TW202321315A - Polypeptide complex of interleukin 15 and interleukin 15 receptor - Google Patents

Abstract

The present invention relates to the field of biomedicine, in particular, to a polypeptide complex of interleukin 15 and interleukin 15 receptor, and application thereof. A non-natural interchain bond is present between IL15 and IL15Rα in the polypeptide complex of the present invention.

Description

Polypeptide complex of interleukin 15 and its receptor

This application claims the Chinese patent application with the filing date of September 24, 2021 (Application No.: 202111123534.X, invention name: a polypeptide complex of interleukin 15 and its receptor) and the patent application with the filing date of September 24, 2021 Priority of Chinese patent applications (application number: 202111121937.0, invention title: bispecific multifunctional fusion polypeptide), the full texts of these two Chinese patent applications are incorporated into this application in their entirety by reference.

The present invention relates to the field of biomedicine, and specifically to a disulfide bond-modified polypeptide complex containing interleukin 15 and its receptor.

Cytokines play an important role in human immune regulation. They also participate in the immune regulation of tumors and are closely related to the occurrence and development of tumors. In immunotherapy, cytokines can directly act on immune effector cells in the tumor microenvironment to enhance the tumor suppressive effect. Through clinical studies and animal experiments, many cytokines have been proven to have significant anti-tumor activity, and several cytokines have been approved by the FDA.

Interleukin 15 (IL-15) is a cytokine of approximately 12-14kD discovered by Grabstein et al. in 1994. It can play a role in the body's normal immune response, such as promoting T cells, B cells, and natural killer ( NK) cell proliferation.

IL-15 is a member of the Small four α-helix bundle family of cytokines. IL-15 needs to bind to its receptor to exert biological activity. The IL-15 receptor is composed of three receptor subunits: IL-15 receptor alpha (IL-15Rα), IL-2 receptor beta (IL-2Rβ, also known as IL-15Rβ or CD122), and γc (also known as CD132). IL-15Rα contains a Sushi domain that can bind to IL-15 and is necessary for the combined IL-15 to exert its biological functions.

In recent years, IL15 and IL15 receptor have been increasingly used to construct fusion proteins. In order to improve the stability of the fusion protein, this application introduces a disulfide bond between interleukin 15 and its receptor.

The invention provides an IL15/IL15Rα polypeptide complex. There is an unnatural interchain bond between IL15 and IL15Rα. The unnatural interchain bond is formed from the first mutated residue of IL15 and the second mutated residue of IL15Rα. Between the bases, the first mutated residue of IL15 is the E at position 90 that is mutated to C, and the second mutated residue of IL15Rα is the P at position 67 that is mutated to C. The amino acid residue mutation site of IL15 is the natural sequence numbering site corresponding to SEQ ID NO: 26, and the amino acid residue mutation site of IL15Rα is the natural sequence numbering site corresponding to SEQ ID NO: 27. In some embodiments, the IL15/IL15Rα polypeptide complex described in any of the preceding items, the IL15 and IL15Rα specifically bind; in some embodiments, wherein,

A) The D at position 61 of IL15 is mutated to N, the E at position 64 is mutated to Q, and/or the N at position 65 is mutated to D; and/or

B) at least one N-glycosylation site of IL15 is absent; in some embodiments, the N-glycosylation site is selected from N71, N79 and/or N112; in some embodiments, the IL15 comprises The following amino acid mutations: N71Q, N79Q and/or N112Q; and/or at least one O-glycosylation site of IL15Rα is absent; in some embodiments, the O-glycosylation site is selected from T2, T81 and/or or T86; In some embodiments, the IL15Rα comprises the following amino acid mutations: T2A, T81A and/or T86A;

The amino acid residue mutation site of IL15 is the natural sequence numbering site corresponding to SEQ ID NO: 26, and the amino acid residue mutation site of IL15Rα is the natural sequence numbering site corresponding to SEQ ID NO: 27.

In some embodiments, the IL15/IL15Rα polypeptide complex described in any one of the preceding items, the IL15 is the amino acid sequence shown in SEQ ID NO. 84 or its mutant sequence; in some embodiments, the mutant sequence includes Selected from D61N, E64Q and/or N65D amino acid mutations, and/or selected from N71Q, N79Q and/or N112Q amino acid mutations; the amino acid residue mutation site of IL15 is the natural sequence numbering position corresponding to SEQ ID NO: 26 point.

In some embodiments, the IL15/IL15Rα polypeptide complex according to any one of the preceding items, the IL15Rα comprises SEQ ID NO.28 or its mutant sequence, SEQ ID NO.77 or its mutant sequence, SEQ ID NO.78 or Its mutant sequence, SEQ ID NO.79 or its mutant sequence, SEQ ID NO.80 or its mutant sequence, or SEQ ID NO.81 or its mutant sequence; in some embodiments, the mutant sequence includes selected from T2A, Amino acid mutations of T81A and/or T86A; in some embodiments, the mutation sequence includes a P67C amino acid mutation; in some embodiments, the mutation sequence includes a P67C amino acid mutation, and also includes an amino acid mutation selected from T2A, T81A, and/or T86A Amino acid mutation; the amino acid residue mutation site of IL15Rα is the natural sequence numbering site corresponding to SEQ ID NO: 27.

In some embodiments, the IL15/IL15Rα polypeptide complex described in any one of the preceding items, the IL15Rα is SEQ ID NO.77 or its mutant sequence, SEQ ID NO.78 or its mutant sequence, SEQ ID NO.79 or Its mutant sequence, SEQ ID NO.80 or its mutant sequence, or SEQ ID NO.81 or its mutant sequence; in some embodiments, the mutant sequence includes P67C amino acid mutation; in some embodiments, the mutant sequence Including P67C amino acid mutation, also includes amino acid mutation selected from T2A, T81A and/or T86A; In some embodiments, the mutation sequence includes amino acid mutation selected from T2A, T81A and/or T86A; The amino acid residue of IL15Rα The base mutation site is the natural sequence numbering site corresponding to SEQ ID NO: 27.

In some embodiments, the IL15/IL15Rα polypeptide complex as described in any of the preceding items further comprises an antibody Fc constant region; in some embodiments, the antibody Fc constant region is a heterodimer; in some embodiments In one embodiment, the antibody Fc constant region associates as a heterodimer based on KiH, hydrophobic interactions, electrostatic interactions, hydrophilic interactions, and/or increased flexibility; in some embodiments, the IL15 or The C-terminus of IL15Rα is connected to the N-terminus of the Fc constant region.

In some embodiments, the IL15/IL15Rα polypeptide complex according to any one of the preceding items, the polypeptide complex is a bispecific fusion polypeptide, and the bispecific fusion polypeptide includes a first antigen-binding portion, wherein,

(A) The first antigen-binding portion comprises: a first polypeptide comprising the first heavy chain variable domain VH1 of the first antibody from the N-terminus to the C-terminus operably linked to to IL15; and a second polypeptide comprising from N-terminus to C-terminus the first light chain variable domain VL1 of the first antibody operably linked to IL15Rα; or

(B) The first antigen-binding portion comprises: a first polypeptide comprising the first heavy chain variable domain VH1 of the first antibody from the N-terminus to the C-terminus operably linked to to IL15Rα; and a second polypeptide comprising the first light chain variable domain VL1 of the first antibody from the N-terminus to the C-terminus operably linked to IL15.

In some embodiments, the IL15/IL15Rα polypeptide complex according to any one of the preceding items, the polypeptide complex further includes a second antigen-binding portion, wherein the second antigen-binding portion includes: a third polypeptide, The third polypeptide comprises from the N-terminus to the C-terminus the second heavy chain variable domain VH2 of the second antibody, which is operably linked to the antibody heavy chain constant region CH1, and a fourth polypeptide, the fourth polypeptide The polypeptide comprises from the N-terminus to the C-terminus the second light chain variable domain VL2 of the second antibody operably linked to the antibody light chain constant region CL.

In some embodiments, for the IL15/IL15Rα polypeptide complex described in any one of the preceding items, the first antigen-binding portion and the second antigen-binding portion bind to different antigens or bind to different epitopes of the same antigen;

In some embodiments, the first antigen-binding moiety targets immune cells and the second antigen-binding moiety targets tumor cells;

In some embodiments, the first antigen binding moiety and the second antigen binding moiety both target tumor cells;

In some embodiments, both the first antigen-binding moiety and the second antigen-binding moiety target immune cells;

In some embodiments, the first antigen binding portion targets human PD-L1 and the second antigen binding portion targets human TIGIT; or the first antigen binding portion targets human TIGIT and the second antigen binding portion targets Human PD-L1.

The invention also relates to an isolated nucleic acid encoding an IL15/IL15Rα polypeptide complex as described in any one of the preceding items. The invention also relates to vectors containing nucleic acids as described above.

The invention also relates to host cells containing nucleic acids as described above or vectors as described above.

The present invention also relates to a method for preparing the IL15/IL15Rα polypeptide complex, which includes the steps of: transforming host cells with the vector as described above; cultivating the transformed host cells; and collecting the IL15/IL15Rα polypeptide complex expressed in the host cells. .

The present invention also relates to a pharmaceutical composition comprising the IL15/IL15Rα polypeptide complex as described above and a pharmaceutically acceptable carrier, excipient or stabilizer.

The present invention also relates to the use of the IL15/IL15Rα polypeptide complex or pharmaceutical composition as described above in the preparation of drugs for treating diseases.

The present invention also relates to any of the preceding IL15/IL15Rα polypeptide complexes or pharmaceutical compositions for use as medicaments for the treatment of diseases or conditions.

The present invention also relates to a method of treating a disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of the IL15/IL15Rα polypeptide complex or pharmaceutical composition described in any one of the preceding items.

Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used in another embodiment, to yield still further embodiments. All documents cited in this disclosure, including publications, patents, and patent applications, are hereby incorporated by reference in their entirety.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which the invention belongs. The terminology used herein in the description of the invention is for the purpose of describing specific embodiments only and is not intended to limit the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The term "antigen-binding portion" or "antigen-binding domain" means that portion of an antigen-binding molecule that confers its binding specificity for an antigenic determinant. In some embodiments, the "antigen binding portion" is a functional fragment of an antibody.

The term "wild type or WT" means the amino acid sequence or nucleotide sequence found in nature, including allelic variations. WT proteins have an amino acid sequence or nucleotide sequence that has not been intentionally modified.

The term "antibody" covers any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody, bispecific (bivalent) antibody or bispecific fusion polypeptide that binds a specific antigen. A natural intact antibody contains two heavy chains and two light chains. Each heavy chain consists of a variable region ("HCVR" or VH) and first, second and third constant regions (CH1, CH2, CH3, respectively), and each light chain consists of a variable region ("LCVR" 》 or VL) and a constant region (CL). Mammalian heavy chains can be classified as α, δ, ε, γ, and μ, and mammalian light chains can be classified as λ or κ.

Antibodies are "Y" shaped, with the backbone consisting of the second (CH2), third (CH3) and optionally fourth (CH4) constant regions of two heavy chains, which are bonded by disulfide bonds. Each arm of the "Y" structure contains the variable domain (VH) and the first constant domain (CH1) of one of the heavy chains, which are combined with the variable domain (VL) and constant domain (CL) of one light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable region of each chain contains three hypervariable regions, called complementarity determining regions (CDRs). The CDRs of the light (L) chain include LCDR1, LCDR2, and LCDR3, and the CDRs of the heavy (H) chain include HCDR1, HCDR2, and HCDR3. . Among them, the three CDRs are separated by a portion called the framework region (FR), which is more highly conserved than the CDRs and forms a scaffold supporting the hypervariable loop. HCVR and LCVR each contain 4 FRs, and the CDRs and FRs are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The constant regions of the heavy and light chains are not involved in antigen binding but have a variety of effector functions. Antibodies can be divided into several categories based on the amino acid sequence of the heavy chain constant region. Antibodies can be divided into five main classifications or isoforms according to whether they contain α, δ, ε, γ and μ heavy chains: IgA, IgD, IgE, IgG and IgM. Several major antibody classes can also be divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain) etc.

The hypervariable region typically contains amino acid residues 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3) from the light chain variable region and 31-35B from the heavy chain variable region (HCDR1) , 50-65 (HCDR2) and 95-102 (HCDR3) amino acid residues (Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th ed. Bethesda, MD Public Health Service, National Institutes of Health, Bethesda, Md. (1991)), or those residues that form hypervariable loops, such as residue 26 in the light chain variable region -32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3) and 26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chain variable region (Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917).

In some embodiments, the antibody is a bispecific antibody (BiAb). The term "bispecific" as used herein refers to two different antigens, or when the two are the same antigen, each having binding specificities for different epitopes. The epitopes may be derived from different antigens or from the same antigen. The terms "bispecific fusion polypeptide" and "bispecific antibody" are used herein to refer to all products produced that have full-length antibodies or fragments with antigen-binding sites. The antibody may be a human antibody, a non-human antibody (such as a mouse-derived antibody), a humanized antibody, or a chimeric antibody (such as a human-mouse chimeric antibody or a chimeric antibody of different subtypes). In some cases, antibody variants are obtained by conservative modifications or conservative substitutions or substitutions on the antibody sequences provided by the invention. "Conservative modification" or "conservative substitution or substitution" refers to the replacement of amino acids in a protein with other amino acids with similar characteristics (such as charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.), such that it can be performed frequently Change without altering the biological activity of the protein. Those skilled in the art are aware that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter the biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., Page 224, (4th ed.)). In addition, substitution of amino acids with similar structure or function is unlikely to destroy biological activity. One skilled in the art will be able to determine suitable variants of the antigen-binding molecules as set forth herein using well-known techniques. For nucleotide and amino acid sequences, the term "identity" indicates the degree of identity between two nucleic acids or two amino acid sequences when optimally aligned and compared with appropriate insertions or deletions.

The term "Fab" refers to a Fab fragment of an immunoglobulin that contains no or a small portion of the residual Fc fragment. For example, a Fab fragment includes the variable regions of the heavy and light chains, and all or part of the first constant domain.

The term "Fc" or "Fc region" or "Fc domain" is meant to include the constant region of an antibody, in some cases excluding all or part of the first constant region immunoglobulin domain (eg, CH1), and in some cases The following further excludes polypeptides that are all or part of the hinge. Thus, Fc may refer to the last two constant region immunoglobulin domains (e.g., CH2 and CH3) of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains (e.g., CH2, CH3, and CH4) of IgE and IgM. , and optionally all or part of the N-terminus of the flexible hinge of these domains. For IgA and IgM, the Fc can contain a J chain. For IgG, the Fc domain consists of the immunoglobulin domains CH2 and CH3 (Cγ2 and Cγ3) and the lower hinge region between CH1 (Cγ1) and CH2 (Cγ2). Although the boundaries of the Fc region can vary, the human IgG heavy chain Fc region is generally defined to include residues E216, C226 or A231 at its carboxyl terminus, where numbering is according to the EU index as in Kabat. In some embodiments, as described more fully below, the Fc region is subject to amino acid modifications, eg, the Fc is a heterodimer.

"Modification" as used herein refers to amino acid substitutions, insertions and/or deletions in a polypeptide sequence or changes in the portion chemically linked to a protein. "Amino acid modification" herein refers to amino acid substitutions, insertions and/or deletions in a polypeptide sequence. For clarity, unless otherwise stated, amino acid modifications are the amino acids encoded by DNA, such as the 20 amino acids with codons in DNA and RNA.

"Epitope" as used herein means a determinant that interacts with a specific antigen-binding domain, such as a variable region (called a paratope) of an antibody molecule. Epitopes are groupings of molecules, such as amino acids or sugar side chains, and often have specific structural characteristics as well as specific charge characteristics. A single molecule can have more than one epitope. An epitope may contain amino acid residues that are directly involved in binding (also known as the immunodominant component of the epitope) and other amino acid residues that are not directly involved in binding, such as amino acid residues that are effectively blocked by specific antigen-binding peptides; In other words, the amino acid residues are within the coverage area of the specific antigen-binding peptide. Epitopes can be conformational or linear. Conformational epitopes result from the spatial juxtaposition of amino acids from different segments of a linear polypeptide chain. Linear epitopes are epitopes resulting from adjacent amino acid residues in a polypeptide chain. Conformal and non-conforming epitopes can be distinguished by the loss of binding to the former but not the latter in the presence of denaturing solvents. Epitopes typically include at least 3, and more typically at least 5 or 8-10 amino acids in a unique spatial conformation. Antigen-binding molecules that recognize the same epitope can be verified in a simple immunoassay showing the ability of one antigen-binding molecule to block the binding of another antigen-binding molecule to the target antigen. As summarized below, the present invention not only includes the antigen-binding molecules and antigen-binding domains listed herein, but also includes antigen-binding molecules and antigen-binding structures that compete for binding to the epitope bound by the listed antigen-binding molecules or antigen-binding domains. area.

The terms "specific binding", "selective binding", "selectively binding" and "specific binding" refer to a specific ligand that is directed and can be competitively blocked by the corresponding substance in vitro or in vivo. Structural site interactions in biological binding processes. Such as the binding between antigen and antibody or receptor and ligand.

The strength or affinity of specific binding can be expressed in terms of the dissociation constant (KD) of the interaction, where a smaller KD indicates greater affinity and a larger KD indicates lower affinity. For example, the KD is at least about 10 ^-4 M, at least about 10 ^-5 M, at least about 10 ^-6 M, at least about 10 ^-7 M, at least about 10 ^-8 M, at least about 10 ^-9 M, alternatively at least about 10 ^-10 M, at least about 10 ^-11 M, at least about 10 ^-12 M, or greater antigen binding capacity. Binding properties can be determined by methods well known in the art, such as biolayer interferometry and surface plasmon resonance based methods. One such method requires measuring the rates of association and dissociation of antigen-binding site/antigen or receptor/ligand complexes, where the rates depend on the concentration of the complex partners, the affinity of the interaction, and equally in both directions. Geometric parameters that affect velocity. Therefore, the association rate (ka) and the dissociation rate (kd) can be determined, and the ratio kd/ka is equal to the dissociation constant KD (Nature 361:186-187 (1993) and Davies et al. (1990) Annual Rev Biochem 59: 439-473).

The term "immune cells" includes cells of the immune system that participate in protecting the body against both infectious diseases and foreign substances. Immune cells may include, for example, neutrophils, eosinophils, basophils, lymphocytes such as B cells and T cells, and monocytes. T cells can include, for example, CD4+, CD8+, T helper cells, cytotoxic T cells, γδ T cells, regulatory T cells, suppressor T cells, and natural killer cells.

The term "multifunctional fusion polypeptide" means a non-naturally occurring binding molecule designed to target two or more antigens. "Multifunctional fusion polypeptides" as described herein are typically genetically engineered fusion proteins, eg, designed to bring two different desired biological functions into a single binding molecule. For example, a multifunctional fusion polypeptide can be a multifunctional binding molecule.

The term "FiBody" refers to a bispecific fusion polypeptide or multifunctional fusion protein obtained by replacing part or all of the constant region of a bispecific antibody with a ligand and its receptor-specific affinity. The "YBody" technology mentioned in the present invention was developed by Wuhan Youzhiyou Company in 2012. This technology is based on the "Knob-into-Holes" technology. One of the heterodimers formed is a normal heavy chain. , and the other is the N-terminal link of the Fc functional region to the scFv, forming an asymmetric bispecific antibody.

"IL15/IL15Rα polypeptide complex" refers to a polypeptide complex including IL15 and IL15Rα, and the IL15 specifically binds to IL15Rα to form a polypeptide complex.

The term "about" or "approximately" means a difference of 30, 25, 20, 25, 10, 9, 8, 7, from a reference quantity, level, value, quantity, frequency, percentage, dimension, size, amount, weight or length. 6, 5, 4, 3, 2 or 1% of quantity, level, value, quantity, frequency, percentage, dimension, size, quantity, weight or length. In certain embodiments, when the term "about" or "approximately" precedes a numerical value, it means the stated value plus or minus a range of 15%, 10%, 5%, or 1%.

Unless the context otherwise requires, the words "comprising", "includes" and "containing" will be understood to mean the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. "Consisting of" means including and being limited to the content following the phrase "consisting of." Thus, the phrase "consisting of" means that the listed elements are required or necessary and that no other elements can be present. "Consisting essentially of" is intended to include any of the elements listed after this phrase and is limited to other elements that contribute to or do not interfere with the activity or role of the listed elements as detailed in this invention. Thus, the phrase "consisting essentially of" means that the listed elements are required or required, but that other elements are optional and may be present depending on whether they affect the activity or action of the listed elements or does not exist.

“One embodiment”, “implementation”, “specific embodiment”, “related embodiment”, “certain embodiment”, “another embodiment” or “further embodiment” mentioned throughout the present invention ” or a combination thereof means that a specific feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Therefore, the aforementioned terms appearing in various places throughout this specification do not necessarily refer to the same implementation. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner in one or more embodiments.

The term "optionally" is used for descriptive purposes only and is not to be construed as indicating or implying relative importance. Thus, a feature qualified with "optionally" may explicitly or implicitly include or exclude that feature.

The terms "first" and "second" in the description and claims are used to distinguish similar elements and are not necessarily used to describe a sequence or temporal order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein. bispecific fusion peptides

The invention provides novel bispecific fusion polypeptides, which comprise a ligand (or a fragment thereof) and a receptor (or a fragment thereof) thereof, which are respectively Independently replace CH1 and CL of Fab on one side of the antibody. Specifically, the bispecific fusion polypeptide includes a first antigen-binding portion, and the first antigen-binding portion includes: a first polypeptide, and the first polypeptide is from The N-terminus to the C-terminus includes the first heavy chain variable domain VH1 of the first antibody operably linked to the first conjugation fragment; and a second polypeptide from the N-terminus to the C-terminus The terminus comprises the first light chain variable domain VL1 of the first antibody, which is operably linked to a second conjugation fragment; wherein the first conjugation fragment is a receptor and the second conjugation fragment is Ligand; or the first conjugate fragment is a ligand, and the second conjugate fragment is a receptor.

In some embodiments, the bispecific fusion polypeptide has: a first polypeptide, which from the N-terminus to the C-terminus is: [VH1]-[Linker 1]-[IL15]-[Linker 2]- [CH2]-[CH3], the second polypeptide, which from the N-terminus to the C-terminus is: [VL1]-[linker 3]-[[IL15RA]; in some embodiments, the bispecific fusion The polypeptide has: a first polypeptide, which from N-terminus to C-terminus is: [VH1]-[Linker 1]-[IL15RA]-[Linker 2]-[CH2]-[CH3], a second polypeptide , from N end to C end: [VL1]-[Connector 3]-[IL15]. Wherein, the CH2 and CH3 are heavy chain constant region subunits, and the linker 1, linker 2 and linker 3 are linkers connecting polypeptides, which may be the same or different; in some embodiments, the The above-mentioned linker 1, linker 2 and linker 3 are independently selected from (GxS) y linkers, where x is selected from an integer of 1-5, and y is selected from an integer of 0-6.

In some embodiments, the bispecific fusion polypeptide further comprises a second antigen-binding portion that is different from the first antigen-binding portion; the second antigen-binding portion can be selected from:

1. CH1 and CL of the Fab on the other side of the antibody are replaced by another ligand (or fragment thereof) and its receptor (or fragment thereof), that is, the second antigen-binding part includes: a third polypeptide, the third polypeptide The peptide comprises from the N-terminus to the C-terminus the second heavy chain variable domain VH2 of the second antibody operably linked to the third conjugation fragment, and a fourth polypeptide from the N-terminus to the C-terminus Comprising the second light chain variable domain VL2 of the second antibody to the C-terminus, which is operably linked to a fourth conjugate fragment; wherein the third conjugate fragment is a receptor, and the fourth conjugate fragment The fragment is a ligand; or the third conjugate fragment is a ligand, and the fourth conjugate fragment is a receptor; the third conjugate fragment/fourth conjugate fragment is conjugated with the first The substance fragment/second conjugate fragment is selected from different receptors/ligands, and the third conjugate fragment and the fourth conjugate fragment are capable of specific binding; or

2. The Fab on the other side of the antibody retains the original CH1 and CL, that is, the second antigen-binding part includes: a third polypeptide, and the third polypeptide includes the second heavy chain of the second antibody from the N-terminus to the C-terminus. Variable domain VH2 operably linked to the antibody heavy chain constant region CH1, and a fourth polypeptide comprising the second light chain variable domain of the second antibody from the N-terminus to the C-terminus VL2, which is operably linked to the antibody light chain constant region CL.

The present invention utilizes the unique specific binding force of the ligand and its receptor to creatively operably connect it to the antigen-binding region (antibody variable region VH/VL). The connection includes binding to one of the antigens. region is connected, and the other antigen-binding region is still connected to CH1 and CL; or both antigen-binding regions are connected to ligands/receptors, but the two antigen-binding regions are connected to different types of ligands/receptors, thereby avoiding different antigens Mismatching occurs between the light and heavy chains in the binding region.

In some embodiments, the bispecific fusion polypeptide provided by the present invention is a multifunctional fusion polypeptide that contains two Fabs, in which CH1 and CL of one Fab are independently replaced by ligands and their receptors, and the other Fab CH1 and CL are not substituted, and the receptor includes both an active site that recognizes and binds ligands and a functional active site that generates a response; the light chain of the first antigen-binding portion does not interact with the second Heavy chain mismatch in the antigen-binding portion. In some embodiments, CH1 and CL of one Fab are independently replaced by the first ligand and its receptor, and CH1 and CL of the Fab on the other side are independently replaced by the second ligand and its receptor, said The first ligand and its receptor are different from the second ligand and its receptor.

The multifunctional fusion protein can not only exert dual target specificity, but also exert biological activity of ligand/receptor transmission. For example, in a specific embodiment, the ligand and its receptor are IL15 and IL15Rα. In addition to the dual-target targeting effect, the multifunctional fusion polypeptide can also present IL-15 to IL15Rα. IL-2/15Rβγ dimer forms a ternary complex, activates JAK and STAT pathways, promotes target cell proliferation and activation, and increases IFN-γ and TNF-α secretion levels; JAK/STAT, Ras/MAPK—enhance proliferation signals ; Up-regulation of Bcl-2 and Bcl-XL (anti-apoptotic proteins), down-regulation of Bim and Puma (pro-apoptotic proteins) - weakening apoptotic signals.

In some embodiments, the bispecific fusion polypeptide has: a first polypeptide, which from the N-terminus to the C-terminus is: [VH1]-[Linker 1]-[IL15]-[Linker 2]- [Fc1], the second polypeptide, from the N terminus to the C terminus is: [VL1]-[Linker 3]-[IL15RA], the third polypeptide, the sequence from the N terminus to the C terminus is: [VH2 ]-[Fc2], and a fourth polypeptide, which in order from N terminus to C terminus is: [VL2]-[CL]; in some embodiments, the bispecific fusion polypeptide has: a first polypeptide, From the N terminus to the C terminus, it is: [VH1]-[Linker 1]-[IL15RA]-[Linker 2]-[Fc1]. The second polypeptide, from the N terminus to the C terminus, is: [ VL1]-[Linker 3]-[IL15], the third polypeptide, which from N-terminus to C-terminus is: [VH2]- -[Fc2], and the fourth polypeptide, which from N-terminus to C-terminus The order is: [VL2]-[CL]. Wherein, the Fc1 and Fc2 are two subunits of the heavy chain constant region Fc, which may be the same or different. Preferably, the Fc constant region is a heterodimer (heterodimer Fc fusion protein); in In some embodiments, the Fc constant regions associate as heterodimers based on KiH, hydrophobic interactions, electrostatic interactions, hydrophilic interactions, and/or increased flexibility. The linker 1, linker 2 and linker 3 are linkers connecting polypeptides, which may be the same or different; in some embodiments, the linker 1, linker 2 and linker 3 are independently selected. From (GxS) y connector, where x is selected from an integer from 1 to 5 and y is selected from an integer from 0 to 6.

In some embodiments, VH1 and VL1 cooperate to form an antigen-binding site that specifically binds TIGIT, and VH2 and VL2 cooperate to form an antigen-binding site that specifically binds PD-L1. In some embodiments, VH1 and VL1 cooperate to form an antigen-binding site that specifically binds PD-L1, and VH2 and VL2 cooperate to form an antigen-binding site that specifically binds TIGIT. In some embodiments, the TIGIT-binding antigen-binding portion includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes or is at least 80% (eg, at least 80%) SEQ ID NO. , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98% or 99%) sequence identity, the light chain variable region includes SEQ ID NO. 74 or has at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85) sequence identity with SEQ ID NO. %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity. In some embodiments, wherein the heavy chain variable region of the antigen-binding portion that binds TIGIT includes HCDR1, HCDR2, and HCDR3 regions, the HCDR1, HCDR2, and HCDR3 respectively include HCDR1, HCDR2, and HCDR3 in SEQ ID NO. 73 , in some embodiments, wherein the light chain variable region includes LCDR1, LCDR2 and LCDR3 regions, and the LCDR1, LCDR2 and LCDR3 respectively include LCDR1, LCDR2 and LCDR3 in SEQ ID NO. 74; in some embodiments , the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT numbering system, or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system. In some embodiments, the antigen-binding portion that binds PD-L1 includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes or has at least 80% the sequence of SEQ ID NO. 36 Identity sequence, the light chain variable region includes SEQ ID NO. 37 or a sequence with at least 80% sequence identity thereto; in some embodiments, the antigen-binding portion that binds PD-L1 includes the heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes SEQ ID NO. 71 or a sequence having at least 80% sequence identity thereto, and the light chain variable region includes SEQ ID NO. 72 or has at least 80% sequence identity thereto Sequences of sequence identity; in some embodiments, the antigen-binding portion that binds PD-L1 includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes SEQ ID NO. 75 or The light chain variable region includes SEQ ID NO. 76 or a sequence having at least 80% sequence identity therewith; the sequence having at least 80% sequence identity may, for example, have At least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98% or 99% sequence identity. In some embodiments, the heavy chain variable region of the antigen-binding portion that binds to PD-L1 includes HCDR1, HCDR2, and HCDR3 regions, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 regions, wherein: 1) the HCDR1 , HCDR2 and HCDR3 respectively include HCDR1, HCDR2 and HCDR3 in SEQ ID NO.36, and the LCDR1, LCDR2 and LCDR3 respectively include LCDR1, LCDR2 and LCDR3 in SEQ ID NO.37; 2) the HCDR1, HCDR2 and HCDR3 respectively include HCDR1, HCDR2 and HCDR3 in SEQ ID NO.71, and the LCDR1, LCDR2 and LCDR3 respectively include LCDR1, LCDR2 and LCDR3 in SEQ ID NO.72; or 3) the HCDR1, HCDR2 and HCDR3 respectively includes HCDR1, HCDR2 and HCDR3 in SEQ ID NO.75, and the LCDR1, LCDR2 and LCDR3 respectively include LCDR1, LCDR2 and LCDR3 in SEQ ID NO.76. In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT numbering system, or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by AbM numbering System definition.

In some embodiments, there is an unnatural interchain bond between IL15 and IL15Rα, and the unnatural interchain bond is formed between a first mutated residue of IL15 and a second mutated residue of IL15Rα, In some technical solutions, the first mutated residue of IL15 is the E mutation at position 90 to C, and the second mutated residue of IL15Rα is the P mutation at position C at position 67; in some technical solutions, the IL15 The first mutated residue is the E mutation at position 93 to C, and the second mutated residue of IL15Rα is the R mutation position C at position 35; the amino acid residue mutation site of IL15 is with reference to SEQ ID NO: 26 The corresponding natural sequence numbering site, the amino acid residue mutation site of IL15Rα is the natural sequence numbering site corresponding to SEQ ID NO: 27. In some embodiments, the IL15 is the amino acid sequence shown in SEQ ID NO. 84 or a mutant sequence thereof. The mutant sequence includes, for example, amino acid mutations selected from D61N, E64Q and/or N65D, and/or selected from N71Q, N79Q and/or N112Q amino acid mutations. The IL15Rα comprises SEQ ID NO.28 or its mutant sequence, SEQ ID NO.77 or its mutant sequence, SEQ ID NO.78 or its mutant sequence, SEQ ID NO.79 or its mutant sequence, SEQ ID NO.80 or Its mutation sequence, or SEQ ID NO. 81 or its mutation sequence; In some embodiments, the mutation sequence includes an amino acid mutation selected from T2A, T81A and/or T86A; In some embodiments, the mutation sequence includes P67C amino acid mutation; in some embodiments, the mutation sequence includes the P67C amino acid mutation, and also includes an amino acid mutation selected from T2A, T81A and/or T86A. In some embodiments, the IL15Rα is SEQ ID NO.77 or a mutant sequence thereof, SEQ ID NO.78 or a mutant sequence thereof, SEQ ID NO.79 or a mutant sequence thereof, SEQ ID NO.80 or a mutant sequence thereof, Or SEQ ID NO.81 or its mutant sequence; in some embodiments, the mutation is a P67C amino acid mutation; in some embodiments, the mutation is P67C and an amino acid mutation selected from T2A, T81A and/or T86A; In some embodiments, the mutation is an amino acid mutation selected from T2A, T81A, and/or T86A. The aforementioned amino acid residue mutation site of IL15Rα refers to the natural sequence numbering site corresponding to SEQ ID NO: 27. In some embodiments, the IL15 is selected from the group consisting of SEQ ID NO. %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity, the IL15RA is selected from the group consisting of SEQ ID NO. .77-81 Any sequence shown has at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity. i. Ligands and receptors

"Receptor" is a substance on the cell membrane or within a cell that can recognize and bind to biologically active molecules. Bioactive substances that can bind to receptors are collectively called "ligands."

According to the location of the receptors in the cell, they are divided into two categories: cell surface receptors and intracellular receptors. The receptor itself contains at least two active sites: one is the active site that recognizes and binds the ligand; the other is the functional active site responsible for generating the response. This site only forms a binary complex with the ligand and is allosteric. Only then can a response be generated, thus initiating a series of biochemical reactions that ultimately lead to biological effects on the target cells.

Receptors are generally glycoproteins. The binding between wild-type receptors and ligands is not mediated by covalent bonds, but mainly relies on ionic bonds, hydrogen bonds, van der Waals forces and hydrophobic interactions. Receptors have characteristics such as saturation, high affinity, and specificity when binding to ligands.

Cooperating receptors and ligands have relatively specific binding affinities and, optionally, biological effects. In some embodiments, the receptor only contains an active site that recognizes and binds a ligand, and does not contain a functional active site that generates a response (eg, a function that activates a biological effect of a downstream signaling pathway). In some embodiments, the receptor and/or ligand is a natural receptor ligand structure. The receptor includes both an active site that recognizes the binding ligand and a functional active site that is responsible for generating a response, and can exert Corresponding biological function, the bispecific fusion protein is a multifunctional fusion protein that not only has bispecificity, but also can function as a ligand receptor.

In some embodiments, the receptor and/or ligand are modified based on the natural sequence, and the modifications include, but are not limited to: truncation, insertion, and/or mutation; the purposes of these modifications include, but are not limited to : Increase or decrease the binding force between ligand and receptor; enhance, decrease or eliminate the biological function of ligand receptor; increase, decrease or eliminate glycosylation sites in receptor and/or ligand protein; decrease or eliminate Ligand toxicity.

In some embodiments, the amino acid sequences of the receptors and/or ligands each independently consist of 10 to 1000 amino acids; in some embodiments, the amino acid sequences of the receptors and/or ligands each independently consist of Composed of 20 to 800 amino acids; in some embodiments, the amino acid sequences of the receptor and/or ligand each independently consist of 30 to 600 amino acids; in some embodiments, the receptor and/or The amino acid sequences of the ligands each independently consist of 40 to 400 amino acids; in some embodiments, the amino acid sequences of the receptor and/or ligand each independently consist of 50 to 300 amino acids; in some embodiments , the amino acid sequences of the receptor and/or ligand each independently consist of 55 to 260 amino acids. For example, the amino acid sequence of the receptor and/or the ligand can also be independently selected from 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900 amino acids.

In some embodiments, the molecular weight of the receptor and/or ligand is each independently selected from 1KD~100KD; In some embodiments, the molecular weight of the receptor and/or ligand is each independently selected from 2KD~80KD; In some embodiments, the molecular weight of the receptor and/or ligand is each independently selected from 3KD~70KD; in some embodiments, the molecular weight of the receptor and/or ligand is each independently selected from 4KD~60KD; In some embodiments, the molecular weight of the receptor and/or ligand is each independently selected from 4KD~50KD; in some embodiments, the molecular weight of the receptor and/or ligand is each independently selected from 4KD~40KD; In some embodiments, the receptor and/or ligand molecular weights are each independently selected from 5 KD to 30 KD. For example, the molecular weight of the receptor and/or ligand can be independently selected from 1KD, 2KD, 3KD, 4KD, 4.5KD, 5KD, 6KD, 7KD, 8KD, 9KD, 10KD, 11KD, 15KD, 18KD, 20KD, 25KD, 30KD , 35KD, 40KD, 45KD, 50KD, 60KD, 70KD, 80KD, 90KD, 100KD.

The binding mode of the receptor (or fragment thereof) and its corresponding ligand (or fragment thereof) may be covalent binding, non-covalent interaction, or a combination thereof; examples of non-covalent bonds include, but are not limited to, hydrogen bonds , hydrophobic bonds, ionic bonds, and van der Waals bonds. In some embodiments, when the affinity between the inserted or replaced conjugated fragments is lower than expected (e.g., the two variable regions in the antigen-binding portion cannot be brought closer to allow them to obtain the function of specifically recognizing the antigen, or Inability to prevent heavy chain mismatching between 2 or more heavy chain constant regions, or inability to prevent mismatching between antigen-binding moieties to achieve a specific VL-VH moiety combination), can be achieved by pairing the antibody with the ligand and/or Or the receptor is modified to increase affinity. In some embodiments, the receptor and the ligand contain at least one non-natural interchain bond, and the non-natural interchain bond can enhance the specific binding force between the receptor and the ligand; in some embodiments wherein the unnatural interchain bond is formed between a first mutated residue comprised by the receptor and a second mutated residue comprised by the ligand; in some embodiments, the first and second mutated residues At least one of the groups is a cysteine residue; in some embodiments, the non-natural interchain bond is a disulfide bond.

"Unnatural interchain linkages" refer to interchain linkages not found in wild-type polypeptide polymers. For example, a non-natural interchain bond can be formed between a mutated amino acid residue of one polypeptide and a mutated amino acid residue of another polypeptide.

In some embodiments, at least one native glycosylation site is absent in the receptor and/or ligand.

In some embodiments, the receptors and ligands are selected from interleukins and their receptors.

The inventor conducted three-dimensional conformation studies on a large number of interleukins and their receptors, and found that the three-dimensional conformations of a large number of interleukins or IFN molecules can be divided into four categories: type A - lift type, type B - bowtie type. , C-baseball hand type, type D-clamp type, as shown in Table 1:

Table 1. Three-dimensional conformation classification Interleukin name Three-dimensional conformation classification Ligand Ligand size receptor Receptor size Interleukin 1α IL-1α 160a.a. IL-1R 310a.a. Interleukin 1β C IL-1β 155a.a. IL1R1+IL1R2 310a.a. interleukin 2 A IL-2 133a.a. IL-2Rα+IL2Rβ+Rγ 220 aa interleukin 3 C IL-3 132a.a. IL-3R 286a.a. interleukin 4 A IL-4 128a.a. IL-4Rα+Rγ 206a.a interleukin 5 D IL-5 115a.a. IL-5R 321a.a. interleukin 6 A IL-6 182a.a. IL-6R 345a.a. interleukin 7 D IL-7 152a.a. IL-7R 218a.a. interleukin 8 IL-8/CXCL8 77a.a. IL-8RA/IL-8RB 350a.a./ 360a.a. interleukin 9 IL-9 136a.a. IL-9R 230a.a. Interleukin 10 B IL-10 160a.a. IL-10R1 224a.a. Interleukin 11 A IL-11 178a.a. IL-11R 346a.a. Interleukin 12 IL-12α/p35 197a.a. IL-12β 306a.a. Interleukin 12 IL-12β/p40 306a.a. IL23A+IL23R+IL12RB1 Interleukin 13 A IL-13 122a.a. IL-13R1 321a.a. Interleukin 15 A IL-15 114a.a. IL-15Rα 175a.a. Interleukin 17 IL-17/CTLA8 132a.a. IL-17R+IL-17RA 287a.a. Interleukin 18 C IL-18 157a.a. IL18R1 164a.a. Interleukin 19 IL-19 177 aa IL20Rα+IL20Rβ Interleukin 20 A IL-20 152a.a. IL20Rα+IL20Rβ Interleukin 21 D IL-21 138a.a. IL21R 213 aa Interleukin 23 D IL-23A/p19 170a.a. IL12B 306a.a. Interleukin 24 A IL24 155a.a. IL20Rα+IL20Rβ Interleukin 27 IL-27A/p28 215a.a. IL-27B 209 aa

In some embodiments, the ligand and its receptor are selected from class A interleukins and their receptors, such as IL15/IL15R, IL2/IL2R, IL4/IL-4Rα+Rγ, IL-6/IL-6R , IL-11/ IL-11R, IL-13/ IL-13R1, IL-20/ IL20Rα+IL20Rβ, IL24/ IL20Rα+IL20Rβ.

In some embodiments, the ligand and its receptor are selected from class D interleukins and their receptors, such as IL7/IL7R, IL21/IL21R, IL23A/IL12B.

In some embodiments, the interleukin and its receptor have the following amino acid sequence in Table 2:

Table 2. Amino acid sequences of interleukins and their receptors Cytokines or their receptors sequence IL2 (human, mature form amino acid sequence) -133a.a. APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO.21) IL2Rα (human, mature form amino acid sequence of IL2Rα extracellular domain)-219a.a. ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTFQIQTEMAATME TSIFTTEYQ（SEQ ID NO.22） IL2Rα (human, mature fully truncated form amino acid sequence of IL2Rα extracellular domain)-166a.a. ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGE (SEQ ID NO. 23) IL2Rα (human, amino acid sequence of mature semi-truncated form of IL2Rα extracellular domain)-191a.a. ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQ ASPEGRPESETS（SEQ ID NO.24） IL-15 (human, mature form amino acid sequence)-114a.a. NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO.25) IL-15Rα (human, extracellular domain amino acid sequence)-175a.a. ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTT (SEQ ID NO.26) IL-15Rαsushi (human, sushi domain amino acid sequence)-77a.a. ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP (SEQ ID NO.27) IL-15Rαsushi (human, sushi domain amino acid sequence)-65a.a. ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO.28) IL-15Rαsushi (human, sushi domain amino acid sequence)-73a.a. ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQR (SEQ ID NO.29) IL-15Rαsushi (human, sushi domain amino acid sequence)-86a.a. ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVT (SEQ ID NO.30) IL-15Rαsushi (human, sushi domain amino acid sequence)-102a.a. ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAAS (SEQ ID NO.31)

In some embodiments, the receptor-ligand combination is selected from IL15 and IL15Rα, and "IL15Rα" and "IL15RA" are interchangeable in this invention. In some embodiments, the IL15 and IL15Rα have the following amino acid sequences:

Table 3. Amino acid sequences of IL15 and IL15Rα Cytokines or their receptors sequence IL-15 (human, mature form amino acid sequence)-114a.a. NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO.25) IL-15Rα (human, extracellular domain amino acid sequence)-175a.a. ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTT (SEQ ID NO.26) IL-15Rαsushi (human, sushi domain amino acid sequence)-77a.a. ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP (SEQ ID NO.27) IL-15Rαsushi (human, sushi domain amino acid sequence)-65a.a. ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO.28) IL-15Rαsushi (human, sushi domain amino acid sequence)-73a.a. ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQR (SEQ ID NO.29) IL-15Rαsushi (human, sushi domain amino acid sequence)-86a.a. ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVT (SEQ ID NO.30) IL-15Rαsushi (human, sushi domain amino acid sequence)-102a.a. ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAAS (SEQ ID NO.31)

Note: In the table, for example, "IL-15Rαsushi (human, sushi domain amino acid sequence)-77a.a." means the polypeptide fragment from the 1st to 77th amino acid residues starting from the N-terminus of the sushi domain of human IL-15Rα. , also labeled as IL15Rαsushi-77a.a., and so on for others.

In some embodiments, the IL15 and IL15Rα comprise at least one non-natural interchain bond. In some embodiments, the non-natural interchain bond is a disulfide bond, and the IL15 and/or the IL15Rα at least comprise One amino acid is mutated to cysteine. In some embodiments, the mutation is located at the contact interface between IL-15 and IL15Rα. In some embodiments, the cysteine mutation of IL15 is selected from E90C. The cysteine mutation of IL15Rα is selected from P67C; in some technical solutions, the IL15 cysteine mutation is selected from E93C, and the cysteine mutation of IL15Rα is selected from R35C. In some embodiments, the IL15 is the mutant sequence of SEQ ID NO.26, and the mutations include E90C mutation and/or E93C mutation; in some embodiments, the IL15Rα is SEQ ID NO.27, 29, 30 Or the mutation sequence of 31, the mutation includes P67C mutation and/or R35C mutation.

In the present invention, the mutation position of the IL15 amino acid residues refers to the natural sequence number corresponding to the amino acid sequence of the mature form of human IL-15 (SEQ ID NO. 26), and the mutation position of the IL15Rα amino acid residues refers to the sushi of human IL-15Rα. The natural sequence number corresponding to the structural domain (SEQ ID NO. 27).

In some embodiments, the D at position 61 of any of the aforementioned IL15 is mutated to N, the E at position 64 is mutated to Q, and/or the N at position 65 is mutated to D. In some embodiments, the IL15 is a mutant sequence of SEQ ID NO. 26, and the mutations include mutations selected from the group consisting of D61N, E64Q, and/or N65D. In some embodiments, the IL15 is a mutant sequence of SEQ ID NO. 26, and the mutations include E90C mutations, and also include D61N, E64Q and/or N65D mutations.

In some embodiments, at least one N-glycosylation site of any of the aforementioned IL15 is absent. In some embodiments, the N-glycosylation site is selected from N71, N79, and/or N112; in some embodiments, In this way, the IL15 contains the following amino acid mutations: N71Q, N79Q and/or N112Q. In some embodiments, the IL15 is a mutated sequence of SEQ ID NO. 26, including mutations selected from N71Q, N79Q and/or N112Q. In some embodiments, the IL15 is a mutant sequence of SEQ ID NO. 26, the mutations include E90C mutations, also include N71Q, N79Q and/or N112Q mutations, and/or also include N71Q, N79Q and/or N112Q mutations .

In some embodiments, at least one O-glycosylation site of any of the aforementioned IL15Rα is absent; in some embodiments, the O-glycosylation site is selected from T2, T81 and/or T86; in some embodiments In this way, the IL15Rα contains the following amino acid mutations: T2A, T81A and/or T86A. In some embodiments, the IL15Rα is a mutant sequence of SEQ ID NO. 27-31, and the mutations include mutations selected from T2A, T81A, and/or T86A; in some embodiments, the P67C mutation is also included.

In some embodiments, the amino acid sequences of IL15 and IL15Rα are as follows in Table 4:

Table 4. Amino acid sequences of IL15 and IL15Rα mutations sequence IL15Rα-175a.a.(P67C) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRD C ALVHORPAPPSTVTTAGVTPOPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPOGHSDTT (SEQ ID NO.77) IL15Rαsushi-77a.a. (P67C) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRD C ALVHQRPAPP (SEQ ID NO.78) IL15Rαsushi-73a.a. (P67C) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRD C ALVHQR (SEQ ID NO.79) IL15Rαsushi-86a.a. (P67C) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRD C ALVHQRPAPPSTVTTAGVT (SEQ ID NO.80) IL15Rαsushi-102a.a. (P67C) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRD C ALVHORPAPPSTVTTAGVTPOPESLSPSGKEPAAS (SEQ ID NO.81) IL15Rα-114a.a. (T2A, T81A, T86A) I A CPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTV A TAGV A PQPESLSPSGKEPAASSPSSNNTAATTA (SEQ ID NO.82) IL15Rαsushi-65a.a. (T2A) I A CPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO.83) IL15-114a.a.(E90C) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECE C LEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO.84) IL15-114a.a.(N71Q, N79Q, N112Q) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISCESGDASIHDTVENLIILA Q NSLSSNG Q VTESGCKECEELEEKNIKEFLQSFVHIVQMFI Q TS (SEQ ID NO.85) IL15-114a.a.(D61N, E64Q, N65D) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIH N TV QD LIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO.86)

Note: The IL15 mutation position refers to the natural sequence number corresponding to the amino acid sequence of the mature form of human IL-15 (SEQ ID NO. 26), and the IL15Rα mutation position refers to the sushi domain of human IL-15Rα (SEQ ID NO. 27 ) corresponds to the natural sequence number.

The insertion or replacement position of the receptor (or its fragment) and the ligand (or its fragment) that cooperate with each other can be located at, for example: the receptor or its fragment is inserted into or replaced with the CL region, and the ligand or its fragment is inserted into or replaced with the CH1 region; Or the receptor or its fragment is inserted into or replaces the CH1 region, and the ligand or its fragment is inserted into or replaces the CL region. ii. Antigen binding part

The bispecific fusion polypeptide provided by the invention includes a first antigen-binding part and a second antigen-binding part, and has two antigen specificities. The first antigen-binding part and the second antigen-binding part are different and may be the first antigen. The binding portion and the second antigen-binding portion bind to different antigens, or the first antigen-binding portion and the second antigen-binding portion bind to different epitopes of the same antigen.

In some embodiments, the target of the bispecific fusion protein is a tumor. In some embodiments, the first antigen-binding portion and the second antigen-binding portion bind to targets expressed on tumor cells; in some embodiments, the first antigen-binding portion binds to a target on tumor cells, and the second antigen-binding portion The target site bound by the moiety is on immune cells; in some embodiments, the target sites bound by the first antigen-binding moiety and the second antigen-binding moiety are both on immune cells.

Redirected killing of T cells is an ideal mechanism of action in many therapeutic areas. Various bispecific antibody formats have been implicated in T cell redirection in preclinical and clinical trials (May C et al (2012) Biochem Pharmacol, 84(9)):1105-1112, pp; Frankel SR, and Baeuerle PA, (2013) CURR OPIN Chemical Biology, Vol. 17(3): pp. 385-92). All T cell retargeting bispecific antibodies or fragments thereof have been engineered to have at least two antigen binding sites, one of which binds to a surface antigen on the target cell and another to which the T cell surface antigen binds . Among T cell surface antigens, the epsilon subunit of human CD3 derived from the TCR protein complex is most commonly targeted as a target for redirecting T cell killing.

Tumor-associated antigens that can be targeted include, but are not limited to: alpha-fetoprotein (AFP), alpha-actinin-4, A3, antigens specific for A33 antibodies, ART-4, B7, Ba 733 , BAGE, BrE3-antigen, CA125, CAMEL, CAP-1, carbonic anhydrase IX, CASP-8/m, CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a- e, CD67, CD70, CD70L, CD74, CD79a, CD80, CD83, CD95, CD126, CD132, CD133, CD138, CD147, CD154, CDC27, CDK-4/m, CDKN2A, CTLA-4, CXCR4, CXCR7, CXCL12, HIF-1α, colon-specific antigen p (CSAp), CEA (CEACAM5), CEACAM6, c-Met, DAM, EGFR, EGFRvIII, EGP-1 (TROP-2), EGP-2, ELF2-M, Ep-CAM , fibroblast growth factor (FGF), Flt-1, Flt-3, folate receptor, G250 antigen, Claudin18.2, GAGE, gp100, GRO-β, HLA-DR, HM1.24, human chorion BCMA gonadotropin (HCG) and its subunits, HER2/neu, HMGB-1, hypoxia-inducible factor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ, IFN- α, IFN-β, IFN-λ, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, insulin-like growth factor-1 (IGF-1), KC4-antigen, KS-1-antigen, KS1-4, Le-Y, LDR/FUT, macrophage migration inhibitory factor (MIF), MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP- 1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1/2, MUM-3, NCA66, NCA95, NCA90, PAM4 antigen, pancreatic cancer mucin, PD-1 receptor, placental growth Factor, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, PlGF, ILGF, ILGF-1R, IL-6, IL-25, RS5, RANTES, T101, SAGE, S100, survivin, survivin-2B , TAC, TAG-72, tenascin, TRAIL receptor, TNF-α, Tn antigen, Thomson-Friedenreich antigen, tumor necrosis antigen, VEGFR, ED-B fibronectin, WT-1, 17-1A-antigen, Complement factors C3, C3a, C3b, C5a, C5, angiogenic markers, bcl-2, bcl-6, Kras, oncogene markers, and oncogene products (see, e.g., Sensi et al., Clin Cancer Res 2006, 12: 5023-32; Parmiani et al. J Immunol 2007, 178: 1975-79; Novellino et al. Cancer Immunol Immunother 2005, 54: 187-207).

Although antibodies or other binding molecules specific for effector T cells preferably bind to the CD3 antigen, other antigens expressed on effector T cells are known and can be targeted by T-cell redirecting complexes. Exemplary T-cell antigens include, but are not limited to, CD2, CD3, CD4, CD5, CD6, CD8, CD25, CD28, CD30, CD40, CD40L, CD44, CD45, CD69, and CD90.

Immune checkpoints are inhibitory pathways in the immune system that are critical for maintaining self-tolerance and regulating the duration and amplitude of physiological immune responses in peripheral tissues to minimize collateral tissue damage. In some embodiments, the targets to which the first antigen-binding portion and the second antigen-binding portion bind are immune checkpoints or their ligands. The immune checkpoint molecules include but are not limited to: TIGIT, PD-1, TIM- 3. LAG3, GTLA4, BTLA, BTN1A1, VISTA, LAIR, CD96, PVRIG, LILRA3, LILRA4, LILRB1, LILRB2, LILRB3, LLRB4, NKG-2A, CD47, CD200R1, CD300, Dectin-1, ICOS, NKp30, CD28, CD28H, CRTAM, DNAM-1, 4-1-BB, BAFF, CD27, CD30, CD40, DR3, GITR, HVEM, LIGHT, OX40, TACI, 2B4, CD2, CD48, CD229, SLAM, SLAMF5, GRAAC, TIM1, TIM4, CD7, DPPIV.

In some embodiments, the target point bound by the first antigen-binding portion is PD-1, and the target point bound by the second antigen-binding portion is PD-L1; in some embodiments, the target point bound by the first antigen-binding portion is PD-1, the target that the second antigen-binding portion binds is TIGIT; in some embodiments, the target that the first antigen-binding portion binds is PD-1, and the target that the second antigen-binding portion binds is GTLA4; in some In embodiments, the target point bound by the first antigen-binding portion is PD-1, and the target point bound by the second antigen-binding portion is LAG3; in some embodiments, the target point bound by the first antigen-binding portion is PD-1, The target that the second antigen-binding portion binds is TIM-3; in some embodiments, the target that the first antigen-binding portion binds is PD-1, and the target that the second antigen-binding portion binds is CD47; in some embodiments, In, the target point bound by the first antigen-binding portion is PD-1, and the target point bound by the second antigen-binding portion is GTLA4; in some embodiments, the target point bound by the first antigen-binding portion is PD-1, and the target point bound by the second antigen-binding portion is PD-1, and the target point bound by the second antigen-binding portion is GTLA4. The target point bound by the antigen-binding portion is 4-1-BB; in some embodiments, the target point bound by the first antigen-binding portion is PD-L1, and the target point bound by the second antigen-binding portion is 4-1-BB; In some embodiments, the first antigen-binding portion binds to a target point that is PD-L1, and the second antigen-binding portion binds to a target point that is TIGIT.

In some embodiments, the first antigen-binding moiety targets a tumor-associated antigen and the second antigen-binding moiety targets an immune checkpoint. In some embodiments, the first antigen-binding moiety targets HER2 and the second antigen-binding moiety targets PD-1; in some embodiments, the first antigen-binding moiety targets VEGF and the second antigen-binding moiety targets PD-1 L1; in some embodiments, the first antigen-binding moiety targets Claudin18.2, and the second antigen-binding moiety targets PD-L1; in some embodiments, the first antigen-binding moiety targets HER2, and the second antigen-binding moiety targets HER2 Targets CTLA-4; in some embodiments, the first antigen binding moiety targets CD20 and the second antigen binding moiety targets CD47; in some embodiments, the first antigen binding moiety targets HER2 and the second antigen binding moiety targets Targeting CD47.

In some embodiments, the first antigen binding moiety and the second antigen binding moiety simultaneously target tumor heterogeneity. Exemplary common targets for tumors include, but are not limited to, HGF and VEGF, IGF-IR and VEGF, Her2 and VEGF, CD19 and CD3, CD20 and CD3, Her2 and CD3, CD19 and FcγRIIIa, CD20 and FcγRIIIa, Her2 and FcγRIIIa. The bispecific fusion polypeptide of the present invention can bind to VEGF and phosphatidylserine; VEGF and ErbB3; VEGF and PLGF; VEGF and ROBO4; VEGF and BSG2; VEGF and CDCP1; VEGF and ANPEP; VEGF and c-MET; HER-2 and ERB3; HER-2 and BSG2; HER-2 and CDCP1; HER-2 and ANPEP; EGFR and CD64; EGFR and BSG2; EGFR and CDCP1; EGFR and ANPEP; IGF1R and PDGFR; IGF1R and VEGF; IGF1R and CD20; CD20 and CD74; CD20 and CD30; CD20 and DR4; CD20 and VEGFR2; CD20 and CD52; CD20 and CD4; HGF and c-MET; HGF and NRP1; HGF and phosphatidylserine; ErbB3 and IGF1R; ErbB3 and IGF1,2; c- Met and Her-2; c-Met and NRP1; c-Met and IGF1R; IGF1,2 and PDGFR; IGF1,2 and CD20; IGF1,2 and IGF1R; IGF2 and EGFR; IGF2 and HER2; IGF2 and CD20; IGF2 and VEGF; IGF2 and IGF1R; IGF1 and IGF2; PDGFRa and VEGFR2; PDGFRa and PLGF; PDGFRa and VEGF; PDGFRa and c-Met; PDGFRa and EGFR; PDGFRb and VEGFR2; PDGFRb and c-Met; PDGFRb and EGFR; RON and c- Met; RON and MTSP1; RON and MSP; RON and CDCP1; VGFR1 and PLGF; VGFR1 and RON; VGFR1 and EGFR; VEGFR2 and PLGF; VEGFR2 and NRP1; VEGFR2 and RON; VEGFR2 and DLL4; VEGFR2 and EGFR; VEGFR2 and ROBO4; VEGFR2 and CD55; LPA and S1P; EPHB2 and RON; CTLA4 and VEGF; CD3 and EPCAM; CD40 and IL6; CD40 and IGF; CD40 and CD56; CD40 and CD70; CD40 and VEGFR1; CD40 and DR5; CD40 and DR4; CD40 and APRIL; CD40 and BCMA; CD40 and RANKL; CD28 and MAPG; CD80 and CD40; CD80 and CD30; CD80 and CD33; CD80 and CD74; CD80 and CD2; CD80 and CD3; CD80 and CD19; CD80 and CD4; CD80 and CD52; CD80 and VEGF; CD80 and DR5; CD80 and VEGFR2; CD22 and CD20; CD22 and CD80; CD22 and CD40; CD22 and CD23; CD22 and CD33; CD22 and CD74; CD22 and CD19; CD22 and DR5; CD22 and DR4; CD22 and VEGF; CD22 and CD52; CD30 and CD20; CD30 and CD22; CD30 and CD23; CD30 and CD40; CD30 and VEGF; CD30 and CD74; CD30 and CD19; CD30 and DR5; CD30 and DR4; CD30 and VEGFR2; CD30 and CD52; CD30 and CD4; CD138 and RANKL; CD33 and FTL3; CD33 and VEGF; CD33 and VEGFR2; CD33 and CD44; CD33 and DR4; CD33 and DR5; DR4 and CD137; DR4 and IGF1,2; DR4 and IGF1R; DR4 and DR5; DR5 and CD40; DR5 and CD137; DR5 and CD20; DR5 and EGFR; DR5 and IGF1,2; DR5 and IGFR; DR5 and HER-2; and EGFR and DLL4. Other target combinations include one or more members of the EGF/erb-2/erb-3 family.

Additionally, exemplary common targets for autoimmune and inflammatory disorders include, but are not limited to, IL-1 and TNFα, IL-6 and TNFα, IL-6 and IL-1, IgE and IL-13, IL-1 and IL-13, IL-4 and IL-13, IL-5 and IL-13, IL-9 and IL-13, CD19 and FcγRIIb, and CD79 and FcγRIIb.

Exemplary targets for treating inflammatory diseases include, but are not limited to: TNF and IL-17A; TNF and RANKL; TNF and VEGF; TNF and SOST; TNF and DKK; TNF and αVβ3; TNF and NGF; TNF and IL- 23p19; TNF and IL-6; TNF and SOST; TNF and IL-6R; TNF and CD-20; IgE and IL-13; IL-13 and IL23p19; IgE and IL-4; IgE and IL-9; IgE and IL-9; IgE and IL-13; IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-9; IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-23p19; IL-13 and IL-9; IL-6R and VEGF; IL-6R and IL-17A; IL-6R and RANKL; IL-17A and IL-1β; IL-1β and RANKL; IL-1β and VEGF; RANKL and CD-20; IL-1α and IL-1β; IL-1α and IL-1β.

Targets involved in rheumatoid arthritis (RA) include, but are not limited to: TNF and IL-18; TNF and IL-12; TNF and IL-23; TNF and IL-1β; TNF and MIF; TNF and IL-17 ; and TNF and IL-15.

Targets for treating systemic lupus erythematosus (SLE) include, but are not limited to: CD20, CD22, CD19, CD28, CD4, CD24, CD37, CD38, CD40, CD69, CD72, CD74, CD79A, CD79B, CD80, CD81, CD83, CD86, IL-4, IL-6, IL10, IL2, IL4, IL11, TNFRSF5, TNFRSF6, TNFRSF8, C5, TNFRSF7, TNFSF5, TNFSF6, TNFSF7, BLR1, HDAC4, HDAC5, HDAC7A, HDAC9, ICOSL, IGBP1, MS4A1, RGSI, SLA2, IFNB1, AICDA, BLNK, GALNAC4S-6ST, INHA, INHBA, KLF6, DPP4, FCER2, R2, ILIR2, ITGA2, ITGA3, MS4A1, ST6GALI, CDIC, CHSTIO, HLA-A, HLA-DRA, NT5E, CTLA4, B7.1, B7.2, BlyS, BAFF, IFN-α and TNF-α.

Targets used to treat multiple sclerosis (MS), including but not limited to: IL-12, TWEAK, IL-23, CXCL13, CD40, CD40L, IL-18, VEGF, VLA-4, TNF, CD45RB, CD200, IFNγ, GM-CSF, FGF, C5, CD52 and CCR2.

Targets used to treat sepsis include, but are not limited to: TNF, IL-1, MIF, IL-6, IL-8, IL-18, IL-12, IL-10, IL-23, FasL, LPS, Toll-like receptors, TLR-4, tissue factor, MIP-2, ADORA2A, IL-1B, CASP1, CASP4, NFκB1, PROC, TNFRSFIA, CSF3, CCR3, ILIRN, MIF, NFκB1, PTAFR, TLR2, TLR4, GPR44 , HMOX1, midkine, IRAK1, NFκB2, SERPINA1, SERPINE1, and TREM1.

To form a bispecific fusion protein of the invention, antibodies may be prepared against any combination of these antigens; i.e., each of these antigens may optionally and independently be included or excluded by the multispecific antibody according to the invention .

In some embodiments, the first antigen binding moiety and the second antigen binding moiety target different epitopes of the same antigen.

In some embodiments, at least one of the two antigen-binding fragments may also include a secretion signal sequence.

The secretion signal sequence refers to a sequence that induces the secretion of the expressed protein or peptide by being connected to the N-terminus of the coding sequence located outside the cell membrane or outside the cell. The signal sequence can be a peptide sequence composed of about 18-30 amino acids. . All proteins that can be transported to the outside of the cell membrane have different signal sequences that are cleaved by signal peptidases on the cell membrane. Generally, for foreign proteins that are not naturally expressed by the host cell, a secretion signal sequence capable of secreting the protein into the periplasm or culture medium, or a modified sequence, may be used.

In some embodiments, VH1 and VL1 cooperate to form an antigen-binding site that specifically binds TIGIT, and VH2 and VL2 cooperate to form an antigen-binding site that specifically binds PD-L1. In some embodiments, VH1 and VL1 cooperate to form an antigen-binding site that specifically binds PD-L1, and VH2 and VL2 cooperate to form an antigen-binding site that specifically binds TIGIT. In some embodiments, the TIGIT-binding antigen-binding portion includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes or is at least 80% (eg, at least 80%) SEQ ID NO. , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98% or 99%) sequence identity, the light chain variable region includes SEQ ID NO. 74 or has at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85) sequence identity with SEQ ID NO. %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity. In some embodiments, wherein the heavy chain variable region of the antigen-binding portion that binds TIGIT includes HCDR1, HCDR2, and HCDR3 regions, the HCDR1, HCDR2, and HCDR3 respectively include HCDR1, HCDR2, and HCDR3 in SEQ ID NO. 73 , in some embodiments, wherein the light chain variable region includes LCDR1, LCDR2 and LCDR3 regions, and the LCDR1, LCDR2 and LCDR3 respectively include LCDR1, LCDR2 and LCDR3 in SEQ ID NO. 74; in some embodiments , the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT numbering system, or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system. In some embodiments, the antigen-binding portion that binds PD-L1 includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes or has at least 80% the sequence of SEQ ID NO. 36 Identity sequence, the light chain variable region includes SEQ ID NO. 37 or a sequence with at least 80% sequence identity thereto; in some embodiments, the antigen-binding portion that binds PD-L1 includes the heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes SEQ ID NO. 71 or a sequence having at least 80% sequence identity thereto, and the light chain variable region includes SEQ ID NO. 72 or has at least 80% sequence identity thereto Sequences of sequence identity; in some embodiments, the antigen-binding portion that binds PD-L1 includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes SEQ ID NO. 75 or The light chain variable region includes SEQ ID NO. 76 or a sequence having at least 80% sequence identity therewith; the sequence having at least 80% sequence identity may, for example, have At least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98% or 99% sequence identity. In some embodiments, the heavy chain variable region of the antigen-binding portion that binds to PD-L1 includes HCDR1, HCDR2, and HCDR3 regions, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 regions, wherein: 1) the HCDR1 , HCDR2 and HCDR3 respectively include HCDR1, HCDR2 and HCDR3 in SEQ ID NO.36, and the LCDR1, LCDR2 and LCDR3 respectively include LCDR1, LCDR2 and LCDR3 in SEQ ID NO.37; 2) the HCDR1, HCDR2 and HCDR3 respectively include HCDR1, HCDR2 and HCDR3 in SEQ ID NO.71, and the LCDR1, LCDR2 and LCDR3 respectively include LCDR1, LCDR2 and LCDR3 in SEQ ID NO.72; or 3) the HCDR1, HCDR2 and HCDR3 respectively includes HCDR1, HCDR2 and HCDR3 in SEQ ID NO.75, and the LCDR1, LCDR2 and LCDR3 respectively include LCDR1, LCDR2 and LCDR3 in SEQ ID NO.76. In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by the IMGT numbering system, or by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by AbM numbering System definition. iii. Heterodimeric Fc fusion protein

In some embodiments, it comprises a heavy chain constant region Fc that is a heterodimer (heterodimeric Fc fusion protein).

The Fc includes but is not limited to the following combinations: CH2; CH2+CH3; CH2+CH3+CH4; Mutations were introduced into the Fc constant region to avoid heavy chain mismatches.

In some embodiments, the introduction of mutations in the Fc constant region is based on KiH technology (Knob-into-Holes), that is, amino acid mutations are introduced in a heavy chain of the Fc constant region, and the introduced amino acid volume is greater than the original amino acid residue volume, A protruding "pestle"-like structure (Knob) is formed, and another mutation is introduced in another chain region of the Fc constant region. The introduced amino acid volume is smaller than the original amino acid residue volume, forming a depression, similar to a "mortar" structure ( Hole), so that the convex heavy chain is more likely to pair with the concave heavy chain, thereby avoiding heavy chain mispairing. This technology was developed by Genentech and is documented in patent application WO1996027011, the full text of which is incorporated into the present invention.

In some embodiments, the introduction of mutations in the Fc constant region is based on electrostatic interactions, such as ART-lg technology, which was developed by Chugai, a subsidiary of Roche, to specifically change the charge of the Fc constant region domain and promote heterologous recombination. The pairing of chains is equivalent to the charge version of KiH technology. This technology is recorded in patent application WO2006106905, the full text of which is incorporated into the present invention.

In some embodiments, the Fc constant region introduces mutations based on SEED technology. SEED heterodimerization is another spatial mutation-based design strategy that utilizes SEED CH3 domains from IgG and IgA (also known as AG SEED CH3 and GA SEED CH3)-derived complementarity of alternating sequences. IgG and IgA CH3 derivatives generate complementary sequences, thus precluding the assembly of homodimers lacking complementarity while assembling two complementary heavy chain heterodimers. This technology is described in patent application WO2007110205, the entire text of which is incorporated into the present invention.

In some embodiments, the mutation introduced into the Fc constant region is based on changes in the isoelectric point, which facilitates the improvement of the heterodimer formation rate and the maintenance of the stability of the Fc region. This technology is recorded in WO2014145806, the full text of which is incorporated into this patent. .

In some embodiments, the Fc constant regions associate as heterodimers based on hydrophilic interactions or increased flexibility.

In some embodiments, the Fc constant region associates as a heterodimer based on any combination of the above techniques, for example, in some embodiments, the Fc constant region is mutated based on a combination of KIH and electrostatic interactions . For example, the XmAb bispecific platform approach can improve the thermal stability of bispecific antibodies by combining electrostatic interactions, CH3 domain conformation, and hydrogen bonding. Specifically, this strategy swaps the Fc side chain mutations of native IgG1 to S364K and K370S heterodimers to form hydrogen bonds between the two, and then performs L368D/K370S substitutions to drive salt bridge interactions to promote heterodimers Formation, patent application WO2014145907, the full text of which is incorporated into this patent.

In some embodiments, all or part of the CH2, CH3 or CH4 region is inserted or replaced with the receptor and its ligand.

In some embodiments, the inserted or replaced region is located independently in the CH2, CH3 or CH4 region, or at any position between adjacent regions (such as the CH1-CH2 junction, CH2-CH3 junction, CH3- CH4 junction);

In some embodiments, when any two of the above constant regions (such as any one of the CL-CH1, CH2-CH2, CH3-CH3 or CH4-CH4 regions) are inserted or replaced, the two matching suffixes of the replacement regions are The affinity between the combined fragments, KD <1×10 ^-3 (M), for example, x×10 ^-4 (M), x×10 ^-5 (M), x×10 ^-6 (M), x×10 ^-7 (M), x×10 ^-8 (M), x×10 ^-9 (M), x×10 ^-10 (M), x×10 ^-11 (M); the value of x can be from 1 to 9, such as 1, 2, 3, 4, 5, 6, 7, 8 or 9.

In some embodiments, the N-terminus and/or C-terminus of the conjugated fragment is linked to the antigen-binding fragment through a linking peptide.

The term "operably linked" means that parts (eg, two polypeptides) are joined by a covalent bond, either directly or via one or more linkers (linking peptides).

In some embodiments, the number of amino acids of the connecting peptide is 1 to 30; it can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30; preferably 5 to 20.

In some embodiments, the amino acids of the connecting peptide are meaningless polypeptides that do not have additional functions other than connecting (such as protein localization, enzyme cleavage sites, etc.).

In some embodiments, the linker peptide is a flexible linker peptide;

In some embodiments, the amino acid sequence of the connecting peptide is selected from one or more of Gly, Ser, Pro, Ala and Glu.

In some embodiments, the amino acid sequence of the connecting peptide is selected from (GGGGS)n, (GGGS)n, (GGS)n, (GS)n or (G)n, where n is selected from 1, 2, 3, 4, 5 or 6.

The linking peptide is usually flexible and can reduce the steric hindrance between the fusion protein and the target protein, which is more conducive to the correct folding of the protein.

In additional embodiments, the linking peptide is a rigid linker peptide; that is, a relatively inflexible peptide linker. Rigid linker peptides do not require a complete lack of flexibility, but rather are less flexible than flexible linker peptides, such as glycine-rich peptide linkers. Due to their relative lack of flexibility, rigid linker peptides reduce the motion of two protein domains (in the present case, the stabilizer protein and the thermostable reverse transcriptase) linked together by the rigid linker peptide. Linking peptides that provide ordered chains (e.g., α-helical structures) can provide rigid linker peptides. For example, arginine, leucine, glutamate, glutamine, and methionine all show a relatively high propensity for helical linker structures. However, nonhelical linkers containing many proline residues can also exhibit significant rigidity. Examples of rigid linker peptides include polylysine and poly-DL-alanine polylysine. A further description of rigid peptide linkers is provided by Wriggers et al., Biopolymers, 80, pp. 736-46 (2005). Furthermore, rigid linker peptides are described in the linker database described by George et al., Protein Engineering, 15, pp. 871-79 (2003). Preferably, the rigid linker peptide is also a non-cleavable linker peptide, ie, a non-cleavable rigid linker peptide. isolated nucleic acid

The invention also relates to isolated nucleic acids encoding bispecific fusion polypeptides or multifunctional fusion proteins as described above.

The term "isolated nucleic acid" as used herein refers to a deoxyribonucleic acid or ribonucleic acid polymer present in single- or double-stranded form. The isolated nucleic acids include RNA genomic sequences, DNA (gDNA and cDNA) or RNA sequences transcribed from DNA, and, unless otherwise specified, the polypeptides also include natural polynucleotides, sugars, or base-altered analogs. According to one aspect of the invention, the polynucleotide is a light chain polynucleotide.

The isolated nucleic acid includes a nucleotide sequence encoding the amino acid sequence of the protein complex, and also includes a nucleotide sequence complementary thereto. The complementary sequence includes a completely complementary sequence and a substantially complementary sequence, which refers to a sequence that hybridizes to the nucleotide sequence encoding the amino acid sequence of the protein complex under stringent conditions known in the art.

Furthermore, the nucleotide sequence encoding the amino acid sequence of the protein complex may be altered or mutated. Such changes include additions, deletions, or non-conservative or conservative substitutions. A polynucleotide encoding an amino acid sequence of a protein complex may be construed as including a nucleotide sequence that is substantially identical to the isolated nucleic acid. The substantial identity is achieved by aligning the nucleotide sequence with another random sequence in a manner that maximizes correspondence between them. When the aligned sequences are analyzed using algorithms common in the art, the sequence may show greater than 80 % homology, greater than 90% homology, or greater than 95% homology. carrier

The invention also relates to vectors containing nucleic acids as described above.

The term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell. Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses , Papilloma vacuolating virus (such as SV40). The vector may contain a selectable marker (such as a tag that facilitates enrichment, such as his tag; or a tag that facilitates detection, such as GFP), and an origin of replication that matches the cell type specified by the cloning vector, and the expression vector then contain regulatory elements necessary to affect expression in a given target cell such as enhancers, promoters, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and polymerization U sequence, etc.). The vectors may be cloning vectors and expression vectors. When expressing or preparing antibodies or fragments, prokaryotic expression vectors and eukaryotic expression vectors are often involved. Prokaryotic expression vectors commonly use PET series and pGEX series, and eukaryotic expression vectors commonly use pcDNA3.1, pcDNA3.4, pcDNA4, pEGFP-N1, pEGFP-N1, pSV2, etc.

In the present invention, the vector can be a composition, for example, a mixture of multiple plasmids, with different plasmids carrying part of the antibody or fragment thereof. host cell

The invention also relates to host cells containing nucleic acids as described above or vectors as described above.

A variety of cultured host cells that can be used include, for example, prokaryotic cells, eukaryotic cells, bacterial cells (such as Escherichia coli or Bacillus stearothermophilus), fungal cells (such as Saccharomyces cerevisiae or Pichia pastoris), insect cells. Cells (such as lepidopteran cells including Spodoptera frugiperda cells) or mammalian cells (such as Chinese hamster ovary (CHO) cells, NS0 cells, baby hamster kidney (BHK) cells, monkey kidney cells, HeLa cells, human liver cells cancer cells or 293 cells etc.).

Methods for preparing bispecific fusion polypeptides or multifunctional fusion proteins

Any method known in the art can be used to prepare the bispecific fusion polypeptide or multifunctional fusion protein of the present invention.

For example: transform host cells with vectors as described above; culturing the transformed host cells; and Collect bispecific fusion polypeptides or multifunctional fusion proteins expressed in host cells.

In particular, the following methods can be used.

Early methods for constructing bispecific antibodies include chemical cross-linking methods or hybrid hybridoma or quadrivalent tumor methods (for example, Staerz UD et al., Nature, 314:628-31, 1985; Milstein C et al., Nature, 305:537 -540, 1983; Karpovsky B et al., J. Exp. Med., 160:1686-1701, 1984). The chemical coupling method is to link two different monoclonal antibodies together through chemical coupling to prepare bispecific monoclonal antibodies. For example the chemical conjugation of two different monoclonal antibodies, or for example the chemical conjugation of two antibody fragments such as two Fab fragments. Hybrid-Hybridoma method is to produce bispecific monoclonal antibodies through cell hybridization or ternary hybridoma. These cell hybridomas or ternary hybridomas are fused through the established hybridomas, or the established hybridomas are fused with each other from childhood. Obtained by fusion of lymphocytes obtained from mice. Although these techniques are used to make BiAbs, various production issues make such complexes difficult to use, such as the generation of mixed populations containing different combinations of antigen binding sites, difficulties with protein expression, the need to purify the target BiAb, low yields, and production costs. higher.

Recent approaches utilize genetically engineered constructs that are capable of producing a homogeneous product of a single BiAb without the need for extensive purification to remove unwanted by-products. Such constructs include tandem scFv, diabodies, tandem diabodies, dual variable domain antibodies and heterodimerization using motifs such as Ch1/Ck domains or DNLTM (Chames & Baty, Curr. Opin. Drug. Discov. Devel., 12:276-83, 2009; Chames & Baty, mAbs, 1:539-47). Relevant purification techniques are well known.

Antibodies can also be produced using single lymphocyte antibody methods by cloning and expressing immunoglobulin variable region cDNAs produced by single lymphocytes selected for production of specific antibodies, for example by Babcook J et al., Proc. Natl. Acad. Sci. USA. 93:7843-7848, 1996; Methods described in WO 92/02551; WO 2004/051268 and WO 2004/106377.

Antigenic polypeptides for use in generating, for example, immunizing a host or for panning for antibodies, such as for phage display (or yeast cell or bacterial cell surface expression) can be obtained from a genetically engineered host containing an expression system by methods well known in the art. Cells are prepared, or they can be recovered from natural biological sources. For example, nucleic acids encoding one or both polypeptide chains of a bispecific antibody can be introduced into cultured host cells by a variety of known methods (eg, transformation, transfection, electroporation, bombardment with nucleic acid-coated microparticles, etc.). In some embodiments, the nucleic acid encoding the bispecific antibody can be inserted into a vector suitable for expression in the host cell before being introduced into the host cell. Typically such vectors may contain sequence elements that enable expression of the inserted nucleic acid at both the RNA and protein levels.

Bispecific antibodies of the invention, or portions thereof, may be used to detect any or all of these antigens by conventional immunological assays, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or tissue immunohistochemistry. (e.g. in biological samples such as serum or plasma). The present invention provides a method for detecting an antigen in a biological sample. The method includes: contacting the biological sample with a bispecific antibody of the present invention that can specifically recognize the antigen, or an antibody partial antigen, and detecting the antigen-binding An antibody (or antibody portion), or a non-binding antibody (or antibody portion), thereby detecting said antigen in said biological sample. The antibodies are directly or indirectly labeled with a detectable substance to facilitate detection of bound or unbound antibodies. Suitable detectable substances include various enzymes, repair groups, fluorescent substances, luminescent substances and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, acetylcholinesterase; examples of suitable repair group complexes include streptavidin/biotin and Avidin/Biotin; examples of suitable fluorescent substances include 7-hydroxycoumarin, fluorescein, fluorescein isothiocyanate, rhodopsin B, dichlorotriazinylamine fluorescein, fluorescein, Sulfonyl chloride or phycoerythrin; examples of luminescent substances include 3-aminophthaloyl cyclohydrazine; examples of suitable radioactive substances include I ¹²⁵ , I ¹³¹ , ³⁵ S or ³ H. pharmaceutical composition

The bispecific fusion polypeptide or multifunctional fusion protein of the present invention or the nucleic acid encoding the same can be used to prepare pharmaceutical compositions or sterile compositions, for example, the bispecific fusion polypeptide or multifunctional fusion protein and a pharmaceutically acceptable carrier , excipients or stabilizers. Pharmaceutical compositions may include one or a combination (eg, two or more different) of the antibodies of the invention and functional fragments thereof. For example, a pharmaceutical composition of the invention may comprise a combination of antibodies or antibody fragments (or immunoconjugates) with complementary activities that bind to different epitopes on the target antigen. Formulations of therapeutic and diagnostic agents can be prepared by mixing with pharmaceutically acceptable carriers, excipients or stabilizers in the form of, for example, lyophilized powders, slurries, aqueous solutions or suspensions.

The bispecific fusion polypeptide or multifunctional fusion protein in the pharmaceutical composition may be in a form combined with a second activator (functional molecule). The second activator can be a random functional molecule that can prevent or treat the target disease, and can include compounds, peptides, polypeptides, nucleic acids, carbohydrates, lipids, or inorganic particles. In the pharmaceutical composition, the bispecific fusion polypeptide or multifunctional fusion protein may itself have therapeutic activity; but it may function to target the second activator to a specific disease area. The disease areas may be those organs, tissues, or cells in which bispecific antibodies that specifically bind to the antigen are accumulated and distributed. Drugs targeting the disease area are present in high concentrations such that the drug effect is increased compared to the amount injected. Therefore, the pharmaceutical composition can be used to treat drug-resistant tumors and can reduce side effects and adverse drug reactions due to non-specific drug distribution.

The activator containing bispecific fusion polypeptide or multifunctional fusion protein in the pharmaceutical composition can be contained in microcapsules, or contained in colloidal drug delivery systems (such as liposomes, albumin globules, microemulsions, nanoparticles and Nanocapsules), or contained in macroemulsions, which can be prepared by techniques such as coacervation or interfacial polymerization, examples being hydroxymethylcellulose or gelatin microcapsules and poly-( Methyl methacrylate) microcapsules. Medical uses and treatments

The present invention also relates to the use of the bispecific fusion polypeptide or multifunctional fusion protein as described above in the preparation of drugs for treating diseases.

The present invention also relates to the bispecific fusion polypeptide or multifunctional fusion protein as described above for use as a medicament; the medicament is used to treat diseases.

According to one aspect of the invention, the disease may be, for example, cancer, immune disorders, metabolic diseases and microbial infections.

The term "cancer" refers to a large group of diseases characterized by the uncontrolled growth of abnormal cells in the body. "Cancer" includes benign and malignant cancers as well as dormant tumors or micrometastases.

In some embodiments, the microorganism in the microbial infection may be a foreign pathogen or a population of cells harboring a foreign pathogen, such as a virus. The invention is applicable to exogenous pathogens such as bacteria, fungi, viruses, mycoplasma and parasites. Pathogens that may be treated with the present invention may be any infectious organism known in the art that causes disease in animals, including organisms such as Gram-negative or Gram-positive cocci or bacilli, DNA viruses, and RNA viruses. , including but not limited to DNA viruses such as papillomaviruses, parvoviruses, adenoviruses, herpesviruses, and vaccinia viruses, and DNA viruses such as arenaviruses, coronaviruses, rhinoviruses, respiratory syncytial viruses, influenza viruses, and picornaviruses. RNA viruses of viruses, paramyxoviruses, reoviruses, retroviruses and rhabdoviruses. Of particular interest are antibiotic-resistant bacteria, such as antibiotic-resistant Streptococcus species and Staphlococcus species, or bacteria that are susceptible to antibiotics but cause recurrent infections treated with antibiotics, leading to the eventual development of resistant organisms. germ. Such organisms can be treated with the ligand-immunogen conjugates of the present invention in combination with lower doses of antibiotics than would normally be administered to patients to avoid the development of these antibiotic-resistant bacterial strains. The present invention also applies to any fungus, mycoplasma species, parasite or other infectious organism that causes disease in animals. Examples of fungi that may be treated by the methods of the present invention include fungi that grow like molds or yeasts, including, for example, fungi that cause diseases such as: ringworm, histoplasmosis, blastomycosis, aspergillosis, cryptococcosis, sporotrichosis Mycosis, coccidioidomycosis, coccidioidomycosis and candidiasis. The present invention can be used to treat parasitic infections, including but not limited to infections caused by the following parasites: tapeworms, schistosomiasis, histosomes, amoebae, and Plasmodium, Trypanosoma, Lechon Leishmania and Toxoplasma species. Parasites of particular interest are those that express folate receptors and bind folate; however, there are numerous references in the literature to ligands that exhibit high affinity for infectious organisms. For example, penicillins and cephalosporins, which are known for their antibiotic activity and which bind specifically to bacterial cell wall precursors, may also be used as ligands in the preparation of ligand-immunogen conjugates for use according to the invention. The ligand-immunogen conjugates of the present invention can also be targeted to cell populations harboring endogenous pathogens, wherein the pathogen-specific antigen is preferentially expressed on the surface of cells harboring the pathogen and serves as a target specific for the antigen. Receptors for sexually binding ligands.

The present invention also relates to a method of preventing and/or treating and administering a therapeutically effective amount of a pharmaceutical composition to prevent and/or treat diseases as described above.

The methods of the invention can be used in human clinical medicine and veterinary medicine applications. Thus, the host animal harboring the pathogenic organism population and treated with the ligand-immunogen conjugate may be a human, or in the case of veterinary applications, a laboratory animal, an agricultural animal, a domestic animal, or a wild animal. The present invention may be applicable to host animals including, but not limited to, humans; laboratory animals such as rodents (e.g. mice, rats, hamsters, etc.), rabbits, monkeys, chimpanzees; domesticated animals such as dogs, cats and rabbits ; Agricultural animals, such as cattle, horses, pigs, sheep, goats; and captive wild animals, such as bears, pandas, lions, tigers, leopards, elephants, zebras, giraffes, gorillas, dolphins and whales.

Pharmaceutical compositions can be injected into entities, including rats, mice, domestic animals, and/or humans, by a variety of routes. All injection methods are contemplated, for example, oral, rectal, intravenous, nasal, abdominal, subcutaneous, or local injections are possible. The compositions may be injected using other methods known in the art.

"Therapeutically effective dose" in this article refers to a sufficient amount that can treat the disease based on a reasonable profit-loss ratio. The therapeutically effective amount may vary for a variety of reasons attributable to the patient, such as disease type, severity, onset, age of the entity, weight, excretion rate, susceptibility to reactions, health status, and/or complications; and /or drug activity, injection route, injection cycle and number of injections, and/or drug combination; can also be appropriately selected according to the purpose of treatment by those of ordinary skill in the art. For example, the injection amount can be randomly divided into multiple times so that the amount is about 0.001-100 mg/kg adult body weight.

Bispecific fusion polypeptides or multifunctional fusion proteins of the invention or nucleic acids or polynucleotides encoding antibodies of the invention may also be administered in combination with, for example, standard cancer treatments (eg, surgery, radiation, and chemotherapy). For example, anti-tumor therapy using the compositions of the invention and/or effector cells equipped with these compositions is used in combination with chemotherapy. Non-limiting examples of antibody combination treatments of the invention include surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and combinations thereof.

The embodiments of the present invention will be described in detail below with reference to examples. Example 1 , FiBody design

In some embodiments, FiBody is a bispecific antibody obtained by recombination by replacing CL and CH1 on one side of the bispecific antibody by utilizing the specific affinity between the ligand and its receptor, which can avoid or reduce the bispecific antibody. A mismatch occurs between the light chain and the heavy chain.

In this embodiment, interleukin and its receptor are used as an example to construct FiBody. Interleukin and its receptor are divided into four categories according to their three-dimensional conformation, as shown in Table 5 below and Figure 5:

Table 5. FiBody classification three-dimensional conformation Classification number Interleukins and their receptors lifting type A IL15/IL15R, IL2/IL2R, IL4/ IL-4Rα+Rγ, IL-6/ IL-6R, IL-11/ IL-11R, IL-13/ IL-13R1, IL-20/ IL20Rα+IL20Rβ, IL24/ IL20Rα+IL20Rβ Bow tie type B IL10/IL10R1, IL22/IL22R baseball hand C IL1β/ILR1, IL3/IL3R, IL5/IL5R, IL18/IL18R1 clamp type D IL7/IL7R, IL21/ IL21R, IL23A/ IL12B

Bispecific antibodies were constructed based on the above four types of interleukins and their receptors. Example 2. Construction of FiBody based on interleukin and its receptor

Select the VH targeting the first antibody to be connected to the receptor protein through a Linker, and then connect to the Fc of the antibody through Hinge; the VL targeting the first antibody is connected to the ligand protein through a Linker to reduce or avoid the interaction between the light chain and the heavy chain. Chain mismatch occurs; the other end is the complete Fab structure targeting the second antibody (anti-TIGIT antibody). The Fc of the first antibody and the Fc of the second antibody have conventional KiH modifications to reduce or avoid heavy chain mismatches. match. or

Select the VH targeting the first antibody and connect it to the ligand protein through a linker, and then connect it to the Fc of the antibody through Hinge; the VL targeting the second antibody is connected to the receptor protein (IL15) through a linker to reduce or avoid mild disease. The chain and heavy chain mismatch occurs; the other end is the complete Fab structure targeting the first antibody. The Fc that makes up the first antibody and the Fc that makes up the second antibody have conventional KiH modifications to reduce or avoid heavy chain mismatches. Some exemplary FiBody molecular structures and sequence lists constructed are shown in Table 6 and Table 7

Table 6. FiBody molecular structure Molecule number Molecule name Antibody structure replacement method Three-dimensional conformation classification Heavy chain CH1 is replaced The light chain CL is replaced Molecule 1 R0950 IL15RA IL15 A Molecule 2 R0951 IL15RA IL15 A Molecule 3 R0952 IL15 IL15RA A Molecule 4 R1115 IL2RA IL2 A Molecule 5 R1116 IL2 IL2RA A Molecule 6 R1117 IL22RA_D1D2 IL22 B Molecule 7 R1118 IL22RA_D1 IL22 B Molecule 8 R1119 IL18R1_D1D2 IL18 C Molecule 9 R1120 IL18R1_D1D2D3 IL18 C Molecule 10 R1123 IL21R IL21 D

Table 7. Exemplary FiBody molecule sequences Molecule 1 R0950 : PD-L1_VH_IL15RA/PD-L1_VL_IL15/TIGIT-Fab ( Figure 7 ) first polypeptide @PD-L1_VH_IL15RA_Fc-Knob (SEQ ID NO.1) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGITCPPPMSVEHADIWVKSYSLYSRERY ICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQ TQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLWCMVTDFMPEDIYVEWTNNGKTELNYDNTEPVLDSDGSYFMYSDLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK second polypeptide @PD-L1_VL_IL15 (SEQ ID NO.2) QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVLGGGGSGGGGSGGGGSGGGGSGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQ VISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS third polypeptide @TIGIT_VH_CH1_Fc-Hole (SEQ ID NO.3) QVQLESEGGLFKPTDTLTLTCTVSGSSLSSSSYMSWVRQAPGKGLEWIGIIGSNGNTYYANWAKGRFTISKTSTTVELKITSPTTEDTATYFCARGGYRTSGMDPWGPGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSD LYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCRVTDFMPEDIYVEWTNNGK TELNYKNTEPVLKSDGSYFMASKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK Fourth polypeptide @TIGIT_VL_CL (SEQ ID NO.4) DIVMTQTPASVEVAVGGTVTIKCQASQSISSYLNWYQQKPGQPPKLLIYDALKLASGVPSRFSGSGSGTEYTLTISGVESADAATYYCQQEHSVGNVDNVFGGGTEVVVKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTY SMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC Molecule 2 R0951 : TIGIT_VH_IL15RA/TIGIT_VL_IL15/PD-L1-Fab first polypeptide @TIGIT_VH_IL15RA_Fc-Knob (SEQ ID NO.5) QVQLESEGGLFKPTDTLTLTCTVSGSSSSSYMSWVRQAPGKGLEWIGIIGSNGNTYYANWAKGRFTISKTSTTVELKITSPTTEDTATYFCARGGYRTSGMDPWGPGTLVTVSSGGGGSGGGGSGGGGSGGGGSGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAG TSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNS TLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLWCMVTDFMPEDIYVEWTNNGKTELNYDNTEPVLDSDGSYFMYSDLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK second polypeptide @TIGIT_VL_IL15 (SEQ ID NO.6) DIVMTQTPASVEVAVGGTVTIKCQASQSISSYLNWYQQKPGQPPKLLIYDALKLASGVPSRFSGSGSGTEYTLTISGVESADAATYYCQQEHSVGNVDNVFGGGTEVVVKGGGGSGGGGSGGGGSGGGGSGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCF LLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS third polypeptide @PD-L1_VH_CH1_Fc-Hole (SEQ ID NO.7) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGV HTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEMTKKQVTLTCRVTDFMPEDI YVEWTNNGKTELNYKNTEPVLKSDGSYFMASKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK Fourth polypeptide @PD-L1_VL_CL (SEQ ID NO.8) QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVLRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSST LTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC Molecule 3 R0952 : TIGIT_VH_IL15/TIGIT_VL_IL15 RA/PD-L1-Fab ( Figure 7 ) first polypeptide @TIGIT_VH_IL15_Fc-Knob (SEQ ID NO.9) QVQLESEGGLFKPTDTLTLTCTVSGSLSSSYMSWVRQAPGKGLEWIGIIGSNGNTYYANWAKGRFTISKTSTTVELKITSPTTEDTATYFCARGGYRTSGMDPWGPGTLVTVSSGGGGSGGGGSGGGGSGGGGSGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAM KCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPE EEMTKKQVTLWCMVTDFMPEDIYVEWTNNGKTELNYDNTEPVLDSDGSYFMYSDLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK second polypeptide @TIGIT_VL_IL15RA (SEQ ID NO.10) DIVMTQTPASVEVAVGGTVTIKCQASQSISSYLNWYQQKPGQPPKLLIYDALKLASGVPSRFSGSGSGTEYTLTISGVESADAATYYCQQEHSVGNVDNVFGGGTEVVVKGGGGSGGGGSGGGGSGGGGSGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTS SLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTT third polypeptide @PD-L1_VH_CH1_Fc-Hole (SEQ ID NO.7) Fourth polypeptide @PD-L1_VL_CL (SEQ ID NO.8) Molecule 4 R1115 : TIGIT_VH_IL2RA/TIGIT_VL_IL2/PD-L1-Fab first polypeptide @TIGIT_VH_IL2RA_Fc-Hole(SEQ ID NO.32) QVQLVQSGAEVKKPGSSVKVSCKASGYSFTSYWMNWVRQAPGQGLEWIGMIRPSDSETRLNQMFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAGIHDYGHGAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGELCDDDPPEIPHATFKAMAYKEGTMLNCEC KRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCRVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK second polypeptide @TIGIT_VL_IL2(SEQ ID NO.33) DIQMTQSPSSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKSPKLLVYAASHLPDGVPSRFSGSGSGTDYSLTISSLQPEDFATYYCQHFWGTPRTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSGAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFY MPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT third polypeptide @PD-L1_VH_CH1_Fc-Knob(SEQ ID NO.34) EVQLQESGPGLVKPSETLSLTCAVYGDSITSGYWNWIRKPPGKGLEYMGYISYTGSTYQNPSLKSRITFSRDTSKNQYYLKLSSVTAADTATYYCARSRAWIRTYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLV KGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Fourth polypeptide @PD-L1_VL_CL(SEQ ID NO.35) EIVLTQSPDFQSVTPKEKVTITCSVSSSISSSNLHWYQQKPDQSPKLLIYGTSNLASGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQWSSYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Molecule 5 R1116 : TIGIT_VH_IL2/TIGIT_VL_IL2RA/PD-L1-Fab first polypeptide @TIGIT_VH_IL2_Fc-Hole(SEQ ID NO.39) QVQLVQSGAEVKKPGSSVKVSCKASGYSFTSYWMNWVRQAPGQGLEWIGMIRPSDSETRLNQMFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAGIHDYGHGAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGAPTSSSTKKTQLQLEHLLLDLQMILNG INNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCRVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK second polypeptide @TIGIT_VL_IL2RA(SEQ ID NO.40) DIQMTQSPSSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKSPKLLVYAASHLPDGVPSRFSGSGSGTDYSLTISSLQPEDFATYYCQHFWGTPRTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSGELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHS SWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTG third polypeptide @PD-L1_VH_CH1_Fc-Knob(SEQ ID NO.34) Fourth polypeptide @PD-L1_VL_CL(SEQ ID NO.35) Molecule 6 R1117 : TIGIT_VH_IL22RA_D1D2/TIGIT_VL_IL22/PD-L1-Fab first polypeptide @TIGIT_VH_IL22RA_D1D2_Fc-Hole(SEQ ID NO.41) QVQLVQSGAEVKKPGSSVKVSCKASGYSFTSYWMNWVRQAPGQGLEWIGMIRPSDSETRLNQMFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAGIHDYGHGAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGTQSTHESLKPQRVQF QSRNFHNILQWQPGRALTGNSSVYFVQYKIMFSCSMKSSHQKPSGCWQHISCNFPGCRTLAKYGQRQWKNKEDCWGTQELSCDLTSETSDIQEPYYGRVRAASAGSYSEWSMTPRFTPWWETKIDEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCRVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK second polypeptide @TIGIT_VL_IL22(SEQ ID NO.42) DIQMTQSPSSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKSPKLLVYAASHLPDGVPSRFSGSGSGTDYSLTISSLQPEDFATYYCQHFWGTPRTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSGAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVS MSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI third polypeptide @PD-L1_VH_CH1_Fc-Knob(SEQ ID NO.34) Fourth polypeptide @PD-L1_VL_CL(SEQ ID NO.35) Molecule 7 R1118 : TIGIT_VH_IL22RA_D1/TIGIT_VL_IL22/PD-L1-Fab first polypeptide @TIGIT_VH_IL22RA_D1_Fc-Hole (SEQ ID NO.43) QVQLVQSGAEVKKPGSSVKVSCKASGYSFTSYWMNWVRQAPGQGLEWIGMIRPSDSETRLNQMFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAGIHDYGHGAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGTQSTHESLKPQRVQFQSR NFHNILQWQPGRALTGNSSVYFVQYKIMFEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCRVKGFYPSDIAVEWES NGQPENNYKTTPPVLKSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK second polypeptide @TIGIT_VL_IL22(SEQ ID NO.42) third polypeptide @PD-L1_VH_CH1_Fc-Knob(SEQ ID NO.34) Fourth polypeptide @PD-L1_VL_CL(SEQ ID NO.35) Molecule 8 R1119 : TIGIT_VH_IL18R1_D1D2/TIGIT_VL_IL18/PD-L1-Fab first polypeptide @TIGIT_VH_IL18R1_D1D2_Fc-Hole : (SEQ ID NO.44) QVQLVQSGAEVKKPGSSVKVSCKASGYSFTSYWMNWVRQAPGQGLEWIGMIRPSDSETRLNQMFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAGIHDYGHGAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGCTSRPHITVVEGEPF YLKHCSCSLAHEIETTTKSWYKSSGSQEHVELNPRSSSSRIALHDCVLEFWPVELNDTGSYFFQMKNYTQKWKLNVIRRNKHSCFTERQVTSKIVEVKKFFQITCENSYYQTLVNSTSLYKNCKKLLLENNKNPTIKKNAEFEDQGYYSCVHFLHHNGKLFNITKTFNITIVEDRSNIVEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCRVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK second polypeptide @TIGIT_VL_IL18(SEQ ID NO.45) DIQMTQSPSSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKSPKLLVYAASHLPDGVPSRFSGSGSGTDYSLTISSLQPEDFATYYCQHFWGTPRTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSGYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFI ISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED third polypeptide @PD-L1_VH_CH1_Fc-Knob(SEQ ID NO.34) Fourth polypeptide @PD-L1_VL_CL(SEQ ID NO.35) Molecule 9 R1120 : TIGIT_VH_IL18R1_D1D2D3/TIGIT_VL_IL18/PD-L1-Fab first polypeptide @TIGIT_VH_IL18R1_D1D2D3_Fc-Hole(SEQ ID NO.46) QVQLVQSGAEVKKPGSSVKVSCKASGYSFTSYWMNWVRQAPGQGLEWIGMIRPSDSETRLNQMFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAGIHDYGHGAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGCTSRPHITVVEGE PFYLKHCSCSLAHEIETTTKSWYKSSGSQEHVELNPRSSSRIALHDCVLEFWPVELNDTGSYFFQMKNYTQKWKLNVIRRNKHSCFTERQVTSKIVEVKKFFQITCENSYYQTLVNSTSLYKNCKKLLLENNKNPTIKKNAEFEDQGYYSCVHFLHHNGKLFNITKTFNITIVEDRSNIVPVLLGPKLNHVAVELGKNVRLNCSALLNEEDVIY WMFGEENGSDPNIHEEKEMRIMTPEGKWHASKVLRIENIGESNLNVLYNCTVASTGGTDTKSFIEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCRVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK second polypeptide @TIGIT_VL_IL18(SEQ ID NO.45) third polypeptide @PD-L1_VH_CH1_Fc-Knob(SEQ ID NO.34) Fourth polypeptide @PD-L1_VL_CL(SEQ ID NO.35) Molecule 10 R1123 : TIGIT_VH_IL21R/TIGIT_VL_IL21/PD-L1-Fab first polypeptide @TIGIT_VH_IL21R_Fc-Hole(SEQ ID NO.47) QVQLVQSGAEVKKPGSSVKVSCKASGYSFTSYWMNWVRQAPGQGLEWIGMIRPSDSETRLNQMFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAGIHDYGHGAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGCPDLVCYTDYLQTVICILEMWNLHP STLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFMADDIFSVQITDQSGQYSQECGSFLLAESIKPAPPFDVTVTFSGQYQISWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKEEPKSSDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCRVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK second polypeptide @TIGIT_VL_IL21(SEQ ID NO.48) DIQMTQSPSSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKSPKLLVYAASHLPDGVPSRFSGSGSGTDYSLTISSLQPEDFATYYCQHFWGTPRTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSGHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVET NCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS third polypeptide @PD-L1_VH_CH1_Fc-Knob(SEQ ID NO.34) Fourth polypeptide @PD-L1_VL_CL(SEQ ID NO.35) Example 3 Bispecific antibodies with disulfide bond modification

3.1 In order to further improve the stability of the bispecific antibody and extend the half-life of the bispecific antibody, the disulfide bond of the bispecific antibody is modified, see Figure 8.

Ligand receptor disulfide bond modification: select the VH targeting the second antibody (anti-TIGIT) and connect it to the receptor protein (IL15RA) through the Linker, and then connect it to the Fc of the antibody through Hinge; the VL targeting the second antibody is connected through the Linker Connected to the ligand protein (IL15); the other end is the complete Fab structure targeting the first antibody. The Fc that makes up the first antibody and the Fc that makes up the second antibody have conventional KiH modifications to avoid heavy chain mismatching. At the same time, the receptor and ligand proteins are mutated in order to form intermolecular disulfide bonds and further improve the stability of the molecules. For example, Table 8 below:

Table 8. Amino acid sequence of disulfide bond modified bispecific antibodies Molecule number R0954 : TIGIT_VH_IL15RA ( D96+C97 ) / TIGIT_VL_IL15 ( E87C ) / PD-L1-Fab first polypeptide @TIGIT_VH_IL15RA（D96+C97）_Fc-Knob (SEQ ID NO.13) QVQLESEGGLFKPTDTLTLTCTVSGSSLSSSYMSWVRQAPGKGLEWIGIIGSNGNTYYANWAKGRFTISKTSTTVELKITSPTTEDTATYFCARGGYRTSGMDPWGPGTLVTVSSGGGGSGGGGSGGGGSGGGGSGITCPPPMSVEHADIWVKSYS LYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVE VHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLWCMVTDFMPEDIYVEWTNNGKTELNYDNTEPVLDSDGSYFMYSDLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK second polypeptide @TIGIT_VL_IL15 (E87C) (SEQ ID NO.14) DIVMTQTPASVEVAVGGTVTIKCQASQSISSYLNWYQQKPGQPPKLLIYDALKLASGVPSRFSGSGSGTEYTLTISGVESADAATYYCQQEHSVGNVDNVFGGGTEVVVKGGGGSGGGGSGGGGSGGGGSGNWVNVISDLKKIEDLIQSMHIDATLYTESDV HPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKCCEELEEKNIKEFLQSFVHIVQMFINTS third polypeptide @PD-L1_VH_CH1_Fc-Hole (SEQ ID NO.7) Fourth polypeptide @PD-L1_VL_CL (SEQ ID NO.8) Molecule number R1085 : TIGIT_VH_IL15RA ( P67C ) /TIGIT_VL_IL15 ( E90C ) /PD-L1-Fab first polypeptide @TIGIT_VH_IL15RA(P67C)_Fc-Knob(SEQ ID NO.49) QVQLVQSGAEVKKPGSSVKVSCKASGYSFTSYWMNWVRQAPGQGLEWIGMIRPSDSETRLNQMFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAGIHDYGHGAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGITCPPPMSVEHADI WVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDCALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCRVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK second polypeptide @TIGIT_VL_IL15 (E90C) (SEQ ID NO.50) DIQMTQSPSSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKSPKLLVYAASHLPDGVPSRFSGSGSGTDYSLTISSLQPEDFATYYCQHFWGTPRTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCK VTAMKCFLLELQVISLESGDASIHNTVQDLIILANNSLSSNGNVTESGCKECECLEEKNIKEFLQSFVHIVQMFINTS third polypeptide @PD-L1_VH_CH1_Fc-Knob(SEQ ID NO.51) QVQLQESGPGLVKPSETLSLTCAVYGDSITSGYWNWIRKPPGKGLEYIGYISYTGSTYQNPSLKSRITMSRDTSKNQYYLKLSSVTAADTAVYYCARSRAWIRTYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL WCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Fourth polypeptide @PD-L1_VL_CL(SEQ ID NO.52) DIQMTQSPSSSLSASVGDRVTITCSVSSSISSSNLHWYQQKPGKAPKPWIYGTSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSSYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Molecule number R1086 : TIGIT_VH_IL15RA ( R35C ) /TIGIT_VL_IL15 ( E93C ) /PD-L1-Fab first polypeptide @TIGIT_VH_IL15RA（R35C）_Fc-Knob(SEQ ID NO.65) QVQLVQSGAEVKKPGSSVKVSCKASGYSFTSYWMNWVRQAPGQGLEWIGMIRPSDSETRLNQMFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAGIHDYGHGAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGITCPPPMSVEHADI WVKSYSLYSRERYICNSGFKCKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCRVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK second polypeptide @TIGIT_VL_IL15 (E93C) (SEQ ID NO.12) DIQMTQSPSSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKSPKLLVYAASHLPDGVPSRFSGSGSGTDYSLTISSLQPEDFATYYCQHFWGTPRTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCK VTAMKCFLLELQVISLESGDASIHNTVQDLIILANNSLSSNGNVTESGCKECEELECKNIKEFLQSFVHIVQMFINTS third polypeptide @PD-L1_VH_CH1_Fc-Knob(SEQ ID NO.51) Fourth polypeptide @PD-L1_VL_CL(SEQ ID NO.52)

3.2 Design disulfide bond modification between VH and VL to form dsFv, which helps to form covalent disulfide linkage between the non-covalently designed ligand heavy and light chains. Disulfide bond modification positions include but are not limited to the following mutation sites:

Table 9. Designed disulfide bond modification positions between VH and VL combination VH VL 1 37C 95C 2 44C 100C 3 44C 101C 4 44C 105C 5 45C 87C 6 45C 98C 7 100C 50C 8 100bC 49C 9 98C 46C 10 101C 46C 11 105C 43C 12 106C 57C 13 108C 43C

Table 10. Amino acid sequence of VH/VL disulfide bond modified bispecific antibodies Molecule number R1081 : TIGIT_VH ( G44C ) _IL15RA/TIGIT_VL(Q100C)_IL15/PD-L1-Fab first polypeptide @TIGIT_VH （ G44C ） _IL15RA_Fc-Knob (SEQ ID NO.53) QVQLVQSGAEVKKPGSSVKVSCKASGYSFTSYWMNWVRQAPGQCLEWIGMIRPSDSETRLNQMFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAGIHDYGHGAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCRVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK second polypeptide @TIGIT_VL（Q100C）_IL15 (SEQ ID NO.54) DIQMTQSPSSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKSPKLLVYAASHLPDGVPSRFSGSGSGTDYSLTISSLQPEDFATYYCQHFWGTPRTFGCGTKLEIKGGGGSGGGGSGGGGSGGGGSGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCK VTAMKCFLLELQVISLESGDASIHNTVQDLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS third polypeptide @PD-L1_VH_CH1_Fc-Knob(SEQ ID NO.51) Fourth polypeptide @PD-L1_VL_CL(SEQ ID NO.52) Molecule number R1082 : TIGIT_VH ( W108C ) _IL15RA/TIGIT_VL(S43C)_IL15/PD-L1-Fab first polypeptide @TIGIT_VH （ W108C ） _IL15RA_Fc-Knob (SEQ ID NO.55) QVQLVQSGAEVKKPGSSVKVSCKASGYSFTSYWMNWVRQAPGQGLEWIGMIRPSDSETRLNQMFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAGIHDYGHGAYCGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCRVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK second polypeptide @TIGIT_VL(S43C)_IL15 (SEQ ID NO.56) DIQMTQSPSSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKCPKLLVYAASHLPDGVPSRFSGSGSGTDYSLTISSLQPEDFATYYCQHFWGTPRTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSC KVTAMKCFLLELQVISLESGDASIHNTVQDLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS third polypeptide @PD-L1_VH_CH1_Fc-Knob(SEQ ID NO.51) Fourth polypeptide @PD-L1_VL_CL(SEQ ID NO.52) Molecule number R1083 : TIGIT_VH ( L45C ) _IL15RA/TIGIT_VL(F98C)_IL15/PD-L1-Fab first polypeptide @TIGIT_VH （ L45C ） _IL15RA_Fc-Knob (SEQ ID NO.57) QVQLVQSGAEVKKPGSSVKVSCKASGYSFTSYWMNWVRQAPGQGCEWIGMIRPSDSETRLNQMFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAGIHDYGHGAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCRVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK second polypeptide @TIGIT_VL（F98C）_IL15 (SEQ ID NO.58) DIQMTQSPSSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKSPKLLVYAASHLPDGVPSRFSGSGSGTDYSLTISSLQPEDFATYYCQHFWGTPRTCGQGTKLEIKGGGGSGGGGSGGGGSGGGGSGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCK VTAMKCFLLELQVISLESGDASIHNTVQDLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS third polypeptide @PD-L1_VH_CH1_Fc-Knob(SEQ ID NO.51) Fourth polypeptide @PD-L1_VL_CL(SEQ ID NO.51) Molecule number R1084 : PD-L1_VH ( G44C ) _IL15RA/ PD-L1_VL(Q101C)_IL15/TIGIT-Fab first polypeptide @PD-L1_VH(G44C)_IL15RA_Fc-Knob (SEQ ID NO.59) QVQLQESGPGLVKPSETLSLTCAVYGDSITSGYWNWIRKPPGKCLEYIGYISYTGSTYQNPSLKSRITMSRDTSKNQYYLKLSSVTAADTAVYYCARSRAWIRTYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGITCPPPMSVEHA DIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCRVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK second polypeptide @PD-L1_VL(Q101C)_IL15 (SEQ ID NO.60) DIQMTQSPSSSLSASVGDRVTITTCSVSSSSSSNLHWYQQKPGKAPKPWIYGTSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSSYPLTFGCGTKLEIKGGGGSGGGGSGGGGSGGGGSGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHP SCKVTAMKCFLLELQVISLESGDASIHNTVQDLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS third polypeptide @TIGIT _VH_CH1_Fc-Knob(SEQ ID NO.61) QVQLVQSGAEVKKPGSSVKVSCKASGYSFTSYWMNWVRQAPGQGLEWIGMIRPSDSETRLNQMFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAGIHDYGHGAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL WCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Fourth polypeptide @TIGIT _VL_CL(SEQ ID NO.62) DIQMTQSPSSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKSPKLLVYAASHLPDGVPSRFSGSGSGTDYSLTISSLQPEDFATYYCQHFWGTPRTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Example 4. Mutational transformation to eliminate glycosylation

The construction method of Fab-IL15/IL15RA_Fc specifically selects the VH targeting the second antibody (anti-TIGIT) to be connected to the receptor protein (IL15RA) through the Linker, and then connects to the Fc of the antibody through Hinge; the VL targeting the second antibody is Connected to the ligand protein (IL15) through a Linker; the other end is the complete Fab structure targeting the first antibody. The Fc that makes up the first antibody and the Fc that makes up the second antibody have conventional KiH modifications to avoid heavy chain mismatching. . At the same time, the receptor and ligand proteins are mutated to form intermolecular disulfide bonds to further improve the stability of the molecules; further, the glycosylation sites on the receptor and ligand proteins are modified to eliminate Molecular heterogeneity, such as:

Table 11. Amino acid sequence of IL15/IL15RA engineered bispecific antibodies Molecule number R1072 : TIGIT_VH_IL15 RA/TIGIT_VL_IL15/PD-L1-Fab first polypeptide @TIGIT_VH_IL15RA_Fc-Knob (SEQ ID NO.63) QVQLVQSGAEVKKPGSSVKVSCKASGYSFTSYWMNWVRQAPGQGLEWIGMIRPSDSETRLNQMFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAGIHDYGHGAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGITCPPPMSVEHADIWVKSYSLY SRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCRVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK second polypeptide @TIGIT_VL_IL15 (SEQ ID NO.64) DIQMTQSPSSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKSPKLLVYAASHLPDGVPSRFSGSGSGTDYSLTISSLQPEDFATYYCQHFWGTPRTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLEL QVISLESGDASIHNTVQDLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS third polypeptide @PD-L1_VH_CH1_Fc-Knob(SEQ ID NO.51) Fourth polypeptide @PD-L1_VL_CL(SEQ ID NO.52) Molecule number R0955 : TIGIT_VH_IL15 RA/TIGIT_VL_IL15 ( N71Q , N79Q , N112Q ) /PD-L1-Fab first polypeptide @TIGIT_VH_IL15RA_Fc-Knob (SEQ ID NO.11) QVQLESEGGLFKPTDTLTLTCTVSGSSSLSSSYMSWVRQAPGKGLEWIGIIGSNGNTYYANWAKGRFTISKTSTTVELKITSPTTEDTATYFCARGGYRTSGMDPWGPGTLVTVSSGGGGSGGGGSGGGGSGGGGSGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRK AGTCSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTEPRGGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDY NSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLWCMVTDFMPEDIYVEWTNNGKTELNYDNTEPVLDSDGSYFMYSDLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK second polypeptide @TIGIT_VL_IL15 (N71Q, N79Q, N112Q) (SEQ ID NO.15) DIVMTQTPASVEVAVGGTVTIKCQASQSISSYLNWYQQKPGQPPKLLIYDALKLASGVPSRFSGSGSGTEYTLTISGVESADAATYYCQQEHSVGNVDNVFGGGTEVVVKGGGGSGGGGSGGGGSGGGGSGNWVNVISDLKKI EDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISCESGDASIHDTVENLIILAQNSLSSNGQVTESGCKECEELEEKNIKEFLQSFVHIVQMFIQTS third polypeptide @PD-L1_VH_CH1_Fc-Hole (SEQ ID NO.7) Fourth polypeptide @PD-L1_VL_CL (SEQ ID NO.8) Molecule number R1109 : TIGIT_VH_IL15 RA ( sushi65 ) /TIGIT_VL_IL15/PD-L1-Fab first polypeptide @TIGIT_VH_IL15RA（sushi65）_Fc-Knob (SEQ ID NO.66) QVQLVQSGAEVKKPGSSVKVSCKASGYSFTSYWMNWVRQAPGQGLEWIGMIRPSDSETRLNQMFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAGIHDYGHGAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGITCPPPMSVEHADI WVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCR VKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK second polypeptide @TIGIT_VL_IL15 (SEQ ID NO.64) third polypeptide @PD-L1_VH_CH1_Fc-Knob(SEQ ID NO.51) Fourth polypeptide @PD-L1_VL_CL(SEQ ID NO.52 Molecule number R1110 : TIGIT_VH_IL15 RA ( sushi65 T2A ) /TIGIT_VL_IL15/PD-L1-Fab first polypeptide @TIGIT_VH_IL15RA (sushi65 T2A)_Fc-Knob (SEQ ID NO.67) QVQLVQSGAEVKKPGSSVKVSCKASGYSFTSYWMNWVRQAPGQGLEWIGMIRPSDSETRLNQMFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAGIHDYGHGAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGIACPPPMS VEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ VSLTCRVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK second polypeptide @TIGIT_VL_IL15 (SEQ ID NO.64) third polypeptide @PD-L1_VH_CH1_Fc-Knob(SEQ ID NO.51) Fourth polypeptide @PD-L1_VL_CL(SEQ ID NO.52) Molecule number R1111 : TIGIT_VH_IL15 RA ( sushi65 T2A , T81A , T86A ) /TIGIT_VL_IL15/PD-L1-Fab first polypeptide @TIGIT_VH_IL15RA (sushi65 T2A, T81A, T86A)_Fc-Knob (SEQ ID NO.68) QVQLVQSGAEVKKPGSSVKVSCKASGYSFTSYWMNWVRQAPGQGLEWIGMIRPSDSETRLNQMFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAGIHDYGHGAYWGQGTLVTVSSGGGGSGGGG SGGGGSGGGGSGIACPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVATAGVAPQPESLSPSGKEPAASSPSSNNTAATTAEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCRVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK second polypeptide @TIGIT_VL_IL15 (SEQ ID NO.64) third polypeptide @PD-L1_VH_CH1_Fc-Knob(SEQ ID NO.51) Fourth polypeptide @PD-L1_VL_CL(SEQ ID NO.52) Example 5 , IL15/IL15RA modified to reduce the affinity of IL15/IL15RA and IL2 /15Rβ/γC complex and bispecific antibodies composed of the same

In some applications, in order to avoid IL15/IL15RA and its component bispecific antibodies from interacting with the IL2/15Rβ/γC complex and causing unwanted non-specific binding, we have designed IL15/IL15RA and its component bispecific antibodies. The specific antibody is modified to reduce or completely lose the affinity of IL15/IL15RA and IL2/15Rβ/γC complex. By examining the crystal structure of the interface between IL15/IL15RA and the IL2/15Rβ/γC complex and modeling using Molecular Operating Environment (MOE; Chemical Computing Group, Montreal, Quebec, Canada) software, we predict that at the IL15/IL15RA interface Amino acid mutations were engineered to reduce or completely lose the affinity of IL15/IL15RA to the IL2/15Rβ/γC complex, as depicted in Figure 9 .

Describe the construction method of Fab-IL15/IL15RA_Fc. Specifically, the VH targeting the second antibody (anti-TIGIT) is connected to the receptor protein (IL15RA) through the Linker, and then connected to the Fc of the antibody through Hinge; the VH targeting the second antibody is VL is connected to the ligand protein (IL15) through a Linker; the other end is a complete Fab structure targeting the first antibody. The Fc that makes up the first antibody and the Fc that makes up the second antibody have conventional KiH modifications to avoid heavy chain misunderstandings. match. At the same time, the ligand protein is mutated in order to reduce or inactivate the biological function of the receptor and ligand complex, for example:

Table 12. Amino acid sequence of IL15/IL15RA engineered bispecific antibodies Molecule number R0960 : TIGIT_VH_IL15 RA/TIGIT_VL_IL15 ( D61N , E64Q , N65D ) /PD-L1-Fab first polypeptide @TIGIT_VH_IL15RA_Fc-Knob (SEQ ID NO.5) QVQLESEGGLFKPTDTLTLTCTVSGSSSSSYMSWVRQAPGKGLEWIGIIGSNGNTYYANWAKGRFTISKTSTTVELKITSPTTEDTATYFCARGGYRTSGMDPWGPGTLVTVSSGGGGSGGGGSGGGGSGGGGSGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAG TSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNS TLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLWCMVTDFMPEDIYVEWTNNGKTELNYDNTEPVLDSDGSYFMYSDLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK second polypeptide @TIGIT_VL_IL15 (D61N, E64Q, N65D) (SEQ ID NO.16) DIVMTQTPASVAVAVGGTVTIKCQASQSISSYLNWYQQKPGQPPKLLIYDALKLASGVPSRFSGSGSGTEYTLTISGVESADAATYYCQQEHSVGNVDNVFGGGTEVVVKGGGGSGGGGSGGGGSGGGGSGNWVNVISDLKKIED LIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHNTVQDLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS third polypeptide @PD-L1_VH_CH1_Fc-Hole (SEQ ID NO.7) Fourth polypeptide @PD-L1_VL_CL (SEQ ID NO.8)

Example 6. Construction of bispecific antibodies based on scFv and CrossMab structures as experimental controls

As described above, scFv and CrossMab are both commonly used bispecific antibody construction technologies. Here they are used as a design control to compare with our molecules:

Based on the construction method of scFv structure double antibody, the VH of the targeting second antibody (anti-TIGIT) is specifically selected to be connected to the VL of the second antibody through the Linker to form the scFv structure, and then connected to the Fc of the antibody through Hinge; the other end is the targeting The complete Fab structure of the first antibody (this dual-antibody platform was developed by Wuhan Youzhiyou and named Ybody). The Fc that makes up the first antibody and the Fc that makes up the second antibody have conventional KiH modifications to avoid heavy chain mismatching. . Specific examples:

Table 13. Y-Body structural double antibody molecule sequence Molecule number R0809 : Y-Body TIGIT_scFv/PD-L1-Fab ( Figure 1, C ) scFv arm @TIGIT_scFv_Fc-Knob (SEQ ID NO.17) QVQLESEGGLFKPTDTLTLTCTVSGSSLSSSSYMSWVRQAPGKGLEWIGIIGSNGNTYYANWAKGRFTISKTSTTVELKITSPTTEDTATYFCARGGYRTSGMDPWGPGTLVTVSSGGGGSGGGGSGGGGSDIVMTQTPASVEVAVGGTVTIKCQASQSISSYLNWYQQKPGQPP KLLIYDALKLASGVPSRFSGSGSGTEYTLTISGVESADAATYYCQQEHSVGNVDNVFGGGTEVVVKGAAAEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYV LPPPEEEMTKKQVTLWCMVTDFMPEDIYVEWTNNGKTELNYDNTEPVLDSDGSYFMYSDLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK Fab arm-VH @PD-L1_VH_CH1_Fc-Hole (SEQ ID NO.7) Fab arm-VL @PD-L1_VL_CL (SEQ ID NO.8)

Describes the construction method of double antibodies based on the scFv structure. Specifically, the VH targeting the second antibody (anti-TIGIT) is connected to the VL of the second antibody through a Linker to form an scFv structure, and then the linker is used with the complete Fc targeting the first antibody. The C-terminal connection; forming a symmetrical structure. Specific examples:

Table 14. scFv structure double antibody molecule sequence Molecule number R0810 : Symmetric scFv PD-L1_VH_CH1_Fc_@TIGIT_scFv/ PD-L1_VL_CL ( Figure 1, D ) heavy chain @PD-L1_VH_CH1_Fc_@TIGIT_scFv (SEQ ID NO.20) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVT LTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVT LTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGAGGGGSGGGGSGGGGSGGGGSQVQLESEGGLFKPTDTLTLTCTVSGSSSLSSSYMSWVRQAPGKGLEWIGIIGSNGNTYYANWAKGRFTISKTSTTVELKITSPTTEDTATYFCARGGYRTSGMDPWGPGTLVTVSSGGGG SGGGGSGGGGSDIVMTQTPASVEVAVGGTVTIKCQASQSISSYLNWYQQKPGQPPKLLIYDALKLASGVPSRFSGSGSGTEYTLTISGVESADAATYYCQQEHSVGNVDNVFGGGTEVVVK light chain @PD-L1_VL_CL (SEQ ID NO.8)

Based on the CrossMab structural double antibody construction method, the VH targeting the second antibody (anti-TIGIT) is specifically selected to be connected to the CL domain, and then connected to the Fc of the antibody through Hinge, and the VL targeting the second antibody (anti-TIGIT) is connected to The CH1 domain forms the light chain; the other end is the complete Fab structure targeting the first antibody. The Fc that makes up the first antibody and the Fc that makes up the second antibody have conventional KiH modifications to avoid heavy chain mismatches. Specific examples:

Table 15. CrossMab structural double antibody molecule sequence Molecule number R0959 : CrossMab TIGIT_VH_CL/TIGIT_VL_CH1/PD-L1-Fab (Figure 1 , B ) first polypeptide @TIGIT_VH_CL_Fc-Knob (SEQ ID NO.18) QVQLESEGGLFKPTDTLTLTCTVSGSSSSSYMSWVRQAPGKGLEWIGIIGSNGNTYYANWAKGRFTISKTSTTVELKITSPTTEDTATYFCARGGYRTSGMDPWGPGTLVTVSSLRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQ DSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLWCMVTDFMPEDI YVEWTNNGKTELNYDNTEPVLDSDGSYFMYSDLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK second polypeptide @TIGIT_VL_CH1 (SEQ ID NO.19) DIVMTQTPASVEVAVGGTVTIKCQASQSISSYLNWYQQKPGQPPKLLIYDALKLASGVPSRFSGSGSGTEYTLTISGVESADAATYYCQQEHSVGNVDNVFGGGTEVVVKAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLY TLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKI third polypeptide @PD-L1_VH_CH1_Fc-Hole (SEQ ID NO.7) Fourth polypeptide @PD-L1_VL_CL (SEQ ID NO.8) Example 7. Antibody heavy - light chain mismatch test

Light chain mismatch is a difficult problem faced by dual-antibody platforms. In order to verify the anti-mismatch performance of this platform, we specially designed a Fab with receptors and ligands distributed on both sides of the antibody, deliberately designed mismatched heavy and light chain structures, and performed expression verification.

Describe the construction method of Fab-IL15/IL15RA_Fc mismatch:

R1042: Specifically select the VH targeting the first antibody (anti-PD-L1 antibody) to be connected to the receptor protein (IL15RA) through Linker, and then connect to the Fc of the antibody through Hinge; the VH targeting the second antibody (anti-TIGIT antibody) VL is connected to the ligand protein (IL15) through Linker; the other end is the VH targeting the second antibody, which is connected to CH1 through a conventional sequence, and then connected to the Fc of the antibody through Hinge, and the VL targeting the first antibody is connected through a conventional sequence In CL, both Fcs have conventional KiH modifications to avoid heavy chain mispairing. Specific examples:

Table 16. Mismatched FiBody antibody sequences Molecule number R1042 : PD-L1_VH_IL15RA/TIGIT_VL_IL15/TIGIT_VH/PD-L1-VL (Figure 10 left) first polypeptide @PD-L1_VH_IL15RA_Fc-Knob (SEQ ID NO.1) second polypeptide @TIGIT_VL_IL15 (SEQ ID NO.6) third polypeptide @TIGIT_VH_CH1_Fc-Hole (SEQ ID NO.3) Fourth polypeptide @PD-L1_VL_CL (SEQ ID NO.8)

R1043: Specifically select the VH targeting the second antibody (anti-TIGIT antibody) to be connected to the receptor protein (IL15RA) through Linker, and then connect to the Fc of the antibody through Hinge; the VH targeting the first antibody (anti-PD-L1 antibody) VL is connected to the ligand protein (IL15) through Linker; the other end is the VH targeting the first antibody connected to CH1 through a conventional sequence, and then connected to the Fc of the antibody through Hinge, and the VL targeting the second antibody is connected through a conventional sequence In CL, both Fcs have conventional KiH modifications to avoid heavy chain mispairing.

Table 17. KiH double antibody sequence Molecule number R1043 : PD-L1_VH_IL15RA/TIGIT_VL_IL15/TIGIT_VH/PD-L1-VL first polypeptide @TIGIT_VH_IL15RA_Fc-Knob (SEQ ID NO:5) second polypeptide @PD-L1_VL_IL15 (SEQ ID NO:2) third polypeptide @ PD-L1_VH_CH1_Fc-Hole (SEQ ID NO.7) Fourth polypeptide @TIGIT _VL_CL (SEQ ID NO.4)

Describe the construction method of Fab-IL21/IL21R_Fc mismatch:

Select the VH targeting the second antibody (anti-PD-L1 antibody) and connect it to the receptor protein (IL21R) through Linker, and then connect it to the Fc of the antibody through Hinge; the VL targeting the first antibody (anti-TIGIT antibody) is connected through Linker Connected to the ligand protein (IL21); the other end is the VL of the targeting second antibody (anti-PD-L1 antibody) connected to CL, and the VH of the targeting structure first antibody (anti-TIGIT antibody) is connected to CH1, Then it is connected to the Fc of the antibody through Hinge, and the Fc at both ends has conventional KiH modification. Specific examples:

Table 18. Fab-IL21/IL21R_Fc mismatch Molecule number R1124 : PD-L1_VH_IL21R/TIGIT_VL_IL21/TIGIT_VH/PD-L1-VL (Figure 10 right) first polypeptide @ PD-L1_VH_IL21R_Fc-Knob (SEQ ID NO:69) EVQLQESGPGLVKPSETLSLTCAVYGDSITSGYWNWIRKPPGKGLEYMGYISYTGSTYQNPSLKSRITFSRDTSKNQYYLKLSSVTAADTATYYCARSRAWIRTYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGCPDLVCYTDYLQTVICILEMWNLHP STLTLTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFMADDIFSVQITDQSGQYSQECGSFLLAESIKPAPPFDVTVTFSGQYQISWRSDYEDPAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSSVSLLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKEEPKSSDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCRVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG second polypeptide @TIGIT _VL_IL21 (SEQ ID NO:48) third polypeptide @TIGIT _VH_CH1_Fc-Hole (SEQ ID NO.70) QVQLVQSGAEVKKPGSSVKVSCKASGYSFTSYWMNWVRQAPGQGLEWIGMIRPSDSETRLNQMFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAGIHDYGHGAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWC LVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Fourth polypeptide @PD-L1 _VL_CL (SEQ ID NO. 35) Example 8 , Preparation of FiBody samples

Protein transient expression:

After the plasmid containing the target gene forms a cationic complex with the transfection reagent PEI, it is introduced into the host cell Expi293. While the plasmid is in the cell, the foreign gene on the plasmid is transcribed and translated in the cell to obtain the target protein.

Expi293 was cultured at 37°C, 8% carbon dioxide, and 130 rpm, and the cells were counted before transfection. The 2E6 cells were inoculated into a 1L shake flask, and the culture system was about 300 ml. Prepare the transfection complex and prepare for transfection: first add 750 μg of target plasmid into a 50 ml centrifuge tube containing 15 ml Opti-MEM reagent, mix gently, and mark it as tube A; add 1.5 mg transfection reagent PEI into a 50 ml centrifuge tube containing 15 ml Opti-MEM reagent in a 50ml centrifuge tube, mix gently, and incubate at room temperature for 5 minutes, label it tube B; add the PEI diluent in tube B dropwise to the DNA diluent in tube A, mix gently, and incubate at room temperature for 15 minutes. , after the incubation, add the PEI-target plasmid complex to Expi293 cells and place them in a 37°C shaker to continue culturing. Samples were collected after D7-D10.

Protein purification:

The transiently transfected cell expression solution was centrifuged at 9000rpm/20min, the supernatant was collected, and then sterilized and filtered through a 0.22μm filter membrane. Purification was performed using ProA affinity chromatography. The process is as follows, use AKTA avant 150 chromatography equipment, equilibrate the chromatography column (such as MabSelectSuRe LX, GE) with at least 5CV equilibrium buffer (10mM PBS), load the sample to the chromatography column, and allow the target protein to be adsorbed on the chromatography column. Other impurities penetrate and separate. After loading, rinse the column again with at least 5CV of equilibration buffer (10mM PBS), and then use elution buffer (20mM NaAc, pH=3.4) to elute the target protein. Neutralization buffer (1M) is pre-added to the collection tube. Tris, pH8.0), the added volume of neutralizing buffer is determined based on the estimated content of the elution sample, generally 10% of the elution volume is added. Example 9 , FiBody physical and chemical detection

After one-step purification, the purity of the sample was tested by HPLC-SEC (analytical column TOSOH, TSKgel G2000). The expression level and purity results of each sample are shown in the table below.

Table 19. Bispecific antibody physical and chemical detection results Double antibody type Sample number Expression level mg/L Purity % FiBody-A R0950 25.1 89.2 R0951 16.1 90.8 R0952 14.6 84.7 R1072 122.1 91.7 R1109 70.2 98.4 R1115 83.9 R1116 89.3 94.0 FiBody-C R1119 22.9 95.3 R1120 18.0 50.7 FiBody-D R1123 49.6 92.9 FiBody-disulfide bond modification R0954 17.1 91.0 R1085 99.6 90.4 R1086 66.13 56.1 FiBody-glycosylation modification R0955 7.6 90.9 R1110 98.4 98.6 R1111 107.6 92.4 FiBody - Affinity Transformation R0960 17.0 90.4 ScFv-Asymmetric (Y-Body) R0809 1.5 36.8 ScFv-Symmetric R0810 10.0 68.9 CrossMab R0959 1.4 41.4 FiBody-mismatch R1042 12.9 41.9 R1043 19.5 86.8 R1124 14.5 65.6

The HPLC-SEC detection results of sample R0951 are shown in Figure 11, the HPLC-SEC detection results of sample R1042 are shown in Figure 12, the HPLC-SEC detection results of sample R0809 are shown in Figure 13, the HPLC-SEC detection results of sample R1110 The results of SEC detection are shown in Figure 14.

The results show that compared with bispecific antibodies with asymmetric scFv (Y-Body, R0809), symmetric ScFv (R0810), and CrossMab (R0959) structures, bispecific antibodies (including various modified and optimized antibodies) prepared on the FiBody platform have better Higher expression and/or higher purity.

Unexpectedly, bispecific antibodies with mispaired forms (samples R1042, R1043, R1124) were also expressed and had expression levels similar to normal molecules, but with significantly lower purity. Example 10 , FiBody antigen affinity detection TIGIT end binding activity analysis

The binding activity between the double antibody molecule (TIGIT end) and CHO-TIGIT cells was detected by FCM experimental method. Configure 3% BSA buffer: weigh 4.5 gBSA into 150mL 1XPBS, mix well and place on ice for later use; antibody dilution: dilute the test antibody and positive control with 3% BSA to an initial concentration of 800nM, and dilute the subtype control to an initial concentration of 800nM. The initial concentration is 20 μg/mL, the volume is 300 μL, 3-fold gradient dilution (100+200), a total of 10 points; Binding activity detection: Cell counting and plating: After counting R0254-3 cells, divide into 100 μL, 2E+05/well into a 96-well V-type plate; first add 50 μL of antibodies of different concentrations to the cells, incubate at 2-8 degrees for 0.5 hours, then add 50 μL of ligand, and incubate at 2-8 degrees for 0.5 hours; centrifuge at 350xg for 5 minutes, remove the supernatant, Add 200 μL/well of 3% BSA; centrifuge at 350xg for 5 minutes, remove the supernatant, prepare fluorescent antibodies PE Goat anti-human IgG Fc and PE Goat anti-mouse IgG Fc (1:500x dilution) in 3% BSA, add 100 μL/well In the corresponding 96-well plate, incubate at 2-8 degrees for 30 minutes; centrifuge at 350g for 5 minutes, remove the supernatant, and wash the cells with 3% BSA; centrifuge at 350xg for 5 minutes, remove the supernatant, and add 1XPBS at 100 μL/well to resuspend the cells; follow CytoFLEX Flow cytometer standard operating procedures for on-machine testing.

The results are shown in Figures 15 to 18; unexpectedly, the binding activity of R0950 (@TIGIT is at the Fab end) is lower than that of R0951~R0960 (@TIGIT is at the IL15/IL15R end); the binding activity of R0951~R0960 is the same as the positive control R0226 (Tigit single The cloned antibody, OMP-313R12, WO2016191643) is similar; after replacing CH1 and CL with class A and class D interleukins and their receptors, the binding ability of the targeting region is not affected, and is similar to the positive control (R0226\R0774 (VH as sequence As shown in SEQ ID NO:73, the VL sequence is as shown in SEQ ID NO:74), Tigit monoclonal antibody) showed considerable affinity; after the replacement of CH1 and CL in class C molecules, the targeting region was affected, and the targeting The binding capacity is significantly lower than that of the positive control (R0226\R0774, Tigit monoclonal antibody).

The tigit end affinity results of the modified molecules of disulfide bond modified optimized samples R1081, R1085 and glycosylated samples are comparable to those of the molecules before modification.

R1042, R1043 and R1124 are mismatched test molecules, and their TIGIT binding activity is significantly reduced; R0810 is a ScFv structural molecule, and its binding activity is also weaker than that of the control molecule R0226. PD-L1 terminal binding activity analysis

The binding activity of the double antibody molecule (PD-L1 end) and CHO-PD-L1 cells was detected by FCM experimental method. Configure 3% BSA buffer: weigh 4.5 gBSA into 150mL 1XPBS, mix well and place on ice for later use; antibody dilution: dilute the test antibody and positive control with 3% BSA to an initial concentration of 800nM, and dilute the subtype control to an initial concentration of 800nM. The initial concentration is 20 μg/mL, the volume is 300 μL, 3-fold gradient dilution (100+200), a total of 10 points; binding activity detection: cell counting and plating: after counting R0254-3 cells, divide into 100 μL, 2E+05/well into a 96-well V-type plate; first add 50 μL of antibodies of different concentrations to the cells, incubate at 2-8 degrees for 0.5 hours, then add 50 μL of ligand, and incubate at 2-8 degrees for 0.5 hours; centrifuge at 350xg for 5 minutes, remove the supernatant, 200 μL/well 3% BSA; after centrifugation at 350xg for 5 minutes, remove the supernatant, prepare fluorescent antibodies PE Goat anti-human IgG Fc and PE Goat anti-mouse IgG Fc (1:500x dilution) in 3% BSA, 100 μL/well Add to the corresponding 96-well plate and incubate at 2-8 degrees for 30 minutes; centrifuge at 350g for 5 minutes, remove the supernatant, and wash the cells with 3% BSA; centrifuge at 350xg for 5 minutes, remove the supernatant, and add 1XPBS at 100 μL/well to resuspend the cells; follow CytoFLEX flow cytometer standard operating procedures for on-machine testing.

The results are shown in Figures 19 to 23. Surprisingly, the activity of @PD-L1 on the IL15 end is better than that on the Fab end; R1042, R1043 and R1124 are corresponding mismatched molecules, and the activity is obviously very weak, and other FiBody all show Comparable affinity to positive antibodies (PD-L1 monoclonal antibodies). Among them, R0802 is (PD-L1 monoclonal antibody, 176F9, the VH sequence is shown in SEQ ID NO: 36, and the VL sequence is shown in SEQ ID NO: 37), R0514 is (PD-L1 monoclonal antibody, Avelumab), and R0919 is ( PD-L1 monoclonal antibody, VH sequence is shown in SEQ ID NO:75, VL sequence is shown in SEQ ID NO:76), R0968 is (PD-L1 monoclonal antibody, VH sequence is shown in SEQ ID NO:71 , the VL sequence is shown in SEQ ID NO:72) Example 11. Binding activity of FiBody receptor ligand complex ( IL15/IL15R )

Antibody dilution: Use FACS buffer to dilute all molecules to an initial concentration of 400nM, volume 180μl, 3-fold gradient dilution (60+120), 10 concentrations; cell counting and plating: R0255-2 (CHO-mTigit)/293T-IL15R -28 Cells were centrifuged at 250g for 5 minutes and the supernatant was discarded. Use FACS buffer to adjust the cell density to 2E+06 and distribute 100 μL/tube evenly into a 96-well V-shaped plate. Add the above diluted antibodies to the cells at 100 μL/tube. well, incubate at 2-8 degrees for 0.5h; take out the 96-well plate, centrifuge at 250g for 5 minutes, carefully remove the supernatant, add 200 μL/well of FACS buffer, centrifuge again at 250g for 5 minutes, carefully remove the supernatant; use FACS buffer to prepare PE fluorescent secondary antibodies (1:500 dilution), add 100 μL/well to the corresponding 96-well plate, resuspend the cells, and incubate at 2-8 degrees for 30 minutes; take out the 96-well plate, centrifuge at 250g for 5 minutes, carefully remove the supernatant, and add 200 μL/FACS buffer well, centrifuge again at 250g for 5 minutes, carefully remove the supernatant; resuspend in 1xPBS 100μL/well, and detect by FACS.

The results are shown in Figure 24 and Figure 25; R0952 (IL15, IL15RA transposition), R0960 (inactivated) molecules have very low IL15 receptor complex binding activity; R0955 (deglycosylated) IL15 activity decreases; R0953, The activity of R0954 after covalent linkage is similar to that of R0951, indicating that it has little impact on the structure.

The activities of the mismatched molecules R1042 and R1043 are equivalent to those of R0951, indicating that no mismatch occurs and even the wrong Fv can be expressed; among them, R0655 is (IL15/IL15RFc fusion protein, see SEQ ID NO: 38) Example 12 , disulfide Key modified molecular electrophoresis detection

SDS-PAGE electrophoresis was performed on the disulfide bond double antibody molecule. The results are shown in Figure 26. The R1072 molecule without disulfide bond modification has a band between the molecular weight of 25KD and 35KD, indicating the presence of free light chain; ligand receptor The disulfide bond modified molecules are R0954, R1085, and R1086. The electrophoresis results of R0954 and R1086 show that there are still non-covalent light chains (bands between 25KD and 35KD), while R1085 has no non-covalent light chains (25KD and 35KD). There is no band between them), indicating that the R1085 disulfide bond transformation was successful.

The light and heavy chain disulfide bond modified molecules are R1081, R1082, and R1084. The electrophoresis results show that there is no non-covalent light chain (no band between 25KD and 35KD), indicating that the disulfide bond modification of R1081, R1082, and R1084 was successful.

The technical features of the above-described embodiments can be combined in any way. To simplify the description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, All should be considered to be within the scope of this manual. Example 13. IL15/IL15Rα complex 1. Design of disulfide bonds between IL15 and IL15Rα

Design disulfide bond modification between IL15 and IL15Rα to help form covalent disulfide linkage between non-covalently linked IL15 and IL15Rα to construct a targeted IL15/IL15Rα complex. Intermolecular disulfide between IL15 and IL15RA The schematic diagram of the bond mutation pairing three-dimensional structure is shown in Figure 27. For example, the mutation sites are as follows:

Table 20. IL15/IL15Rα disulfide bond modification combination IL15 IL15Rα 1 E87C D96+C97 2 E90C P67C 3 E93C R35C

An exemplary usage scenario of selecting an IL15/IL15Rα complex: the heavy chain variable region (VH) variable region targeting the antigen is connected to IL15Rα through a linker, and then connected to the Fc of the antibody through Hinge; targeting the same antigen The light chain variable region (VL) is connected to IL15 through a linker to prepare a targeted IL15/IL15Rα complex. The schematic structural diagram of the IL15/IL15Rα complex is shown in Figure 28. As an example, the amino acid sequence of the IL15/IL15Rα complex is shown in Table 21 below:

Table 21. Amino acid sequence of IL15/IL15Rα complex Complex 1 @TIGIT_VH_IL15Rα (P67C)_Fc (SEQ ID NO.49) QVQLVQSGAEVKKPGSSVKVSCKASGYSFTSYWMNWVRQAPGQGLEWIGMIRPSDSETRLNQMFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAGIHDYGHGAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGITCPPPMSVEHADIWVK SYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDCALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCRVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK @TIGIT_VL_IL15 (E90C) (SEQ ID NO.87) DIQMTQSPSSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKSPKLLVYAASHLPDGVPSRFSGSGSGTDYSLTISSLQPEDFATYYCQHFWGTPRTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCK VTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECECLEEKNIKEFLQSFVHIVQMFINTS Complex 2 @PD-L1_VH_IL15Rα(P67C)_Fc (SEQ ID NO.88) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGITCPPPMSVEHADIWVKSYS LYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDCALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVE VHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLWCMVTDFMPEDIYVEWTNNGKTELNYDNTEPVLDSDGSYFMYSDLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK @ PD-L1_VL_IL15 (E90C) (SEQ ID NO.89) QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVLGGGGSGGGGSGGGGSGGGGSGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCK VTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECECLEEKNIKEFLQSFVHIVQMFINTS Complex 3 @TIGIT_VH_IL15Rα（D96+C97）_Fc (SEQ ID NO.13) QVQLESEGGLFKPTDTLTLTCTVSGSSSLSSSYMSWVRQAPGKGLEWIGIIGSNGNTYYANWAKGRFTISKTSTTVELKITSPTTEDTATYFCARGGYRTSGMDPWGPGTLVTVSSGGGGSGGGGSGGGGSGGGGSGITCPPPMSVEHADIWVKSYSLYSRE RYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTA QTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLWCMVTDFMPEDIYVEWTNNGKTELNYDNTEPVLDSDGSYFMYSDLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK @TIGIT_VL_IL15 (E87C) (SEQ ID NO.14) DIVMTQTPASVEVAVGGTVTIKCQASQSISSYLNWYQQKPGQPPKLLIYDALKLASGVPSRFSGSGSGTEYTLTISGVESADAATYYCQQEHSVGNVDNVFGGGTEVVVKGGGGSGGGGSGGGGSGGGGSGNWVNVISDLKKIEDLIQSMHIDATLYTESDV HPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKCCEELEEKNIKEFLQSFVHIVQMFINTS Complex 4 @TIGIT_VH_IL15RA (R35C)_Fc-Knob (SEQ ID NO.65) QVQLVQSGAEVKKPGSSVKVSCKASGYSFTSYWMNWVRQAPGQGLEWIGMIRPSDSETRLNQMFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAGIHDYGHGAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGITCPPPMSVEHADI WVKSYSLYSRERYICNSGFKCKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCRVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK @TIGIT_VL_IL15 (E93C) (SEQ ID NO.90) DIQMTQSPSSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKSPKLLVYAASHLPDGVPSRFSGSGSGTDYSLTISSLQPEDFATYYCQHFWGTPRTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCK VTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELECKNIKEFLQSFVHIVQMFINTS two. Preparation of complex samples

Protein transient expression:

After the plasmid containing the target gene forms a cationic complex with the transfection reagent PEI, it is introduced into the host cell Expi293. While the plasmid is in the cell, the foreign gene on the plasmid is transcribed and translated in the cell to obtain the target protein.

Expi293 was cultured at 37°C, 8% carbon dioxide, and 130 rpm, and the cells were counted before transfection. The 2E6 cells were inoculated into a 1L shake flask, and the culture system was about 300 ml. Prepare the transfection complex and prepare for transfection: first add 750 μg of target plasmid into a 50 ml centrifuge tube containing 15 ml Opti-MEM reagent, mix gently, and mark it as tube A; add 1.5 mg transfection reagent PEI into a 50 ml centrifuge tube containing 15 ml Opti-MEM reagent in a 50ml centrifuge tube, mix gently, and incubate at room temperature for 5 minutes, label it tube B; add the PEI diluent in tube B dropwise to the DNA diluent in tube A, mix gently, and incubate at room temperature for 15 minutes. , after the incubation, add the PEI-target plasmid complex to Expi293 cells and place them in a 37°C shaker to continue culturing. Samples were collected after D7-D10.

Purification of complex samples:

The transiently transfected cell expression solution was centrifuged at 9000rpm/20min, the supernatant was collected, and then sterilized and filtered through a 0.22μm filter membrane. Purification was performed using ProA affinity chromatography. The process is as follows, use AKTA avant 150 chromatography equipment, equilibrate the chromatography column (such as MabSelectSuRe LX, GE) with at least 5CV equilibrium buffer (10mM PBS), load the sample to the chromatography column, and allow the target protein to be adsorbed on the chromatography column. Other impurities penetrate and separate. After loading, rinse the column again with at least 5CV of equilibration buffer (10mM PBS), and then use elution buffer (20mM NaAc, pH=3.4) to elute the target protein. Neutralization buffer (1M) is pre-added to the collection tube. Tris, pH8.0), the added volume of neutralizing buffer is determined based on the estimated content of the elution sample, generally 10% of the elution volume is added. three. Gel electrophoresis detection of IL15/IL15Rα complex

The disulfide bond modified IL15/IL15Rα complex was detected by SDS-PAGE electrophoresis. The detection results are shown in Figure 29. There are bands between the molecular weight of 25KD and 35KD, indicating the presence of free light chains; Complex 1 and Complex 2 ( The disulfide bond modified position IL15(E90C)/IL15Rα(P67C)) has no band between the molecular weight of 25KD and 35KD, indicating that the disulfide bond modified position is successful. Complex 3 (disulfide bond modified position IL15(E87C)/IL15Rα(D96) +C97)) and complex 4 (disulfide bond modification position IL15(E93C)/IL15Rα(R35C)) have bands between the molecular weight of 25KD and 35KD, indicating the presence of free light chains and failure of disulfide bond modification. Four. Targeted moiety affinity detection TIGIT end binding activity analysis

The binding activity between the double antibody molecule (TIGIT end) and CHO-TIGIT cells was detected by FCM experimental method. Configure 3% BSA buffer: weigh 4.5 gBSA into 150mL 1XPBS, mix well and place on ice for later use; antibody dilution: dilute the test antibody and positive control with 3% BSA to an initial concentration of 800nM, and dilute the subtype control to an initial concentration of 800nM. The initial concentration is 20 μg/mL, the volume is 300 μL, 3-fold gradient dilution (100+200), a total of 10 points; binding activity detection: cell counting and plating: after counting R0254-3 cells, divide into 100 μL, 2E+05/well into a 96-well V-type plate; first add 50 μL of antibodies of different concentrations to the cells, incubate at 2-8 degrees for 0.5 hours, then add 50 μL of ligand, and incubate at 2-8 degrees for 0.5 hours; centrifuge at 350xg for 5 minutes, remove the supernatant, Add 200 μL/well of 3% BSA; centrifuge at 350xg for 5 minutes, remove the supernatant, prepare fluorescent antibodies PE Goat anti-human IgG Fc and PE Goat anti-mouse IgG Fc (1:500x dilution) in 3% BSA, add 100 μL/well In the corresponding 96-well plate, incubate at 2-8 degrees for 30 minutes; centrifuge at 350g for 5 minutes, remove the supernatant, and wash the cells with 3% BSA; centrifuge at 350xg for 5 minutes, remove the supernatant, and add 1XPBS at 100 μL/well to resuspend the cells; follow CytoFLEX The flow cytometer standard operating procedures were tested on the machine. The test results are shown in Figure 30. Compared with the molecules without disulfide bond modification, the affinity is equivalent, indicating that the disulfide bond modification will not affect the affinity of the target region. PD-L1 terminal binding activity analysis

The binding activity between the double antibody molecule (PD-L1 end) and CHO-PD-L1 cells was detected by FCM experimental method. Configure 3% BSA buffer: weigh 4.5 gBSA into 150mL 1XPBS, mix well and place on ice for later use; antibody dilution: dilute the test antibody and positive control with 3% BSA to an initial concentration of 800nM, and dilute the subtype control to an initial concentration of 800nM. The initial concentration is 20 μg/mL, the volume is 300 μL, 3-fold gradient dilution (100+200), a total of 10 points; Binding activity detection: Cell counting and plating: After counting R0254-3 cells, divide into 100 μL, 2E+05/well into a 96-well V-type plate; first add 50 μL of antibodies of different concentrations to the cells, incubate at 2-8 degrees for 0.5 hours, then add 50 μL of ligand, and incubate at 2-8 degrees for 0.5 hours; centrifuge at 350xg for 5 minutes, remove the supernatant, Add 200 μL/well of 3% BSA; centrifuge at 350xg for 5 minutes, remove the supernatant, prepare fluorescent antibodies PE Goat anti-human IgG Fc and PE Goat anti-mouse IgG Fc (1:500x dilution) in 3% BSA, add 100 μL/well In the corresponding 96-well plate, incubate at 2-8 degrees for 30 minutes; centrifuge at 350g for 5 minutes, remove the supernatant, and wash the cells with 3% BSA; centrifuge at 350xg for 5 minutes, remove the supernatant, and add 1XPBS at 100 μL/well to resuspend the cells; follow CytoFLEX Flow cytometer standard operating procedures for on-machine testing. The test results are shown in Figure 31. Compared with the molecule without disulfide bond modification, the affinity is equivalent, indicating that disulfide bond modification will not affect the affinity of the targeting region.

Sequences of other related proteins mentioned in the examples of the present invention:

Sequence 36:@PD-L1:VH (SEQ ID NO:36) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSS

Sequence 37:@PD-L1:VL (SEQ ID NO:37) QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVL

R0655 amino acid sequence: (SEQ ID NO:38) EPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVER NSYSCSVVHEGLHNHHTTKSFSRTPGAGGGGSGGGGSGGGGSGGGGSGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTTSGGSG GGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

R0968 PD-L1 VH amino acid sequence: (SEQ ID NO:71) QVQLQESGPGLVKPSETLSLTCAVYGDSITSGYWNWIRKPPGKCLEYIGYISYTGSTYQNPSLKSRITMSRDTSKNQYYLKLSSVTAADTAVYYCARSRAWIRTYFDYWGQGTLVTVSS

R0968 PD-L1 VL amino acid sequence: (SEQ ID NO:72) DIQMTQSPSSSLSASVGDRVTITCSVSSSISSSNLHWYQQKPGKAPKPWIYGTSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQWSSYPLTFGCGTKLEIK

R0774 TIGIT VH amino acid sequence: (SEQ ID NO:73) QVQLVQSGAEVKKPGSSVKVSCKASGYSFTSYWMNWVRQAPGQGLEWIGMIRPSDSETRLNQMFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYCAGIHDYGHGAYWGQGTLVTVSS

R0774 TIGIT VL amino acid sequence: (SEQ ID NO:74) DIQMTQSPSSSLSASVGDRVTITCRASENIYSNLAWYQQKPGKSPKLLVYAASHLPDGVPSRFSGSGSGTDYSLTISSLQPEDFATYYCQHFWGTPRTFGQGTKLEIK

R0919 PD-L1 VH amino acid sequence: (SEQ ID NO:75) EVQLQESGPGLVKPSETLSLTCAVYGDSITSGYWNWIRKPPGKGLEYMGYISYTGSTYQNPSLKSRITFSRDTSKNQYYLKLSSVTAADTATYYCARSRAWIRTYFDYWGQGTLVTVSS

R0919 PD-L1 VL amino acid sequence: (SEQ ID NO:76) EIVLTQSPDFQSVTPKEKVTITCSVSSSISSSNLHWYQQKPDQSPKLLIYGTSNLASGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQWSSYPLTFGQGTKLEIK

without

Figure 1 shows four classic double antibody platforms: Figure 1A shows KiH heterodimeric Fc transformation technology; Figure 1B shows CrossMab bispecific antibody technology; Figure 1C shows Wuhan Youzhiyou YBody double antibody technology (asymmetric scFv double antibody technology). Antibody); Figure 1D shows symmetric scFv double antibody; Figure 2 is a novel bispecific antibody FiBody provided by the present invention, which replaces CH1 and CL of one side of the Fab with a ligand with specific affinity; Figure 3 is an example showing the four possible solutions of FiBody: Figure 3-1 is a modified ligand receptor with non-naturally occurring inter-chain bonds between the ligand receptors; Figure 3-2 shows the CH1 and CH1 of the Fab on both sides. CL is replaced by receptors and ligands, and both sides are selected from different ligands; Figure 3-3 shows the CH1 and CL of the Fab on one side of the antibody are replaced by ligands, and the CH3 segment in the Fc dimer is also liganded. Receptor replacement; Figure 3-4 shows that CH1 and CL of the Fab on one side of the antibody are replaced by ligand receptors, and CH2 in the Fc dimer is also replaced by ligand receptors; there are many other feasible transformation methods; Figure 4 is an exemplary example. When the bispecific antibody of the present invention is used to treat tumors, the target binding of the antigen-binding portion of the bispecific antibody includes three exemplary types: Figure 4-A First antigen-binding portion target To T cells, the second antigen-binding part targets tumor cells; Figure 4-B The first antigen-binding part and the second antigen-binding part both target tumor cells; Figure 4-C The first antigen-binding part and the second antigen-binding part Both target T cells; Figure 4-D illustrates the exemplary embodiment of the bispecific antibody of the present invention, which can be a trifunctional fusion protein. In addition to exerting different antigen binding functions, it can also activate the ligand receptor pathway and stimulate the biological activity of the ligand receptor. ; Figure 5 shows the three-dimensional conformation of interleukins and their receptors, which can be divided into four categories: type A is the lift type, type B is the bowtie type, type C is the baseball hand type, and type D is the clamp type; Figure 6 shows examples of four types of interleukins and their receptors in three-dimensional configurations. Type A push-up type is IL2/IL2R, type B bow-tie type is IL22/IL22R, type C bow-tie type is IL18/IL18R, and type D clamp type is IL21/IL21R; Figure 7 is a FiBody design based on IL15 (ligand) and IL15RA (receptor) in an embodiment of the present invention. The second antigen-binding region VH is connected to IL15RA, and the second antigen-binding region VL is connected to IL15; Figure 8 is a schematic diagram of the structure optimized by disulfide bond modification; Figure 9 is a schematic diagram of the three-dimensional structure of the interaction between IL15/IL15RA and IL2/15Rβ/γC complex; Figure 10 is a schematic structural diagram of the mismatched molecule R1042/R1124 in an embodiment of the present invention; Figure 11 is the HPLC-SEC detection result of sample R0951 in the embodiment of the present invention; Figure 12 is the HPLC-SEC detection result of sample R1042 in the embodiment of the present invention; Figure 13 is the HPLC-SEC detection result of sample R0809 in the embodiment of the present invention; Figure 14 is the HPLC-SEC detection result of sample R1110 in the embodiment of the present invention; Figure 15 shows the FCM method in the embodiment of the present invention to detect the binding activity of the double antibody TIGIT end to CHO-Tigit cells (R0950, R0951, R0952, R0954, R0955, R0960); Figure 16 shows the FCM method in the embodiment of the present invention to detect the binding activity of the double antibody TIGIT end to CHO-Tigit cells (R1123/R1119/R1120/R1124); Figure 17 shows the FCM method in the embodiment of the present invention to detect the binding activity of the TIGIT end of the double antibody to CHO-Tigit cells (R1042/R1043); Figure 18 shows the FCM method in the embodiment of the present invention to detect the binding activity between the TIGIT end of the double antibody and CHO-Tigit cells (R0810); Figure 19 shows the FCM method in the embodiment of the present invention to detect the binding activity of the dual antibody PD-L1 end to CHO-PD-L1 cells (R0950, R0951, R0952, R0954, R0955, R0960); Figure 20 shows the FCM method in the embodiment of the present invention to detect the binding activity of the dual antibody PD-L1 end to CHO-PD-L1 cells (R1072, R1115-R1120, R1123-R1124); Figure 21 shows the FCM method in the embodiment of the present invention to detect the binding activity of the dual antibody PD-L1 terminal to CHO-PD-L1 cells (R0950, R1042, R1043); Figure 22 shows the FCM method in the embodiment of the present invention to detect the binding activity of the dual antibody PD-L1 terminal to CHO-PD-L1 cells (R1072, R1081-R1086); Figure 23 shows the FCM method in the embodiment of the present invention to detect the binding activity of the dual antibody PD-L1 terminal to CHO-PD-L1 cells (R1072, R1109-R1111); Figure 24 shows the binding activity (R0950, R0951, R0952, R0954, R0955, R0960) of the sample receptor ligand complex (IL15/IL15R) in the example of the present invention; Figure 25 shows the binding activity (R1042, R1043) of the sample receptor ligand complex (IL15/IL15R) in the example of the present invention; Figure 26 shows the gel electrophoresis detection results of disulfide bond modified and optimized samples in the embodiment of the present invention (R1072, R1081, R1082, R0954, R1084-R1086) Figure 27 is a schematic diagram of the three-dimensional structure of the mutated disulfide bond between IL15 (ligand) and IL15RA (receptor) molecules; Figure 28 is a schematic structural diagram of an exemplary IL15/IL15Rα complex; Figure 29 shows the gel electrophoresis detection results of complex 1-4; Figure 30 shows the detection results of the binding ability of IL15/IL15Rα complex to the targeting region (@TIGIT); Figure 31 shows the detection results of the binding ability of IL15/IL15Rα complex to the targeting region (@PD-L1).

TW202321315A_111136248_SEQL.xml

Claims (13)

An IL15/IL15Rα polypeptide complex, wherein there is an unnatural interchain bond between IL15 and IL15Rα, and the unnatural interchain bond is formed between the first mutated residue of IL15 and the second mutated residue of IL15Rα. During the period, the first mutated residue of IL15 is E at position 90 mutated to C, and the second mutated residue of IL15Rα is P mutated into C at position 67; the amino acid residue mutation site of IL15 is a reference The natural sequence numbering site corresponding to SEQ ID NO. 26, and the amino acid residue mutation site of IL15Rα is referred to the natural sequence numbering site corresponding to SEQ ID NO. 27. The IL15/IL15Rα polypeptide complex as described in claim 1, wherein the IL15 and IL15Rα specifically bind; optionally, wherein A) The D at position 61 of IL15 is mutated to N, the E at position 64 is mutated to Q, and/or the N at position 65 is mutated to D; and/or B) At least one N-glycosylation site of IL15 does not exist. Preferably, the N-glycosylation site is selected from N71, N79 and/or N112; more preferably, the IL15 contains the following amino acid mutation: N71Q , N79Q and/or N112Q; and/or at least one O glycosylation site of IL15Rα does not exist; preferably, the O glycosylation site is selected from T2, T81 and/or T86; more preferably, the O glycosylation site The IL15Rα contains the following amino acid mutations: T2A, T81A and/or T86A; The amino acid residue mutation site of IL15 is the natural sequence numbering site corresponding to SEQ ID NO. 26, and the amino acid residue mutation site of IL15Rα is the natural sequence numbering site corresponding to SEQ ID NO. 27. The IL15/IL15Rα polypeptide complex according to claim 1, wherein the IL15 is the amino acid sequence shown in SEQ ID NO. 84 or a mutant sequence thereof; preferably, the mutant sequence includes D61N, E64Q and/or N65D amino acid mutation, and/or amino acid mutation selected from N71Q, N79Q and/or N112Q; the amino acid residue mutation site of IL15 is the natural sequence numbering site corresponding to SEQ ID NO. 26. The IL15/IL15Rα polypeptide complex according to any one of claims 1 to 3, wherein the IL15Rα comprises SEQ ID NO.28 or its mutant sequence, and the IL15Rα comprises SEQ ID NO.77 or its mutant sequence, SEQ ID NO.78 or its mutant sequence, SEQ ID NO.79 or its mutant sequence, SEQ ID NO.80 or its mutant sequence, or SEQ ID NO.81 or its mutant sequence; Preferably, the mutant sequence includes selected from Amino acid mutations of T2A, T81A and/or T86A; the amino acid residue mutation site of IL15Rα is the natural sequence numbering site corresponding to SEQ ID NO. 27. The IL15/IL15Rα polypeptide complex according to any one of claims 1 to 4, which further comprises an antibody Fc constant region; optionally, the antibody Fc constant region is a heterodimer; optionally, the The Fc constant region of the antibody associates into a heterodimer based on KiH, hydrophobic interaction, electrostatic interaction, hydrophilic interaction and/or increased flexibility; optionally, the C-terminus of IL15 or IL15Rα and Fc N-terminal linkage of the constant region. The IL15/IL15Rα polypeptide complex according to any one of claims 1 to 5, wherein the polypeptide complex is a bispecific fusion polypeptide, and the polypeptide complex includes a first antigen-binding portion, wherein (A) The first antigen-binding portion comprises: a first polypeptide comprising the first heavy chain variable domain VH1 of the first antibody from the N-terminus to the C-terminus operably linked to to IL15; and a second polypeptide comprising from N-terminus to C-terminus the first light chain variable domain VL1 of the first antibody operably linked to IL15Rα; or (B) The first antigen-binding portion comprises: a first polypeptide comprising the first heavy chain variable domain VH1 of the first antibody from the N-terminus to the C-terminus operably linked to to IL15Rα; and a second polypeptide comprising the first light chain variable domain VL1 of the first antibody from the N-terminus to the C-terminus operably linked to IL15. The IL15/IL15Rα polypeptide complex of claim 6, wherein the polypeptide complex includes a second antigen-binding portion, wherein the second antigen-binding portion includes: a third polypeptide comprising from N-terminus to C-terminus the second heavy chain variable domain VH2 of the second antibody operably linked to the antibody heavy chain constant region CH1, and A fourth polypeptide comprising the second light chain variable domain VL2 of the second antibody from the N-terminus to the C-terminus, which is operably linked to the antibody light chain constant region CL. The IL15/IL15Rα polypeptide complex as described in claim 6 or 7, wherein the first antigen-binding portion and the second antigen-binding portion bind different antigens or bind different epitopes of the same antigen; Optionally, the first antigen-binding moiety targets immune cells, and the second antigen-binding moiety targets tumor cells; Optionally, both the first antigen-binding moiety and the second antigen-binding moiety target tumor cells; Optionally, both the first antigen-binding portion and the second antigen-binding portion target immune cells; Alternatively, the first antigen-binding portion targets human PD-L1 and the second antigen-binding portion targets human TIGIT; or the first antigen-binding portion targets human TIGIT and the second antigen-binding portion targets human PD. -L1. An isolated nucleic acid encoding the IL15/IL15Rα polypeptide complex according to any one of claims 1 to 8. A vector containing the nucleic acid according to claim 9. A host cell containing the nucleic acid according to claim 9 or the vector according to claim 10. A pharmaceutical composition comprising the IL15/IL15Rα polypeptide complex as described in any one of claims 1 to 8, and a pharmaceutically acceptable carrier, excipient or stabilizer. Use of an IL15/IL15Rα polypeptide complex as described in any one of claims 1 to 8 or a pharmaceutical composition as described in claim 12 in the preparation of drugs for treating diseases.

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