Serum albumin binding protein and use thereof

Abstract

Disclosed in the present invention are a serum albumin binding protein and a fusion protein containing the serum albumin binding protein. The present invention further provides uses of the serum albumin binding protein and the fusion protein.

Description

Serum albumin binding protein and its application

[0001] This application claims priority to the Chinese invention patent application with application date of March 28, 2024, application number CN202410370122.3, and invention name “Serum albumin binding protein and its application”. Technical Field

[0002] The present application relates to the field of biomedicine, and in particular to a binding protein for serum albumin, a fusion protein comprising the binding protein, and applications thereof. Background Art

[0003] Human serum albumin (HSA) is a soluble carrier protein with a molecular weight of approximately 67 kilodaltons. It is synthesized by the liver in the human body and is the most abundant protein in plasma, accounting for approximately 50%-60% of the total plasma protein content. The content in the human body is 40-50 grams per liter of blood. It has high stability, solubility and a long circulation half-life, and can bind to a variety of endogenous and exogenous substances.

[0004] Human serum albumin has two drug-binding sites, of which Site II is more flexible and has a higher affinity for drug molecules. Antibodies that bind to serum albumin have been developed to increase the circulation half-life of therapeutic proteins. Despite significant progress, limitations such as limited selectivity and weak affinity persist, necessitating a continued need for novel and useful anti-serum albumin antibodies and multispecific binding proteins with improved pharmacokinetic properties. Summary of the Invention

[0005] The present invention provides novel antibodies that bind to serum albumin and have cross-binding activity to serum albumin from humans and cynomolgus monkeys. Based on this novel antibody that binds to serum albumin, the present application also provides multispecific antibodies that comprise a first domain that binds to a first target protein expressed on target cells, such as B7H4, and / or a second domain that binds to a second target protein expressed on immune effector cells, such as CD3, and a third domain that binds to serum albumin. The third domain is derived from the novel antibody of the present invention. The first, second, and third domains of the multispecific antibody are linked in a specific manner to obtain a favorable therapeutic effect and in vivo half-life. The multispecific binding protein can be used to treat diseases and conditions associated with abnormal cells expressing the first target protein, such as breast cancer, ovarian cancer, endometrial cancer, non-small cell lung cancer, and renal cancer that overexpress B7H4.

[0006] In one aspect, the present invention provides a serum albumin binding protein comprising a heavy chain variable region (VH), wherein:

[0007] (1) the VH comprises CDR1, CDR2, and CDR3 of the VH shown in SEQ ID NO: 60;

[0008] (2) the VH comprises CDR1, CDR2, and CDR3 of the VH shown in SEQ ID NO: 61;

[0009] (3) the VH comprises CDR1, CDR2, and CDR3 of the VH shown in SEQ ID NO: 59; or

[0010] (4) The VH comprises CDR1, CDR2 and CDR3 of the VH shown in SEQ ID NO: 58.

[0011] In some embodiments, CDR1, CDR2, and CDR3 are defined according to the following systems: Kabat, AbM, Chothia, or IMGT. In a preferred embodiment, CDR1, CDR2, and CDR3 are defined according to the Chothia system.

[0012] In some embodiments, the present invention provides a serum albumin binding protein comprising a heavy chain variable region (VH), wherein:

[0013] (1) the VH comprises CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 11, 24, and 37, respectively;

[0014] (2) the VH comprises CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 12, 25, and 37, respectively;

[0015] (3) the VH comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 10, 23 and 36, respectively; or

[0016] (4) The VH comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 9, 23 and 35, respectively.

[0017] In some embodiments, the present invention provides a serum albumin binding protein comprising a heavy chain variable region (VH), wherein:

[0018] (1) the VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 60;

[0019] (2) the VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 61;

[0020] (3) the VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 59; or

[0021] (4) The VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:58.

[0022] In some embodiments, the binding protein does not comprise a light chain.In preferred embodiments, the binding protein is a single domain antibody or a heavy chain antibody.

[0023] The constant region of the heavy chain antibody of the present application can be derived from IgA, IgD, IgE, IgG or IgM isotype. In some embodiments, the constant region of the heavy chain antibody of the present application can be derived from IgG1, IgG2, IgG3 or IgG4 subtype.

[0024] In a more preferred embodiment, the binding protein is a single domain antibody.

[0025] In some embodiments, the binding protein is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

[0026] In another aspect, the present invention provides a fusion protein comprising:

[0027] (i) a serum albumin binding region comprising the above-mentioned binding protein; and

[0028] (ii) a second functional domain selected from the group consisting of an antibody, a ligand, a complement, a cytokine, an enzyme, and a hormone.

[0029] In some embodiments, the second functional region is a monoclonal antibody or a bispecific antibody that binds to one or more antigens selected from tumor-associated antigens and immune cell antigens.

[0030] In some embodiments, the second functional region is a bispecific antibody targeting a tumor-associated antigen and CD3.

[0031] In some embodiments, the tumor-associated antigen is an immune checkpoint molecule. In a preferred embodiment, the tumor-associated antigen is B7H4.

[0032] In some embodiments, the bispecific antibody comprises a B7H4 binding region and a CD3 binding region, wherein the B7H4 binding region comprises a VH and a VL, the VH comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 6, 20, and 32, respectively, and the VL comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 43, 47, and 51, respectively.

[0033] In some embodiments, the B7H4 binding region comprises a VH and a VL, the VH comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:55, and the VL comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:62.

[0034] In some embodiments, the bispecific antibody comprises a B7H4 binding region and a CD3 binding region, wherein the CD3 binding region comprises a VH and a VL, the VH comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 8, 22, and 34, respectively, and the VL comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 44, 48, and 52, respectively.

[0035] In some embodiments, the CD3 binding region comprises a VH and a VL, the VH comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:57, and the VL comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:63.

[0036] In some embodiments, the second functional region does not include an immunoglobulin Fc region.

[0037] In some embodiments, the fusion protein comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, and the serum albumin binding region is located at the C-terminus of at least one of the polypeptide chains.

[0038] In some embodiments, the first polypeptide chain comprises VL_A-CL-VH_B-CH1-VH_C from N-terminus to C-terminus; the second polypeptide chain comprises VH_A-CH1 from N-terminus to C-terminus; and the third polypeptide chain comprises VL_B-CL from N-terminus to C-terminus. VH_A represents the VH of the B7H4-binding region, VL_A represents the VL of the B7H4-binding region, VH_B represents the VH of the CD3-binding region, VL_B represents the VL of the CD3-binding region, and VH_C represents the VH of the serum albumin-binding region. "-" represents that the domains on both sides are directly connected or connected via a linker.

[0039] In some embodiments, the fusion protein comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein:

[0040] (a) the first polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:77; the second polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:73; and the third polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:72;

[0041] (b) the first polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:78; the second polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:73; and the third polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:72;

[0042] (c) the first polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:76; the second polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:73; and the third polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:72; or

[0043] (d) the first polypeptide chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:75; the second polypeptide chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:73; and the third polypeptide chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:72.

[0044] In another aspect, the present invention provides a nucleic acid comprising a nucleotide sequence encoding the serum albumin binding protein, the multispecific antibody or the fusion protein.

[0045] In another aspect, the present invention provides a vector comprising the above-mentioned nucleic acid.

[0046] In another aspect, the present invention provides a host cell comprising the above nucleic acid or the above vector.

[0047] In another aspect, the present invention provides a pharmaceutical composition comprising: the serum albumin binding protein, the multispecific antibody or the fusion protein; and a pharmaceutically acceptable carrier or excipient.

[0048] In some embodiments, the composition further comprises a second therapeutic agent selected from an antibody, a chemotherapeutic agent, a small molecule drug, an siRNA, and an antisense oligonucleotide.

[0049] In another aspect, the present invention provides a method for extending the serum half-life of a drug, comprising linking the drug to the serum albumin binding protein of the present invention.

[0050] In some embodiments, the drug is a polypeptide. In a specific embodiment, the drug is an antibody.

[0051] In some embodiments, the antibody does not have an immunoglobulin Fc region.

[0052] In some embodiments, the antibodies target one or more antigens selected from tumor-associated antigens and immune cell antigens.

[0053] In some embodiments, the antibody is a bispecific antibody that targets a tumor-associated antigen and CD3.

[0054] In some embodiments, the tumor-associated antigen is an immune checkpoint molecule, such as B7H4.

[0055] In another aspect, the present invention provides use of the multispecific antibody described above in the preparation of a medicament for treating cancer in a subject.

[0056] In another aspect, the present invention provides a method of treating cancer in a subject, comprising administering to the subject an effective amount of the multispecific antibody described above.

[0057] In another aspect, the present invention provides the above-mentioned multispecific antibody for use in treating cancer in a subject.

[0058] In some embodiments, the cancer is selected from breast cancer, ovarian cancer, endometrial cancer, non-small cell lung cancer, and renal cancer. In a preferred embodiment, the cancer is breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] FIG1 is a schematic diagram of the structure of the multispecific antibody BiTE-HLE.

[0060] FIG2 is a schematic diagram of the structure of the multispecific antibody constructed in the example.

[0061] FIG3 is a schematic diagram of the structure of the multispecific antibody constructed in the example.

[0062] FIG4 shows the results of the binding of anti-serum albumin single domain antibody (sdAb) to serum albumin.

[0063] FIG5 shows the results of the binding of anti-serum albumin multispecific antibody (BiTE-HLE) to serum albumin.

[0064] FIG6 shows the results of binding of anti-serum albumin multispecific antibody (BiTE-HLE) to CHO-K1 / B7H4 cells.

[0065] FIG7 shows the results of binding of anti-serum albumin multispecific antibody (BiTE-HLE) to Pan-T cells.

[0066] FIG8 shows the results of a cytotoxicity experiment dependent on a multispecific anti-serum albumin antibody (BiTE-HLE).

[0067] FIG9 shows the results of extending the serum half-life of a multispecific anti-serum albumin antibody (BiTE-HLE). DETAILED DESCRIPTION

[0068] The above features and advantages of the present invention and additional features and advantages will be more clearly understood from the following detailed description of embodiments taken in conjunction with the accompanying drawings.

[0069] The embodiments described herein with reference to the accompanying drawings are illustrative, exemplary, and are used for a general understanding of the present invention. The embodiments should not be construed as limiting the scope of the present invention. Identical or similar elements and elements with identical or similar functions are represented by the same reference numerals throughout the specification.

[0070] Unless otherwise mentioned or defined, all terms used have the ordinary meaning in the art that is clear to those skilled in the art. Reference is made, for example, to standard manuals, such as Leuenberger, HGW, Nagel, B. and Klbl, H. eds., "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Helvetica Chimica Acta (1995), CH-4010 Basel, Switzerland; Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et al., eds., "Current protocols in molecular biology", Green Publishing and Wiley InterScience, New York (1987); Roitt et al., "Immunology (6th Ed.), Mosby / Elsevier, Edinburgh (2001); and Janeway et al., "Immunobiology" (6th Ed.), Garland Science Publishing / Churchill Livingstone, New York (2005), and the general background art cited above.

[0071] definition

[0072] Unless otherwise stated or defined, the term “comprise” and variations such as “include” and “comprising” will be understood to imply the inclusion of stated elements or steps or groups of elements or steps but not the exclusion of any other elements or steps or groups of elements or steps.

[0073] As used herein, the term "immunoglobulin" has the ability to specifically bind to a specific antigen. Such molecules typically contain two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Unless otherwise specified, typically, each heavy chain of an immunoglobulin consists of a heavy chain variable region (or domain) (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (or domain) (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The variable regions of the heavy and light chains of immunoglobulins contain binding domains that interact with antigens. The constant region of an immunoglobulin can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system, such as C1q (the first component in the classical pathway of complement activation).

[0074] The heavy chain of an immunoglobulin can be divided into three functional regions: the Fd region, the hinge region, and the Fc region (crystallizable fragment). The Fd region comprises the VH and CH1 domains and combines with the light chain to form the Fab (antigen binding fragment). The Fc fragment is responsible for immunoglobulin effector functions, including, for example, complement fixation and binding to the cognate Fc receptors of effector cells. The hinge region found in IgG, IgA, and IgD immunoglobulin classes acts as a flexible spacer, allowing the Fab portion to move freely in space relative to the Fc region. The hinge domain is structurally diverse, with different sequences and lengths between immunoglobulin classes and subclasses.

[0075] According to crystallographic studies, the immunoglobulin hinge region can be further divided into three regions in terms of structure and function: the upper hinge, the core hinge, and the lower hinge (Shin et al., Immunological Reviews 130:87, 1992). The upper hinge includes the amino acid residues from the carboxyl terminus of CH1 to the first residue in the hinge that restricts movement, usually the first cysteine ​​residue that forms an interchain disulfide bond between the two heavy chains. The length of the upper hinge region is related to the flexibility of the immunoglobulin fragment. The core hinge region contains inter-heavy chain disulfide bonds. The lower hinge region connects the amino terminus of the CH2 domain and includes residues in the CH2 domain. The conformational changes allowed by the structure and flexibility of the immunoglobulin hinge region polypeptide sequence can affect the effector function of the immunoglobulin Fc portion.

[0076] A "light chain variable region" (VL) or "heavy chain variable region" (VH) consists of a "framework" region separated by three "complementarity determining regions" or "CDRs." The framework regions are used to align the CDRs that specifically bind to an antigenic epitope. The CDRs include the amino acid residues in the immunoglobulin that are primarily responsible for antigen binding. Both the VL and VH domains contain the following framework regions (FRs) and CDR regions from amino-terminus to carboxyl-terminus: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, and FWR4. The CDR1, CDR2, and CDR3 of the VL domain are also referred to herein as LCDR1, LCDR2, and LCDR3, respectively; the CDR1, CDR2, and CDR3 of the VH domain are also referred to herein as HCDR1, HCDR2, and HCDR3, respectively.

[0077] The amino acid arrangement of each VL domain and VH domain is consistent with any conventional definition of CDR. Conventional definitions include the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991)), the Chothia definition (Chothia and Lesk, J. Mol. Biol. 196: 901-917, 1987; Chothia et al., Nature 342: 878-883, 1989); a composite of the Chothia Kabat CDR, wherein CDR-H1 is a composite of the Chothia CDR and the Kabat CDR; the AbM definition used by Oxford Molecular's immunoglobulin modeling software; and the CONTACT definition of Martin et al. (world wide web bioinfo.org.uk / abs). Kabat provides a widely used numbering convention (Kabat numbering system), in which corresponding residues between different heavy chains or between different light chains are given the same number. The present disclosure may use CDRs defined according to any of these numbering systems, but preferred embodiments use the Chothia defined CDRs.

[0078] Based on the amino acid sequence of the heavy chain constant region, immunoglobulin molecules can be divided into five classes (isotypes): IgA, IgD, IgE, IgG, and IgM, and can be further divided into different subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc. Based on the amino acid sequence of the light chain, the light chain of immunoglobulins can be divided into lambda (λ) chain and kappa (κ) chain.

[0079] As used herein, the term "antibody" should be understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, and multispecific antibodies (e.g., bispecific antibodies) containing at least two antigen-binding regions. Antibodies may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations in glycosylation sites. Antibodies also include post-translationally modified antibodies, fusion proteins containing antigenic determinants of antibodies, and immunoglobulin molecules containing any other modifications to antigen recognition sites, as long as these antibodies exhibit the desired biological activity.

[0080] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, except for a small number of naturally occurring mutations, each antibody comprising the population is identical. Monoclonal antibodies are highly specific and are directed against a single antigen. The term "monoclonal antibody" herein is not limited to antibodies produced by hybridoma technology, nor should it be construed as requiring antibodies produced by any particular method.

[0081] The term "bispecific antibody" should be understood in the context of the present invention as an antibody having two different antigen-binding regions defined by different antibody sequences. This can be understood as binding to different targets, but also includes binding to different epitopes of a target. The term "bispecific antibody" as used herein should be understood in its broadest sense, including full-length bispecific antibodies and antigen-binding fragments thereof. Bispecific antibodies may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations in glycosylation sites. Bispecific antibodies also include post-translationally modified antibodies, fusion proteins containing the antigenic determinants of antibodies, and immunoglobulin molecules containing any other modifications to the antigen recognition site, as long as these antibodies exhibit the desired biological activity.

[0082] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0083] Examples of antigen-binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) a Fab' fragment, which is essentially a Fab with a partial hinge region; (iv) a Fd fragment, which consists of the VH and CH1 domains; (v) a Fd' fragment, which has the VH and CH1 domains and one or more cysteine ​​residues at the C-terminus of the CH1 domain; (vi) an Fv fragment, which consists of the VL and VH domains of a single arm of an antibody; (vii) a dAb fragment, which consists of the VH domain; (viii) isolated complementarity determining regions (CDRs); and (ix) nanobodies, heavy chain variable regions containing a single variable domain and two constant domains. In addition, although the two domains VL and VH of the Fv fragment are encoded by different genes, they can be connected by synthetic linkers using recombinant methods so that they can be made into a single protein chain, in which the VL and VH regions are paired to form a monovalent molecule (called single-chain Fv (scFv)). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. In addition, the term also includes a "straight-chain antibody" comprising a pair of tandem Fd fragments (VH-CH1-VH-CH1), which together with a complementary light chain polypeptide form an antigen-binding region, as well as modified forms of any of the aforementioned fragments that retain antigen-binding activity.

[0084] These antigen-binding fragments can be obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0085] The term "therapeutic polypeptide" refers to a polypeptide for treating a disease or condition. In some cases, therapeutic polypeptides can be used to treat diseases in which the subject lacks or is deficient in polypeptides (e.g., insulin). In some cases, therapeutic polypeptides can be used to treat diseases by inhibiting or initiating biological processes. For example, therapeutic antibodies (e.g., monoclonal antibodies) can treat diseases by inhibiting cell growth rate (e.g., tumor cells) or triggering an immune response. Examples of potential therapeutic polypeptides include, but are not limited to, any therapeutic polypeptide with known crystallization conditions. Exemplary categories of therapeutic polypeptides include antibodies, fusion proteins, anticoagulants, blood factors, bone morphogenesis production proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytic agents, etc.

[0086] As used herein, the term "binding" or "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its target antigen. The binding specificity of an antibody can be determined based on affinity and / or avidity. Avidity represents the equilibrium constant (KD) for the dissociation of an antigen from an antibody and is a measure of the binding strength between an antigenic determinant and the antigen binding site of an antibody: the smaller the value of KD, the stronger the binding strength between the antigenic determinant and the antibody. Alternatively, affinity can also be expressed as an affinity constant (KA), which is 1 / KD.

[0087] Avidity is a measure of the strength of binding between an antibody and its associated antigen. Avidity is related to the affinity between an antigenic determinant and the antigen-binding site of an antibody and the number of associated binding sites present on the antibody. Typically, an antibody binding to an antigen will bind to the antigen with the following dissociation constant (KD): 10 -5 M to 10 -12 M or less, and preferably 10 -7 M to 10 - 12 M or less, and more preferably 10 -8 M to 10 -12 M, and / or have the following binding affinity: at least 10 7 M -1 , preferably at least 10 8 M -1 , more preferably at least 10 9 M -1 , such as at least 10 12 M -1 It is generally believed that any value greater than 10 -4 The KD value of M represents nonspecific binding. Specific binding of an antibody to an antigen or antigenic determinant can be determined in any known suitable manner, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competition assays, and different variations thereof known in the art.

[0088] The term "epitope" refers to the site on an antigen to which an antibody binds. An epitope can be formed by continuous amino acids or by non-continuous amino acids juxtaposed by the tertiary folding of one or more proteins. Epitopes formed by continuous amino acids (also referred to as linear epitopes) are typically retained in exposure to denaturing solvents, while epitopes formed by tertiary folding (also referred to as conformational epitopes) are typically lost in the treatment of denaturing solvents. An epitope typically includes at least 3, more typically at least 5 or 8-10 amino acids in a unique spatial conformation. An epitope defines the minimum binding site of an antibody and is therefore the specific target of an antibody or its antigen-binding fragment.

[0089] As used herein, the term "sequence identity" refers to the degree to which two sequences (amino acids) have identical residues at the same position after alignment. For example, "an amino acid sequence is X% identical to SEQ ID NO: Y" means that the amino acid sequence is X% identical to SEQ ID NO: Y and is stated as X% of the residues in the amino acid sequence being identical to the residues in the sequence disclosed in SEQ ID NO: Y. Typically, such calculations are performed using computer programs. Exemplary programs for comparing and aligning sequence pairs include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990), and gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984).

[0090] In addition, when determining the degree of sequence identity between two amino acid sequences, one of skill in the art may consider so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of a similar chemical structure, which has little or essentially no effect on the function, activity, or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art.

[0091] Such conservative substitutions are preferably substitutions in which one amino acid from the following groups (a) to (e) is replaced by another amino acid residue from the same group: (a) small aliphatic, nonpolar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0092] Particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met to Leu, to Tyr or to Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to Ile or to Leu.

[0093] As used herein, the term "tumor-associated antigen (TAA)" refers to an antigen that is differentially expressed in cancer cells compared to normal cells and, therefore, can be used to target cancer cells.

[0094] As used herein, the term "CD3" refers to the human CD3 protein complex, which has five peptide chains: γ, δ, ε, ζ, and η, and associates with the T cell receptor α and β chains to form the TCR-CD3 complex. The term includes any CD3 variants, subtypes, and species homologs, which may be naturally expressed by cells, including T cells, or expressed by cells transfected with genes or cDNAs encoding the aforementioned chains.

[0095] As used herein, the term "bispecific T cell engager" or "BiTE" refers to a polypeptide chain molecule having two antigen-binding domains, one of which binds to a T cell antigen and the second binds to an antigen presented on the surface of a target cell (see PCT Publication WO 05 / 061547; Baeuerle et al., 2008, Drugs of the Future 33:137-147; Bargou et al., 2008, Science 321:974-977, which are incorporated herein by reference in their entireties). Thus, the BiTEs of the present disclosure have one antigen-binding region that binds to CLL1 and a second antigen-binding region that is directed to a T cell antigen.

[0096] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.

[0097] As used herein, the term "host cell" refers to a cell into which an expression vector has been introduced.

[0098] The term "pharmaceutically acceptable" means that the carrier or excipient is compatible with the other ingredients of the composition and not substantially deleterious to the recipient thereof, and / or such carrier or excipient is approved or available for inclusion in pharmaceutical compositions for parenteral administration to humans.

[0099] As used herein, the terms "treat," "therapy," "treatment," and the like refer to the administration of an agent or the performance of a procedure for the purpose of obtaining an effect. These effects may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of achieving a partial or complete cure of a disease and / or disease symptoms. As used herein, "treatment" may include treating a disease or condition (e.g., cancer) in a mammal, particularly a human, and includes: (a) preventing the occurrence of a disease or disease symptom in a subject who may be susceptible to the disease (e.g., including a disease that may be associated with or caused by the primary disease) but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., arresting its development; and (c) alleviating the disease, i.e., causing regression of the disease. Treatment may refer to any indicator of success in the treatment, improvement, or prevention of cancer, including any objective or subjective parameter, such as a reduction in symptoms; relief; elimination of disease symptoms or making the disease condition more tolerable for the patient; slowing the rate of deterioration or decline; or reducing the final stage of deterioration. Treatment or improvement of symptoms is based on one or more objective or subjective parameters; including the results of a doctor's examination. Thus, the term "treatment" includes the administration of an antibody, composition, or conjugate disclosed herein to prevent or delay, alleviate, or arrest or inhibit the development of symptoms or conditions associated with a disease (e.g., cancer). The term "therapeutic effect" refers to the reduction, elimination, or prevention of a disease, disease symptom, or disease side effect in a subject.

[0100] As used herein, the term "effective amount" refers to the amount administered to a subject for treating a disease that is sufficient to effect treatment for the disease.

[0101] As used herein, the term "subject" refers to any mammalian subject for whom diagnosis, treatment, or therapy is desired. "Mammal" for therapeutic purposes refers to any animal classified as a mammal, including humans, livestock, and laboratory, zoo, sports, or pet animals such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, and the like.

[0102] serum albumin binding protein

[0103] The present invention provides binding proteins to serum albumin.

[0104] In some embodiments, the present invention provides a serum albumin binding protein comprising a heavy chain variable region (VH), wherein:

[0105] (1) the VH comprises CDR1, CDR2, and CDR3 of the VH shown in SEQ ID NO: 60;

[0106] (2) the VH comprises CDR1, CDR2, and CDR3 of the VH shown in SEQ ID NO: 61;

[0107] (3) the VH comprises CDR1, CDR2, and CDR3 of the VH shown in SEQ ID NO: 59; or

[0108] (4) The VH comprises CDR1, CDR2 and CDR3 of the VH shown in SEQ ID NO: 58.

[0109] In some embodiments, CDR1, CDR2 and CDR3 are defined according to the following systems: Kabat, AbM, Chothia or IMGT.In a preferred embodiment of the invention, the CDRs of the binding protein are defined according to the Chothia system.

[0110] In some embodiments, the serum albumin binding proteins disclosed herein specifically bind to domain II of human serum albumin.

[0111] In some embodiments, the present invention provides a serum albumin binding protein comprising a heavy chain variable region (VH), wherein:

[0112] (1) the VH comprises CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 11, 24, and 37, respectively;

[0113] (2) the VH comprises CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 12, 25, and 37, respectively;

[0114] (3) the VH comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 10, 23 and 36, respectively; or

[0115] (4) The VH comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 9, 23 and 35, respectively.

[0116] In some embodiments, the present invention provides a serum albumin binding protein comprising a heavy chain variable region (VH), wherein:

[0117] (1) the VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 60;

[0118] (2) the VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 61;

[0119] (3) the VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 59; or

[0120] (4) The VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:58.

[0121] In a preferred embodiment, the present invention provides a serum albumin binding protein comprising a heavy chain variable region (VH), wherein:

[0122] (1) the amino acid sequence of the VH is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 60;

[0123] (2) the amino acid sequence of the VH is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 61;

[0124] (3) the amino acid sequence of the VH is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 59; or

[0125] (4) The amino acid sequence of the VH is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:58.

[0126] In some embodiments, the binding protein does not include a light chain. In a preferred embodiment, the binding protein is a single domain antibody or a heavy chain antibody. In a more preferred embodiment, the binding protein is a single domain antibody.

[0127] In some embodiments, the binding protein is a bispecific antibody or a multispecific antibody.

[0128] In some embodiments, the present invention provides a single domain antibody (sdAb) targeting serum albumin, the heavy chain variable region sequence of which is shown in Table 1.

[0129] Table 1 Single domain antibody heavy chain variable region sequences

[0130] In some embodiments, the single domain antibody (sdAb) targeting serum albumin of the present invention is prepared by recombinant expression, and the full-length polypeptide sequence of the recombinantly expressed single domain antibody is shown in Table 2.

[0131] Table 2 Full-length sequences of single-domain antibodies

[0132] In some embodiments, the present invention uses antibodies targeting serum albumin known in the art as control antibodies, the sequences of which are shown in Table 3:

[0133] Table 3 Control single domain antibody PR000929 sequence

[0134] Multispecific antibodies

[0135] The present invention provides multispecific antibodies.

[0136] In some embodiments, the present invention provides a multispecific antibody comprising:

[0137] (i) a serum albumin binding region comprising the serum albumin binding protein described above; and

[0138] (ii) a second antigen-binding region that binds to one or more antigens selected from tumor-associated antigens and immune cell antigens.

[0139] In some embodiments, the second antigen binding region is a bispecific antibody that targets a tumor-associated antigen and CD3.

[0140] Many tumor-associated antigens relevant to specific cancers have been identified in this area. In some embodiments, tumor-associated antigens are antigens that can stimulate a significant tumor-specific immune response. Some of these antigens are encoded by normal cells, but are not necessarily expressed by normal cells. These antigens can be characterized as antigens that are usually silent (i.e., not expressed) in normal cells, antigens that are only expressed at certain stages of differentiation, and antigens expressed over time such as embryonic and fetal antigens. Other cancer cell antigens are encoded by mutant cell genes such as oncogenes (e.g., activated ras oncogenes), suppressor genes (e.g., P53 mutants), and fusion proteins produced by internal deletions or chromosomal translocations. Other cancer antigens can be encoded by genes carried by viral genes such as RNA and DNA tumor viruses. Many other tumor-associated antigens and antibodies against them are known and / or commercially available, and can also be prepared by those skilled in the art.

[0141] Examples of tumor-associated antigens include, but are not limited to, B7H4, 5T4, alpha-fetoprotein, CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD40, CD56, CD79, CD78, CD123, CD138, c-Met, CSPG4, IgM, AXL, EGFR, EGFRvIII, epithelial tumor antigen, ERBB2, FLT3, folate binding protein, GD2, GD3, HIV -1 envelope glycoprotein gp41, HIV-1 envelope glycoprotein gp120, melanoma-associated antigen, MUC-1, mutated p53, mutated ras, ROR1, GPC3, VEGFR2, CTLA-4, 4-1BB (CD137), 4-1BBL (CD137L), PD1, B7H3, B7H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, SIGLEC9 and 2B4, and combinations thereof.

[0142] In some embodiments, the tumor-associated antigen is an immune checkpoint molecule, for example, including but not limited to: CTLA-4, 4-1BB (CD137), 4-1BBL (CD137L), PD1, B7H3, B7H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, SIGLEC9 and 2B4. In a preferred embodiment, the tumor-associated antigen is B7H4.

[0143] In some embodiments, the second antigen binding region comprises a B7H4 binding region and a CD3 binding region, wherein the B7H4 binding region comprises VH and VL, VH comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 6, 20, and 32, respectively, and VL comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 43, 47, and 51, respectively.

[0144] In some embodiments, the B7H4 binding region comprises a VH and a VL, the VH comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:55, and the VL comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:62.

[0145] In some embodiments, the second antigen binding region comprises a B7H4 binding region and a CD3 binding region, wherein the CD3 binding region comprises VH and VL, VH comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 8, 22, and 34, respectively, and VL comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 44, 48, and 52, respectively.

[0146] In some embodiments, the CD3 binding region comprises a VH and a VL, the VH comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:57, and the VL comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:63.

[0147] In some embodiments, the second antigen binding region does not include an immunoglobulin Fc region.

[0148] In some embodiments, the multispecific antibody comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the serum albumin binding region is located at the C-terminus of at least one of the polypeptide chains. In some embodiments, the N-terminus of the serum albumin binding region is connected to the CD3 binding region.

[0149] In some embodiments, the first polypeptide chain comprises VL_A-CL-VH_B-CH1-VH_C from N-terminus to C-terminus; the second polypeptide chain comprises VH_A-CH1 from N-terminus to C-terminus; and the third polypeptide chain comprises VL_B-CL from N-terminus to C-terminus. VH_A represents the VH of the B7H4-binding region, VL_A represents the VL of the B7H4-binding region, VH_B represents the VH of the CD3-binding region, VL_B represents the VL of the CD3-binding region, and VH_C represents the VH of the serum albumin-binding region. "-" represents that the domains on both sides are directly connected or connected via a linker.

[0150] In some embodiments, the multispecific antibody comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein:

[0151] (a) the first polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:77; the second polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:73; and the third polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:72;

[0152] (b) the first polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:78; the second polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:73; and the third polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:72;

[0153] (c) the first polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:76; the second polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:73; and the third polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:72; or

[0154] (d) the first polypeptide chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:75; the second polypeptide chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:73; and the third polypeptide chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:72.

[0155] In a preferred embodiment, the multispecific antibody comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein:

[0156] (a) the first polypeptide chain is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:77; the second polypeptide chain is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:73; and the third polypeptide chain is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:72;

[0157] (b) the first polypeptide chain is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:78; the second polypeptide chain is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:73; and the third polypeptide chain is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:72;

[0158] (c) the first polypeptide chain is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:76; the second polypeptide chain is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:73; and the third polypeptide chain is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:72; or

[0159] (d) the first polypeptide chain is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:75; the second polypeptide chain is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:73; and the third polypeptide chain is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:72.

[0160] In some embodiments, the present invention provides a multispecific antibody (BiTE-HLE), which comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, and the sequence information thereof is shown in Table 4.

[0161] Table 4 Multispecific antibody sequences

[0162] In some embodiments, the sequences of the multispecific antibodies (BiTE-HLE) used as controls of the present invention are shown in Table 5.

[0163] Table 5 Comparative multispecific antibody sequences

[0164] Fusion protein

[0165] The present invention also provides a fusion protein. In another aspect, the present invention provides a fusion protein comprising:

[0166] (i) a serum albumin binding region comprising the above-mentioned binding protein; and

[0167] (ii) a second functional domain selected from the group consisting of an antibody, a ligand, a complement, a cytokine, an enzyme, and a hormone.

[0168] In some embodiments, in addition to the serum albumin binding protein, the fusion protein further comprises one or more other biologically active proteins. The biologically active proteins can be any proteins having biological, therapeutic, preventive, or diagnostic significance or function, which, when administered to a subject, mediate biological activity that can prevent or alleviate a disease, disorder, or condition. Specifically, the biologically active proteins can be agonists, antagonists, modulators, ligands, complement, cytokines, enzymes, or hormones, and can be particularly useful in preventing and / or treating cancer.

[0169] In some embodiments, the second functional region is a monoclonal antibody or a bispecific antibody that binds to one or more antigens selected from tumor-associated antigens and immune cell antigens.

[0170] In some embodiments, the second functional region is a bispecific antibody targeting a tumor-associated antigen and CD3.

[0171] Many tumor-associated antigens relevant to specific cancers have been identified in this area. In some embodiments, tumor-associated antigens are antigens that can stimulate a significant tumor-specific immune response. Some of these antigens are encoded by normal cells, but are not necessarily expressed by normal cells. These antigens can be characterized as antigens that are usually silent (i.e., not expressed) in normal cells, antigens that are only expressed at certain stages of differentiation, and antigens expressed over time such as embryonic and fetal antigens. Other cancer cell antigens are encoded by mutant cell genes such as oncogenes (e.g., activated ras oncogenes), suppressor genes (e.g., P53 mutants), and fusion proteins produced by internal deletions or chromosomal translocations. Other cancer antigens can be encoded by genes carried by viral genes such as RNA and DNA tumor viruses. Many other tumor-associated antigens and antibodies against them are known and / or commercially available, and can also be prepared by those skilled in the art.

[0172] Examples of tumor-associated antigens include, but are not limited to, B7H4, 5T4, alpha-fetoprotein, CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD40, CD56, CD79, CD78, CD123, CD138, c-Met, CSPG4, IgM, AXL, EGFR, EGFRvIII, epithelial tumor antigen, ERBB2, FLT3, folate binding protein, GD2, GD3, HIV -1 envelope glycoprotein gp41, HIV-1 envelope glycoprotein gp120, melanoma-associated antigen, MUC-1, mutated p53, mutated ras, ROR1, GPC3, VEGFR2, CTLA-4, 4-1BB (CD137), 4-1BBL (CD137L), PD1, B7H3, B7H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, SIGLEC9 and 2B4, and combinations thereof.

[0173] In some embodiments, the tumor-associated antigen is an immune checkpoint molecule, for example, including but not limited to: CTLA-4, 4-1BB (CD137), 4-1BBL (CD137L), PD1, B7H3, B7H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, SIGLEC9 and 2B4. In a preferred embodiment, the tumor-associated antigen is B7H4.

[0174] In some embodiments, the second functional region comprises a B7H4 binding region and a CD3 binding region, wherein the B7H4 binding region comprises VH and VL, VH comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 6, 20, and 32, respectively, and VL comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 43, 47, and 51, respectively.

[0175] In some embodiments, the B7H4 binding region comprises a VH and a VL, the VH comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:55, and the VL comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:62.

[0176] In some embodiments, the second functional region comprises a B7H4 binding region and a CD3 binding region, wherein the CD3 binding region comprises VH and VL, VH comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 8, 22, and 34, respectively, and VL comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 44, 48, and 52, respectively.

[0177] In some embodiments, the CD3 binding region comprises a VH and a VL, the VH comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:57, and the VL comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:63.

[0178] In some embodiments, the second functional region does not include an immunoglobulin Fc region.

[0179] In some embodiments, the fusion protein comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, and the serum albumin binding region is located at the C-terminus of at least one of the polypeptide chains.

[0180] In some embodiments, the first polypeptide chain comprises VL_A-CL-VH_B-CH1-VH_C from N-terminus to C-terminus; the second polypeptide chain comprises VH_A-CH1 from N-terminus to C-terminus; and the third polypeptide chain comprises VL_B-CL from N-terminus to C-terminus. VH_A represents the VH of the B7H4-binding region, VL_A represents the VL of the B7H4-binding region, VH_B represents the VH of the CD3-binding region, VL_B represents the VL of the CD3-binding region, and VH_C represents the VH of the serum albumin-binding region. "-" represents that the domains on both sides are directly connected or connected via a linker.

[0181] In some embodiments, the fusion protein comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein:

[0182] (a) the first polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:77; the second polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:73; and the third polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:72;

[0183] (b) the first polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:78; the second polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:73; and the third polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:72;

[0184] (c) the first polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:76; the second polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:73; and the third polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:72; or

[0185] (d) the first polypeptide chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:75; the second polypeptide chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:73; and the third polypeptide chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:72.

[0186] In a preferred embodiment, the fusion protein comprises a first polypeptide chain, a second polypeptide chain and a third polypeptide chain, wherein:

[0187] (a) the first polypeptide chain is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:77; the second polypeptide chain is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:73; and the third polypeptide chain is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:72;

[0188] (b) the first polypeptide chain is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:78; the second polypeptide chain is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:73; and the third polypeptide chain is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:72;

[0189] (c) the first polypeptide chain is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:76; the second polypeptide chain is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:73; and the third polypeptide chain is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:72; or

[0190] (d) the first polypeptide chain is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:75; the second polypeptide chain is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:73; and the third polypeptide chain is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:72.

[0191] Nucleic Acids

[0192] The present invention provides a nucleic acid comprising a nucleotide sequence encoding the serum albumin binding protein of the present invention, the multispecific antibody of the present invention, or the fusion protein of the present invention.

[0193] The term "nucleic acid" includes single-stranded and double-stranded nucleotide polymers. Nucleic acids can be modified forms of ribonucleotides or deoxyribonucleotides or any type of nucleotide. The modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2', 3'-dideoxyribose, internucleotide linkage modifications such as phosphorothioates, phosphorodithioates, selenophosphates, diselenphosphates, phenylthiophosphates, aniline phosphates, and phosphoramidates.

[0194] For example, the present invention provides nucleic acid molecules encoding any of the heavy chain variable region sequences disclosed herein. The present invention also provides nucleic acid molecules that are at least 90%, at least 95%, at least 98%, or at least 99% identical to the nucleic acid encoding any of the heavy chain variable region sequences disclosed herein.

[0195] For example, the present invention provides a nucleic acid molecule encoding a heavy chain variable region sequence comprising a CDR sequence of any one of the heavy chain variable region sequences disclosed herein. The present invention also provides a nucleic acid molecule encoding a heavy chain variable region sequence comprising a CDR sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to a CDR sequence of any one of the heavy chain variable region sequences disclosed herein.

[0196] In some embodiments, the nucleic acid is ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In some embodiments, the present invention provides ribonucleic acid (RNA) comprising a nucleotide sequence encoding an antibody disclosed herein. In some embodiments, the present invention provides deoxyribonucleic acid (DNA) comprising a deoxynucleotide sequence encoding an antibody disclosed herein.

[0197] In some embodiments, the deoxyribonucleic acid (DNA) can be introduced into human cells in vivo. In some embodiments, the deoxyribonucleic acid (DNA) of the present invention is contained in a carrier or delivery agent. In some embodiments, the deoxyribonucleic acid (DNA) of the present invention is integrated into the genome of the cell.

[0198] In some embodiments, the ribonucleic acid (RNA) can be introduced into human cells in vivo.In some embodiments, the ribonucleic acid (RNA) of the present invention is contained in a vector or delivery agent.

[0199] carrier

[0200] The present invention provides vectors comprising the nucleic acids disclosed herein.

[0201] In some embodiments, the vector is an expression vector capable of expressing a polypeptide comprising the heavy chain or light chain variable region of an antibody.For example, the present invention provides an expression vector comprising any of the above-mentioned nucleic acid molecules.

[0202] Any vector may be suitable for use in the present disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, a SFG vector, a plasmid, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein Barr virus vector, a papovaviral vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector (AAV), a lentiviral vector, or any combination thereof. Suitable exemplary vectors include, for example, pGAR, pBABE-puro, pBABE-neo largeT cDNA, pBABE-hygro-hTERT, pMKO.1GFP, MSCV-IRES-GFP, pMSCV PIG (Puro IRES GFP empty plasmid), pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES luciferase, pMIG, MDH1-PGK-GFP_2.0, TtRMPVIR, pMSCV-IRES-mCherry FP, pRetroX GFP T2A Cre, pRXTN, pLncEXP, and pLXIN-Luc.

[0203] The expression vector can be any suitable recombinant expression vector. Suitable vectors include vectors designed for propagation and amplification or for expression or both, such as plasmids and viruses. For example, vectors can be selected from pUC series (Fermentas Life Sciences, Glen Burnie, Md.), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden) and pEX series (Clontech, Palo Alto, Calif.). Phage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4 and λNM1149 can also be used. Examples of plant expression vectors that can be used for the present disclosure include pBI01, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animal expression vectors that can be used in the present disclosure include pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech).

[0204] Recombinant expression vectors can be prepared using standard recombinant DNA techniques as described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Circular or linear expression vector constructs can be prepared to contain replication systems functional in prokaryotic or eukaryotic host cells. Replication systems can be derived, for example, from COLEL, 2μ plasmids, lambda, SV40, bovine papilloma virus, and the like.

[0205] For example, the vector can be an adenovirus vector comprising a nucleotide sequence encoding a binding protein or antibody disclosed herein. The vector can be administered to a subject and then enter the subject's cells, thereby integrating the nucleotide sequence encoding the antibody disclosed herein into the genome of the cell, which then expresses the antibody disclosed herein.

[0206] host cells

[0207] The present invention provides host cells comprising a nucleic acid disclosed herein or a vector disclosed herein.

[0208] Any cell can be used as the host cell of nucleic acid or vector of the present disclosure. In some embodiments, the cell can be a prokaryotic cell, a fungal cell, a yeast cell or a higher eukaryotic cell such as a mammalian cell. Suitable prokaryotic cells include but are not limited to true bacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae (Enterobactehaceae), such as Escherichia (Escherichia), such as Escherichia coli (E.coli); Enterobacter (Enterobacter); Erwinia (Erwinia); Klebsiella (Klebsiella); Proteus (Proteus); Salmonella (Salmonella), such as Salmonella typhimurium (Salmonella typhimurium); Serratia (Serratia), such as Serratia marcescens (Serratia marcescens). marcescans and Shigella; Bacilli, such as Bacillus subtilis and Bacillus licheniformis; Pseudomonas, such as Pseudomonas aeruginosa; and Streptomyces. In some embodiments, the cell is a human cell. In some embodiments, the cell is an immune cell. In some embodiments, the host cell includes, for example, CHO cells, such as CHOS cells and CHO-K1 cells, or HEK293 cells, such as HEK293A, HEK293T, and HEK293FS.

[0209] The host cells of the present invention are prepared by introducing the vectors disclosed herein or the nucleic acids disclosed herein in vitro or ex vivo. The host cells of the present invention can be administered to a subject, and the host cells express the binding proteins or antibodies disclosed herein in vivo.

[0210] Pharmaceutical composition

[0211] The present invention provides a pharmaceutical composition comprising the serum albumin binding protein of the present invention, the multispecific antibody of the present invention, or the fusion protein of the present invention; and a pharmaceutically acceptable carrier or excipient.

[0212] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration. Preferred examples of such carriers or excipients include, but are not limited to, water, saline, Ringer's solution, and dextrose solution. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active compound, their use in the composition is contemplated. Supplementary active compounds may also be incorporated into the composition.

[0213] In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, a small molecule drug, siRNA, and an antisense oligonucleotide.

[0214] The pharmaceutical composition of the present invention can be formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous application may include the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; an antibacterial agent, such as benzyl alcohol or methyl paraben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as tetraethylammonium oxalate (EDTA); a buffer, such as acetate, citrate, or phosphate; and an agent for adjusting tension, such as sodium chloride or dextrose. The pH value can be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral formulations can be packaged in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0215] Pharmaceutical compositions suitable for injectable applications include sterile aqueous solutions (if water-soluble) or dispersions and sterile powders for the temporary preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be a fluid that is easy to inject. It must be stable under preparation and storage conditions and must prevent the contamination of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium, and the solvent or dispersion medium contains, for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Suitable mobility can be maintained, for example, by using a coating such as lecithin, by keeping the particle size required in the case of dispersions and by using a surfactant. The effects of microorganisms can be prevented by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0216] Sterile injectable solutions can be prepared by mixing the required amount of the active compound with one or a combination of ingredients listed above (as needed) in an appropriate solvent, followed by filtration sterilization. Typically, dispersions are prepared by introducing the active compound into a sterile vehicle containing a basic dispersion medium and other ingredients required from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation method is vacuum drying and freeze drying to produce a powder of the active ingredient plus any other desired ingredients from a previously sterile-filtered solution thereof.

[0217] Oral compositions typically include an inert diluent or edible carrier. They can be encapsulated in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be mixed with excipients and used in the form of tablets, lozenges, or capsules. Oral compositions can also be prepared using a fluid carrier used as a mouthwash, wherein the compound in the fluid carrier is administered orally and swished into the mouth and expectorated or swallowed. Pharmaceutically compatible binders and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds of similar properties: binders such as microcrystalline cellulose, tragacanth, or gelatin; excipients such as starch or lactose; disintegrants such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavorings such as peppermint oil, methyl salicylate, or orange flavoring.

[0218] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser or nebulizer which contains a suitable propellant, eg, a gas such as carbon dioxide.

[0219] Systemic administration can also be carried out by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants suitable for the barrier to be penetrated are used in the formulation. Such penetrants are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be accomplished by using nasal sprays or suppositories. For transdermal administration, the active compound is formulated into ointments, salves, gels, or creams well known in the art.

[0220] The active compounds can also be formulated in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0221] In one embodiment, the active compound is prepared with a carrier that will protect the compound from rapid elimination from the body (such as a controlled release formulation, including implants and microencapsulated delivery systems). Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art.

[0222] The present invention provides therapeutic compositions comprising antibodies of the present invention or their antigen-binding fragments. According to the therapeutic compositions of the present invention, the transfer, delivery, tolerance, etc. of the present invention will be provided together with suitable carriers, excipients, and other medicaments incorporated into the formulation. Many suitable formulations are found in prescriptions known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, vesicle lipids (cations or anions) (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, polyethylene glycol emulsions (polyethylene glycol of various molecular weights), semisolid gels, and semisolid mixtures containing polyethylene glycol. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52: 238-311.

[0223] Treatment

[0224] The present disclosure provides methods of treating cancer in a subject, comprising administering to the subject an effective amount of a multispecific antibody disclosed herein, a fusion protein disclosed herein, or a pharmaceutical composition disclosed herein.

[0225] Cancer can include hematologic malignancies and solid tumors. Solid tumors can include, but are not limited to, neuroblastoma, Wilms' tumor, hepatoblastoma, pancreatic blastoma, rhabdomyosarcoma, osteosarcoma, chondrosarcoma, pulmonary blastoma, germ cell tumors, lung cancer, liver cancer, breast cancer, ovarian cancer, endometrial cancer, non-small cell lung cancer, kidney cancer, intestinal cancer, stomach cancer, gallbladder cancer, pancreatic cancer, prostate cancer, thyroid cancer, central nervous system tumors, and the like. Hematologic malignancies may include, but are not limited to, acute myeloid leukemia, chronic myeloid leukemia, mast cell leukemia, plasma cell leukemia, myeloma, chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL) (including B-cell acute lymphoblastic leukemia and T-cell acute lymphoblastic leukemia), chronic lymphocytic leukemia (CLL), acute promyelocytic leukemia (APL), hairy cell leukemia (HCL), Hodgkin lymphoma (such as diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma), non-Hodgkin lymphoma (Burkitt lymphoma, lymphoblastic lymphoma, anaplastic large cell lymphoma).

[0226] In some embodiments, the cancer is a cancer that overexpresses B7H4, such as breast cancer (e.g., triple-negative breast cancer), ovarian cancer, endometrial cancer, non-small cell lung cancer, and renal cancer. In a preferred embodiment, the cancer is breast cancer.

[0227] In some embodiments, the dosage administered to a subject may vary with the embodiment, the drug used, the method of administration, and the site and subject to be treated. However, the dosage should be sufficient to provide a therapeutic response. A clinician can determine the effective amount to administer to a human or other subject to treat a medical condition. The precise amount required for effective treatment may depend on many factors, such as the activity of the antibody and the route of administration.

[0228] The dosage of the antibodies or compositions described herein can be administered to a mammal at once or in a series of sub-doses over an appropriate time period, for example, daily, semi-weekly, weekly, bi-weekly, monthly, bimonthly, semi-annually or annually as needed. A dosage unit comprising an effective amount of an antibody, composition or conjugate can be administered in a single daily dose, or the total daily dose can be administered in two, three, four or more divided doses administered daily as needed.

[0229] Suitable modes of administration can be selected by a physician. The route of administration can be parenteral administration, for example, by injection, nasal administration, pulmonary administration or transdermal administration. Systemic or local administration can be performed by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection. In some embodiments, the antibody, composition or conjugate is selected for parenteral delivery, for inhalation or for delivery through the digestive tract, for example, orally. The dosage and method of administration can vary according to the weight, age, condition, etc. of the subject and can be appropriately selected.

[0230] In some embodiments, the method further comprises administering to the subject a second therapeutic agent.In certain embodiments, the antibody, composition, or conjugate disclosed herein is administered prior to, substantially simultaneously with, or after administration of the second therapeutic agent.

[0231] In some embodiments, the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, a small molecule drug, an siRNA, and an antisense oligonucleotide.

[0232] Medical uses

[0233] The present invention provides use of the multispecific antibody disclosed herein, the fusion protein disclosed herein, or the pharmaceutical composition disclosed herein in the preparation of a medicament for treating cancer in a subject.

[0234] The present invention provides a multispecific antibody disclosed herein, a fusion protein disclosed herein, or a pharmaceutical composition disclosed herein for use in treating cancer in a subject.

[0235] Cancer can include hematological tumors and solid tumors. Solid tumors can include but are not limited to neuroblastoma, Wilms' tumor, hepatoblastoma, pancreatic blastoma, rhabdomyosarcoma, osteosarcoma, chondrosarcoma, pulmonary blastoma, germ cell tumors, lung cancer, liver cancer, breast cancer, ovarian cancer, endometrial cancer, non-small cell lung cancer, kidney cancer, intestinal cancer, gastric cancer, gallbladder cancer, pancreatic cancer, prostate cancer, thyroid cancer, central nervous system tumors and other solid tumors. Hematologic malignancies may include, but are not limited to, acute myeloid leukemia, chronic myeloid leukemia, mast cell leukemia, plasma cell leukemia, myeloma, chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL) (including B-cell acute lymphoblastic leukemia and T-cell acute lymphoblastic leukemia), chronic lymphocytic leukemia (CLL), acute promyelocytic leukemia (APL), hairy cell leukemia (HCL), Hodgkin lymphoma (such as diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma), non-Hodgkin lymphoma (Burkitt lymphoma, lymphoblastic lymphoma, anaplastic large cell lymphoma).

[0236] In some embodiments, the cancer is a cancer that overexpresses B7H4, such as breast cancer (e.g., triple-negative breast cancer), ovarian cancer, endometrial cancer, non-small cell lung cancer, and renal cancer. In a preferred embodiment, the cancer is breast cancer.

[0237] Methods for extending serum half-life

[0238] The present invention provides a method for extending the serum half-life of a drug, comprising linking the drug to a serum albumin binding protein disclosed herein.

[0239] In some embodiments, the pharmaceutical polypeptide. In some embodiments, the pharmaceutical is a biologically active protein. A biologically active protein can be any protein having biological, therapeutic, preventive, or diagnostic significance or function, which, when administered to a subject, mediates a biological activity that can prevent or alleviate a disease, disorder, or condition. Specifically, the biologically active protein can be an agonist, antagonist, modulator, ligand, cytokine, enzyme, or hormone, and is particularly useful for preventing and / or treating cancer.

[0240] In some embodiments, the drug is a polypeptide. In a specific embodiment, the drug is an antibody.

[0241] In some embodiments, the antibody does not have an immunoglobulin Fc region.

[0242] In some embodiments, the antibodies target one or more antigens selected from tumor-associated antigens and immune cell antigens.

[0243] In some embodiments, the antibody is a bispecific antibody that targets a tumor-associated antigen and CD3.

[0244] In some embodiments, the tumor-associated antigen is an immune checkpoint molecule, such as B7H4.

[0245] Example

[0246] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0247] Example 1 Acquisition of fully human single-domain antibodies and construction of multispecific antibodies

[0248] 1.1 Immunization of Harbour HCAb Mice

[0249] The Harbour HCAb mouse (Harbour Antibodies BV, WO 2002 / 085945A3) is a transgenic mouse carrying a human immunoglobulin repertoire, capable of producing heavy chain-only antibodies that are half the size of traditional IgG antibodies. The antibodies it produces possess only the human heavy chain variable domain and the mouse Fc constant domain. By lacking light chains, the antibodies virtually eliminate the problems of light chain mispairing and heterodimerization, enabling the development of products that are difficult to achieve with traditional technology platforms.

[0250] Ten 6- to 8-week-old Harbour HCAb transgenic mice were immunized with recombinant human HSA serum albumin (Sigma, #A9731) as an antigen. Each mouse was injected subcutaneously into the inguinal region or intraperitoneally. In the first round of immunization, mice were immunized with an immunogen prepared by mixing 50 μg of antigen protein with complete Freund's adjuvant (Sigma, #F5881) at a 1:1 volume ratio. In each subsequent booster round, each mouse received an immunogen prepared by mixing 25 μg of antigen protein with Ribi adjuvant (Sigma, #S6322). Immunizations were performed on days 0, 21, 42, 63, 84, and 105; serum antibody titers were measured on days 49, 70, 91, 111, and 132.

[0251] Mouse serum was collected and diluted to six concentrations (1:100, 1:1K, 1:10K, 1:100K, 1:1000K, 1:10000K, and 1:10000K). ELISA plates were coated with human serum albumin (HSA) (Sigma, #A9731) or cynomolgus monkey serum albumin (CSA) (abcam, #ab184894) fusion proteins, and the anti-serum albumin titer in mouse serum was measured by ELISA. The blank control group (PB) consisted of serum from pre-immunized mice.

[0252] 1.2 Screening of anti-serum albumin antibody sequences by mammalian cell expression

[0253] When serum albumin-specific antibody titers in mouse serum reached a certain level, spleen cells and bone marrow were removed, and B cells were isolated. RNA from the B cells was extracted and reverse-transcribed into cDNA (SuperScript IV First-Strand Synthesis System, Invitrogen, 18091200). The human VH gene was then amplified by PCR using specific primers. The amplified VH fragment was then constructed into the mammalian cell expression plasmid pCAG vector containing a FLAG epitope tag and a polyhistidine tag.

[0254] The constructed plasmid was transfected into mammalian host cells (such as human embryonic kidney cells HEK293) for expression to obtain single-domain antibodies. The supernatant expressing the single-domain antibody was tested for binding to human serum albumin HSA (Sigma, #A9731), cynomolgus monkey serum albumin CSA (abcam, #ab184894), or mouse serum albumin MSA (innov-research, #IRMSA_319) by ELISA screening. 77 sequences that specifically bind to human serum albumin (HSA) were obtained, and the nucleotide sequence encoding the variable domain of the antibody molecule and the corresponding amino acid sequence were obtained using conventional sequencing methods. 18 fully human serum albumin single-domain antibodies with unique sequences were selected and tested for affinity to serum albumin of three species: HSA-his / MSA-his / RAS-his using Octet. Based on the results of the human cell binding ability experiment, the top 17 antibodies were selected for recombinant expression. Fourteen purified monoclonal antibodies were obtained and further tested for their binding abilities to human serum albumin (HSA) and cynomolgus monkey serum albumin (CSA). Two single-domain antibody VHHs (PR003451 and PR003455) with the highest overall ranking were selected as candidate molecules. The amino acid sequences of the heavy chain variable domains of PR003451 and PR003455, as well as the control antibody PR000929, and their CDRs defined according to Chothia rules are shown in Table 6.

[0255] 1.3 Screening and obtaining anti-serum albumin single domain antibody sequences by phage display

[0256] After serum albumin-specific antibody titers in mouse serum reached a certain level, splenocytes and bone marrow were removed and B cells were isolated. B cell RNA was extracted and reverse-transcribed into cDNA (SuperScript IV First-Strand synthesis system, Invitrogen, 18091200). Human VH genes were then amplified by PCR using specific primers. The resulting ligated products were transformed into SS320 cells (Lucigen, 60512-1), and a phage library was prepared. Three rounds of biopanning (procedures described below) were performed, followed by screening using mouse serum albumin (MSA) and human serum albumin (HSA) by ELISA. All hits were sequenced (procedures described below) and specific clones were generated.

[0257] A. Bio-Panning

[0258] The phage library (6.8E10 phage particles) was depleted of nonspecific binding using streptavidin (SA)-coated magnetic beads (SA beads, Thermofisher, 11206). The cells were then incubated with SA-Bio-MSA magnetic beads at room temperature (refer to the manufacturer's instructions) and washed 14 times with 1xPBST. After washing, 20 ml of MC106F cells were infected at 37°C for 30 minutes. The infected cells were plated with limiting dilutions of 2×YT (containing 100 μg / ml ampicillin) and incubated overnight at 37°C to generate colonies. Then, 50 μl of 2E12 / ml M13K07 helper phage (HBM) was added to the infected cells and incubated at 37°C for 30 minutes. Finally, 80 ml of 2×YT (containing 100 μg / ml ampicillin, 50 μg / ml kanamycin, and 0.5 mM IPTG) was added and incubated overnight at 30°C. 100 ml of the overnight culture was centrifuged and filtered through a 0.22 μm filter. 20 ml of 20% PEG6000 / 2.5 M NaCl was added to the supernatant, followed by centrifugation at 4°C for 30 minutes. The resulting phage pellet was resuspended in 1 ml of 1× PBS and centrifuged at high speed for 5 minutes to discard the pellet. The resulting supernatant (phage particles) was transferred to a fresh tube and prepared for the second and third rounds of panning. The procedures for the second and third rounds of panning were identical to those described above.

[0259] B. Preliminary Screening

[0260] Overnight colonies were picked into 96-well plates supplemented with 2×YT medium (with ampicillin and 2% glucose) (Sangon Biotech, A507016-0250) and grown at 37°C for 4 hours. The supernatant was discarded by centrifugation, resuspended in fresh 2×YT medium (with ampicillin and 1 mM IPTG), and induced overnight at 30°C. After centrifugation, the supernatant was collected for ELISA. Supernatants expressing serum albumin antibodies were tested for binding to human serum albumin (HSA) (Sigma, #A9731) or mouse serum albumin (MSA) (Innov-Research, #IRMSA_319), yielding 297 positive monoclonal antibodies. Clones with the best binding were selected for sequencing, resulting in 20 fully human serum albumin (HCAb) monoclonal antibodies with unique sequences. Comprehensive analysis selected 10 antibodies for recombinant expression and purification to yield the corresponding monoclonal antibodies. Further testing by ELSIA revealed binding to human serum albumin (HSA), cynomolgus monkey serum albumin (CSA), and mouse serum albumin (MSA). Two single-domain antibodies (PR003960 and PR004133) were identified as candidate molecules by comparison with the PR000929 control molecule. The amino acid sequences of the heavy chain variable domains of PR003960, PR004133, and the control antibody PR000929, as well as the CDRs defined according to Chothia rules, are shown in Table 6.

[0261] Table 6 Sequences of serum albumin single domain antibodies (sdAb) SEQ ID NO

[0262] 1.4 Construction of multispecific antibodies

[0263] The first domain is a tumor-associated antigen (TAA) binding domain. The TAA involved in this embodiment is B7H4, and the first domain is derived from PR000157. The second domain is a CD3 binding domain derived from PR001848. The third domain is an anti-serum albumin binding domain, i.e., the antigen binding structure VH of the above-mentioned PR003451, PR003455, PR003960, PR004133, and the control antibody PR000929 serves as the third domain. A multispecific antibody BiTE-HLE was constructed in the form of TAA-Fab-CD3-Fab-VH, and its structure is shown in Figure 1. The constructed multispecific antibodies are numbered PR004684, PR004686, PR004687, PR004688, and PR004683 (control), and the specific structures of the multispecific antibodies are shown in Figures 2 and 3. The antibody sequence information of the first domain and the second domain is shown in Table 7, and the relevant domains and sequence information of the multispecific antibody are shown in Tables 8 to 10.

[0264] Table 7. B7H4 and CD3 binding antibody sequences SEQ ID NO

[0265] Table 8 Multispecific antibody domains

[0266] Table 9 SEQ ID NO numbers of bispecific antibody CDR sequences

[0267] Table 10 Multispecific (BiTE-HLE) antibody sequence SEQ ID NO

[0268] Example 2 Preparation of single-domain antibodies and multispecific antibodies against serum albumin

[0269] 2.1 Antibody Expression and Purification

[0270] Conventional recombinant DNA technology was used to express the above-mentioned single-domain antibodies and multispecific antibodies, respectively. Regarding the expression of single-domain antibodies, the heavy chain variable domain VH sequence was added with a flag tag and a 6xHis tag sequence, and then fusion expression was performed to obtain a recombinant antibody molecule. The expression full-length polypeptide chain sequence containing the flag tag and the 6xHis tag is shown in Table 6 above. Regarding the multispecific antibody (BiTE-HLE), the sequence shown in Table 8 above was successfully constructed into a mammalian cell expression plasmid vector by gene synthesis to encode the antibody.

[0271] The antibody-encoding plasmid is transfected into mammalian host cells (such as human embryonic kidney cells HEK293), and purified anti-serum albumin fully human recombinant single domain antibodies can be obtained using conventional recombinant and purification techniques. TMExpand the culture in F17 Expression Medium (Thermo, A1383504). Adjust the cell concentration to 6E5 cells / ml and culture at 37°C in an 8% CO2 shaker for 24 hours, at which point the cell concentration is approximately 1.2E6 cells / ml. Prepare 30 ml of the above cells and dissolve the plasmids encoding the single-domain and multispecific antibodies in 1.5 ml of Opti-MEM Reduced Serum Medium (Thermo, 31985088). Add 12 μl of 1 mg / ml PEI (Polysciences, #23966-2) to 1.5 ml of Opti-MEM and let it stand for 5 minutes. Slowly add PEI to the plasmid and incubate at room temperature for 10 minutes. Slowly add the plasmid-PEI mixture dropwise to the 30 ml of the above cells while mixing. Culture at 37°C in an 8% CO2 shaker for 5 days. After 5 days, observe cell viability. The culture was collected and centrifuged at 3300G for 10 minutes to obtain the supernatant, which was then centrifuged at high speed to remove impurities. TM (GE Healthcare Life Science, #71-5020-91AE) gravity column (Bio-Rad, #7311550), 2-5 times the volume of the gravity column. The supernatant sample was passed through the column and the column was rinsed with 5-10 times the gravity column volume of PBS. The target protein was then eluted with 0.1M glycine at pH 3.5, followed by adjustment to neutrality with Tris-HCl at pH 8.0, and finally concentrated and exchanged with an ultrafiltration tube (Millipore, UFC901024) to obtain a purified antibody solution. The antibody concentration was obtained by measuring absorbance at a wavelength of 280 nm using NanoDrop, and the antibody purity was determined by SEC-HPLC and SDS-PAGE.

[0272] 2.2 Analysis of protein purity and aggregates using SEC-HPLC

[0273] Size exclusion chromatography (SEC) was used to determine and analyze the purity and aggregate form of the protein sample. First, the analytical column TSKgel G3000SWxl (Tosoh Bioscience, 08541, 5 μm, 7.8 mm × 30 cm) was connected to a high-pressure liquid chromatograph (HPLC) (Agilent Technologies, Agilent 1260 Infinity II) and equilibrated with PBS at room temperature for more than 1 hour. The protein concentration was adjusted to 1 mg / ml, and at least 10 micrograms of protein sample was filtered with a 0.22 μm filter membrane before being injected into the system. The injection method was set as follows: the protein sample was passed through the column at a flow rate of 1 ml / min through PBS (pH 7.4), the running time did not exceed 20 minutes, and the detection wavelength was 280 nm. After the sample was collected, the chromatogram was integrated and the relevant data were calculated using ChemStation software.

[0274] 2.3 Analysis of protein purity and hydrophobicity using HIC-HPLC

[0275] The purity and hydrophobicity of protein samples were determined and analyzed using hydrophobic interaction chromatography (HIC). An analytical column, TSKgel Butl1-NPR (Tosoh Bioscience, 12947, 4.6 mm × 3.5 cm), was connected to a high-pressure liquid chromatograph (HPLC) (Agilent Technologies, Agilent 1260 Infinity II) and equilibrated with PBS at room temperature for at least 1 hour. The protein concentration was adjusted to 1 mg / ml, and the injection volume was 20 μl. The protein sample was injected at 0.7 ml / min from 100% mobile phase A (20 nM histidine, 1.8 M ammonium sulfate, pH 6.0) to 100% mobile phase B (20 mM histidine, pH 6.0), with a run time of no more than 16 minutes and a detection wavelength of 280 nm. After sample collection, the chromatograms were integrated and the relevant data were calculated using ChemStation software.

[0276] 2.4 Determination of the thermal stability of antibody molecules using DSF

[0277] Differential scanning fluorimetry (DSF) is used to determine the thermal stability of proteins. This method uses a real-time fluorescence quantitative PCR instrument to monitor changes in fluorescence intensity when unfolded protein molecules bind to a dye, reflecting the process of protein denaturation and thus the thermal stability of the protein, expressed as the thermal denaturation temperature (Tm). In this example, 10 μg of protein was added to a 96-well PCR plate (Thermo, AB-0700 / W), followed by 2 μl of a 100-fold diluted dye, SYPRO™ (Invitrogen, 2008138), and buffer was added to a total volume of 40 μl per well. The PCR plate was sealed and placed in a real-time fluorescence quantitative PCR instrument (Bio-Rad CFX96 PCR System). The plate was incubated at 25°C for 5 minutes, followed by a temperature ramp of 0.2°C / 0.2 minutes from 25°C to 95°C. After the test, the temperature was lowered to 25°C. Data were analyzed using Bio-Rad CFX Maestro software in FRET scanning mode, and the sample Tm was calculated. The expression information and physicochemical properties of the obtained antibodies are shown in Tables 11 and 12.

[0278] Table 11 Expression and physicochemical properties of anti-serum albumin single domain antibodies (sdAb)

[0279] Table 12 Expression and physicochemical properties of anti-serum albumin multispecific antibody (BiTE-HLE)

[0280] Example 3 Binding of fully human serum albumin antibodies to serum albumin

[0281] In order to study the in vitro binding activity of the single-domain antibodies and multispecific antibodies prepared in Example 2 to human, cynomolgus monkey, rat, and mouse serum albumin, this example uses the enzyme-linked immunosorbent assay (ELISA) method to measure the binding of the above antibodies to serum albumin at the protein level.

[0282] Coat a 96-well plate with high-absorption serum albumin from different species at 4°C overnight. Dilute serum albumin from different species to 1-2 μg / ml and add 100 μl to each well. The next day, wash the coated 96-well plate three times with 1X PBST. Then, add 100 μl of 2% BSA to each well and block at 37°C for 1 hour. Wash the plate three more times with 1X PBST and add serum albumin antibody, serially diluting each antibody 3-fold. Add 100 μl to each well and block at 37°C for 1 hour. Wash the plate three more times with 1X PBST and add the desired anti-flag HRP or anti-his HRP secondary antibody (diluted 1:4000 in 1X PBST). Add 100 μl to each well and block at 37°C for 1 hour. Wash the 96-well plate three times with 1X PBST. Add 100 μl of TMB colorimetric solution to each well. Allow to react for approximately 5-8 minutes before adding the reaction stop solution. Read the plate at OD 450 nm.

[0283] As shown in FIG4 , the anti-serum albumin single domain antibody (sdAb) of the present invention strongly binds to both human serum albumin (HSA) and cynomolgus monkey serum albumin (CSA).

[0284] As shown in Figure 5, the multispecific anti-serum albumin antibody (BiTE-HLE) of the present invention binds to both human serum albumin (HSA) and cynomolgus monkey serum albumin (CSA). Compared to the binding of the reference antibody PR004683 to rodent rat serum albumin (RSA) and mouse serum albumin (MSA), the PR004687 molecule exhibited similar cross-binding reactions.

[0285] Example 4 Binding of a fully human serum albumin multispecific antibody (BiTE-HLE) to cell surface B7H4 and CD3

[0286] To investigate the in vitro binding activity of the first domain of a fully human serum albumin multispecific antibody (BiTE-HLE) to the B7H4 target protein, this example employed a CHO-K1 / huB7H4 (Genwizhi) stable cell line overexpressing human B7H4, and performed antibody binding experiments at the cellular level using flow cytometry. Simultaneously, the in vitro binding activity of the second domain of the serum albumin multispecific antibody (BiTE-HLE) to the CD3 target protein was also examined. In this example, primary T cells were isolated from PBMCs using the Miltenyi T Cell Isolation Kit (Miltenyi, #130-096-535) according to the manufacturer's instructions, and antibody binding experiments were performed at the cellular level using flow cytometry.

[0287] After harvesting CHO-K1 / huB7H4 or Pan-T cells, adjust the cell concentration to 1E6 / ml. 100 μL of cells were plated in a 96-well V-bottom plate (Corning, 3894) and centrifuged at 500 g for 3 minutes at 4°C. The supernatant was discarded. 100 μL of serially diluted test antibody was added to each well and incubated at 4°C for 1 hour. The cells were then washed three times with 200 μL / well of pre-chilled FACS buffer (2% FBS in PBS), centrifuged at 500 g for 3 minutes at 4°C, and the supernatant was discarded. Next, a fluorescent secondary antibody (Jackson Immunology, 109-546-006) was added and incubated at 4°C in the dark for 1 hour. Finally, the cells were washed twice with pre-chilled FACS buffer, centrifuged at 500 g for 3 minutes at 4°C, and the supernatant was discarded. The cells were resuspended in 150 μL / well of pre-chilled FACS buffer and the fluorescence signal was read using an ACEA_NovoCyte.

[0288] As shown in FIG6 , the first domains of the multispecific antibodies (BiTE-HLE) of the present invention can bind to CHO-K1 / B7H4, and the binding ability of the antibodies to cells is positively correlated with the concentration of the antibodies.

[0289] As shown in FIG7 , the second domains of the multispecific antibodies (BiTE-HLE) of the present invention can bind to Pan-T cells, and the binding ability of the antibodies to the cells is positively correlated with the concentration of the antibodies.

[0290] Example 5 Affinity test of antibodies and serum albumin

[0291] KD values ​​for single-domain antibodies (sdAbs) and multispecific antibodies (BiTE-HLEs) were determined using conventional Octet methods. Candidate antibodies were immobilized onto SA sensors at a height of 0.2 nm. Binding to diluted biotinylated serum albumin (biotin-HSA), biotin-CSA, or biotin-MSA took 200 seconds, followed by 600 seconds of dissociation, and then regeneration with 10 mM glycine HCl (pH 1.5) for 15 seconds. Association rates (kOn) and dissociation rates (kDis) were calculated using a simple one-to-one Languir binding model (Octet Red96 software). The equilibrium dissociation constant (KD) was calculated as the ratio kDis / kOn. As shown in Tables 13 and 14, the affinity of the experimental antibodies was evaluated against the single-domain reference antibody (sdAb) PR000929 (Ablynx Alb23).

[0292] Table 13 Single domain antibody affinity determination results

[0293] Table 14 Multispecific antibody affinity determination results

[0294] Example 6 Epitope Identification of Antigen Binding Proteins

[0295] The present invention uses a protein recombination method to construct domains I, II and III (domain1-3) of human serum albumin (HSA) into 5 recombinant proteins containing different domains, namely: PR004215 (domain1+2-6His), PR004216 (domain1-6His), PR004217 (domain2+3-6His), PR004218 (domain2-6His), PR004219 (domain3-6His), and the constructed plasmids are transfected into mammalian host cells (such as human embryonic kidney cells HEK293) for expression, and the corresponding recombinant proteins are purified. The single-domain antibody (sdAb) against serum albumin obtained in Example 2 was epitope identified by ELISA. The binding epitope of the antibody was determined by the OD450nm reading of the antibody binding to different recombinant proteins. The results showed that all four single-domain antibodies (sdAb) bound to all recombinant proteins containing domain2, so all four single-domain antibodies bound to the domain2 epitope of HSA.

[0296] Example 7 Multispecific (BiTE-HLE) Antibody T Cell Killing Experiment

[0297] This experiment used human primary T cells as effector cells and MDA-MB-468 (human breast cancer) cells that highly express B7H4 as target cells. The conductivity of target cells was measured using an ACEA RTCA instrument to reflect the killing efficiency. The 96-well e-plate was first equilibrated with 50 μl of complete culture medium. The target cells were digested, resuspended in RPM1640 complete culture medium containing 10% fetal bovine serum, and diluted to 4 × 10 5 / mL, plate 50 μl / well in e-plate 96 plate, i.e. 2×10 4 Each well was incubated at 37°C overnight. The next day, primary T cells were isolated using the Miltenyi T cell isolation kit (Miltenyi, #130-096-535) according to the instructions. 50 μl of fresh T cells containing 2×10 5 The T cell culture medium was then supplemented with 50 μl of a 4x serially diluted antibody, with a maximum final concentration of 10 nM. Eight concentrations of each antibody were measured in duplicate. The conductivity of the target cells was monitored in real time. Data from the 24-hour time point were generally used to calculate target cell killing efficiency (target cell killing efficiency = (1 - sample / blank control) x 100%).

[0298] As shown in FIG8 , the multispecific antibody (BiTE-HLE) of the present invention has a high killing effect on MDA-MB-468 cells that highly express B7H4.

[0299] Example 8 Prolonging the serum half-life of a multispecific antibody (BiTE-HLE) using serum albumin

[0300] Because the "κ / λ" Fab-Fab structure lacks an Fc region, its serum half-life is expected to be shorter than that of normal IgG. Currently, various technical approaches have been developed to increase the serum half-life of protein molecules, such as using PEGylation to increase the molecular hydration radius to further increase serum half-life, or coupling to human serum albumin (HSA) or anti-serum albumin (ALB) antibodies to further increase serum half-life through FcRn-mediated recycling. For example, the single-domain antibody Alb23 is known to specifically bind to serum albumin and exhibit cross-reactivity with human, cynomolgus macaque, and mouse species. To test its serum half-life extension effect, as described in Example 2, the present application fused the variable region of Alb23 to the second polypeptide chain (long chain) of the "κ / λ" Fab-Fab structure to construct a multispecific antigen-binding protein containing a "κ / λ" Fab-Fab structure (as shown in Figures 2-3). This is the PR004683 antibody of the present application, which is a B7H4×CD3×ALB multispecific control antibody containing a "κ / λ" Fab-Fab structure and a "half-life extension module" (serum albumin binding domain). PR004687 and PR004688 are test samples of the present application.

[0301] The protocol was as follows: 3 female SD rats of appropriate weight were selected and divided into 3 groups. The test drug was administered intravenously at a dose of 3 mg / kg. Whole blood was collected before administration and at 5 minutes, 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 24 hours (1 day), and on days 2, 3, 4, and 7 after administration (see Table 15 below for sampling times for each group). Whole blood was allowed to clot for 30 minutes, then centrifuged at 2,000 rpm for 5 minutes at 4°C. The separated serum samples were frozen at -80°C until analysis. ELISA was used to quantify drug concentrations in serum: B7H4 recombinant protein (Human B7-H4 Protein, His Tag, ACRO Biosystems, #B74-H5222) was coated at 1 μg / ml, 50 μl / well, in a 96-well plate to capture the test molecules containing the anti-B7H4 Fab end in serum. HRP-labeled goat anti-human IgG (Fab-specific) secondary antibody (Sigma-Aldrich, #A0293) was then added for detection. Pharmacokinetic parameters were analyzed using Phoenix WinNonlin software version 8.2, employing a non-compartmental model (NCA) (see Table 16 below).

[0302] Table 15 Serum half-life schedule for pharmacokinetic studies

[0303] Table 16 Pharmacokinetic parameters in rats

[0304] As shown in FIG9 , the pharmacokinetics in rats showed that PR004687 with rat cross-reactivity had a significantly longer serum half-life than PR004688 without rat cross-reactivity.

[0305] In summary, the four single-domain antibodies (sdAbs) of this invention can be used as HLE modules in bispecific antibodies (BiTEs) in future studies to increase the serum half-life of bispecific antibodies (BiTEs) in humans, cynomolgus monkeys, or humanized HSA / hFcRn transgenic mice. The "κ / λ" Fab-Fab structure of the bispecific antibody molecule in this example offers excellent flexibility and versatility. Bispecific antibodies can be prepared from any two conventional IgG antibodies and can be expanded to a variety of other structures, offering a "plug-and-play" advantage.

Claims

1. A serum albumin binding protein comprising a heavy chain variable region (VH), wherein: (1) the VH comprises CDR1, CDR2, and CDR3 of the VH shown in SEQ ID NO: 60; (2) the VH comprises CDR1, CDR2, and CDR3 of the VH shown in SEQ ID NO: 61; (3) the VH comprises CDR1, CDR2, and CDR3 of the VH shown in SEQ ID NO: 59; or (4) The VH comprises CDR1, CDR2 and CDR3 of the VH shown in SEQ ID NO:

58.

2. The binding protein of claim 1, wherein: (1) the VH comprises CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 11, 24, and 37, respectively; (2) the VH comprises CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 12, 25, and 37, respectively; (3) the VH comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 10, 23 and 36, respectively; or (4) The VH comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 9, 23 and 35, respectively.

3. The binding protein of claim 1 or 2, wherein: (1) the VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 60; (2) the VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 61; (3) the VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 59; or (4) The VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

58. The binding protein according to any one of claims 1 to 3, which does not comprise a light chain, preferably the antibody is a single domain antibody or a heavy chain antibody. The binding protein according to any one of claims 1 to 4, which is a monoclonal antibody, a bispecific antibody or a multispecific antibody.

6. A fusion protein comprising: (i) a serum albumin binding region comprising the binding protein of any one of claims 1 to 5; and (ii) a second functional domain selected from the group consisting of an antibody, complement, a ligand, a cytokine, an enzyme, and a hormone. 7 . The fusion protein of claim 6 , wherein the second functional region is a monoclonal antibody or a bispecific antibody that binds to one or more antigens selected from tumor-associated antigens and immune cell antigens. The fusion protein of claim 7 , wherein the second functional region is a bispecific antibody targeting a tumor-associated antigen and CD3.

9. The fusion protein of claim 8, wherein the tumor-associated antigen is an immune checkpoint molecule.

10. The fusion protein of claim 9, wherein the tumor-associated antigen is B7H4.

11. The fusion protein of claim 10, wherein the bispecific antibody comprises a B7H4 binding region and a CD3 binding region, wherein: The B7H4 binding region comprises a VH comprising HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 6, 20, and 32, respectively, and a VL comprising LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 43, 47, and 51, respectively; and / or The CD3 binding region comprises a VH comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 8, 22 and 34, respectively, and a VL comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 44, 48 and 52, respectively.

12. The fusion protein of claim 11, wherein the B7H4 binding region comprises a VH and a VL, wherein the VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:55, and the VL comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

62.

13. The fusion protein of claim 11 or 12, wherein the CD3 binding region comprises a VH and a VL, wherein the VH comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 57, and the VL comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

63.

14. The fusion protein of any one of claims 7 to 13, wherein the second functional region does not comprise an immunoglobulin Fc region.

15. The fusion protein of claim 14, comprising a first polypeptide chain, a second polypeptide chain and a third polypeptide chain, wherein the serum albumin binding region is located at the C-terminus of at least one of the polypeptide chains.

16. The fusion protein of claim 15, wherein the first polypeptide chain comprises VL_A-CL-VH_B-CH1-VH_C from N-terminus to C-terminus; the second polypeptide chain comprises VH_A-CH1 from N-terminus to C-terminus; and the third polypeptide chain comprises VL_B-CL from N-terminus to C-terminus. Among them, VH_A represents the VH of the B7H4 binding region, VL_A represents the VL of the B7H4 binding region, VH_B represents the VH of the CD3 binding region, VL_B represents the VL of the CD3 binding region, and VH_C represents the VH of the serum albumin binding region. "-" represents that the domains on both sides are directly connected or connected through a linker.

17. The fusion protein of claim 16, wherein: (a) the first polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 77; the second polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 73; and the third polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 72; (b) the first polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:78; the second polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:73; and the third polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:72; (c) the first polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:76; the second polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:73; and the third polypeptide chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:72; or (d) the first polypeptide chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:75; the second polypeptide chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:73; and the third polypeptide chain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

72.

18. A nucleic acid comprising a nucleotide sequence encoding the serum albumin binding protein according to any one of claims 1 to 5 or the fusion protein according to any one of claims 6 to 17.

19. A vector comprising the nucleic acid of claim 18.

20. A host cell comprising the nucleic acid of claim 18 or the vector of claim 19.

21. A pharmaceutical composition comprising the binding protein according to any one of claims 1 to 5 or the fusion protein according to any one of claims 6 to 17, and a pharmaceutically acceptable carrier or excipient.

22. The pharmaceutical composition of claim 21, further comprising a second therapeutic agent selected from the group consisting of an antibody, a chemotherapeutic agent, a small molecule drug, siRNA, and an antisense oligonucleotide.

23. A method for extending the serum half-life of a drug, comprising linking the drug to the serum albumin binding protein according to any one of claims 1 to 5.

24. The method of claim 23, wherein the drug is a polypeptide, such as an antibody.

25. The method of claim 23 or 24, wherein the antibody targets one or more antigens selected from the group consisting of tumor-associated antigens and immune cell antigens.

26. The method of claim 25, wherein the antibody is a bispecific antibody targeting a tumor-associated antigen and CD3.

27. The method of claim 26, wherein the tumor-associated antigen is an immune checkpoint molecule, such as B7H4.

28. The method of any one of claims 23-27, wherein the antibody does not comprise an immunoglobulin Fc region.

29. Use of the fusion protein of any one of claims 10 to 17 in the preparation of a medicament for treating cancer in a subject.

30. The use of claim 29, wherein the cancer is selected from breast cancer, ovarian cancer, endometrial cancer, non-small cell lung cancer, and renal cancer.

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