SIRPALPHA-TARGETING ANTIBODY OR ANTIGEN BINDING FRAGMENT THEREOF, AND PREPARATION AND APPLICATION THEREOF

Abstract

Disclosed is a SIRPα-targeting antibody or an antigen-binding fragment thereof, comprising a light chain variable region and/or a heavy chain variable region. The antibody or the antigen-binding fragment thereof binds to human SIRPα-V1 and human SIRPα-V2, but weakly or does not bind to human SIRPβ and SIRPγ, does not bind to human T cells, and has the function of blocking the binding of SIRPα to CD47. Further disclosed are a bispecific antibody comprising same, a method for preparing the antibody or the antigen-binding fragment thereof and an application thereof. The unique properties of the disclosed antibody or the antigen-binding fragment thereof enable same to be more suitable for the development of drugs for an antibody or antigen-binding fragment against a human SIRPα target. As a candidate drug, same can be administered alone or in combination, providing a new or even better choice for the combined immunotherapy of tumors.

Claims

1. An Sirpα-targeting antibody or an antigen-binding fragment thereof, comprising a light chain variable region and a heavy chain variable region, wherein the antibody or the antigen-binding fragment thereof binds to human Sirpα-V1 and human Sirpα-V2, but weakly binds to or does not bind to human Sirpβ and Sirpγ, and does not bind to human T cells, and has the function of blocking the binding of Sirpα to CD47; the antibody or the antigen-binding fragment thereof also binds to one or more of Cyno Sirpα L932, L933, L936 and L937, but does not bind to Cyno Sirpα L938 and L939; wherein the amino acid sequence of the L932 has a NCBI sequence number of NP_001271679.1, the amino acid sequence of the L933 has a NCBI sequence number of XP_015313155.1, the amino acid sequence of the L936 is as shown in SEQ ID NO: 3, the amino acid sequence of the L937 is as shown in SEQ ID NO: 4, the amino acid sequence of the L938 is as shown in SEQ ID NO: 5, and the amino acid sequence of the L939 is as shown in SEQ ID NO: 6; the light chain variable region comprises the following CDRs: VL CDR1 as shown in the amino acid sequence of SEQ ID NO: 11; VL CDR2 as shown in the amino acid sequence of SEQ ID NO: 12; and VL CDR3 as shown in the amino acid sequence of SEQ ID NO: 13; and the heavy chain variable region comprises the following CDRs: VH CDR1 as shown in the amino acid sequence of SEQ ID NO: 14; VH CDR2 as shown in the amino acid sequence of SEQ ID NO: 15; and VH CDR3 as shown in the amino acid sequence of SEQ ID NO: 16.

2. The Sirpα-targeting antibody or the antigen-binding fragment thereof of claim 1, wherein the Sirpα-targeting antibody is a murine antibody; preferably, the light chain variable region of the murine antibody is the amino acid sequence as shown in SEQ ID NO: 9 or a mutation thereof, or, the heavy chain variable region of the murine antibody is the amino acid sequence as shown in SEQ ID NO: 10 or a mutation thereof, more preferably, the light chain variable region of the murine antibody is encoded by a nucleotide as shown in SEQ ID NO: 7; or, the heavy chain variable region of the murine antibody is encoded by a nucleotide as shown in SEQ ID NO: 8; the mutation is the deletion, substitution or insertion of one or more amino acid residues on the amino acid sequence of the light chain variable region or the heavy chain variable region, and the mutated amino acid sequence has at least 85% sequence identity with the amino acid sequence of the light chain variable region or the heavy chain variable region, and maintains or improves the binding of the antibody or the antigen-binding fragment thereof to Sirpα; the at least 85% sequence identity is preferably at least 90% sequence identity; more preferably at least 95% sequence identity; and most preferably at least 99% sequence identity.

3. The Sirpα-targeting antibody or the antigen-binding fragment thereof of claim 2, wherein the Sirpα-targeting antibody or the antigen-binding fragment thereof further comprises a constant region of the murine antibody or a constant region of a human antibody; the constant region of the murine antibody comprises the heavy chain constant region of murine IgG1, IgG2a, IgG2b or IgG3 and κ or λ type light chain constant region, and the constant region of the human antibody comprises the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 and κ or λ type light chain constant region; preferably, when the Sirpα-targeting antibody or the antigen-binding fragment thereof comprises the variable region of the murine antibody and the constant region of the human antibody, the constant region of the human antibody comprises the heavy chain constant region of human IgG4 and κ type light chain constant region of amino acid sequences as shown in SEQ ID NO: 28 and SEQ ID NO: 27 respectively.

4. The Sirpα-targeting antibody or the antigen-binding fragment thereof of claim 1, wherein the Sirpα-targeting antibody is a humanized antibody; preferably, a framework region of the humanized antibody comprises a heavy chain framework region of a human antibody and a light chain framework region of a human antibody; more preferably, the light chain variable region of the humanized antibody comprises the amino acid sequence as shown in any one of SEQ ID NOs: 29-34 or a mutation thereof, or, the heavy chain variable region sequence of the humanized antibody comprises the amino acid sequence as shown in any one of SEQ ID NOs: 35-41 or a mutation thereof, the mutation is the deletion, substitution or insertion of one or more amino acid residues on the amino acid sequence of the light chain variable region or the heavy chain variable region, and the mutated amino acid sequence has at least 85% sequence identity with the amino acid sequence of the light chain variable region or the heavy chain variable region, and maintains or improves the binding of the antibody or the antigen-binding fragment thereof to Sirpα; the at least 85% sequence identity is preferably at least 90% sequence identity; more preferably at least 95% sequence identity; and most preferably at least 99% sequence identity; furthermore preferably, the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 29, and the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 35; or alternatively, the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 30, and the heavy chain variable region comprises the amino acid sequence as shown in any one of SEQ ID NOs: 36-41; or alternatively, the light chain variable region comprises the amino acid sequence as shown in any one of SEQ ID NOs: 31-34, and the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 36; or alternatively, the light chain variable region comprises the amino acid sequence as shown in any one of SEQ ID NO: 29 or 31-34, and the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 39.

5. The Sirpα-targeting antibody or the antigen-binding fragment thereof of claim 4, wherein the light chain of the antibody or the antigen-binding fragment thereof comprises a κ or λ type light chain constant region of a human antibody or a mutation thereof, or the heavy chain of the antibody or the antigen-binding fragment thereof comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a mutation thereof, preferably, the light chain of the antibody or the antigen-binding fragment thereof comprises the κ type light chain constant region of a human antibody; or the heavy chain of the antibody or the antigen-binding fragment thereof comprises the heavy chain constant region of human IgG4; more preferably, light chain of the antibody or the antigen-binding fragment thereof comprises the amino acid sequence as shown in SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 or SEQ ID NO: 48 or a mutation thereof, or heavy chain of the antibody or the antigen-binding fragment thereof comprises the amino acid sequence as shown in SEQ ID NO: 43 or a mutation thereof, furthermore preferably, the Sirpα-targeting antibody or the antigen-binding fragment thereof comprising the following light and heavy chains: the light chain shown in any one of the amino acid sequences of SEQ ID NO: 42 or 44-48, and the heavy chain shown in the amino acid sequence of SEQ ID NO: 43.

6. (canceled)

7. The Sirpα-targeting antibody or the antigen-binding fragment thereof of claim 1, wherein the Sirpα-targeting antibody or the antigen-binding fragment thereof comprises immunoglobulin, Fab, Fab′, F(ab′).sub.2, Fv or scFv, a bispecific antibody, a multispecific antibody, a single domain antibody, a single-domain antibody or any other antibody that retains the partial ability of the antibody of specifically binding to an antigen, or a monoclonal or polyclonal antibody prepared from the aforementioned antibodies.

8. A bispecific antibody comprising a first protein functional region and a second protein functional region, wherein the first protein functional region is the Sirpα-targeting antibody or the antigen-binding fragment thereof of claim 1; and the second protein functional region is an antibody targeting a non-Sirpα antigen or an antigen-binding fragment thereof, preferably, the non-Sirpα antigen is an immune checkpoint antigen or a tumor therapy target, and the immune checkpoint antigen or the tumor therapy target antigen preferably comprises PD-1, PD-L1, Tim3, LAG3 or CLDN18.2; more preferably, the second protein functional region is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-Tim3 antibody, an anti-LAG3 antibody or an anti-CLDN18.2 antibody; and most preferably, the anti-PD-1 antibody is Nivolumab or Pembrolizumab, and the anti-PD-L1 antibody is Atezolumab, Avelumab or Durvalumab.

9. The bispecific antibody of claim 8, wherein the first protein functional region is immunoglobulin, and the second protein functional region is one or more, preferably two scFvs; or alternatively, the second protein functional region is immunoglobulin, and the first protein functional region is one or more, preferably two scFvs; wherein, the scFv comprises a heavy chain variable region and a light chain variable region that are linked by a linker; the scFv is linked to the immunoglobulin through a linker, the linker is preferably (G.sub.4S).sub.w; and the w is preferably an integer between 0-10, and more preferably 1, 2, 3 or 4.

10. The bispecific antibody of claim 9, wherein the structure of the scFv is light chain variable region-linker-heavy chain variable region, and the N-terminus of the light chain variable region or the C-terminus of the heavy chain variable region of the structure is accordingly linked to the C-terminus or N-terminus of the light chain or heavy chain of the immunoglobulin through the linker respectively; or alternatively, the structure of the scFv is heavy chain variable region-linker-light chain variable region, and the N-terminus of the heavy chain variable region or the C-terminus of the light chain variable region of the structure is accordingly linked to the C-terminus or N-terminus of the light chain or heavy chain of the immunoglobulin through the linker respectively; and the two scFvs are symmetrically connected at the C-terminus or N-terminus of the light chain or heavy chain of the immunoglobulin; preferably, the bispecific antibody is: (1) the first protein functional region is scFv, and the second protein functional region is immunoglobulin; wherein, the scFv of the first protein functional region comprises the light chain variable region and heavy chain variable region of any one of claims 1-4; or alternatively, (2) the first protein functional region is immunoglobulin and the second protein functional region is scFv: wherein, the immunoglobulin comprises the amino acid sequence having the light chain as shown in SEQ ID NO: 48 and the heavy chain as shown in SEQ ID NO: 43; the sequence of the light chain variable region of the scFv is the light chain variable region of Pem, and the heavy chain variable region of the scFv is the heavy chain variable region of Pem; more preferably, when the bispecific antibody is (1), the immunoglobulin comprises the light chain variable region of Pem, the κ type chain as the light chain constant region, the heavy chain variable region of Pem, and the amino acid sequence of hIgG4 as the heavy chain constant region; or alternatively, the immunoglobulin comprises the light chain variable region of Nivo, the κ type chain as the light chain constant region, the heavy chain variable region of Nivo, and the amino acid sequence of hIgG4 as the heavy chain constant region; furthermore preferably, the C-terminuses of the heavy chain variable regions of the two scFvs are symmetrically linked to the N-terminuses of the two heavy chains of the immunoglobulin through the linker; and, the light chain variable region of the scFv is a light chain variable region having the amino acid sequence as shown in SEQ ID NO: 29, and the heavy chain variable region of the scFv is a heavy chain variable region having the amino acid sequence as shown in SEQ ID NO: 39; or alternatively, the C-terminuses of the heavy chain variable regions of the two scFvs are symmetrically linked to the N-terminuses of the two light chain variable regions of the immunoglobulin through the linker; and, the light chain variable region of the scFv is a light chain variable region having the amino acid sequence as shown in SEQ ID NO: 29, and the heavy chain variable region of the scFv is a heavy chain variable region having the amino acid sequence as shown in SEQ ID NO: 39; or alternatively, the N-terminuses of the heavy chain variable regions of the two scFvs are symmetrically linked to the C-terminuses of the two heavy chains of the immunoglobulin through the linker; and, the light chain variable region of the scFv is a light chain variable region having the amino acid sequence as shown in SEQ ID NO: 29, and the heavy chain variable region of the scFv is a heavy chain variable region having the amino acid sequence as shown in SEQ ID NO: 39; or alternatively, the N-terminuses of the heavy chain variable regions of the two scFvs are symmetrically linked to the C-terminuses of the two light chains of the immunoglobulin through the linker; and, the light chain variable region of the scFv is a light chain variable region having the amino acid sequence as shown in SEQ ID NO: 29, and the heavy chain variable region of the scFv is a heavy chain variable region having the amino acid sequence as shown in SEQ ID NO: 39.

11. The bispecific antibody of claim 8, wherein the bispecific antibody comprises the following light chain amino acid sequence and heavy chain amino acid sequence: the light chain amino acid sequence as shown in SEQ ID NO: 50, and the heavy chain amino acid sequence as shown in SEQ ID NO: 51; or alternatively, the light chain amino acid sequence as shown in SEQ ID NO: 52, and the heavy chain amino acid sequence as shown in SEQ ID NO: 53; or alternatively, the light chain amino acid sequence as shown in SEQ ID NO: 54, and the heavy chain amino acid sequence as shown in SEQ ID NO: 51; or alternatively, the light chain amino acid sequence as shown in SEQ ID NO: 52, and the heavy chain amino acid sequence as shown in SEQ ID NO: 55; or alternatively, the light chain amino acid sequence as shown in SEQ ID NO: 56, and the heavy chain amino acid sequence as shown in SEQ ID NO: 57.

12. An isolated nucleic acid, wherein the isolated nucleic acid encodes the Sirpα-targeting antibody or the antigen-binding fragment thereof of claim 1 or a bispecific antibody; wherein the bispecific antibody comprising a first protein functional region and a second protein functional region, wherein the first protein functional region is a Sirpα-targeting antibody or a antigen-binding fragment thereof, and the second protein functional region is an antibody targeting a non-Sirpα antigen or an antigen-binding fragment thereof, preferably, the non-Sirpα antigen is an immune checkpoint antigen or a tumor therapy target, and the immune checkpoint antigen or the tumor therapy target antigen preferably comprises PD-1, PD-L1, Tim3, LAG3 or CLDN18.2; more preferably, the second protein functional region is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-Tim3 antibody, an anti-LAG3 antibody or an anti-CLDN18.2 antibody; and most preferably, the anti-PD-1 antibody is Nivolumab or Pembrolizumab, and the anti-PD-L1 antibody is Atezolumab, Avelumab or Durvalumab; wherein the Sirpα-targeting antibody or an antigen-binding fragment thereof, comprising a light chain variable region or a heavy chain variable region, wherein the antibody or the antigen-binding fragment thereof binds to human Sirpα-V1 and human Sirpα-V2, but weakly binds to or does not bind to human Sirpβ and Sirpγ, and does not bind to human T cells, and has the function of blocking the binding of Sirpα to CD47; preferably, the antibody or the antigen-binding fragment thereof also binds to one or more of Cyno Sirpα L932, L933, L936 and L937, but does not bind to Cyno Sirpα L938 and L939; wherein the amino acid sequence of the L932 has a NCBI sequence number of NP_001271679.1, the amino acid sequence of the L933 has a NCBI sequence number of XP_015313155.1, the amino acid sequence of the L936 is as shown in SEQ ID NO: 3, the amino acid sequence of the L937 is as shown in SEQ ID NO: 4, the amino acid sequence of the L938 is as shown in SEQ ID NO: 5, and the amino acid sequence of the L939 is as shown in SEQ ID NO: 6; more preferably, the light chain variable region comprises the following CDRs: VL CDR1 as shown in the amino acid sequence of SEQ ID NO: 11; VL CDR2 as shown in the amino acid sequence of SEQ ID NO: 12; and VL CDR3 as shown in the amino acid sequence of SEQ ID NO: 13; or the heavy chain variable region comprises the following CDRs: VH CDR1 as shown in the amino acid sequence of SEQ ID NO: 14; VH CDR2 as shown in the amino acid sequence of SEQ ID NO: 15; and VH CDR3 as shown in the amino acid sequence of SEQ ID NO: 16; or alternatively, the light chain variable region has 3, 2 or 1 amino acid mutations on the amino acid sequences of the VL CDR1, VL CDR2 and VL CDR3 respectively, or the heavy chain variable region has 3, 2 or 1 amino acid mutations on the amino acid sequences of the VH CDR1, VH CDR2 and VH CDR3 respectively.

13. A recombinant expression vector comprising the isolated nucleic acid of claim 12; preferably, the expression vector comprises a eukaryotic expression vector or a prokaryotic expression vector.

14. A transformant comprising the recombinant expression vector of claim 13 in a host cell; preferably, the host cell is a prokaryotic or eukaryotic cell, the prokaryotic cell is preferably E. coli cell such as TG1 and BL21, and the eukaryotic cell is preferably HEK293 cell or CHO cell.

15. A method for preparing a Sirpα-targeting antibody or an antigen-binding fragment thereof, comprising culturing the transformant of claim 14, and obtaining the Sirpα-targeting antibody or the antigen-binding fragment thereof from a culture.

16. A pharmaceutical composition comprising the Sirpα-targeting antibody or the antigen-binding fragment thereof of claim 1, and a pharmaceutically acceptable carrier; preferably: the pharmaceutical composition further comprises other anti-tumor antibodies as active ingredients; or, the pharmaceutically acceptable carrier comprises a His-HCl buffer at a concentration of 5 mM-50 mM, glycine at a concentration of 0 mM-200 mM, trehalose at a concentration of 0 mM-300 mM or Tween 80 with a volume ratio of 0.01%-1% of the pharmaceutical composition; more preferably: the concentration of the Sirpα-targeting antibody or the antigen-binding fragment thereof is 5 mg/mL-100 mg/mL, and preferably 5 mg/mL; or, the concentration of the His-HCl buffer is 20 mM; or, the concentration of the glycine is 125 mM; or, the concentration of the trehalose is 125 mM; or, the volume ratio of the Tween 80 to the pharmaceutical composition is 0.02%; or, the pH of the pharmaceutical composition is 5.0-7.0, e.g., 5.5, 6 or 6.5.

17. A method for diagnosing, preventing or treating a tumor in a subject in need thereof, comprising: administering an effective amount of the Sirpα-targeting antibody or the antigen-binding fragment thereof of claim 1 to the subject.

18. A pharmaceutical composition comprising the bispecific antibody of claim 8, and a pharmaceutically acceptable carrier; preferably: the pharmaceutical composition further comprises other anti-tumor antibodies as active ingredients; or, the pharmaceutically acceptable carrier comprises a His-HCl buffer at a concentration of 5 mM-50 mM, glycine at a concentration of 0 mM-200 mM, trehalose at a concentration of 0 mM-300 mM or Tween 80 with a volume ratio of 0.01%-1% of the pharmaceutical composition; more preferably: the concentration of the bispecific antibody is 5 mg/mL-100 mg/mL, and preferably 5 mg/mL; or, the concentration of the His-HCl buffer is 20 mM; or, the concentration of the glycine is 125 mM; or, the concentration of the trehalose is 125 mM; or, the volume ratio of the Tween 80 to the pharmaceutical composition is 0.02%; or, the pH of the pharmaceutical composition is 5.0-7.0, e.g., 5.5, 6 or 6.5; or, in the bispecific antibody, the amino acid sequence of the light chain is as shown in SEQ ID NO: 52, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 55; furthermore preferably, the pharmaceutical composition consists of: the bispecific antibody at a concentration of 5 mg/mL and with amino acid sequences of light and heavy chains as shown in SEQ ID NO: 52 and 55 respectively, the His-HCl buffer at a concentration of 20 mM, glycine at a concentration of 125 mM, trehalose at a concentration of 125 mM, and Tween 80 with a volume ratio of 0.02% of the pharmaceutical composition.

19. A method for diagnosing, preventing or treating a tumor in a subject in need thereof, comprising: administering an effective amount of the bispecific antibody of claim 8 to the subject.

20. A method for diagnosing, preventing or treating a tumor in a subject in need thereof, comprising: administering an effective amount of the pharmaceutical composition of claim 16 to the subject.

21. A method for diagnosing, preventing or treating a tumor in a subject in need thereof, comprising: administering an effective amount of the pharmaceutical composition of claim 18 to the subject.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0140] FIG. 1 shows the binding activity of the human-mouse chimeric anti-human Sirpα antibody mab14c of the present disclosure to human T cells, wherein 1a, 1b, 1c, and id are diagrams of the results of a negative control, a control antibody 1, a control antibody 2 and the antibody of the present disclosure, respectively.

[0141] FIG. 2 shows the specific binding activities of the humanized anti-human Sirpα antibody of the present disclosure to human Sirpβ (left panel) and Sirpγ (right panel).

[0142] FIG. 3 shows the binding activity of the humanized anti-human Sirpα antibody of the present disclosure to (different polymorphisms of) Cyno Sirpα.

[0143] FIG. 4 shows the in vivo pharmacodynamic activity of the Sirpα and PD-1 bispecific antibody of the present disclosure.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0144] The present disclosure is further described hereafter by way of examples, but the present disclosure is not limited to the scope of the described examples. The experimental methods that do not specify specific conditions in the following examples are selected according to conventional methods and conditions, or according to the description of products.

Example 1 Cloning, Expression and Purification of Antigen and Antibody

[0145] The antigens used in the present disclosure are purchased from various different companies as follows.

[0146] Sino Biological Inc.: human Sirpα-V1-his (Cat. No.: 11612-H08H), human Sirpα-V1-mFc (Cat. No.: 11612-H38H), mouse Sirpα-his (Cat. No.: 50956-M08H), and human Sirpγ (Cat. No.: 11828-H08H); or Acrobiosystems Co., Ltd.: human Sirpβ-hFc (Cat. No.: SIA-H5257), human Sirpγ-hFc (Cat. No.: SIG-H5253); or Biointron: human CD47-his (Cat. No.: B2048) or obtained by expression and purification of the present disclosure.

[0147] The sequence of human Sirpα-V1 protein (his, or Fc Tag) is NCBI Reference Sequence: NP_001035111, with a full length of 504-amino-acid, of which position 1-position 30 is a signal peptide; and the extracellular domain (ECD) is the amino-acid of position 31-position 373. The amino-acid of position 31-position 137 of the ECD is the Ig-like-V-type region, the amino-acid of position 148-position 247 is the Ig-like C1-type1 region, and the amino-acid of position 254-position 348 is the Ig-like C1-type2 region.

[0148] The sequence of human Sirpα-V2 protein (his, or Fc tag) is NCBI Reference Sequence: NP_001317657.1, with a full length of 508-amino-acid, of which position 1-position 30 is a signal peptide; and the ECD is the amino-acid of position 31-position 373. In the extracellular domain, the amino-acid sequence of position 35-position 145 is the Ig-like 1 region, the amino-acid of position 147-position 250 is the Ig-like 2 region, and the amino-acid of position 252-position 333 is the Ig-like 3 region.

[0149] The sequence of human Sirpβ protein (his tag) is NCBI Reference Sequence: NP_006056.2, with a full length of 398-amino-acid, of which position 1-position 29 is a signal peptide; and the ECD region is the amino-acid of position 30-position 371. In the ECD, the amino-acid of position 37-position 144 is the Ig-like-V-type region, the amino-acid of position 142-position 253 is the Ig-like region, and the amino-acid of position 265-position 344 is the Ig C region.

[0150] The sequence of human Sirpγ protein (his tag) is NCBI Reference Sequence: AAH64532.1, with a full length of 387-amino-acid, of which position 1-position 28 is a signal peptide; and the ECD is the amino-acid of position 29-position 360. In the ECD, the amino-acid of position 29-position 137 is the Ig-like-V-type region, the amino-acid of position 146-position 245 is the Ig-like C1-type1 region, and the amino-acid of position 252-position 340 is the Ig-like C1-type2 region.

[0151] The sequence of NOD mouse Sirpα protein (his tag) is referred to the article polymorphism in Sirpα modulates engraftment of human hematopoietic stem cells (Nature Immunology, 2007).

[0152] Cyno Sirpα (cynomolgus Sirpα, Macaca mulatta Sirpα) has different forms of polymorphism. In addition to two sequences from the database published by NCBI, the amino acid sequence of the Cyno Sirpα protein of the present disclosure includes 4 polymorphic sequences which are different from the Cyno Sirpα published by NCBI and obtained by collecting different cynomolgus monkeys. Also, the binding activities of the antibody of the present disclosure to these different polymorphic forms of the Cyno Sirpα protein are evaluated.

[0153] The protein sequence of Cyno Sirpα (his tag) is derived from NCBI database (see https://www.ncbi.nlm.nih.gov/), Reference Sequence: NP_001271679.1, with a full length of 503-amino-acid, of which position 1-position 30 is a signal peptide; and the ECD is the amino-acid of position 31-position 370. In the ECD, the amino-acid of position 38-position 144 is the Ig-like-V-type region, the amino-acid of position 142-position 253 is the Ig region, and the amino-acid of position 265-position 345 is the Ig-C region. NCBI Reference Sequence: XP_015313155.1 has a full length of 503 amino acids. The two ECD sequences differ by 3 amino acids (polymorphism).

[0154] Moreover, in the present disclosure, blood samples of cynomolgus monkeys (cynos) were purchased from Zhaoqing Experimental Animal Center, human peripheral blood mononuclear cells (PBMCs) were separated by using SEPMATE 50 (Beijing Office of STEMCELL Technologies Inc., Canada, Cat. No. 86450), then the PBMCs were cultured with RPMI1640 (HYCLONE, Cat. No. SH30809.01) containing 10% FBS (Shanghai Biosun Sci&Tech Co., Ltd., Cat. No. BS-0002-500) until confluence for 3 h, and the adherent macrophages were digested by pancreatin (Shanghai Basalmedia Technologies Co., Ltd., Cat. No. S310JV), then RNA was extracted by a Trizol method for reverse transcription, then the fragment of interest was amplified by PCR, wherein the primers used for PCR were SI-2F: taaacggatctctaGCGAATTCatggagcccgccggcccggcccccg (SEQ ID NO: 1) and SI-2R: cggccttgccggcctcGAGCGGCCGCtgtctgattcggacgaggtagag (SEQ ID NO: 2), and the purified PCR product was cloned into a vector for sequencing, wherein the sequencing primer is p63a-SEQ: cacaggtgtccactcccaggt (SEQ ID NO: 49). Finally, two Cyno Sirpα sequences (polymorphisms) different from those published by NCBI were obtained. Meanwhile, 2 other different Cyno Sirpα sequences (polymorphisms) were obtained from the blood samples of cynomolgus monkeys purchased from Hainan Animal Center by the same method.

[0155] The base sequence of each Cyno Sirpα protein was subjected to whole gene synthesis (C-terminus linked to 6×his) and cloned into a pTT5 vector (Biovector, Cat #: 102762) for expression, so as to obtain different Cyno Sirpα proteins through purification.

TABLE-US-00001 TABLE 1 Different polymorphic molecules of Cyno Sirpα No. of Cyno Sirpα protein Sequence number L932 NP_001271679.1 L933 XP_015313155.1 L936 RB3-3 L937 RB3-5 L938 RB6-1 L939 RB6-2

[0156] The amino acid sequence of the Cyno Sirpα protein (polymorphism) found in the present disclosure:

TABLE-US-00002 RB3-3 sequence: (SEQ ID NO: 3) EEELQVIQPEKSVSVAAGDSATLNCTVTSLIPVGPIQWFRGAGPGRELI YHQKEGHFPRVTSVSESTKRNNMDFSIHISNITPADAGTYYCVKFRKGS PDVEVKSGAGTELSVRAKPSAPVVSGPAVRATAEHTVSFTCESHGFSPR DITLKWFKNGNELSDFQTNVDPAGKSVSYSIRSTARVVLTRRDVHSQVI CEVAHVTLQGDPLRGTANLSEAIRVPPFLEVTQQSMRADNQVNVTCQVT KFYPQRLQLTWLENGNVSRTEMASALPENKDGTYNWTSWLLVNVSAHRD DVKLTCQVEHDGQPAVNKSFSVKVSAHPKEQGSNTAAENTGTNERN RB3-5 sequence: (SEQ ID NO: 4) EEELQVIQPEKSVSVAAGDSATLNCTVSSLIPVGPIQWFRGAGPGRELI YNLKEGHFPRVTPVSDPTKRNNMDFSIRISNITPADAGTYYCVKFRKGS PDVELKSGAGTELSVRAKPSAPVVSGPAVRATAEHTVSFTCESHGFSPR DITLKWFKNGNELSDVQTNVDPAGKSVSYSIRSTARVLLTRRDVHSQVI CEVAHVTLQGDPLRGTANLSEAIRVPPFLEVTQQSMRADNQVNVTCQVT KFYPQRLQLTWLENGNVSRTEMASALPENKDGTYNWTSWLLVNVSAHRD DVKLTCQVEHDGQPAVNKSFSVKVSAHPKEQGSNTAAENTGTNERN RB6-1 sequence: (SEQ ID NO: 5) EEELQVIQPEKSVSVAAGESATLNCTATSLIPVGPIQWFRGVGPGRELI YHQKEGHFPRVTPVSDPTKRNNMDFSIRISNITPADAGTYYCVKFRKGS PDVELKSGAGTELSVRAKPSAPVVSGPAVRATAEHTVSFTCESHGFSPR DITLKWFKNGNELSDFQTNVDPAGKSVSYSIRSTARVVLTRRDVHSQVI CEVAHVTLQGDPLRGTANLSEAIRVPPFLEVTQQSMRADNQVNVTCQVM KFYPQRLQLTWLENGNVSRTEMASALPENKDGTYNWTSWLLVNVSAHRD DVKLTCQVEHDGQPAVNKSFSVKVSAHPKEQGSNTAAENTGTNERN RB6-2 sequence: (SEQ ID NO: 6) EEELQVIQPEKSVSVAAGESATLNCTATSLIPVGPIQWFRGVGPGRELI YSQKEGHFPRVTPVSDPTKRNNMDFSIRISNITPADAGTYYCVKFRKGS PDVELKSGAGTELSVRAKPSAPVVSGPAVRATAEHTVSFTCESHGFSPR DITLKWFKNGNELSDFQTNVDPAGKSVSYSIRSTARVVLTRRDVHSQVI CEVAHVTLQGDPLRGTANLSEAIRVPPFLEVTQQSMRADNQVNVTCQVT KFYPQRLQLTWLENGNVSRTEMASALPENKDGTYNWTSWLLVNVSAHRD DVKLTCQVEHDGQPAVNKSFSVKVSAHPKEQGSNTAAENTGTNERN

[0157] The human CD47 (hFc/his tag) protein sequence was NCBI Reference Sequence: NP_001768.1, with a full length of 323 amino acids, of which position 1-position 18 is a signal peptide; and the ECD includes amino-acid of position 19-position 141.

[0158] The human PD-1 (hFc/his tag) protein sequence was NCBI Reference Sequence: NP_005009.2, with a full length of 288 amino acids, of which position 1-position 20 is a signal peptide; and the ECD includes amino-acid of position 21-position 167.

[0159] The human PD-L1 (hFc/his tag) protein sequence was NCBI Reference Sequence: NP_054862.1, with a full length of 290 amino acids, of which position 1-position 18 is a signal peptide; and the ECD includes amino-acid of position 19-position 239.

[0160] The proteins with hFc tag used in the present disclosure were expressed with a IgG1 Fc region at the C-terminus, and the proteins with his tag were expressed with 6×his at the C-terminus.

[0161] The antibodies used in the present disclosure, including a positive control antibody 1 (referred to as Ref1 for short, namely OSE-172, with a sequence from WO2017178653A2, #55 light chain, and #42 heavy chain, wherein # represents the sequence number in the reference) and a positive control antibody 2 (referred to as Ref2 for short, with a sequence from SIRP29 in US20140242095 A1, #6 light chain, and #12 heavy chain, acting as a positive control when binding to Sirpγ), were all expressed and purified by the present disclosure.

[0162] A pTT5 vector (Biovector, Cat #: 102762) was used as an expression vector. The expressed recombinant protein, and light and heavy chain sequences of the antibody were cloned into the pTT5 vector, transiently transfected into HEK293F cells (Life Technologies Cat. No. 11625019, hereinafter referred to as 293F cells for short) for expression, and then purified.

[0163] Specifically, expanding culture of the 293F cells was conducted in a Gibco FreeStyle 293 Expression Medium (Gibco, Cat #: 12338018) medium. Before the start of the transiently transfection, the cell concentration was adjusted to 8×10.sup.5 cells/mL, and cultured in 1% FBS (AusGeneX FBS Excellent, Supplier: AusGeneX, China, Cat #: FBSSA500-S) in a shaker at 37° C. under 8% CO.sub.2 for 24 h, with the survival rate being >95% by microscopic examination again, and the cell concentration being 1.2×10.sup.6 cells/mL.

[0164] A 300 mL culture system of cells was prepared, wherein 150 μg each of (the amount of a single plasmid was 300 μg if it was a recombinant protein) the heavy chain, light chain plasmids or fused protein plasmids was dissolved in 15 mL of Opti-MEM (Gibco, Cat #: 31985070), and filtered through a 0.22 μm filter for sterilization. Then 600 μL of 1 mg/mL PEI (Polysciences, Inc, Cat #: 23966-2) was dissolved into another 15 mL of Opti-MEM, and the mixture was left standing for 5 min. The PEI was slowly added into the plasmids and incubated at room temperature for 10 min, and the mixed solution of the plasmid-PEI was slowly added dropwise while shaking the culture flask. A sample was collected after culture in the shaker at 37° C. under 8% CO.sub.2 for 5 days and centrifuged at 3300 g for 10 min, and the supernatant was taken for purification.

[0165] Purification of antibody or -Fc fusion protein: The sample was centrifuged at a high speed to remove impurities, and a gravity column (Sangon, Cat #: F506606-0001) containing protein A (bselect, GE Healthcare Life Science, Cat #: 71-5020-91 AE) was equilibrated by rinsing with 2-5 times the volume of the column of PBS pH7.4. The sample passed through the column. The column was rinsed with 5-10 times the volume of the column of PBS (Sangon, CAT #: B548117-0500). Then the protein of interest was eluted with 0.1 M acetic acid at pH3.5, subsequently adjusted to neutrality with Tris-HCl at pH8.0, determined for the concentration with a microplate reader, subpackaged and stored for later use.

[0166] Purification of His Tagged protein: The sample was centrifuged at high speed to remove impurities. Equilibration of nickel column (Ni smart beads 6FF, Changzhou Smart-Lifesciences Biotechnology Co., Ltd., Cat #: SA036010): the nickel column was equilibrated by rinsing with 2-5 times the volume of the column of a PBS pH7.4 solution containing 10 mM imidazole and 0.5 M NaCl. The supernatant of the sample passed through the column.

[0167] Rinsing of impurity protein: the chromatographic column was rinsed with a PBS pH7.4 solution containing 10 mM imidazole and 0.5 M NaCl to remove non-specifically bound impurity proteins, and the effluent was collected. The protein of interest was eluted with PBS pH7.4 containing 250 mM imidazole and 0.5 M NaCl. Buffer replacement: the eluted protein of interest was centrifuged at 12,000 g for 10 min through an ultrafiltration tube (ultrafiltration tube, Merck Millipore, Cat #: UFC500308), then added with 1 mL of PBS, determined for the concentration, subpackaged and stored for later use.

Example 2 Construction of High Expression Cell Line and Detection of Cell Activity (ELISA)

[0168] The high expression cell line used in the present disclosure were all constructed by the inventors themselves through the stable cell line construction platform of our company. The construction process would be explained below by taking the construction of a human Sirpα high expression strain as an example. The specific steps were as follows:

[0169] On the 1st day of the experiment, 293T cells (The cell bank of National Collection of Authenticated Cell Cultures of Chinese Academy of Sciences, Cat #: GNHu17) were inoculated into two 6 cm culture dishes, with the number of cells in each culture dish reaching 7.5×10.sup.5. On the 2nd day, each 4 μg of a packaging plasmid (BioVector plasmid vector strain cell gene collection centers pGag-pol, pVSV-G and the like) and a plasmid pBabe-hSirpα cloned with a human Sirpα gene were added to Opti-MEM (Thermo Fisher Scientific, Cat #: 31985070) to make a final volume of 200 μL. Another 200 μL of OPTI-MEM was prepared, added with 36 μL of a transfection reagent fectin (Shanghai Basalmedia Technologies Co., Ltd., CAT #: F210), evenly mixed and allowed to stand at room temperature for 5 min. Then, the mixture (200 μl per dish) was added dropwise into the cultured 293T cells. On the 3rd day, the 293T cell culture medium was replaced with 4 mL of a DMEM high-sugar medium (Shanghai Basalmedia Technologies Co., Ltd., Cat #: L130KJ). On the 4th day, CHO-K1 cells (The cell bank of National Collection of Authenticated Cell Cultures of Chinese Academy of Sciences, CAT #: SCSP-507) were inoculated into a 10 cm culture dish to make the number of cells reach 5×10.sup.5. On the 5th day, the supernatant of 293T cells (viruses) was collected, filtered through a 0.45 μm filter membrane into cultured CHO-K1 cells, meanwhile added with 10 μg/mL of polybrene (Yeasen Biotechnology (Shanghai) Co., Ltd., Cat #: 40804ES76), mixed uniformly and placed into an incubator for 3-4 h, and then the medium was replaced with a DMEM/F12 10% FBS medium (Shanghai Basalmedia Technologies Co., Ltd., Cat #: L310KJ). On the 7th day, CHO-K1 cells were passaged, and the passaged cells were added with 10 μg/mL of puromycin on the 8th day for screening (Shanghai Basalmedia Technologies Co., Ltd., Cat #: S250J0). After 2-3 days, a large number of cells died, the medium was replaced to continue the culture until the cells no longer died, a large number of cells were expanded, and monoclonal cell strains were screened, subjected to expanding culture, and cryopreserved for seed preservation.

[0170] The amino acid sequence of human Sirpα (pBabe-hSirpα-V1) used in this example was NP_001035111, with a full length of amino-acids from position 1-position 504, of which position 1-position 30 was a signal peptide; and position 31-position 504 was the protein sequence expressed by the CHO-K1 hSirpα+ cell line constructed by the present disclosure.

[0171] hSirpα+ Cell Binding Activity (ELISA) Assay:

[0172] The cells obtained above, i.e., the monoclonal cell strains with high expression of human Sirpα, were subjected to expanding culture, then plated onto a 96-well plate in accordance with 10×10.sup.4/well, and incubated overnight in an incubator at 37° C. Then the supernatant was discarded, and the cell pellet was fixed with 100 μL/well of an immunostaining fix solution (Shanghai beyotime Biotechnology Co., Ltd., CAT #: P098) at room temperature for half an hour. The cells were washed once with PBS (Shanghai Basalmedia Technologies Co., Ltd., CAT #: B320), then blocked in 230 μL of 5% milk at 37° C. for 2 hours, and washed with PBST for three times. Each well was added with 50 μL of 5-fold gradient dilution of the sample to be tested at 10 μg/mL. After incubation at 37° C. for 1 hour, the well was washed with PBST for 5 times. The well was added with Anti-human HRP (Jackson Immuno Research, Cat #: 109-035-003) 1:2500 at 50 μL/well, incubated at 37° C. for 1 hour, and then washed with PBST for 5 times. Each well was added with 50 μL of TMB (Surmodic, Cat #: TTMB-1000-01) for color development, and added with 1 M H.sub.2SO.sub.4 at 50 μL/well to stop the reaction. The readings on a microplate reader (MultiskanGO Thermo, Model 51119200) were subjected to data analysis by GraphPad prism 5.

Example 3 Assay of Binding of Anti-Sirpα Antibody to Antigen (ELISA)

[0173] Different antigens (recombinant proteins) such as the human Sirpα-V1-hFc, Sirpα-V1-his, Sirpα-V2-his, Sirpβ-his, Sirpγ-his, monkey Sirpα-his (cynoSirpα-his) or NOD-mSirpα-his described in Example 1 were diluted with a PBS buffer at pH7.4 to concentrations of 1 μg/mL, 2 μg/mL or 5 μg/mL, added into a 96-well ELISA plate (Corning, CLS3590-100EA) at a volume of 50 μL/well, and placed in an incubator at 37° C. for 2 hours. After the liquid was discarded, the wells were added with a blocking solution of 5% skimmed milk (skimmed milk powder available from Bright Dairy) diluted with PBS at 230 μL/well, and incubated in an incubator at 37° C. for 3 hours, or placed overnight at 4° C. (for 16-18 hours) for blocking. The blocking solution was discarded, and the plate was washed with a PBST buffer (pH7.4 PBS containing 0.05% Tween-20) for 5 times, then added with 50 μL/well of the supernatant (containing a detection antibody) or 10 μg/mL of the starting 5-fold gradient dilution of the antibody to be tested, and incubated at 37° C. for 1 hour. The plated was washed with PBST for 5 times, added with 50 μL/well of Anti-mouse or human HRP secondary antibody (Jackson Immuno Research, Cat #: 115-035-003 or 109-035-003) diluted at 1:2500, and incubated at 37° C. for 1 hour. The plated was washed with PBST for 5 times, then added with 50 μL/well of TMB chromogenic substrate (KPL, Cat #: 52-00-03), incubated at room temperature for 10-15 min, added with 50 μL/well of 1 M H.sub.2SO.sub.4 to stop the reaction, and read by a MULTISKAN Go microplate reader (ThermoFisher, Cat #: 51119200) for the absorbance value at 450 nm, and then a clone with high binding activity was selected or a EC.sub.50 value was calculated according to the OD value.

Example 4 Activity of Anti-Sirpα Antibody in Preventing the Binding of Sirpα to CD47

[0174] The recombinant proteins such as human Sirpα-V1-hFc/Sirpα-V1-his were diluted to concentrations of 5 μg/mL or 2 μg/mL respectively with a PBS buffer at pH 7.4, and then added into a 96-well ELISA plate (Corning, CLS3590-100EA) at a volume of 50 μL/well, and placed in an incubator at 37° C. for 2 hours. After the liquid was discarded, the wells were added with a blocking solution of 5% skimmed milk (skimmed milk powder available from Bright Dairy) diluted with PBS at 230 μL/well, and incubated in an incubator at 37° C. for 3 hours, or placed overnight at 4° C. (for 16-18 hours) for blocking. The blocking solution was discarded, and the plate was washed with a PBST buffer (pH7.4 PBS containing 5% Tween-20) for 5 times, then added with 25 μL/well of the supernatant (containing a detection antibody) or 100 μg/mL of the starting 3-fold gradient dilution of the antibody to be tested, and 25 μL/well of Biotin-labeled CD47-hFc at a concentration of 4 μg/mL or 3 μg/mL of CD47-his, mixed uniformly, and then incubated in an incubator at 37° C. for 1 hour. The plate was washed for 5 times, added with 50 μL/well of streptavidin-HRP (GenScript, M00091) or anti-his-HRP secondary antibody (GenScript, A00612) diluted at 1:1000, incubated at 37° C. for 1 hour, and then washed with PBST for 5 times. Each well was added with 50 μL of TMB (Surmodic, Cat #: TTMB-1000-01) for color development, and added with 50 μL/well of 1 M H.sub.2SO.sub.4 to stop the reaction. The readings on a microplate reader (MultiskanGO Thermo, Model 51119200) were subjected to data analysis by GraphPad prism 5.

Example 5 Binding of Anti-Human Sirpα Antibody to Human T Cells

[0175] The T cells used in the present disclosure were isolated from the peripheral blood of healthy volunteers with SEPMATE 50 (Beijing Office of STEMCELL Technologies Inc., Cat. No. 86450), and cryopreserved with a cryopreservation solution (RPMI1640 medium: FBS:DMSO=5:4:1) in a liquid nitrogen tank for later use. The cryopreserved PBMC cells were taken, thawed at 37° C., then added into 3 mL of a FACS buffer (1×PBS+2% FBS), and centrifuged at 1,000 rpm for 5 min, the supernatant was discarded, the cells were resuspended in the FACS buffer for counting, and the density was adjusted to 1×10.sup.6 cells/mL. PBMCs were added into a U-shaped 96-well plate at 100 μL cells/well, and added with the antibody to be detected so that the final concentrations thereof are 10 μg/mL, 1 μg/mL and 0 μg/mL respectively. They were mixed uniformly, then incubated at room temperature for 20 min, and centrifuged at 2,000 rpm for 5 min. The supernatant was discarded, and the cell pellet was resuspended by adding 100 μL of the FACS buffer, and centrifuged at 2,000 rpm for 5 min. The supernatant was discarded, and the cell pellet was added with 50 μL of PE anti-human IgG Fc (Biolegend, Cat. No.: 409304) diluted at 1:200, mixed uniformly, and incubated with protected from light at room temperature for 20 min. The mixture was centrifuged at 2,000 rpm for 5 min. The supernatant was discarded, and the cell pellet was washed once with 100 μL of the FACS buffer, resuspended with 100 μL of the FACS buffer, and read by a flow cytometer (Novosampler™ pro, model: NS200). The assay data was processed with NovoExpress. Taking the sample well with the antibody concentration of 0 μg/mL as the negative control, based on this, the proportion of T cells specifically bound with the antibody to be tested was calculated.

Example 6 Discovery of Anti-Human Sirpα Antibody

[0176] In the present disclosure, human Sirpα-V1-mFc (Sino Biological Inc., Cat. No.: 11612-H38H) and hSirpα-V2-hFc (as expressed in Example 1) recombinant proteins were used as antigens. After the mice were subjected to cross immunization with a Freund's complete adjuvant for 4 times, the cells were subjected to electrofusion and screened for fused hybridomas, so that the clones with good binding activity to hSirpα-V1 were screened out from tens of thousands of hybridoma clones. After further screening, it was unexpectedly found that a clone could simultaneously bind to human Sirpα-V1 and human Sirpα-V2, had blocking activity, and did not bind to human PBMCs (T cells). Further, monoclonal cell strains are obtained from these unexpectedly discovered hybridoma clones through multiple times of subcloning, antibody sequences are extracted from the monoclonal cell strains, and expressed and purified to obtain the murine antibody of the present disclosure.

[0177] Specifically, the 4 weeks old female SJL mice for experimental use, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., with the animal production license number: SCXK (Beijing) 2016-0011. After the mice were purchased, they were reared in a laboratory environment for 1 week under adjustment of a daytime light/night dark cycle, at a temperature of 20-25° C. and humidity of 40-60%. The mice were divided into 3 mice/group/cage. The mice were immunized with the antigen prepared in Example 1. The immune adjuvant used at the first time was a Freund's complete adjuvant (Sigma-Aldrich, SIGMA F5506-10M), and the immune adjuvants used started from the second time were Freund's incomplete adjuvants (Sigma-Aldrich, SIGMA F5881-10M). The ratio of the antigen to the adjuvant was 1:1 (v/v). The mice were subjected to first immunization at 100 μL/25 μg/mouse, and second, third, and fourth immunization respectively at 100 μL/12.5 μg/mouse, through intramuscular injection in a lower leg. 3 days before fusion, the mice were subjected to booster immunization at 100 μL/25 μg/mouse. The immunization times were days 0, 14, 28, 42, and 56 (booster immunization). On days 36 and 50, the serum antibody titer of the mice was detected by the ELISA method of Example 3, and the mice with high serum antibody titer which was at a plateau were selected for spleen cell fusion. Spleen lymphocytes were fused with myeloma cells Sp2/0 (ATCC® CRL-8287™) to obtain hybridoma cells. The hybridoma cells were plated onto a 96-well plate, and then screened to obtain preferred clones.

[0178] The hybridoma cell strains were subjected to primary screening, wherein the binding activity of the antibody in the supernatant secreted by the hybridoma cell strains to human Sirpα was detected by the ELISA method of Example 3 (partial binding data was shown in Table 2), and the clones with good activity were selected. The supernatant was taken, and the activity of the secreted antibody in blocking the binding of hSirpα to hCD47 (Blocking activity) was detected by the method of Example 4, and meanwhile the binding activity of it to human Sirpα-V2 was also detected. The preferred clones were further subjected to limiting dilution to obtain monoclonal antibody cell strains, and partial of the results were shown in Table 3.

TABLE-US-00003 TABLE 2 The binding activity of the supernatant of the hybridoma fusion cells (clones) to hSirpα-V1 (OD.sub.450 value) Initial clone number ELISA value 1E6 1.58 2D9 1.52 3C6 1.22 3B8 1.6 3C11 2.49 4C1 3.0 5G7 1.54 5E 9 1.56 5B10 1.71 6H1 1.57 6F12 1.59 7G4 1.32 7H4 1.85 8C4 2.2 8H4 2.14 8H7 2.11 9H1 2.48 9F8 1.25 9G8 2.25 9H10 2.02 10A3 1.12 10G4 1.85 10Q5 1.98 10C11 2.39 11H1 1.4 12C1 1.57 12E 2 1.22 12C3 1.56 12D7 1.54 13F10 1.0 13A11 1.24 13C11 1.7 14A4 1.41 14H6 1.81 15Q6 1.53 16G12 1.28

TABLE-US-00004 TABLE 3 Data of the binding activity of the supernatant of the hybridoma fusion to hSirpα-V2, and blocking activity of the same (OD.sub.450 value) Initial ELISA value activity of blocking clone (activity of binding the binding of number to hSirpα-V2) hSirpα to hCD47 1E6 1.09 0.85 3C11 1.02 0.83 4C1 0.92 0.73 5G7 1.24 0.65 5B10 0.07 0.24 6H1 1.09 0.79 6F12 1.05 0.68 8C4 1.25 0.79 8H4 1.27 0.75 9H1 1.0 0.75 9F8 0.99 0.75 9G8 1.0 0.72 9H10 1.0 0.69 10C11 1.0 0.73 12C1 0.89 0.69 12D7 1.03 0.78 14A4 0.87 0.75 14H6 1.25 0.33 15Q6 0.87 0.68 16G12 0.99 0.78

[0179] Partial of the primary screening data was listed in Table 2. The data showed that many hybridoma fusion cells exhibited very high binding activity in the primary screening and very high ELISA reading values (OD.sub.450 above 2), such as 4C1, 8C4, 8H-7, 9H1 and the like clones. However, it had been found by further screening that almost all of the clones either had low binding activities to hSirpα-V2, or did not have activities of blocking the binding of hSirpα to hCD47 (when the OD.sub.450 value of the blocking activity was lower and had the greater difference from the binding ELISA value, the better blocking activity was indicated). See Table 3.

[0180] Very surprisingly, it was found that the clone 14H6 with better binding activity to hSirpα-V1 (Table 2, with the OD.sub.450 value of 1.81) showed very strong binding activity to hSirpα-V2 (Table 3, with the OD.sub.450 value of 1.25), and was able to block the binding of hSirpα to hCD47 (with the OD.sub.450 value of 0.33) very well.

[0181] This clone was subjected to multiple times of limiting dilution, with each time of dilution being conducted for 7-10 days. After the clones were proliferated, the antibody (supernatant) secreted by each clone was re-detected by the ELISA method for the binding activities to different Sirpαs and the blocking activity of the same. The results were shown in Table 4.

TABLE-US-00005 TABLE 4 Screening activity of preferred hybridoma cell subclones Activity of Activity of blocking the blocking the binding of binding of Activity of Activity of Activity of hSirpα to hSirpα binding to binding to binding to CD47 (dimer) to Subclone number hSirpα-V1 hSirpα-V2 hSirpγ (dimer) CD47 14H6E5H9F6E3 1.1 1.64 0.11 0.48 0.075 14H6E5H9F6G4 1.2 1.4 0.081 0.49 0.10 14H6E5H9F6E5 1.11 1.3 0.089 0.44 0.071 14H6E5H9F6E11 1.15 1.4 0.092 0.46 0.072 14H6E5B5C8G2 1.27 1.25 0.099 0.42 0.069 14H6E5B5C8G4 1.23 1.29 0.082 0.43 0.083 14H6E5B5C8F5 1.3 1.2 0.091 0.41 0.067 14H6E5B5C8G5 1.37 1.34 0.085 0.42 0.08

[0182] The aforementioned results indicated that the antibody secreted by the monoclonal cell strain obtained from 14H6 through multiple times of subcloning retained the activities of binding to hSirpα-V1 and hSirpα-V2, and exhibited the activities of blocking the binding of hSirpα-V1-his to hCD47-Fc (dimer) and blocking the binding of hSirpα-V1-hFc (dimer) to hCD47-his.

[0183] Even more unexpectedly, the antibodies secreted by these monoclonal cell strains did not bind to Sirpγ (in Table 4, the value of the activity of binding to hSirpγ was below 0.1, i.e., the background value). It indicated that the antibody of the present disclosure had very good selectivity to Sirpα and Sirpγ.

[0184] In the present disclosure, the antibody sequence was further extracted from 14H6E5B5C8G2, one of the subclones of 14H6, to obtain the preferred murine mab14 antibody sequence of the present disclosure, which was described specifically in the following examples.

Example 7 the Extraction, Analysis and Identification of Antibody Sequence of Murine Anti-Human Sirpα Antibody Mab14

[0185] The process of extracting an antibody sequence from the preferred monoclonal cell strain obtained from the aforementioned hybridoma 14H6 subclones is a method commonly used by those skilled in the art. Specifically, the aforementioned monoclonal cell strains were collected and subjected to expanding culture, then 1×10.sup.6 cells were taken, and RNAs were extracted with Trizol (Invitrogen, 15596-018) (according to the steps of the instructions of the kit). The extracted RNAs were reverse transcribed into cDNAs, and the reverse transcription kit was purchased from Sangon (Shanghai) Co., Ltd., Cat #: B532435. PCR amplification was performed by using the cDNAs obtained by reverse transcription as templates. The amplified products were sequenced, and the base/coding sequences of the light and heavy chain variable regions of the mab14 antibody were obtained respectively (as follows). The primers as used are referred to the manual TB326 Rev. C0308 published by Novagen.

[0186] The nucleotide sequence of the light chain variable region of the murine monoclonal antibody mab14 obtained in the preferred hybridoma cell strain of the present disclosure (the underlined part was the coding sequence):

TABLE-US-00006 (SEQ ID NO: 7) Taatggtgtccctcagctcagttccttggtctcctgttgctctgttttc aaggtaccagatgtgatatccagatgacacagactacatcctccctgtc tgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggac attaggaattatttaaactggtatcagcagaagccagatggaactgtta aactcctgatctacttcacatcaacattacactcaggagtcccatcaag gttcagtggcagtgggtctggaacagattattctctcaccattagcaac ctggaacaagaagatattgccacttacttttgccaacagggtaatacgc ttccgtggacgttcggtggaggcaccaagctggaaatcaaacgggctga tgctgcaccaactgtatccatcttcccaccatccagtgagcagttaaca tctggaggtgcctcagtcgtgtgcttctgaacaactctaccccaaagac atcaaggacct

[0187] The nucleotide sequence of the heavy chain variable region of the murine monoclonal antibody mab14 obtained in the preferred hybridoma cell strain of the present disclosure (the underlined part was the coding sequence):

TABLE-US-00007 (SEQ ID NO: 8) Tcatgggatggagctgtatcatgttctttttggtagccgcagctacagg tgtccactcccaggtccatctgcagcagcctggggctgagcttgtgaag cctggggcttcagtgaagttgtcctgcaaggcttctggctacaatttca acatctactggataaattgggtgaagcagaggcctggacaaggccttga gtggattggaaatatttatcctagtagtattagtactaactacaatgag aagttcaagacgaaggccacactgactgtagacaaatcctccaacacag tctacatgcagttcagcagcctgacatctgaggactctgcggtctatta ttgtgcgcgatcggagggaacttactatggtggtcgctacgagggggac tggtttggttactggggccaagggactctggtcactgtctctgcagcca aaacaacacccccatcagtctatccactggcccctgggtgtggagatac aactggttcctccgtgactctgggatgcctggtcaagggctactgccga gtcgaagttcc

[0188] The amino acid sequences encoded by the nucleotide sequences of the light and heavy chain variable regions of the murine monoclonal antibody mab14 obtained by the present disclosure were as shown in SEQ ID NO: 9 and SEQ ID NO: 10 below.

[0189] The amino acid sequence of the light chain variable region of the murine monoclonal antibody mab14 obtained in the preferred hybridoma cell strain of the present disclosure:

TABLE-US-00008 (SEQ ID NO: 9) DIQMTQTTSSLSASLGDRVTISCRASQDIRNYLNWYQQKPDGTVKLLIY FTSTLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTF  GGGTKLEIK

[0190] The amino acid sequence of the heavy chain variable region of the murine monoclonal antibody mab14 obtained in the preferred monoclonal hybridoma cell strain of the present disclosure:

TABLE-US-00009 (SEQ ID NO: 10) QVHLQQPGAELVKPGASVKLSCKASGYNFNIYWINWVKQRPGQGLEWIG NIYPSSISTNYNEKFKTKATLTVDKSSNTVYMQFSSLTSEDSAVYYCAR SEGTYYGGRYEGDWFGYWGQGTLVTVSA

[0191] The light and heavy chain variable region sequences of the aforementioned antibody of the present disclosure and the constant regions of different IgG types, for example human IgG1 (hIgG1), human IgG2 (hIgG2), human IgG3 (hIgG3) or human IgG4 (hIgG4), human light chain κ and λ types; murine mIgG1, mIgG2 or mIgG3, murine light chain κ and λ types, etc. were recombined, expressed and purified to obtain an entire human-murine chimeric antibody and a murine antibody. In the present disclosure, taking the heavy chain constant region being hIgG4 and the light chain being of the κ type as an example, the human-murine chimeric antibody mab14c was obtained according to the expression and purification methods of Example 1. The binding activities of mab14c to hSirpα-V1, hSirpα-V2 and hSirpα+ cells and the activity of the same in blocking the binding of hSirpα to CD47 were detected by the methods of Example 2, Example 3 and Example 4, and compared with those of the control antibody 1. The results were shown in Table 5.

TABLE-US-00010 TABLE 5 Activity analysis of the human-murine chimeric antibody mab14c of the present disclosure Blocking Blocking binding of binding of hSirpα to hSirpα hSirpα+ hCD47 (dimer) to Sample hSirpα-V1, hSirpα-V2, cell, (dimer), hCD47, name EC.sub.50, nM EC.sub.50, nM EC.sub.50, nM IC.sub.50, nM IC50, nM mab14c 0.16 0.14 0.29 5.54 5.7 Ref1 0.17 No binding 0.37 4.58 4.55

[0192] The aforementioned results showed that it was unexpectedly found by the present disclosure the human-murine chimeric antibody mab14c could not only block the binding of Sirpα to human CD47, but also have good binding activity to all of hSirpα-V1, hSirpα-V2 and hSirpα+ cells. This was different from Ref1, which did not bind to hSirpα-V2.

[0193] Furthermore, the binding activities of mab14c to the muSirpα of NOD mice and different polymorphisms of Cyno Sirpαs (including L932, L933 and L936-L939) were detected by the method of Example 4, and the results were shown in Table 6.

TABLE-US-00011 TABLE 6 Analysis of the binding activity of the antibody mab14c of the present disclosure to murine and Cyno Sirpαs (EC.sub.50, nM) Sample name NOD-muSirpα L932 L933 L936 L937 L938 L939 mab14c No 0.10 0.14 0.099 0.099 No No binding binding binding Ref1 No No No No faint faint weak binding binding binding binding binding binding binding Note: it was faint binding when the EC.sub.50 was greater than 10 nM and less than 50 nM (10 nM ≤ EC.sub.50 < 50 nM); it was weak binding when the EC.sub.50 was between 2-10 nM (i.e., 2 nM ≤ EC.sub.50 < 10 nM; and it was no binding when the EC.sub.50 was greater than 50 nM (EC.sub.50 ≥ 50 nM) or no signal value was detected.

[0194] The aforementioned results showed that the antibody mab14c of the present disclosure had very good binding to 4 of 6 polymorphic proteins of cynomolgus (cyno) Sirpα, which was significantly different from that of Ref1, which did not bind to any of these 6 Cyno Sirpαs. In the preclinical research stage of new drug development, it was necessary to select related primate species for preclinical research. If it did not bind to the protein of the primate such as cyno (the most commonly used primate), the primate could not be selected for preclinical research, which would bring great inconvenience to the preclinical research. Therefore, it brought great convenience to the preclinical research since the antibody of the present disclosure bound to various polymorphic proteins of Cyno Sirpα.

[0195] Furthermore, since human T cells expressed Sirpγ, in order to evaluate the binding activity (selectivity) of the antibody of the present disclosure to human T cells, the binding activity of the antibody mab14c of the present disclosure to human T cells was detected by the method of Example 5, and the results were shown in FIG. 1. 1a was a negative control, that was, only 0.39% of the cells (background level) had fluorescence intensity above 103 (positive cells) in the absence of the antibody. That was, no bound cells were detected. In the presence of 10 μg/mL of the control antibody 1 (Ref1) (1b), the proportion of cells with fluorescence intensity above 103 was 0.49% (background level), that was, the antibody did not bind to T cells. Under the same conditions, the proportion of the detected cells bound to the control antibody 2 (Ref2) was 51.3%, that was, the antibody bound to T cells (1c). Under the same conditions, the proportion of cells bound to the antibody of the present disclosure was 0.9% (background level, 1d), that was, the antibody of the present disclosure did not bind to T cells.

[0196] The aforementioned results indicated that the antibody mab14c of the present disclosure was anew antibody which was different from both Ref1 and Ref2. Compared with Ref1, it had better selectivity, could bind to both V1 and V2 forms of Sirpα, and could be targeted at more patient populations (those who expressed V1 and those who expressed V2) clinically. Compared with Ref1, it had better activity of binding to Cyno Sirpα, which brought great convenience for preclinical pharmacological and toxicological research. Furthermore, the antibody of the present disclosure was also different from Ref2 and it had better selectivity than Ref2: it did not bind to Sirpγ or T cells (expressing Sirpγ), could be developed as a drug, and avoided the side effects caused by non-specific targeting brought about by the binding to T cells clinically.

Example 8 Humanization of the Murine Antibody of the Present Disclosure

[0197] In order to avoid the risks of immunogenicity and the like aspects in the process of drug development, in the present disclosure, humanization design and screening, and sequence optimization were conducted on the murine antibody mab14. The specific process was described as follows.

[0198] Regarding the definition of the CDR of the antibody, there were many different methods in the art. These methods of labeling CDRs could be summarized in the Table 7 below.

TABLE-US-00012 TABLE 7 Summary of different methods for defining the CDR of the antibody in the art* Definition Definition Definition Definition Definition Loop of CCG of Kabat of AbM of Chothia of Contact Light chain L24-L34 L24-L34 L24-L34 L24-L34 L30-L36 CDR1 Light chain L50-L56 L50-L56 L50-L56 L50-L56 L45-L55 CDR2 Light chain L89-L97 L89-L97 L89-L97 L89-L97 L89-L96 CDR3 Heavy chain H26-H35 H31-H35 H26-H35 H26-H32 H30-H35 CDR1 Heavy chain H50-H65 H50-H65 H50-H58 H52-H56 H47-H58 CDR2 Heavy chain H95-H102 H95-H102 H95-H102 H95-H102 H93-H101 CDR3 *Please refer to the website for more information: http://www.bioinf.org.uk/abs/#cdrdef
http://www.bioinforg.uk/abs/#cdrdef

[0199] Laa-Lbb in Table 7 could refer to the amino acid sequence from position aa to position bb starting from the N-terminus of the light chain of the antibody; and Haa-Hbb could refer to the amino acid sequence from position aa to position bb starting from the N-terminus of the heavy chain of the antibody. For example, L24-L34 could refer to the amino acid sequence from position 24 to position 34 starting from the N-terminus of the light chain of the antibody, according to a CCG coding rule; and H26-H32 could refer to the amino acid sequence from position 26 to position 35 starting from the N-terminus of the heavy chain antibody, according to the CCG coding rule. It was well known to those skilled in the art that insertion and/or site deletion sometimes occurred in some positions when coding CDRs.

[0200] The aforementioned variable region of the murine anti-human Sirpα antibody mab14 was according to various definition methods in Table 7, and its CDR sequence was labeled/annotated as follows.

TABLE-US-00013 TABLE 8 CDR sequences of the anti-hSirpa antibody mab14 of the present disclosure defined according to the CCG numbering rule Antibody mab14 CDRs Light chain RASQDIRNYLN (SEQ ID NO: 11) CDR1 Light chain FTSTLHS (SEQ ID NO: 12) CDR2 Light chain QQGNTLPWT (SEQ ID NO: 13) CDR3 Heavy chain GYNFNIYWIN (SEQ ID NO: 14) CDR1 Heavy chain NIYPSSISTNYNEKFKT (SEQ ID NO: 15) CDR2 Heavy chain SEGTYYGGRYEGDWFGY (SEQ ID NO: 16) CDR3

TABLE-US-00014 TABLE 9 The CDR sequences of the anti-hSirpα antibody of the present disclosure defined according to the  Kabat numbering rule Antibody mab14 CDRs Light chain RASQDIRNYLN (SEQ ID NO: 11) CDR1 Light chain FTSTLHS (SEQ ID NO: 12) CDR2 Light chain QQGNTLPWT (SEQ ID NO: 13) CDR3 Heavy chain IYWIN (SEQ ID NO: 17) CDR1 Heavy chain NIYPSSISTNYNEKFKT (SEQ ID NO: 15) CDR2 Heavy chain SEGTYYGGRYEGDWFGY (SEQ ID NO: 16) CDR3

TABLE-US-00015 TABLE 10 The CDR sequences of the antibody of the present disclosure defined according to the AbM  numbering rule Antibody mab14 CDRs Light chain RASQDIRNYLN (SEQ ID NO: 11) CDR1 Light chain FTSTLHS (SEQ ID NO: 12) CDR2 Light chain QQGNTLPWT (SEQ ID NO: 13) CDR3 Heavy chain GYNFNIYWIN (SEQ ID NO: 14) CDR1 Heavy chain NIYPSSIST (SEQ ID NO: 18) CDR2 Heavy chain SEGTYYGGRYEGDWFGY (SEQ ID NO: 16) CDR3

TABLE-US-00016 TABLE 11 The CDR sequences of the antibody of the present disclosure defined according the Chothia numbering rule Antibody mab14 CDRs Light chain RASQDIRNYLN (SEQ ID NO: 11) CDR1 Light chain FTSTLHS (SEQ ID NO: 12) CDR2 Light chain QQGNTLPWT (SEQ ID NO: 13) CDR3 Heavy chain GYNFNIY (SEQ ID NO: 19) CDR1 Heavy chain YPSSI (SEQ ID NO: 20) CDR2 Heavy chain SEGTYYGGRYEGDWFGY (SEQ ID NO: 16) CDR3

TABLE-US-00017 TABLE 12 The CDR sequences of the antibody of the present disclosure defined according to the Contact numbering rule Antibody mab14 CDRs Light chain RNYLNWY (SEQ ID NO: 21) CDR1 Light chain KLLIYFTSTLH (SEQ ID NO: 22) CDR2 Light chain QQGNTLPW (SEQ ID NO: 23) CDR3 Heavy chain NIYWIN (SEQ ID NO: 24) CDR1 Heavy chain WIGNIYPSSIST (SEQ ID NO: 25) CDR2 Heavy chain ARSEGTYYGGRYEGDWFG (SEQ ID NO: 26) CDR3

[0201] After the aforementioned analysis, labeling and definition of the CDR sequence of the murine antibody mab14 of the present disclosure, it was humanized according to the methods published in many literatures in the art. The murine antibody sequence was compared with a human antibody germline database (v-base) to find out the light and heavy chain germlines of the human antibody with high homology. On this basis, computer modeling was carried out to simulate the sites in the antibody structure that might affect the binding to the antigen, key sites of back mutation and a combination thereof, so as to screen out a humanized antibody molecule with the preferred activity.

[0202] Specifically, through the comparative analysis of sequence homology, it was found that the human antibody germline with better homology with the light chain of mab14 included IGKV1-27*01, IGKV1-33*01, IGKV1-39*01, IGKV1-NL1*01, IGKV1/OR10-1*01, IGKV1D-33*01, IGKV1D-39*01, IGKV1-12*01, IGKV1-12*02, IGKV1-17*02, etc. After further comparison and analysis, the human antibody germline light chain IGKV1-39*01 was preferred. It had been found through sequence alignment that the J gene region of the light chain of mab14 had high homology with human antibody germlines hJk1, hJk2.1, hJk2.2, hJk2.3, hJk2.4, hJk3, hJk4.1, hJk4.2, and hJk5. After further comparison and analysis, hJh4.1 was preferred for the humanized human antibody germline J region of the light chain of mab14, and subjected to humanized design, screening and sequence optimization.

[0203] Through the comparative analysis of sequence homology, it was found that the human antibody germline with better homology to the heavy chain of mab14 included IGHV1-46*01, IGHV1-46*02, IGHV1-46*03, IGHV1-69*02, IGHV1-69*04, IGHV1-69*06, IGHV1-69*08, IGHV1-69*09, IGHV1-69*10, IGHV1-69*14, etc. After further comparison and analysis, the sequence of the human germline heavy chain IGHV1-46*01 was preferred for humanization of the antibody of the present disclosure. It had been found through sequence alignment that the J gene region of the heavy chain of mab14 had high homology with the human antibody germline heavy chain J genes hJh1, hJh2, hJh3.1, hJh3.2, hJh4.1, hJh4.2, hJh4.3, hJh5.1, hJh5.2, hJh6.1, hJh6.2, hJh6.3 etc. After further comparison and analysis, hJh4.1 was preferred for the humanized human antibody germline J region of the heavy chain of mab14, and subjected to humanized design, screening and sequence optimization.

[0204] The antibody of the present disclosure was transplanted with the CDR of mab14 (see the definition of CDR above, which was mainly defined by CCG in this example) to the selected humanized light and heavy chain human antibody germline template, and then recombined with IgG light and heavy chain constant regions. Then, based on the three-dimensional structure of the murine antibody, the embedded residues, residues that directly interacted with CDR, and residues that had important influence on the conformations of VL and VH are subjected to back mutation, and these mutations and mutation combinations were screened to see the influence on the antibody activity, and the chemically unstable amino acid residues of the CDR were optimized to obtain an antibody molecular sequence with optimization in structure, activity and the like. That was, the humanization of the murine antibody of the present disclosure was completed.

[0205] Combined with the specific sequence of mab14, the hIgG4 heavy chain and the κ type light chain (with the sequences as shown below) were taken as examples for explanation hereafter.

TABLE-US-00018 Light chain constant region κ chain of human antibody: (SEQ ID NO: 27) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC Heavy chain constant region of human IgG4: (SEQ ID NO: 28) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS QEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDK SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0206] The preferred sequence of the humanized light chain variable region of the present disclosure was as follows:

TABLE-US-00019 >mab14-hL1 (SEQ ID NO: 29) DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIY FTSTLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTF GGGTKVEIK >mab14-hL2 (SEQ ID NO: 30) DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGGTVKLLIY FTSTLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTF GGGTKVEIK >mab14-hL3 (SEQ ID NO: 31) DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGGTPKLLIY FTSTLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTF GGGTKVEIK >mab14-hL4 (SEQ ID NO: 32) DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGGAPKLLIY FTSTLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTF GGGTKVEIK >mab14-hL5 (SEQ ID NO: 33) DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGGTPKLLIY FTSTLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTF GGGTKVEIK >mab14-hL6 (SEQ ID NO: 34) DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKTPKLLIY FTSTLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTF GGGTKVEIK

[0207] The preferred sequence of the humanized heavy chain variable region of the present disclosure:

TABLE-US-00020 mab14-hH1 (SEQ ID NO: 35) QVQLVQSGAEVKKPGASVKVSCKASGYNFNIYWINWVRQAPGQGLEWMG NIYPSSISTNYNEKFKTRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR SEGTYYGGRYEGDWFGYWGQGTLVTVSS mab14-hH2 (SEQ ID NO: 36) QVQLVQSGAEVKKPGASVKVSCKASGYNFNIYWINWVKQAPGQGLEWIG NIYPSSISTNYNEKFKTKATLTVDKSTSTVYMEFSSLRSEDTAVYYCAR SEGTYYGGRYEGDWFGYWGQGTLVTVSS mab14-hH3 (SEQ ID NO: 37) QVQLVQSGAEVKKPGASVKVSCKASGYNFNIYWINWVRQAPGQGLEWMG NIYPSSISTNYNEKFKTRATLTVDTSTSTVYMELSSLRSEDTAVYYCAR SEGTYYGGRYEGDWFGYWGQGTLVTVSS mab14-hH4 (SEQ ID NO: 38) QVQLVQSGAEVKKPGASVKVSCKASGYNFNIYWINWVRQAPGQGLEWIG NIYPSSISTNYNEKFKTRATLTVDTSTSTVYMELSSLRSEDTAVYYCAR SEGTYYGGRYEGDWFGYWGQGTLVTVSS mab14-hH5 (SEQ ID NO: 39) QVQLVQSGAEVKKPGASVKVSCKASGYNFNIYWINWVRQAPGQGLEWIG NIYPSSISTNYNEKFKTRATLTVDKSTSTVYMELSSLRSEDTAVYYCAR SEGTYYGGRYEGDWFGYWGQGTLVTVSS mab14-hH6 (SEQ ID NO: 40) QVQLVQSGAEVKKPGASVKVSCKASGYNFNIYWINWVKQAPGQGLEWIG NIYPSSISTNYNEKFKTRATLTVDTSTSTVYMELSSLRSEDTAVYYCAR SEGTYYGGRYEGDWFGYWGQGTLVTVSS mab14-hH7 (SEQ ID NO: 41) QVQLVQSGAEVKKPGASVKVSCKASGYNFNIYWINWVKQAPGQGLEWIG NIYPSSISTNYNEKFKTKATLTVDTSTSTVYMELSSLRSEDTAVYYCAR SEGTYYGGRYEGDWFGYWGQGTLVTVSS

[0208] The humanized sequence of the light chain of the murine antibody of the present disclosure contained different back mutations, and the number of the back mutation sites could be 10 or more, and preferably 0-10, as for the sequences listed above. These arbitrary sequences were combined with the constant region sequences of the light chain constant region κ or λ chain of the human antibody to obtain the light chain sequence of the antibody of the present disclosure, such as the κ type light chain constant region for the light chain of the present disclosure, as for the sequences listed above. Likewise, the heavy chain variable regions used for humanization also had different numbers of back mutations, and the number of back mutation sites could be 10 or more, and preferably 0-10, as for the heavy chain variable region sequences listed above. These heavy chain variable region sequences containing different numbers of back mutations were recombined with optionally the constant region sequences of human IgG1, human IgG2, human IgG3, and human IgG4 chains to obtain the heavy chain sequences of the present disclosure. For example, the heavy chain of the present disclosure was illustrated by taking hIgG4 which was used as a constant region sequence as an example.

[0209] The present disclosure partially optimized the humanized antibody sequence, and the results of the expression level and activity evaluation of the expressed antibody (by the ELISA detection method of Example 3 of the present disclosure) were as follows.

TABLE-US-00021 TABLE 13 Humanized antibody sequences of the present disclosure (human κ type light chain, taking the hIgG4 heavy chain constant region as an example) Light chain Heavy chain Activity of Chimeric and Constant Constant Expression binding to Humanized Variable region (κ Variable region level hSirpα-V1, Antibodies region chain) region (hIgG4) (mg/L) ELISA, nM mab14c SEQ ID SEQ ID SEQ ID SEQ ID 49.3 0.093 NO: 9  NO: 27 NO: 10 NO: 28 mab14-h1 SEQ ID SEQ ID 227.9 0.148 NO: 29 NO: 35 mab14-h2 SEQ ID SEQ ID 195.5 0.073 NO: 30 NO: 36 mab14-h3 SEQ ID SEQ ID 206.6 0.103 NO: 30 NO: 37 mab14-h4 SEQ ID SEQ ID 224.3 0.088 NO: 30 NO: 38 mab14-h5 SEQ ID SEQ ID 217.8 0.061 NO: 30 NO: 39 mab14-h6 SEQ ID SEQ ID 191.8 0.095 NO: 30 NO: 40 mab14-h7 SEQ ID SEQ ID 143.9 0.081 NO: 30 NO: 41 mab14-h8 SEQ ID SEQ ID 190.8 0.067 NO: 31 NO: 36 mab14-h9 SEQ ID SEQ ID 164.9 0.062 NO: 32 NO: 36 mab14-h10 SEQ ID SEQ ID 199.5 0.068 NO: 33 NO: 36 mab14-h11 SEQ ID SEQ ID 219.2 0.073 NO: 34 NO: 36 mab14-h12 SEQ ID SEQ ID 105.34 0.092 NO: 31 NO: 39 mab14-h13 SEQ ID SEQ ID 103.8 0.089 NO: 32 NO: 39 mab14-h14 SEQ ID SEQ ID 103.6 0.057 NO: 33 NO: 39 mab14-h15 SEQ ID SEQ ID 76.29 0.077 NO: 34 NO: 39 mab14-h16 SEQ ID SEQ ID 275 0.087 NO: 29 NO: 39 Ref1 SEQ ID SEQ ID 44.9 0.072 NO: 55 of NO: 42 of WO20171 WO201717 78653A2 8653A2

[0210] The aforementioned results showed that the binding activity of the human-murine chimeric antibody mab14c of the present disclosure to hSirpα-v1 was almost the same as that of the control molecule Ref1 (0.093 VS 0.072). The aforementioned humanized antibody molecules obtained by 3 rounds of different combinations of the light and heavy chain sequences at different humanized degrees of the human-murine chimeric antibody sequence mab14c of the present disclosure all retained the binding activity that was almost consistent with that of the chimeric antibody.

[0211] More preferably, there were significant differences in the expression levels of different combined antibodies between sequences with different degrees of humanization, wherein the expression levels of mab14-h1, mab14-h11, mab14-h16 and the like antibodies were up to 200 mg/L, wherein the expression level of mab14-h16 was the highest, at 275 mg/L, which was 4.5 times higher than that of the chimeric antibody mab14c (275 mg/L VS 9 mg/L).

[0212] More specifically, the effects of the humanized antibody of the present disclosure in blocking the binding of hSirpα (dimer) to hCD47 and blocking the binding of hSirpα to hCD47 (dimer) were detected by the experimental method of Example 4. The results were shown in Table 14, and the humanized antibody of the present inventor retained the characteristic of the chimeric antibody mab14c of blocking the binding of hSirpα to hCD47 well. This result showed that the chimeric antibody and humanized antibody of the present disclosure not only bound to the hSirpα protein, but also effectively blocked the binding of hSirpα to hCD47.

TABLE-US-00022 TABLE 14 Activity of humanized antibody of the present disclosure (human κ type light chain, taking the hIgG4 heavy chain constant region as an example) Blocking Blocking binding of binding of h Sirpa hSirpα to (dimer) to hCD47 Humanized hCD47 (ELISA), (dimer) (ELISA), antibody IC.sub.50, nM IC.sub.50, nM mab14c 5.48 9.98 mab14-h5 8.99 8.47 mab14-h12 3.49 8.08 mab14-h13 5.31 9.56 mab14-h14 4.75 9.1 mab14-h15 5.13 7.37 mab14-h16 5.92 10.14

[0213] The light and heavy chain amino acid (including constant regions) sequences of the humanized antibody (some representative molecules) were listed as follows.

[0214] The amino acid sequence of the humanized mab14-h5 antibody:

TABLE-US-00023 Light chain: (SEQ ID NO: 42) DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGGTVKLLIY FTSTLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTF GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC Heavy chain: (SEQ ID NO: 43) QVQLVQSGAEVKKPGASVKVSCKASGYNFNIYWINWVRQAPGQGLEWIG NIYPSSISTNYNEKFKTRATLTVDKSTSTVYMELSSLRSEDTAVYYCAR SEGTYYGGRYEGDWFGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSES TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH YTQKSLSLSLGK

[0215] The amino acid sequence of the humanized mab14-h12 antibody:

TABLE-US-00024 Light chain: (SEQ ID NO: 44) DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGGTPKLLIY FTSTLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTF GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC 

[0216] The heavy chain sequence of the humanized antibody mab14-h12 was the same as SEQ ID NO: 43.

[0217] The amino acid sequence of the humanized mab14-h13 antibody:

[0218] Light chain:

TABLE-US-00025 (SEQ ID NO: 45) DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGGAPKLLIY FTSTLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTF GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC 

[0219] The heavy chain sequence of the humanized antibody mab14-h13 was the same as SEQ ID NO: 43.

[0220] The amino acid sequence of the humanized mab14-h14 antibody

TABLE-US-00026 Light chain: (SEQ ID NO: 46) DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGGTPKLLIY FTSTLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTF GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC 

[0221] The heavy chain sequence of the humanized antibody mab14-h14 was the same as SEQ ID NO: 43.

[0222] The amino acid sequence of the humanized mab14-h15 antibody:

TABLE-US-00027 Light chain: (SEQ ID NO: 47) DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKTPKLLIY FTSTLHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTF GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC 

[0223] The heavy chain sequence of the humanized antibody mab14-h15 was the same as SEQ ID NO: 43.

[0224] The amino acid sequence of the humanized mab14-h16 antibody:

[0225] Light chain:

TABLE-US-00028 (SEQ ID NO: 48) DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIY FTSTLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTF GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC 

[0226] The heavy chain sequence of the humanized antibody mab14-h16 was the same as SEQ ID NO: 43.

Example 9 Comprehensive Evaluation of Binding Activity of Humanized Anti-Human Sirpα Antibody and Preferred Antibody of the Present Disclosure

[0227] In order to further evaluate the binding activity of the humanized antibody of the present disclosure, taking the preferred humanized antibody mab14-h16 of the present disclosure as an example, it was subjected to evaluation of parallel/repeated binding activity (ELISA) compared with the reference antibody Ref. The binding activities of the antibody to hSirpα-V1, hSirpα-V2, hSirpβ, hSirpγ and different polymorphic Cyno Sirpαs were detected. The experimental method was the same as that of Example 3, and the results were shown in Table 15 below and FIGS. 2 and 3.

TABLE-US-00029 TABLE 15 Comprehensive evaluation of the binding activity of the antibody of the present disclosure to Sirpα (EC.sub.50, nM) Antigen mab14-h16 Ref1 Ref2 hSirpα-V1 0.065 0.057 0.159 hSirpα-V2 0.094 ND 0.37 hSirpβ faint 0.126 0.167 binding hSirpγ ND ND 1.46 NOD mouse mu-Sirpα ND ND Weak binding (2.82) Cyno Sirpα polymorphism 1 0.181 ND 0.209 (L932) Cyno Sirpα polymorphism 2 0.159 ND 0.164 (L933) Cyno Sirpα polymorphism 3 0.143 ND 0.198 (L936) Cyno Sirpα polymorphism 4 0.121 faint 0.186 (L937) binding Cyno Sirpα polymorphism 5 ND faint 0.175 (L938) binding Cyno Sirpα polymorphism 6 ND faint 0.167 (L939) binding

[0228] ND, the binding signal was not detected, i.e. no binding; faint binding referred to that EC.sub.50 was higher than 10 nM; and weak binding referred to that EC.sub.50 was between 2-10 nM.

[0229] The aforementioned results showed that, in the present disclosure it was unexpectedly found that the murine anti-human Sirpα antibody and humanized anti-human Sirpα antibody were very specific and novel antibodies. Unlike Ref1, this antibody could bind to human Sirpα-V1, V2 and various Cyno Sirpαs (polymorphism) simultaneously, but not to Sirpβ.

[0230] Not only that, the novel antibody of the present disclosure was also significantly different from Ref2. Ref2 could also bind to both Sirpα-V1 and V2, but the binding activity of it was weaker than that of the antibody of the present disclosure. The binding activity of the antibody mab14-h16 of the present disclosure to Sirpα-V1 was 1.4 times stronger than that of Ref2 (0.065 nM vs 0.159 nM in the above table), and the binding activity of it to Sirpα-V2 was 2.9 times stronger than that of Ref2 (0.094 nM vs 0.37 nM). A more significant difference was that Ref2 had relatively stronger binding to both Sirpβ and Sirpγ, with EC.sub.50 of 0.167 nM and 1.46 nM, respectively. The antibody of the present disclosure bound only faintly or not at all to Sirpβ and Sirpγ. The unique selectivity of the present disclosure (specifically binding to Sirpα, but not to Sirpβ and Sirpγ) given it an outstanding advantage in the process of clinical development, that was, it could avoid the safety problem brought about by off-target.

[0231] Moreover, Ref2 could bind to Cyno Sirpα polymorphisms 5 and 6. This was also different from the antibody of the present disclosure.

[0232] These characteristics indicated that the binding sites (epitopes) of the antibody of the present disclosure were different from those of Ref1 and Ref2.

[0233] In order to further evaluate the cell-binding activity of the antibody of the present disclosure and the activity of the same in blocking the binding of Sirpα to a corresponding ligand CD47, in the present disclosure, taking the preferred humanized antibody mab14-h16 as an example, the binding activity of it to cell strains with high expression of human Sirpα was detected by ELISA of a plated coated by the cell strains with high expression of human Sirpα+, and the detection method was as shown in Example 2. The ability of the humanized antibody of the present disclosure to block the binding of human Sirpα to CD47 was detected by the method of Example 4, and the binding of the humanized antibody to human T cells was detected by the method of Example 5. The results were shown in Table 16a.

[0234] Moreover, the binding of human Sirpα-V2 to human CD47 as blocked by the antibody of the present disclosure was further detected. Specifically, the recombinant protein of human Sirpα-V2-his was diluted to a concentration of 2 μg/mL with a PBS buffer at pH7.4, then added into a 96-well ELISA plate (Corning, CLS3590-100EA) at a volume of 50 μL/well, and placed in an incubator at 37° C. for 2 hours. After the liquid was discarded, the wells were added with a blocking solution of 5% skimmed milk (skimmed milk powder available from Bright Dairy) diluted with PBS at 230 μL/well, and incubated in an incubator at 37° C. for 3 hours, or placed overnight at 4° C. (for 16-18 hours) for blocking. The blocking solution was discarded, and the plate was washed with a PBST buffer (pH7.4 PBS containing 0.05% tween-20) for 5 times, then added with 25 μL/well of the 3-fold gradient dilution of the antibody to be tested at a starting concentration of 100 μg/mL and 25 μL/well of Biotin labeled CD47-hFc or CD47-his at a concentration of 4 μg/mL, mixed uniformly, and then incubated in an incubator at 37° C. for 1 hour. The plate was washed for 5 times, added with 50 μL/well of a streptavidin-HRP secondary antibody (GenScript, M00091) diluted at 1:1000, incubated at 37° C. for 1 hour, and then washed with PBST for 5 times. Each well was added with 50 μL TMB (Surmodic, Cat #TTMB-1000-01) for color development, and added with 50 μL/well of 1 M H.sub.2SO.sub.4 to stop the reaction. The readings on a microplate reader (MultiskanGO Thermo, Model: 51119200) were subjected to data analysis by GraphPad prism 5. The results were shown in Table 16b.

TABLE-US-00030 TABLE 16a Evaluation of cell binding and blocking activities of the antibody of the present disclosure Antibody sample mab14-h16 Ref1 Ref2 T-cell binding activity (% binding) 1.95 1.79 47.6 hSirpα-v1+ cell strain binding 0.124 0.084 0.106 activity (EC.sub.50, nM) Blocking the binding of hSirpα (dimer) 4.81 3.74 4.79 to CD47 (IC.sub.50, nM) Blocking the binding of hSirpα to CD47 5.28 4.16 5.44 (dimer), IC.sub.50 (nM)

TABLE-US-00031 TABLE 16b Evaluation of the activity of the antibody of the present disclosure in blocking the binding of human Sirpα-V2 to human CD47 Antibody sample mab14-h16 Ref1 Ref2 Blocking the binding of hSirpα-V2 to 2.39 ND 1.29 hCD47 (IC.sub.50, nM) Blocking the binding of hSirpα-V2 to 6.29 ND 3.09 human CD47 (dimer), IC.sub.50 (nM) ND: the blocking signal was not detected.

[0235] The results in the aforementioned table showed that the humanized antibody and preferred antibody mab14-h16 of the present disclosure did not bind to human T cells (the binding percentage of the negative control in this test was 1.67%, which was close to the binding ratio 1.95% of mab14-h16, i.e., the background level). Its activities of binding to a hSirpα-v1+ cell strain and blocking the binding of hSirpα-v1 to human CD47 were close to those of the controls Ref1 and Ref2, and it could effectively block the binding of hSirpα-V2 to human CD47.

[0236] The data of the aforementioned examples showed that, in the present disclosure, it was unexpectedly found that the murine anti-human Sirpα antibody and humanized anti-human Sirpα antibody can bind to human Sirpα-V1 and Sirpα-V2 simultaneously, and have stronger binding activities to human Sirpα-V1 and human Sirpα-V2 and bind to various Cyno Sirpα (polymorphism), and did not bind to human Sirpβ and Sirpγ as well as human T cells.

[0237] Moreover, the antibody of the present disclosure had a very good activity of blocking the binding of human Sirpα (including Sirpα-V1 and Sirpα-V2) to human CD47, so that the antibody can be developed as a new drug targeting the binding of Sirpα to CD47, so as to achieve the purpose of treating a tumor.

[0238] These outstanding characteristics enabled the antibody of the present disclosure to show unique clinical advantages, which was presented as that the antibody could target more patient populations (the population expressing V1 and the population expressing V2). The antibody did not bind to Sirpβ, Sirpγ and T cells, and had very good specificity, thereby avoiding the side effects caused by off-target in clinical. Meanwhile, the antibody of the present disclosure could bind to a variety of Cyno Sirpαs very strongly, and the primate cyno could be selected in the preclinical safety evaluation research, which provided convenience for the preclinical research.

Example 10 Humanized Anti-Human Sirpα Antibody PTM of the Present Disclosure

[0239] By means of MOE (molecular operating environment), https://www.chemcomp.com/Products.htm; Schrodinger, https://www.schrodinger.com/; or DS (Discovery Studio) and the like computer analysis software, the antibody of the present disclosure was subjected to Post-translational Modification (PTM) site analysis. As a result, it was found that among M4, C23, W35, C88, and W96 of the light chain and C22, W33, W36, W47, M81, C96, and W112 of the heavy chain (H Chain) of the preferred antibody of the present disclosure, only M4 was a low-risk oxidation site, while the others were non-oxidation hot spots. There was no other deamination site or hot spot in the whole sequence, except a slight risk of deamination in the N92 of the light chain. There was no N-glycosylation hot spot and no Asp isomerization site or hot spot in the whole sequence. The humanized sequence of the present disclosure was therefore the preferred sequence for PTM analysis.

Example 11 Design of Bispecific Antibody Against Sirpα Target

[0240] Based on the anti-Sirpα antibody found above, the design of various bispecific antibodies had carried out in the present disclosure. The general formula of the designed bispecific antibody was as follows.

TABLE-US-00032 TABLE 17 Bispecific design based on the anti-Sirpα antibody of the present disclosure (general formula 1) Light chain-containing Heavy chain-containing Protocol sequence sequence 1 T2 (scFv).sub.n1-T1VL-LC-T2 T2 (scFv).sub.n3-T1VH-HC-T2 (scFv)n.sub.2 (scFv).sub.n4 2 T1 (scFv).sub.n1-T2VL-LC-T1 T1 (scFv).sub.n3-T2VH-HC-T1 (scFv).sub.n2 (scFv).sub.n4 3 T2 (scFv).sub.n1-T1VL-LC-T1 T2 (scFv).sub.n3-T1VH-HC-T1 (scFv).sub.n2 (scFv).sub.n4 4 T1 (scFv).sub.n1-T2VL-LC-T2 T1 (scFv).sub.n3-T2VH-HC-T2 (scFv).sub.n2 (scFv).sub.n4

[0241] In Table 17, a light chain-containing sequence means that the sequence may include, in addition to the light chain sequence, a scFv linked to the light chain sequence; and a heavy chain-containing sequence means that the sequence may include, in addition to the heavy chain sequence, a scFv linked to the heavy chain sequence. T1 represents the first protein functional region against the target 1 (e.g., Sirpα), and T2 represents the second protein functional region against the target 2 (not Sirpα). T1 (scFv) represents the scFv sequence of the antibody against target 1; and T2 (scFv) represents the scFv sequence against target 2.

[0242] n1, n2, n3 and n4 in (scFv).sub.n1, (scFv).sub.n2, (scFv).sub.n3 and (scFv).sub.n4 are respectively natural numbers, which can be 0, 1, 2, 3, etc. In a specific embodiment of the present disclosure, the value of at least one of the n1, n2, n3 and n4 is 1, and the rest are 0. VL represents the light chain variable region sequence of the antibody against the target 1 or 2; and VH represents the heavy chain variable region sequence of the antibody against the target 1 or 2. LC represents the constant region sequence of the light chain (κ or λ), preferably the human light chain constant region sequence; and HC represents the constant region sequence of the heavy chain including IgG1, IgG2, IgG3, IgG4, etc. (abbreviated as HC-IgG1, HC-IgG2, HC-IgG3, and HC-IgG4), preferably human heavy chain constant region sequence (HC-hIgG). When scFv or other protein sequences are linked to the C-terminus of the heavy chain constant region, the last amino acid K at the C-terminus of the heavy chain constant region can be mutated, preferably mutated to A. Therefore, in scheme 1, T1 is immunoglobulin, and T2 is scFv; in scheme 2, T2 is immunoglobulin, and T1 is scFv; the targets of the scFvs are the same; and in schemes 3 and 4, the scFvs at two ends target two different targets.

[0243] In Table 17, the scFv is alight chain variable region-linker-heavy chain variable region, and the N-terminus of the light chain variable region or the C-terminus of the heavy chain variable region is accordingly linked to the C-terminus or N-terminus of the light and/or heavy chain of the immunoglobulin through the linker; or the scFv is heavy chain variable region-linker-light chain variable region, and the N-terminus of the heavy chain variable region or the C-terminus of the light chain variable region is accordingly linked to the C-terminus or N-terminus of the light and/or heavy chain of the immunoglobulin through the linker.

[0244] It should be noted that when the aforementioned scFv is light chain variable region-linker-heavy chain variable region, the linking mode of it is that the C-terminus of the light chain variable region is linked with the linker, and the linker is then linked with the N-terminus of the heavy chain variable region, thereby exposing the N-terminus of the light chain variable region and the C-terminus of the heavy chain variable region in the scFv, so that it can be linked to the light and/or heavy chain of the immunoglobulin through a linker. In the present disclosure, when it is linked to the light chain of the immunoglobulin, in some specific embodiments, preferably the C-terminus of the heavy chain variable region of the scFv is linked to the N-terminus of the heavy chain of the immunoglobulin through a linker; and when it is linked to the heavy chain of the immunoglobulin, in some specific embodiments, preferably the N-terminus of the light chain variable region of the scFv is linked to the C-terminus of the heavy chain of the immunoglobulin.

[0245] When the scFv is heavy chain variable region-linker-light chain variable region, the linking mode of it is that the N-terminus of the light chain variable region is linked with the linker, and the linker is then linked with the C-terminus of the heavy chain variable region, thereby exposing the C-terminus of the light chain variable region and the N-terminus of the heavy chain variable region in the scFv, so that it can be linked to the light and/or heavy chain of the immunoglobulin through a linker. In this case, when it is linked to the light chain of the immunoglobulin, in some specific embodiments, preferably the C-terminus of the light chain variable region of the scFv is linked to the N-terminus of the heavy chain of the immunoglobulin; and when it is linked to the heavy chain of the immunoglobulin, in some specific embodiments, preferably the N-terminus of the heavy chain variable region of the scFv is linked to the C-terminus of the heavy chain of the immunoglobulin.

[0246] The linker was preferably (G.sub.4S).sub.m, and the m was preferably an integer between 0-10. Further preferably, the linker was (Gly-Gly-Gly-Gly-Ser).sub.3, and/or the number of the scFvs was a pair of scFvs which were symmetrically linked to the C-terminus and/or N-terminus of the light and/or heavy chain of the immunoglobulin.

[0247] For the sequences against various targets involved in the aforementioned bispecific design, in addition to the anti-Sirpα antibody sequence of the present disclosure, other antibody sequences against the targets are derived from published antibody sequences. It included anti-PD-1 antibodies Nivolumab/Opidivo (referred to as Nivo for short) and Pembrolizumab/Keytruda (referred to as Pem for short). Sequences such as Nivolumab and Pembrolizumab could be found from public resources such as www.drugbank.ca.

Example 12 Antigen-Antibody Binding (ELISA) Assay

[0248] The self-expressed human PD-1, Sirpα and the like antigens of the present disclosure were diluted with a PBS buffer at pH7.4 to a concentration of 2 μg/mL according to different assays, and then added into a 96-well ELISA plate (Corning, CLS3590-100EA) at a volume of 50 μL/well, and placed in an incubator at 37° C. for 2 hours. After the liquid was discarded, the wells were added with a blocking solution of 5% skimmed milk (Sangon Biotech (Shanghai) Co., Ltd., A600669-0250) diluted with PBS at 200 μL/well, and incubated in an incubator at 37° C. for 3 hours, or placed overnight at 4° C. (for 16-18 hours) for blocking. The blocking solution was discarded, and the plate was washed with a PBST buffer (pH7.4 PBS containing 0.05% tween-20) for 5 times, then added with 50 μL/well of 5-fold serial dilution of the antibody to be tested in 1% BSA, and incubated at 37° C. for 1 hour. The plated was washed with PBST for 5 times, added with 50 μL/well of HRP-labeled secondary antibody (Jackson Immuno Research, 115-035-003) diluted at 1:2500, and incubated at 37° C. for 1 hour. The plated was washed with PBST for 5 times, then added with 50 μL/well of TMB chromogenic substrate (KPL, 52-00-03), incubated at room temperature for 5-10 min, added with 50 μL/well of 1 M H.sub.2SO.sub.4 to stop the reaction, and read by a MULTISKAN Go microplate reader (ThermoFisher, 51119200) for the absorbance value at 450 nm, and then EC.sub.50 was calculated according to the OD value.

Example 13 Assay of Blocking Antigen-Antibody Binding by Antibody

[0249] The antigens PD-1 and Sirpα expressed according to the method of Example 1 were diluted with a PBS buffer at pH7.4 to a concentration of 2 μg/mL, added into a 96-well ELISA plate (Corning, CLS3590-100EA) at a volume of 50 μL/well, and incubated at 37° C. for 2 hours. After the liquid was discarded, the wells were added with a blocking solution of 5% skimmed milk (Sangon Biotech (Shanghai) Co., Ltd., A600669-0250) formulated with PBS at 200 μL/well, and incubated at 37° C. for 3 hours for blocking. The blocking solution was discarded, and the plate was washed with a PBST buffer (pH7.4 PBS containing 0.05% tween-20) for 5 times, then each well was added with 25 μL of 3-fold serial dilution of the antibody to be tested in 1% BSA and 25 μL of biotin-labeled ligands (CD47-his/CD47-hFc, PD-L1, etc., which were expressed and purified by the present disclosure) with a final concentration of 1 μg/mL or 12 μg/mL, and incubated at 37° C. for 1 hour. The plate was washed with PBST for 5 times, added with 50 μL/well of HRP-labeled secondary antibody (GenScript Biotechnology Co., Ltd., M00091) diluted at 1:1000, and incubated at 37° C. for 1 hour. The plated was washed with PBST for 5 times, then added with 50 μl/well of TMB chromogenic substrate (KPL, 52-00-03), incubated at room temperature for 5-10 min, added with 50 μl/well of 1 M H.sub.2SO.sub.4 to stop the reaction, and read by a MULTISKAN Go microplate reader (ThermoFisher, 51119200) for the absorbance value at 450 nm, and then IC.sub.50 was calculated according to the OD value.

[0250] The Biotin-labeled kit was Biotin Labeling Kit-NH2, which was purchased from Dojindo Chemical Technology (Shanghai) Co., Ltd., with the Cat. No. LK03. The operation method was carried out according to the instructions, and the labeled antibody was used after concentration detection with a Multiskan GO (ThermoFisher) microplate reader.

Example 14 Design of Bispecific Antibody Against Dual Targets Sirpα and PD-1, and Activity Evaluation

[0251] In the present disclosure, bispecific antibodies with different sequence structures against two targets Sirpα and PD-1 had been designed, as shown in the table below.

TABLE-US-00033 TABLE 18 Bispecific antibodies designed against dual targets Sirpα and PD-1 Antibody number Light chain sequence Heavy chain sequence LB501 mab14-h16VL-(G4S).sub.3- PemVH-HC (hIgG4) mab14-h16VH-(G4S).sub.3- PemVL-LC (κ chain) LB502 PemVL-LC (κ chain) mab14-h16VL-(G4S).sub.3- mab14-h16VH-(G4S).sub.3- PemVH-HC (hIgG4) LB503 PemVL-LC-(G4S).sub.3- PemVH-HC (hIgG4) mab14-h16VH-(G4S).sub.3- mab14-h16VL(κ chain) LB504 PemVL-LC (κ chain) PemVH-HC(hIgG4)-(G4S).sub.3- mab14-h16VH-(G4S).sub.3- mab14-h16VL LB505 NivoVL-LC (κ chain) mab14-h16VL-(G4S).sub.3- mab14-h16VH-(G4S).sub.3- NivoVH-HC (hIgG4) LB506 NivoVL-LC (κ chain) NivoVH-HC(hIgG4)-(G4S).sub.3- mab14-h16VH-(G4S).sub.3- mab14-h16VL LB507 mab14-h16VL-LC PemVL-(G4S).sub.3- (κ chain) PemVH-(G4S).sub.3- mab14-h16VH-HC (hIgG4) LB508 mab14-h16VL-LC mab14-h16VH- (κ chain) HC(hIgG4)-(G4S).sub.3- PemVH-(G4S).sub.3- PemVL * κ chain indicated that the light chain was the κ type light chain constant region of human IgG. #: when a linker was linked to the C-terminus of IgG4, the last amino acid K of IgG4 was mutated to A. The design of introducing scFv at the C-terminus of the heavy chain all mutated the last amino acid K to A.
all mutated the last amino acid K to A.

[0252] The aforementioned bispecific antibodies were cloned, expressed and purified according to the cloning, expression and purification methods of Example 1 of the present disclosure. The binding activities of these designed bispecific molecules to human Sirpα and PD-1 were detected by the methods of Examples 12 and 13, respectively, and it was found that LB501, LB502, LB503, LB504 and LB506 all could retain the binding activities to the two target antigens. The results were shown in the table below.

TABLE-US-00034 TABLE 19 Evaluation of binding activities of bispecific antibodies designed against dual targets Sirpα and PD-1 Activity of binding to Activity of binding to human Sirpα-V1 human PD-1 multiple of multiple of Antibody change of change of number EC.sub.50, nM EC.sub.50* EC.sub.50, nM EC.sub.50* LB501 0.059 (0.077) 0.77 0.075 (0.15) 0.50 LB502 0.066 (0.077) 0.86 0.074 (0.15) 0.49 LB503 0.075 (0.077) 0.97 0.063 (0.15) 0.42 LB504 0.055 (0.077) 0.71 0.061 (0.15) 0.41 LB506 0.064 (0.077) 0.83 0.053 (0.043) 1.23 #: the value in parentheses was the binding activities EC.sub.50 of the monoclonal antibodies corresponding to the same target under the same experimental conditions. *the ratio of binding activities EC.sub.50 of the bispecific antibody and the corresponding monoclonal antibody under the same experimental conditions. When the ratio was larger, it indicated that the decrease in the binding force of the designed bispecific antibody to a single target was larger. For example, if the ratio was 2, it indicated that the binding activity of the designed bispecific antibody to the target was reduced by one time compared with the corresponding monoclonal antibody. When the ratio was within 2 (experimental error range), it indicated that the binding activity was not affected.

[0253] In the above table were bispecific molecules designed by placing the scFv of the anti-Sirpα antibody mab14-h16 of the present disclosure at the N-terminus of the heavy chain, the C-terminus of the heavy chain; the N-terminus of the light chain and the C-terminus of the light chain of the PD-1 antibody Pem, or bispecific molecules designed by placing the scFv of the anti-Sirpα antibody mab14-h16 of the present disclosure at the N-terminus of the heavy chain and the C-terminus of the heavy chain of the PD-1 antibody Nivo, or bispecific molecules designed from the scFv of the PD-1 antibody Pem and the anti-Sirpα antibody mab14-h16 of the present disclosure.

[0254] The results showed that for bispecific antibodies designed from the same Sirpα antibody and scFv at different positions, the effect on the activity of Pem was much less than that on Nivo, such as LB501, LB502, LB503 and LB504 (the scFv linked on the Pem), the binding activities of them to the two targets were all close to those of the corresponding monoclonal antibodies, and compared with the bispecific antibody in which scFv was linked on the Nivo, only LB506 had similar binding activities to the two targets as those of the corresponding monoclonal antibody. It showed that linking the scFv of the Sirpα antibody at different positions had little effect on the activity of Pem, which was different for the effect on the activity of Nivo. Similarly, the binding activities of bispecific antibodies formed by linking the scFv of Pem to the N-terminus of the heavy chain (LB507) and C-terminus of the heavy chain (LB508) of the anti-Sirpα antibody mab14-h16 of the present disclosure were also different. The binding activity of LB507 to Sirpα was 1-fold weaker than that of the corresponding monoclonal antibody and the binding activity to PD-1 was close to that of Pem, while the binding activities of LB508 to both targets were significantly reduced.

[0255] The aforementioned data of these designed bispecific antibodies of the present disclosure showed that when the Sirpα antibody (the present disclosure) and scFv were the same, the designing manner was the same, but the PD-1 antibody sequence was different, the designed bispecific antibody molecules had huge differences in activity. When the scFv of the Sirpα antibody (the present disclosure) and the PD-1 antibody were the same, but the position of the scFv of the Sirpα antibody (the present disclosure) was different, the designed bispecific antibody molecules had huge differences in activity. When the scFv of the PD-1 antibody and the Sirpα antibody (the present disclosure) were the same, but the position of the scFv of the PD-1 antibody was different, and the difference in the activity was also very huge.

[0256] These data showed that the bispecific antibodies designed based on the Sirpα antibody sequence of the present disclosure had different sequences, different positions of the scFv and antibodies, and thus different activities. With proper positions and proper sequence design, bispecific antibodies with good activity against dual targets could be obtained. These bispecific antibodies were similar in structure to conventional IgGs and had an entire Fc. In the present disclosure, it was called sequence-based IgG like bispecific antibody format (SBody). These bispecific antibody molecules have the same entire Fc as normal antibodies, so that their purification process can be carried out according to those of normal antibodies, and thus the process is simple and has the advantage of low production cost.

[0257] The aforementioned SBodies which retained the activity against the two targets, were evaluated for its functions against the two targets (by an assay of blocking the binding of an antigen to a corresponding ligand) respectively, and the results were shown in the table below.

TABLE-US-00035 TABLE 20 Evaluation of functional activities of bispecific antibodies designed against dual targets Sirpα and PD-1 Activity of blocking the Activity of blocking the Activity of blocking binding of Sirpα to binding of Sirpα (dimer) the binding of PD- CD47 (dimer) to CD47 1/PD-L1 multiple multiple multiple of Antibody of change of change change number IC.sub.50, nM of IC.sub.50* IC.sub.50, nM of IC.sub.50* IC.sub.50, nM of IC.sub.50* LB501 5.42 (6.14) 0.88 2.55 (3.88) 0.66 1.30 (2.53) 0.51 LB502 5.37 (6.14) 0.87 3.78 (3.88) 0.97 2.00 (2.53) 0.79 LB503 7.63 (6.14) 1.24 4.80 (3.88) 1.24 1.45 (2.53) 0.57 LB504 6.63 (6.14) 1.08 4.62 (3.88) 1.19 1.09 (2.53) 0.43 LB506 6.51 (6.14) 1.06 4.53 (3.88) 1.17 1.39 (1.33) 1.05 #: the value in parentheses was the IC.sub.50 of the activity of the monoclonal antibody corresponding to the same target to block the binding of an antigen to a ligand under the same experimental conditions. *: the multiple of change of IC.sub.50, that was, the IC.sub.50 ratio of the bispecific antibody and the corresponding monoclonal antibody (control antibody). When the ratio was larger, it indicated that the decrease in the functional activity of the designed bispecific antibody to a single target was larger. For example, if the ratio was 2, it indicated that the functional activity of the designed bispecific antibody to the target was reduced by one time compared with the corresponding monoclonal antibody. When the ratio was within 2 as the experimental error range, that was the activity was not affected. ND: No activity of the molecule of blocking the binding of Sirpα to Daudi cells was detected.

[0258] The aforementioned functional activity results showed that the change of the activity of the bispecific antibody (SBody) designed in the present disclosure in blocking the binding of an antigen with a corresponding ligand was consistent with the change of the binding activity thereof, such as LB507, of which the activities of binding to human Sirpα and human PD-1 were slightly weakened and the activities of blocking the binding of human Sirpα to human CD47 and blocking the binding of human PD-1 to human PD-L1 were slightly weakened (compared with the corresponding monoclonal antibodies, the multiples of change were 2.46, 2.59 and 2.20 respectively). Other designs LB501, LB502, LB503, LB504, and LB506 all retained the functional activity against the dual targets.

[0259] To evaluate the expression levels of the bispecific antibodies SBodies of the present disclosure, the SBodies were transiently transfected in the same expression system (293F cells) by the same method, and purified by conventional Protein A to obtain the respective expression levels. The results were shown in the table below.

TABLE-US-00036 TABLE 21 Evaluation of expression levels of bispecific antibodies designed against dual targets Sirpα and PD-1 Antibody Expression number level (mg/L) LB501 2.24 LB502 2.43 LB503 3.83 LB504 7.35 LB506 44.29

[0260] The aforementioned result showed that that the design of the present disclosure had a great difference in the expression yield of the Sirpα and PD1 bispecific antibody (SBody). In conclusion based on the aforementioned data, for SBodies with the same design mode, the same scFv of the Sirpα antibody and different PD-1 antibody sequences, the expression levels were different, and the expression level of the SBody corresponding to Nivo was 5 times or even 17 times higher than that of Pem. For example, the yield of LB506 was 17 times higher than that of LB502 (44.29/2.43); and the yield of LB506 was 5 times higher than that of LB504 (44.29/7.35). For SBodies with the same design mode, the same PD-1 sequence, the same scFv sequence of the Sirpα antibody and different scFv positions, the expression levels also differed by more than 2 times, such as LB504 (7.35 mg/L) vs LB501 (2.24 mg/L) vs LB502 (2.43 mg/L).

[0261] These data showed that the bispecific antibodies-SBodies designed from the anti-Sirpα antibody mab14-h16 of the present disclosure and the PD1 antibody was sequence-specific not only in activity, function, but also in expression level.

[0262] Partial sequences of the bispecific antibodies-SBodies designed from the anti-Sirpα antibody mab14-h16 of the present disclosure and the PD1 antibody were as follows:

TABLE-US-00037 Light chain sequence of LB501: (SEQ ID NO: 50) DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIY FTSTLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTF GGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKAS GYNFNIYWINWVRQAPGQGLEWIGNIYPSSISTNYNEKFKTRATLTVDK STSTVYMELSSLRSEDTAVYYCARSEGTYYGGRYEGDWFGYWGQGTLVT VSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASKGVST SGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTIS SLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Heavy chain sequence (Pem heavy chain) of LB501: (SEQ ID NO: 51) QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWM GGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCA RRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQ PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGK Light chain sequence (Pem light chain) of LB502: (SEQ ID NO: 52) EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPR LLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDL PLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC Heavy chain sequence of LB502: (SEQ ID NO: 53) DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIY FTSTLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTF GGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKAS GYNFNIYWINWVRQAPGQGLEWIGNIYPSSISTNYNEKFKTRATLTVDK STSTVYMELSSLRSEDTAVYYCARSEGTYYGGRYEGDWFGYWGQGTLVT VSSGGGGSGGGGSGGGGSQVQLVQSGVEVKKPGASVKVSCKASGYTFTN YYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAY MELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVF PLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP PCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLGK  Light chain sequence of LB503: (SEQ ID NO: 54) EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPR LLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDL PLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGECAGGGGSGGGGSGGGGSQVQLVQSGAEV KKPGASVKVSCKASGYNFNIYWINWVRQAPGQGLEWIGNIYPSSISTNY NEKFKTRATLTVDKSTSTVYMELSSLRSEDTAVYYCARSEGTYYGGRYE GDWFGYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGD RVTITCRASQDIRNYLNWYQQKPGKAPKLLIYFTSTLHSGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGGGTKVEIK

[0263] Heavy chain sequence (Pem heavy chain) of LB503: the same as SEQ ID NO: 51.

[0264] Light chain sequence (Pem light chain) of LB504: the same as SEQ ID NO: 52.

TABLE-US-00038 Heavy chain sequence of LB504: (SEQ ID NO: 55) QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWM GGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCA RRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQ PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGAGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGY NFNIYWINWVRQAPGQGLEWIGNIYPSSISTNYNEKFKTRATLTVDKST STVYMELSSLRSEDTAVYYCARSEGTYYGGRYEGDWFGYWGQGTLVTVS SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRNYL NWYQQKPGKAPKLLIYFTSTLHSGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQGNTLPWTFGGGTKVEIK Light chain sequence (Nivo light chain) of LB506: (SEQ ID NO: 56) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIY DASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTF GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC Heavy chain sequence of LB506: (SEQ ID NO: 57) QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVA VIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCAT NDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC NVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGAG GGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYNFNIYWIN WVRQAPGQGLEWIGNIYPSSISTNYNEKFKTRATLTVDKSTSTVYMELS SLRSEDTAVYYCARSEGTYYGGRYEGDWFGYWGQGTLVTVSSGGGGSGG GGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPG KAPKLLIYFTSTLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ GNTLPWTFGGGTKVEIK

Example 15 Evaluation of Stability of Sirpα and PD-1 Bispecific Antibody of the Present Disclosure in Different Formulation Recipes

[0265] The buffer of the bispecific antibody LB504 of the aforementioned in Example 14 was replaced by each formulation recipe by a desalting centrifugal column (Thermo, Cat #89890), and the formulation recipe scheme was as shown in Table 22. The replacement process of each formulation buffer was as follows: the desalting centrifugal column was pretreated, wherein the desalting centrifugal column was centrifuged at 1,000 g for 2 min, the stock solution was removed, the desalting centrifugal column was added with 1 mL of each formulation buffer and centrifuged at 1,000 g for 2 min for 3 times, and the buffer in the collection tube was discarded; the desalting centrifugal column was placed in anew collection tube, slowly added with an appropriate amount of LB504, added with 20 μL of the formulation buffer for a hydraulic layer, and centrifuged at 1,000 g for 2 min to collect the centrifuged samples, and the samples were mixed evenly and filtered with a 0.2 μm filter membrane; the filtered LB504 formulation samples were subpackaged at 200 μL/tube, 3 of the tubes were placed in a 40° C. water bath kettle, and the samples were detected by SEC-HPLC and SDS-PAGE on days 9, 20 and 30 respectively, i.e., detection of the samples treated at 40° C. for 9 days, 20 days and 30 days; and another tube was taken and detected by SEC-HPLC and SDS-PAGE after sterile subpackaging, i.e., detection of the samples treated at 40° C. for 0 day. The SEC-HPLC results of different formulation recipes were as shown in Table 23.

TABLE-US-00039 TABLE 22 Formulation recipe scheme of bispecific antibody LB504 of the present disclosure Experimental Recipe Composition of formulation conditions and number recipe formulation buffer sampling time points 1 20 mM citric acid-sodium citrate, Protein concentration 125 mM glycine, 125 mM trehalose, of 5 ± 0.2 mg/mL, 0.02% ps80, pH 5.0 placed at 40 ± 2° C., 2 20 mM acetic acid-sodium acetate, under accelerated 125 mM glycine, 125 mM trehalose, conditions, sampled on 0.02% ps80, pH 5.0 days 0, 9, 20, and 30 3 20 mM His-HCl, 125 mM glycine, respectively 125 mM trehalose, 0.02% ps80, pH 5.5 4 20 mM citric acid-sodium citrate, 125 mM glycine, 125 mM trehalose, 0.02% ps80, pH 5.5 5 20 mM His-HCl, 125 mM glycine, 125 mM trehalose, 0.02% ps80, pH 6.0 6 20 mM citric acid-sodium citrate, 125 mM glycine, 125 mM trehalose, 0.02% ps80, pH 6.0 7 20 mM His-HCl, 125 mM glycine, 125 mM trehalose, 0.02% ps80, pH 6.5 8 20 mM PB (phosphate buffer), 125 mM glycine, 125 mM trehalose, 0.02% ps80, pH 7.0

TABLE-US-00040 TABLE 23 Detection results of formulation recipe samples of the bispecific antibody LB504 of the present disclosure by SEC-HPLC Recipe Treatment % % % number Condition days aggregation monomer fragment 1 40° C. 0 4.74 95.26 0 9 7.89 92.11 0 20 12.3 87.3 0.4 30 24.56 74.89 0.55 2 40° C. 0 0.69 99.31 0 9 0.73 99.27 0 20 2.34 97.37 0.3 30 3.59 96.05 0.36 3 40° C. 0 0.14 99.86 0 9 0.16 99.78 0.06 20 2.16 97.63 0.2 30 4.9 94.77 0.33 4 40° C. 0 6.05 93.95 0 9 10.1 89.9 0 20 11.93 87.75 0.33 30 18.75 80.85 0.4 5 40° C. 0 0.13 99.87 0 9 0.2 99.8 0 20 3.62 96.14 0.24 30 5.03 94.54 0.44 6 40° C. 0 6.77 93.23 0 9 9.45 90.55 0 20 11.12 88.68 0.2 30 16.23 83.39 0.37 7 40° C. 0 0.15 99.85 0 9 0 100 0 20 0.83 98.6 0.57 30 2.1 97.1 0.8 8 40° C. 0 5.57 94.43 0 9 8.73 91.05 0.22 20 12.91 86.22 0.86 30 19.09 79.85 1.07

[0266] The aforementioned results showed that when the bispecific antibody LB504 of the present disclosure was in a citric acid buffer system (pH5.0, pH5.5, pH6.0) and a phosphate buffer system at a medium concentration (5 mg/mL), the aggregation was increased after buffer replacement, and with the increase of the treatment time at 40° C., the increase of the aggregation was obvious and meanwhile a few fragments were produced, and the purity detected by SEC-HPLC was decreased to about 80%. LB504 exhibited as relatively stable in a acetic acid buffer system (pH5.0), and after 30 days of treatment at 40° C., the aggregation was increased by 3.59%, the fragments were increased by 0.36%, and the decrease of the total purity detected by SEC-HPLC was within 5%. In a histidine buffer system (including pH5.5, pH6.0, pH6.5), LB504 had a high purity after buffer replacement, and after 30 days of treatment at 40° C., it exhibited as relatively stable with little increase of aggregations, and with the increase of the pH of the buffer, the generation of the aggregation was decreased. When LB504 was treated in recipe 7 (20 mM His-HCl, 125 mM glycine, 125 mM trehalose, 0.02% ps80, pH6.5) at 40° C. for 30 days, the purity detected by SEC-HPLC was decreased by 2.1%, the fragments were increased by 0.8%, and the change trend of the purity was small. More preferably, when LB504 was treated in recipe 7 at 40° C. for 20 days, almost no aggregation and fragment were produced, and the purity was decreased by 1.4% (0.83% of the aggregation and 0.57% of the fragments were produced respectively). The results of SDS-PAGE also showed that LB504 treated at 40° C. for 20 days in recipe 7 had almost no generation of aggregations and degraded fragments.

[0267] The aforementioned results showed that the bispecific antibody LB504 of the present disclosure had the best stability in the formulation buffer (20 mM His-HCl, 125 mM glycine, 125 mM trehalose, 0.02% ps80, pH6.5) at a high concentration (5 mg/mL), and it could remain stable after being treated at 40° C. for 30 days, and the purity detected by SEC-HPLC was decreased only by 2.9%, which indicated that LB504 had good stability.

Example 16 Evaluation of In Vivo Pharmaceutical Effect of the Optimally Designed Molecule of Sirpα and PD-1 Bispecific Antibody of the Present Disclosure in an Animal

[0268] An animal pharmacodynamic model was established with human PD-1/Sirpα double transgenic Balb/cJGPt strain mice Balb/cJGPt-hPD-1/hSirpα to conduct in vivo pharmaceutical effect evaluation of the bispecific antibody LB504 of the present disclosure. The mice were purchased from Jiangsu GemPharmatech Co., Ltd., with the production license number: SCXK (Su) 2018-0008.

[0269] Using the method of Example 2, the full-length sequence of human CD47 (see Example 1) was overexpressed on the surfaces of CT26 cells (purchased from the cell bank of National Collection of Authenticated Cell Cultures of Chinese Academy of Sciences) to obtain mouse colon cancer cell line CT26-920 overexpressing human CD47. The constructed CT26-920 cells were cultured in a RPMI1640 medium (Hyclone, Cat #SH3080901) containing 10% fetal bovine serum (Gibco, Cat #10270-106), and continuously cultured in a 37° C. cell incubator containing 5% CO.sub.2. Balb/cJGPt-hPD-1/hSirpα female mice were raised at 5 mice/cage in a SPF-level environment, at a temperature of 20-25° C. and humidity of 40%-60%, the mice had access to food and water ad libitum, and the litter was changed regularly. When the CT26-920 cells were cultured to the logarithmic growth phase (with the confluence of 80%-90%), they were digested with 0.25% pancreatin, and the cells were collected, washed twice with a RPMI1640 medium, and resuspended with the RPMI1640 medium and counted. After that, the RPMI1640 medium and Matrigel were mixed at a ratio of 2:1 to finally adjust the cell density to 6×10.sup.6 cells/mL with the mixture. 100 μL of CT26 cell suspension was inoculated subcutaneously in the left flank of the mice (0.6×10.sup.6 cells/mouse), and the mice with a tumor volume of about 100-120 mm.sup.3 were selected for random grouping, with 8 mice in each group.

[0270] In a sterile environment, the samples to be tested and the control samples were formulated with PBS. PBS was used as blank, the groups administrated with the PD-1 antibody (a-PD1, i.e. anti-PD1 antibody Pembrolizumab/Keytruda (referred to as Pem for short), cloned and expressed by the method of Example 1 of the present disclosure) and Ref1 were respectively control groups of individual medications. LB504 was the drug to be tested. The route of administration was intraperitoneal injection, the administrated dosage of the groups of individual medications was 10 mg/kg, the administrated dosage of LB504 was 13.3 mg/kg, and the injection volume of each group was 200 μL/mouse (LB504 and each control antibody were equimolar). The dosing frequency was 2 time/week, and the dose was administered continuously for 2 weeks.

[0271] The first dosing day was Day 0. The body weight and tumor size were measured before each administration, and the data was recorded. The actual administration period of this experiment was 2 weeks, and the measurement period was 16 days. After completion of tumor measurement, the tumor volume, the relative tumor volume and the tumor inhibition rate were calculated. The results were as shown in FIG. 4 and Table 24.

[0272] Calculation formula of tumor size: The tumor volume TV (mm.sup.3)=0.5×(tumor long diameter×tumor short diameter.sup.2); the relative tumor volume (RTV)=T/T.sub.0 or C/C.sub.0. The relative tumor growth rate (T/C %)=100%×(T−T.sub.0)/(C−C.sub.0); the tumor inhibition rate (TGI)=(1−T/C)×100%; wherein T.sub.0 and T were the tumor volumes at the beginning and end of the experiment in each administration group, respectively; and C.sub.0 and C were the tumor volumes of the control group at the beginning and end of the experiment, respectively.

[0273] The results in FIG. 4 and Table 24 showed that in the Balb/cJGPt-hPD-1/hSirpα double knock-in mouse CT26 colon cancer animal model, the bispecific antibody molecule LB504 of the present disclosure showed obvious inhibition effect on tumor growth, and the in vivo pharmaceutical effect in the mice was significantly better than that of equal molar doses of Ref1 and a-PD1, and even better, the tumor inhibition rate of the group administrated with LB504 could reach 71%.

TABLE-US-00041 TABLE 24 Results of relative tumor volume analysis and TGI calculation after 16 days of administration Mean tumor volume Mean tumor volume Number (mm.sup.3) (mm.sup.3) TGI % mice Group D0 SD D16 SD D16 P of dead PBS 108.46 33.21 3316.05 2816.48 — 0 a-PD1-10 109.05 28.56 2813.69 2737.19 16% 0.3659  0 Ref1-10 107.20 25.54 1711.72 1435.57 50% 0.0866  0 LB504-13.3 106.72 24.98 1044.75 1536.81 71% 0.0325* 0 *represents p < 0.05

[0274] Meanwhile, on day 19 after administration, 5 mice were taken from each group for TILs analysis. Specifically, the tumor of each mouse was taken out and put into a dish, cut into small pieces of 2-4 mm, then transferred into a centrifuge tube containing an enzyme digestion buffer (collagenase IV+DNase I), digested at 37° C., then filtered with a filter screen, and centrifuged (400 g, 5 min). The supernatant was discarded, and the pellet was added with red blood cell lysis buffer for lysis of red blood cells, and centrifuged. The pellet was added with PBS to resuspend the cells and cell counting was conducted, and then 1×10.sup.6 cells/sample were taken for subsequent staining and labeling. The cells were taken, added with Fc Block antibodies, incubated for 10-15 min, then added into each mixed solution of fluorescent antibodies respectively, incubated at 4° C. for 30 min, and washed twice with 200 μL of FACS buffer. The supernatant was discarded, and the pellet was added with 100 μL of the FACS buffer to resuspend cells, and then the resuspended cells were detected on a machine. The results were as shown in Table 25.

TABLE-US-00042 TABLE 25 Analysis results of tumor TILs in mice of each administration group (mean) % of % of lives CD45+ M2 % of CD3+ % of CD3+ Group CD45+ (CD206+) CD4+ CD8+ CD25+CD4+ G1: PBS 38.09 11.10 22.27 56.12 13.87 G2: Ref1 23.96 12.17 22.18 54.63 12.34 G3: a-PD1 36.61 10.52 19.24 60.42 10.02 G4: LB504 44.93 4.25 16.83 59.95 8.83

[0275] The results of TILs analysis showed that the proportion of total lymphocytes and CD8+ T lymphocytes in the tumor microenvironment of mice in each administration group had little change, but the proportion of CD4+ T lymphocytes decreased, and decreased significantly in the group administrated with LB504, which was mainly caused by the decrease of CD4+CD25+ cells (Treg). The proportion of CD206+ cells (M2-type macrophages) in the dosage group administrated with LB504 also decreased significantly by 61.7% (compared with the PBS group, 4.25% vs 11.1%). The proportions of CD11+/F4-80+ cells and CD206− cells in each administration group were basically the same as those in the PBS group. The results indicated that the bispecific antibody LB504 of the present disclosure exerted an antitumor pharmaceutical effect by reducing the proportions of suppressor T cells (Treg) and M2 macrophages.

Example 17 PK Evaluation of the Sirpα and PD-1 Bispecific Antibody of the Present Disclosure

[0276] PK evaluation of the bispecific antibody of the present disclosure was carried out with the same human Sirpα and PD-1 double transgenic mice under the same feeding conditions as in Example 16. 3 mice were randomly selected to form a group. The mice were injected with LB504 via tail vein, with an injection dose being 13.3 mg/kg and an injection volume being 200 μL/mice. Blood samples were drawn from the orbit at 0 hours before the injection and 0.25, 0.5, 1, 5, 24, 48, 72, 101, 120, 144, 168, 192, 216, and 288 hours after injection, respectively. The blood samples were centrifuged, and the supernatant was taken and stored at −20° C. After blood samples at all time points were collected, the PK characteristics of LB504 were evaluated by double-antigen sandwich ELISA detection of the binding of LB504 to PD-1 and Sirpα (the bispecific antibody could bind to PD-1 and Sirpα simultaneously). PK data was analyzed with EXCEL software, and the T.sub.1/2 of LB504 was calculated. The results were shown in Table 26.

TABLE-US-00043 TABLE 26 PK evaluation of the PD-1 and Sirpα bispecific antibody of the present disclosure Antibody LB504 Antigen PD-1/Sirpα Serial number of mouse 1 2 3 Mean T.sub.max (h) 0.25 0.25 0.25 0.25 C.sub.max (g/mL) 224.6 187.86 161.1 191.19 T.sub.1/2 (101 h) 35.36 20.38 38.2 31.31 AUC.sub.0-101 h 3874 1671 1978 2507.67 (μg/mL*h)

[0277] The aforementioned results showed that after a single tail injection of the bispecific antibody LB504 of the present disclosure into the mice, the concentration reached the highest value at 0.25 h, and the Cmax and AUC.sub.0-101 h were 191.19 g/mL and 2507.67 μg/mL*h, respectively. T.sub.1/2 was 31.31 hours. The results showed that the in vivo PK parameters of the bispecific antibody LB504 of the present disclosure in the mice are in a normal range, and thus the bispecific antibody was exploitable.

[0278] In conclusion of the aforementioned data of the present disclosure, it indicated that through innovative screening, the inventor had accidentally discovered an anti-human Sirpα antibody, which had good binding activity to Sirpα and could bind to human Sirpα-V1 and human SIRP α-V2 simultaneously; and had very good binding activities to various of polymorphic proteins of Sirpα of the non-human primate cynomolgus monkey. It could effectively block the binding of human Sirpα to human CD47. It had better activity than the currently clinical antibodies (control antibodies Ref1 and Ref2); and did not bind to human Sirpβ and human Sirpγ, and also did not bind to human T cells, so that it had very good selectivity, could avoid the off-target effect in clinical, and could avoid side effects more effectively. Furthermore, the sequence of the molecule itself had a low PTM risk. The humanized antibody had a high expression level, which provided convenience and cost savings for downstream production and processes. Furthermore, the bispecific antibody designed based on the Sirpα antibody sequence of the present disclosure could retain the functional activity of the antibody against dual targets, the binding activity of it to the two targets was close to that of its corresponding monoclonal antibody, and the activity of it in blocking the binding of the antigen to a corresponding ligand was also consistent with that of the corresponding monoclonal antibody; and it could effectively inhibit tumor growth; had good stability, and was relatively stable in both a acetic acid buffer system and a histidine buffer system. These bispecific antibodies (called SBodies in the present disclosure), which are similar in structure to conventional IgGs, have the same entire Fc as normal antibodies, so that their purification process can be carried out according to those of normal antibodies, and thus the process is simple and has the advantage of low production cost. The unique characteristics of the antibody of the present disclosure made it more suitable for the development of antibody drugs against the human Sirpα target, and as a candidate drug, it could be administered alone or in combination, especially providing a new and even better option for the treatment of tumors in combination with the PD-1 antibody, and the preferred bispecific antibody of the present disclosure provided another option for multi-target therapy of tumors.