TW202430569A - Bi-specific molecules targeting sirpa and claudin 18.2 - Google Patents

Abstract

The present disclosure relates to an anti-SIRP[alpha] antibodies or antigen binding fragment thereof, a bi-specific molecules targeting SIRP[alpha] and Claudin 18.2, pharmaceutical compositions, and the uses of such molecules in preventing, diagnosing, or treating a cancer disease.

Description

Bispecific molecules targeting SIRPA and claudin 18.2

The present disclosure relates to anti-SIRPα antibodies or antigen-binding fragments thereof and bispecific molecules targeting SIRPα (signal regulatory protein α) and claudin 18.2 (claudin 18 isoform 2) and uses thereof.

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

With the continuous deepening of research in the field of cancer treatment, the research, development and application of molecular targeted therapeutic drugs for cancer have received more and more attention. Antibody drugs have quickly become a hot topic in cancer targeted therapy due to their advantages of strong targeting, few side effects and significant therapeutic effects.

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 around the tumor tissue, thereby continuing to divide and grow. 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 a therapeutic target for human solid tumors. Proceedings of the National Academy of Sciences of the United States of America. 2012, 109(17): 6662-6667).

Signal regulatory protein alpha (SIRPα, SIRPA) is a regulatory membrane glycoprotein of the SIRP family. It is expressed primarily by myeloid cells, but also by stem cells or 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.

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.

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 the treatment of such cancers.

In general, new tumor-targeting molecules need to be developed.

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 drug compositions based on anti-SIRPα single-chain variable fragments, and uses thereof.

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%, 100% identical to the amino acid sequences shown in the following: (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. 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 NOs: 24, 26 and 28, respectively; (2) SEQ ID NOs: 25, 27 and 28, respectively.

In some embodiments, HCDR1 has the amino acid sequence shown in SEQ ID NO: 20, HCDR2 has the amino acid sequence shown in X ₁ DPEDX ₂ ETK (SEQ ID NO: 60), HCDR3 has the amino acid sequence shown in DRGLX ₃ Y (SEQ ID NO: 61), LCDR1 has the amino acid sequence shown in X ₄ ASX ₅ SVSSSYLY (SEQ ID NO: 45), LCDR2 has the amino acid sequence shown in YSX ₆ SNX ₇ AS (SEQ ID NO: 46), and LCDR3 has the amino acid sequence shown in SEQ ID NO: 28, X ₁ is V or I, X ₂ is A or G, X ₃ is V or A, X ₄ is R or S, X ₅ is Q or S, X ₆ is A or T, and X ₇ is R or L.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, the VH of the anti-SIRPα antibody or its antigen-binding fragment 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 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.

In some embodiments, the VL of the anti-SIRPα antibody or its antigen-binding fragment 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 NOs: 34, 35, 36 and 37, respectively; (2) SEQ ID NOs: 34, 35, 36 and 38, respectively; (3) SEQ ID NOs: 34, 52, 53 and 37, respectively.

In some embodiments, the anti-SIRPα antibody or its antigen-binding fragment 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

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

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

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

In another aspect, provided herein are bispecific molecules comprising (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%, such as about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more 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%, such as about 80%, 85%, 90%, 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; and (2) a second domain that binds to a target antigen, the second domain being fused to the SIRPα binding domain.

In some embodiments, in the SIRPα binding domain, HCDR1 has the amino acid sequence shown in SEQ ID NO: 20, HCDR2 has the amino acid sequence shown in X ₁ DPEDX ₂ ETK (SEQ ID NO: 60), HCDR3 has the amino acid sequence shown in DRGLX ₃ Y (SEQ ID NO: 61), LCDR1 has the amino acid sequence shown in X ₄ ASX ₅ SVSSSYLY (SEQ ID NO: 45), LCDR2 has the amino acid sequence shown in YSX ₆ SNX ₇ AS (SEQ ID NO: 46), and LCDR3 has the amino acid sequence shown in SEQ ID NO: 28, X ₁ is V or I, X ₂ is A or G, X ₃ is V or A, X ₄ is R or S, X ₅ is Q or S, X ₆ is A or T, and X ₇ is R or L.

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.

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 the following: (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; 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%, 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 NOs: 34, 35, 36 and 37, respectively; (2) SEQ ID NOs: 34, 35, 36 and 38, respectively; (3) SEQ ID NOs: 34, 52, 53 and 37, respectively.

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.

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 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; (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 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; (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 shown in SEQ ID NO: (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 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.

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

In some embodiments, the second domain binds to a target antigen expressed on the surface of a cancer cell. The target antigen may 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.

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 that is 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 that is about 80% to about 100% identical to the amino acid sequence shown in SEQ ID NOs: 42, 43 and 44, respectively.

In some embodiments, the second domain comprises a VH and a VL, wherein the VH has 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 the VL has 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.

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

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

In some embodiments, the bispecific molecule is a symmetric 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.

In some embodiments, the bispecific molecule comprises a heavy chain and a light chain, 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: 56, 58 or 59; 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: 57.

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.

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.

In some embodiments, the bispecific molecule blocks the interaction of CD47 and SIRPα, removes SHP-1/2 inhibition, and/or engages with Fc receptors on immune effector cells (e.g., macrophages) to activate phagocytosis.

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.

In another aspect, the present disclosure provides isolated polynucleotides encoding the above-mentioned anti-SIRPα antibodies or antigen-binding fragments or bispecific molecules.

In another aspect, the present disclosure provides a construct comprising the above-mentioned polynucleotide.

In another aspect, the present disclosure provides an antibody expression system, which comprises the above-mentioned construct or has a genome integrated with the above-mentioned exogenous polynucleotide. Preferably, the expression system is a cell expression system.

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 expressing the antibody.

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

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

In another aspect, the disclosure provides the use of an anti-SIRPα antibody or antigen-binding fragment thereof, a bispecific molecule or a 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 cancer cells express claudin 18.2, preferably human claudin 18.2.

In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer includes esophageal cancer, liver cancer, lung cancer, melanoma, gastric cancer, pancreatic cancer, ovarian cancer, colon cancer, kidney cancer, bladder cancer, breast cancer, classical Hodgkin's lymphoma, hematological malignancies, head and neck cancer and nasopharyngeal cancer, gallbladder cancer and metastasis, Krukenberg's tumor, peritoneal metastasis and/or lymph node metastasis.

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

In another aspect, the present disclosure provides a method for 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.

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

The disclosure is explained in more detail below. This specification is not intended to be an exhaustive catalog of all the different ways in which the invention may be implemented or all the features that may be added to the 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 one of ordinary skill in the art to which the invention pertains, considering the disclosure without departing from the invention. Therefore, the following description is intended to illustrate some specific embodiments of the invention, rather than to exhaustively specify all permutations, combinations, and variations thereof.

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 this disclosure, preferred materials and methods are described herein. The following terms will be used in describing and claiming protection of this disclosure.

Other features and advantages of the present disclosure will become apparent from the following detailed description and drawings and claims.

Terminology The term "antibody" (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 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, bispecific antibodies, nanoantibodies, 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. Immunoglobulins can be divided into different classes based on the antibody amino acid sequence of their heavy chain constant domains. 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 different classes of immunoglobulins are well known.

A typical antibody molecule contains a heavy chain variable region (VH) and a light chain variable region (VL). The variable region is a region with 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 divided into highly variable regions, also called "complementary determining regions" (CDRs), interspersed with more conserved regions called "framework regions" (FRs). Each VH and VL is usually composed of three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

As used herein, the term "single chain variable fragment" or "scFv" is a fusion protein in which the heavy chain (VH) and light chain (VL) variable regions 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 linked directly or through a linker or spacer encoding a peptide that links the N-terminus of VH to the C-terminus of VL, or the C-terminus of VH to the N-terminus of VL. A person of ordinary skill in the art will be able to select the appropriate configuration for use in the present invention.

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.

"Fc region" (fragment crystallizable region) or "Fc domain" or "Fc fragment" refers to the C-terminal region of the 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. Therefore, the Fc region includes the constant region of the antibody and does not include the first constant region immunoglobulin domain (e.g., CH1 or CL). Fc can also refer to this region alone or in the context of a protein polypeptide comprising Fc.

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 into a single amino acid sequence (e.g., by chemical bonding or recombinant means). The fused amino acid sequence can be produced by genetic recombination of two encoding polynucleotide sequences and can be expressed by introducing a construct containing the recombinant polynucleotide into a host cell.

The term "percentage (%) of sequence identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a 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 the 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.

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 rest of the 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 complementary determining regions (CDRs) transplanted into appropriate framework regions in 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 camel). Other forms have one or more CDRs that have been altered relative to the original antibody.

A "chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, for example, an antibody in which the variable region is derived from a mouse or camel antibody and the constant region is derived from a human antibody.

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

The term "pharmaceutically acceptable carrier" refers to a component of a drug composition other than the active ingredient that is non-toxic to a subject. Pharmaceutically acceptable carriers include but are not limited to buffers, excipients, stabilizers or preservatives.

The term "treatment/prevention" (and its grammatical variants) refers to an attempt to alter the natural progression of the disease in the individual being treated, and may be prevention 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 the disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or reducing the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies disclosed herein may be used to delay the development of a disease or delay the progression of a disorder.

As used herein, the term "about" or "approximately" refers 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, when preceding a numerical value, the term "about" or "approximately" means that value plus or minus a range of 20%, 15%, 10%, or 5%.

In this disclosure, "amino acid" refers to an organic compound containing amine (-NH ₂ ) 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: 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

Generally, the terms and techniques related 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 traditional methods well known in the art and as described in various general and more specific references 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, N.Y. (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).

Anti-SIRPα antibodies or antigen-binding fragments thereof The present disclosure provides examples of novel anti-SIRPα antibodies or antigen-binding fragments thereof, which include a heavy chain variable domain (VH) and a heavy chain variable domain (VL). VH includes complementary determining regions (CDRs) 1, 2, and 3, and VL includes CDR1, CDR2, and CDR3.

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

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

In some embodiments, the VH of the anti-SIRPα antibody or its antigen-binding fragment 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 NOs: 20, 21 and 23, respectively; (2) SEQ ID NOs: 20, 22 and 23, respectively; (3) SEQ ID NOs: 20, 47 and 48, respectively.

In some embodiments, the CDR1, CDR2 and CDR3 of the VH of the anti-SIRPα antibody or its antigen-binding fragment have the amino acid sequences shown below: 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.

In some embodiments, the VL of the anti-SIRPα antibody or its antigen-binding fragment comprises CDR1, CDR2 and CDR3, the amino acid sequences of which are at least 80%, such as 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 NOs: 24, 26 and 28, respectively; (2) SEQ ID NOs: 25, 27 and 28, respectively.

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

In some embodiments, HCDR1 has the amino acid sequence shown in SEQ ID NO: 20, HCDR2 has the amino acid sequence shown in X ₁ DPEDX ₂ ETK (SEQ ID NO: 60), HCDR3 has the amino acid sequence shown in DRGLX ₃ Y (SEQ ID NO: 61), LCDR1 has the amino acid sequence shown in X ₄ ASX ₅ SVSSSYLY (SEQ ID NO: 45), LCDR2 has the amino acid sequence shown in YSX ₆ SNX ₇ AS (SEQ ID NO: 46), and LCDR3 has the amino acid sequence shown in SEQ ID NO: 28, X ₁ is V or I, X ₂ is A or G, X ₃ is V or A, X ₄ is R or S, X ₅ is Q or S, X ₆ is A or T, and X ₇ is R or L.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

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.

In some embodiments, FR1, FR2, FR3 and FR4 of the VH of the anti-SIRPα antibody or its antigen-binding fragment have an amino acid sequence that is approximately 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence shown in the following: 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.

In some embodiments, FR1, FR2, FR3 and FR4 of VH of the anti-SIRPα antibody or its antigen-binding fragment have the amino acid sequences shown below: 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.

In some embodiments, the VL of the anti-SIRPα antibody or antigen-binding fragment thereof comprises FR1, FR2, FR3 and FR4. The FR1 of 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 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 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 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.

In some embodiments, FR1, FR2, FR3 and FR4 of the VL of the anti-SIRPα antibody or its antigen-binding fragment have an amino acid sequence that is approximately 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence shown in the following: 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.

In some embodiments, FR1, FR2, FR3 and FR4 of the VL of the anti-SIRPα antibody or its antigen-binding fragment have the amino acid sequences shown below: 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.

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.

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.

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.

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.

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.

In some embodiments, 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 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.

In some embodiments, 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 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.

In some embodiments, 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 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.

In some embodiments, 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 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.

In some embodiments, the anti-SIRPα antibody or its antigen-binding fragment 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.

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

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

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

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

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 the phagocytosis of unwanted host cells (such as tumor cells).

Based on the use of the specific complementation determining region in the present disclosure, humanized antibodies designed according to the technology in the field are all included in the present disclosure. Humanized antibodies further improve drug safety and effectively reduce the immunogenicity of antibodies. The humanized antibodies with modified framework regions obtained in the present disclosure still maintain high affinity, making them useful for practical clinical applications.

Bispecific molecules 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.

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 shown in the following: 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, HCDR1, HCDR2 and HCDR3 of the SIRPα binding domain have the amino acid sequences shown below: 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.

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, 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.

In some embodiments, in the SIRPα binding domain, HCDR1 has the amino acid sequence shown in SEQ ID NO: 20, HCDR2 has the amino acid sequence shown in X ₁ DPEDX ₂ ETK (SEQ ID NO: 60), HCDR3 has the amino acid sequence shown in DRGLX ₃ Y (SEQ ID NO: 61), LCDR1 has the amino acid sequence shown in X ₄ ASX ₅ SVSSSYLY (SEQ ID NO: 45), LCDR2 has the amino acid sequence shown in YSX ₆ SNX ₇ AS (SEQ ID NO: 46), and LCDR3 has the amino acid sequence shown in SEQ ID NO: 28, X ₁ is V or I, X ₂ is A or G, X ₃ is V or A, X ₄ is R or S, X ₅ is Q or S, X ₆ is A or T, and X ₇ is R or L.

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.

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 shown in the following: 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, VH FR1, FR2, FR3 and FR4 of the SIRPα binding domain have the amino acid sequences shown 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 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 shown in the following: 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, VL FR1, FR2, FR3 and FR4 of the SIRPα binding domain have the amino acid sequences shown 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, 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.

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.

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.

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.

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 shown 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 shown in SEQ ID NOs: 15, 17 and 19, respectively.

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.

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.

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.

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.

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.

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, wherein VH and VL are linked via a first polypeptide linker.

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

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

The second domain binds to a target antigen expressed on the surface of a cancer cell. The target antigen may 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.

In some embodiments, the second domain comprises: a) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3, wherein VH CDR1 has an amino acid sequence that is at least 80%, such as 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 39; VH CDR2 has an amino acid sequence that is at least 80%, such as 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 40; VH CDR3 has an amino acid sequence that is at least 80%, such as 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 41; and b) a light chain variable region (VL) comprising CDR1, CDR2 and CDR3, wherein VL CDR1 has an amino acid sequence that is at least 80%, such as 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 42; VL CDR2 has an amino acid sequence that is at least 80%, such as 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 43; VL CDR3 has an amino acid sequence that is at least 80%, such as 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 44.

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

In some embodiments, the second domain comprises a VH and a VL, the 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 the 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. In some embodiments, the second domain comprises a VH having the amino acid sequence shown in SEQ ID NO: 9 and a VL having the amino acid sequence shown in SEQ ID NO: 10.

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 an amino acid sequence of SEQ ID NO: 12, and the light chain constant region (λ) has an amino acid sequence of SEQ ID NO: 13.

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 an amino acid sequence of SEQ ID NO: 11.

In some embodiments, the bispecific molecule is a symmetric 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.

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 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.

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.

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.

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.

In some embodiments, the heavy chain has the amino acid sequence shown in SEQ ID NO: 7; and the light chain has the amino acid sequence shown in SEQ ID NO: 8.

In some embodiments, the heavy chain has the amino acid sequence shown in SEQ ID NO: 5; and the light chain has the amino acid sequence shown in SEQ ID NO: 6.

In some embodiments, the bispecific molecule blocks the interaction of CD47 and SIRPα, removes SHP-1/2 inhibition, and/or engages with 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, cytotoxic T cells.

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.

The bispecific molecule of this application has the advantages of high dual-target binding affinity and specificity, thereby further enhancing anti-tumor immune function.

The provided bispecific molecules 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-7 M, or no more than 9.9E-8 M, as detected by Octet rules. Typically, the KD value or affinity constant is the equilibrium dissociation constant or Kd/Ka ratio between the antibody and its target antigen. Kd refers to the dissociation constant, and Ka refers to the association constant.

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 (fluorescent flow cytometry).

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 (such as 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, such as 48°C, 50°C, 55°C or 57°C, indicating that the antibodies have good thermal stability.

Good stability under low pH conditions is important for antibodies because the purification process usually involves exposure to acidic solution conditions and common low pH virus inactivation. Low pH usually leads to soluble, insoluble aggregates and accelerates fragmentation. The bispecific molecules provided herein have good stability under pH 3.0-3.5 conditions.

The bispecific molecules provided herein have 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 main degradation pathways of freeze-thaw are aggregates, including precipitates, granules, and soluble particles.

The bispecific molecules provided herein have good stability, and the purity of the bispecific molecules changes little at least 7, 14, 28, and 28 days at 4°C, and at least 14 days at 40°C.

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

A polynucleotide is a polymer of DNA, RNA, a 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. DNA or RNA encoding the above-mentioned anti-SIRPα antibodies or their antigen-binding fragments or bispecific molecules can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibodies). Coding DNA or RNA can also be obtained by synthetic methods.

Using recombinant techniques known in the art, the isolated polynucleotide encoding the above-mentioned anti-SIRPα antibody or antigen-binding fragment thereof or bispecific molecule can be inserted into a construct for further replication (DNA amplification) or expression.

There are many constructs 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 transcriptional termination sequence.

Constructs The constructs provided herein include the isolated polynucleotides provided above. Methods for constructing constructs are known to those of ordinary skill in the art to which this case belongs. For example, the constructs can be obtained by in vitro recombinant DNA technology, DNA synthesis technology, or in vivo recombination technology. More specifically, the constructs can be constructed by inserting the isolated polynucleotides into multiple replication sites 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 bacterioplasms, bacteriophages, yeast plasmids, viruses that infect plant cells, viruses that infect mammalian cells such as adenoviruses, retroviruses, or other vectors. The vector may also include one or more regulatory sequences operably linked to the polynucleotide sequence, wherein the regulatory sequence may include a suitable promoter sequence. The promoter sequence is usually operably linked to a sequence encoding the amino acid sequence to be expressed. The promoter can be any nucleotide sequence that exhibits transcriptional activity in a selected host cell, including mutant, truncated and hybrid promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell. The regulatory sequence may also include a suitable transcriptional terminator sequence that is recognized by the host cell to terminate transcription. The terminator sequence is linked to the 3' end or end of the nucleotide sequence encoding the polypeptide, and any terminator that functions in the selected host cell can be used in the present disclosure.

Generally, suitable vectors may contain a replication origin, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers that are capable of at least one organism. For example, these promoters may include, but are not limited to, the lac or trp promoter of Escherichia coli (E. coli); the λ phage PL promoter; and eukaryotic promoters (including the CMV immediate-early promoter, the HSV thymidine kinase promoter, the 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 cells or eukaryotic cells or viruses. Marker genes can be used to provide phenotypic characteristics for selecting transformed host cells. For example, marker genes may 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 Escherichia coli. When the polynucleotide is expressed, the expression vector may further comprise an enhancer sequence. If the enhancer sequence is inserted into the vector, transcription will be enhanced. The enhancer is a cis-acting factor of DNA, typically comprising about 10 to 300 base pairs. The enhancer acts on the promoter to enhance gene transcription.

Antibody expression system The present disclosure provides an antibody expression system, which comprises the construct provided above or incorporates 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, and Salmonella typhimurium; or a fungal cell, such as yeast and filamentous fungi; a plant cell; an insect cell derived from Drosophila S2 or Sf9; an animal cell, such as CHO, COS, HEK293 cell or Bowes melanoma cell, or a combination thereof. The method for constructing the expression system should be known to those with ordinary knowledge in the technical field to which this case belongs, for example, including but not limited to microinjection, gene gun method, electroporation, virus-mediated transformation, electron bombardment, calcium phosphate precipitation or a combination thereof.

Pharmaceutical composition The present disclosure relates to a pharmaceutical composition, which comprises the above-mentioned anti-SIRPα antibody or its antigen-binding fragment or the above-mentioned bispecific molecule and a pharmaceutically acceptable carrier. Preferably, the composition is a pharmaceutical composition, which contains the above-mentioned anti-SIRPα antibody or its antigen-binding fragment or the above-mentioned bispecific molecule and a pharmaceutically acceptable carrier.

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 may vary depending on the nature of the substance being formulated and the disease condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including, but not limited to, intratumoral administration, intraperitoneal administration, intravenous administration or topical administration.

The drug composition disclosed herein contains a safe and effective amount (e.g., 0.001% by weight to 99% by weight, preferably 0.01% by weight to 95% by weight, and more preferably 0.1% by weight to 90% by weight) of the provided single-domain antibody or fusion protein and a pharmaceutically acceptable carrier or formulation. Such carriers include (but are not limited to) saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The drug formulation should match the mode of administration. The drug composition of this application can be prepared in the form of an injection, for example, the drug composition is prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Drug 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 anti-SIRPα antibody or antigen-binding fragment thereof or the bispecific molecule may also be used together with other therapeutic agents.

Kits The present disclosure provides kits comprising the anti-SIRPα antibodies or antigen-binding fragments thereof, bispecific molecules, isolated polynucleotides, constructs and/or pharmaceutical compositions provided herein. If desired, such kits may further include one or more of a variety of traditional pharmaceutical kit components, such as containers with one or more pharmaceutically acceptable carriers, additional containers, etc., as would be apparent to one of ordinary skill in the art. Instructions such as inserts or labels indicating the amounts of components to be administered, administration instructions, and/or instructions for mixing the components may also be included in the kit.

Uses The present disclosure provides the use of the above-mentioned anti-SIRPα antibodies or antigen-binding fragments thereof, bispecific molecules or drug compositions in the manufacture of therapeutic agents for the prevention, diagnosis or treatment of diseases, disorders or symptoms.

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 called cancer).

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 expressing 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's lymphoma, hematological malignancies, head and neck cancer, nasopharyngeal cancer, gallbladder cancer and its metastasis, Krukenberg's tumor, peritoneal metastasis and lymph node metastasis, which may be early, intermediate or advanced, such as metastatic cancer.

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

A "therapeutically effective amount" of the above-mentioned anti-SIRPα antibody or its antigen-binding fragment, bispecific molecule or drug composition provided in the present disclosure preferably causes a reduction in the severity of disease symptoms and an increase in the frequency and duration of symptom-free periods of the disease, disorder or condition, or prevents damage or disability caused by the disease or affliction. 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" is preferably an inhibition of 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%, and more preferably at least about 80% relative to an untreated subject. 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 inhibition can be measured in vitro by assays known to those of ordinary skill in the art. A therapeutically effective amount of the above-mentioned anti-SIRPα antibodies or their antigen-binding fragments, bispecific molecules and drug compositions can generally reduce tumor size or otherwise alleviate the subject's symptoms. A person with ordinary knowledge in the technical field to which this case belongs can select an appropriate therapeutically effective dose according to the actual situation (for example, the size of the subject's tumor, the severity of the subject's symptoms, and the specific composition or route of administration selected). The treatment prescription (for example, dosage determination, etc.) can be determined by the doctor, and factors generally considered include but are not limited to the disease being treated, the patient's condition, the delivery site, the route of administration and other factors. A preventively effective amount refers to an effective amount that achieves the expected preventive effect within a certain dose and the required time period. Typically, but not necessarily, a "prophylactically effective amount" is lower than a "therapeutically effective amount" because a prophylactic dose is administered to a subject before disease onset or at an early stage of disease.

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

The present disclosure provides a method for reducing tumor growth rate, which comprises contacting tumor cells with an effective amount of the above-mentioned anti-SIRPα antibody or its antigen-binding fragment, bispecific molecule or drug composition.

The present disclosure provides a method for killing tumor cells, which comprises contacting the tumor cells with an effective amount of the above-mentioned anti-SIRPα antibody or its antigen-binding fragment, bispecific molecule or drug composition.

Embodiments The present invention is further described in the following embodiments, which do not limit the scope of the present invention described in the patent application.

Example 1: Generation of reagents 1.1. Wild-type antibodies Table 1 shows the amino acid sequences of wild-type antibodies anti-claudin 18.2 antibody and anti-SIRPα scFv (Seq. 2534m1v3), which were generated by hybridoma and humanized through in-house development.

Table 1 Amino acid sequences of wild-type antibodies (single letter codes) VH in the claudin 18.2 antibody QVQLVQSGAEVKKPGASVKVSCKASGYTFTNWVHWVRQAPGQGLEWMGEINPTNARSNYNEKFKKRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARIYYGNSFAHWGQGTLVTVSS (SEQ ID NO: 1) VL in the claudin 18.2 antibody DIVMTQSPDSLAVSLGERATINCKSSQSLLNAGNQKNYLTWYQQKPGQPPKLLIYWSSTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYHCQNNYYYPLTFGGGTKLEIK (SEQ ID NO: 2) Anti-SIRPα scFv (2534m1V3) EIVLTQSPATLSLSPGERATLSCRASQSVSSSYLYWYQQKPGQAPKLLIYSASNRASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCHQWSSYPYTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGATVKISCKVSGFNIKDYYMHWVQQAPGKGLEWIGRVDPEDAETKYAEKFQGRVTITADTSTDTAY MELSSLRSEDTAVYYCDRGLAYWGQGTLVTVSS (SEQ ID NO: 3)

1.2. Stable cell lines A cell line CHOK1/SIRPα that stably expresses human SIRPα has been generated for chimeric antibody in vitro assays. To generate the cell line, CHOK1 cells were transfected with 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 replicates were then isolated by limiting dilution and screened by FACS to obtain a single cell line that stably expresses SIRPα.

1.3. Recombinant protein Human SIRPα extracellular domain (ECD; Uniprot accession number P78324, AA Met1-Arg369) recombinant protein with or without biotinylation was purchased from SinoBiological (Catalog number: 30014-H08H-B and Catalog number: 30014-H08H).

Example 2: Library construction and antibody screening 2.1. Random mutation library construction Mutations were randomly introduced into the anti-SIRPα scFv 2534m1V3 template. First, random mutation PCR was performed using the GeneMorph II random induction kit. The PCR products were then separated on 1% agarose gel. DNA marker DL2000 was used to indicate the length of the DNA band. The DNA band of about 750 bp was excised and purified by the NucleoSpin gel and PCR cleanup kit according to its protocol. In the next step, the mutant DNA was amplified using the same primers using the PrimeSTAR Max DNA polymerase kit. PCR products were separated on 1% agarose gel and purified by NucleoSpin Gel and PCR Cleanup Kit according to its protocol. 12 ug of mutant DNA was mixed with 8 ug of linearized yeast display vector and then concentrated into 20 ul double distilled water. The concentrated DNA mixture was transferred into yeast BEY100 by electroporation. A mutant pool with 2.8E8 diversity was generated and expanded in SDCAA medium.

2.2. Enrichment of stable colonies by MACS The yeast pool was induced overnight at 30°C in SGRCAA medium and then used for the first round of magnetic-activated cell sorting (MACS).

2.8E9 induced yeast from the initial pool were washed twice with 0.1% phosphate NaCl buffer (PBSA) and incubated with biotinylated SIRPα (bio-SIRPα) in 50 mL 0.1% PBSA at R.T. (room temperature) for 2 hours. The mixture was then washed with 0.1% PBSA and incubated with streptavidin-labeled magnetic beads in 50 mL 0.1% PBSA at R.T. for 1 hour. Yeast displaying SIRPα-binding complexes were captured by magnetic beads, and yeast-magnetic bead complexes were separated from the pool by a magnet set. Bead-captured yeast were expanded in SDCAA medium and induced in SG medium to form a new pool, labeled Pool 1.

In the second round of MACS, 2.3E8 yeast from pool 1 were washed and resuspended in 3 M 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 5 nM bio-SIRPα in 0.5 M guanidine solution for 2 h at R.T. The mixture was then washed with 0.1% PBSA and incubated with streptavidin-labeled magnetic beads for 1 h at R.T. Yeast displaying SIRPα-binding complexes were captured by magnetic beads, and yeast-magnetic bead complexes were separated from pool 1 by a magnet set. The bead-captured yeast were then expanded in SDCAA medium and induced in SG medium to form a new pool, labeled Pool 2.

2.3. Enrichment of stable colonies by sorting In the first round of sorting, 3E7 yeast from pool 2 were washed and resuspended in 3 M guanidine and incubated overnight at 4°C on a rotator at an appropriate speed. The next day, the yeast were washed and incubated with 5 nM bio-SIRPα in 0.5 M guanidine for 2 h at R.T. 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-tagged antibody) and incubated for 0.5 h at R.T. Wild-type 2534m1V3 (anti-SIRPα scFv) was displayed on yeast as a control (wild-type control below) and treated identically. 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 in Gate 1 in Figure 1 as a control, the desired yeast population in library 2, Gate R2, was collected into a FACS tube. The yeast was then expanded in SDCAA medium and induced in SG medium to form a new library, labeled as Library 3.

In the second round of sorting, 3E7 yeast from pool 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 at R.T. on a rotator at the appropriate speed for 2 hours. The mixture was then washed and resuspended in 0.1% PBSA containing PE-streptavidin and FITC-anti-HA antibody and incubated at R.T. for 0.5 hours. Wild-type controls were 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 as Gate 1, the desired yeast population in Library 3, Gate 2, was collected into FACS tubes. The yeast was then expanded in SDCAA medium and plated onto SD (Sabouraud Dextrose) agar plates.

2.4. Screening of stable colonies Colonies on the plate were picked and induced in SG medium in 96-deep-well plates at 30°C overnight. Wild-type replicates 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 1 M guanidine and 1 nM bio-SIRPα and incubated at R.T. for 2 h. 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 R.T. for 0.5 h. The yeast were washed and injected into BD Celesta for signal collection. 380 colonies were picked and screened.

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. As shown in Figure 3, copy 201 was improved in both scFv display levels and antigen binding potency. The sequence of copy 201 is shown as SEQ ID NO: 4.

Example 3: Bispecific Antibody (BsAb) Generation and Characterization 3.1. BsAb Generation As shown in Figure 4A, the bispecific antibody is a symmetric structure with anti-SIRPa scFv (Seq. 2534m1v3) fused to the C-terminus of the Fc of the claudin 18.2 antibody in Example 1.1. Wild-type bispecific antibodies have been generated with IgG1 isotype and used as a control.

The sequence of copy 201 was synthesized and copied to the C-terminus of the Fc fragment of the claudin 18.2 antibody using a (G4S)3 linker, and co-electroporated with the light chain into a CHO cell line 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 the 5th day 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, the purity of AE016_201 in SEC characterization was higher than 90%.

Table 2 Amino acid sequence of AE016_201 (single letter code) AE016_201 Heavy Chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTNWVHWVRQAPGQGLEWMGEINPTNARSNYNEKFKKRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARIYYGNSFAHWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK GGGGSGGGGSGGGGS EIVLTQSPATLSLSPGERATLSCRASQSVSSSYLYWYQQKPGQAPKLLIYSASNRASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCHQWSSYPYTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGATVKISCKVSGFNIKDYYMHWVRQAPGKGLEWIGRVDPEDAETKYAEKFQGRVTITADTSTDTAYME LSSLSEDTAVYYCDRGLVYWGQGTLVTVSS (SEQ ID NO: 5) AE016_201 Light Chain DIVMTQSPDSLAVSLGERATINCKSSQSLLNAGNQKNYLTWYQQKPGQPPKLLIYWSSTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYHCQNNYYYPLTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC (SEQ ID NO: 6)

Table 3 Productivity and one-step purity of mutants in the 30 mL expression system Mutant ID Yield (mg/L) Purity 005-08 20.7 AE016_201 107 100%

3.2. BsAb characterization 3.2.1. Binding affinity The binding affinity of AE016_201 and wt BsAb to human SIRPα ECD recombinant protein was 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. The binding curves are shown in Figure 5. AE016_201 showed an affinity comparable to that of wt BsAb.

Table 4 Affinity of AE016_201 and SIRPα detected by Octet Mutant ID Affinity KD (M) Kon（1/Ms） Koff（1/s） 005-08 1.2E-09 4.8E+05 5.5E-04 AE016_201 1.1E-09 5.2E+05 5.8E-04

3.2.2. Cell Line Binding SIRPα overexpressing cells CHOK1/SIRPα were harvested by TrypLE digestion and centrifugation. Cells were resuspended in FACS buffer containing DPBS (Dulbecco's phosphate buffered saline) and 2% fetal bovine serum (FBS) 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 100 nM, while the remaining wells were diluted 5-fold. After incubation at 4°C for 60 minutes, 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 in Figure 6, the binding curve and _EC50 were calculated by GraphPad Prism 9 software.

3.2.3. Tm value test Thermal stability is the ability of a protein to maintain its structural and functional integrity under different temperature environments. It is also an inherent property of antibodies and can affect product stability (such as aggregation) during manufacturing and storage.

The melting temperature (Tm) values of AE016_201 in 10 mM histidine and 10 mM glycine buffer (HG buffer), PBS or 20 mM histidine buffer were measured to predict its thermal stability. Briefly, the hit antibody AE016_201 was dissolved in HGS or PBS or 20 mM histidine buffer. The Tm value was then detected by differential scanning fluorimetry (DSF) using the QuantStudio 7 Flex 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.

Table 5 Tm1 values of AE016_201 in different buffers Buffer Tm1 (°C) PBS 48.9 HG Buffer 56.5 20 mM histidine buffer 55.6

3.2.4. Long-term stability test at 4°C 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 on days 7, 14, 21, and 28 were loaded onto the SEC column at 0.5 ml/min. The purity of the samples at different times is shown in Figure 7. The results showed that AE016_201 was stable after long-term storage at 4°C.

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

Table 6 Purity changes of AE016_201 before and after 3xFT treatment Mutant ID SEC Purity T0 (%) After 3xFT (%) △Single% 005-08 96.1 95.2 -0.9 AE016_201 100 98.6 -1.4 △The single body is the purity change of the sample before and after 3xFT treatment.

3.2.6. Low pH stability test To determine the stability of the variant after low pH treatment, 1 μL of citric acid was added to 50 μL of sample, and the pH of the sample solution was 3.63 under this condition. The sample was kept at RT for 2 hours and then neutralized with 15 μL Tris 9.0. Then 17 μL PBS was added to 33 μL 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. The purity before and after treatment with low pH AE016_201 is shown in Table 7.

Table 7 Purity changes of AE016_201 before and after low pH treatment Mutant ID SEC Purity T0 (%) After low pH treatment (%) △Single% AE016_201 98.09 97.16 -0.93 △The monomer is the purity change of the sample before and after low pH treatment.

3.2.7. Accelerated stability testing Thermal stress at temperatures above normal storage conditions accelerates degradation, thereby increasing the detectability of potential degradation pathways to provide information on long-term degradation under expected storage conditions.

Thermal stress testing of AE016_201 in HGS was performed at 40°C for 14 days. SEC purity was tested to monitor insoluble or soluble aggregates. As shown in Table 8, the purity of AE016_201 in HG buffer changed little after 14 days of incubation at 40°C.

Table 8 Purity changes of AE016_201 at 40°C for 1 week and 2 weeks Mutant ID Purity (%) T0 40°C - 1 week 40°C - 2 weeks AE016_201 97.5 96.9 95.2

Example 4: Reengineering AE016_201 4.1. Reengineering AE016_201 and characterization Based on the structural comparison and sequence alignment of the scFv part in BsAb 005-08 and AE016_201, AE016_201 was reengineered by reverse mutagenesis to generate a new mutant AE016_201.003. The mutations designed in AE016_201.003 are 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 the purity was 97.3% after one-step purification. Affinity, Tm value and freeze-thaw stability (FT) were evaluated as described above. The results are summarized in Table 10.

Table 9 Amino acid sequence of AE016_201.003 (single letter code) AE016_201.003 Heavy Chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTNWVHWVRQAPGQGLEWMGEINPTNARSNYNEKFKKRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARIYYGNSFAHWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK GGGGSGGGGSGGGGS EIVLTQSPATLSLSPGERATLSCSASSSVSSSYLYWYQQKPGQAPKLLIYSTSNLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCHQWSSYPYTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGATVKISCKVSGFNIKDYYMHWVRQAPGKGLEWIGRIDPEDGETKYAEKFQGRVTITADTSTDTAYMELSSLRS EDTAVYYCDRGLVYWGQGTLVTVSS (SEQ ID NO: 7) AE016_201.003 Light Chain DIVMTQSPDSLAVSLGERATINCKSSQSLLNAGNQKNYLTWYQQKPGQPPKLLIYWSSTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYHCQNNYYYPLTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC (SEQ ID NO: 8)

Table 10 Characteristics of mutant AE016_201.003 Mutant ID Yield (mg/L) Purity (%) Affinity Tm 1 (°C, DSF) 3 Purity after FT stabilization (%) KD (M) Kon (1/Ms) Koff (1/s) AE016_201.003 91 97.3 1.2E-09 3.7E+05 4.5E-04 60.6 96.7

Example 5: Anti-Claudin 18.2 Antibody Anti-Claudin 18.2 antibody was generated from a hybridoma 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 in separate expression vectors. The culture medium was harvested, and the fusion protein was purified by protein A agarose column. Table 11 shows the VH and VL of hu26.H1L2.

Table 11. Amino acid sequences of the variable regions of the anti-claudin 18.2 antibody hu26.H1L2 antibody VH V L hu26.H1L2 EVQLLESGGGLVQPGGSLRLSCAAS GFTLSSYALS WVRQAPGKGLEWVS YISNLGGSTFYPDTVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK HLYNYDAFAS WGQGTLVTVSS (SEQ ID NO: 9) DIQLTQSPSFLSASVGDRVTITC RASSSVNYIH WYQQKPGKAPKALIY ATSNLAS GVPSRFSGSGSGTEYTLTISSLQPEDFATYYC QQWNA NPLTF GQGTKLEIK (SEQ ID NO: 10) CDRs are underlined and defined using the Kabat definition.

Example 6: Anti-SIRPα Antibodies Anti-SIRPα antibodies were generated from hybridomas and humanized by in-house 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.

Table 12. Amino acid sequences of variable regions of anti-SIRPα antibodies or scFv antibody VH V L hu025.060 EVQLVQSGAEVKKPGATVKISCKA SGFNIKDYYMH WVQQAPGKGLEWIGR IDPEDAETK YAPKFQGRVTITADTSTNTAYMELSSLRSEDTAVYYC DRGLAY WGQGTLVTVSS (SEQ ID NO: 54) EIVLTQSPATLSLSPGERATLSC SA SSSVSSSYLY WYQQKPGQAPKLWI YSTSNLAS GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC HQWSSYPYT FGQGTKLEIK (SEQ ID NO: 55) hu025.201 EVQLVQSGAEVKKPGATVKISCKV SGFNIKDYYMH WVRQAPGKGLEWIGR VDPEDAETK YAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYC DRGLVY WGQGTLVTVSS (SEQ ID NO: 14) EIVLTQSPATLSLSPGERATLSC RASQSVSSSYLY WYQQKPGQAPKLLI YSASNRAS GIPARFSGSGSGTDYTLTISSLEPEDFAVYYC HQWSSYPYT FGQGTKLEIK (SEQ ID NO: 15) hu025.003 EVQLVQSGAEVKKPGATVKISCKV SGFNIKDYYMH WVRQAPGKGLEWIGR IDPEDGETK YAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYC DRGLVY WGQGTLVTVSS (SEQ ID NO: 16) EIVLTQSPATLSLSPGERATLSC SA SSSVSSSYLY WYQQKPGQAPKLLI YSTSNLAS GIPARFSGSGSGTDYTLTISSLEPEDFAVYYC HQWSSYPYT FGQGTKLEIK (SEQ ID NO: 17) hu025.003.SS EVQLVQSGAEVKKPGATVKISCKV SGFNIKDYYMH WVRQAPGKCLEWIGR IDPEDGETK YAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYC DRGLVY WGQGTLVTVSS (SEQ ID NO: 18) EIVLTQSPATLSLSPGERATLSC SA SSSVSSSYLY WYQQKPGQAPKLLI YSTSNLAS GIPARFSGSGSGTDYTLTISSLEPEDFAVYYC HQWSSYPYT FGCGTKLEIK (SEQ ID NO: 19) CDRs are underlined and defined using the Kabat definition.

Example 7: Construction and characterization of anti-claudin 18.2/SIRPα bispecific antibody 7.1 Construction and expression of anti-claudin 18.2/SIRPα bispecific antibody The anti-claudin 18.2/SIRPα bispecific antibody was constructed as an anti-claudin 18.2 antibody (hu26.H1L2) with 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. A schematic diagram of the anti-claudin 18.2/SIRPα bispecific antibody is shown in FIG4A. The amino acid sequence of the resulting bispecific antibody is shown below:

Table 13 Amino acid sequence of ES028.26.201 (single letter code) ES028.26.201 Rechain EVQLLESGGGLVQPGGSLRLSCAAS GFTLSSYALS WVRQAPGKGLEWVS YI SNLGGSTFYPDTVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK HLYNYDAFAS WGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK GGGGSGGGGSGGGGS EIVLTQSPATLSLSPGERATLSC RASQSVSSSYLY WYQQKPGQAPKLLI YSAS NRAS GIPARFSGSGSGTDYTLTISSLEPEDFAVYYC FGQGTKLEIK GGGGSGGGGSGGGGSGGGGS EVQLVQSGAEVKKPGATVKISCKV SGFNIKDYYMH WVRQAPGKGLEWIGR VDPEDAETK YAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYC DR GLVY WGQGTLVTVSS (SEQ ID NO: 56) ES028.26.201 Light chain DIQLTQSPSFLSASVGDRVTITC RASSSVNYIH WYQQKPGKAPKALIY ATSNLAS GVPSRFSGSGSGTEYTLTISSLQPEDFATYYC QQWNANPLTF GQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC (SEQ ID NO: 57)

Table 14. Amino acid sequence of ES028.26.003 (single letter code) ES028.26.003 Heavy Chain EVQLLESGGGLVQPGGSLRLSCAAS GFTLSSYALS WVRQAPGKGLEWVS Y ISNLGGSTFYPDTVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK HLYNYDAFAS WGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK GGGGSGGGGSGGGGS EIVLTQSPATLSLSPGERATLSC SASSSVSSSYLY WYQQKPGQAPKLLI YSTSNLAS GIPARFSGSGSGTDYTLTISSLEPEDFAVYYC FGQGTKLEIK GGGGSGGGGSGGGGSGGGGS EVQLVQSGAEVKKPGATVKISCKV SGFNIKDYYMH WVRQAPGKGLEWIGR IDPEDGETK YAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYC DRGLVY WGQGTLVTVSS (SEQ ID NO: 58 ) ES028.26.003 Light chain DIQLTQSPSFLSASVGDRVTITC RASSSVNYIH WYQQKPGKAPKALIY ATSNLAS GVPSRFSGSGSGTEYTLTISSLQPEDFATYYC QQWNANPLTF GQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC (SEQ ID NO: 57)

Table 15. Amino acid sequence of ES028.26.003.SS (single letter code) ES028.26.003.SS Relink EVQLLESGGGLVQPGGSLRLSCAAS GFTLSSYALS WVRQAPGKGLEWVS YISNLGGSTFYPDTVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK HLYNYDAFAS WGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK GGGGSGGGGSGGGGS EIVLTQSPATLSLSPGERATLSC SASSSVSSSYLY WYQQKPGQAPKLLI YST SNLAS GIPARFSGSGSGTDYTLTISSLEPEDFAVYYC HQWSSYPYT FGCGTKLEIK GGGGSGGGGSGGGGSGGGGS EVQLVQSGAEVKKPGATVKISCKV SGFNIKDYYMH WVRQAPGKCLEWIGR IDPEDGETK YAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYC DRGLVY WGQGTLVTVSS （SEQ ID NO: 59） ES028.26.003.SS Light Chain DIQLTQSPSFLSASVGDRVTITC RASSSVNYIH WYQQKPGKAPKALIY ATS NLAS GVPSRFSGSGSGTEYTLTISSLQPEDFATYYC QQWNANPLTF GQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC (SEQ ID NO: 57)

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

7.2 Anti-Claudin 18.2/SIRPα Bispecific Antibody Binding Affinity The binding affinity of anti-Claudin 18.2/SIRPα bispecific protein to human Claudin 18.2 or SIRPα, respectively, was characterized using the Octet assay (ForeBio) according to the manufacturer's manual. Briefly, the antibodies were complexed on the sensor, and then the sensor was immersed in a gradient of Claudin 18.2 or SIRPα protein (starting from 200 nM, diluted 2-fold, 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 tested antibodies.

Table 13. Binding affinity of bispecific antibodies to human SIRPα Sample KD(M) Kon（1/Ms） Koff（1/s） Anti-SIRPα mAb hu025.003 2.07E-09 5.32E+05 1.10E-03 ES028.26.201 8.4E-09 3.9E+05 3.3E-03 ES028.26.003 1.9E-09 3.1E+05 6.0E-04 ES028.26.003.SS 2.52E-09 2.09E+05 5.27E-04

Table 14. Binding affinity of bispecific antibodies to human claudin 18.2 Sample KD (M) Kon（1/Ms） Koff（1/s） Anti-claudin 18.2 mAb, hu26.H1L2 7.25E-10 2.42E+05 1.75E-04 ES028.26.003.SS 8.33E-10 1.70E+05 1.42E-04

7.3 Binding of anti-claudin 18.2/SIRPα bispecific antibody to claudin 18.2 and SIRPα by FACS analysis Approximately 100,000 Raji lymphoma cells overexpressing human claudin 18.2 (Raji/human claudin 18.2) generated by lentiviral stable transfection were washed with wash buffer and incubated with 100 μl of serial dilutions of claudin 18.2/SIRPα bispecific protein on ice for 30 min. The cells were then washed twice with wash buffer and incubated with 100 μl of AF647 anti-human Fc antibody on ice for 30 min. The cells were then washed twice with wash buffer and analyzed on a FACS Canto II analyzer (BD Biosciences). As shown in Figure 8A, the anti-claudin 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.

CHOK1 cells expressing human SIRPα (CHOK1/SIRPα) were washed with wash buffer and incubated with 100 μl of serial dilutions of claudin 18.2/SIRPα bispecific protein on ice for 30 min. Cells were then washed twice with wash buffer and incubated with 100 μl of AF647 anti-human Fc antibody on ice for 30 min. Cells were then washed twice with wash buffer and analyzed on a FACS Canto II analyzer (BD Biosciences). As shown in FIG8B , similar to the anti-SIRPα monoclonal antibody hu025.060, the anti-claudin 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.

7.4 Anti-claudin 18.2/SIRPα bispecific antibodies enhance macrophage phagocytosis of claudin ^18.2+ cancer cells in vitro

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

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

7.5 Anti-claudin 18.2/SIRPα bispecific antibodies enhance in vivo antitumor efficacy 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 by Shanghai Model Organisms Center, Inc.). When the average tumor volume reached about 60-100 ^mm3 , the mice were divided into 5 groups according to the tumor volume. The mice were ip-administered with hu26.H1L2, ES028.26.201, ES028.26.003, ES028.26.003.SS or vector at the same molar concentration. The dosing regimen was BIW for a total of 5 doses. Tumor volumes were measured twice a week. Mice were sacrificed 3 days after the fifth administration, and tumors were weighed. Two-way ANOVA was used to compare the mean tumor volumes of different treatment groups with those of the control group.

The relative tumor inhibition rate TGI (%) is calculated as follows: TGI% = (1-T/C) × 100%. (T and C are the relative tumor volume (RTV) or tumor weight (TW) of the treatment group and the control group at a specific time point, respectively).

T/C % = TRTV / CRTV × 100% (TRTV: average RTV of the treatment group; CRTV: average RTV of the vehicle control group; RTV = Vt/V0, V0 is the tumor volume of the animal at the time of grouping, Vt is the tumor volume of the animal after treatment); T/C can also be calculated based on tumor weight as follows: T/C % = TTW / CTW × 100% (TTW: average tumor weight of the treatment group at the end; CTW: average tumor weight of the vehicle control group at the end).

As shown in Figure 10, monotherapy with the anti-claudin 18.2 antibody hu26.H1L2 failed to inhibit tumor growth, similar to vector therapy. Compared with monotherapy or vector therapy, the anti-claudin 18.2/SIRPα bispecific antibodies ES028.26.201, ES028.26.003, and ES028.26.003.SS induced strong tumor growth inhibition.

Although particular embodiments have been described, alternatives, modifications, variations, improvements, and substantial equivalents may occur that were not or may not currently be foreseen by the applicant or others of ordinary skill in the art to which the present invention pertains. Accordingly, the appended claims as filed and as they may be amended are intended to cover all such alternatives, modifications, variations, improvements, and substantial equivalents.

without

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.

Figure 1 shows the antigen binding spectra of the wild-type control group and library 2 in the first round of yeast library sorting detected by fluorescence-activated cell sorter (FACS). Figure 2 shows the antigen binding spectra of the wild-type control group and library 2 in the first round of yeast library sorting detected by fluorescence flow cytometry (FACS). Figure 3 shows the comparison of binding potency between the wild-type control group and replicate 201 detected by fluorescence flow cytometry (FACS). Figure 4A is a schematic diagram of the 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. Figure 4B shows the size-exclusion chromatograph (SEC) spectrum of AE016_201, and the purity of each component in the protein sample is listed in the table below the SEC spectrum. Figure 5 shows the binding curve of AE016_201 to SIRPα detected by Octet. Figure 6 shows the CHO-K1 SIRPα binding curve of AE016_201 and anti-SIRPα antibody of IgG4 isotype detected by FACS. EC50 values are listed in the table below the binding curve. Figure 7 shows the long-term stability of AE016_201 at 4°C. Figure 8A shows the binding curves of representative bispecific antibodies to Raji/hClaudin 18.2 cells detected by FACS. Anti-Claudin 18.2 antibody hu26.H1L2 was used as a control. Figure 8B 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. Figure 9 shows that anti-Claudin 18.2/SIRPα bispecific antibodies stimulated BMDM phagocytosis of MC38/hCD47/hClaudin 18.2 cells better than single or combined treatments. Figure 10 shows the in vivo anti-tumor efficacy of anti-claudin 18.2/SIRPα in the MC38/human claudin 18.2/hSIRPα syngeneic model.

TW202430569A_113102013_SEQL.xml

Claims (52)

An anti-SIRPα antibody or an antigen-binding fragment thereof comprises 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 shown in the following: (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 the following: (1) SEQ ID NOs: 24, 26 and 28, respectively; (2) SEQ ID NOs: 25, 27 and 28, respectively. The anti-SIRPα antibody or antigen-binding fragment thereof as described in 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 X ₁ DPEDX ₂ ETK (SEQ ID NO: 60), the HCDR3 has the amino acid sequence shown in DRGLX ₃ Y (SEQ ID NO: 61), the LCDR1 has the amino acid sequence shown in X ₄ ASX ₅ SVSSSYLY (SEQ ID NO: 45), the LCDR2 has the amino acid sequence shown in YSX ₆ SNX ₇ AS (SEQ ID NO: 46), and the LCDR3 has the amino acid sequence shown in SEQ ID NO: 28, X ₁ is V or I, X ₂ is A or G, X ₃ is V or A, X ₄ is R or S, X ₅ is Q or S, X ₆ is A or T, and X ₇ is R or L. An anti-SIRPα antibody or antigen-binding fragment thereof as described in 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: The amino acid sequences shown in 25, 27 and 28. An anti-SIRPα antibody or antigen-binding fragment thereof as described in 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 the following: (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 NOs: 34, 35, 36 and 37, respectively; (2) SEQ ID NOs: 34, 35, 36 and 38, respectively; (3) SEQ ID NOs: 34, 52, 53 and 37. An anti-SIRPα antibody or antigen-binding fragment thereof as described in 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 shown 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 shown 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 shown 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 shown in SEQ ID NOs: 34, 35, 36 and 38; 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. An anti-SIRPα antibody or antigen-binding fragment thereof as described in 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: 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. An anti-SIRPα antibody or antigen-binding fragment thereof as described in any one of claims 1-6, 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. An anti-SIRPα antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 7, wherein the anti-SIRPα antibody or antigen-binding fragment binds to human and/or mouse SIRPα. The anti-SIRPα antibody or antigen-binding fragment thereof as described in any one of claims 1 to 8, wherein the anti-SIRPα antibody or antigen-binding fragment is Fab, Fab', F(ab') ₂ , Fv fragment or single-chain variable fragment (scFv). An anti-SIRPα antibody or antigen-binding fragment thereof as described in any one of claims 1-9, wherein the anti-SIRPα antibody or antigen-binding fragment is a scFv, wherein the VH and VL are linked via a first peptide linker, optionally wherein the first peptide linker comprises (Gly4Ser)4. An anti-SIRPα antibody or an antigen-binding fragment thereof as described in claim 10, wherein the N-terminus of the VH is connected to the C-terminus of the VL. The anti-SIRPα antibody or antigen-binding fragment thereof as described in any one of claims 1 to 11, wherein the anti-SIRPα antibody or antigen-binding fragment is a chimeric antibody or a humanized antibody. A bispecific molecule comprising: (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% identical to the amino acid sequences shown in the following: (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 the following: (1) SEQ ID NOs: 24, 26 and 28, respectively; (2) SEQ ID NOs: 25, 27 and 28; and (2) a second domain that binds to a target antigen, wherein the second domain is fused to the SIRPα binding domain. The bispecific molecule of claim 13, wherein in the SIRPα binding domain, the HCDR1 has the amino acid sequence shown in SEQ ID NO: 20, the HCDR2 has the amino acid sequence shown in X ₁ DPEDX ₂ ETK (SEQ ID NO: 60), the HCDR3 has the amino acid sequence shown in DRGLX ₃ Y (SEQ ID NO: 61), the LCDR1 has the amino acid sequence shown in X ₄ ASX ₅ SVSSSYLY (SEQ ID NO: 45), the LCDR2 has the amino acid sequence shown in YSX ₆ SNX ₇ AS (SEQ ID NO: 46), and the LCDR3 has the amino acid sequence shown in SEQ ID NO: 28, X ₁ is V or I, X ₂ is A or G, X ₃ is V or A, X ₄ is R or S, X ₅ is Q or S, X ₆ is A or T, and X ₇ is R or L. A bispecific molecule as described in 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: The amino acid sequences shown in 25, 27 and 28. A bispecific molecule as described in any 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 shown in the following: (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 about 80% to about 100% identical to the amino acid sequences shown in the following: (1) SEQ ID NOs: 34, 35, 36 and 37, respectively; (2) SEQ ID NOs: 34, 35, 36 and 38; (3) SEQ ID NOs: 34, 52, 53 and 37, respectively. A bispecific molecule as described in any one of claim 13-16, 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 shown 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 shown 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 shown in SEQ ID NOs: 29, 31, 32 and 33; 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, 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. A bispecific molecule as described in any one of claim 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 NOs: 15, 17, 19 and 55. A bispecific molecule as described in any 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. A bispecific molecule as described in any of claim items 13-19, wherein the VH of the SIRPα binding domain comprises the amino acid sequence shown in SEQ ID NO: 14, and the VL of the SIRPα binding domain comprises the amino acid sequence shown 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 SEQ ID NO: The amino acid sequence shown in 55. The bispecific molecule of any one of claims 13-20, wherein the SIRPα binding domain is a scFv, wherein the VH and VL are linked via a first polypeptide linker, optionally comprising (Gly4Ser)4. The bispecific molecule of any one of claims 13-21, wherein in the SIRPα binding domain, the N-terminus of the VH is connected to the C-terminus of the VL. 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. The bispecific molecule of any one of claims 13-23, wherein the second domain binds to claudin 18.2 and/or CD47, preferably claudin 18.2. A bispecific molecule as described in 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 that are 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 that are about 80% to about 100% identical to the amino acid sequences shown in SEQ ID NOs: 42, 43 and 44, respectively. The bispecific molecule of any one of claims 13-25, wherein the second domain comprises VH and VL, wherein the VH has an amino acid sequence that is at least about 80% identical to the amino acid sequence shown in SEQ ID NO: 9, and the VL has an amino acid sequence that is at least about 80% identical to the amino acid sequence shown in SEQ ID NO: 10. 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. The bispecific molecule of claim 27, wherein the CL comprises the amino acid sequence shown in SEQ ID NO: 12 or 13. 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. The bispecific molecule of any one of claims 13 to 29, wherein the bispecific molecule is a symmetric bispecific molecule. A bispecific molecule as described in any 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. 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. 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. A bispecific molecule as described in 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 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; 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. The bispecific molecule of any one of claims 13-34, wherein the bispecific molecule blocks the interaction between CD47 and SIRPα, removes SHP-1/2 inhibition, and/or binds to Fc receptors on immune effector cells (e.g., macrophages) to activate phagocytosis. 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. The bispecific molecule of any one of claims 13-36, wherein the bispecific molecule binds to human claudin 18.2 and human SIRPα. An isolated polynucleotide encoding an anti-SIRPα antibody or an antigen-binding fragment thereof as described in any one of claims 1-12 or encoding a bispecific molecule as described in any one of claims 13-37. A construct comprising the polynucleotide of claim 38. An antibody expression system comprising a construct containing the isolated polynucleotide as described in claim 38 or a genome integrated with the exogenous polynucleotide as described in claim 38, wherein preferably, the expression system is a cell expression system. A method for producing an anti-SIRPα antibody or an antigen-binding fragment thereof as described in any one of claims 1-12 or a bispecific molecule as described in any one of claims 13-37, the method comprising: expressing the antibody or protein using the antibody expression system as described in claim 40 under conditions suitable for expressing the antibody. A pharmaceutical composition comprising an anti-SIRPα antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 12 or a bispecific molecule as described in any one of claims 13 to 37 and a pharmaceutically acceptable carrier. A kit comprising the anti-SIRPα antibody or antigen-binding fragment thereof as described in any one of claims 1-2 or the bispecific molecule as described in any one of claims 13-37, the isolated polynucleotide as described in claim 38, or the construct as described in claim 39. Use of an anti-SIRPα antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 12, or a bispecific molecule as described in any one of claims 13 to 37, or a pharmaceutical composition as described in claim 42 in the manufacture of a therapeutic agent for preventing, diagnosing or treating a disease, disorder or condition. The use of claim 44, wherein the disease, disorder or condition is a tumor. The use as described in claim 45, wherein the tumor cells at least express claudin 18.2, preferably human claudin 18.2. The use of claim 45 or 46, wherein the tumor is a solid tumor. The use as described in any of claims 44-47, wherein the tumor includes esophageal cancer, liver cancer, lung cancer, melanoma, gastric cancer, pancreatic cancer, ovarian cancer, colon cancer, kidney cancer, bladder cancer, breast cancer, classical Hodgkin's lymphoma, hematological malignancies, head and neck cancer and nasopharyngeal cancer, gallbladder cancer and its metastasis, Krukenberg's tumor, peritoneal metastasis and/or lymph node metastasis. A method for treating a subject suffering from cancer, the method comprising administering to the subject a therapeutically effective amount of an anti-SIRPα antibody or an antigen-binding fragment thereof as described in any one of claims 1-12, or a bispecific molecule as described in any one of claims 13-37, or a pharmaceutical composition as described in claim 42. A method as described in claim 49, wherein the subject is a mammal, including humans, mice and cynomolgus monkeys. A method for reducing tumor growth rate, the method comprising contacting tumor cells with an effective amount of an anti-SIRPα antibody or antigen-binding fragment thereof as described in any one of claims 1-12, or a bispecific molecule as described in any one of claims 13-37, or a pharmaceutical composition as described in claim 42. A method for killing tumor cells, the method comprising contacting the tumor cells with an effective amount of an anti-SIRPα antibody or an antigen-binding fragment thereof as described in any one of claims 1-12, or a bispecific molecule as described in any one of claims 13-37, or a pharmaceutical composition as described in claim 42.

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