ANTI-SIRP ALPHA ANTIBODIES

Abstract

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

Claims

1. A method for treating human solid tumours or haematological malignancies, which comprises administering an anti-SIRPα antibody, or an antigen-binding fragment thereof, to a patient in need thereof, wherein the anti-SIRPα antibody or the antigen-binding fragment thereof comprises heavy chain (HC) and light chain (LC) variable region (VR) complementarity determining regions (CDRs) selected from the group consisting of: (a) CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:3 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:4; (b) CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:5 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:6; (c) CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:7 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO: 8; (d) CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:9 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO: 10; (e) CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:11 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO: 12; (f) CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO: 13 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO: 14; (g) CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO: 15 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:16; and (h) CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO: 17 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO: 18; and wherein the CDRs are determined according to Kabat numbering.

2. The method according to claim 1, wherein the anti-SIRPα antibody is chimeric, humanized or human.

3. The method according to claim 2, wherein the anti-SIRPα antibody comprises HCVR and LCVR CDRs selected from the group consisting of: (a) CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:7 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO: 8; and (b) CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:13 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO: 14; and wherein the antibody is humanized.

4. The method according to claim 3, wherein the anti-SIRPα antibody comprises: (a) HCVR amino acid sequence of SEQ ID NO:30 and LCVR amino acid sequence of SEQ ID NO:31; (b) HCVR amino acid sequence of SEQ ID NO:32 and LCVR amino acid sequence of SEQ ID NO:33; (c) HCVR amino acid sequence of SEQ ID NO:34 and LCVR amino acid sequence of SEQ ID NO:8; (d) HCVR amino acid sequence of SEQ ID NO:35 and LCVR amino acid sequence of SEQ ID NO:36; (e) HCVR amino acid sequence of SEQ ID NO:35 and LCVR amino acid sequence of SEQ ID NO:37; (f) HCVR amino acid sequence of SEQ ID NO:13 and LCVR amino acid sequence of SEQ ID NO:38; or (g) HCVR amino acid sequence of SEQ ID NO:13 and LCVR amino acid sequence of SEQ ID NO:37.

5. The method according to claim 1, wherein the anti-SIRPα antibody comprises 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.

6. The method according to claim 1, wherein the anti-SIRPα antibody comprises a modified human IgG.sub.1 Fc region comprising one or more amino acid substitutions at one or more positions selected from the group consisting of L234, L235, G237, D265, D270, N297, A327, P328, and P329 according to Eu numbering.

7. The method according to claim 6, wherein the anti-SIRPα antibody comprises the amino acid substitutions L234A and L235A; L234E and L235A; L234A, L235A and P329A; or L234A, L235A and P329G.

8. The method according to claim 7, wherein the anti-SIRPα antibody comprises the amino acid substitutions L234A and L235A; or L234E and L235A.

9. The method according to claim 1, which further comprises administering to said patient one or more other anti-cancer therapeutics for use in the treatment of human solid tumours or haematological malignancies.

10. The method according to claim 9, wherein the one or more anti-cancer therapeutics are targeted therapeutics or immunotherapeutic agents.

11. The method according to claim 10, wherein the targeted therapeutic is a therapeutic antibody or an antibody-drug conjugate.

12. The method according to claim 11, wherein the therapeutic antibody is a therapeutic antibody against a membrane-bound target on the surface of tumour cells which comprises a human Fc region that binds to activating Fc receptors present on human immune effector cells.

13. The method according to claim 12, wherein the therapeutic antibody is a monospecific or bispecific antibody or antibody fragment comprising at least one HCVR and LCVR binding to a target selected from the group consisting of annexin Al, B7H3, B7H4, CA6, CA9, CA15-3, CA19-9, CA27-29, CA125, CA242, CCR2, CCR5, CD2, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD40, CD44, CD47, CD56, CD70, CD74, CD79, CD115, CD123, CD138, CD203c, CD303, CD333, CEA, CEACAM, CLCA-1, CLL-1, c-MET, Cripto, CTLA-4, DLL3, EGFL, EGFR, EPCAM, Eph, endothelin B receptor (ETBR), FAP, FcRL5 (CD307), FGF, FGFR, FOLR1, GCC, GPNMB, HER2, HMW-MAA, integrin α, IGF1R, TM4SF1, Lewis A, Lewis X, Lewis Y, LIV1, mesothelin, MUC1, MUC16, NaPi2b, Nectin-4, PD-1, PD-L1, PSMA, PTK7, SLC44A4, STEAP-1, 5T4 antigen, TF (tissue factor), Thomsen-Friedenreich antigen (TF-Ag), Tag72, TNF, TNFR, TROP2, VEGF, VEGFR, and VLA.

14. The method according to claim 13, wherein the therapeutic antibody is selected from the group consisting of alemtuzumab, bevacizumab, cetuximab, panitumumab, rituximab, and trastuzumab.

15. The method according to claim 11, wherein the antibody-drug conjugate is trastuzumab emtansine or brentuximab vedotin.

16. The method according to claim 1, wherein the human solid tumour is selected from the group consisting of breast cancer, renal cancer, or melanoma.

17. The method according to claim 1, wherein the haematological malignancy is Acute Myeloid Leukaemia.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1. Comparison of the ADCC measured in % cytotoxicity of trastuzumab (Tmab) alone, trastuzumab in combination with the murine 12C4 anti-SIRPα antibody (mu12C4), trastuzumab in combination with an antibody wherein murine 12C4 variable regions are grafted onto the human IgG.sub.1 constant region (12C4huIgG.sub.1), and trastuzumab in combination with an antibody wherein murine 12C4 variable regions are grafted onto the human IgG.sub.1 constant region comprising the amino acid substitutions L234A and L235A (12C4huIgG.sub.1LALA), measured on SKBR3 HER2-positive breast cancer cells using human neutrophils as effector cells.

[0017] FIG. 2. Comparison of % ADCC relative to trastuzumab (set to 100%) of trastuzumab in combination with the anti-SIRPα antibodies 1-9 having a human IgG.sub.1 constant region comprising the amino acid substitutions L234A and L235A, anti-SIRPα antibody 12C4huIgG.sub.1LALA (12C4LALA) and anti-CD47 antibody B6H12huIgG.sub.1LALA (B6H12LALA) on SKBR3 cells. Filled squares, (.square-solid.), are the values measured with neutrophils of donors having the SIRPα.sub.BIT variant, open circles, (∘), are the values measured with neutrophils of donors having the SIRPα.sub.1 variant. Columns are the average of all donors; error bars represent the standard deviation.

[0018] FIG. 3. Comparison of % ADCC relative to trastuzumab alone and trastuzumab in combination with the anti-SIRPα antibodies 4, 7, 10, 14 in various concentrations (dose response curves) having a human IgG.sub.1 constant region comprising the amino acid substitutions L234A and L235A, and anti-SIRPα antibody 12C4huIgG.sub.1LALA (12C4LALA) on SKBR3 cells. Neutrophils of two donors (Δ, ∘) having the SIRPα.sub.BIT variant. Columns are the average of the two donors.

[0019] FIG. 4. Comparison of % ADCC relative to trastuzumab alone and trastuzumab in combination with the anti-SIRPα antibodies 4, 7, 10, 13, 14, 15 and 16 having a human IgG1 constant region comprising the amino acid substitutions L234A and L235A, and anti-SIRPα antibody 12C4huIgG1LALA (12C4LALA) on SKBR3 cells. Neutrophils of donors having the SIRPα.sub.BIT variant (Δ, , ∇, ⋄), having the SIRPα.sub.1 variant (◯,) and neutrophils of a donor which variant was not determined (□) were used. Columns are the average of the donors.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0020] No approved therapeutics directed against SIRPα are available, although this target has been shown to play an important role in tumour immune evasion mechanisms. In addition, SIRPα is expressed on various malignant cells, rendering it a potential tumour associated antigen.

[0021] The present invention relates to antagonistic anti-SIRPα antibodies which exhibit specific binding to the two predominant SIRPα polymorphic variants SIRPα.sub.BIT and SIRPα.sub.1, that do not bind to SIRPγ and that increase the ADCC and/or ADCP of therapeutic antibodies.

[0022] The term “antibody” as used throughout the present specification refers to a monoclonal antibody (mAb) comprising two heavy chains and two light chains. Antibodies may be of any isotype such as IgA, IgE, IgG, or IgM antibodies. Preferably, the antibody is an IgG antibody, more preferably an IgG.sub.1 or IgG.sub.2 antibody. The antibodies may be chimeric, humanized or human. Preferably, the antibodies of the invention are humanized. Even more preferably, the antibody is a humanized or human IgG antibody, most preferably a humanized or human IgG.sub.1 mAb. The antibody may have κ (kappa) or λ (lambda) light chains, preferably κ (kappa) light chains, i.e., a humanized or human IgG.sub.1-κ antibody. The antibodies may comprise a constant region that is engineered, i.e. one or more mutations may be introduced to e.g. increase half-life, and/or increase or decrease effector function.

[0023] The terms “monoclonal antibody” and “mAb” as used herein refer to an antibody obtained from a population of substantially homogenous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Antibodies may be generated by immunizing animals with a mixture of peptides representing the desired antigen. B-lymphocytes are isolated and fused with myeloma cells or single B-lymphocytes are cultivated for several days in the presence of conditioned medium and feeder cells. The myeloma or B-lymphocyte supernatants containing the produced antibodies are tested to select suitable B-lymphocytes or hybridomas. Monoclonal antibodies may be prepared from suitable hybridomas by the hybridoma methodology first described by Kohler et al. Nature 1975, 256, 495-497. Alternatively, the RNA of suitable B-cells or lymphoma may be lysed, RNA may be isolated, reverse transcripted and sequenced. Antibodies may be made by recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in the art, e.g. in Clackson et al. Nature 1919, 352, 624-628 and Marks et al. J. Mol. Biol. 1991, 222, 581-597.

[0024] The term “antigen-binding fragment” as used throughout the present specification includes a Fab, Fab′ or F(ab′).sub.2 fragment, a single chain (sc) antibody, a scFv, a single domain (sd) antibody, a diabody, or a minibody.

[0025] In humanized antibodies, the antigen-binding complementarity determining regions (CDRs) in the variable regions (VRs) of the heavy chain (HC) and light chain (LC) are derived from antibodies from a non-human species, commonly mouse, rat or rabbit. These non-human CDRs are combined with human framework regions (FR1, FR2, FR3 and FR4) of the variable regions of the HC and LC, in such a way that the functional properties of the antibodies, such as binding affinity and specificity, are retained. Selected amino acids in the human FRs may be exchanged for the corresponding original non-human species amino acids to improve binding affinity, while retaining low immunogenicity. Alternatively, selected amino acids of the original non-human species FRs are exchanged for their corresponding human amino acids to reduce immunogenicity, while retaining the antibody's binding affinity. The thus humanized variable regions are combined with human constant regions.

[0026] The CDRs may be determined using the approach of Kabat (in Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., NIH publication no. 91-3242, pp. 662, 680, 689 (1991)), Chothia (Chothia et al., Nature 1989, 342, 877-883) or IMGT (Lefranc, The Immunologist 1999, 7, 132-136). In the context of the present invention, Eu numbering is used for indicating the positions in the heavy chain and light chain constant regions of the antibody. The expression “Eu numbering” refers to the Eu index as in Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., NIH publication no. 91-3242, pp. 662, 680, 689 (1991).

[0027] Antagonistic antibodies have affinity for a specific antigen, and binding of the antibody to its antigen inhibits the function of an agonist or inverse agonist at receptors. In the present case, binding of an antagonistic anti-SIRPα antibody to SIRPα will either prevent binding of CD47 to SIRPα or disrupt the inhibitory signal that is triggered by the CD47-SIRPα binding.

[0028] Antagonistic anti-SIRPα antibodies may bind to the same site where CD47 binds, preventing ligation of SIRPα by CD47 and consequently inhibiting the signalling that negatively regulates the Fc-receptor-dependent action of immune effector cells. Antagonistic anti-SIRPα antibodies may also bind to a site of SIRPα that is different from the binding site of CD47, i.e. an allosteric site, and inhibit the inhibitory signalling of SIRPα without direct interference with the physical CD47-SIRPα interaction, e.g. a change in the three-dimensional shape of SIRPα. This change in the three-dimensional shape prevents (downstream) signalling upon binding to CD47. When SIRPα is bound at an allosteric site, CD47 may still be bound by SIRPα, which might cause CD47 to be less available for binding to thrombospondin-1 (TSP-1). Ligation of TSP-1 to CD47 plays a role in e.g. negative regulation of T-cell activation (Soto-Pantoja et al. Crit. Rev. Biochem. Mol. Biol. 2015, 50(3), 212-230).

[0029] The term “binding affinity” as used throughout the present specification, refers to the dissociation constant (K.sub.D) of a particular antigen-antibody interaction. The K.sub.D is the ratio of the rate of dissociation (k.sub.off) to the association rate (k.sub.on). Consequently, K.sub.D equals k.sub.off/k.sub.on and is expressed as a molar concentration (M). It follows that the smaller the K.sub.D, the stronger the affinity of binding. Typically, K.sub.D values are determined by using surface plasmon resonance (SPR), typically using a biosensor system (e.g. Biacore®) using methods known in the art (e.g. E. S. Day et al. Anal. Biochem. 2013, 440, 96-107). The term “binding affinity” may also refer to the concentration of antibody that gives half-maximal binding (EC.sub.50) determined with e.g. an ELISA assay or as determined by flow cytometry.

[0030] The term “specific binding” as used throughout the present specification relates to binding between an antibody and its antigen with a K.sub.D of typically less than 10.sup.−7 M, such as 10.sup.−8 M, 10.sup.−9 M, 10.sup.−10 M, 10.sup.−11 M or even lower as determined by SPR at 25° C.

[0031] The term “low affinity” as used throughout the present specification is interchangeable with the phrases “does/do not bind” or “is/are not binding to”, and refers to a binding affinity between an antibody and its antigen with an EC.sub.50 larger than 1500 ng/ml as determined using an ELISA assay, or where no specific binding is observed between the immobilized antigen and the antibody as determined by SPR.

[0032] The term “high affinity” as used throughout the present specification and refers to a binding affinity between an antibody and its antigen with a K.sub.D of typically less than 10.sup.−10 M, 10.sup.−11 M or even lower as determined by SPR at 25° C.

[0033] In particular, the present invention relates to an anti-SIRPα antibody or an antigen-binding fragment thereof comprising heavy chain (HC) and light chain (LC) variable region (VR) complementarity determining regions (CDRs) selected from the group consisting of: [0034] a. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:1 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:2; [0035] b. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:3 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:4; [0036] c. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:5 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:6; [0037] d. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:7 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:8; [0038] e. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:9 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:10; [0039] f. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:11 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:12; [0040] g. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:13 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:14; [0041] h. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:15 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:16; and [0042] i. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:17 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:18,
wherein the CDRs are determined according to Kabat numbering.

[0043] Preferably, the present invention relates to an anti-SIRPα antibody or an antigen-binding fragment thereof comprising heavy chain (HC) and light chain (LC) variable region (VR) complementarity determining regions (CDRs) selected from the group consisting of: [0044] a. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:3 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:4; [0045] b. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:5 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:6; [0046] c. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:7 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:8; [0047] d. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:9 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:10; [0048] e. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:11 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:12; [0049] f. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:13 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:14; [0050] g. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:15 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:16; and [0051] h. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:17 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:18,
wherein the CDRs are determined according to Kabat numbering.

[0052] More preferably, the present invention relates to an anti-SIRPα antibody or an antigen-binding fragment thereof comprising HCVR and LCVR CDRs selected from the group consisting of: [0053] a. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:3 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:4; [0054] b. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:5 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:6; [0055] c. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:7 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:8; [0056] d. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:9 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:10; and [0057] e. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:13 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:14,
wherein the CDRs are determined according to Kabat numbering.

[0058] Even more preferably, the present invention relates to an anti-SIRPα antibody or an antigen-binding fragment thereof comprising HCVR and LCVR CDRs selected from the group consisting of: [0059] a. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:5 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:6; [0060] b. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:7 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:8; and [0061] c. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:13 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:14,
wherein the CDRs are determined according to Kabat numbering.

[0062] Most preferably, the present invention relates to an anti-SIRPα antibody or an antigen-binding fragment thereof comprising HCVR and LCVR CDRs selected from the group consisting of: [0063] a. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:7 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:8; and [0064] b. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:13 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:14,
wherein the CDRs are determined according to Kabat numbering.

[0065] In a preferred embodiment, the present invention relates to an anti-SIRPα antibody or an antigen-binding fragment thereof as defined hereinabove, wherein the antibody shows specific binding to both SIRPα.sub.BIT and SIRPα.sub.1 and does not bind to SIRPγ.

[0066] In a more preferred embodiment, the anti-SIRPα antibody or an antigen-binding fragment thereof specifically binds SIRPα.sub.BIT with a K.sub.D below 10.sup.−9 M and binds SIRPα.sub.1 with a K.sub.D below 10.sup.−7 M, wherein the K.sub.D is measured with SPR at 25° C. Preferably, the anti-SIRPα antibody or an antigen-binding fragment thereof binds SIRPα.sub.1 with a K.sub.D below 10.sup.−8 M.

[0067] In another more preferred embodiment, the anti-SIRPα antibody or an antigen-binding fragment thereof specifically binds SIRPα.sub.BIT and SIRPα.sub.1 with a K.sub.D below 10.sup.−9 M, wherein the K.sub.D is measured with SPR at 25° C.

[0068] In an even more preferred embodiment, the anti-SIRPα antibody or an antigen-binding fragment thereof specifically binds SIRPα.sub.BIT and SIRPα.sub.1 with a K.sub.D below 10.sup.−10 M. Preferably, the anti-SIRPα or an antigen-binding fragment thereof antibody specifically binds SIRPα.sub.BIT with a K.sub.D below 10.sup.−10 M and SIRPα.sub.1 with a K.sub.D below 10.sup.−11 M. Typically, the anti-SIRPα antibody as defined hereinabove is a chimeric, humanized or human antibody. Preferably, the anti-SIRPα antibody is a humanized or human antibody. More preferably, the anti-SIRPα antibody is a humanized antibody. In a particular embodiment, the humanized anti-SIRPα antibody or an antigen-binding fragment thereof according to the invention comprises a HCVR and a LCVR selected from the group consisting of: [0069] a. HCVR amino acid sequence of SEQ ID NO:30 and LCVR amino acid sequence of SEQ ID NO:31; [0070] b. HCVR amino acid sequence of SEQ ID NO:32 and LCVR amino acid sequence of SEQ ID NO:33; [0071] c. HCVR amino acid sequence of SEQ ID NO:34 and LCVR amino acid sequence of SEQ ID NO:8; [0072] d. HCVR amino acid sequence of SEQ ID NO:35 and LCVR amino acid sequence of SEQ ID NO:36; [0073] e. HCVR amino acid sequence of SEQ ID NO:35 and LCVR amino acid sequence of SEQ ID NO:37; [0074] f. HCVR amino acid sequence of SEQ ID NO: 13 and LCVR amino acid sequence of SEQ ID NO:38; and [0075] g. HCVR amino acid sequence of SEQ ID NO: 13 and LCVR amino acid sequence of SEQ ID NO:37.

[0076] In a preferred embodiment, the humanized anti-SIRPα antibody or an antigen-binding fragment thereof comprises HCVR amino acid sequence of SEQ ID NO:30 and LCVR amino acid sequence of SEQ ID NO:31.

[0077] In another preferred embodiment, the humanized anti-SIRPα antibody or an antigen-binding fragment thereof comprises HCVR amino acid sequence of SEQ ID NO:32 and LCVR amino acid sequence of SEQ ID NO:33.

[0078] In yet another preferred embodiment, the humanized anti-SIRPα antibody or an antigen-binding fragment thereof comprises HCVR amino acid sequence of SEQ ID NO:34 and LCVR amino acid sequence of SEQ ID NO:8.

[0079] In yet another preferred embodiment, the humanized anti-SIRPα antibody or an antigen-binding fragment thereof comprises HCVR amino acid sequence of SEQ ID NO:35 and LCVR amino acid sequence of SEQ ID NO:36.

[0080] In yet another preferred embodiment, the humanized anti-SIRPα antibody or an antigen-binding fragment thereof comprises HCVR amino acid sequence of SEQ ID NO:35 and LCVR amino acid sequence of SEQ ID NO:37.

[0081] In yet another preferred embodiment, the humanized anti-SIRPα antibody or an antigen-binding fragment thereof comprises HCVR amino acid sequence of SEQ ID NO:13 and LCVR amino acid sequence of SEQ ID NO:38.

[0082] In yet another preferred embodiment, the humanized anti-SIRPα antibody or an antigen-binding fragment thereof comprises HCVR amino acid sequence of SEQ ID NO:13 and LCVR amino acid sequence of SEQ ID NO:37.

[0083] Besides binding to both human (hu)SIRPα.sub.BIT and (hu)SIRPα.sub.1, the antibodies according to the invention may also bind to cynomolgus monkey (cy)SIRPα, enabling in vivo studies in a relevant animal model.

[0084] The antibodies according to the invention may bind to a site of SIRPα that is different from the binding site of CD47, i.e. an allosteric site and inhibit the inhibitory signalling of SIRPα without direct interference with the physical CD47-SIRPα interaction. Alternatively, the antibodies may bind to the same site where CD47 binds, preventing ligation of SIRPα by CD47 and consequently inhibiting the signalling that negatively regulates the Fc-receptor-dependent action of immune effector cells.

[0085] The anti-SIRPα antibodies or antigen-binding fragments thereof as described hereinabove are more specific than known anti-SIRPα antibodies, and show excellent affinity for both SIRPα.sub.BIT and SIRPα.sub.1. As well, the anti-SIRPα antibodies according to the invention do not bind to SIRPγ.

[0086] In one particular embodiment, the anti-SIRPα antibody according to the invention comprises an Fc region that binds to activating Fc receptors present on human immune effector cells. Such anti-SIRPα antibody is suitable for monotherapy of SIRPα-positive human solid tumours and haematological malignancies as it can induce ADCC and/or ADCP. Human immune effector cells possess a variety of activating Fc receptors, which upon ligation trigger phagocytosis, cytokine release, ADCC and/or ADCP, etc. Examples of these receptors are Fcγ receptors, e.g. FcγRI (CD64), FcγRIIA (CD32), FcγRIIIA (CD16a), FcγRIIIB (CD16b), FcγRIIC and the Fcα receptor FcαRI (CD89). The various natural antibody isotypes bind to these receptors. E.g. IgG.sub.1 binds to FcγRI, FcγRIIA, FcγRIIC, FcγRIIIA, FcγRIIIB; IgG.sub.2 binds to FcγRIIA, FcγRIIC, FcγRIIIA; IgG.sub.3 binds to FcγRI, FcγRIIA, FcγRIIC, FcγRIIIA, FcγRIIIB; IgG.sub.4 binds to FcγRI, FcγRIIA, FcγRIIC, FcγRIIIA; and IgA binds to FcαRI.

[0087] In a preferred embodiment, the anti-SIRPα antibody according to the invention comprises an Fc region of the IgA or IgG isotype. More preferred is an anti-SIRPα antibody comprising an Fc region of the IgG.sub.1, IgG.sub.2, IgG.sub.3 or IgG.sub.4 isotype; the IgG.sub.1, IgG.sub.2 or IgG.sub.4 isotype is even more preferred. Most preferred is an anti-SIRPα antibody comprising an Fc region of the IgG.sub.1 isotype.

[0088] Although the anti-SIRPα antibodies comprising an Fc region that binds to activating Fc receptors present on human immune effector cells may be suitable to treat cancers expressing SIRPα, chimeric anti-SIRPα IgG.sub.1 antibodies did not show the expected results when tested in vitro in combination with other antibodies that comprise a human Fc region that binds to activating Fc receptors present on human immune effector cells (i.e. antibodies that are able to induce ADCC and/or ADCP). Results of in vitro ADCC assays showed that a chimeric IgG.sub.1 anti-SIRPα antibody does not increase the ADCC of such other antibody as much as expected on the basis of earlier results using murine antibodies.

[0089] Therefore, the invention relates to anti-SIRPα antibodies that exhibit reduced binding to or low affinity for activating Fc receptors present on human immune effector cells. Such anti-SIRPα antibodies comprise a modified Fc region in which one or more amino acids have been substituted by (an)other amino acid(s) when compared to a similar unmodified Fc region. Reduced binding means that the affinity of the anti-SIRPα antibody comprising a modified Fc region for the activating Fc receptors is less than the affinity of an anti-SIRPα antibody with the same variable regions comprising a similar unmodified Fc region. The binding affinity of antibodies for activating Fc receptors is typically measured using Surface Plasmon Resonance (SPR) or flow cytometry using methods known in the art, e.g. the method of Harrison et al. in J. Pharm. Biomed. Anal. 2012, 63, 23-28. Antibodies exhibiting reduced binding to or low affinity for the human Fcα or Fcγ receptor in combination with a therapeutic antibody are especially effective in cellular destruction of cancer cells by increasing ADCC and/or ADCP of effector immune effector cells. Typically, the Fc region of the anti-SIRPα antibody according to the invention is modified to reduce binding to activating Fc receptors present on human immune effector cells.

[0090] Therefore, the anti-SIRPα antibody according to the invention comprises a modified Fc region that exhibits reduced binding to or low affinity for a human Fcα or Fcγ receptor. For instance, the IgG.sub.1 binding to an Fcγ receptor can be reduced by substituting one or more IgG.sub.1 amino acids selected from the group consisting of L234, L235, G237, D265, D270, N297, A327, P328, and P329 (Eu numbering); the IgG.sub.2 binding can be reduced by introducing e.g. one or more of the following amino acid substitutions V234A, G237A, P238S, H268A, V309L, A330S, and P331S; or H268Q, V309L, A330S, and P331S (numbering analogue to IgG.sub.1 Eu numbering) (Vafa et al. Methods 2014, 65, 114-126); the IgG.sub.3 binding can be reduced by introducing e.g. the amino acid substitutions L234A and L235A, or the amino acid substitutions L234A, L235A and P331S (Leoh et al. Mol. Immunol. 2015, 67, 407-415); and the IgG.sub.4 binding can be reduced by introducing e.g. the amino acid substitutions S228P, F234A and L235A ((numbering analogue to IgG.sub.1 Eu numbering) (Parekh et al. mAbs 2012, 4(3), 310-318). IgA binding to the Fcα receptor can be reduced by introducing e.g. one or more of the amino acid substitutions L257R, P440A, A442R, F443R, and P440R (sequential numbering, Pleass et al. J. Biol. Chem. 1999, 271(33), 23508-23514).

[0091] Preferably, the anti-SIRPα antibody according to the invention comprises a modified Fc region that exhibits reduced binding to or low affinity for a human Fcγ receptor. More preferably, the modified Fc region is an Fc region of the IgG isotype. Even more preferably, the modified Fc region is an Fc region of the IgG.sub.1, IgG.sub.2 or IgG.sub.4 isotype.

[0092] In a preferred embodiment, the anti-SIRPα antibody according to the invention comprises a modified human IgG.sub.1 Fc region comprising one or more amino acid substitutions at one or more positions selected from the group consisting of L234, L235, G237, D265, D270, N297, A327, P328, and P329 (Eu numbering).

[0093] Preferably, the anti-SIRPα antibody comprises a modified Fc IgG.sub.1 region, which does not comprise either amino acid substitution N297A or N297G. More preferably, the anti-SIRPα antibody comprises a modified Fc IgG.sub.1 region, which does not comprise an amino acid substitution at position N297.

[0094] In one embodiment, the modified human IgG.sub.1 Fc region comprises one or more amino acid substitutions selected from the group consisting of L234A, L234E, L235A, G237A, D265A, D265E, D265N, D270A, D270E, D270N, N297A, N297G, A327Q, P328A, P329A and P329G. Preferably, the one or more amino acid substitutions are selected from the group consisting of L234A, L234E, L235A, G237A, D265A, D265E, D265N, N297A, P328A, P329A and P329G.

[0095] In another embodiment, the modified human IgG.sub.1 Fc region comprises one or more amino acid substitutions selected from the group consisting of L234A, L234E, L235A, G237A, D265A, D265E, D265N, D270A, D270E, D270N, A327Q, P328A, P329A and P329G. Preferably, the one or more amino acid substitutions are selected from the group consisting of L234A, L234E, L235A, G237A, D265A, D265E, D265N, P328A, P329A and P329G. More preferably, the modified Fe IgG.sub.1 region does not comprise either amino acid substitution N297A or N297G. Even more preferably, the modified Fc IgG.sub.1 region does not comprise an amino acid substitution at position N297.

[0096] In a preferred embodiment, the modified human IgG.sub.1 Fc region comprises the amino acid substitutions L234A and L235A, L234E and L235A, L234A, L235A and P329A or L234A, L235A and P329G. Preferably, the modified Fc IgG.sub.1 region does not comprise either amino acid substitution N297A or N297G. More preferably, the modified Fc IgG.sub.1 region does not comprise an amino acid substitution at position N297.

[0097] In another preferred embodiment, the anti-SIRPα antibody according to the invention comprises a modified human IgG.sub.1 Fc region comprising the amino acid substitutions L234A and L235A or L234E and L235A, preferably amino acid substitutions L234A and L235A. More preferably, the modified Fc IgG.sub.1 region does not comprise either amino acid substitution N297A or N297G. Even more preferably, the modified Fc IgG.sub.1 region does not comprise an amino acid substitution at position N297.

[0098] The present invention further relates to a pharmaceutical composition comprising an anti-SIRPα antibody as described hereinabove and one or more pharmaceutically acceptable excipients. Typical pharmaceutical formulations of therapeutic proteins such as antibodies take the form of lyophilized cakes (lyophilized powders), which require (aqueous) dissolution (i.e. reconstitution) before intravenous infusion, or frozen (aqueous) solutions, which require thawing before use.

[0099] Typically, the pharmaceutical composition is provided in the form of a lyophilized cake. Suitable pharmaceutically acceptable excipients for inclusion into the pharmaceutical composition (before freeze-drying) in accordance with the present invention include buffer solutions (e.g. citrate, histidine or succinate containing salts in water), lyoprotectants (e.g. sucrose, trehalose), tonicity modifiers (e.g. sodium chloride), surfactants (e.g. polysorbate), and bulking agents (e.g. mannitol, glycine). Excipients used for freeze-dried protein formulations are selected for their ability to prevent protein denaturation during the freeze-drying process as well as during storage.

[0100] The present invention further relates to an anti-SIRPα antibody or pharmaceutical composition as described hereinabove for use as a medicament.

[0101] In one embodiment, the present invention relates to an anti-SIRPα antibody or pharmaceutical composition as described hereinabove for use in the treatment of human solid tumours and haematological malignancies. The anti-SIRPα antibodies of the invention may be used in the treatment of solid tumours, such as breast cancer, renal cancer, or melanoma, or haematological malignancies, such as Acute Myeloid Leukaemia (AML).

[0102] In a second embodiment, the invention relates to an anti-SIRPα antibody comprising an Fc region that binds to activating Fc receptors present on human immune effector cells for use in the treatment of SIRPα-positive human solid tumours and haematological malignancies. Preferably, the Fc region that binds to activating Fc receptors present on human immune effector cells is of the IgA or IgG isotype. More preferred is an anti-SIRPα antibody comprising an Fc region of the IgG.sub.1, IgG.sub.2, IgG.sub.3 or IgG.sub.4 isotype; the IgG.sub.1, IgG.sub.2 or IgG.sub.4 isotype is even more preferred. Most preferred is an anti-SIRPα antibody comprising an Fc region of the IgG.sub.1 isotype for use in the treatment of SIRPα-positive human solid tumours and haematological malignancies.

[0103] In a third embodiment, the present invention relates to an anti-SIRPα antibody or pharmaceutical composition as described hereinabove for use in the treatment of human solid tumours and haematological malignancies in combination with the use of one or more other anti-cancer therapies. Suitable anti-cancer therapies are surgery, chemotherapy, radiation therapy, hormonal therapy, targeted therapy and immunotherapy. The anti-SIRPα antibody or pharmaceutical composition as described hereinabove may be for concomitant or sequential use in the treatment of human solid tumours and haematological malignancies in combination with the use of one or more other anti-cancer therapies. In particular, the anti-SIRPα antibody or pharmaceutical composition as described hereinabove may be for use in the treatment of human solid tumours and haematological malignancies after the use of one or more other anti-cancer therapies.

[0104] Preferably, the present invention relates to an anti-SIRPα antibody or pharmaceutical composition as described hereinabove for use in the treatment of human solid tumours and haematological malignancies in combination with the use of one or more other anti-cancer therapeutics. In particular, the anti-SIRPα antibody or pharmaceutical composition as described hereinabove may be for use in the treatment of human solid tumours and haematological malignancies after the use of one or more other anti-cancer therapeutics.

[0105] Suitable anti-cancer therapeutics include chemotherapeutics, radiation therapeutics, hormonal therapeutics, targeted therapeutics and immunotherapeutic agents. Suitable chemotherapeutics include alkylating agents, such as nitrogen mustards, nitrosoureas, tetrazines and aziridines; anti metabolites, such as anti-folates, fluoropyrimidines, deoxynucleoside analogues and thiopurines; anti-microtubule agents, such as vinca alkaloids and taxanes; topoisomerase I and II inhibitors; and cytotoxic antibiotics, such as anthracyclines and bleomycins.

[0106] Suitable radiation therapeutics include radio isotopes, such as .sup.131I-metaiodobenzylguanidine (MIBG), .sup.32P as sodium phosphate, .sup.223Ra chloride, .sup.89Sr chloride and .sup.153Sm diamine tetramethylene phosphonate (EDTMP).

[0107] Suitable agents to be used as hormonal therapeutics include inhibitors of hormone synthesis, such as aromatase inhibitors and GnRH analogues; and hormone receptor antagonists, such as selective oestrogen receptor modulators and antiandrogens.

[0108] Targeted therapeutics are therapeutics that interfere with specific proteins involved in tumorigenesis and proliferation and may be small molecule drugs; proteins, such as therapeutic antibodies; peptides and peptide derivatives; or protein-small molecule hybrids, such as antibody-drug conjugates. Examples of targeted small molecule drugs include mTor inhibitors, such as everolimus, temsirolimus and rapamycin; kinase inhibitors, such as imatinib, dasatinib and nilotinib; VEGF inhibitors, such as sorafenib and regorafenib; and EGFR/HER2 inhibitors such as gefitinib, lapatinib and erlotinib. Examples of peptide or peptide derivative targeted therapeutics include proteasome inhibitors, such as bortezomib and carfilzomib.

[0109] Immunotherapeutic agents include agents that induce, enhance or suppress an immune response, such as cytokines (IL-2 and IFN-α); immuno modulatory imide drugs, such as thalidomide, lenalidomide and pomalidomide; therapeutic cancer vaccins, such as talimogene laherparepvec; cell based immunotherapeutic agents, such as dendritic cell vaccins, adoptive T-cells and chimeric antigen receptor-modified T-cells); and therapeutic antibodies that can trigger ADCC/ADCP or CDC via their Fc region when binding to membrane bound ligands on a cancer cell.

[0110] Preferably, the invention relates to an anti-SIRPα antibody or pharmaceutical composition as described hereinabove for use in the treatment of human solid tumours and haematological malignancies in combination with one or more other anti-cancer therapeutics, wherein the anti-cancer therapeutic is a targeted therapeutic or an immunotherapeutic agent. A preferred targeted therapeutic in accordance with the invention is a therapeutic antibody or an antibody-drug conjugate (ADC). The most preferred targeted therapeutic is a therapeutic antibody.

[0111] The term “therapeutic antibody” as used throughout the present specification refers to an antibody or an antigen-binding fragment thereof as defined hereinabove, which is suitable for human therapy. Antibodies suitable for human therapy are of sufficient quality, safe and efficacious for treatment of specific human diseases. Quality may be assessed using the established guidelines for Good Manufacturing Practice; safety and efficacy are typically assessed using established guidelines of medicines regulatory authorities, e.g. the European Medicines Agency (EMA) or the United States Food and Drug Administration (FDA). These guidelines are well-known in the art.

[0112] Preferably, the therapeutic antibody is an antibody approved by a medicines regulatory authority, such as the EMA or FDA. Online databases of most Regulatory Authorities can be consulted to find whether an antibody is approved.

[0113] The term “ADC” as used throughout the present specification refers to a cytotoxic drug conjugated to an antibody or an antigen-binding fragment thereof as defined hereinabove via a linker. Typically, the cytotoxic drugs are highly potent, e.g. a duocarmycin, calicheamicin, pyrrolobenzodiazepine (PBD) dimer, maytansinoid or auristatin derivative. The linker may be cleavable, e.g. comprising the cleavable dipeptide valine-citrulline (vc) or valine-alanine (va), or non-cleavable, e.g. succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC).

[0114] Typically, the therapeutic antibody for use in combination with an anti-SIRPα antibody according to the invention is a monospecific or bispecific antibody or antibody fragment comprising at least one HCVR and LCVR binding to a target selected from the group consisting of annexin Al, B7H3, B7H4, CA6, CA9, CA15-3, CA19-9, CA27-29, CA125, CA242, CCR2, CCR5, CD2, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD40, CD44, CD47, CD56, CD70, CD74, CD79, CD115, CD123, CD138, CD203c, CD303, CD333, CEA, CEACAM, CLCA-1, CLL-1, c-MET, Cripto, CTLA-4, DLL3, EGFL, EGFR, EPCAM, EPh (e.g. EphA2 or EPhB3), endothelin B receptor (ETBR), FAP, FcRL5 (CD307), FGF, FGFR (e.g. FGFR3), FOLR1, GCC, GPNMB, HER2, HMW-MAA, integrin α (e.g. αvβ3 and αvβ5), IGF1R, TM4SF1 (or L6 antigen), Lewis A like carbohydrate, Lewis X, Lewis Y, LIV1, mesothelin, MUC1, MUC16, NaPi2b, Nectin-4, PD-1, PD-L1, PSMA, PTK7, SLC44A4, STEAP-1, 5T4 antigen (or TPBG, trophoblast glycoprotein), TF (tissue factor), Thomsen-Friedenreich antigen (TF-Ag), Tag72, TNF, TNFR, TROP2, VEGF, VEGFR, and VLA.

[0115] Preferred is a monospecific therapeutic antibody. More preferred is an antibody against a membrane-bound target on the surface of tumour cells.

[0116] Suitable therapeutic antibodies for use in combination with an anti-SIRPα antibody according to the invention include alemtuzumab, bevacizumab, cetuximab, panitumumab, rituximab, and trastuzumab.

[0117] Suitable ADCs for use in combination with an anti-SIRPα antibody according to the invention include trastuzumab emtansine and brentuximab vedotin.

[0118] In a preferred embodiment, the present invention relates to an anti-SIRPα antibody as described hereinabove for the aforementioned use in combination with a therapeutic antibody against a membrane-bound target on the surface of tumour cells which comprises a human Fc region that binds to activating Fc receptors present on human immune effector cells.

[0119] Via binding to these activating Fc receptors, described hereinabove, a therapeutic antibody comprising a human Fc region that binds to activating Fc receptors present on human immune effector cells can induce ADCC and/or ADCP. Therapeutic antibodies of the human IgG, IgE, or IgA isotype comprise a human Fc region that binds to activating Fc receptors present on human immune effector cells.

[0120] A preferred therapeutic antibody for use according to the invention is a therapeutic antibody of the IgG or IgA isotype. More preferred is a therapeutic antibody of the IgG isotype, such as IgG.sub.1, IgG.sub.2, IgG.sub.3, and IgG.sub.4 antibodies. Even more preferred is a therapeutic antibody of the IgG.sub.1 or IgG.sub.2 isotype. Most preferred is a therapeutic antibody of the IgG.sub.1 isotype.

[0121] Preferably, the present invention relates to a humanized anti-SIRPα antibody comprising HCVR and LCVR CDRs selected from the group consisting of: [0122] a. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:1 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:2; [0123] b. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:3 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:4; [0124] c. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:5 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:6; [0125] d. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:7 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:8; [0126] e. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:9 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:10; [0127] f. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:11 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:12; [0128] g. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:13 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:14; [0129] h. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:15 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:16; and [0130] i. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:17 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:18,
for use in the treatment of human solid tumours and haematological malignancies in combination with the use of a therapeutic antibody against a membrane-bound target on the surface of tumour cells, which comprises a human Fc region that binds to activating Fc receptors present on human immune effector cells, wherein the anti-SIRPα antibody comprises a modified Fc region that exhibits reduced binding to a human Fcα or Fcγ receptor, when compared to the same anti-SIRPα antibody comprising a wild-type Fc region.

[0131] In a preferred embodiment, the humanized anti-SIRPα antibody for use in the treatment of human solid tumours and haematological malignancies in combination with the therapeutic antibody, comprises a modified human IgG.sub.1 Fc region comprising one or more amino acid substitutions at one or more positions selected from the group consisting of L234, L235, G237, D265, D270, N297, A327, P328, and P329 (Eu numbering).

[0132] Preferably, the humanized anti-SIRPα antibody for use in the treatment of human solid tumours and haematological malignancies in combination with the therapeutic antibody comprises a modified Fe IgG.sub.1 region, which does not comprise either amino acid substitution N297A or N297G. More preferably, the anti-SIRPα antibody comprises a modified Fe IgG.sub.1 region, which does not comprise an amino acid substitution at position N297.

[0133] In one embodiment, the modified human IgG.sub.1 Fc region comprises one or more amino acid substitutions selected from the group consisting of L234A, L234E, L235A, G237A, D265A, D265E, D265N, D270A, D270E, D270N, N297A, N297G, A327Q, P328A, P329A, and P329G.

[0134] In another embodiment, the humanized anti-SIRPα antibody for use in the treatment of human solid tumours and haematological malignancies in combination with the therapeutic antibody comprises a modified Fe IgG.sub.1 region comprising one or more amino acid substitutions selected from the group consisting of L234A, L234E, L235A, G237A, D265A, D265E, D265N, D270A, D270E, D270N, A327Q, P328A, P329A and P329G. Preferably, the one or more amino acid substitutions are selected from the group consisting of L234A, L234E, L235A, G237A, D265A, D265E, D265N, P328A, P329A and P329G. More preferably, the modified Fe IgG.sub.1 region does not comprise either amino acid substitution N297A or N297G. Even more preferably, the modified Fe IgG.sub.1 region does not comprise an amino acid substitution at position N297.

[0135] In a preferred embodiment, the modified human IgG.sub.1 Fc region comprises the amino acid substitutions L234A and L235A, L234E and L235A, L234A, L235A and P329A or L234A, L235A and P329G. Preferably, the modified Fe IgG.sub.1 region does not comprise either amino acid substitution N297A or N297G. More preferably, the modified Fe IgG.sub.1 region does not comprise an amino acid substitution at position N297.

[0136] In another preferred embodiment, the humanized anti-SIRPα antibody for use in the treatment of human solid tumours and haematological malignancies in combination with the therapeutic antibody comprises a modified human IgG.sub.1 Fc region comprising the amino acid substitutions L234A and L235A or L234E and L235A, preferably amino acid substitutions L234A and L235A. More preferably, the modified Fe IgG.sub.1 region does not comprise either amino acid substitution N297A or N297G. Even more preferably, the modified Fe IgG.sub.1 region does not comprise an amino acid substitution at position N297.

[0137] In a preferred embodiment, the humanized anti-SIRPα antibody for use in the treatment of human solid tumours and haematological malignancies in combination with the use of a therapeutic antibody against a membrane-bound target on the surface of tumour cells which comprises a human Fc region that binds to activating Fc receptors present on human immune effector cells, comprises an Fc region comprising the amino acid substitutions L234A and L235A, and HCVR and LCVR CDRs selected from the group consisting of: [0138] a. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:3 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:4; [0139] b. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:5 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:6; [0140] c. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:7 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:8; [0141] d. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:9 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:10; and [0142] e. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:13 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:14.

[0143] In a second preferred embodiment, the humanized anti-SIRPα antibody for use as defined hereinabove comprises an Fc region comprising the amino acid substitutions L234A and L235A, and HCVR and LCVR CDRs selected from the group consisting of: [0144] a. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:5 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:6; [0145] b. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:7 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:8; and [0146] c. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:13 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:14.

[0147] In a third preferred embodiment, the humanized anti-SIRPα antibody for use as defined hereinabove comprises an Fc region comprising the amino acid substitutions L234A and L235A, and HCVR and LCVR CDRs selected from the group consisting of: [0148] a. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:7 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:8; and [0149] b. CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:13 and CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO:14.

[0150] In a fourth preferred embodiment, the humanized anti-SIRPα antibody for use as defined hereinabove comprises an Fc region comprising the amino acid substitutions L234A and L235A, and [0151] a. HCVR amino acid sequence of SEQ ID NO:30 and LCVR amino acid sequence of SEQ ID NO:31; [0152] b. HCVR amino acid sequence of SEQ ID NO:32 and LCVR amino acid sequence of SEQ ID NO:33; [0153] c. HCVR amino acid sequence of SEQ ID NO:34 and LCVR amino acid sequence of SEQ ID NO:8; [0154] d. HCVR amino acid sequence of SEQ ID NO:35 and LCVR amino acid sequence of SEQ ID NO:36; [0155] e. HCVR amino acid sequence of SEQ ID NO:35 and LCVR amino acid sequence of SEQ ID NO:37; [0156] f. HCVR amino acid sequence of SEQ ID NO: 13 and LCVR amino acid sequence of SEQ ID NO:38; or [0157] g. HCVR amino acid sequence of SEQ ID NO: 13 and LCVR amino acid sequence of SEQ ID NO:37.

[0158] In one preferred embodiment, the humanized anti-SIRPα antibody for use as defined hereinabove comprises an Fc region comprising the amino acid substitutions L234A and L235A, and HCVR amino acid sequence of SEQ ID NO:30 and LCVR amino acid sequence of SEQ ID NO:31. More preferably, the modified Fc IgG.sub.1 region does not comprise either amino acid substitution N297A or N297G. Even more preferably, the modified Fc IgG.sub.1 region does not comprise an amino acid substitution at position N297.

[0159] In another preferred embodiment, the humanized anti-SIRPα antibody for use as defined hereinabove comprises an Fc region comprising the amino acid substitutions L234A and L235A, and HCVR amino acid sequence of SEQ ID NO:32 and LCVR amino acid sequence of SEQ ID NO:33.

[0160] In yet another preferred embodiment, the humanized anti-SIRPα antibody for use as defined hereinabove comprises an Fc region comprising the amino acid substitutions L234A and L235A, and HCVR amino acid sequence of SEQ ID NO:34 and LCVR amino acid sequence of SEQ ID NO:8.

[0161] In yet another preferred embodiment, the humanized anti-SIRPα antibody for use as defined hereinabove comprises an Fc region comprising the amino acid substitutions L234A and L235A, and HCVR amino acid sequence of SEQ ID NO:35 and LCVR amino acid sequence of SEQ ID NO:36.

[0162] In yet another preferred embodiment, the humanized anti-SIRPα antibody for use as defined hereinabove comprises an Fc region comprising the amino acid substitutions L234A and L235A, and HCVR amino acid sequence of SEQ ID NO:35 and LCVR amino acid sequence of SEQ ID NO:37.

[0163] In yet another preferred embodiment, the humanized anti-SIRPα antibody for use as defined hereinabove comprises an Fc region comprising the amino acid substitutions L234A and L235A, and HCVR amino acid sequence of SEQ ID NO:13 and LCVR amino acid sequence of SEQ ID NO:38.

[0164] In yet another preferred embodiment, the humanized anti-SIRPα antibody for use as defined hereinabove comprises an Fc region comprising the amino acid substitutions L234A and L235A, and HCVR amino acid sequence of SEQ ID NO:13 and LCVR amino acid sequence of SEQ ID NO:37.

[0165] More preferably, the humanized anti-SIRPα antibodies as defined hereinabove for use as defined hereinabove comprising an Fc region comprising the amino acid substitutions L234A and L235A, the modified Fc IgG.sub.1 region do not comprise either amino acid substitution N297A or N297G. Even more preferably, the modified Fc IgG.sub.1 region does not comprise an amino acid substitution at position N297.

[0166] The anti-SIRPα antibodies comprising a modified Fc region that exhibits reduced binding to a human Fcα or Fcγ receptor, when compared to the same anti-SIRPα antibody comprising a wild-type Fc region as described hereinabove enhance the in vitro ADCC of a therapeutic antibody using neutrophils as effector cells from different donors homozygous for either SIRPα.sub.BIT or SIRPα.sub.1. All of these antibodies increase the in vitro ADCC using neutrophils of most donors, the preferred antibodies even increase in vitro ADCC using neutrophils of all donors.

Examples

[0167] Immunization Protocol and Selection

[0168] Rabbits were repeatedly immunized with a mixture of peptides representing the extra cellular domain region of human (hu)SIRPα.sub.BIT, human (hu)SIRPα.sub.1 and cynomolgus (cy)SIRPα. Blood was collected at different time points and enriched with lymphocytes.

[0169] Single B-cells were deposited into single wells of microtiter plates. These B-cells were cultivated for several days in the presence of conditioned medium and feeder cells. During this time they produced and released monoclonal antibodies into the cultivation medium (B-cell supernatants). The supernatants of these single B-cells were analyzed for IgG production; subsequently the specific binding huSIRPα.sub.BIT and huSIRPα.sub.1, to cySIRPα and to an anti-Fc antibody was determined. Suitable supernatants were those binding to both huSIRPα.sub.BIT and huSIRPα.sub.1 and to cySIRPα. After a hit picking step binding to mouse (mu) SIRPα and to huSIRPβ.sub.1v1, huSIRPβ.sub.1v2 and huSIRPγ (as anti-targets) was measured. In addition, the binding to SIRPα.sub.BIT and SIRPα.sub.1-over expressing CHO cells was determined. Binding to parental CHO cells was applied as a control assay.

[0170] Suitable B-cell lysates were selected for RNA isolation, reverse transcription and sequencing. The unique variable regions of antibody light and heavy chains were gene synthesized and cloned in front of the antibody constant region sequence (kappa LC SEQ ID NO:26 and human IgG.sub.1 HC-LALA format SEQ ID NO:27), respectively.

[0171] HEK 293 cells were transiently transfected with the antibody sequence containing plasmids using an automated procedure on a Tecan Freedom Evo platform. Immunoglobulins were purified from the cell supernatant using affinity purification (Protein A) on a Dionex Ultimate 3000 HPLC system with a plate autosampler. The produced antibodies were tested in ELISA-type assays (ELISA: huSIRPα.sub.1, huSIRPα.sub.BIT, cySIRPα, muSIRPα, huSIRPβ.sub.1v1/β.sub.1v2/γ; cell binding assays: huSIRPα.sub.1, huSIRPα.sub.BIT).

[0172] Transient Expression of Antibodies

[0173] a) Preparation of cDNA Constructs and Expression Vectors

[0174] The HCVR amino acid sequences of the antibodies were each joined_at the N-terminus to a leader sequence (SEQ ID NO:28 for antibodies 1-9, 15, 16; SEQ ID NO:39 for antibodies 10-14), and at the C-terminus to the constant domain of a human IgG.sub.1 HC LALA according to SEQ ID NO:27. The HCVR amino acid sequences of antibodies 12C4huIgG.sub.1LALA, 12C4huIgG.sub.1 or 29AM4-5huIgG.sub.1LALA were each joined at the N-terminus to a HAVT20 leader sequence (SEQ ID NO:29) and at the C-terminus to the constant domain of a human IgG.sub.1 HC LALA according to SEQ ID NO:27 or a wild type human IgG.sub.1 HC (SEQ ID NO:25). The resulting chimeric amino acid sequences were back-translated into a cDNA sequence codon-optimized for expression in human cells (Homo sapiens). Similarly, the chimeric cDNA sequence for the LC of the construct was obtained by joining the sequences of a leader sequence (SEQ ID NO:28 for antibodies 1-9, 12; SEQ ID NO:40 for antibodies 10, 11, 13-16, SEQ ID NO:29 for 12C4huIgG.sub.1LALA, 12C4huIgG.sub.1 and 29AM4-5huIgG.sub.1LALA) to the LCVR of antibodies 1-16, 12C4huIgG.sub.1LALA and 12C4huIgG.sub.1 and 29AM4-5huIgG.sub.1LALA at the N-terminus and at the C-terminus to a human IgG.sub.1 κ light chain constant region (SEQ ID NO:26). The HCVR and LCVR sequences according to Table 1 were used.

TABLE-US-00001 TABLE 1 HCVR and LCVR sequences of the antibodies and reference antibodies Antibody HCVR LCVR 1 SEQ ID NO: 1 SEQ ID NO: 2 2 SEQ ID NO: 3 SEQ ID NO: 4 3 SEQ ID NO: 5 SEQ ID NO: 6 4 SEQ ID NO: 7 SEQ ID NO: 8 5 SEQ ID NO: 9 SEQ ID NO: 10 6 SEQ ID NO: 11 SEQ ID NO: 12 7 SEQ ID NO: 13 SEQ ID NO: 14 8 SEQ ID NO: 15 SEQ ID NO: 16 9 SEQ ID NO: 17 SEQ ID NO: 18 29AM4-5huIgG.sub.1LALA SEQ ID NO: 19 SEQ ID NO: 20 12C4huIgG.sub.1LALA SEQ ID NO: 21 SEQ ID NO: 22 12C4huIgG.sub.1 SEQ ID NO: 21 SEQ ID NO: 22 KWAR23 SEQ ID NO: 23 SEQ ID NO: 24 10 humanized SEQ ID NO: 30 SEQ ID NO: 31 11 humanized SEQ ID NO: 32 SEQ ID NO: 33 12 humanized SEQ ID NO: 34 SEQ ID NO: 8 13 humanized SEQ ID NO: 35 SEQ ID NO: 36 14 humanized SEQ ID NO: 35 SEQ ID NO: 37 15 humanized SEQ ID NO: 13 SEQ ID NO: 38 16 humanized SEQ ID NO: 13 SEQ ID NO: 37

[0175] b) Vector Construction and Cloning Strategy

[0176] For expression of the antibody chains a mammalian expression vector was used, which contains a CMV:BGHpA expression cassette. The final vectors containing either the HC or the LC expression cassette (CMV:HC:BGHpA and CMV:LC-BGHpA, respectively) were transferred to and expanded in E. coli NEB 5-alpha cells. Large-scale production of the final expression vectors for transfection was performed using Maxi- or Megaprep kits (Qiagen).

[0177] c) Transient Expression in Mammalian Cells

[0178] Commercially available Expi293F cells (Thermo Fisher) were transfected with the expression vectors using the ExpiFectamine transfection agent according to the manufacturer's instructions as follows: 75×10.sup.7 cells were seeded in 300 mL FortiCHO medium, 300 μg of the expression vector was combined with 800 μl of ExpiFectamine transfection agent and added to the cells. One day after transfection, 1.5 ml Enhancer 1 and 15 ml Enhancer 2 were added to the culture. Six days post transfection, the cell culture supernatant was harvested by centrifugation at 4,000 g for 15 min and filtering the clarified harvest over PES bottle filters/MF 75 filters (Nalgene).

[0179] Antibody Binding and Specificity

[0180] Experimental

[0181] ELISA assay: Solutions of huSIRPα.sub.1, huSIRPα.sub.BIT, huSIRPβ.sub.1v1, huSIRPβ.sub.1v2, huSIRPγ and cySIRPα in phosphate buffered saline (PBS) were each added to a multiple well black polystyrene plate for ELISA and allowed to adhere for 1 h at RT. Unbound protein was removed with three washing steps using standard washing buffer. Subsequently, blocking buffer was added to the wells. After 1 h incubation at RT, the wells were washed three times with standard washing buffer. The antibodies in buffer at various concentrations were added to the wells and incubated at RT for 1 h. Unbound antibodies were removed with three washing steps using standard washing buffer. Goat anti human IgG (Fab′).sub.2:horse radish peroxidase (HRP) in buffer was added to the wells and incubated at RT for 1 h. 3,3′,5,5′-Tetramethylbenzidine (TMB) was added and after sufficient colour development HCl was added. Absorbance was read at 450 nm/620 nm.

[0182] Surface Plasmon Resonance (SPR) assay: Affinity analysis was performed by single cycle kinetics analysis on a Surface Plasmon Resonance apparatus (Biacore T200 system, GE Life Sciences) at 25° C. Biotinylated SIRP antigens were captured on the surface of a chip suitable for biotinylated molecules (Sensor Chip CAP, GE Life Sciences) by injecting 5 μg/ml of the SIRP antigen in running buffer (10 mM HEPES buffer at pH 7.4 with 150 mM NaCl, 3 mM EDTA and 0.005% v/v polyoxyethylene (20) sorbitan monolaurate (Surfactant P20) for 60 sec at 10 μL/min after injection of a streptavidin conjugate (20× diluted biotin CAPture reagent, GE Life Sciences) for 60 sec at 10 μl/min. Baseline stabilization was set at 1 min after which five increasing concentrations of an anti-SIRP antibody in running buffer (10 mM HEPES buffer at pH 7.4 with 150 mM NaCl, 3 mM EDTA and 0.005% v/v polyoxyethylene (20) sorbitan monolaurate) were injected. For each step an association time of 150 sec was used, followed by a dissociation time of 1200 sec at the highest concentration only, all at a flow rate of 30 μL/min. Regeneration was performed with 6 M guanidine-HCl, 0.25 M NaOH solution (60 sec with flow rate of 30 μL/min). Double blank subtraction was performed on the observed sensorgrams using a non anti-SIRP (blank) immobilized reference flow channel and running buffer injection. Sensorgrams were fitted with a 1:1 Langmuir model for all tested anti-SIRP antibodies. The kinetic parameters (k.sub.a, k.sub.d and K.sub.D) were calculated using the Biacore T200 evaluation software (v3.1).

[0183] Flow Cytometry: U937 cells endogenously expressing human SIRPα.sub.BIT antigen and cells derived from a non-engineered subclone that has been screened and isolated from CHO-S Chinese hamster ovary cells (ExpiCHO-S) cells expressing human SIRPα.sub.1, SIRPα.sub.BIT or cySIRPα antigen (100,000 cells/well in a 96-well plate) were washed three times with ice-cold FACS buffer (1×PBS (LONZA) containing 0.2% v/w BSA (Sigma-Aldrich, St. Louis, Mo.) and 0.02% v/w NaN.sub.3 (Sigma-Aldrich), followed by the addition of a concentration range of each primary mAb (50 μL/well) diluted in ice-cold FACS buffer. After an incubation time of 30 min at 4° C., the cells were washed three times with ice-cold FACS buffer and 50 μL/well secondary mAb (AffiniPure F(ab′).sub.2 fragment Goat-anti-human IgG-APC, 1:6,000 dilution, Jackson Immuno Research) was added. After 30 min at 4° C., cells were washed twice and resuspended in 150 μL FACS buffer. Fluorescence intensities were determined by flow cytometry (BD FACSVerse, Franklin Lakes, N.J.) and indicated as the median fluorescence intensity (MFI-Median) for U937 cells and ExpiCHO-S cells. Curves were fitted by nonlinear regression using the sigmoidal dose-response equation with variable slope (four parameters) in GraphPad Prism (version 7.02 for Windows, GraphPad, San Diego, Calif.). EC.sub.50 values were calculated as the concentration in μg/mL that gives a response half way between bottom and top of the curve, when using a 4-parameter logistic fit.

[0184] Results

[0185] ELISA assay: The EC.sub.50 values for binding to huSIRPα.sub.1, huSIRPα.sub.BIT, huSIRPβ.sub.1, huSIRPβ.sub.1v2, huSIRPγ, cySIRPα obtained with ELISA for antibodies 1-9 and reference antibodies are summarized in Table 2. All antibodies bind to huSIRPα.sub.1 and to huSIRPα.sub.BIT. Antibodies 29AM4-5huIgG.sub.1LALA and 12C4huIgG.sub.1LALA, bind to huSIRPβ.sub.1v1, huSIRPβ.sub.1v2, and huSIRPγ. The antibodies 2-6, 8 and 9 show a low affinity for huSIRPβ.sub.1v1 and for huSIRPγ. Antibody 7 binds to huSIRPβ.sub.1v1, but has low affinity for huSIRPβ.sub.1v2 and huSIRPγ. Antibody 1 binds to huSIRPβ.sub.1v2 and huSIRPγ.

TABLE-US-00002 TABLE 2 Specificity of the anti-SIRPα antibodies and reference antibodies huSIRPα.sub.1 huSIRPα.sub.BIT huSIRPß.sub.1v1 huSIRPß.sub.1v2 huSIRPγ cySIRPα Antibody EC.sub.50 (ng/ml) EC.sub.50 (ng/ml) EC.sub.50 (ng/ml) EC.sub.50 (ng/ml) EC.sub.50 (ng/ml) EC.sub.50 (ng/ml) 1 39 21 100,000 58 43 305 2 33 27 100,000 28 100,000 38 3 15 24 100,000 89 5,216 36 4 53 25 100,000 92 100,000 99 5 31 21 3,518 110 100,000 123 6 21 20 100,000 24 100,000 33 7 23 20 14 100,000 100,000 335 8 19 20 100,000 19 100,000 26 9 23 26 100,000 47 100,000 30 29AM4-5* 9 9 13 17 34 11 12C4* 7 5 8 6 6 5 *huIgG.sub.1LALA EC.sub.50 values > 100,000 have been adjusted to 100,000.

[0186] SPR assay: The K.sub.D values for binding to huSIRPα.sub.1, huSIRPα.sub.BIT and huSIRPγ of antibodies 4, 7, 10-14 in comparison with reference antibodies KWAR23, huIgG.sub.112C4LALA and SE5A5 (purchased from a commercial supplier) are summarized in Table 3. Antibodies 4, 7, 10-14 bind to both huSIRPα.sub.1 and huSIRPα.sub.BIT, and do not bind to huSIRPγ. All reference antibodies do bind to huSIRPγ.

TABLE-US-00003 TABLE 3 SPR data (K.sub.D in M) Antibody K.sub.D (hUSIRPα.sub.BIT) K.sub.D (huSIRPα.sub.1) K.sub.D (huSIRPγ) KWAR23 <1.0E−11.sup.1 <1.0E−11  <1.0E−11 mouse IgG2a KWAR23 <1.0E−11.sup.1 1.1E−11 <1.0E−11 huIgG.sub.1LALA 12C4huIgG.sub.1LALA  1.5E−11 8.7E−11  1.6E−11 SE5A5 2.6E−9 2.2E−9  4.9E−8 4 <1.0E−11 2.6E−11  N.sup.2 7 <1.0E−11 <1.0E−11  N 10 humanized <1.0E−11 3.2E−9  N 11 humanized  1.4E−10 4.1E−8  N 12 humanized <1.0E−11 5.9E−11 N 13 humanized  1.2E−11 <1.0E−11  N 14 humanized  8.9E−11 <1.0E−11  N .sup.1<1.0E−11: K.sub.D is outside the range which means high affinity .sup.2N: No specific binding found

[0187] Flow Cytometry assay: The binding of various antibodies to huSIRPα.sub.1, huSIRPβ.sub.BIT, and/or cySIRPα expressed on cells was determined by flow cytometry. The binding is indicated in EC.sub.50 values, which are shown in Table 4. Antibodies 2, 4, 5, 7, 8, 10-14 bind to huSIRPα.sub.1, huSIRPα.sub.BIT and cySIRPα. Antibodies 2, 4, 5, 7, 8, 10-14 bind to cySIRPα in the low μg/mL range.

TABLE-US-00004 TABLE 4 Flow Cytometry data U937 cells ExpiCHO-S ExpiCHO-S ExpiCHO-S (SIRPα.sub.BIT) (huSIRPα.sub.1) (huSIRPα.sub.BIT) (cySIRPα) EC.sub.50 EC.sub.50 EC.sub.50 EC.sub.50 Antibody (μg/mL) (μg/mL) (μg/mL) (μg/mL) 1 — — — — 2 0.14 0.19 0.27 0.16 3 0.22 — — — 4 0.12 0.41 0.23 0.18 5 0.16 0.27 0.22 0.26 6 — — — — 7 0.17 0.23 0.21 0.07 8 0.12 0.22 0.18 0.15 9 0.11 — — — 29AM4-5 0.25 — — — huIgG.sub.1LALA 12C4huIgG.sub.1 0.19 — — — LALA KWAR23 0.09 — — — huIgG.sub.1LALA 10 0.17 0.38 0.2 0.27 11 0.13 1.05 0.3 0.32 12 0.2 0.1 0.46 0.17 13 0.14 0.36 0.23 0.44 14 0.22 0.37 0.29 0.38 15 0.16 — — — 16 0.23 — — — — value not determined

[0188] Antibody Blocking of CD47-SIRPα Binding

[0189] Experimental

[0190] CHO cells transfected with either SIRPα.sub.1 or SIRPα.sub.BIT or parental CHO cells as control were seeded in 20 μl cell medium in a well plate with clear bottom and incubated overnight. Antibodies 1-9, 29AM4-5huIgG.sub.1LALA or 12C4huIgG.sub.1LALA reference antibodies together with a mixture of His Tag® CD47 and anti-His Tag® fluorescent detection antibody were added to the wells and incubated for 2 h. After incubation, the cells were washed with cell wash buffer. Fluorescence was determined using a screening system (CellInsight®, Thermo Scientific®) and total fluorescence per cell was determined.

[0191] Results

[0192] Antibodies 29AM4-5huIgG.sub.1LALA, 12C4huIgG.sub.1LALA, 3 and 7 block binding of CD47 to both CHO cells expressing huSIRPα.sub.1 and CHO cells expressing huSIRPα.sub.BIT completely, antibodies 1, 2, 4-6, 8 and 9 do neither block binding of CD47 to CHO cells expressing huSIRPα.sub.1 nor to CHO cells expressing huSIRPα.sub.BIT.

[0193] ADCC Assay

[0194] Neutrophils of donors homozygous for either SIRPα.sub.1 or SIRPα.sub.BIT were isolated and cultured according to the method in Chao et al. PNAS 2011, 108(45), 18342-18347. ADCC was determined using the .sup.51Cr release assay or the non-radioactive Europium TDA (EuTDA) cytotoxicity assay (DELFIA, PerkinElmer). SKBR3 cells were used as target cells and labelled with 100 μCi .sup.51Cr (Perkin-Elmer) for 90 min at 37° C., or with bis (acetoxymethyl) 2,2′:6′,2″-terpyridine-6,6″-dicarboxylate) (BATDA reagent Delfia), for 5 min at 37° C. After 2 washes with PBS, 5×10.sup.3 target cells per well were incubated in IMDM culture medium supplemented with 10% (v/v) foetal calf serum (FCS) for 4 hours at 37° C. and 5% CO.sub.2 in a 96-well U-bottom plate together with neutrophils in an effector to target cell ratio of 50:1 in the presence of the appropriate antibodies. After the incubation, supernatant was harvested and analyzed for radioactivity in a gamma counter (Wallac) or was added to europium solution (DELFIA, PerkinElmer) and the europium 2,2′:6′,2″-terpyridine-6,6″-dicarboxylic acid (EuTDA) fluorescence was determined using a spectrofluorometer (Envision, PerkinElmer). The percentage of cytotoxicity was calculated as [(experimental release−spontaneous release)/(total release−spontaneous release)]×100%. All conditions were measured in duplicate and/or triplicate.

[0195] ADCC Data 12C4huIgG.sub.1LALA Versus 12C4IgG.sub.1

[0196] FIG. 1 shows the results of the ADCC assay as cytotoxicity in %. The % cytotoxicity measured on SKBR3 cells using neutrophils as effector cells and trastuzumab alone is less than the % cytotoxicity of trastuzumab in combination with the murine 12C4 antibody (mu12C4). Trastuzumab in combination with an antibody wherein 12C4 variable regions are grafted onto a human IgG.sub.1 constant region (12C4huIgG.sub.1) shows similar % cytotoxicity as compared to trastuzumab alone at low concentrations of 12C4huIgG.sub.1. At higher concentrations 12C4huIgG.sub.1, a decrease in % cytotoxicity is observed. Trastuzumab in combination with an antibody wherein 12C4 variable regions are grafted onto a human IgG.sub.1 constant region comprising amino acid substitutions L234A and L235A (12C4huIgG.sub.1LALA) shows increased % cytotoxicity compared to the % cytotoxicity of trastuzumab alone, and increased % cytotoxicity compared to the combination of 12C4huIgG.sub.1 and trastuzumab.

[0197] ADCC Data

[0198] FIG. 2 compares the % ADCC by human neutrophils relative to trastuzumab (set to 100%) in the presence of antibody 1-9 having a human IgG.sub.1 constant region comprising amino acid substitutions L234A and L235A (LALA) in combination with trastuzumab in comparison with 12C4huIgG.sub.1LALA. B6H12IgG.sub.1LALA, having the VR of a murine anti-CD47 antibody and a human IgG.sub.1 constant region comprising amino acid substitutions L234A and L235A, and vehicle (no trastuzumab) were used as positive and negative control, respectively. Filled squares, (.square-solid.), are the values measured with neutrophils of donors having the SIRPα.sub.BIT variant (homozygous for SIRPα.sub.BIT), open circles (∘) are the values measured with neutrophils of donors having the SIRPα.sub.1 variant (homozygous for SIRPα.sub.1). For all antibodies the average ADCC was increased in comparison to trastuzumab alone. For antibodies 1, 2, 4, 5, 7 and 8 the average ADCC increase was enhanced even more than the 12C4huIgG.sub.1LALA-induced ADCC increase. When the ADCC increase per donor per antibody is compared, antibodies 1, 3-6, 8 and 9 show less variation in % increase in ADCC than 12C4huIgG.sub.1LALA.

[0199] FIG. 3 compares the % ADCC by human neutrophils in the presence of various concentrations of chimeric antibodies 4 and 7 and humanized antibodies 10 and 14 having a human IgG.sub.1 constant region comprising amino acid substitutions L234A and L235A (LALA) in combination with trastuzumab in comparison with trastuzumab alone and trastuzumab in combination with various concentrations of 12C4huIgG.sub.1LALA. Neutrophils of two donors homozygous for SIRPα.sub.BIT were used. Even at low concentrations antibodies 4, 7, 10 and 14 increase ADCC. The ADCC increase is concentration dependent.

[0200] FIG. 4 compares the % ADCC by human neutrophils in the presence of antibodies 4, 7, 10, 13, 14, 15 and 16 in combination with trastuzumab (Tmab) in comparison with the % ADCC trastuzumab alone and 12C4huIgG.sub.1LALA. All antibodies increase the ADCC in comparison with trastuzumab alone. The ADCC increase by neutrophils of most donors in the presence of antibodies 4, 7, 10, 13, 14, 15 and 16 in combination with trastuzumab is similar or increased in comparison with 12C4huIgG.sub.1LALA in combination with trastuzumab.

TABLE-US-00005 Sequence listings with underlined CDR1, CDR2 and CDR3 amino acid sequences in heavy chain (HC) and light chain (LC) variable region (VR) amino acid sequences (determined using the method of Kabat) SEQ ID NO: 1 (HC VR 1) 1 QSVEESGGRL VTPGTPLTLT CTVSGIDLSS YAMSWVRQAP GKGLEWIGII 51 SSGGITYYAS WAKGRFTISK TSTTVDLKIP SPTTEDTATY FCARSLWAAS 101 NYYMALWGPG TLVTVSS SEQ ID NO: 2 (LC VR 1) 1 AIKMTQTPAS VSAAVGGTVS INCQASEDIE SYLAWYQQKP GQPPKLLIYR 51 ASTLASGVSS RFKGSGSGTQ FTLTISDLES ADAATYYCLG DYYSSSGDTG 101 AFGGGTEVVV K SEQ ID NO: 3 (HC VR 2) 1 QSVEESGGRL VTPGTPLTLT CTVSGFSLSN YAMHWVRQAP GKGLEWIGII 51 YTGGATSYAT WAKGQFTISK TSTTVDLKIT SPTTEDTATY FCARGDRDGY 101 AYFNIWGPGT LVTVSL SEQ ID NO: 4 (LC VR 2) 1 QIVMTQTPFS VSAVVGGTVT IKCQASHNIG SWLAWYQQKP GQRPKLLIYD 51 ASTLASGVSS RFKGSGSGTE FTLTISGVES ADAATYYCQQ GYGISYVHNV 101 FGGGTEVVVK SEQ ID NO: 5 (HC VR 3) 1 QSVEESGGRL VTPGTPLTLA CTVSGFSLIS YYISWVRQAP EKGLEYIGII 51 NIGGGASYAS WAKGRFTISK TSTTVDLKIT SPTPEDTATY FCAMSYGMDT 101 GAFNIWGPGT LVTVSL SEQ ID NO: 6 (LC VR 3) 1 AQVLTQTPAS VSAAVGGTVT ISCQSSESVY KNNFLSWYQQ KPGKPPKLLI 51 YGASTLASGV PSRFKGSGSG TQFTLTISDL ESDDAATYFC QGGYRTDIYP 101 FGGGTEVVVK SEQ ID NO: 7 (HC VR 4) 1 QSVEESGGRL GTPGTPLTLT CTVSGFSLSS YVMGWFRQAP GKGLEYIGII 51 SSSGSPYYAS WVNGRFTISK TSTTMDLKMN SPTTEDTATY FCARVGPLGV 101 DYFNIWGPGT LVTVSL SEQ ID NO: 8 (LC VR 4) 1 DIVMTQTPSS VEAAVGGTVT IKCQAGQSIN SYLAWYQQKP GQRPKLLIYY 51 ASTLESGVPS RFKGSGSGTD YTLTISDLES ADAATYYCQS WHYISRSYAF 101 GGGTEVVVK SEQ ID NO: 9 (HC VR 5) 1 QSVEESGGRL VTPGTPLTLT CTVSGFSLSS YVMGWFRQAA GKGLEYIGYI 51 NADGSPYYAT WVNGRFTISK TPTTMDLKIN SPTTEDTATY FCARVGPLGV 101 DYFNIWGPGT LVTVSL SEQ ID NO: 10 (LC VR 5) 1 DIVMTQTPAS VEAAVGGTVT IKCQASQSIN RYLTWYQQKP GQRPKLLIYY 51 ASTLESGVPS RFEGSGSGTD YTLTISDLES ADAATYYCQS YYYISRTYAF 101 GGGTEV VVK SEQ ID NO: 11 (HC VR 6) 1 QSVEESGGRL VTPGTPLTLT CTVSGIDLSS YTMTWVRQAP GKGLEWIGII 51 YAGGSTAYAS WAKGRFTISK TSTTVDLKIT SPTTEDTATY FCARSSSDGY 101 DYFNIWGPGT LVTVS L SEQ ID NO: 12 (LC VR 6) 1 GVVMTQTPSS VSAAVGGTVT INCQASQSIG SWLAWYQQKP GQPPKLLIYQ 51 ASKLASGVPS RFSGRGSGTH FTLTISDVQS DDAATYYCQQ TVTAASNVDNA 101 FGGGTEVVVK SEQ ID NO: 13 (HC VR 7) 1 RSVEESGGRL VTPGTPLTLT CTVSGFSLSS HGISWVRQAP GKGLEYIGTI 51 GTGVITYFAS WAKGRFTGSK TSTTVDLKIT SPTTEDTATY FCARGSAWND 101 PFDPWGPGTL VTVSS SEQ ID NO: 14 (LC VR 7) 1 ALVMTQTPAS VSAAVGGTVT TKCQASQSVY GNNDLAWYQH KPGQPPKLLI 51 YLASTLATGV PSRFSGSGSG TQFTLTITGV QSDDAATYYC LGGGDDEADN 101 VFGGGTEVVV K SEQ ID NO: 15 (HC VR 8) 1 QSLEESGGRL VTPGTPLTLT CTASGVDLSN YAMGWVRQAP GKGLEWIGII 51 YAGGSTSYAT WAKGRFTISK TSTTMDLKMT SPTTEDTATY FCARHRSDGY 101 DYFHLWGPGT LVTVSL SEQ ID NO: 16 (LC VR 8) 1 AIDMTQTPAS VSEPVGGTVT IKCQASQSIS SWLAWYQQKP GQRPKLLIYD 51 ASKLASGVPS RFSGSGSGTE FTLTISGVQS DDAAAYYCQQ GYAVSYVENI 101 FGGGTEVVVK SEQ ID NO: 17 (HC VR 9) 1 QSMEESGGRL VTPGTPLTLT CTASGFSLSN YGVSWVRQAP GKGLEWIGII 51 YGGSDITAYA SWAKGRFTIS KTSTTVDLTI TSPTTEDTAT YFCAKSYTNG 101 MDYYNIWGPG TLVTVSL SEQ ID NO: 18 (LC VR 9) 1 AFDLTQTPSS VEAPVGGTVI IKCQASQSIS SYLAWYQQKP GQPPKLLIYS 51 ASTLASGVSS RFKGSGSETQ FPLTISDLES ADAATYYCQS YYGSRSNVFG 101 GGTEVVVK SEQ ID NO: 19 (HC VR 29AM4-5) 1 EVQLVESGGG LVQPGGSLRL SCAASGFNIS YYFIHWVRQA PGKGLEWVAS 51 VYSSFGYTYY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARFT 101 FPGLFDGFFG AYLGSLDYWG QGTLVTVSS SEQ ID NO: 20 (LC VR 29AM4-5) 1 DIQMTQSPSS LSASVGDRVT ITCRASQSVS SAVAWYQQKP GKAPKLLIYS 51 ASSLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQQ AVNWVGALVT 101 FGQGTKVEIK SEQ ID NO: 21 (HC VR 12C4) 1 EVKLEESGGG LMQPGGSMKL SCVASGFTFS NYWMNWVRQS PEKGLEWVAE 51 IRLKSNNYAT HYAESVKGRF TISRDDSKSS VYLQMNNLRA EDTGIYYCIR 101 DYDYDAYFDY WGQGTTLTVS S SEQ ID NO: 22 (LC VR 12C4) 1 DIVLTQSPAS LAVSLGQRAT ISCRASKSVS TSGYNYMYWY QQKPGQPPKL 51 LIYLASNLES GVPARFSGSG SGTDFTLNIH PVEEEDAATY YCQHSGELPY 101 TFGGGTKLEI K SEQ ID NO: 23 (HC VR KWAR23 ) 1 EVQLQQSGAE LVKPGASVKL SCTASGFNIK DYYIHWVQQR TEQGLEWIGR 51 IDPEDGETKY APKFQDKATI TADTSSNTAY LHLSSLTSED TAVYYCARWG 101 AYWGQGTLVT VSS SEQ ID NO: 24 (LC VR KWAR23) 1 QIVLTQSPAI MSASPGEKVT LTCSASSSVS SSYLYWYQQK PGSSPKLWIY 51 STSNLASGVP ARFSGSGSGT SYSLTISSME AEDAASYFCH QWSSYPRTFG 101 AGTKLELK SEQ ID NO: 25 (human IgG.sub.1 antibody HC constant region) 1 ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV 51 HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP 101 KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS 151 HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK 201 EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC 251 LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 301 QQGNVFSCSV MHEALHNHYT QKSLSLSPGK SEQ ID NO: 26 (human IgG.sub.1 antibody LC κ constant region) 1 RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG 51 NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK 101 SFNRGEC SEQ ID NO: 27 (human IgGi antibody HC constant region LALA mutant (mutations underlined) 1 ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV 51 HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP 101 KSCDKTHTCP PCPAPEAAGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS 151 HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK 201 EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC 251 LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 301 QQGNVFSCSV MHEALHNHYT QKSLSLSPGK SEQ ID NO: 28 (leader sequence HC 1-9, 15 + 16, LC 1-9 + 12) 1 MGWSCIILFL VATATGVHS SEQ ID NO: 29 (HAVT20 leader sequence) 1 MACPGFLWAL VISTCLEFSMA SEQ ID NO: 30 (HC VR 10) 1 KVEESGGGLV QPGGSLRLSC AASGFSLSSY VMGWVRQAPG KGLEWVSIIS 51 SSGSPYYASW VNGRFTISKD NSEGMVYLQM NSLRAEDTAV YYCARVGPLG 101 VDYFNIWGQG TTVTVSS SEQ ID NO: 31 (LC VR 10) 1 DIVMTQSPDS LAVSLGERAT INCQAGQSIN SYLAWYQQKP GQPPKLLIYY 51 ASTLESGVPD RFSGSGSGTD FTLTISSLQA EDVAVYYCQS WHYISRSYAF 101 GGGTKLEIK SEQ ID NO: 32 (HC VR 11) 1 EVKVEESGGG LVQPGGSLRL SCAASGFSLS SYVMGWVRQA PGKGLEWVSI 51 ISSSGSPYYA SWVNGRFTIS KTSTTMDLQM NSLRAEDTAV YYCARVGPLG 101 VDYFNIWGQG TTVTVSS SEQ ID NO: 33 (LC VR 11) 1 DIQMTQSPSS LSASVGDRVT ITCQAGQSIN SYLAWYQQKP GKVPKLLIYY 51 ASTLESGVPS RFSGSGSGTD FTLTISSLQP EDVATYYCQS WHYISRSYAF 101 GQGTKVEIK SEQ ID NO: 34 (HC VR 12) 1 VQLVESGGRL VQPGTPLTLS CTVSGFSLSS YVMGWFRQAP GKGLEYIGII 51 SSSGSPYYAS WVNGRFTISK TSTTMDLKMN SLRSEDTATY FCARVGPLGV 101 DYFNIWGPGT LVTVSS SEQ ID NO: 35 (HC VR 13 + 14) 1 RQLVESGGGL VQPGGSLRLS CTASGFSLSS HGISWVRQAP GKGLEYIGTI 51 GTGVITYFAS WAKGRFTGSK TSSTAYMELS SLRSEDTAVY FCARGSAWND 101 PFDPWGQGTL VTVSS SEQ ID NO: 36 (LC VR 13) 1 AIQMTQSPSS LSASVGDRVT ITCQASQSVY GNNDLAWYQQ KPGKAPKLLI 51 YLASTLATGV PSRFSGSGSG TDFTLTISSL QPEDFATYYC LGGGDDEADN 101 VFGGGTKVEI K SEQ ID NO: 37 (LC VR 14 + 16) 1 DIEMTQSPSS VSASVGDRVT LTCQASQSVY GNNDLAWYQQ KPGQAPKLLI 51 YLASTLATGV PSRFSGSGSG TDFTLTISSL QPEDFATYYC LGGGDDEADN 101 VFGGGTKVEI K SEQ ID NO: 38 (LC VR 15) 1 ELVMTQSPSS LSASVGDRVT ITCQASQSVY GNNDLAWYQQ KPGEAPKLLI 51 YLASTLATGV PSRFSGSGSG TDFTLTISGL QSEDFATYYC LGGGDDEADN 101 VFGQGTKVEI K SEQ ID NO: 39 (leader sequence heavy chains 10-14) 1 MGWTLVFLFL LSVTAGVHS SEQ ID NO: 40 (leader sequence light chains 10, 11, 13-16) 1 MVSSAQFLGL LLLCFQGTRC