position corresponding to position 92 of SEQ ID NO;

6 or SEQ ID NO;

8 selected from V92A, V92C, V92D, V92E, V92G, V92I, V92M, V92N, V92Q, V92R, V92S, or V92T; and

/or

a substitution at a position corresponding to position 94 wherein the substitution is F94L.

66. A nucleic acid or nucleic acids encoding the immunoglobulin fusion protein of any one of claim 1.

67. A cell comprising the nucleic acid or nucleic acids of claim 66.

68. A method of producing an immunoglobulin fusion protein by maintaining the cell of claim 67 under conditions that permit expression of the nucleic acid or nucleic acids.

69. A pharmaceutical composition comprising a pharmaceutically effective amount of the immunoglobulin fusion protein of claim 1 and a pharmaceutically acceptable carrier.

70. A method of treating cancer in a mammal comprising administering an effective amount the immunoglobulin fusion protein of claim 1 to a mammal in need thereof.

10-19. -19. (canceled)

22. (canceled)

24-40. -40. (canceled)

41. A SIRPα

immunoglobulin fusion protein comprising;

an IgV extracellular domain of SIRPα

or of a SIRPα

variant comprising an amino acid sequence at least 80% identical to residues 1-115 of SEQ ID NO;

190; and

an immunoglobulin molecule or portion thereof that binds to a surface antigen on a disease promoting cell.

42-54. -54. (canceled)

55. An immunoglobulin fusion protein comprising:

an immunoglobulin molecule or portion thereof that binds a tumor cell antigen; and

a CD47 binding moiety comprising an IgV extracellular domain of SIRPα

or a SIRPα

variant comprising an amino acid sequence at least 80% identical to residues 3-115 of SEQ ID NO;

6 or 3-114 of SEQ ID NO;

8;

wherein the fusion protein has a % red blood cell (RBC) binding mean fluorescence intensity (MFI) of 35% or less when % RBC binding MFI to an anti-CD47 antibody is calibrated at 100%, wherein the anti-CD47 antibody is B6H12/huIgG1, and

wherein the fusion protein also binds to a CD47 antigen on a non-red blood cell.

56-65. -65. (canceled)

71. (canceled)