A multispecific and / or multivalent binding protein for preventing and / or treating HIV infection

Abstract

The present application relates to a kind of multispecific and / or multivalent binding protein for preventing and / or treating HIV infection, it includes at least 3 antigen binding sites, part of antigen binding site is specifically bound HIV-1-gp41 or HIV-1-gp120 or HIV-1-gp160, still part of antigen binding site is specifically bound human CD4 receptor or human CCR5 receptor;The binding protein of the present application has excellent broad-spectrum neutralizing ability in the treatment or prevention of HIV infection, provides a new solution for the problem of drug resistance escape caused by HIV virus variation, in addition, the present application inventor also unexpectedly found that the binding protein of the present application can treat or prevent the tumor-related disease caused by HIV infection.

Description

Technical Field

[0001] This invention relates to a multispecific and / or multivalent binding protein for the prevention and / or treatment of HIV infection, belonging to the field of biomedicine. Background Technology

[0002] AIDS is short for Acquired Immunodeficiency Syndrome, a chronic systemic infectious disease caused by infection with the human immunodeficiency virus (HIV). HIV primarily infects and destroys CD4+ T cells and lymphocytes, leading to damage or even defects in the body's immune cells and / or their function, ultimately resulting in various serious opportunistic infections and malignant tumors.

[0003] Highly active antiretroviral therapy (HAART) is currently the most effective clinical treatment for AIDS, suppressing HIV replication. However, HIV has a latent infection characteristic, and HAART cannot completely eradicate the virus. Eliminating the latent HIV reservoir is a key focus of AIDS treatment research.

[0004] The process of HIV-1 infecting target cells begins with the binding of its envelope protein surface subunit gp120 to the target cell's major receptor CD4 and co-receptors CXCR4 and / or CCR5. This binding process causes a conformational change in the transmembrane subunit gp41 of the envelope protein, exposing a highly hydrophobic fusion peptide (FP) located at the N-terminus, which inserts into the target cell membrane. Subsequently, the N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR) backfold, forming a core structure consisting of three NHRs surrounded by a six-helix bundle (6HB) of three CHRs. This structure shortens the distance between the viral membrane and the cell membrane, ultimately promoting membrane fusion.

[0005] Currently, four of the clinically approved anti-HIV drugs are invasion inhibitors: iMab (ibalizumab), a monoclonal antibody targeting the CD4 receptor; maraviroc, a small molecule compound targeting the CCR5 co-receptor; fostemsavir, a small molecule compound targeting the gp120 protein; and enfuvirtide (T20), a peptide targeting the gp41 subunit.

[0006] The small molecule compound maraviroc, which targets the CCR5 co-receptor, can only inhibit HIV viruses that infect the corresponding cellular receptor, but is ineffective against dual / mixed viral strains.

[0007] Peptide invasion inhibitors are mainly derived from the NHR or CHR sequences of the virus. They inhibit the formation of homologous 6HB by competitively binding to the natural NHR or CHR, thereby inhibiting the fusion of the viral membrane and the cell membrane.

[0008] However, because HIV has a strong ability to mutate, long-term use of these four invasion inhibitors alone can lead to drug-resistant mutations in the virus and cannot eliminate the latent viral reservoir.

[0009] Researchers have discovered several novel, fully human broadly neutralizing antibodies (bNAb) from HIV patients who produce these antibodies using newly developed B-cell culture techniques or probe-labeled cell sorting methods. These neutralizing antibodies can not only effectively block viral infection of host cells but also clear the viral reservoir.

[0010] In animal studies, broad-spectrum neutralizing antibodies have been shown to be effective in treating and preventing HIV infection. However, in terms of treatment, because HIV has many strains and high variability, using only one bNAb in clinical practice can lead to drug resistance and escape, thus failing to achieve the desired HIV prevention and treatment effect. Summary of the Invention

[0011] To address the aforementioned technical problems, the first aspect of the present invention provides a binding protein, wherein the binding protein comprises at least two antigen-binding sites;

[0012] One of the antigen-binding sites specifically binds to an HIV target protein, wherein the HIV target protein is selected from HIV-gp120, HIV-gp41, or HIV-gp160.

[0013] One of the antigen-binding sites specifically binds to the human CD4 receptor or specifically binds to the human CCR5 receptor.

[0014] In one or more embodiments, the binding protein comprises at least three antigen-binding sites;

[0015] One of the said antigen-binding sites specifically binds to HIV-1-gp41, HIV-1-gp120, or HIV-1-gp160; wherein two of the said antigen-binding sites specifically bind to the human CD4 receptor and the human CCR5 receptor, respectively; or...

[0016] One of the said antigen-binding sites specifically binds to the human CD4 receptor or the human CCR5 receptor, wherein two of the said antigen-binding sites specifically bind to HIV-1-gp41 and HIV-1-gp120, respectively; or,

[0017] One of the antigen-binding sites specifically binds to the human CD4 receptor or the human CCR5 receptor, while two of the antigen-binding sites specifically bind to two different epitopes of HIV-1-gp120, respectively; or...

[0018] One of the antigen-binding sites specifically binds to the human CD4 receptor or the human CCR5 receptor, and the two antigen-binding sites specifically bind to two different epitopes of HIV-1-gp41, respectively.

[0019] Preferably, the antigen binding site is selected from any one of Fv, Fab, Fab', dsFv, scFv or a peptide inhibitor.

[0020] In one or more embodiments, the binding protein comprises at least one polypeptide chain, each polypeptide chain forming three antigen-binding sites;

[0021] One of the said antigen-binding sites specifically binds to HIV-1-gp41, HIV-1-gp120, or HIV-1-gp160; wherein two of the said antigen-binding sites specifically bind to the human CD4 receptor and the human CCR5 receptor, respectively; or...

[0022] One of the said antigen-binding sites specifically binds to the human CD4 receptor or the human CCR5 receptor, wherein two of the said antigen-binding sites specifically bind to HIV-1-gp41 and HIV-1-gp120, respectively; or,

[0023] One of the antigen-binding sites specifically binds to the human CD4 receptor or the human CCR5 receptor, and two of the antigen-binding sites specifically bind to two different epitopes of HIV-1-gp120, respectively; or...

[0024] One of the antigen-binding sites specifically binds to the human CD4 receptor or the human CCR5 receptor, and the two antigen-binding sites specifically bind to two different epitopes of HIV-1-gp41, respectively.

[0025] Preferably, the antigen binding site is selected from any one of Fv, Fab, Fab', dsFv, scFv or a peptide inhibitor.

[0026] In one or more embodiments, the binding protein comprises at least one polypeptide chain, each polypeptide chain comprising three single-chain antibody-binding fragments forming the three antigen-binding sites, respectively.

[0027] In an alternative embodiment of the invention, the binding protein of the invention may also include more antigen binding sites / antigen binding modules, which may be the same as or different from the three antigen binding sites, for example, they may be antigen binding sites / antigen binding modules that bind to other antigen targets.

[0028] In an alternative embodiment of the invention, the three antigen-binding sites are selected from animal-derived antibodies, such as mouse antibodies or their immunoglobulins, camel antibodies or their immunoglobulins, humanized antibodies, or chimeric antibodies.

[0029] In one or more embodiments, the polypeptide chain comprises a structure of ScFv1-L1-ScFv2-L2-ScFv3 from the N-terminus to the C-terminus;

[0030] ScFv1, ScFv2, and ScFv3 are the three single-chain antibody-binding fragments, respectively.

[0031] The single-chain antibody-binding fragment scFv consists of a light chain variable region VL, a linker peptide (GGGGS)n, and a heavy chain variable region VH. Its molecular structure from the N-terminus to the C-terminus is VL—(GGGGS)n—VH; or VH—(GGGGS)n—VL, where the value of n falls in the range of 1 to 10.

[0032] L1 and L2 each possess (GGGGS) n The connection sequence, where the value of n falls within the range of 1 to 10;

[0033] Preferably, the C-terminus of the ScFv3 is linked to a tag sequence; preferably, the tag sequence is a small molecule polypeptide tag sequence; more preferably, the tag sequence is glutathione S-transferase GST, polyhistidine (Poly-His), streptavidin (Strep), FLAG tag, or maltose-binding protein (MBP).

[0034] In one or more embodiments, the polypeptide chain includes a constant region; preferably, the constant region is a heavy chain constant region and / or a light chain constant region of a human immunoglobulin;

[0035] The preferred heavy chain constant region is the heavy chain constant region of human IgG1, 2, 3, and 4;

[0036] The preferred constant region of the light chain is the constant region of the human κ or λ light chain;

[0037] Preferably, the C-terminus of the ScFv3 is connected to the constant region sequentially via a linker peptide and a hinge peptide; preferably, the linker peptide is selected from GS, GGS, GGGS, GGGGS, GGGG, and (GGGGS). n (GGGGS)n G、(GGGGS) n GS, GS (GGGGS) n GS (GGGGS) n GS, GSGGSG, GGSGGGSGGGSGGSGGG, GGGGSGGG, (GGGGSGG) n Wherein, the value of n falls within the range of 1 to 10; preferably, the hinge peptide is selected from EPKSCDKTHTCPPCP, EPKCCVECPPCP, ELKTPLGDTTHTCPRCP (EPKSCDTPPPCPRCP). n ESKYGPPCPSCP, where the value of n falls within the range of 1 to 10; or, the hinge peptide may be selected from the α1 chain, α2 chain, γ1 chain, γ2 chain, γ3 chain, γ4 chain, or δ chain of human immunoglobulin.

[0038] In one or more embodiments, the polypeptide chain comprises a ScFv1-L1-ScFv2 structure from the N-terminus to the C-terminus, wherein the C-terminus of the ScFv2 is connected to a constant region via a linker peptide L3 and a hinge peptide, and the C-terminus of the constant region is connected to a polypeptide inhibitor via a linker peptide L4.

[0039] The constant region is the heavy chain constant region and / or light chain constant region of human immunoglobulin;

[0040] The preferred heavy chain constant region is the heavy chain constant region of human IgG1, 2, 3, and 4;

[0041] The preferred constant region of the light chain is the constant region of the human κ or λ light chain;

[0042] The L1 is characterized by (GGGGS) n The connection sequence, where the value of n falls within the range of 1 to 10;

[0043] Preferably, the linker peptide L3 is selected from GS, GGS, GGGS, GGGGS, GGGG, (GGGGS) n (GGGGS) n G、(GGGGS) n GS, GS (GGGGS) n GS (GGGGS) n GS, GSGGSG, GGSGGGSGGGSGGSGGG, GGGGSGGG, (GGGGSGG) n Wherein, the value of n falls within the range of 1 to 10; preferably, the hinge peptide is selected from EPKSCDKTHTCPPCP, EPKCCVECPPCP, ELKTPLGDTTHTCPRCP (EPKSCDTPPPCPRCP).n ESKYGPPCPSCP, where the value of n falls within the range of 1 to 10; or, the hinge peptide may be selected from the α1 chain, α2 chain, γ1 chain, γ2 chain, γ3 chain, γ4 chain, or δ chain of human immunoglobulin;

[0044] The linker peptide L4 has (GGGGS) n The connection sequence, where the value of n falls within the range of 1 to 10;

[0045] Preferably, the peptide inhibitor is selected from membrane fusion inhibitors targeting gp41.

[0046] In one or more embodiments, the domains of the constant region undergo homodimerization to form homodimers, and the binding protein comprises two polypeptide chains;

[0047] Preferably, the structural domains of the heavy chain constant region undergo homodimerization to form homodimers. Preferably, the amino acids in the structural domains of the hinge region and the heavy chain constant region interact to form disulfide bonds.

[0048] A second aspect of the present invention provides a nucleic acid molecule, wherein the nucleic acid molecule encodes a binding protein as described above.

[0049] Optionally, the nucleic acid molecule can be a nucleic acid molecule formed from deoxyribonucleic acid or a nucleic acid molecule formed from ribonucleic acid.

[0050] A third aspect of the present invention provides a vector comprising the above-mentioned nucleic acid molecules; preferably, the vector is an expression vector; preferably, the vector is a viral vector; preferably, the vector is a lentiviral vector, an adenovirus vector, or an adeno-associated virus vector.

[0051] Preferably, the vector can be transcribed, translated, or modified to form the aforementioned multi-binding specific antibody or its antigen-binding molecule that has the functional activity of inhibiting coronavirus infection or inhibiting respiratory allergic inflammatory response.

[0052] The term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted, thereby enabling the expression of that protein. Vectors can express their carried genetic material elements within host cells through transformation, transduction, or transfection. Vectors can contain various elements controlling expression, such as promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain a replication initiation site. Vectors may also include components that facilitate their entry into cells, such as viral particles, liposomes, or protein coats, but are not limited to these substances. In embodiments of the present invention, the vector may be selected from, but is not limited to: plasmids, phage particles, Cos plasmids, artificial chromosomes (such as yeast artificial chromosome YAC, bacterial artificial chromosome BAC, or P1-derived artificial chromosome PAC), bacteriophages (such as λ phage or M13 phage), and animal viruses used as vectors, such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40).

[0053] Preferably, the vector is a viral vector; more preferably, the viral vector is an adenovirus, lentivirus, or adeno-associated virus vector (e.g., AAV8), including its recombinant form. The term "recombinant" means that the composition is manipulated in a manner not normally found in nature (i.e., engineered). In embodiments of recombinant adeno-associated virus vectors, a nucleic acid sequence encoding the antibody or antigen-binding molecule of the present invention will be inserted into the viral genome.

[0054] In one or more embodiments of the present invention, the vector can be used to produce the multispecific antibody or its antigen-binding molecule of the present invention in vitro. For example, the vector containing the antibody gene of the present invention is delivered to a suitable host cell in an in vitro infection manner, and the host cell expresses and secretes the antibody.

[0055] In one or more embodiments of the present invention, the vector can be used as a gene therapy drug, for example, a gene therapy drug (e.g., liposomes or other lipid-containing complexes) delivered in a recombinant viral system (lentiviral system or adeno-associated virus system) or a non-viral vector, to stably express the antibody or antigen-binding molecule of the present invention in vivo (e.g., in a patient) in a genome-integrated or non-integrated manner for a long period of time.

[0056] A fourth aspect of the present invention provides a host cell comprising the above-described carrier; preferably, the host cell is a mammalian cell.

[0057] Regarding the "host cell," options include, but are not limited to: prokaryotic cells such as *Escherichia coli* or *Bacillus subtilis*, fungal cells such as yeast or *Aspergillus*, insect cells such as S2 *Drosophila* or Sf9, or animal cell models such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, and HEK293 cells; or cells in an animal (e.g., in a human body) that can express the protein molecule. Preferably, the host cell is HEK293 cell.

[0058] In one or more embodiments of the present invention, the host cell may be a cell isolated from an animal and cultured in vitro. The cell may be modified to express the aforementioned binding protein in a membrane-fused or soluble form, and reinfusion into the patient may achieve the prevention or treatment of HIV infection.

[0059] The fifth aspect of the present invention provides a method for producing the binding protein as described above, wherein the protein is produced by transfecting a host cell with the vector or vector system described above; preferably, a lentiviral vector or vector system comprising the nucleic acid molecules described above is used; preferably, an adeno-associated virus vector or vector system comprising the nucleic acid molecules described above is used.

[0060] The binding protein of the present invention can be produced by the above-described recombinant method, by a stable expression cell line, or by a hybridoma method.

[0061] A sixth aspect of the present invention provides a recombinant protein, wherein the recombinant protein comprises the binding protein as described above.

[0062] A seventh aspect of the present invention provides an immune conjugate comprising the binding protein as described above, preferably, one or more heterologous molecules conjugated to the binding protein as described above; more preferably, the heterologous molecule is a cytotoxin.

[0063] Other aspects of the present invention also provide glycosylated variants of the aforementioned binding proteins, antibody variants engineered with cysteine ​​or the like, antibody derivatives, etc.

[0064] An eighth aspect of the present invention provides a pharmaceutical composition comprising, as described above, a binding protein, or a nucleic acid molecule, or a carrier, or a host cell, or a recombinant protein, or an immunoconjugate, and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition is in the form of an injection or a mucosal application; more preferably, the pharmaceutical composition is in the form of an intraperitoneal injection, an intravenous injection, or an intramuscular injection; even more preferably, the pharmaceutical composition is in the form of a gel, spray, drops, oral liquid, cream, film, tablet, vaginal ring, microsphere, liposome, nanoparticle, or in situ gel.

[0065] Preferably, the dosage form is typically administered by injection, including but not limited to intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intra-capsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion; more preferably, the pharmaceutical composition is administered via non-standard routes, such as local, epidermal, or mucosal administration, for example, intranasal, oral, vaginal, rectal, sublingual, or local administration; even more preferably, the pharmaceutical composition is in the form of a gel, nasal spray gel, nasal drops, nebulized inhalation, nasal irrigation solution, oral liquid, mouthwash, cream, film, tablet, vaginal ring, microsphere, liposome, nanoparticle, or in situ gel.

[0066] A ninth aspect of the present invention provides a detection product, wherein the detection product comprises the binding protein as described above, or comprises the nucleic acid molecule as described above, or comprises the carrier as described above, or comprises the host cell as described above, or comprises the recombinant protein as described above, or comprises the immunoconjugate as described above.

[0067] The detection product is used to detect the presence or level of HIV virus in a sample.

[0068] In one or more embodiments, the detection product includes, but is not limited to, detection reagents, detection kits, detection chips, test strips, detection instruments, etc.

[0069] The binding proteins (antibodies or their antigen-binding molecules) of the present invention can be labeled by chemical methods or genetic engineering methods, and can be used for detection after labeling; the labeled binding proteins (antibodies or their antigen-binding molecules) fall within the protection scope of the present invention.

[0070] The specific detection method can adopt the following steps: 1) provide a sample; 2) contact the sample with the binding protein of the present invention; 3) detect the immune reaction between the sample and the binding protein.

[0071] The tenth aspect of the present invention provides the use of the above-described binding protein, or the above-described nucleic acid molecule, or the above-described carrier, or the above-described host cell, or the above-described recombinant protein, or the above-described immune conjugate in the preparation of a medicament for treating or preventing AIDS or tumors caused by HIV infection; preferably, the HIV virus is HIV-1 virus.

[0072] In one or more embodiments of the present invention, AIDS caused by HIV infection refers to sexual immunodeficiency syndrome (AIDS) caused by human immunodeficiency virus (HIV) infection. HIV primarily infects the host through blood, sexual contact, and mother-to-child transmission, gradually damaging the body's immune system, particularly CD4+ T cells, thereby reducing an individual's resistance to various infections and tumors. After HIV infection, patients typically experience three stages: the acute infection phase, the asymptomatic phase, and the AIDS phase. During the acute infection phase, patients may experience flu-like symptoms, while the asymptomatic phase can last for many years, with the virus continuously replicating in the body and causing gradual damage to the immune system. Ultimately, patients enter the AIDS phase, at which point the CD4+ T cell count significantly decreases, leading to opportunistic infections (such as pneumonia, tuberculosis, and yeast infections) and malignancies (such as Kaposi's sarcoma, lymphoma, cervical cancer, and anal cancer, all HIV-related malignancies). Kaposi's sarcoma is a common tumor in AIDS patients, primarily caused by human herpesvirus 8 (HHV-8), which typically presents as purple patches on the skin and mucous membranes. Lymphoma, especially central nervous system lymphoma, is also more common in HIV-infected individuals and is often exacerbated by Epstein-Barr virus (EBV) infection. Furthermore, the incidence of cervical and anal cancers is significantly higher in HIV-infected individuals, primarily associated with human papillomavirus (HPV) infection.

[0073] The eleventh aspect of the present invention also provides a method for treating AIDS or tumors caused by HIV infection, by administering a therapeutically effective amount of the above-mentioned binding protein to a patient, or by administering a pharmaceutical composition containing a therapeutically effective amount of the above-mentioned binding protein to a patient.

[0074] It should be understood that one, a portion, or all of the features of the various embodiments described herein can be combined to form other embodiments of the invention. These and other alternative embodiments of the invention will be apparent to those skilled in the art. These and other alternative embodiments of the invention are further detailed below. Attached Figure Description

[0075] Figure 1 This is a schematic diagram of the expected molecular structure of the multispecific binding proteins in Examples 1-8 of the present invention;

[0076] Figure 2This is a schematic diagram of the expected molecular structure of the multispecific binding protein in Example 9 of the present invention.

[0077] Figure 3 The images show the expression plasmid maps of the multispecific binding proteins in Examples 1-8 of this invention.

[0078] Figure 4 The image shown is a Reduced SDS-PAGE gel electrophoresis image of the multispecific binding proteins in Examples 1-8 of this invention.

[0079] Figure 5 The image shown is an SDS-PAGE gel electrophoresis image of the multispecific binding proteins of Examples 1-8 after immobilizing the dimer conformation by cross-linking with glutaraldehyde. Detailed Implementation

[0080] The present invention will now be described in detail with reference to the specific embodiments shown in the accompanying drawings. However, these embodiments do not limit the present invention, and any structural, methodological, or functional modifications made by those skilled in the art based on these embodiments are included within the scope of protection of the present invention.

[0081] The binding protein provided by this invention contains at least three antigen-binding sites, some of which specifically bind to HIV-1-gp41, HIV-1-gp120, or HIV-1-gp160, and others specifically bind to human CD4 receptors or human CCR5 receptors. The binding protein of this invention exhibits excellent broad-spectrum neutralizing ability in the treatment or prevention of HIV infection, providing a new solution to the problem of drug resistance escape caused by HIV mutations. Furthermore, the inventors of this application have unexpectedly discovered that the binding protein of this invention can treat or prevent tumor-related diseases caused by HIV infection.

[0082] To explain this specification, the following definitions will be used, and terms used in the singular may also include plural forms, where appropriate. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive.

[0083] The term “about” when used in conjunction with a numeric value means to cover a range of numeric values ​​that have a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value.

[0084] As used herein, the terms “comprising” or “including” mean that the stated elements, integers or steps are included, but do not exclude any other elements, integers or steps.

[0085] The term “antibody” is used in the broadest sense herein to encompass natural and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies), single-chain antibodies, intact antibodies, and antigen-binding molecules, antigen-binding fragments, (antigen)-binding proteins, fusion proteins, recombinant proteins, etc., that exhibit the desired antigen-binding activity.

[0086] The terms "antigen-binding molecule" and "antibody-binding fragment" are used interchangeably in this article, referring to a molecule that is not a complete antibody but rather the specific part of a complete antibody that binds to the antigen. Antigen-binding molecules can be prepared using recombinant DNA technology or by enzymatic or chemical cleavage of complete antibodies.

[0087] The term "multispecific" antibody refers to an antibody having at least two antigen-binding sites / antigen-binding modules, each of which binds to a different epitope of the same antigen or to a different epitope of a different antigen.

[0088] The term "trivalent binding protein" refers to a binding protein having three binding sites. In a particular embodiment, a trivalent binding protein binds to one target antigen. In other embodiments, a trivalent binding protein binds to two target antigens. In still other embodiments, a trivalent binding protein binds to three target antigens.

[0089] The terms "antigen binding site" and "antigen binding module" refer to the region of an antibody molecule that actually binds to an antigen, including, for example, a VH / VL pair consisting of a variable domain (VL) of the antibody light chain and a variable domain (VH) of the antibody heavy chain. In some embodiments of the invention, a trivalent binding protein is provided having three antigen binding sites / antigen binding modules capable of binding to three different target antigens (epitopes).

[0090] In some embodiments of the present invention, the antigen binding site / antigen binding module is selected from any one of Fv, Fab, Fab', dsFv or scFv.

[0091] Regarding the combination of the three antigen-binding sites of trispecific antibodies, depending on the different structures of the antigen-binding sites, the main combinations include Fab-Fab-Fab, Fab-Fab-Fv, and Fv-Fv-Fv combinations.

[0092] Among them, the Fab-Fab-Fab combination of trispecific antibodies mainly includes: Triomab generated by the hybrid hybridomas re-fusion technology of rat and mouse hybridoma cells; various bsIgG generated by various Fc heterodimer technologies (such as Knob-in-Hole, charge pairing, SEED, BEAT, LUZ-Y and Duobody, etc.) and Fab mismatch avoidance technologies (such as CrossMab, shared light chain, single chain Fab, κλ-body, Orthogonal Fab, Duetmab and TCR-CαCβ, etc.); IgG-like molecules generated by multiple Fab tandem methods (such as Tandem orthogonal Fab-IgG, FIT-IgG and BiXAb, etc.); IgG-IgG generated by chemical cross-linking technology; and Fab linking molecules generated by various Fab cross-linking technologies (such as F(ab')2, Dockand Lock, etc.).

[0093] In a Fab-Fab-Fv combination of trispecific antibodies, two binding domains that recognize the antigen or epitope are Fab, and the other is Fv. Structurally, the "Fab-Fab-Fv combination" in this application includes Fab-Fab-Fv, Fab-Fv-Fab, and Fv-Fab-Fab cases. Broadly defined, Fv can include single-chain variable region antibodies (scFv), engineered peptides or protein domains with specific recognition functions (such as anticalins, bicyclic peptides, DARPins, finomers, etc.), ligands or receptor molecules, and engineered ligands or receptor molecules.

[0094] In trispecific antibodies using the Fv-Fv-Fv combination, the binding domain that recognizes the antigen or epitope is Fv. Representative structures include scFv-scFv-scFv-Fc, etc. This combination method is very flexible and can easily construct multivalent and multispecific binding molecules.

[0095] The structural stability of the trispecific antibodies with different combinations mentioned above may vary, but their affinity for antigens / epitaxes and their ability to neutralize viruses mainly depend on the "antigen-binding module / site" of the trispecific antibody; in other words, trispecific antibodies with a determined "antigen-binding module / site" can exist in any of the known combinations mentioned above.

[0096] In one specific embodiment of the present invention, a trispecific antibody scheme of scFv-scFv-scFv-Fc is provided; after those skilled in the art learn about the trispecific antibody scheme of the present invention and the sequence of its antigen binding site, they can use existing trispecific antibody technology to modify it into any of the above-mentioned known combinations, such as Fab-Fab-Fab combination, Fab-Fab-Fv combination or other Fv-Fv-Fv combination trispecific antibodies.

[0097] In one specific embodiment of the present invention, the antigen binding site is scFv, which includes a heavy chain variable region (VH region) and a light chain variable region (VL region).

[0098] The heavy chain variable region (VH region) and light chain variable region (VL region) can be further divided into complementarity-determining regions (CDR) and framework regions (FR); the CDR is a hypervariable region, interspersed with more conserved FR regions. Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In a given VH or VL amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of many known schemes, including, for example: Chothia (Chothia et al. (1989) Nature 342:877-883), Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., USDapartment of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath) and Contact (University College London), and the international ImMunoGeneTics database (IMGT) (https: / / www.imgt.org / ). The CDR boundaries of the antibodies of this invention can be determined according to any scheme or combination thereof in the art and human evaluation.

[0099] In one specific embodiment of the present invention, one of the antigen binding sites is a polypeptide inhibitor; specifically, a polypeptide inhibitor refers to a polypeptide inhibitor that binds to a target on the surface of the HIV virus to prevent the HIV virus from entering the host cell, such as a membrane fusion inhibitor that targets gp41 on the surface of HIV.

[0100] In a preferred embodiment of the present invention, a trispecific antibody regimen for the scFv-scFv-Fc-peptide inhibitor is provided. Those skilled in the art, upon learning of the trispecific antibody regimen of the present invention and the sequence of its antigen-binding site, can modify it using existing trispecific antibody technologies to any of the known combinations described above.

[0101] The term "Fc domain" or "Fc region" is used herein to define the carboxyl-terminal region of a human immunoglobulin heavy chain containing at least a portion of a constant region. This term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" contains two or three constant domains: a CH2 domain, a CH3 domain, and optionally a CH4 domain. For example, in native antibodies, the immunoglobulin Fc domain contains the second and third constant domains (CH2 and CH3 domains) of two heavy chains derived from IgG, IgA, and IgD antibodies; or it contains the second, third, and fourth constant domains (CH2, CH3, and CH4 domains) of two heavy chains derived from IgM and IgE antibodies. Unless otherwise stated herein, amino acid residues in the Fc region or heavy chain constant region are numbered according to the EU numbering system (also known as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0102] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibody provided herein to create Fc region variants for enhanced efficacy. For example, mutating methionine at position 428 of the Fc domain to leucine and asparagine at position 434 of the Fc domain to serine can prolong the antibody's half-life in vivo. Modifications to the Fc region include amino acid changes (substitution, deletion, and insertion), glycosylation or deglycosylation, and the addition of multiple Fc groups. Modifications to the Fc can alter the half-life of the antibody in therapeutic antibodies, thereby enabling less frequent dosing and thus increased convenience and reduced material usage. Changes to the Fc can also enhance the antibody's activity in vivo, allowing it to exert its effector function through multiple pathways for more effective and rapid disease relief.

[0103] The term "linker peptide" refers to a linker peptide composed of amino acids, such as glycine and / or serine residues used alone or in combination, to link various variable domains in an antibody. In some embodiments, the linker peptide may be about 1 to about 100 amino acids long, for example, about 1 to 50 amino acids long. In one embodiment, the linker peptide is a G / S linker peptide, non-limiting examples of which are disclosed in the literature (Shen et al., Anal. Chem. 80(6): 1910-1917(2008)) and patent (WO2014 / 087010), the entire contents of which are incorporated herein by reference.

[0104] As used herein, the terms “antigen” or “target antigen” or “antigen target” or “target antigen” refer to a molecule or part of a molecule that can be bound by a binding protein and, additionally, can be used in animals to generate antibodies that bind to an epitope of that antigen. A target antigen may have one or more epitopes. Regarding individual target antigens recognized by a binding protein, the binding protein can compete with the intact antibody that recognizes the target antigen.

[0105] The term "epitope" includes any determinant cluster, preferably a polypeptide determinant cluster capable of specifically binding to immunoglobulins or T-cell receptors. In some embodiments, epitope determinants comprise chemically active surface clusters of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in some embodiments, may have specific three-dimensional structural features and / or specific charge features. An epitope is a region of an antigen that is bound by an antibody or binding protein. In some embodiments, antigen-specific binding is defined as when a binding protein preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. In some embodiments, when the equilibrium dissociation constant is ≤10... -8 M, more preferably when the equilibrium dissociation constant is ≤10 -9 M, the optimal choice is when the dissociation constant ≤ 10 -10 When M occurs, the binding protein is referred to as binding specifically to the antigen.

[0106] As used herein, the terms “binding” or “specific binding” mean that the binding is selective for the antigen and can be distinguished from unwanted or nonspecific interactions. The ability of an antigen-binding module / site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art.

[0107] As used herein, “binding protein” refers to a molecule that can inhibit or significantly reduce the effector function of the target antigen it binds to. As used herein, “significantly reduce” means a reduction of at least about 60%, preferably at least about 70%, more preferably at least about 75%, more preferably at least about 80%, more preferably at least about 85%, and most preferably at least about 90% of the effector function of the target antigen.

[0108] The term "specific binding" as used in this article refers to binding of a protein or its antigen-binding fragment at a distance of at least about 1 x 10-1. -6 M, 1X10 -7 M, 1X10 -8 M, 1X10 -9 M, 1X10 -10 M, 1X10 -11 M, 1X10 -12 M or larger Kd binds to antigens containing epitopes and / or binds to epitopes with an affinity at least twice that of nonspecific antigens.

[0109] "Affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The affinity of molecule X for its partner Y can usually be represented by the dissociation constant (KD), which is the ratio of the dissociation rate constant (kdis) to the association rate constant (kon). Affinity can be measured by common methods known in the art. One specific method used to measure affinity is biomembrane interferometry to detect the association dissociation rate between two molecules.

[0110] "Immune conjugates" are products in which antibodies are conjugated with one or more heterologous molecules (including but not limited to cytotoxic agents).

[0111] The percentage of "sequence homology" of amino acid sequences is calculated by determining the number of matching positions based on the number of amino acid residues present in both sequences, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence homology percentage. Optimal alignment for determining the percentage of sequence homology can be performed in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the full-length sequence being compared or within the target sequence region.

[0112] In this invention, the percentage of amino acid sequence homology for antibody sequences is determined by optimally aligning the candidate antibody sequence with a reference antibody sequence, preferably according to the Kabat numbering rules. In some embodiments, sequence homology for antibodies may be distributed across the entire heavy chain variable region and / or the entire light chain variable region, or the percentage of sequence homology may be limited to the framework region, while the sequence corresponding to the CDR region remains 100% identical.

[0113] Similarly, in terms of antibody sequences, based on alignment, candidate antibodies that have amino acid alterations in the target antibody region relative to a reference antibody can be identified.

[0114] In this invention, "conservative substitution" refers to an amino acid change that results in the replacement of a certain amino acid with a chemically similar amino acid. Amino acid modifications, such as substitutions, can be introduced into the antibodies of this invention using standard methods known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0115] Providing a conservative substitution table of functionally similar amino acids is well known in the art.

[0116] Using well-known techniques, those skilled in the art can identify suitable variants of the polypeptide chain that binds to the protein. For example, by targeting regions considered unimportant to activity, those skilled in the art can identify suitable regions of the polypeptide chain that can be altered without disrupting activity. Alternatively, those skilled in the art can identify conserved residues and molecular moieties in similar polypeptides. Furthermore, even conserved amino acid substitutions can be made in regions that may be important for biological activity or structure without disrupting biological activity or adversely affecting the polypeptide structure.

[0117] The term "N-terminus" refers to the last amino acid residue of a protein or peptide being the amino terminus, while the term "C-terminus" refers to the last amino acid residue of a protein or peptide being the carboxyl terminus.

[0118] The antibody CDR region, also known as the complementarity-determining region or simply "CDR" (which can be used interchangeably with "hypervariant region" or "HVR" in this text), is the amino acid region in the antibody variable region that is primarily responsible for binding to the antigen epitope. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. CDRs located within the variable domain of the antibody heavy chain are called HCDR1, HCDR2, and HCDR3, while those located within the variable domain of the antibody light chain are called LCDR1, LCDR2, and LCDR3.

[0119] Various schemes for determining the CDR sequence within a given VH or VL amino acid sequence are well-known in the art. For example, the Kabat complementarity-determining region (CDR) is determined based on sequence variability and is the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia, on the other hand, refers to the location of a structural loop (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). AbM HVR is a compromise between Kabat HVR and Chothia structural loops and is used by the AbM antibody modeling software from Oxford Molecular. "Contact" HVR is based on the analysis of available complex crystal structures.

[0120] Unless otherwise stated, in this invention, when referring to the position of residues in the antibody variable region (including heavy chain variable region residues and light chain variable region residues), it means the numbering position according to the Kabat numbering system.

[0121] Antibodies with different specificities (i.e., different binding sites against different antigens) have different core binding receptors (CDRs). However, although CDRs differ between antibodies, only a limited number of amino acid sites within a CDR are directly involved in antigen binding. Minimal overlapping regions can be determined using at least two of the Kabat, Chothia, AbM, and Contact methods, thus providing a “minimum binding unit” for antigen binding. The minimum binding unit can be a sub-part of a CDR. As will be apparent to those skilled in the art, the residues of the remaining CDR sequence can be determined by the antibody’s structure and protein folding. Therefore, the present invention also contemplates any variants of the CDRs given herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia may be substituted with conserved amino acid residues.

[0122] The term "hinge peptide" or "hinge region" typically refers to amino acids Glu216 to Pro230 of human IgG1 (see Burton, Molec. Immunol. 22:161-206 (1986)). In some embodiments, the hinge regions of other immunoglobulin heavy chains can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form the inter-heavy chain SS bond in the same position.

[0123] As used herein, the term "vector" refers to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer coding information into a host cell. The term "vector" includes nucleic acid molecules capable of transporting another nucleic acid to which they are linked. One type of vector is the "plasmid," which is a circular double-stranded DNA molecule into which an additional DNA segment can be inserted. Another type of vector is a viral vector, into which an additional DNA segment can be inserted into the viral genome. Some vectors are capable of autonomous replication in the host cell to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the host cell's genome upon introduction into the host cell, thereby replicating along with the host genome. Furthermore, some vectors can direct the expression of the genes to which they are effectively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors used in recombinant DNA technology are often in the form of plasmids. The terms "plasmid" and "vector" are used interchangeably herein, as plasmids are the most commonly used form of vector. However, the present invention is intended to include other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which serve an equivalent function.

[0124] As used herein, the phrase “recombinant host cell” (or “host cell”) refers to a cell in which a recombinant expression vector has been introduced. Recombinant host cell or host cell refers not only to the specific test cell but also to the progeny of such cells. Because certain modifications due to mutations or environmental influences can occur in the progeny, such cells are included within the scope of the term “host cell” as used herein, even though they may not be physically identical to the parent cells. Various host cell expression systems can be used to express the binding protein, including bacterial, yeast, baculovirus, and mammalian expression systems (as well as phage display expression systems). An example of a suitable bacterial expression vector is pUC19. For recombinant expression of the binding protein, a host cell is transformed or transfected with one or more recombinant expression vectors carrying DNA fragments encoding a polypeptide chain encoding the binding protein, such that the polypeptide chain is expressed in the host cell and preferably secreted into the culture medium in which the host cell is cultured, from which the binding protein can be recovered.

[0125] As used herein, the term "transformation" refers to an alteration of a cell's genetic characteristics. A cell is transformed when it is modified to contain new DNA. For example, a cell is transformed when it is genetically modified from its native state. After transformation, the transformed DNA may recombine with the cell through physical integration into the cell's chromosome, or it may transiently remain as an adjunct element without replication, or it may replicate independently as a plasmid. A cell is considered stably transformed when its DNA replicates with cell division. The term "transfection" as used herein refers to the uptake of foreign or exogenous DNA by a cell. A cell is "transfected" when the exogenous DNA is introduced into the cell membrane. Various transfection techniques are well known in the art. These techniques can be used to introduce one or more exogenous DNA molecules into a suitable host cell.

[0126] The term "patient" as used in this article includes both human and animal subjects.

[0127] As used herein, the term "treatment" or "treat" refers to both therapeutic treatment and preventative or preventative measures. Subjects requiring treatment include those with the condition, those susceptible to the condition, or those seeking prevention of their condition. In specific implementations, the binding protein can be used to treat or prevent HIV infection in individuals or patients with tumors caused by HIV infection. The binding protein can also be used to prevent HIV infection in human patients or to prevent tumor development in HIV-infected patients.

[0128] As used herein, the terms “pharmaceutical composition” or “therapeutic composition” refer to any compound or composition that, when properly administered to a patient, can produce the desired therapeutic effect.

[0129] As used in this article, “pharmaceutically acceptable carrier” or “physiologically acceptable carrier” refers to one or more formulation materials suitable for achieving or enhancing the delivery of binding proteins.

[0130] The terms "effective amount" and "therapeutic effective amount," when used to modify pharmaceutical compositions comprising one or more binding proteins, refer to an amount or dose sufficient to produce the desired therapeutic outcome. More specifically, a therapeutic effective amount is an amount of binding protein sufficient to inhibit one or more clinically established pathological processes associated with the condition to be treated for a certain period of time. The effective amount can vary depending on the specific binding protein used and can also depend on various factors and conditions associated with the patient being treated and the severity of the condition. For example, if the binding protein is administered in vivo, factors such as the patient's age, weight, and health status, as well as dose-response curves and toxicity data obtained in preclinical animal studies, will be considered. Determining the effective amount or therapeutic effective amount of a given pharmaceutical composition is entirely within the competence of those skilled in the art.

[0131] The term "HIV virus" refers to the Human Immunodeficiency Virus, a virus that attacks the immune system, particularly CD4+ T cells, leading to impaired immune function. HIV can be transmitted through blood, sexual contact, and from mother to child. Long-term infection can lead to acquired immunodeficiency syndrome (AIDS). HIV belongs to the Retroviridae family, has an RNA genome, and transcribes its RNA into DNA through reverse transcription to integrate into the host cell's genome, thereby replicating and spreading.

[0132] The term "HIV-1-gp160" refers to a precursor glycoprotein in human immunodeficiency virus type 1 (HIV-1), which is the precursor form of gp120 and gp41. gp160 plays an important role in viral infection. It is translated from the environmental genome of HIV-1 during viral synthesis and then broken down into two functional glycoproteins: gp120 and gp41.

[0133] The term "HIV-1-gp120" refers to a glycoprotein in human immunodeficiency virus type 1 (HIV-1), a crucial component of the viral envelope. gp120 is responsible for binding to the CD4 receptor and its co-receptors (such as CCR5 or CXCR4) on the surface of host cells, a primary step in HIV infection. By binding to the CD4 receptor, gp120 triggers subsequent membrane fusion, allowing the virus to efficiently enter the host cell. The high variability of gp120 makes it a challenge in vaccine development, but it also provides a potential target for antibody therapy and vaccine design. Due to its crucial role, gp120 has received widespread attention in HIV research.

[0134] The term "HIV-1-gp41" refers to a glycoprotein in human immunodeficiency virus type 1 (HIV-1), which is an important component of the viral envelope. gp41 works in conjunction with gp120 to participate in the process of viral infection of host cells.

[0135] During HIV infection, gp120 first binds to the CD4 receptor and co-receptors (such as CCR5 or CXCR4) on the surface of host cells. This binding leads to structural changes in gp41, enabling it to mediate the fusion of the viral membrane with the host cell membrane, thus allowing the virus to enter the host cell. The function of gp41 is crucial for viral infection, therefore it is considered a potential vaccine and therapeutic target. Research on gp41 contributes to the development of antibodies and treatments against HIV.

[0136] The term "human CD4 receptor" refers to a glycoprotein expressed on the surface of human immune cells, primarily found on T lymphocytes, monocytes, and macrophages. The CD4 receptor plays a crucial role in the immune system, mainly acting as a surface marker for helper T cells and participating in intercellular signaling.

[0137] HIV enters host cells and causes infection by binding to the CD4 receptor. This binding is a crucial step in HIV infection, making the CD4 receptor an important target for HIV vaccine and treatment research. The expression level of the CD4 receptor is also closely related to the health of the immune system and plays a vital role in the progression of AIDS.

[0138] The term "human CCR5 receptor" refers to a chemokine receptor located on the surface of immune cells, belonging to the chemokine receptor family. The CCR5 receptor is primarily expressed on CD4+ T cells, macrophages, and other immune cells. In HIV infection, the CCR5 receptor, as a core receptor, binds to the gp120 receptor of the HIV-1 virus, facilitating viral entry into host cells. Certain HIV strains are particularly dependent on CCR5 for infection, making this receptor an important target for anti-HIV therapy. Studies have found that individuals carrying CCR5 gene mutations (such as CCR5-Δ32) possess natural resistance to HIV infection, providing new insights for vaccine development and gene therapy. Furthermore, CCR5 plays a crucial role in regulating immune responses and inflammation.

[0139] Binding proteins in Examples 1-8

[0140] The construct structures (from N-terminus to C-terminus) of the binding proteins in Examples 1-8 are all as follows:

[0141] Signal peptide—scFv1—linker peptide L1—scFv2—linker peptide L2—scFv3—linker peptide L3-hinge peptide of human IgG1—CH2 of human IgG1—CH3 of human IgG1;

[0142] The structural schematic diagrams of the binding proteins in Examples 1-8 are shown below. Figure 1 As shown.

[0143] Binding protein of Example 9

[0144] The construct structures of the binding protein in Example 9 (from N-terminus to C-terminus) are as follows:

[0145] Signal peptide —scFv1 —linker peptide L1 —scFv2 —linker peptide L3 —hinge peptide of human IgG1 —CH2 of human IgG1 —CH3 of human IgG1 —linker peptide L4 —peptide inhibitor.

[0146] The structural schematic diagram of the binding protein in Example 9 is shown below. Figure 2As shown.

[0147] Table 1 below shows the specific binding of antigen targets 1-3 at the three antigen binding sites in the binding proteins of Examples 1-9:

[0148] Table 1

[0149] binding protein Target 1 Target 2 Target 3 Example 1 HIV-1-gp41 human CD4 receptor Human CCR5 receptor Example 2 HIV-1-gp41 Human CCR5 receptor human CD4 receptor Example 3 human CD4 receptor Human CCR5 receptor HIV-1-gp120 Example 4 HIV-1-gp41 human CD4 receptor HIV-1-gp120 Example 5 HIV-1-gp41 human CD4 receptor HIV-1-gp120 Example 6 HIV-1-gp120 human CD4 receptor HIV-1-gp41 Example 7 Human CCR5 receptor HIV-1-gp41 HIV-1-gp120 Example 8 HIV-1-gp120 human CD4 receptor HIV-1-gp120 Example 9 HIV-1-gp41 human CD4 receptor HIV-1-gp41

[0150] The sequence sources of the binding proteins scFv1-3 in Examples 1-8, the sequence source of the binding protein scFv1-2 in Example 9, the sequence source of the peptide inhibitor, and the sequence of the linker peptide L1-2 are shown in Table 2 below:

[0151] Table 2

[0152]

[0153] In a preferred embodiment of the present invention, the linker peptide L3 is selected from GS, GGS, GGGS, GGGGS, GGGG, (GGGGS). n (GGGGS) n G、(GGGGS) n GS, GS (GGGGS) n GS (GGGGS) n GS, GSGGSG, GGSGGGSGGGSGGSGGG, GGGGSGGG, (GGGGSGG) n ; where n takes values ​​from 1 to 10.

[0154] Specifically, in the constructs of the binding proteins in Examples 1-9, the sequence of the linker peptide L3 is GS.

[0155] In a preferred embodiment of the present invention, the hinge peptide linked to the constant region is selected from EPKSCDKTHTCPPCP, EPKCCVECPPCP, ELKTPLGDTTHTCPRCP (EPKSCDTPPPCPRCP). n ESKYGPPCPSCP, where the value of n falls within the range of 1 to 10; or, the hinge peptide may be selected from the α1 chain, α2 chain, γ1 chain, γ2 chain, γ3 chain, γ4 chain, or δ chain of human immunoglobulin.

[0156] Specifically, in the constructs of the binding proteins in Examples 1-9, the hinge peptide linked to the constant region is the hinge peptide of human IgG1, the sequence of which is shown in SEQ ID NO. 31. The sequence of CH2 of human IgG1 in the constant region is shown in SEQ ID NO. 32; the sequence of CH3 of human IgG1 is shown in SEQ ID NO. 33.

[0157] In a preferred embodiment of the present invention, the linker peptide L4 is selected from GS, GGS, GGGS, GGGGS, GGGG, (GGGGS). n (GGGGS) n G、(GGGGS) n GS, GS (GGGGS) n GS (GGGGS) n GS, GSGGSG, GGSGGGSGGGSGGSGGG, GGGGSGGG, (GGGGSGG) n ; where n takes values ​​from 1 to 10.

[0158] Specifically, in the construct structure of the binding protein in Example 9, the sequence of the linker peptide L4 is (GGGGS)4.

[0159] In Table 2, the amino acid sequence of polypeptide F is the sequence shown in SEQ ID NO.34; the light chain and heavy chain variable region sequences of monoclonal antibodies A to E in Table 2 are shown in Table 3 below;

[0160] Table 3

[0161]

[0162]

[0163] Binding proteins in Examples 1-8

[0164] The construct structures (from N-terminus to C-terminus) of the binding proteins in Examples 1-8 are all as follows:

[0165] Signal peptide—scFv1—linker peptide L1—scFv2—linker peptide L2—scFv3—linker peptide L3-hinge peptide of human IgG1—CH2 of human IgG1—CH3 of human IgG1;

[0166] The molecular structures of scFv1, scFv2, and scFv3 (from the N-terminus to the C-terminus) are: VL—(GGGGS)n—VH or VH—(GGGGS)n—VL, where n is a positive integer from 1 to 10.

[0167] Specifically, in Examples 1-8, the linker peptide (GGGGS)n between VL and VH of scFv1, scFv2 and scFv3 is (GGGGS)4.

[0168] Preparation of binding proteins in Examples 1-8

[0169] Step 1) Synthesize the sequence of the multispecific binding protein.

[0170] The gene sequence in Example 1 comprises five parts: a secretory protein signal peptide gene SP, three antigen-binding proteins scFv-1, scFv-2, and scFv-3, and the Fc sequence of human IgG1. A linker with the amino acid sequence (GGGGS)n is added between the antigen-binding proteins scFv, and a hinge peptide expressing human IgG1 is added as a linker between the antigen-binding proteins scFv and the Fc sequence of human IgG1.

[0171] As described above, the VL-1 of the antigen-binding protein scFv-1 in Example 1 uses the light chain variable region of monoclonal antibody A, and the VH-1 of scFv-1 uses the heavy chain variable region of monoclonal antibody A, with the two linked by (GGGGS)4.

[0172] Correspondingly, the VL-2 region of the antigen-binding protein scFv-2 in Example 1 uses the light chain variable region of monoclonal antibody B, and the VH-2 region of scFv-2 uses the heavy chain variable region of monoclonal antibody B. The two are linked by (GGGGS)4.

[0173] Correspondingly, the VL-3 region of the antigen-binding protein scFv-3 in Example 1 uses the light chain variable region of monoclonal antibody C, and the VH-3 region of scFv-3 uses the heavy chain variable region of monoclonal antibody C. The two are linked by (GGGGS)4.

[0174] In Example 1 of this application, the nucleotide sequence from the 5' end to the 3' end is as follows: signal peptide SP gene—scFv of monoclonal antibody A—(GGGGS)n—scFv of monoclonal antibody B—(GGGGS)n—scFv of monoclonal antibody C—GS—human IgG1 hinge peptide—human IgG1 Fc;

[0175] The corresponding nucleotide sequences were synthesized by Nanjing GenScript Biotech Co., Ltd. using conventional methods.

[0176] Step 2) Construct expression vectors for multispecific binding protein genes

[0177] The nucleotide sequence of the multispecific binding protein synthesized in step 1) above was cloned into the protein expression vector pcDNA3.4 using homologous recombination, a method well-known to those in the field. Sequencing confirmed the sequence's correctness after cloning. The plasmid map is shown below. Figure 3 .

[0178] Step 3) Expression of multispecific binding proteins in mammalian 293F cells

[0179] The plasmid containing the multispecific binding protein from step 2) above was amplified by transforming *E. coli* DH5α and purified using an endotoxin-free plasmid extraction kit (MGEN, catalog number P1112-03) according to the product instructions. Subsequently, the plasmid was transiently transfected into 293F cells, resulting in the expression of the secreted protein. Transfection of the plasmid into cells was performed using EZ trans cell transfection reagent (Liji Biotechnology, catalog number AC04L092). The transfection procedure was performed according to the EZ trans cell transfection reagent product instructions, i.e., the transfection reagent and the protein expression plasmid were diluted separately in serum-free Opti-MEM (Gibco, catalog number 31985070) at a volume-to-weight ratio of 3:1. The diluted plasmid and transfection reagent were then thoroughly mixed, allowed to stand for 15 minutes, and then the plasmid-transfection reagent mixture was slowly and evenly added to the 293F cells in a droplet-like manner. Six days after transfection, the culture supernatant of 293F cells was collected at 3500 rpm for 10 minutes. The culture supernatant was used to purify the multispecific binding proteins secreted in the supernatant.

[0180] Step 4) Extraction and purification of multispecific binding proteins

[0181] The cell culture supernatant collected in step 3) above was mixed 1:1 with PBS, and Protein G agarose microspheres (Changzhou Tiandi Renhe, catalog number SA016100) were added. The antibody was purified by affinity according to the product instructions. That is, 1 ml of Protein G beads was added to every 100 mL of cell culture supernatant, and the mixture was mixed in a shaker. After incubation at room temperature for 4 hours, the agarose microspheres were collected using a gravity collection column, and 0.1 M Glycine-HCl (pH = 2.8) was added to elute the target protein bound to the microspheres. The eluted protein was added to an ultrafiltration tube, centrifuged at room temperature for concentration, and the replacement buffer was PBS.

[0182] The absorbance at 280 nm was measured using a Nanodrop 2000 (ThermoFisher) to calculate antibody concentration. The purity and identification of the affinity-purified antibody were analyzed by SDS-PAGE. 5 μl of purified sample was mixed with 20 μl of 5× loading buffer and heated in a 100°C metal water bath for 10 minutes. Then, 10 μl of the heated sample was loaded onto a PAGE gel (Nanjing Genscript Biotech Co., Ltd.) for electrophoresis to separate samples according to molecular weight. After separation, the gel was stained with Coomassie Brilliant Blue R250 for 3 hours and destained with destaining solution. Finally, the SDS-PAGE results were captured using a GelDoc Go gel imaging system (BIO-RAD) to evaluate the expression and purification efficiency of the multispecific binding protein.

[0183] The preparation methods of the binding proteins in Examples 2-8 are the same as those for the binding proteins in Example 1 above.

[0184] Binding protein of Example 9

[0185] The construct structures of the binding protein in Example 9 (from N-terminus to C-terminus) are as follows:

[0186] Signal peptide —scFv1 —linker peptide L1 —scFv2 —linker peptide L3 —hinge peptide of human IgG1 —CH2 of human IgG1 —CH3 of human IgG1 —linker peptide L4 —peptide inhibitor.

[0187] The molecular structures of scFv1 and scFv2 (from the N-terminus to the C-terminus) are both: VL—(GGGGS)n—VH, where n is a positive integer from 1 to 10.

[0188] Specifically, in this embodiment 9, the linker peptide (GGGGS)n between VL and VH of scFv1 and scFv2 is (GGGGS)4.

[0189] Specifically, in Example 9, the peptide inhibitor used is the gp41 peptide inhibitor (gp41 peptide membrane fusion inhibitor), which is peptide F (amino acid sequence shown in SEQ ID NO.34).

[0190] The preparation method of the binding protein in Example 9 is the same as that of the binding protein in Example 1.

[0191] Results data

[0192] I. Preparation of HIV-1 pseudovirus strains

[0193] The preparation of HIV-1 pseudovirus involved two plasmids: the HIV envelope protein plasmid preserved in our laboratory and the pNL4-3.Luc.RE-backbone plasmid (NIH AIDS Reagent Program). These two plasmids were first transformed into DH5α E. coli for amplification and then purified using an endotoxin-free plasmid extraction kit (MAGEN, catalog number p1112-02).

[0194] Next, pseudovirus particles were prepared by transfecting 293T cells with these two plasmids. The procedure was as follows: First, 5 × 10⁶ HEK293T cells were seeded in 10 cm diameter culture dishes. 24 hours later, the pNL4-3.Luc.RE backbone plasmid and HIV envelope protein particles were co-transfected into these cells using EZTrans cell transfection reagent (Liji Biotechnology, catalog number C4058L1092). This step was performed according to the EZTrans reagent instructions. 48 hours after transfection, cell debris was removed by centrifugation, and the culture supernatant containing pseudoviruses was collected. The supernatant was aliquoted and stored at -80°C for subsequent experiments to evaluate the neutralizing activity of multispecific binding proteins.

[0195] II. Detection of neutralizing activity of multispecific binding proteins

[0196] We selected 30 common HIV-1 pseudovirus strains prevalent in China and worldwide for antibody neutralization experiments, testing the neutralizing activity of the multispecific binding proteins described in Examples 1-9 against these viral strains. Monoclonal antibodies corresponding to the heavy / light chain variable regions of the multispecific binding proteins were used as controls. The 30 strains covered nine serotypes: Clade A, Clade AC, Clade AE, Clade AG, Clade B, Clade BC, Clade C, Clade D, and Clade G.

[0197] After mixing serially diluted multispecific binding protein / monoclonal antibody with pseudovirus, the mixture was incubated at 37°C for 30 minutes. U87 cells were then added to the antibody-virus mixture at a rate of 10,000 cells / well. Wells without multispecific binding protein / antibody and pseudovirus were designated as negative controls, and wells with pseudovirus alone were designated as positive controls. 24 hours after infection, 130 μl of fresh culture medium was added to each well. After another 24 hours of incubation, the supernatant was discarded, and 50 μl of cell lysis buffer was added to lyse the cells. 30 μl of cell lysis buffer was mixed with 40 μl of Luciferase Assay System (Promega Cat.#E1500), and the fluorescence signal (RLU) was detected using a Perkin Elmer microplate reader. The inhibition rate of HIV pseudovirus infection by adding multispecific binding protein was calculated based on the RLU. Inhibition rate (%) = (sample wells - negative control wells) / (positive control wells - negative control wells), and the half-maximal inhibitory concentration (IC50) of the multispecific binding protein inhibiting viral infection was calculated using PRISM7 software (GraphPad) (unit μg / ml).

[0198] Table 4. Neutralizing effect of the binding proteins in Examples 1-3 against pseudoviruses

[0199]

[0200]

[0201] Table 5. Neutralizing effect of the binding proteins in Examples 4-7 against pseudoviruses.

[0202]

[0203]

[0204]

[0205] Table 6. Neutralizing effect of the binding protein against pseudovirus in Example 8.

[0206]

[0207]

[0208] Table 7 shows the neutralizing effect of the binding protein in Example 9 against the pseudovirus.

[0209]

[0210]

[0211] As can be clearly seen from the data in Tables 4, 5, 6, and 7, the multispecific binding proteins in Examples 1-9 exhibit significant improvements in both the neutralizing broad spectrum and neutralizing activity of HIV-1 pseudoviruses.

[0212] 1. Neutralizes broad spectrum:

[0213] Against 30 pseudovirus strains from different HIV-1 subtypes (including 10 AE strains circulating in the Chinese homosexual community, which are highly pathogenic and rapidly spreading), the multispecific binding proteins in Examples 1-9 all exhibited 100% inhibition of these strains infecting target cells, demonstrating strong broad-spectrum neutralizing ability, especially effective in curbing the AE strains circulating in China. In contrast, while Comparative Examples C and F also neutralized these pseudoviruses 100%, their neutralizing activity was significantly lower than that in Example 4, by 100-fold and 12-fold, respectively. The neutralizing broad-spectrum neutralization of the remaining comparative examples ranged only from 46.7% to 96.7%. These results indicate that the multispecific binding proteins in Examples 1-9 exhibit stronger broad-spectrum neutralizing activity against different HIV-1 subtypes, and show better adaptability and tolerance, especially in responding to HIV viral mutations.

[0214] This broad-spectrum neutralization result demonstrates that the multispecific binding proteins of Examples 1-9 can effectively recognize and neutralize multiple HIV-1 subtypes, which is particularly crucial for addressing HIV-1 subtypes prevalent in different regions and populations worldwide. Their ability to effectively combat rapid viral mutations provides a potential broad-spectrum anti-HIV treatment strategy.

[0215] 2. Neutralizing activity:

[0216] Regarding neutralizing activity, the median neutralizing IC50 values ​​of the multispecific binding proteins in Examples 1-9 against 30 pseudovirus strains were all less than 0.02 μg / mL, significantly lower than the neutralizing IC50 values ​​of the comparative examples. For example, the median IC50 value of Example 4 was only 0.00306 μg / mL, demonstrating effective inhibition of HIV-1 pseudovirus infection at lower doses. In contrast, the median IC50 values ​​of comparative examples A, B, and D constituting Example 4 were 0.2819 μg / mL, 0.066 μg / mL, and 0.034 μg / mL, respectively. Compared to comparative examples A, B, and D, the neutralizing activity of Example 4 was increased by 92-fold, 22-fold, and 11-fold, respectively. This result further confirms that the multispecific binding proteins in Examples 1-9 have higher activity in inhibiting HIV-1 viral infection.

[0217] These data highlight the significant inhibitory effects of the multispecific binding proteins in Examples 1-9 even at low concentrations. This not only means they can more effectively prevent HIV-1 infection, thereby reducing clinical dosage and treatment costs, but also suggests their enormous potential for clinical application.

[0218] In summary, the multispecific binding proteins in Examples 1-9 demonstrate significant advantages over monoclonal antibodies or peptides targeting single epitopes in terms of broad-spectrum neutralization and neutralizing activity, particularly exhibiting superior adaptability and highly efficient inhibitory effects in the face of HIV-1 mutations and diversity. These characteristics suggest their potential in future anti-HIV treatment, providing broad-spectrum and highly effective antiviral therapy options for different regions and populations worldwide, especially in environments with frequent viral mutations and diverse prevalent subtypes.

[0219] It should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This way of describing the specification is only for clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

[0220] The detailed descriptions listed above are merely specific descriptions of feasible embodiments of the present invention, and are not intended to limit the scope of protection of the present invention. All equivalent embodiments or modifications made without departing from the spirit of the present invention should be included within the scope of protection of the present invention.

Claims

1. A binding protein, characterized in that: The binding protein contains at least two antigen-binding sites; One of the antigen-binding sites specifically binds to an HIV target protein, wherein the HIV target protein is selected from HIV-gp120, HIV-gp41, or HIV-gp160. One of the antigen-binding sites specifically binds to the human CD4 receptor or specifically binds to the human CCR5 receptor.

2. The binding protein as described in claim 1, characterized in that: The binding protein contains at least three antigen-binding sites; One of the said antigen-binding sites specifically binds to HIV-1-gp41, HIV-1-gp120, or HIV-1-gp160; wherein two of the said antigen-binding sites specifically bind to the human CD4 receptor and the human CCR5 receptor, respectively; or... One of the said antigen-binding sites specifically binds to the human CD4 receptor or the human CCR5 receptor, wherein two of the said antigen-binding sites specifically bind to HIV-1-gp41 and HIV-1-gp120, respectively; or, One of the antigen-binding sites specifically binds to the human CD4 receptor or the human CCR5 receptor, while two of the antigen-binding sites specifically bind to two different epitopes of HIV-1-gp120, respectively; or... One of the antigen-binding sites specifically binds to the human CD4 receptor or the human CCR5 receptor, and the two antigen-binding sites specifically bind to two different epitopes of HIV-1-gp41, respectively. Preferably, the antigen binding site is selected from any one of Fv, Fab, Fab', dsFv, scFv or a peptide inhibitor.

3. The binding protein as described in claim 2, characterized in that: The binding protein comprises at least one polypeptide chain, and each polypeptide chain forms three antigen-binding sites; One of the said antigen-binding sites specifically binds to HIV-1-gp41, HIV-1-gp120, or HIV-1-gp160; wherein two of the said antigen-binding sites specifically bind to the human CD4 receptor and the human CCR5 receptor, respectively; or... One of the said antigen-binding sites specifically binds to the human CD4 receptor or the human CCR5 receptor, wherein two of the said antigen-binding sites specifically bind to HIV-1-gp41 and HIV-1-gp120, respectively; or, One of the antigen-binding sites specifically binds to the human CD4 receptor or the human CCR5 receptor, while two of the antigen-binding sites specifically bind to two different epitopes of HIV-1-gp120, respectively; or... One of the antigen-binding sites specifically binds to the human CD4 receptor or the human CCR5 receptor, and the two antigen-binding sites specifically bind to two different epitopes of HIV-1-gp41, respectively. Preferably, the antigen binding site is selected from any one of Fv, Fab, Fab', dsFv, scFv or a peptide inhibitor.

4. The binding protein as described in claim 3, characterized in that: The polypeptide chain comprises, from the N-terminus to the C-terminus, the structure ScFv1-L1-ScFv2-L2-ScFv3; ScFv1, ScFv2, and ScFv3 are the three single-chain antibody-binding fragments, respectively. L1 and L2 each possess (GGGGS) n The connection sequence, where the value of n falls within the range of 1 to 10; Preferably, the C-terminus of the ScFv3 is linked to a tag sequence; preferably, the tag sequence is a small molecule polypeptide tag sequence; more preferably, the tag sequence is glutathione S-transferase GST, polyhistidine (Poly-His), streptavidin (Strep), FLAG tag, or maltose-binding protein (MBP). Preferably, any one of ScFv1, ScFv2 and ScFv3 is composed of a light chain variable region VL, a linker peptide (GGGGS)n and a heavy chain variable region VH, and its molecular structure from the N-terminus to the C-terminus is VL—(GGGGS)n—VH; or VH—(GGGGS)n—VL, wherein the value of n falls in the range of 1 to 10.

5. The binding protein as described in claim 4, characterized in that: The polypeptide chain includes a constant region; preferably, the constant region is the heavy chain constant region and / or the light chain constant region of human immunoglobulin. The preferred heavy chain constant region is the heavy chain constant region of human IgG1, 2, 3, and 4; The preferred constant region of the light chain is the constant region of the human κ or λ light chain; Preferably, the C-terminus of the ScFv3 is connected to the constant region sequentially via a linker peptide and a hinge peptide; preferably, the linker peptide is selected from GS, GGS, GGGS, GGGGS, GGGG, and (GGGGS). n (GGGGS) n G、(GGGGS) n GS, GS (GGGGS) n GS (GGGGS) n GS, GSGGSG, GGGGSGGGSGGSGGG, GGGGSGGG, (GGGGSGG) n Wherein, the value of n falls within the range of 1 to 10; preferably, the hinge peptide is selected from EPKSCDKTHTCPPCP, EPKCCVECPPCP, ELKTPLGDTTHTCPRCP (EPKSCDTPPPCPRCP). n ESKYGPPCPSCP, where the value of n falls within the range of 1 to 10; or, the hinge peptide may be selected from the α1 chain, α2 chain, γ1 chain, γ2 chain, γ3 chain, γ4 chain, or δ chain of human immunoglobulin.

6. The binding protein as described in claim 3, characterized in that: The polypeptide chain comprises a ScFv1-L1-ScFv2 structure from the N-terminus to the C-terminus, wherein the C-terminus of the ScFv2 is connected to a constant region via a linker peptide L3 and a hinge peptide, and the C-terminus of the constant region is connected to a polypeptide inhibitor via a linker peptide L4. The constant region is the heavy chain constant region and / or light chain constant region of human immunoglobulin; The preferred heavy chain constant region is the heavy chain constant region of human IgG1, 2, 3, and 4; The preferred constant region of the light chain is the constant region of the human κ or λ light chain; The L1 is characterized by (GGGGS) n The connection sequence, where the value of n falls within the range of 1 to 10; Preferably, the linker peptide L3 is selected from GS, GGS, GGGS, GGGGS, GGGG, (GGGGS) n (GGGGS) n G、(GGGGS) n GS, GS (GGGGS) n GS (GGGGS) n GS, GSGGSG, GGGGSGGGSGGSGGG, GGGGSGGG, (GGGGSGG) n Wherein, the value of n falls within the range of 1 to 10; preferably, the hinge peptide is selected from EPKSCDKTHTCPPCP, EPKCCVECPPCP, ELKTPLGDTTHTCPRCP (EPKSCDTPPPCPRCP). n ESKYGPPCPSCP, where the value of n falls within the range of 1 to 10; or, the hinge peptide may be selected from the α1 chain, α2 chain, γ1 chain, γ2 chain, γ3 chain, γ4 chain, or δ chain of human immunoglobulin; The linker peptide L4 has (GGGGS) n The connection sequence, where the value of n falls within the range of 1 to 10; Preferably, the peptide inhibitor is selected from membrane fusion inhibitors targeting gp41.

7. The binding protein as described in claim 5 or 6, characterized in that: The structural domains of the constant region undergo homodimerization to form homodimers, and the binding protein comprises two polypeptide chains; Preferably, the structural domains of the heavy chain constant region undergo homodimerization to form homodimers.

8. A nucleic acid molecule, characterized in that: The nucleic acid molecule encodes the binding protein as described in any one of claims 1 to 7.

9. A vector comprising the nucleic acid molecule as described in claim 8; preferably, the vector is an expression vector; preferably, the vector is a viral vector; preferably, the vector is a lentiviral vector, an adenovirus vector, or an adeno-associated virus vector.

10. A host cell comprising the vector as described in claim 9; preferably, the host cell is a mammalian cell.

11. A method for producing the binding protein as described in any one of claims 1 to 7, characterized in that: It is produced by transfecting host cells with the vector or vector system described in claim 9; preferably, it uses a lentiviral vector or vector system containing the nucleic acid molecules described in claim 8; preferably, it uses an adeno-associated virus vector or vector system containing the nucleic acid molecules described in claim 8.

12. A recombinant protein, characterized in that: The recombinant protein comprises the binding protein as described in any one of claims 1 to 7.

13. An immunoconjugate comprising a binding protein as described in any one of claims 1 to 7, preferably, one or more heterologous molecules conjugated to the binding protein as described in any one of claims 1 to 7; more preferably, the heterologous molecule is a cytotoxin.

14. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises a binding protein as described in any one of claims 1 to 7, or a nucleic acid molecule as described in claim 8, or a carrier as described in claim 9, or a host cell as described in claim 10, or a recombinant protein as described in claim 12, or an immunoconjugate as described in claim 13, and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition is in the form of an injection or a mucosal application; more preferably, the pharmaceutical composition is in the form of an intraperitoneal injection, an intravenous injection, or an intramuscular injection; even more preferably, the pharmaceutical composition is in the form of a spray, a drop, or an oral liquid.

15. A testing product, characterized in that: The detection product comprises a binding protein as described in any one of claims 1 to 7, or a nucleic acid molecule as described in claim 8, or a vector as described in claim 9, or a host cell as described in claim 10, or a recombinant protein as described in claim 12, or an immunoconjugate as described in claim 13.

16. Use of the binding protein as described in any one of claims 1 to 7, or the nucleic acid molecule as described in claim 8, or the vector as described in claim 9, or the host cell as described in claim 10, or the recombinant protein as described in claim 12, or the immunoconjugate as described in claim 13, in the preparation of a medicament for the treatment or prevention of AIDS or tumors caused by HIV infection; preferably, the HIV virus is HIV-1.

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