Bispecific molecules targeting SIRPA and CLAUDIN 18.2

Abstract

The present disclosure relates to anti-SIRP [alpha] antibodies or antigen binding fragments thereof, bispecific molecules targeting SIRP [alpha] and Claudin 18.2, pharmaceutical compositions, and the use of such molecules in the prevention, diagnosis or treatment of cancer diseases.

Description

Technical Field

[0001] The present disclosure relates to anti-SIRPα antibodies or antigen-binding fragments thereof, as well as bispecific molecules targeting SIRPα (signal regulatory protein α) and claudin 18.2 (claudin 18 isoform 2), and uses thereof. Background Art

[0002] The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.

[0003] As research in the field of cancer treatment continues to deepen, the research, development, and application of molecularly targeted cancer therapeutics are gaining increasing attention. Antibody drugs have rapidly become a hot topic in cancer targeted therapy due to their strong targeting, minimal side effects, and significant therapeutic effects.

[0004] However, tumor cells can evade the surveillance, recognition, and attack of the innate immune system (so-called tumor immune escape) by modifying their own surface antigens and changing the microenvironment surrounding the tumor tissue, thereby continuously dividing and growing. For example, tumor cells suppress the immune function of macrophages and significantly inhibit the activity of immune cells by highly expressing CD47, which binds to the inhibitory receptor signal regulatory protein α (SIRPα or SIRPA) on the surface of macrophages. (Willingham SB et al. The CD47-signal regulatory protein Alpha (SIRPα) interaction is atherapeutic target for human solid tumors. [CD47 signal regulatory protein α (SIRPα) interaction is a therapeutic target for human solid tumors] Proceedings of the National Academy of Sciences of the United States of America [Proceedings of the National Academy of Sciences of the United States of America]. 2012, 109 (17): 6662-6667).

[0005] SIRPα (signal regulatory protein α, SIRPA) is a regulatory membrane glycoprotein of the SIRP family. It is primarily expressed by myeloid cells, but also by stem cells and neurons. SIRPα acts as an inhibitory receptor and interacts with the ubiquitously expressed transmembrane protein CD47. This interaction negatively controls the effector functions of innate immune cells, such as host cell phagocytosis.

[0006] Therefore, the inhibitory effect of CD47 on immune cells can be alleviated by blocking the CD47-SIRPα signaling pathway (such as SIRPα-Fc fusion protein), and exhibit certain anti-tumor activity. Wild-type SIRPα-Fc fusion protein does not have effective efficacy due to its low affinity for CD47.

[0007] Claudin 18 (CLDN 18.2) belongs to the claudin family, which has at least 27 members in mammals (Furuse M. et al., J Cell Biol., 1998, 141, 1539). Claudin 18 has two different splice variants, claudin 18.1 or CLDN 18.1 and claudin 18.2 or CLDN 18.2 (Sanada Y. et al., J Pathol., 2006, 208, 633). CLDN 18.2 is a CD20-like differentiation protein that is overexpressed in various types of cancer, such as gastric cancer, esophageal cancer, pancreatic cancer, and non-small cell lung cancer. Therefore, this molecule is a valuable target for treating such cancers.

[0008] In general, there is a need to develop novel tumor-targeting molecules. Summary of the Invention

[0009] For the above purposes, the present disclosure relates to anti-SIRPα antibodies as described herein. Specifically, the present disclosure relates to anti-SIRPα single-chain variable fragments, bispecific molecules and pharmaceutical compositions based on the anti-SIRPα single-chain variable fragments, and uses thereof.

[0010] Provided herein are novel anti-SIRPα antibodies or antigen-binding fragments thereof, comprising a heavy chain variable domain (VH) and a heavy chain variable domain (VL). The VH of the anti-SIRPα antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, and HCDR3, the amino acid sequences of which are at least 80%, such as about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequences set forth in: (1) SEQ ID NOs: 20, 21, and 23, respectively; (2) SEQ ID NOs: 20, 22, and 23, respectively; and (3) SEQ ID NOs: 20, 47, and 48, respectively. The VL of the anti-SIRPα antibody or its antigen-binding fragment comprises LCDR1, LCDR2 and LCDR3, the amino acid sequences of which are at least 80%, for example, about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequences shown in the following: (1) SEQ ID NO: 24, 26 and 28, respectively; (2) SEQ ID NO: 25, 27 and 28, respectively.

[0011] In some embodiments, HCDR1 has the amino acid sequence set forth in SEQ ID NO:20, HCDR2 has the amino acid sequence set forth as X1DPEDX2ETK (SEQ ID NO:60), HCDR3 has the amino acid sequence set forth as DRGLX3Y (SEQ ID NO:61), LCDR1 has the amino acid sequence set forth as X4ASX5SVSSSYLY (SEQ ID NO:45), LCDR2 has the amino acid sequence set forth as YSX6SNX7AS (SEQ ID NO:46), and LCDR3 has the amino acid sequence set forth as SEQ ID NO:28, X1 is V or I, X2 is A or G, X3 is V or A, X4 is R or S, X5 is Q or S, X6 is A or T, and X7 is R or L.

[0012] In some embodiments, HCDR1, HCDR2, and HCDR3 have the amino acid sequences set forth in SEQ ID NOs: 20, 21, and 23, respectively; and LCDR1, LCDR2, and LCDR3 have the amino acid sequences set forth in SEQ ID NOs: 24, 26, and 28, respectively.

[0013] In some embodiments, HCDR1, HCDR2, and HCDR3 have the amino acid sequences set forth in SEQ ID NOs: 20, 22, and 23, respectively; and LCDR1, LCDR2, and LCDR3 have the amino acid sequences set forth in SEQ ID NOs: 25, 27, and 28, respectively.

[0014] In some embodiments, HCDR1, HCDR2, and HCDR3 have the amino acid sequences set forth in SEQ ID NOs: 20, 47, and 48, respectively; and LCDR1, LCDR2, and LCDR3 have the amino acid sequences set forth in SEQ ID NOs: 25, 27, and 28, respectively.

[0015] In some embodiments, the VH of the anti-SIRPα antibody or antigen-binding fragment thereof comprises FR1, FR2, FR3 and FR4, the amino acid sequences of which are at least 80%, for example, about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequences shown in the following: (1) SEQ ID NO: 29, 30, 32 and 33, respectively; (2) SEQ ID NO: 29, 31, 32 and 33, respectively; (3) SEQ ID NO: 49, 50, 51 and 33, respectively.

[0016] In some embodiments, the VL of the anti-SIRPα antibody or antigen-binding fragment thereof comprises FR1, FR2, FR3 and FR4, the amino acid sequences of which are at least 80%, for example, about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequences shown in the following: (1) SEQ ID NO: 34, 35, 36 and 37, respectively; (2) SEQ ID NO: 34, 35, 36 and 38, respectively; (3) SEQ ID NO: 34, 52, 53 and 37, respectively.

[0017] In some embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof comprises a VH and / or a VL, wherein the amino acid sequence of the VH is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to one of the amino acid sequences shown in SEQ ID NOs: 14, 16, 18 and 54; and the amino acid sequence of the VL is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to one of the amino acid sequences shown in SEQ ID NOs: 15, 17, 19 and 55.

[0018] In some embodiments, the VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 14; and the VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 15.

[0019] In some embodiments, the VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 16; and the VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 17.

[0020] In some embodiments, the VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 18; and the VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 19.

[0021] In some embodiments, the VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:54; and the VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:55.

[0022] In some embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof binds to human and / or mouse SIRPα.

[0023] In some embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof is a Fab, Fab', F(ab')2, Fv fragment or single-chain variable fragment (scFv). In some embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof is a scFv, wherein VH and VL are connected via a first peptide linker, optionally comprising (Gly4Ser)4. In some embodiments, in the anti-SIRPα antibody or antigen-binding fragment thereof, the N-terminus of VH is connected to the C-terminus of VL.

[0024] In some embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof is a chimeric antibody or a humanized antibody.

[0025] In another aspect, provided herein are bispecific molecules comprising

[0026] (1) A SIRPα binding domain comprising a heavy chain variable domain (VH) and a heavy chain variable domain (VL), wherein VH comprises HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are about 80% to about 100%, for example, about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more to about 100% identical to the amino acid sequences shown in the following: (1) SEQ ID NO: 20, 21 and 23, respectively; (2) SEQ ID NO: 20, 22 and 23, respectively; (3) SEQ ID NO: 20, 47 and 48, respectively. VL comprises LCDR1, LCDR2, and LCDR3, the amino acid sequences of which are about 80% to about 100%, e.g., about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequences set forth in: (1) SEQ ID NOs: 24, 26, and 28, respectively; (2) SEQ ID NOs: 25, 27, and 28, respectively; and

[0027] (2) A second domain that binds to the target antigen, wherein the second domain is fused to the SIRPα binding domain.

[0028] In some embodiments, in the SIRPα binding domain, HCDR1 has the amino acid sequence set forth in SEQ ID NO:20, HCDR2 has the amino acid sequence set forth as X1DPEDX2ETK (SEQ ID NO:60), HCDR3 has the amino acid sequence set forth as DRGLX3Y (SEQ ID NO:61), LCDR1 has the amino acid sequence set forth as X4ASX5SVSSSYLY (SEQ ID NO:45), LCDR2 has the amino acid sequence set forth as YSX6SNX7AS (SEQ ID NO:46), and LCDR3 has the amino acid sequence set forth in SEQ ID NO:28, X1 is V or I, X2 is A or G, X3 is V or A, X4 is R or S, X5 is Q or S, X6 is A or T, and X7 is R or L.

[0029] In some embodiments, in the SIRPα binding domain, (1) HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 20, 21 and 23, respectively; and LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NOs: 24, 26 and 28, respectively; (2) HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 20, 22 and 23, respectively; and LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NOs: 25, 27 and 28, respectively; or (3) HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 20, 47 and 48, respectively; and LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NOs: 25, 27 and 28, respectively.

[0030] In some embodiments, in the bispecific molecule, the VH of the SIRPα binding domain comprises FR1, FR2, FR3, and FR4, the amino acid sequences of which are at least 80%, such as about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequences shown in: (1) SEQ ID NO: 29, 30, 32, and 33, respectively; (2) SEQ ID NO: 29, 31, 32, and 33, respectively; (3) SEQ ID NO: 29, 31, 32, and 33, respectively. NO:49, 50, 51 and 33; and / or the VL of the SIRPα binding domain comprises FR1, FR2, FR3 and FR4, the amino acid sequences of which are at least 80%, for example, about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequences shown in the following: (1) SEQ ID NO:34, 35, 36 and 37, respectively; (2) SEQ ID NO:34, 35, 36 and 38, respectively; (3) SEQ ID NO:34, 52, 53 and 37, respectively.

[0031] In some embodiments, in the bispecific molecule, the VH of the SIRPα binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to one of the amino acid sequences shown in SEQ ID NOs: 14, 16, 18 and 54, respectively; and / or the VL of the SIRPα binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to one of the amino acid sequences shown in SEQ ID NOs: 15, 17, 19 and 55, respectively.

[0032] In some embodiments, in the SIRPα binding domain, (1) VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 14; and VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 15; (2) VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 16; and VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: NO:17; (3) VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:18; and VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:19; or (4) VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:54; and VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:55. An amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in NO:55.

[0033] In some embodiments, in the bispecific molecule, the SIRPα binding domain is a scFv, wherein VH and VL are connected via a first polypeptide linker, optionally comprising (Gly4Ser)4. In some embodiments, the N-terminus of VH is linked to the C-terminus of VL.

[0034] In some embodiments, the second domain binds to a target antigen expressed on the surface of a cancer cell. The target antigen can be an immune checkpoint molecule, such as CD47, PD1, claudin 18.2, or CTLA-4. In some embodiments, the second domain binds to claudin 18.2 and / or CD47, preferably claudin 18.2.

[0035] In some embodiments, the second domain comprises: a) a heavy chain variable region (VH) comprising CDR1, CDR2, and CDR3, wherein the VH CDR1, CDR2, and CDR3 have an amino acid sequence about 80% to about 100% identical to the amino acid sequence shown in SEQ ID NOs: 39, 40, and 41, respectively; and b) a light chain variable region (VL) comprising CDR1, CDR2, and CDR3, wherein the VL CDR1, CDR2, and CDR3 have an amino acid sequence about 80% to about 100% identical to the amino acid sequence shown in SEQ ID NOs: 42, 43, and 44, respectively.

[0036] In some embodiments, the second domain comprises a VH having an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO:9, and a VL having an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO:10.

[0037] In some embodiments, the second domain comprises a light chain constant region (CL), which is a kappa or lambda light chain. In some embodiments, the CL comprises the amino acid sequence set forth in SEQ ID NO: 12 or 13.

[0038] In some embodiments, the second domain comprises a heavy chain constant region (CH) having the amino acid sequence shown in SEQ ID NO:11.

[0039] In some embodiments, the bispecific molecule is a symmetrical bispecific molecule in which the SIRPα binding domain is fused to the C-terminus of the heavy chain constant region of the second domain at the N-terminus of the heavy chain variable domain of the SIRPα binding domain. Preferably, the SIRPα binding domain and the second domain are covalently linked via a second peptide linker having the formula (Gly4Ser)n, wherein n is an integer from 1 to 5, such as 1, 2, 3, 4, 5. In some embodiments, the first domain and the second domain are covalently linked via (Gly4Ser)3.

[0040] In some embodiments, the bispecific molecule comprises a heavy chain comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:56, 58 or 59; and a light chain comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:57.

[0041] In some embodiments, the heavy chain of the bispecific molecule comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:7; and the light chain of the bispecific molecule comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:8.

[0042] In some embodiments, the heavy chain of the bispecific molecule comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:5; and the light chain of the bispecific molecule comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:6.

[0043] In some embodiments, the bispecific molecules block the interaction of CD47 and SIRPα, remove SHP-1 / 2 inhibition, and / or engage Fc receptors on immune effector cells (eg, macrophages) to activate phagocytosis.

[0044] In some embodiments, the bispecific molecules enhance phagocytosis of cancer cells expressing a target antigen bound to the second domain (eg, human claudin 18.2 and human SIRPα) by immune effector cells.

[0045] In another aspect, the present disclosure provides an isolated polynucleotide encoding the above-mentioned anti-SIRPα antibody or antigen-binding fragment or bispecific molecule.

[0046] In another aspect, the present disclosure provides a construct comprising the above-described polynucleotide.

[0047] In another aspect, the present disclosure provides an antibody expression system comprising the above construct or having a genome integrated with the above exogenous polynucleotide. Preferably, the expression system is a cell expression system.

[0048] In another aspect, the present disclosure provides a method for producing the above-mentioned anti-SIRPα antibody or antigen-binding fragment thereof or bispecific molecule, the method comprising: expressing the antibody or protein using the above-mentioned antibody expression system under conditions suitable for antibody expression.

[0049] In another aspect, the present disclosure provides a pharmaceutical composition comprising the above-mentioned anti-SIRPα antibody or antigen-binding fragment or bispecific molecule, and a pharmaceutically acceptable carrier.

[0050] In another aspect, the present disclosure provides kits comprising an anti-SIRPα antibody or antigen-binding fragment thereof or bispecific molecule, isolated polynucleotide, or construct provided herein.

[0051] In another aspect, the present disclosure provides the use of an anti-SIRPα antibody, or antigen-binding fragment thereof, bispecific molecule, or pharmaceutical composition provided herein, in the manufacture of a therapeutic agent for preventing, diagnosing, or treating a disease, disorder, or condition. In some embodiments, the disease, disorder, or condition is cancer. In some embodiments, at least the cancer cells express claudin 18.2, preferably human claudin 18.2.

[0052] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer comprises esophageal cancer, liver cancer, lung cancer, melanoma, gastric cancer, pancreatic cancer, ovarian cancer, colon cancer, kidney cancer, bladder cancer, breast cancer, classical Hodgkin lymphoma, hematological malignancies, head and neck cancer and nasopharyngeal cancer, gallbladder cancer and metastasis thereof, Kukenberg tumor, peritoneal metastasis and / or lymph node metastasis.

[0053] In another aspect, the present disclosure provides a method for treating a subject having cancer, comprising administering to the subject a therapeutically effective amount of an anti-SIRPα antibody, or antigen-binding fragment thereof, bispecific molecule, or pharmaceutical composition provided herein. In some embodiments, the subject is a mammal, including humans, mice, and cynomolgus monkeys.

[0054] In another aspect, the present disclosure provides a method of reducing tumor growth rate, comprising contacting tumor cells with an effective amount of an anti-SIRPα antibody, or antigen-binding fragment thereof, bispecific molecule, or pharmaceutical composition provided herein.

[0055] In another aspect, the present disclosure provides a method of killing a tumor cell, comprising contacting the tumor cell with an effective amount of an anti-SIRPα antibody, or antigen-binding fragment thereof, bispecific molecule, or pharmaceutical composition provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] The following is a brief description of the drawings, which are presented for the purpose of illustrating exemplary embodiments disclosed herein and not for the purpose of limiting the same.

[0057] Figure 1 Shown are the antigen binding profiles of the wild-type control and library 2 in the first round of yeast library sorting detected by FACS (fluorescence activated cell sorting).

[0058] Figure 2 Shown are the antigen binding profiles of the wild-type control and library 2 in the first round of yeast library sorting detected by FACS (fluorescence activated cell sorting).

[0059] Figure 3 Comparison of binding efficacy between wild-type control and clone 201 detected by FACS (fluorescence activated cell sorting) is shown.

[0060] Figure 4A Schematic diagram of an anti-claudin 18.2 / SIRPα bispecific molecule, in which two anti-SIRPα scFvs are fused to the C-terminus of the anti-claudin 18.2 antibody heavy chain via a linker.

[0061] Figure 4B The SEC (size exclusion chromatography) spectrum of AE016_201 is shown, and the purity of each component in the protein sample is listed in the table below the SEC spectrum.

[0062] Figure 5 The binding curve of AE016_201 to SIRPα detected by Octet is shown.

[0063] Figure 6 The CHO-K1 SIRPα binding curves of AE016_201 and IgG4 isotype anti-SIRPα antibodies detected by FACS are shown. EC50 values ​​are listed in the table below the binding curves.

[0064] Figure 7 The long-term stability of AE016_201 at 4°C was demonstrated.

[0065] Figure 8A Binding curves of representative bispecific antibodies to Raji / hClaudin 18.2 cells detected by FACS are shown. The anti-hClaudin 18.2 antibody hu26.H1L2 was used as a control.

[0066] Figure 8B Figure 2 shows the binding curves of representative bispecific antibodies to CHOK1 / SIRPα cells detected by FACS. Anti-SIRPα antibody hu025.060 was used as a control.

[0067] Figure 9 It was shown that the anti-claumin 18.2 / SIRPα bispecific antibody stimulated phagocytosis of MC38 / hCD47 / human claudin 18.2 cells by BMDM better than single or combination treatments.

[0068] Figure 10 In vivo anti-tumor efficacy of anti-claulogen18.2 / SIRPα in the MC38 / human claudin18.2 / hSIRPα syngeneic model was demonstrated. DETAILED DESCRIPTION

[0069] The present disclosure will be explained in more detail below. This specification is not intended to be an exhaustive list of all the different ways in which the present invention can be implemented or all the features that can be added to the present invention. For example, features described with respect to one embodiment may be incorporated into other embodiments, and features described with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art without departing from the present invention by considering the present disclosure. Therefore, the following description is intended to illustrate some specific embodiments of the present invention, rather than to exhaustively specify all permutations, combinations, and variations thereof.

[0070] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the testing practice of the present disclosure, preferred materials and methods are described herein. In describing and claiming the present disclosure, the following terminology will be used.

[0071] Other features and advantages of the disclosure will be apparent from the following detailed description and drawings, and from the claims.

[0072] the term

[0073] The term "antibody" (which can be used interchangeably in the plural) is an immunoglobulin molecule that is capable of specifically binding to a target (e.g., carbohydrate, polynucleotide, lipid, polypeptide, etc.) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" includes not only complete (i.e., full-length) polyclonal or monoclonal antibodies, but also includes antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single chains (scFv), mutants thereof, fusion proteins comprising antibody portions, humanized antibodies, chimeric antibodies, diabodies, nanobodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified constructs of immunoglobulin molecules comprising antigen recognition sites with desired specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Antibodies include antibodies of any class, such as IgD, IgE, IgG, IgA, or IgM (or its subclass), and antibodies do not need to be of any particular class. Based on the antibody amino acid sequence of its heavy chain constant domain, immunoglobulins can be divided into different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0074] A typical antibody molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL). The variable region is a region with relatively large variations in amino acid composition and arrangement at the N-terminus of the antibody molecule. The specific binding site (i.e., antigen binding site) is used to determine the specificity of antibody recognition. The VH and VL regions can be further subdivided into hypervariable regions, also known as "complementarity determining regions" (CDRs), interspersed with more conserved regions known as "framework regions" (FRs). Each VH and VL is typically composed of three CDRs and four FRs, which are arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0075] As used herein, the term "single-chain variable fragment" or "scFv" is a fusion protein in which the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin (e.g., mouse or human) are covalently linked to form a VH::VL heterodimer. The heavy chain (VH) and light chain (VL) are directly linked or connected through a linker or spacer encoding a peptide that connects the N-terminus of VH to the C-terminus of VL, or connects the C-terminus of VH to the N-terminus of VL. Those skilled in the art will be able to select the appropriate configuration for use in the present invention.

[0076] As used herein, the term "bispecific molecule" refers to an antibody that exhibits dual binding specificity and affinity for two specific epitopes, or an antibody composition in which all antibodies exhibit dual binding specificity and affinity for two specific epitopes.

[0077] "Fc region" (fragment crystallizable region) or "Fc domain" or "Fc fragment" refers to the C-terminal region of an antibody heavy chain that mediates the binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or binding to the first component (Clq) of the classical complement system. Thus, the Fc region comprises the constant region of an antibody, excluding the first constant region immunoglobulin domain (e.g., CH1 or CL). Fc may also refer to this region alone or in the context of a protein polypeptide comprising Fc.

[0078] The term "fusion" or "fused" when applied to amino acid sequences (e.g., peptides, polypeptides, or proteins) refers to the combination of two or more amino acid sequences (e.g., by chemical bonding or recombinant means) into a single amino acid sequence. A fused amino acid sequence can be produced by the recombination of two genes encoding polynucleotide sequences, and can be expressed by introducing a construct containing the recombinant polynucleotide into a host cell.

[0079] The term "percentage (%) of sequence identity" is defined as the percentage of amino acid residues in the candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percentage of sequence identity, and not considering any conservative substitutions as part of sequence identity. Alignment for determining percentage of amino acid sequence identity can be achieved in various ways using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. For example, the BLAST program of the NCBI database can be used to determine identity.

[0080] The term "humanized antibody" refers to a molecule having an antigen binding site that is substantially derived from an immunoglobulin from a non-human species, wherein the remaining immunoglobulin structure of the molecule is based on the structure and / or sequence of a human immunoglobulin. The antigen binding site may comprise a complete variable domain fused to a constant domain, or may comprise only the complementary determining regions (CDRs) in the appropriate framework regions transplanted into the variable domain. The antigen binding site may be wild type or modified by one or more amino acid substitutions. For example, modifications are made to make the antibody more similar to a human immunoglobulin. Certain forms of humanized antibodies retain all CDR sequences (e.g., a humanized single domain antibody comprises all three CDRs from an alpaca). Other forms have one or more CDRs that have been altered relative to the original antibody.

[0081] A "chimeric antibody" refers to an antibody whose variable region is derived from one species and whose constant region is derived from another species, for example, an antibody whose variable region is derived from a mouse or alpaca antibody and whose constant region is derived from a human antibody.

[0082] A "therapeutically effective amount" of a pharmaceutical agent (e.g., a pharmaceutical composition) is an amount effective to achieve the desired therapeutic or preventive effect at a dosage level and over a period of time. For example, a therapeutically effective amount of an agent can eliminate, reduce, delay, minimize, or prevent the adverse effects of a disease.

[0083] The term "pharmaceutically acceptable carrier" refers to ingredients in a pharmaceutical composition other than the active ingredient that are non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0084] The term "treatment / prevention" (and grammatical variations thereof) refers to an attempt to alter the natural progression of a disease in a treated individual and can be a preventive or clinical intervention performed during the course of clinical pathology. Desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies of the present disclosure can be used to delay the development of a disease or delay the progression of a disorder.

[0085] As used herein, the terms "about" or "approximately" refer to an amount, level, value, quantity, frequency, percentage, identity, dimension, size, number, weight, or length that varies by up to 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to a reference amount, level, value, quantity, frequency, percentage, identity, dimension, size, number, weight, or length. In specific embodiments, the terms "about" or "approximately" when preceding a numerical value refer to that value plus or minus a range of 20%, 15%, 10%, or 5%.

[0086] In this disclosure, "amino acid" refers to an organic compound containing amino (-NH2) and carboxyl (-COOH) functional groups and side chains unique to each amino acid. The names of amino acids are represented by standard single-letter or three-letter codes as follows:

[0087] Amino acid name Three-letter code Single-letter code Alanine Ala A Cysteine Cys C Aspartic acid Asp D glutamate Glu E Phenylalanine Phe F Glycine Gly G Histidine His H Isoleucine Ile I Lysine Lys K Leucine Leu L Methionine Met M Asparagine Asn N Proline Pro P Glutamine Gln Q Arginine Arg R Serine Ser S Threonine Thr T Valine Val V Tryptophan Trp W Tyrosine Tyr Y

[0088] Generally, the nomenclature and techniques relating to cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this specification. See, e.g., Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012); Therapeutic Monoclonal Antibodies: From Bench to Clinic, Zhiqiang An (ed.), Wiley, (2009); and Antibody Engineering, 2nd ed., Vols. 1 and 2, Ontermann and Dubel, eds., Springer-Verlag, Heidelberg (2010).

[0089] Anti-SIRPα antibody or antigen-binding fragment thereof

[0090] The present disclosure provides examples of novel anti-SIRPα antibodies or antigen-binding fragments thereof, which comprise a heavy chain variable domain (VH) and a heavy chain variable domain (VL). VH comprises complementarity determining regions (CDRs) 1, 2, and 3, and VL comprises CDR1, CDR2, and CDR3.

[0091] In some embodiments, the CDR1 (i.e., HCDR1) of VH differs by no more than 5, 4, 3, 2, 1, or 0 amino acids from the amino acid sequence of SEQ ID NO: 20. The CDR2 (i.e., HCDR2) of VH differs by no more than 5, 4, 3, 2, 1, or 0 amino acids from the amino acid sequence of SEQ ID NO: 21, 22, or 47. The CDR3 (i.e., HCDR3) of VH differs by no more than 3, 2, 1, or 0 amino acids from the amino acid sequence of SEQ ID NO: 23 or 48.

[0092] In some embodiments, the CDR1 (i.e., LCDR1) of VL differs by no more than 5, 4, 3, 2, 1, or 0 amino acids from the amino acid sequence of SEQ ID NO: 24 or 25. The CDR2 (i.e., LCDR2) of VL differs by no more than 5, 4, 3, 2, 1, or 0 amino acids from the amino acid sequence of SEQ ID NO: 26 or 27. The CDR3 (i.e., LCDR3) of VL differs by no more than 5, 4, 3, 2, 1, or 0 amino acids from the amino acid sequence of SEQ ID NO: 28.

[0093] In some embodiments, the VH of the anti-SIRPα antibody or antigen-binding fragment thereof comprises CDR1, CDR2, and CDR3, the amino acid sequences of which are at least 80%, for example, about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequences shown in the following: (1) SEQ ID NO: 20, 21, and 23, respectively; (2) SEQ ID NO: 20, 22, and 23, respectively; (3) SEQ ID NO: 20, 47, and 48, respectively.

[0094] In some embodiments, the CDR1, CDR2, and CDR3 of the VH of the anti-SIRPα antibody or antigen-binding fragment thereof have the amino acid sequences set forth in SEQ ID NOs: 20, 21, and 23, respectively; SEQ ID NOs: 20, 22, and 23, respectively; or SEQ ID NOs: 20, 47, and 48, respectively.

[0095] In some embodiments, the VL of the anti-SIRPα antibody or antigen-binding fragment thereof comprises CDR1, CDR2, and CDR3, the amino acid sequences of which are at least 80%, for example, about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequences shown in: (1) SEQ ID NOs: 24, 26, and 28, respectively; (2) SEQ ID NOs: 25, 27, and 28, respectively.

[0096] In some embodiments, the CDR1, CDR2, and CDR3 of the VL of the anti-SIRPα antibody or antigen-binding fragment thereof have the amino acid sequences set forth in SEQ ID NOs: 24, 26, and 28, respectively; or SEQ ID NOs: 25, 27, and 28, respectively.

[0097] In some embodiments, HCDR1 has the amino acid sequence set forth in SEQ ID NO:20, HCDR2 has the amino acid sequence set forth as X1DPEDX2ETK (SEQ ID NO:60), HCDR3 has the amino acid sequence set forth as DRGLX3Y (SEQ ID NO:61), LCDR1 has the amino acid sequence set forth as X4ASX5SVSSSYLY (SEQ ID NO:45), LCDR2 has the amino acid sequence set forth as YSX6SNX7AS (SEQ ID NO:46), and LCDR3 has the amino acid sequence set forth as SEQ ID NO:28, X1 is V or I, X2 is A or G, X3 is V or A, X4 is R or S, X5 is Q or S, X6 is A or T, and X7 is R or L.

[0098] In some embodiments, HCDR1, HCDR2, and HCDR3 have the amino acid sequences set forth in SEQ ID NOs: 20, 21, and 23, respectively; and LCDR1, LCDR2, and LCDR3 have the amino acid sequences set forth in SEQ ID NOs: 24, 26, and 28, respectively.

[0099] In some embodiments, HCDR1, HCDR2, and HCDR3 have the amino acid sequences set forth in SEQ ID NOs: 20, 22, and 23, respectively; and LCDR1, LCDR2, and LCDR3 have the amino acid sequences set forth in SEQ ID NOs: 25, 27, and 28, respectively.

[0100] In some embodiments, HCDR1, HCDR2, and HCDR3 have the amino acid sequences set forth in SEQ ID NOs: 20, 47, and 48, respectively; and LCDR1, LCDR2, and LCDR3 have the amino acid sequences set forth in SEQ ID NOs: 25, 27, and 28, respectively.

[0101] In some embodiments, the VH of the anti-SIRPα antibody or antigen-binding fragment thereof comprises FR1, FR2, FR3, and FR4. The FR1 of VH differs from the amino acid sequence of SEQ ID NO: 29 or 49 by no more than 3, 2, 1, or 0 amino acids. The FR2 of VH differs from the amino acid sequence of SEQ ID NO: 30, 31, or 50 by no more than 5, 4, 3, 2, 1, or 0 amino acids. The FR3 of VH differs from the amino acid sequence shown in SEQ ID NO: 32 or 51 by no more than 3, 2, 1, or 0 amino acids. The FR4 of VH differs from the amino acid sequence of SEQ ID NO: 33 by no more than 3, 2, 1, or 0 amino acids.

[0102] In some embodiments, FR1, FR2, FR3, and FR4 of the VH of the anti-SIRPα antibody or antigen-binding fragment thereof have an amino acid sequence that is about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence shown in: SEQ ID NO: 29, 30, 32, and 33, respectively; SEQ ID NO: 29, 31, 32, and 33, respectively; or SEQ ID NO: 49, 50, 51, and 33, respectively.

[0103] In some embodiments, FR1, FR2, FR3, and FR4 of the VH of the anti-SIRPα antibody or antigen-binding fragment thereof have the amino acid sequences set forth in SEQ ID NOs: 29, 30, 32, and 33, respectively; SEQ ID NOs: 29, 31, 32, and 33, respectively; or SEQ ID NOs: 49, 50, 51, and 33, respectively.

[0104] In some embodiments, the VL of the anti-SIRPα antibody or antigen-binding fragment thereof comprises FR1, FR2, FR3, and FR4. The FR1 of the VL differs from the amino acid sequence of SEQ ID NO: 34 by no more than 3, 2, 1, or 0 amino acids. The FR2 of the VL differs from the amino acid sequence of SEQ ID NO: 35 or 52 by no more than 3, 2, 1, or 0 amino acids. The FR3 of the VL differs from the amino acid sequence shown in SEQ ID NO: 36 or 53 by no more than 3, 2, 1, or 0 amino acids. The FR4 of the VL differs from the amino acid sequence of SEQ ID NO: 37 or 38 by no more than 5, 4, 3, 2, 1, or 0 amino acids.

[0105] In some embodiments, FR1, FR2, FR3, and FR4 of the VL of the anti-SIRPα antibody or antigen-binding fragment thereof have an amino acid sequence that is about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence shown in: SEQ ID NO: 34, 35, 36, and 37, respectively; SEQ ID NO: 34, 35, 36, and 38, respectively; or SEQ ID NO: 34, 52, 53, and 37, respectively.

[0106] In some embodiments, FR1, FR2, FR3, and FR4 of the VL of the anti-SIRPα antibody or antigen-binding fragment thereof have the amino acid sequences set forth in SEQ ID NOs: 34, 35, 36, and 37, respectively; SEQ ID NOs: 34, 35, 36, and 38, respectively; or SEQ ID NOs: 34, 52, 53, and 37, respectively.

[0107] In some embodiments, VH comprises FR1, FR2, FR3 and FR4, the amino acid sequences of which are about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NOs: 29, 30, 32 and 33, respectively; and VL comprises FR1, FR2, FR3 and FR4, the amino acid sequences of which are about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NOs: 34, 35, 36 and 37, respectively.

[0108] In some embodiments, VH comprises FR1, FR2, FR3 and FR4, the amino acid sequences of which are about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NOs: 29, 31, 32 and 33, respectively; and VL comprises FR1, FR2, FR3 and FR4, the amino acid sequences of which are about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NOs: 34, 35, 36 and 38, respectively.

[0109] In some embodiments, VH comprises FR1, FR2, FR3 and FR4, the amino acid sequences of which are about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NOs: 49, 50, 51 and 33, respectively; and VL comprises FR1, FR2, FR3 and FR4, the amino acid sequences of which are about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NOs: 34, 52, 53 and 37, respectively.

[0110] In some embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof comprises a VH whose amino acid sequence is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to one of the amino acid sequences shown in SEQ ID NOs: 14, 16, 18 and 54.

[0111] In some embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof comprises a VL whose amino acid sequence is 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to one of the amino acid sequences shown in SEQ ID NOs: 15, 17, 19 and 55.

[0112] In some embodiments, the VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 14; and the VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 15.

[0113] In some embodiments, the VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 16; and the VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 17.

[0114] In some embodiments, the VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 18; and the VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 19.

[0115] In some embodiments, the VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:54; and the VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:55.

[0116] In some embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof comprises a VH having the amino acid sequence shown in SEQ ID NO: 14 and a VL having the amino acid sequence shown in SEQ ID NO: 15; or a VH having the amino acid sequence shown in SEQ ID NO: 16 and a VL having the amino acid sequence shown in SEQ ID NO: 17; or a VH having the amino acid sequence shown in SEQ ID NO: 18 and a VL having the amino acid sequence shown in SEQ ID NO: 19; or a VH having the amino acid sequence shown in SEQ ID NO: 54 and a VL having the amino acid sequence shown in SEQ ID NO: 55.

[0117] In some embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof binds to human and / or mouse SIRPα, preferably binds to human SIRPα.

[0118] In some embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof is a Fab, Fab', F(ab')2, Fv fragment or single-chain variable fragment (scFv), preferably scFv, and VH and VL are connected via a first peptide linker. In certain embodiments, the N-terminus of VH is fused to the C-terminus of VL.

[0119] In some embodiments, the first peptide linker comprises (Gly4Ser)4. In some embodiments, the first peptide linker is (Gly4Ser)4.

[0120] In some embodiments, the anti-SIRPα antibody or antigen-binding fragment thereof is a chimeric antibody or a humanized antibody.

[0121] The anti-SIRPα antibodies or antigen-binding fragments thereof provided herein specifically bind to human SIRPα, thereby blocking the interaction between CD47 and SIRPα, upregulating the immune response and enhancing phagocytosis of unwanted host cells (such as tumor cells).

[0122] Based on the use of specific complementary determining regions in this disclosure, humanized antibodies designed according to the techniques of the art are encompassed by this disclosure. Humanized antibodies further improve drug safety and effectively reduce the immunogenicity of antibodies. The humanized antibodies obtained in this disclosure with modified framework regions still maintain high affinity, making them suitable for practical clinical applications.

[0123] Bispecific molecules

[0124] The present disclosure relates to bispecific molecules based on the above-mentioned anti-SIRPα antibodies or antigen-binding fragments thereof. The bispecific molecules comprise a SIRPα binding domain and a second domain that binds to a target antigen, wherein the second domain is fused to the SIRPα binding domain.

[0125] The SIRPα binding domain comprises a heavy chain variable domain (VH) and a heavy chain variable domain (VL). The VH of the SIRPα binding domain comprises HCDR1, HCDR2, and HCDR3, the amino acid sequences of which are at least 80%, such as 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequences set forth in SEQ ID NOs: 20, 21, and 23, respectively; or SEQ ID NOs: 20, 22, and 23, respectively; or SEQ ID NOs: 20, 47, and 48, respectively. In some embodiments, the HCDR1, HCDR2, and HCDR3 of the SIRPα binding domain have the amino acid sequences set forth in SEQ ID NOs: 20, 21, and 23, respectively; or SEQ ID NOs: 20, 22, and 23, respectively; or SEQ ID NOs: 20, 47, and 48, respectively.

[0126] The VL of the SIRPα binding domain comprises LCDR1, LCDR2, and LCDR3, the amino acid sequences of which are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequences set forth in SEQ ID NOs: 24, 26, and 28, respectively; or SEQ ID NOs: 25, 27, and 28, respectively. In some embodiments, the LCDR1, LCDR2, and LCDR3 of the SIRPα binding domain have the amino acid sequences set forth in SEQ ID NOs: 24, 26, and 28, respectively; or SEQ ID NOs: 25, 27, and 28, respectively.

[0127] In some embodiments, in the SIRPα binding domain, HCDR1 has the amino acid sequence set forth in SEQ ID NO:20, HCDR2 has the amino acid sequence set forth as X1DPEDX2ETK (SEQ ID NO:60), HCDR3 has the amino acid sequence set forth as DRGLX3Y (SEQ ID NO:61), LCDR1 has the amino acid sequence set forth as X4ASX5SVSSSYLY (SEQ ID NO:45), LCDR2 has the amino acid sequence set forth as YSX6SNX7AS (SEQ ID NO:46), and LCDR3 has the amino acid sequence set forth in SEQ ID NO:28, X1 is V or I, X2 is A or G, X3 is V or A, X4 is R or S, X5 is Q or S, X6 is A or T, and X7 is R or L.

[0128] In some embodiments, in the SIRPα binding domain, (1) HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 20, 21 and 23, respectively; and LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NOs: 24, 26 and 28, respectively; (2) HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 20, 22 and 23, respectively; and LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NOs: 25, 27 and 28, respectively; or (3) HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 20, 47 and 48, respectively; and LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NOs: 25, 27 and 28, respectively.

[0129] In some embodiments, the VH of the SIRPα binding domain comprises FR1, FR2, FR3, and FR4, the amino acid sequences of which are at least 80%, such as about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequences set forth in SEQ ID NOs: 29, 30, 32, and 33, respectively; or SEQ ID NOs: 29, 31, 32, and 33, respectively; or SEQ ID NOs: 49, 50, 51, and 33, respectively. In some embodiments, the VH FR1, FR2, FR3, and FR4 of the SIRPα binding domain have the amino acid sequences set forth in SEQ ID NOs: 29, 30, 32, and 33, respectively; or SEQ ID NOs: 29, 31, 32, and 33, respectively; or SEQ ID NOs: 49, 50, 51, and 33, respectively.

[0130] In some embodiments, the VL of the SIRPα binding domain comprises FR1, FR2, FR3, and FR4, the amino acid sequences of which are at least 80%, such as about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequences set forth in SEQ ID NOs: 34, 35, 36, and 37, respectively; or SEQ ID NOs: 34, 35, 36, and 38, respectively; or SEQ ID NOs: 34, 52, 53, and 37, respectively. In some embodiments, the VL FR1, FR2, FR3, and FR4 of the SIRPα binding domain have the amino acid sequences set forth in SEQ ID NOs: 34, 35, 36, and 37, respectively; or SEQ ID NOs: 34, 35, 36, and 38, respectively; or SEQ ID NOs: 34, 52, 53, and 37, respectively.

[0131] In some embodiments, in the SIRPα binding domain, VH comprises FR1, FR2, FR3 and FR4, the amino acid sequences of which are about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NOs: 29, 30, 32 and 33, respectively; and VL comprises FR1, FR2, FR3 and FR4, the amino acid sequences of which are about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NOs: 34, 35, 36 and 37, respectively.

[0132] In some embodiments, in the SIRPα binding domain, VH comprises FR1, FR2, FR3 and FR4, the amino acid sequences of which are about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NOs: 29, 31, 32 and 33, respectively; and VL comprises FR1, FR2, FR3 and FR4, the amino acid sequences of which are about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NOs: 34, 35, 36 and 38, respectively.

[0133] In some embodiments, in the SIRPα binding domain, VH comprises FR1, FR2, FR3 and FR4, the amino acid sequences of which are about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NOs: 49, 50, 51 and 33, respectively; and VL comprises FR1, FR2, FR3 and FR4, the amino acid sequences of which are about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NOs: 34, 52, 53 and 37, respectively.

[0134] In some embodiments, the VH of the SIRPα binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to one of the amino acid sequences shown in SEQ ID NOs: 14, 16, 18, and 54, respectively. In some embodiments, the VH of the SIRPα binding domain has the amino acid sequence shown in SEQ ID NOs: 14, 16, 18, and 54, respectively.

[0135] In some embodiments, the VL of the SIRPα binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to one of the amino acid sequences set forth in SEQ ID NOs: 15, 17, 19, and 55, respectively. In some embodiments, the VL of the SIRPα binding domain has the amino acid sequence set forth in SEQ ID NOs: 15, 17, and 19, respectively.

[0136] In some embodiments, in the SIRPα binding domain, VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:14; and VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:15.

[0137] In some embodiments, in the SIRPα binding domain, VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:16; and VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:17.

[0138] In some embodiments, in the SIRPα binding domain, VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:18; and VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:19.

[0139] In some embodiments, in the SIRPα binding domain, VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:54; and VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:55.

[0140] In some embodiments, the VH of the SIRPα binding domain comprises or has the amino acid sequence shown in SEQ ID NO: 14, and the VL of the SIRPα binding domain comprises or has the amino acid sequence shown in SEQ ID NO: 15; or the VH of the SIRPα binding domain comprises or has the amino acid sequence shown in SEQ ID NO: 16, and the VL of the SIRPα binding domain comprises or has the amino acid sequence shown in SEQ ID NO: 17; or the VH of the SIRPα binding domain comprises or has the amino acid sequence shown in SEQ ID NO: 18, and the VL of the SIRPα binding domain comprises or has the amino acid sequence shown in SEQ ID NO: 19; or the VH of the SIRPα binding domain comprises or has the amino acid sequence shown in SEQ ID NO: 54, and the VL of the SIRPα binding domain comprises or has the amino acid sequence shown in SEQ ID NO: 55.

[0141] In some embodiments, the SIRPα binding domain is a Fab, Fab', F(ab')2, Fv fragment or a single-chain variable fragment (scFv). Preferably, the SIRPα binding domain is a scFv in which VH and VL are connected via a first polypeptide linker.

[0142] In some embodiments, the first polypeptide linker comprises or is (Gly4Ser)4.

[0143] In some embodiments, the N-terminus of VH is linked to the C-terminus of VL.

[0144] The second domain binds to a target antigen expressed on the surface of cancer cells. The target antigen can be an immune checkpoint molecule, such as CD47, PD1, claudin 18.2, or CTLA-4. In some embodiments, the second domain binds to claudin 18.2 and / or CD47, preferably claudin 18.2.

[0145] In some embodiments, the second domain comprises:

[0146] a) a heavy chain variable region (VH) comprising CDR1, CDR2, and CDR3, wherein the VH CDR1 has an amino acid sequence that is at least 80%, e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 39; the VH CDR2 has an amino acid sequence that is at least 80%, e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 40; and the VH CDR3 has an amino acid sequence that is at least 80%, e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 41; and

[0147] b) a light chain variable region (VL) comprising CDR1, CDR2, and CDR3, wherein the VL CDR1 has an amino acid sequence that is at least 80%, e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:42; the VL CDR2 has an amino acid sequence that is at least 80%, e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:43; and the VL CDR3 has an amino acid sequence that is at least 80%, e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:44.

[0148] In some embodiments, the second domain comprises VH CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 39, 40, and 41, respectively; and VL CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 42, 43, and 44, respectively.

[0149] In some embodiments, the second domain comprises a VH having an amino acid sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 9, and a VL having an amino acid sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the second domain comprises a VH having the amino acid sequence set forth in SEQ ID NO: 9 and a VL having the amino acid sequence set forth in SEQ ID NO: 10.

[0150] In some embodiments, the second domain comprises a light chain constant region (CL), which is a kappa or lambda light chain. The light chain constant region (κ) has the amino acid sequence of SEQ ID NO: 12, and the light chain constant region (λ) has the amino acid sequence of SEQ ID NO: 13.

[0151] In some embodiments, the second domain comprises a heavy chain constant region (CH), preferably a human IgG1 CH. In some embodiments, the human IgG1 CH has the amino acid sequence of SEQ ID NO: 11.

[0152] In some embodiments, the bispecific molecule is a symmetrical bispecific molecule in which the N-terminus of the heavy chain variable domain of the SIRPα binding domain is fused to the C-terminus of the heavy chain constant region of the second domain. Preferably, the SIRPα binding domain and the second domain are covalently linked via a second peptide linker having the formula (Gly4Ser)n, wherein n is an integer from 1 to 5, such as 1, 2, 3, 4, 5. In some embodiments, the SIRPα binding domain and the second domain are covalently linked via (Gly4Ser)3.

[0153] The bispecific molecule comprises a heavy chain and a light chain. In some embodiments, the heavy chain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 56, 58, or 59; and the light chain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 57.

[0154] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:7; and the light chain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:8.

[0155] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:5; and the light chain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:6.

[0156] In some embodiments, the heavy chain has the amino acid sequence shown in SEQ ID NO:56, 58, or 59; and the light chain has the amino acid sequence shown in SEQ ID NO:57.

[0157] In some embodiments, the heavy chain has the amino acid sequence set forth in SEQ ID NO:7; and the light chain has the amino acid sequence set forth in SEQ ID NO:8.

[0158] In some embodiments, the heavy chain has the amino acid sequence set forth in SEQ ID NO:5; and the light chain has the amino acid sequence set forth in SEQ ID NO:6.

[0159] In some embodiments, the bispecific molecules block the interaction of CD47 and SIRPα, remove SHP-1 / 2 inhibition, and / or engage Fc receptors on immune effector cells (e.g., macrophages) to activate phagocytosis. "SHP" refers to a tyrosine phosphatase inhibitor. "Effector cells" are immune cells that can perform immune effector functions, such as macrophages, natural killer cells, monocytes, and cytotoxic T cells.

[0160] In some embodiments, the bispecific molecules enhance phagocytosis of cancer cells expressing a target antigen bound to the second domain (eg, human claudin 18.2 and human SIRPα) by immune effector cells.

[0161] The bispecific molecules of the present application have the advantages of high dual-target binding affinity and specificity, thereby further enhancing anti-tumor immune function.

[0162] The bispecific molecules provided show a higher affinity for SIRPα (e.g., human SIRPα). In some embodiments, the bispecific molecules bind to human SIRPα with a KD of no more than 9.9E-7M, or no more than 9.9E-8M, as determined by the Octet rule. Typically, the KD value or affinity constant is the ratio of the equilibrium dissociation constant or Kd / Ka between an antibody and its target antigen. Kd refers to the dissociation constant, and Ka refers to the association constant.

[0163] In some embodiments, the bispecific molecule binds to SIRPα (e.g., human SIRPα, CHOK1 cells overexpressing human SIRPα) with an EC50 of no more than 3 nM, 2 nM, 1.5 nM, or 1 nM as detected by FACS (fluorescence activated cell sorting).

[0164] Thermal stability is the ability of a protein to maintain its structural and functional integrity under different temperature conditions, and is an inherent property of antibodies that can affect product stability (e.g., aggregation) during manufacturing and storage. Melting temperature (Tm) values ​​can generally predict their thermal stability. The Tm values ​​of the bispecific molecules provided herein range from 40°C to 70°C, preferably from 55°C to 60°C, e.g., 48°C, 50°C, 55°C, or 57°C, indicating that the antibodies have good thermal stability.

[0165] Good stability under low pH conditions is important for antibodies because purification processes typically involve exposure to acidic solution conditions and commonly used low pH virus inactivation. Low pH typically results in soluble, insoluble aggregates and accelerates fragmentation. The bispecific molecules provided herein have good stability under pH 3.0-3.5 conditions.

[0166] The bispecific molecules provided herein exhibit good freeze-thaw stability. Freeze-thaw stability is often used to determine the susceptibility of an antibody to temperature cycling to which the product is frequently exposed. For example, drug substances are often frozen to enable long-term storage. As part of the lyophilization process, drug products may be exposed to freezing temperatures. The primary degradation pathway upon freeze-thaw is aggregate formation, including precipitates, particles, and soluble particles.

[0167] The bispecific molecules provided herein have good stability, with minimal change in purity at 4°C for at least 7, 14, 28, and 28 days, and at 40°C for at least 14 days.

[0168] polynucleotides

[0169] The present disclosure provides isolated polynucleotides encoding the above-mentioned anti-SIRPα antibodies or antigen-binding fragments or bispecific molecules.

[0170] The polynucleotide is a polymer of DNA, RNA, DNA / RNA hybrid or a modified form thereof. In some embodiments, the polynucleotide is a polymer of DNA. The polynucleotide is a polymer of RNA. Using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody), the DNA or RNA encoding the above-mentioned anti-SIRPα antibody or its antigen-binding fragment or bispecific molecule can be easily isolated and sequenced. The encoding DNA or RNA can also be obtained by synthetic methods.

[0171] Using recombinant techniques known in the art, the isolated polynucleotide encoding the above-described anti-SIRPα antibodies, or antigen-binding fragments thereof, or bispecific molecules can be inserted into a construct for further cloning (DNA amplification) or expression.

[0172] Many constructs are available. Construct components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence.

[0173] Construct

[0174] The constructs provided herein comprise the isolated polynucleotides provided above. Methods for constructing the constructs are known to those skilled in the art. For example, the constructs can be obtained by in vitro recombinant DNA techniques, DNA synthesis techniques, or in vivo recombination techniques. More particularly, the constructs can be constructed by inserting the isolated polynucleotides into the multiple cloning site of an expression vector. The expression vectors in this disclosure generally refer to various commercially available expression vectors well known in the art, such as bacterial plasmids, bacteriophages, yeast plasmids, viruses that infect plant cells, viruses that infect mammalian cells such as adenoviruses, retroviruses, or other vectors. The vectors may also comprise one or more regulatory sequences operably linked to the polynucleotide sequence, wherein the regulatory sequences may comprise suitable promoter sequences. The promoter sequence is typically operably linked to a sequence encoding the amino acid sequence to be expressed. The promoter may be any nucleotide sequence that exhibits transcriptional activity in a selected host cell, including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell. The regulatory sequence may also comprise a suitable transcription terminator sequence that is recognized by the host cell to terminate transcription. The terminator sequence is linked to the 3' end or terminus of the nucleotide sequence encoding the polypeptide, and any terminator that is functional in the host cell of choice may be used in the present disclosure.

[0175] Typically, suitable vectors can include a replication origin, a promoter sequence, a convenient restriction enzyme site, and one or more selective markers that can be used in at least one organism. For example, these promoters can include, but are not limited to, the lac or trp promoters of Escherichia coli (E. coli); the lambda phage PL promoter; and eukaryotic promoters (including CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoters, the methanol oxidase promoter of Pichia pastoris), as well as some other known promoters that can control gene expression in prokaryotic or eukaryotic cells or viruses. Marker genes can be used to provide phenotypic characteristics for selecting transformed host cells. For example, marker genes can include, but are not limited to, dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline resistance or ampicillin resistance for E. coli. When the polynucleotide is expressed, the expression vector can further include an enhancer sequence. If the enhancer sequence is inserted into the vector, transcription will be enhanced. Enhancers are cis-acting elements of DNA, typically comprising about 10 to 300 base pairs, that act on promoters to increase gene transcription.

[0176] Antibody expression system

[0177] The present disclosure provides an antibody expression system comprising the construct provided above or incorporating the exogenous polynucleotide provided above into the genome. Any cell suitable for expression of the expression vector can be used as a host cell. For example, the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell, particularly including but not limited to Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; or fungal cells, such as yeast and filamentous fungi; plant cells; insect cells derived from Drosophila S2 or Sf9; animal cells, such as CHO, COS, HEK293 cells or Bowes melanoma cells, or a combination thereof. Methods for constructing an expression system should be known to those skilled in the art, for example, including but not limited to microinjection, gene gun method, electroporation, virus-mediated transformation, electron bombardment, calcium phosphate precipitation or a combination thereof.

[0178] Pharmaceutical composition

[0179] The present disclosure relates to pharmaceutical compositions comprising the aforementioned anti-SIRPα antibody or antigen-binding fragment thereof, or the aforementioned bispecific molecule, and a pharmaceutically acceptable carrier. Preferably, the composition is a pharmaceutical composition comprising the aforementioned anti-SIRPα antibody or antigen-binding fragment thereof, or the aforementioned bispecific molecule, and a pharmaceutically acceptable carrier.

[0180] Typically, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH can be varied depending on the nature of the substance being formulated and the disease condition to be treated. The formulated pharmaceutical compositions can be administered by conventional routes, including but not limited to intratumoral administration, intraperitoneal administration, intravenous administration or topical administration.

[0181] The pharmaceutical compositions disclosed herein contain a safe and effective amount (e.g., 0.001% to 99% by weight, preferably 0.01% to 95% by weight, more preferably 0.1% to 90% by weight) of the provided single-domain antibody or fusion protein and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to) saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should match the mode of administration. The pharmaceutical composition of the present application can be prepared in the form of an injection, for example, the pharmaceutical composition is prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 μg / kg body weight to about 100 mg / kg body weight per day. In addition, the above-mentioned anti-SIRPα antibodies or antigen-binding fragments thereof or the above-mentioned bispecific molecules can also be used with other therapeutic agents.

[0182] Reagent test kit

[0183] The present disclosure provides a kit comprising an anti-SIRPα antibody or antigen-binding fragment thereof, a bispecific molecule, an isolated polynucleotide, a construct and / or a pharmaceutical composition provided herein. If desired, such a kit may further include one or more of a variety of traditional pharmaceutical kit components, such as a container with one or more pharmaceutically acceptable carriers, an additional container, etc., which will be apparent to those skilled in the art. Instructions, administration instructions and / or mixing instructions as inserts or labels indicating the amount of the components to be administered may also be included in the kit.

[0184] use

[0185] The present disclosure provides use of the above-mentioned anti-SIRPα antibody or antigen-binding fragment thereof, bispecific molecule or pharmaceutical composition in the manufacture of a therapeutic agent for preventing, diagnosing or treating a disease, disorder or condition.

[0186] In some embodiments, the disease, disorder or condition is a tumor, and the tumor cells express at least SIRPα, preferably human SIRPα. In some embodiments, the tumor cells express at least claudin 18.2, preferably human claudin 18.2. In certain embodiments, the tumor is a solid tumor. Generally speaking, tumors include benign tumors and malignant tumors (also known as cancer).

[0187] Examples of diseases associated with cells expressing claudin 18.2 that can be diagnosed, treated, and / or prevented in the present disclosure may include all cancers and tumor entities that express claudin 18.2. In some embodiments, the diseases include, but are not limited to, esophageal cancer, liver cancer, lung cancer, melanoma, gastric cancer, pancreatic cancer, ovarian cancer, colon cancer, kidney cancer, bladder cancer, breast cancer, classical Hodgkin lymphoma, hematological malignancies, head and neck cancer, nasopharyngeal cancer, gallbladder cancer and metastases thereof, Kukenberg tumor, peritoneal metastasis, and lymph node metastasis, which may be early, intermediate, or advanced, such as metastatic cancer.

[0188] In another aspect, provided herein are methods of treating a subject having cancer, comprising administering to the subject a therapeutically effective amount of an anti-SIRPα antibody, or antigen-binding fragment thereof, bispecific molecule, or pharmaceutical composition provided herein.

[0189] A “therapeutically effective amount” of the above-mentioned anti-SIRPα antibodies or antigen-binding fragments thereof, bispecific molecules or pharmaceutical compositions provided herein preferably causes a reduction in the severity of disease symptoms and an increase in the frequency and duration of asymptomatic periods of the disease, disorder or condition, or prevents injury or disability caused by the disease or suffering. For example, for the treatment of tumors (including, for example, melanoma, lymphoma, bladder cancer, non-small cell lung cancer, head and neck cancer and colon cancer), a “therapeutically effective amount” preferably inhibits cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, relative to untreated subjects. The ability to inhibit tumor growth can be assessed in an animal model system that predicts efficacy against human tumors, or by detecting the ability to inhibit cell growth. Such inhibitory effects can be determined in vitro by assays well known to those skilled in the art. A therapeutically effective amount of the above-mentioned anti-SIRPα antibodies or antigen-binding fragments thereof, bispecific molecules and pharmaceutical compositions are generally capable of reducing tumor size or otherwise alleviating symptoms in a subject. Those skilled in the art can select a suitable therapeutically effective dose based on actual conditions (e.g., the size of the subject's tumor, the severity of the subject's symptoms, and the specific composition or administration route selected). The treatment prescription (e.g., dosage determination, etc.) can be determined by a doctor, and factors generally considered include, but are not limited to, the disease being treated, the patient's condition, the delivery site, the administration route, and other factors. A prophylactic effective dose refers to an effective amount that achieves the desired prophylactic effect within a certain dose and the required time period. Typically, but not necessarily, a prophylactic dose is administered to a subject before the onset of the disease or in the early stages of the disease, so a "prophylactic effective dose" is generally lower than a "therapeutically effective dose."

[0190] The subjects are mammals, such as humans, mice, and cynomolgus monkeys.

[0191] The present disclosure provides a method for reducing tumor growth rate, comprising contacting tumor cells with an effective amount of the above-described anti-SIRPα antibody or antigen-binding fragment thereof, bispecific molecule, or pharmaceutical composition.

[0192] The present disclosure provides a method for killing tumor cells, comprising contacting the tumor cells with an effective amount of the above-mentioned anti-SIRPα antibody or antigen-binding fragment thereof, bispecific molecule or pharmaceutical composition.

[0193] Example

[0194] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0195] Example 1: Generation of Reagents

[0196] Wild-type antibodies

[0197] Table 1 shows the amino acid sequences of the wild-type antibody anti-claumin 18.2 antibody and anti-SIRPα scFv (Seq. 2534m1v3), which were produced by hybridomas and humanized by in-house development.

[0198] Table 1 Amino acid sequence of wild-type antibody (single letter code)

[0199]

[0200] Stable cell lines

[0201] A CHOK1 / SIRPα cell line stably expressing human SIRPα was generated for in vitro assays with chimeric antibodies. To generate the cell line, CHOK1 cells were transfected with a human SIRPα (Uniprot accession number P78324, AA Met1-Lys504)-expressing lentivirus and selectively cultured in medium containing 10 μg / mL puromycin for 2 weeks. Single cell clones were then isolated by limiting dilution and screened by FACS to obtain a monoclonal cell line stably expressing SIRPα.

[0202] Recombinant proteins

[0203] Human SIRPα extracellular domain (ECD, Uniprot accession number P78324, AAMet1-Arg369) recombinant protein with or without biotinylation was purchased from SinoBiological (Cat. No.: 30014-H08H-B and Cat. No.: 30014-H08H).

[0204] Example 2: Library construction and antibody screening

[0205] 2.1. Construction of random mutation library

[0206] Mutations were randomly introduced into the anti-SIRPα scFv 2534m1V3 template. First, random mutation PCR was performed using the GeneMorph II Random Mutagenesis Kit. The PCR products were then separated on a 1% agarose gel. DNA marker DL2000 was used to indicate the length of the DNA band. The DNA band of approximately 750 bp was excised and purified using the NucleoSpin Gel and PCR Cleanup Kit according to its protocol. In the next step, the mutated DNA was amplified using the same primers using the PrimeSTAR Max DNA Polymerase Kit. The PCR products were separated on a 1% agarose gel and purified using the NucleoSpin Gel and PCR Cleanup Kit according to its protocol. 12 μg of mutant DNA was mixed with 8 μg of linearized yeast display vector and then concentrated into 20 μl of double-distilled water. The concentrated DNA mixture was transferred to yeast BEY100 by electroporation. A mutant library with 2.8E8 diversity was generated and amplified in SDCAA medium.

[0207] 2.2. Enrichment of stable colonies by MACS

[0208] The yeast library was induced overnight at 30°C in SGRCAA medium and then used for the first round of MACS (magnetic activated cell sorting).

[0209] 2.8E9 induced yeast from the initial library were washed twice with 0.1% PBSA (phosphate NaCl buffer) and incubated with biotinylated SIRPα (bio-SIRPα) in 50 mL 0.1% PBSA at RT (room temperature) for 2 h. The mixture was then washed with 0.1% PBSA and incubated with streptavidin-labeled magnetic beads in 50 mL 0.1% PBSA at RT for 1 h. Yeast showing SIRPα-binding substances were captured by magnetic beads, and the yeast-magnetic bead complex was separated from the library by a magnet set. The yeast captured by the beads was amplified in SDCAA medium and induced in SG medium to form a new library, labeled library 1.

[0210] In the second round of MACS, 2.3E8 yeast from library 1 were washed and resuspended in 3M guanidine solution and incubated overnight at 4°C on a rotator at an appropriate speed. The next day, the yeast were washed and incubated with 5nM bio-SIRPα in 0.5M guanidine solution at room temperature for 2 hours. The mixture was then washed with 0.1% PBSA and incubated with streptavidin-labeled magnetic beads at room temperature for 1 hour. Yeast displaying SIRPα-binding substances were captured by magnetic beads, and the yeast-magnetic bead complex was separated from library 1 by a magnet set. The bead-captured yeast were then expanded in SDCAA medium and induced in SG medium to form a new library, labeled library 2.

[0211] 2.3. Enrichment of stable colonies by sorting

[0212] In the first round of sorting, 3E7 yeast from library 2 were washed and resuspended in 3M guanidine and incubated overnight at 4°C on a rotator at an appropriate speed. The next day, the yeast were washed and incubated with 5nM bio-SIRPα in 0.5M guanidine for 2h at RT. The mixture was then washed and resuspended in 0.1% PBSA containing PE-streptavidin (PE-conjugated streptavidin) and FITC-anti-HA antibody (fluorescein isothiocyanate-conjugated hemagglutinin tag antibody) and incubated at RT for 0.5h. Wild-type 2534m1V3 (anti-SIRPαscFv) was displayed on yeast as a control (wild-type control below) and treated the same way. Both groups of yeast were washed and injected into the S3e cell sorter. Based on the antigen binding spectrum of the wild-type control group, as shown Figure 1 Gate 1 in the library was used as a control, and the desired yeast population, gate R2, from library 2 was collected into a FACS tube. The yeast was then expanded in SDCAA medium and induced in SG medium to form a new library, which was labeled library 3.

[0213] In the second round of sorting, 3E7 yeast from library 3 were harvested and washed twice with 0.1% PBSA. The yeast were resuspended in 2 ml PBS (phosphate buffered saline) containing 1 M guanidine and 1 nM bio-SIRPα and incubated on a rotator at an appropriate speed for 2 h at RT. The mixture was then washed and resuspended in 0.1% PBSA containing PE-streptavidin and FITC-anti-HA antibody and incubated at RT for 0.5 h. The wild-type control was treated with the same procedure. Both groups of yeast were injected into the S3e cell sorter. Based on the antigen binding spectrum of the wild-type control group, as shown in Figure 2, the yeast was resuspended in 2 ml PBS (phosphate buffered saline) containing 1 M guanidine and 1 nM bio-SIRPα and incubated on a rotator at an appropriate speed for 2 h at RT. Figure 2As a control, the desired yeast population from library 3, gate 2, was collected into a FACS tube. The yeast was then expanded in SDCAA medium and plated onto SD (Sabouraud Dextrose) agar plates.

[0214] 2.4. Screening of stable colonies

[0215] Colonies on the plate were picked and induced overnight at 30°C in SG medium in a 96-well deep-well plate. Wild-type clones were also induced as controls. The induced yeast were aliquoted into new 96-well low-absorbance plates and washed twice with 0.1% PBSA. The yeast were resuspended in PBS containing 1M guanidine and 1nM bio-SIRPα and incubated at room temperature for 2 hours. The mixture was then washed with 0.1% PBSA and resuspended in 0.1% PBSA containing PE-streptavidin and FITC-anti-HA antibody and incubated at room temperature for 0.5 hours. The yeast were washed and injected into the BD Celesta for signal collection. 380 colonies were picked and screened.

[0216] Comparison between mutant colonies and wild-type controls was based on scFv display levels and SIRPα binding potency. Colonies with improved scFv display levels and binding potency were defined as improved colonies, and all improved colonies were sequenced. Figure 3 As shown, Clone 201 showed improvements in both scFv display levels and antigen binding potency. The sequence of Clone 201 is shown as SEQ ID NO: 4.

[0217] Example 3: Bispecific Antibody (BsAb) Generation and Characterization

[0218] 3.1.BsAb Generation

[0219] like Figure 4A As shown, the bispecific antibody is a symmetrical structure with an anti-SIRPa scFv (Seq. 2534m1v3) fused to the C-terminus of the Fc of the claudin 18.2 antibody in Example 1.1. A wild-type bispecific antibody has been generated using the IgG1 isotype and used as a control.

[0220] The sequence of clone 201 was synthesized and cloned into the C-terminus of the Fc fragment of the claudin 18.2 antibody with a (G4S)3 linker and co-electroporated into a CHO cell line with a light chain and expressed in 30 mL of culture medium to obtain the BsAb antibody AE016_201. As a control, wt (abbreviation of wild type) BsAb 005-08 was also expressed in 30 ml of culture medium. The sequence of AE016_201 is listed in Table 2. The expressed protein was harvested on day 5 and purified by one-step protein-A affinity chromatography. The protein was measured and the productivity is shown in Table 3. Compared with the wt control, the mutant increased the product yield by 7 times. The purity of the product after one-step purification was also analyzed by SEC. As shown in Table 3 and Figure 4B As shown, the purity of AE016_201 in SEC characterization was higher than 90%.

[0221] Table 2 Amino acid sequence of AE016_201 (single letter code)

[0222]

[0223] Table 3 Productivity and one-step purity of mutants in 30 mL expression system

[0224]

[0225] BsAb Characterization

[0226] Binding affinity

[0227] The binding affinities of AE016_201 and wt BsAb to human SIRPα ECD recombinant protein were determined using biolayer interferometry (Octet). The association and dissociation curves were fitted with a 1:1 binding model, and the Kon / Koff / KD values ​​of AE016_201 were calculated and summarized in Table 4. Figure 5 Binding curves are shown in . AE016_201 showed comparable affinity to the wt BsAb.

[0228] Table 4 Affinity of AE016_201 and SIRPα detected by Octet

[0229]

[0230] 3.2.2. Cell line binding

[0231] SIRPα overexpressing cells CHOK1 / SIRPα were harvested by TrypLE digestion and centrifugation. The cells were resuspended in FACS buffer containing DPBS (Dulbecco's phosphate-buffered saline) and 2% FBS (fetal bovine serum) for 30 minutes. 1E5 cells were aliquoted into each well of a 96-well plate containing diluted antibodies. The concentration of the antibody used in the first well was 100nM, while the remaining wells were diluted 5-fold. After incubation at 4°C for 60 minutes, the cells were washed twice with FACS buffer and resuspended with the secondary antibody AF647 anti-human Fc antibody. The cells were then incubated at 4°C in the dark for 30 minutes. Afterwards, the cells were washed twice with FACS buffer, resuspended in FACS buffer, and analyzed on a flow cytometer. The anti-SIRPα antibody 2534m1V3 of the IgG4 isotype was used as a control. As shown Figure 6 Binding curves and EC were calculated using GraphPad Prism 9 software. 50 .

[0232] 3.2.3. Tm value test

[0233] Thermal stability is the ability of a protein to maintain its structural and functional integrity under different temperature environments, and is an inherent property of antibodies that can affect product stability (e.g., aggregation) during manufacturing and storage.

[0234] The melting temperature (Tm) values ​​of AE016_201 in 10mM histidine and 10mM glycine buffer (HG buffer), PBS or 20mM histidine buffer were measured to predict its thermal stability. In brief, the hit antibody AE016_201 was dissolved in HGS or PBS or 20mM histidine buffer. The Tm value was then detected by DSF (differential scanning fluorimetry) using the QuantStudio 7Flex real-time PCR system. The Tm1 values ​​of AE016_201 in different buffers are listed in Table 5, indicating that it has good thermal stability in different buffers.

[0235] Table 5 Tm1 values ​​of AE016_201 in different buffers

[0236]

[0237]

[0238] 3.2.4.4 Long-term stability test at 4°C

[0239] To determine the long-term stability of the variants at 4°C, 200 μl of protein was kept at 4°C and 20 μl of samples from days 7, 14, 21 and 28 were loaded onto the SEC column at 0.5 ml / min. Figure 7The purity of samples at different times is shown in Figure 2. The results show that AE016_201 is stable after long-term storage at 4°C.

[0240] 3.2.5. Freeze-thaw stability test

[0241] To determine the stability of variants after repeated freeze-thaw cycles, 50 μl of protein at a concentration of 1 mg / ml was first frozen and stored at -80°C for 1 hour, then kept at RT for 1 hour. This procedure was repeated 3 times (3xFT), and then 20 μl of sample was loaded at a rate of 0.5 ml / min and analyzed by SEC. Figure 6 The purity of the samples after 3xFT is shown in . The results show that AE016_201 is stable after 3xFT treatment.

[0242] Table 6 Purity changes of AE016_201 before and after 3xFT treatment

[0243]

[0244] △The purity change of the monomer is the sample before and after 3xFT treatment.

[0245] 3.2.6. Low pH stability test

[0246] To determine the stability of the variant after low pH treatment, 1 μL of citric acid was added to 50 μL of sample, under which the pH of the sample solution was 3.63. The sample was kept at RT for 2 hours and then neutralized with 15 μL of Tris 9.0. 17 μL of PBS was then added to 33 μL of sample to make a 2 mg / mL stock solution for SEC testing. 20 μL of the stock solution was loaded onto the SEC column and run at 0.5 ml / min. Table 7 shows the purity before and after treatment with low pH AE016_201.

[0247] Table 7 Purity changes of AE016_201 before and after low pH treatment

[0248]

[0249] △The monomer shows the change in sample purity before and after low pH treatment.

[0250] 3.2.7. Accelerated stability testing

[0251] Thermal stress at temperatures exceeding normal storage conditions accelerates degradation, thereby increasing the detectability of potential degradation pathways to provide information about long-term degradation under expected storage conditions.

[0252] AE016_201 was heat-stressed in HGS at 40°C for 14 days. SEC purity was monitored to detect insoluble or soluble aggregates. As shown in Table 8, after 14 days of incubation at 40°C, the purity of AE016_201 in HG buffer showed minimal change.

[0253] Table 8 Purity changes of AE016_201 at 40°C for 1 week and 2 weeks

[0254]

[0255] Example 4: Reengineering AE016_201

[0256] 4.1. Reengineering AE016_201 and Characterization

[0257] Based on the structural comparison and sequence alignment of the scFv portions of BsAb 005-08 and AE016_201, AE016_201 was reengineered by reverse mutagenesis to generate a new mutant, AE016_201.003. The mutations designed into AE016_201.003 were R486S, Q489S, A514T, R517L, V641I, and A646G in the CDR and / or FR regions of the anti-SPRPα antibody. The sequence of AE016_201.003 is shown in Table 9. The mutant AE016_201.003 was expressed in a CHO cell line in 30 ml of culture medium. The yield was 91 mg / L, and after one-step purification, the purity was 97.3%. Affinity, Tm value, and freeze-thaw stability (FT) were evaluated as described above. The results are summarized in Table 10.

[0258] Table 9 Amino acid sequence of AE016_201.003 (single letter code)

[0259]

[0260]

[0261] Table 10 Characteristics of mutant AE016_201.003

[0262]

[0263] Example 5: Anti-clauin 18.2 Antibodies

[0264] The anti-clauin 18.2 antibody was produced by a hybridoma and humanized through internal development. For expression, CHO cells were transfected with DNA encoding the light and heavy chains in the same or separate expression vectors. The culture medium was harvested and the fusion protein was purified on a protein A agarose column. Table 11 shows the VH and VL of hu26.H1L2.

[0265] Table 11. Amino acid sequences of the variable regions of the anti-clauin 18.2 antibody hu26.H1L2

[0266]

[0267] CDRs are underlined and defined using the Kabat definition.

[0268] Example 6: Anti-SIRPα Antibodies

[0269] Anti-SIRPα antibodies were produced by hybridomas and humanized by internal development. For expression, CHO cells were transfected with DNA encoding the light and heavy chains in the same expression vector or separate expression vectors. The culture medium was harvested and the fusion protein was purified by protein A agarose column. Table 12 shows the VH and VL of hu025.060 (ES0040025), hu025.201, hu025.003 and hu025.003.SS.

[0270] Table 12. Amino acid sequences of the variable regions of anti-SIRPα antibodies or scFv

[0271]

[0272]

[0273] CDRs are underlined and defined using the Kabat definition.

[0274] Example 7: Construction and characterization of anti-clauin 18.2 / SIRPα bispecific antibodies

[0275] 7.1 Construction and Expression of Anti-Claudin 18.2 / SIRPα Bispecific Antibody

[0276] The anti-clauin 18.2 / SIRPα bispecific antibody was constructed as an anti-clauin 18.2 antibody (hu26.H1L2) with an anti-SIRPα scFv (hu025.201, hu025.003, hu025.003.SS) at the C-terminus of the heavy chain. A flexible (Gly4Ser)3 linker was genetically linked to the N-terminus of the anti-SIRPα scFv. Figure 4A A schematic diagram of the anti-clauin 18.2 / SIRPα bispecific antibody is shown in FIG. The amino acid sequence of the resulting bispecific antibody is shown below:

[0277] Table 13 Amino acid sequence of ES028.26.201 (single letter code)

[0278]

[0279]

[0280] Table 14. Amino acid sequence of ES028.26.003 (single letter code)

[0281]

[0282] Table 15. Amino acid sequence of ES028.26.003.SS (single letter code)

[0283]

[0284]

[0285] For expression, CHO-K1 cells were transfected with DNA encoding the light and heavy chains in the same or separate expression vectors. The culture medium was harvested and the fusion protein was purified by protein A agarose column.

[0286] 7.2 Binding Affinity of Anti-Claudin 18.2 / SIRPα Bispecific Antibody

[0287] According to the manufacturer's manual, the binding affinity of the anti-sealing protein 18.2 / SIRPα bispecific protein to human sealing protein 18.2 or SIRPα was characterized using the Octet assay (ForeBio). In brief, the antibody was coupled to the sensor, and the sensor was then immersed in a sealing protein 18.2 or SIRPα protein gradient (starting from 200nM, diluted 2 times, a total of 8 doses). Their binding reactions were measured in real time, and the results were globally fitted. Tables 13 and 14 summarize the affinity data of the test antibodies.

[0288] Table 13. Binding affinity of bispecific antibodies to human SIRPα

[0289]

[0290] Table 14. Binding affinity of bispecific antibodies to human claudin 18.2

[0291]

[0292] 7.3 Binding of Anti-Claudin 18.2 / SIRPα Bispecific Antibody to Claudin 18.2 and SIRPα by FACS Analysis

[0293] About 100,000 Raji lymphoma cells (Raji / human claudin 18.2) overexpressing human claudin 18.2 generated by lentiviral stable transfection were washed with wash buffer and incubated on ice for 30 minutes with 100 μl of serial dilutions of claudin 18.2 / SIRPα bispecific protein. The cells were then washed twice with wash buffer and incubated on ice for 30 minutes with 100 μl of AF647 anti-human Fc antibody. The cells were then washed twice with wash buffer and analyzed on a FACS Canto II analyzer (BD Biosciences). Figure 8A As shown, the anti-claulin 18.2 / SIRPα bispecific antibody bound to Raji / human claudin 18.2 cells in a dose-dependent manner. Similar to the anti-claudin 18.2 monoclonal antibody hu26.H1L2, the bispecific antibodies ES028.26.201, ES028.26.003, and ES028.26.003.SS bound to Raji / human claudin 18.2.

[0294] CHOK1 cells overexpressing human SIRPα (CHOK1 / SIRPα) were washed with wash buffer and incubated on ice for 30 minutes with 100 μl of serial dilutions of claudin 18.2 / SIRPα bispecific protein. The cells were then washed twice with wash buffer and incubated on ice for 30 minutes with 100 μl of AF647 anti-human Fc antibody. The cells were then washed twice with wash buffer and analyzed on a FACS Canto II analyzer (BD Biosciences). Figure 8B As shown, similar to the anti-SIRPα monoclonal antibody hu025.060, the anti-claumin 18.2 / SIRPα bispecific antibodies ES028.26.201, ES028.26.003, and ES028.26.003.SS bound to CHOK1 / SIRPα cells in a dose-dependent manner.

[0295] 7.4 Anti-claulin 18.2 / SIRPα bispecific antibody enhances macrophage response to claudin 18.2 + In vitro phagocytosis of cancer cells

[0296] Mouse MC38 colon tumor cells expressing human CD47 and human claudin 18.2 were labeled with the fluorescent dye CFSE, and in the presence of anti-claudin 18.2 antibody hu26.H1L2, anti-SIRPα antibody hu025.060, anti-claudin 18.2 and anti-SIRPα antibody combination, anti-claudin 18.2 / SIRPα bispecific antibody, and mouse bone marrow-derived macrophages (BMDM) prepared from C57BL6 / hCD47 / hSIRPα knock-in mice were incubated together. After 2 hours, macrophages were harvested, stained with fluorescently labeled anti-mouse macrophage antibodies, and analyzed by flow cytometry. CD11b+CFSE+ double positive events identify macrophages that engulf CFSE-labeled tumor cells. The phagocytic index of three separate samples is shown.

[0297] like Figure 9 As shown, the anti-sealanin 18.2 antibody hu28H1L2 induced approximately 25% phagocytosis by antibody-dependent cellular phagocytosis (ADCP), while the anti-SIRPα antibody induced almost no phagocytosis. The combination of anti-sealanin 18.2 and anti-SIRPα antibodies significantly improved phagocytosis. Compared with combination therapy and single therapy, the anti-sealanin 18.2 / SIRPα bispecific antibodies ES028.26.201, ES028.26.003, and ES028.26.003.SS induced stronger phagocytosis in a dose-dependent manner.

[0298] 7.5 Anti-clauin 18.2 / SIRPα bispecific antibody enhances anti-tumor efficacy in vivo

[0299] Human SIRPα / CD47 double knock-in mice (Shanghai Model Organisms Center, Inc.) were inoculated with hCD47 / hCLDN18.2 overexpressing MC38 cells (stable transfected with lentivirus, Shanghai Model Organisms Center, Inc.). When the average tumor volume reached approximately 60-100 mm 3 Mice were divided into five groups based on tumor volume. Mice were administered ip with equal molar concentrations of hu26.H1L2, ES028.26.201, ES028.26.003, ES028.26.003.SS, or vehicle. The dosing schedule was biweekly for five doses. Tumor volume was measured twice weekly. Three days after the fifth dose, mice were sacrificed and tumors were weighed. Two-way analysis of variance was performed to compare the mean tumor volume between the different treatment groups with the control group.

[0300] The relative tumor inhibition rate TGI (%) was calculated as follows:

[0301] TGI%=(1-T / C)×100%. (T and C are the relative tumor volume (RTV) or tumor weight (TW) of the treatment and control groups, respectively, at a specific time point.)

[0302] T / C%=TRTV / CRTV×100% (TRTV: mean RTV of the treatment group; CRTV: mean RTV of the vehicle control group;

[0303] RTV = Vt / V0, V0 is the tumor volume of the animal at the time of grouping, and Vt is the tumor volume of the animal after treatment);

[0304] T / C can also be calculated based on tumor weight as follows:

[0305] T / C%=TTW / CTW×100% (TTW: average tumor weight of the treatment group at the end of treatment; CTW: average tumor weight of the vehicle control group at the end of treatment).

[0306] like Figure 10 As shown, monotherapy with the anti-claumin-18.2 antibody hu26.H1L2 failed to inhibit tumor growth similar to vehicle treatment. Compared with monotherapy or vehicle treatment, the anti-claumin-18.2 / SIRPα bispecific antibodies ES028.26.201, ES028.26.003, and ES028.26.003.SS induced strong tumor growth inhibition.

[0307] While particular embodiments have been described, alternatives, modifications, variations, improvements, and substantial equivalents may occur that are not or may not presently be foreseen by the applicant or others skilled in the art. Accordingly, the appended claims as filed and as they may be amended are intended to encompass all such alternatives, modifications, variations, improvements, and substantial equivalents.

Claims

1. An anti-SIRPα antibody or an antigen-binding fragment thereof, comprising a heavy chain variable domain (VH) and a heavy chain variable domain (VL), wherein VH comprises HCDR1, HCDR2, and HCDR3, the amino acid sequences of which are about 80% to about 100% identical to the amino acid sequences set forth in: (1) SEQ ID NOs: 20, 21, and 23, respectively; (2) SEQ ID NOs: 20, 22, and 23, respectively; (3) SEQ ID NOs: 20, 47, and 48, respectively; VL comprises LCDR1, LCDR2, and LCDR3, the amino acid sequences of which are about 80% to about 100% identical to the amino acid sequences shown in: (1) SEQ ID NOs: 24, 26, and 28, respectively; and (2) SEQ ID NOs: 25, 27, and 28, respectively.

2. The anti-SIRPα antibody or antigen-binding fragment thereof according to claim 1, wherein the HCDR1 has the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 has the amino acid sequence shown in X1DPEDX2ETK (SEQ ID NO: 60), the HCDR3 has the amino acid sequence shown in DRGLX3Y (SEQ ID NO: 61), the LCDR1 has the amino acid sequence shown in X4ASX5SVSSSYLY (SEQ ID NO: 45), the LCDR2 has the amino acid sequence shown in YSX6SNX7AS (SEQ ID NO: 46), and the LCDR3 has the amino acid sequence shown in SEQ ID NO: 28, X1 is V or I, X2 is A or G, X3 is V or A, X4 is R or S, X5 is Q or S, X6 is A or T, and X7 is R or L.

3. The anti-SIRPα antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein (1) the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 20, 21 and 23, respectively; and the LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NOs: 24, 26 and 28, respectively; (2) the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 20, 22 and 23, respectively; and the LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NOs: 25, 27 and 28, respectively; or (3) The HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 20, 47 and 48, respectively; and the LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NOs: 25, 27 and 28, respectively.

4. The anti-SIRPα antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the VH comprises FR1, FR2, FR3, and FR4, the amino acid sequences of which are about 80% to about 100% identical to the amino acid sequences shown in: (1) SEQ ID NOs: 29, 30, 32, and 33, respectively; (2) SEQ ID NOs: 29, 31, 32, and 33, respectively; (3) SEQ ID NOs: 49, 50, 51, and 33, respectively; and / or The VL comprises FR1, FR2, FR3 and FR4, the amino acid sequences of which are about 80% to about 100% identical to the amino acid sequences shown in the following: (1) SEQ ID NO: 34, 35, 36 and 37, respectively; (2) SEQ ID NO: 34, 35, 36 and 38, respectively; (3) SEQ ID NO: 34, 52, 53 and 37, respectively.

5. The anti-SIRPα antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein (1) the VH comprises FR1, FR2, FR3, and FR4, the amino acid sequences of which are about 80% to about 100% identical to the amino acid sequences set forth in SEQ ID NOs: 29, 30, 32, and 33, respectively; and the VL comprises FR1, FR2, FR3, and FR4, the amino acid sequences of which are about 80% to about 100% identical to the amino acid sequences set forth in SEQ ID NOs: 34, 35, 36, and 37, respectively; (2) the VH comprises FR1, FR2, FR3, and FR4, the amino acid sequences of which are about 80% to about 100% identical to the amino acid sequences set forth in SEQ ID NOs: 29, 31, 32, and 33, respectively; and the VL comprises FR1, FR2, FR3, and FR4, the amino acid sequences of which are about 80% to about 100% identical to the amino acid sequences set forth in SEQ ID NOs: 34, 35, 36, and 38, respectively; or (3) the VH comprises FR1, FR2, FR3 and FR4, the amino acid sequences of which are about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NOs: 49, 50, 51 and 33, respectively; and the VL comprises FR1, FR2, FR3 and FR4, the amino acid sequences of which are about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NOs: 34, 52, 53 and 37, respectively.

6. The anti-SIRPα antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein (1) the VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 14; and the VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 15; (2) the VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 16; and the VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 17; (3) the VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 18; and the VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 19; (4) the VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO:54; and the VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:

55.

7. The anti-SIRPα antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein The VH comprises the amino acid sequence shown in SEQ ID NO: 14, and the VL comprises the amino acid sequence shown in SEQ ID NO: 15; or The VH comprises the amino acid sequence shown in SEQ ID NO: 16, and the VL comprises the amino acid sequence shown in SEQ ID NO: 17; or The VH comprises the amino acid sequence shown in SEQ ID NO: 18, and the VL comprises the amino acid sequence shown in SEQ ID NO: 19; or The VH comprises the amino acid sequence shown in SEQ ID NO:54, and the VL comprises the amino acid sequence shown in SEQ ID NO:

55. 8 . The anti-SIRPα antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 , wherein the anti-SIRPα antibody or antigen-binding fragment binds to human and / or mouse SIRPα.

9. The anti-SIRPα antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the anti-SIRPα antibody or antigen-binding fragment is a Fab, Fab', F(ab')2, Fv fragment or single-chain variable fragment (scFv).

10. The anti-SIRPα antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the anti-SIRPα antibody or antigen-binding fragment is a scFv, wherein the VH and VL are connected via a first peptide linker, optionally comprising (Gly4Ser)4. The anti-SIRPα antibody or antigen-binding fragment thereof according to claim 10 , wherein the N-terminus of the VH is connected to the C-terminus of the VL. 12 . The anti-SIRPα antibody or antigen-binding fragment thereof according to claim 1 , wherein the anti-SIRPα antibody or antigen-binding fragment is a chimeric antibody or a humanized antibody.

13. A bispecific molecule comprising: (1) a SIRPα binding domain comprising a heavy chain variable domain (VH) and a heavy chain variable domain (VL), wherein the VH comprises HCDR1, HCDR2, and HCDR3, the amino acid sequences of which are about 80% to about 100% identical to the amino acid sequences set forth in: (1) SEQ ID NOs: 20, 21, and 23, respectively; (2) SEQ ID NOs: 20, 22, and 23, respectively; (3) SEQ ID NOs: 20, 47, and 48, respectively; and the VL comprises LCDR1, LCDR2, and LCDR3, the amino acid sequences of which are about 80% to about 100% identical to the amino acid sequences set forth in: (1) SEQ ID NOs: 24, 26, and 28, respectively; (2) SEQ ID NOs: 25, 27, and 28, respectively; and (2) a second domain that binds to a target antigen, wherein the second domain is fused to the SIRPα binding domain.

14. The bispecific molecule of claim 13, wherein in the SIRPα binding domain, the HCDR1 has the amino acid sequence set forth in SEQ ID NO: 20, the HCDR2 has the amino acid sequence set forth in X1DPEDX2ETK (SEQ ID NO: 60), the HCDR3 has the amino acid sequence set forth in DRGLX3Y (SEQ ID NO: 61), the LCDR1 has the amino acid sequence set forth in X4ASX5SVSSSYLY (SEQ ID NO: 45), the LCDR2 has the amino acid sequence set forth in YSX6SNX7AS (SEQ ID NO: 46), and the LCDR3 has the amino acid sequence set forth in SEQ ID NO: 28, X1 is V or I, X2 is A or G, X3 is V or A, X4 is R or S, X5 is Q or S, X6 is A or T, and X7 is R or L.

15. The bispecific molecule of claim 13 or 14, wherein in the SIRPα binding domain, (1) the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 20, 21 and 23, respectively; and the LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NOs: 24, 26 and 28, respectively; (2) the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 20, 22 and 23, respectively; and the LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NOs: 25, 27 and 28, respectively; or (3) The HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 20, 47 and 48, respectively; and the LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NOs: 25, 27 and 28, respectively.

16. The bispecific molecule of any one of claims 13-15, wherein the VH of the SIRPα binding domain comprises FR1, FR2, FR3, and FR4, the amino acid sequences of which are about 80% to about 100% identical to the amino acid sequences set forth in: (1) SEQ ID NOs: 29, 30, 32, and 33, respectively; (2) SEQ ID NOs: 29, 31, 32, and 33, respectively; (3) SEQ ID NOs: 49, 50, 51, and 33, respectively; and / or The VL of the SIRPα binding domain comprises FR1, FR2, FR3 and FR4, the amino acid sequences of which are approximately 80% to about 100% identical to the amino acid sequences shown below: (1) SEQ ID NO: 34, 35, 36 and 37, respectively; (2) SEQ ID NO: 34, 35, 36 and 38, respectively; (3) SEQ ID NO: 34, 52, 53 and 37, respectively.

17. The bispecific molecule of any one of claims 13 to 16, wherein In the SIRPα binding domain, (1) the VH comprises FR1, FR2, FR3, and FR4, the amino acid sequences of which are about 80% to about 100% identical to the amino acid sequences set forth in SEQ ID NOs: 29, 30, 32, and 33, respectively; and the VL comprises FR1, FR2, FR3, and FR4, the amino acid sequences of which are about 80% to about 100% identical to the amino acid sequences set forth in SEQ ID NOs: 34, 35, 36, and 37, respectively; (2) the VH comprises FR1, FR2, FR3, and FR4, the amino acid sequences of which are about 80% to about 100% identical to the amino acid sequences set forth in SEQ ID NOs: 29, 31, 32, and 33, respectively; and the VL comprises FR1, FR2, FR3, and FR4, the amino acid sequences of which are about 80% to about 100% identical to the amino acid sequences set forth in SEQ ID NOs: 34, 35, 36, and 38, respectively; or (3) the VH comprises FR1, FR2, FR3 and FR4, the amino acid sequences of which are about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NOs: 49, 50, 51 and 33, respectively; and the VL comprises FR1, FR2, FR3 and FR4, the amino acid sequences of which are about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NOs: 34, 52, 53 and 37, respectively.

18. The bispecific molecule of any one of claims 13-17, wherein the VH of the SIRPα binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to one of the amino acid sequences shown in SEQ ID NOs: 14, 16, 18 and 54; and / or The VL of the SIRPα binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to one of the amino acid sequences shown in SEQ ID NO: 15, 17, 19 and 55.

19. The bispecific molecule of any one of claims 13-18, wherein in the SIRPα binding domain, (1) the VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 14; and the VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 15; (2) the VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 16; and the VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 17; (3) the VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 18; and the VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 19; (4) the VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO:54; and the VL comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:

55.

20. The bispecific molecule of any one of claims 13-19, wherein the VH of the SIRPα binding domain comprises the amino acid sequence set forth in SEQ ID NO: 14, and the VL of the SIRPα binding domain comprises the amino acid sequence set forth in SEQ ID NO: 15; or The VH of the SIRPα binding domain comprises the amino acid sequence shown in SEQ ID NO: 16, and the VL of the SIRPα binding domain comprises the amino acid sequence shown in SEQ ID NO: 17; or The VH of the SIRPα binding domain comprises the amino acid sequence shown in SEQ ID NO: 18, and the VL of the SIRPα binding domain comprises the amino acid sequence shown in SEQ ID NO: 19; or The VH of the SIRPα binding domain comprises the amino acid sequence shown in SEQ ID NO: 54, and the VL of the SIRPα binding domain comprises the amino acid sequence shown in SEQ ID NO:

55.

21. The bispecific molecule of any one of claims 13-20, wherein the SIRPα binding domain is a scFv, wherein the VH and VL are connected via a first polypeptide linker, optionally comprising (Gly4Ser)4.

22. The bispecific molecule of any one of claims 13-21, wherein in the SIRPα binding domain, the N-terminus of the VH is linked to the C-terminus of the VL.

23. The bispecific molecule of any one of claims 13-22, wherein the second domain binds to a target antigen expressed on the surface of a cancer cell.

24. The bispecific molecule of any one of claims 13 to 23, wherein the second domain binds to claudin 18.2 and / or CD47, preferably claudin 18.

2.

25. The bispecific molecule of any one of claims 13-24, wherein the second domain comprises: a) a heavy chain variable region (VH) comprising CDR1, CDR2, and CDR3, wherein the VH CDR1, CDR2, and CDR3 have amino acid sequences about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NOs: 39, 40, and 41, respectively; and b) a light chain variable region (VL) comprising CDR1, CDR2, and CDR3, wherein the VL CDR1, CDR2, and CDR3 have amino acid sequences about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NOs: 42, 43, and 44, respectively.

26. The bispecific molecule of any one of claims 13-25, wherein the second domain comprises a VH having an amino acid sequence at least about 80% identical to the amino acid sequence shown in SEQ ID NO: 9 and a VL having an amino acid sequence at least about 80% identical to the amino acid sequence shown in SEQ ID NO:

10.

27. The bispecific molecule of any one of claims 13-26, wherein the second domain comprises a light chain constant region (CL), wherein the light chain is a kappa or lambda light chain.

28. The bispecific molecule of claim 27, wherein the CL comprises the amino acid sequence shown in SEQ ID NO: 12 or 13.

29. The bispecific molecule of any one of claims 13-28, wherein the second domain comprises a heavy chain constant region (CH) having the amino acid sequence shown in SEQ ID NO:

11.

30. The bispecific molecule of any one of claims 13-29, wherein the bispecific molecule is a symmetric bispecific molecule.

31. The bispecific molecule of any one of claims 27-30, wherein the N-terminus of the heavy chain variable domain of the SIRPα binding domain is fused to the C-terminus of the heavy chain constant region of the second domain.

32. The bispecific molecule of any one of claims 13-31, wherein the SIRPα binding domain and the second domain are covalently linked via a second peptide linker having the formula (Gly4Ser)n, wherein n is an integer from 1 to 5.

33. The bispecific molecule of any one of claims 13-32, wherein the SIRPα binding domain and the second domain are covalently linked via (Gly4Ser)3.

34. The bispecific molecule of any one of claims 13-33, comprising a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 56, 58 or 59; and the light chain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO: 57; or the heavy chain comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:7; and the light chain comprising an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:8; The heavy chain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:5; and the light chain comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

6.

35. The bispecific molecule of any one of claims 13-34, wherein the bispecific molecule blocks the interaction of CD47 and SIRPα, removes SHP-1 / 2 inhibition, and / or engages Fc receptors on immune effector cells (e.g., macrophages) to activate phagocytosis.

36. The bispecific molecule of any one of claims 13-35, wherein the bispecific molecule enhances phagocytosis of cancer cells expressing a target antigen bound to the second domain by immune effector cells.

37. The bispecific molecule of any one of claims 13-36, wherein the bispecific molecule binds to human claudin 18.2 and human SIRPα.

38. An isolated polynucleotide encoding the anti-SIRPα antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 or encoding the bispecific molecule according to any one of claims 13 to 37.

39. A construct comprising the polynucleotide of claim 38.

40. An antibody expression system comprising a construct containing the isolated polynucleotide according to claim 38 or a genome integrated with the exogenous polynucleotide according to claim 38, wherein preferably, the expression system is a cell expression system.

41. A method for producing the anti-SIRPα antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 or the bispecific molecule according to any one of claims 13 to 37, the method comprising: The antibody or protein is expressed using the antibody expression system according to claim 40 under conditions suitable for expressing the antibody.

42. A pharmaceutical composition comprising the anti-SIRPα antibody or antigen-binding fragment thereof according to any one of claims 1-12 or the bispecific molecule according to any one of claims 13-37 and a pharmaceutically acceptable carrier.

43. A kit comprising the anti-SIRPα antibody or antigen-binding fragment thereof according to any one of claims 1-2 or the bispecific molecule according to any one of claims 13-37, the isolated polynucleotide according to claim 38, or the construct according to claim 39.

44. Use of the anti-SIRPα antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, or the bispecific molecule according to any one of claims 13 to 37, or the pharmaceutical composition according to claim 42 in the manufacture of a therapeutic agent for preventing, diagnosing or treating a disease, disorder or condition.

45. The use of claim 44, wherein the disease, disorder or condition is a tumor.

46. ​​The use according to claim 45, wherein the tumor cells express at least claudin 18.2, preferably human claudin 18.

2.

47. The use of claim 45 or 46, wherein the tumor is a solid tumor.

48. The method of any one of claims 44-47, wherein the tumor comprises esophageal cancer, liver cancer, lung cancer, melanoma, gastric cancer, pancreatic cancer, ovarian cancer, colon cancer, kidney cancer, bladder cancer, breast cancer, classical Hodgkin lymphoma, hematological malignancies, head and neck cancer and nasopharyngeal cancer, gallbladder cancer and metastases thereof, Kukenberg tumor, peritoneal metastasis and / or lymph node metastasis.

49. A method of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of the anti-SIRPα antibody or antigen-binding fragment thereof according to any one of claims 1-12, the bispecific molecule according to any one of claims 13-37, or the pharmaceutical composition according to claim 42.

50. The method of claim 49, wherein the subject is a mammal, including humans, mice, and cynomolgus monkeys.

51. A method for reducing tumor growth rate, comprising contacting tumor cells with an effective amount of the anti-SIRPα antibody or antigen-binding fragment thereof according to any one of claims 1-12, or the bispecific molecule according to any one of claims 13-37, or the pharmaceutical composition according to claim 42.

52. A method of killing tumor cells, comprising contacting the tumor cells with an effective amount of the anti-SIRPα antibody or antigen-binding fragment thereof according to any one of claims 1-12, or the bispecific molecule according to any one of claims 13-37, or the pharmaceutical composition according to claim 42.

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