Anti-4-1BB-anti-PD-L1 bispecific antibody, as well as its pharmaceutical composition and use

Abstract

The present invention relates to an anti-4-1BB-anti-PD-L1 bispecific antibody, as well as pharmaceutical compositions and uses thereof. Specifically, the present invention relates to a bispecific antibody comprising a first protein functional region targeting 4-1BB and a second protein functional region targeting PD-L1 or PD-1, wherein the first protein functional region is a single-domain antibody against 4-1BB; and the second protein functional region is an anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA-4 antibody, or anti-HER-2 antibody, or an antigen-binding fragment thereof. The bispecific antibody has the function of blocking the binding of PD-L1 to its receptor PD-1 and binds to 4-1BB on immune cells, thereby activating immune cell activity in the tumor microenvironment and more effectively inhibiting tumor initiation and progression.

Description

Detailed description of the invention

[0001] Technical field This invention belongs to the field of biomedicine and relates to an anti-4-1BB-anti-PD-L1 bispecific antibody, as well as its pharmaceutical composition and use.

[0002] Background technology The tumor necrosis factor receptor superfamily member 4-1BB, also known as CD137 or TNFRF9, is a member of the TNF receptor family. 4-1BB is a type I transmembrane protein with 255 amino acids (NCBI: NP_001552), consisting of a 17-amino acid N-terminal signal peptide, a 169-amino acid extracellular domain, a 27-amino acid transmembrane domain, and a 42-amino acid C-terminal intracellular domain. 4-1BB is primarily expressed in activated T cells, NK cells, regulatory T cells, dendritic cells, monocytes, neutrophils, and eosinophils, and it has also been reported that tumor vascular endothelial cells express 4-1BB. Therefore, 4-1BB is also a potential target in the treatment of tumors and certain autoimmune diseases. Specifically, in preclinical animal models of colorectal cancer, lung cancer, breast cancer, and melanoma, agonist molecules targeting 4-1BB show significant antitumor activity, either as monotherapy or in combination with additional antibodies such as anti-PD-1, anti-PD-L1, anti-CTLA-4, and anti-HER-2 (Etxeberria I et al., ESMO Open 2020;4:e000733).

[0003] Programmed death ligand 1 (PD-L1), also known as CD274, is a member of the B7 family and a ligand for PD-1. PD-L1 is a type I transmembrane protein with a total of 290 amino acids, including an IgV-like domain, an IgC-like domain, a transmembrane hydrophobic domain, and an intracellular domain consisting of 30 amino acids. Unlike other B7 family molecules, PD-L1 negatively regulates the immune response. Studies have shown that PD-L1 is primarily expressed in activated T cells, B cells, macrophages, and dendritic cells. In addition to lymphocytes, PD-L1 is expressed in other endothelial cells in many tissues such as the thymus, heart, and placenta, as well as in various non-lymphoid systems such as melanoma, liver cancer, gastric cancer, renal cell carcinoma, ovarian cancer, colon cancer, breast cancer, esophageal cancer, and head and neck cancer (Akintunde Akinleye & Zoaib Rasool, Journal of Hematology & Oncology, Vol. 12, Article No. 92 (2019)). PD-L1 has a certain degree of versatility in regulating self-reactive T and B cells as well as immune tolerance, and is involved in the response of T and B cells in peripheral tissues. High expression of PD-L1 in tumor cells is associated with poor prognosis in cancer patients.

[0004] Most antibody drugs currently on the market are monoclonal antibodies. Therapeutic monoclonal antibodies are used to treat cancer, autoimmune diseases, inflammation, and other diseases, and most are specific to a single target. However, patients may become resistant to or unresponsive to monoclonal antibody therapy. Furthermore, some diseases are influenced by multiple factors in the body, including different signaling pathways, different regulatory mechanisms of cytokines and receptors, and single-target immunotherapy is thought to be insufficient to destroy cancer cells. Therefore, this needs to be achieved using combinations of different drugs or multi-target strategies using multispecific antibodies.

[0005] While bifunctional antibodies represent a promising direction in antibody drug development, they face numerous challenges, including difficulties with preclinical evaluation models, low expression rates, poor stability, complex manufacturing processes, and significant differences in quality control. Therefore, the development of bifunctional antibodies has always been considered challenging.

[0006] Therefore, it is necessary to develop a bispecific antibody that targets PD-L1 and 4-1BB, which has high specificity, high therapeutic efficacy, and is easy to prepare.

[0007] Contents of the present invention Through meticulous and creative research, the inventors have obtained an anti-4-1BB-anti-PD-L1 bispecific antibody (hereinafter also referred to as anti-PD-L1 / 4-1BB bispecific antibody or anti-4-IBB / PD-L1 bispecific antibody). Surprisingly, the inventors have found that the bispecific antibody of the present invention has the function of blocking the binding of PD-L1 to its receptor PD-1; and simultaneously, the bispecific antibody also binds to 4-1BB on immune cells, activating immune cells in the tumor microenvironment, thereby more effectively improving the effect of inhibiting tumor development and growth, and thus exhibiting excellent antitumor activity and good safety. Therefore, the following invention is provided:

[0008] One aspect of the present invention is, The first protein functional region targeting 4-1BB, and A bispecific antibody comprising a second protein functional domain targeting PD-L1 or PD-1; The first protein functional domain is an anti-4-1BB single-domain antibody; The second protein functional domain relates to bispecific antibodies, which are anti-PD-L1 antibodies, anti-PD-1 antibodies, anti-CTLA-4 antibodies, or anti-HER-2 antibodies, or their antigen-binding fragments.

[0009] In some embodiments of the present invention, in a bispecific antibody, the anti-4-1BB single-domain antibody includes a heavy chain variable region, the heavy chain variable region includes CDR1 having the amino acid sequence described in SEQ ID NO: 16, CDR2 having the amino acid sequence described in SEQ ID NO: 17, and CDR3 having the amino acid sequence described in SEQ ID NO: 18; Preferably, the anti-4-1BB single-domain antibody has the amino acid sequence described in SEQ ID NO: 2.

[0010] In some embodiments of the present invention, in a bispecific antibody, the second protein functional region is an anti-PD-L1 monoclonal antibody, an anti-PD-1 monoclonal antibody, an anti-CTLA-4 monoclonal antibody, or an anti-HER-2 monoclonal antibody.

[0011] In some embodiments of the present invention, in a bispecific antibody, the second protein functional region is an anti-PD-L1 single-chain antibody, an anti-PD-1 single-chain antibody, an anti-CTLA-4 single-chain antibody, or an anti-HER-2 single-chain antibody.

[0012] In some embodiments of the present invention, in a bispecific antibody, the second protein functional domain is an anti-PD-L1 single-domain antibody, an anti-PD-1 single-domain antibody, an anti-CTLA-4 single-domain antibody, or an anti-HER-2 single-domain antibody; Preferably, the anti-PD-L1 single-domain antibody includes a heavy chain variable region, the heavy chain variable region comprising CDR1 having the amino acid sequence described in SEQ ID NO: 19, CDR2 having the amino acid sequence described in SEQ ID NO: 20, and CDR3 having the amino acid sequence described in SEQ ID NO: 21; Preferably, the anti-PD-L1 single-domain antibody has the amino acid sequence described in SEQ ID NO: 4.

[0013] In this invention, the CDRs of the anti-4-1BB single-domain antibody and the anti-PD-L1 single-domain antibody are defined in the IMGT numbering system. See Ehrenmann F, Kaas Q, Lefranc MP, IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF[J], Nucleic acids research, 2009;38 (suppl_1):D301-D307.

[0014] In some embodiments of the present invention, in a bispecific antibody, The anti-4-1BB single-domain antibody includes a heavy chain variable region, which comprises CDR1 having the amino acid sequence described in SEQ ID NO: 16, CDR2 having the amino acid sequence described in SEQ ID NO: 17, and CDR3 having the amino acid sequence described in SEQ ID NO: 18. The anti-PD-L1 single-domain antibody includes a heavy chain variable region, which comprises CDR1 having the amino acid sequence described in SEQ ID NO: 19, CDR2 having the amino acid sequence described in SEQ ID NO: 20, and CDR3 having the amino acid sequence described in SEQ ID NO: 21.

[0015] In some embodiments of the present invention, in a bispecific antibody, The anti-4-1BB single-domain antibody has the amino acid sequence described in SEQ ID NO: 2, and The anti-PD-L1 single-domain antibody has the amino acid sequence described in SEQ ID NO: 4.

[0016] In some embodiments of the present invention, a bispecific antibody further comprises one or more (e.g., two or three) Fc fragments of IgG, such as Fc fragments of IgG1, IgG2, IgG3, or IgG4; Alternatively, a bispecific antibody may further include one or more (e.g., two or three) constant regions of IgG, heavy chain constant regions of IgG1, IgG2, IgG3, or IgG4, etc. Preferably, the Fc fragment of IgG or the constant region of IgG is located between the first protein functional region and the second protein functional region.

[0017] In some embodiments of the present invention, the bispecific antibody comprises a first protein functional region, a second protein functional region, an Fc fragment of IgG, and an optional linker.

[0018] In some embodiments of the present invention, the bispecific antibody consists of a first protein functional region, a second protein functional region, an Fc fragment of IgG1, and an optional linker.

[0019] In some embodiments of the present invention, the bispecific antibody contains one Fc fragment of IgG1, and the Fc fragment of IgG1 is located between the first protein functional region and the second protein functional region.

[0020] In some embodiments of the present invention, in the bispecific antibody, the Fc fragment of IgG or the constant region of the heavy chain of IgG is ligated to the C-terminus of the first protein functional region and / or ligated to the C-terminus of the second protein functional region; The Fc fragment of IgG or the constant region of the heavy chain of IgG is ligated to the first protein functional region directly or via a linker; the Fc fragment of IgG or the constant region of the heavy chain of IgG is ligated to the second protein functional region directly or via a linker; Preferably, the linker has an amino acid sequence independently selected from SEQ ID NO: 5 and SEQ ID NO: 22.

[0021] In some embodiments of the present invention, in the bispecific antibody, the Fc fragment of IgG1 is ligated to the C-terminus of the first protein functional region and / or ligated to the C-terminus of the second protein functional region; The Fc fragment of IgG1 is ligated to the first protein functional region directly or via a linker; the Fc fragment of IgG1 is ligated to the second protein functional region directly or via a linker; Preferably, the amino acid sequence of the connecting fragment is independently selected from SEQ ID NO: 5 and SEQ ID NO: 22.

[0022] In some embodiments of the present invention, in a bispecific antibody, according to the EU numbering system, the Fc fragment of IgG or the heavy chain constant region of IgG comprises the L234A and L235A mutations; optionally, the Fc fragment of IgG also comprises the G237A mutation; Preferably, the IgG Fc fragment is an IgG1 Fc fragment containing the L234A and L235A mutations; optionally, the IgG1 Fc fragment further contains the G237A mutation; Preferably, the Fc fragment of IgG1 has the amino acid sequence described in SEQ ID NO: 3; Preferably, the Fc fragment of IgG further contains a knob-in-hole mutation; Preferably, the Fc fragment of IgG1 further contains a knob-in-hole mutation; preferably, the Fc fragment of IgG1 has the amino acid sequence described in SEQ ID NO: 8 or SEQ ID NO: 9.

[0023] In some embodiments of the present invention, in a bispecific antibody, the bispecific antibody comprises the following first peptide chain: The peptide comprises a first protein functional region, an IgG Fc fragment or an IgG heavy chain constant region, an optional linker, and a second protein functional region in order from the N-terminus to the C-terminus; or a second protein functional region; and a first peptide chain comprising an IgG Fc fragment or an IgG heavy chain constant region, an optional linker, and the first protein functional region. Preferably, the linker has an amino acid sequence independently selected from SEQ ID NO: 5 and SEQ ID NO: 22.

[0024] In some embodiments of the present invention, in a bispecific antibody, the bispecific antibody comprises the following first peptide chain: The peptide comprises a first protein functional region, an Fc fragment of IgG1 or the heavy chain constant region of IgG1, an optional linker, and a second protein functional region in order from the N-terminus to the C-terminus; or it comprises a first peptide chain comprising a second protein functional region, an Fc fragment of IgG1 or the heavy chain constant region of IgG1, an optional linker, and the first protein functional region. Preferably, the linker has an amino acid sequence independently selected from SEQ ID NO: 5 and SEQ ID NO: 22.

[0025] In some embodiments of the present invention, in a bispecific antibody, the Fc fragment of IgG1 is an Fc fragment of IgG1 containing the L234A mutation and the L235A mutation (hereinafter referred to as the "LaLa mutation"); optionally, the Fc fragment of IgG1 further contains the G237A mutation; preferably, the Fc fragment of IgG1 has the amino acid sequence described in SEQ ID NO: 3.

[0026] In some embodiments of the present invention, in a bispecific antibody, the Fc fragment of IgG1 contains a knob-in-hole mutation; preferably, according to the EU numbering system, “knob” means that Fc receives the S354C and T366W mutations, and “hole” means that Fc receives the Y349C, T366S, L368A, and Y407V mutations.

[0027] In some embodiments of the present invention, in a bispecific antibody, the Fc fragment of IgG1 comprises an L234A mutation, an L235A mutation, and a knob-in-hole mutation; preferably, the Fc fragment of IgG1 has the amino acid sequence described in SEQ ID NO: 8 or SEQ ID NO: 9.

[0028] In some embodiments of the present invention, in a bispecific antibody, the first peptide chain has the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 6.

[0029] In some embodiments of the present invention, the bispecific antibody is a dimer formed from two first peptide chains, preferably a dimer formed from the first peptide chains described in SEQ ID NO: 1 and / or SEQ ID NO: 6; Preferably, the two first peptide chains are linked by two or three pairs of disulfide bonds; Optionally, the two first peptide chains also contain knob-in-hole mutations.

[0030] In some embodiments of the present invention, in a bispecific antibody, one of the two first peptide chains contains a knob mutation, and the other peptide chain contains a hole mutation.

[0031] In some embodiments of the present invention, the bispecific antibody is a dimer formed from two first peptide chains as described in SEQ ID NO: 1.

[0032] In some embodiments of the present invention, the bispecific antibody is a dimer formed from two first peptide chains as described in SEQ ID NO: 6.

[0033] In some embodiments of the present invention, the bispecific antibody is a dimer formed from one first peptide chain described in SEQ ID NO: 1 and one first peptide chain described in SEQ ID NO: 6.

[0034] In some embodiments of the present invention, the bispecific antibody comprises the following second peptide chain: The molecule further comprises a second peptide chain containing an Fc fragment of IgG or a heavy chain constant region of IgG; preferably, the N-terminus and / or C-terminus of the second peptide chain are ligated to the first and / or second protein functional regions, either directly or via a linker.

[0035] In some embodiments of the present invention, the bispecific antibody comprises the following second peptide chain: The molecule further comprises a second peptide chain containing an Fc fragment of IgG1 or a heavy chain constant region of IgG1; preferably, the N-terminus and / or C-terminus of the second peptide chain are ligated to the first and / or second protein functional regions, either directly or via a linker.

[0036] In some embodiments of the present invention, in a bispecific antibody, the second peptide chain has the amino acid sequence described in SEQ ID NO: 9 or SEQ ID NO: 10.

[0037] In some embodiments of the present invention, in a bispecific antibody, the first peptide chain and the second peptide chain form a dimer; Preferably, the first peptide chain and the second peptide chain are linked by two or three pairs of disulfide bonds; Preferably, the first and second peptide chains further contain knob-in-hole mutations; Preferably, the first peptide chain has the amino acid sequence described in SEQ ID NO: 7, and the second peptide chain has the amino acid sequence described in SEQ ID NO: 9 or SEQ ID NO: 10.

[0038] In some embodiments of the present invention, a bispecific antibody comprises a first peptide chain containing a knob mutation and a second peptide chain containing a hole mutation.

[0039] In some embodiments of the present invention, a bispecific antibody comprises a first peptide chain containing a hole mutation and a second peptide chain containing a knob mutation.

[0040] Another aspect of the present invention relates to an isolated nucleic acid molecule, which encodes a bispecific antibody as described in any one of the items of the present invention.

[0041] The present invention further relates to a vector, which comprises an isolated nucleic acid molecule according to the present invention.

[0042] The present invention also relates to host cells, which include isolated nucleic acid molecules according to the present invention, or vectors according to the present invention.

[0043] Another aspect of the present invention relates to a method for preparing a bispecific antibody according to any one of the items of the present invention, comprising the steps of culturing host cells according to the present invention under appropriate conditions and recovering a bispecific antibody from the cell culture.

[0044] Another aspect of the present invention relates to a conjugate comprising a bispecific antibody and a coupling moiety, wherein the bispecific antibody is a bispecific antibody according to any one of the items of the present invention, and the coupling moiety is a detectable label; preferably, the coupling moiety is a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

[0045] Other aspects of the present invention relate to a kit comprising a bispecific antibody as described in any one of the items of the present invention, or a kit comprising a conjugate as described in the present invention; Preferably, the kit further comprises a second antibody capable of specifically binding to a bispecific antibody; optionally, the second antibody further comprises a detectable label such as a radioisotope, fluorescent substance, luminescent substance, colored substance, or enzyme.

[0046] Another aspect of the present invention relates to the use of a bispecific antibody according to any one of the items of the present invention in the preparation of a kit for detecting the presence or level of 4-1BB and / or PD-L1 in a sample.

[0047] Another aspect of the present invention relates to a pharmaceutical composition comprising a bispecific antibody according to any one of the items of the present invention, or a conjugate according to the present invention; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0048] Other aspects of the present invention relate to the use of a bispecific antibody or conjugate according to any one of the items of the present invention in the manufacture of a pharmaceutical for the prevention and / or treatment of malignant tumors; preferably, the malignant tumor is selected from the group consisting of rectal cancer, colon cancer, lung cancer, melanoma, liver cancer, gastric cancer, renal cell carcinoma, ovarian cancer, esophageal cancer, and head and neck cancer.

[0049] Another aspect of the present invention relates to a method for the treatment and / or prevention of malignant tumors, comprising the step of administering an effective amount of a bispecific antibody or conjugate according to any one of the items of the present invention to a subject in need; preferably, the malignant tumor is rectal cancer, colon cancer Lung cancer, melanoma, liver cancer, stomach cancer, renal cell carcinoma, ovarian cancer, esophageal cancer, and head and neck cancer. It is selected from the group consisting of the following.

[0050] In some embodiments of the present invention, the step of administering an effective amount of a bispecific antibody described in any one of the items of the present invention to a subject in need thereof is performed before or after surgical treatment and / or before or after radiotherapy.

[0051] In some embodiments of the present invention, in the method, The bispecific antibody of the present invention is administered in a single dose of 0.1 to 100 mg per kg of body weight, preferably 4.8 to 24 mg or 1 to 10 mg; or the bispecific antibody of the present invention is administered in a single dose of 10 to 1,000 mg, preferably 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg or 200 mg; Preferably, administration is performed once every 3, 4, 5, 6, 10 days, 1 week, 2 weeks, or 3 weeks; Preferably, administration is performed by intravenous drip or intravenous injection.

[0052] A bispecific antibody or conjugate described in any one of the items of the present invention is used for the treatment and / or prevention of malignant tumors, preferably selected from the group consisting of rectal cancer, colon cancer, lung cancer, melanoma, liver cancer, gastric cancer, renal cell carcinoma, ovarian cancer, esophageal cancer, and head and neck cancer.

[0053] As used herein, the term “antibody” generally refers to an immunoglobulin molecule composed of two pairs of polypeptide chains, each pair having one “light” (L) chain and one “heavy” (H) chain. Antibody light chains can be classified into κ light chains and λ light chains. Heavy chains may be classified into μ, δ, γ, α, or ε, and the antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by “J” regions of about 12 or more amino acids, and the heavy chain further contains “D” regions of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The antibody constant region mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq). The VH and VL regions can be subdivided into highly variable regions called complementarity-determining regions (CDRs), within which more conserved regions called framework regions (FRs) are scattered. The VH and VL each consist 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, and FR4. The variable regions (VH and VL) of each heavy / light chain pair each form an antibody binding site.For amino acid assignments to regions or domains, refer to the definitions in Bethesda Md, Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 1987; 196: 901-917; Chothia et al., Nature 1989; 342: 878-883, or Ehrenmann F, Kaas Q, Lefranc M P. IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF[J]. Nucleic acids research, 2009; 38(suppl_1): D301-D307, following the IMGT numbering system.

[0054] The term "antibody" is not limited to a specific method of antibody production. Examples include recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies may also be of different isotypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgAl, IgA2, IgD, IgE, or IgM antibodies.

[0055] As used herein, the terms “mAb” and “monoclonal antibody” refer to a highly homologous group of antibody molecules, i.e., antibodies or antibody fragments derived from the same group of antibody molecules, apart from spontaneously occurring innate mutations. mAbs are highly specific to a single epitope on an antigen. Compared to monoclonal antibodies, polyclonal antibodies typically contain at least two different antibodies, and these different antibodies typically recognize different epitopes on an antigen. Monoclonal antibodies can usually be obtained using hybridoma technology, first reported by Kohler et al. (Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity [J], Nature, 1975;256(5517):495), and can also be obtained by recombinant DNA technology (see, e.g., U.S. Patent No. 4,816,567).

[0056] As used herein, the term “humanized antibody” refers to an antibody or antibody fragment obtained by substituting all or part of the CDR region of a human immunoglobulin (recipient antibody) with the CDR region of a non-human antibody (donor antibody), where the donor antibody may be a non-human (e.g., mouse, rat, or rabbit) antibody having the desired specificity, affinity, and reactivity. Furthermore, some amino acid residues in the framework region (FR) of the recipient antibody may be substituted with corresponding amino acid residues of a non-human antibody or other antibody to further improve or optimize the performance of the antibody. For more information on humanized antibodies, see, for example, Jones et al., Nature 1986;321:522-525; Reichmann et al., Nature, 1988;332:323-329; Presta, Curr. Op. Struct. Biol. 1992;2:593-596; and Clark, Immunol. Today 2000;21:397-402. In some cases, the antigen-binding fragment of the antibody is the diabody, and in this case, V H and V LThe domain is expressed on a single polypeptide chain, but the linker used is too short to allow pairing between two domains on the same chain. As a result, the domain pairs with a complementary domain on another chain, forming two antigen-binding sites (see, for example, Holliger P. et al., Proc.Natl.Acad.Sci.USA 1993;90:6444-6448 and Poljak RJ et al., Structure 1994;2:1121-1123).

[0057] The fusion proteins described herein are protein products co-expressed by two genes through DNA recombination. Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art (e.g., Cold Spring Harbor's Antibody Laboratory Technique Guide, Chapters 5-8 and 15).

[0058] As used herein, the terms “isolated” or “isolated” mean acquisition from its natural state by artificial means. When an “isolated” substance or component occurs naturally, it means that either the natural environment in which it exists has been altered, the substance has been isolated from its natural environment, or both. For example, with respect to an unisolated polynucleotide or polypeptide that exists naturally in a living animal, a high-purity polynucleotide or polypeptide isolated from its natural state is called isolated. The terms “isolated” or “isolated” do not preclude the presence of mixtures of artificial or synthetic substances and other impurities that do not affect the substance’s activity.

[0059] As used herein, the term “vector” refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. A vector is called an expression vector if it can achieve the expression of a protein encoded by the inserted polynucleotide. A vector may be introduced into a host cell by transformation, transduction, or transfection, and its genetic material elements can be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); phages such as lambda phages or M13 phages; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). A vector may contain various elements that control expression, including but not limited to promoter sequences, transcription start sequences, enhancer sequences, selection elements, and reporter genes. Furthermore, a vector may also contain an origin of replication.

[0060] As used herein, the term “host cell” refers to a cell that can be used for vector introduction, and includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as Drosophila S2 cells or Sf9 cells, or animal cells such as fibroblasts, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

[0061] As used herein, the term “pharmaceutically acceptable excipients” means carriers and / or excipients that are pharmaceutically and / or physiologically compatible with the subject and active ingredients, which are well known in the art (see, for example, Remington's Pharmaceutical Sciences, Gennaro AR, 19th edition, Pennsylvania, Mack Publishing Company, 1995), and include, but are not limited to, pH modifiers, surfactants, adjuvants, and ionic strength enhancers. For example, pH modifiers include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0062] As used herein, the term “effective dose” means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective dose for preventing a disease (e.g., a tumor) means an amount sufficient to prevent, inhibit, or delay the onset of the disease (e.g., a tumor), and an effective dose for treating a disease means an amount sufficient to cure or at least partially prevent the disease and its complications in a patient with the disease. Determining such an effective dose is well within the capabilities of those skilled in the art. For example, an effective dose for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient’s own immune system, the patient’s general condition such as age, weight, and sex, the mode of administration of the drug, and any other treatments administered in combination.

[0063] In the present invention, unless otherwise specified, "first" (e.g., first protein functional region) and "second" (e.g., second protein functional region) are used for the purpose of reference distinction or clarification of expression and do not have a typical orderly meaning.

[0064] Furthermore, the present invention relates to any one of the following items 1 to 10: 1. It is a bispecific antibody: (a) Anti-PD-L1 single-domain antibody: and (b) Anti-4-1BB single-domain antibody A bispecific antibody characterized by containing [the specified ingredient]. 2. An isolated polynucleotide characterized by encoding the bispecific antibody described in item 1. 3. A vector characterized by containing the polynucleotide described in item 2. 4. The vector described in item 3, or the polynucleotide described in item 2, is incorporated into the host cell's genome; Alternatively, a host cell characterized by expressing the bispecific antibody described in item 1. 5. A method for producing the bispecific antibody described in item 1: (a) The step of culturing the host cells described in item 4 under appropriate conditions to obtain a culture containing bispecific antibodies; and (b) A method comprising the step of purifying and / or separating the culture obtained in step (a) to obtain a bispecific antibody. 6. (a) Bispecific antibodies as described in item 1; and (b) An immunoconjugate characterized by comprising a coupling moiety selected from the group consisting of a detectable label, drug, toxin, cytokine, radionuclide, or enzyme, gold nanoparticles / nanorods, nanomagnetic particles, viral coated proteins or VLPs, or a combination thereof. 7. Use of the bispecific antibody described in item 1 or the immunoconjugate described in item 6 in the manufacture of a pharmaceutical, reagent, detection plate or kit; the reagent, detection plate or kit is used to detect PD-L1 and / or 4-1BB in a sample; the pharmaceutical is used to treat or prevent a tumor expressing PD-L1 (i.e., PD-L1 positive). 8. (i) Bispecific antibodies as described in item 1, or immunoconjugates as described in item 6; and (ii) A pharmaceutically acceptable carrier A pharmaceutical composition characterized by containing the following: 9. Below: (i) Use for detecting human PD-L1 molecules and / or 4-1BB molecules; (ii) Use for flow detection; (iii) Use for cellular immunofluorescence detection; (iv) Use for treating tumors; (v) Use for diagnosing tumors; (vi) Use for blocking the interaction between PD-1 and PD-L1; and (vii) Use for binding to 4-1BB and activating immune cells. Use of one or more bispecific antibodies listed in item 1, selected from the group including the above. 10. A recombinant protein comprising (i) a bispecific antibody as described in item 1; and (ii) an optional tag sequence to aid in expression and / or purification.

[0065] The present invention further relates to any one of the following first to fourteen aspects; In a first embodiment of the present invention, a bispecific antibody is provided, and the bispecific antibody is; (a) Anti-PD-L1 single-domain antibody; and (b) Anti-4-1BB single-domain antibody Includes.

[0066] In other preferred embodiments, the bispecific antibody comprises 1 to 3 anti-PD-L1 single-domain antibodies, preferably 1 or 2 anti-PD-L1 single-domain antibodies.

[0067] In another preferred embodiment, a PD-L1 single-domain antibody can block the interaction between PD-1 and PD-L1.

[0068] In other preferred embodiments, the bispecific antibody comprises 1 to 3 anti-4-1BB single-domain antibodies, preferably 1 or 2 anti-4-1BB single-domain antibodies.

[0069] In another preferred embodiment, a 4-1BB single-domain antibody may activate immune cells.

[0070] In another preferred embodiment, the bispecific antibody further comprises an Fc region derived from human immunoglobulin.

[0071] In another preferred embodiment, the human immunoglobulin is selected from the group consisting of IgG1, IgG2, IgG3, IgG4 or a combination thereof; preferably IgG1.

[0072] In another preferred embodiment, the Fc region of the bispecific antibody is selected from the group consisting of a CH1+CL1 domain, a human IgG domain, or a combination thereof.

[0073] In other preferred embodiments, the Fc region is an engineered mutant, preferably an LALA mutant, and includes a knob-in-hole mutant.

[0074] In another preferred embodiment, the bispecific antibody is a dimer composed of peptide chain i and peptide chain ii, where peptide chain i and peptide chain ii are Equations I and II, A-L1-Fc1-L2-B(Formula I) A-L3-Fc2-L4-B (Formula II) It has the structure shown in the formula, in which, A and B are, independently, non-existent anti-PD-L1 single-domain antibodies or anti-4-1BB single-domain antibodies; L1, L2, and L3 are either independent, non-existent, or linked elements; L4 is a connecting element; Fc1 and Fc2 are, independently, Fc regions of human immunoglobulin (preferably LALA variants); and The hyphen "-" represents a peptide bond; Here, the bispecific antibody comprises at least one anti-PD-L1 single-domain antibody and at least one anti-4-1BB single-domain antibody; Polypeptides represented by formula I and those represented by formula II form heterodimers through disulfide bond interactions and knob-in-hole structures.

[0075] In other preferred embodiments, the bispecific antibody is a homodimer or a heterodimer.

[0076] In other preferred embodiments, the Fc region is an LALA variant Fc, having the amino acid sequence described in SEQ ID NO: 3, or having 85% (preferably 90%, more preferably 95%) or more sequence identity with SEQ ID NO: 3.

[0077] In another preferred embodiment, Fc1 and Fc2 have a knob mutation and a hole mutation, respectively.

[0078] In another preferred embodiment, the amino acid sequence of Fc1 has an S132C mutation at position 132 and a T144W mutation at position 144, based on the amino acid sequence described in Sequence ID No. 3.

[0079] In another preferred embodiment, the amino acid sequence of Fc2 has a Y127C mutation at position 127, a T144S mutation at position 144, and an L146A mutation at position 146, based on the amino acid sequence described in Sequence ID No. 3.

[0080] In another preferred embodiment, Fc1 has the amino acid sequence described in SEQ ID NO: 8, or has 85% (preferably 90%, more preferably 95%) or more sequence identity with the sequence described in SEQ ID NO: 8.

[0081] In another preferred embodiment, Fc2 has the amino acid sequence described in SEQ ID NO: 9, or has 85% (preferably 90%, more preferably 95%) or more sequence identity with the sequence described in SEQ ID NO: 9.

[0082] In another preferred embodiment, the anti-4-1BB single-domain antibody has the amino acid sequence described in SEQ ID NO: 2, or has 85% (preferably 90%, more preferably 95%) or more sequence identity to the sequence described in SEQ ID NO: 2.

[0083] In another preferred embodiment, the anti-PD-L1 single-domain antibody has the amino acid sequence described in SEQ ID NO: 4, or has 85% (preferably 90%, more preferably 95%) or more sequence identity to the sequence described in SEQ ID NO: 4.

[0084] In another preferred embodiment, the linker arrangement is (G4S) n The formula is such that n is a positive integer (for example, 1, 2, 3, 4, 5, or 6), and preferably n is 2 or 4.

[0085] In another preferred embodiment, the linker has the amino acid sequence described in SEQ ID NO: 5, or has 85% (preferably 90%, more preferably 95%) or more sequence identity with respect to the sequence described in SEQ ID NO: 5.

[0086] In another preferred embodiment, the bispecific antibody is a homodimer, which is formed by two identical peptide chains via a disulfide bond, and the peptide chains have the amino acid sequence described in SEQ ID NO: 1 (i.e., Bi-400) or have 85% (preferably 90%, more preferably 95%) or more sequence identity to the sequence described in SEQ ID NO: 1.

[0087] In another preferred embodiment, the bispecific antibody is a homodimer, which is formed by two identical peptide chains via a disulfide bond, and the peptide chains have the amino acid sequence described in SEQ ID NO: 6 (i.e., Bi-088) or have 85% (preferably 90%, more preferably 95%) or more sequence identity to the sequence described in SEQ ID NO: 6.

[0088] In another preferred embodiment, the bispecific antibody is a heterodimer formed by peptide chain i and peptide chain ii via a knob-in-hole interaction (knob-in-hole); where peptide chain i has the amino acid sequence described in SEQ ID NO: 7 or has 85% (preferably 90%, more preferably 95%) or more sequence identity to the sequence described in SEQ ID NO: 7; and peptide chain ii has the amino acid sequence described in SEQ ID NO: 9 or has 85% (preferably 90%, more preferably 95%) or more sequence identity to the sequence described in SEQ ID NO: 9 (i.e., Bi-401-091).

[0089] In another preferred embodiment, the bispecific antibody is a heterodimer formed by peptide chain i and peptide chain ii via a knob-in-hole interaction (knob-in-hole); where peptide chain i has the amino acid sequence described in SEQ ID NO: 7 or has 85% (preferably 90%, more preferably 95%) or more sequence identity to the sequence described in SEQ ID NO: 7; and peptide chain ii has the amino acid sequence described in SEQ ID NO: 10 or has 85% (preferably 90%, more preferably 95%) or more sequence identity to the sequence described in SEQ ID NO: 10 (i.e., Bi-061-091).

[0090] In a second embodiment of the present invention, an isolated polynucleotide is provided, which encodes a bispecific antibody according to a first embodiment of the present invention.

[0091] In another preferred embodiment, when the bispecific antibody is a heterodimer, the polynucleotide sequence encoding peptide chain i and the polynucleotide sequence encoding peptide chain ii are in a 1:1 ratio within the polynucleotide.

[0092] In a third embodiment of the present invention, a vector is provided, the vector comprising a polynucleotide according to a second embodiment of the present invention.

[0093] In other preferred embodiments, the vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof; preferably, the expression vector includes viral vectors such as lentiviruses, adenoviruses, AAV viruses, retroviruses, or combinations thereof.

[0094] In a fourth embodiment of the present invention, a host cell is provided, the host cell having a vector according to a third embodiment of the present invention, or a polynucleotide according to a second embodiment of the present invention incorporated into its genome: or The host cell expresses a bispecific antibody according to the first aspect of the present invention.

[0095] In other preferred embodiments, the host cells include prokaryotic cells or eukaryotic cells.

[0096] In other preferred embodiments, the host cells are selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.

[0097] A fifth aspect of the present invention is a method for producing a bispecific antibody according to the first aspect of the present invention: (a) The step of culturing host cells according to the fourth aspect of the present invention under appropriate conditions to obtain a culture containing bispecific antibodies; and (b) A method is provided which includes the step of purifying and / or separating the culture obtained in step (a) to obtain a bispecific antibody.

[0098] In another preferred embodiment, purification can be performed for the target antibody by protein A affinity column purification and separation.

[0099] In other preferred embodiments, the purified target antibody has a purity of over 95%, over 96%, over 97%, over 98%, over 99%, preferably 100%.

[0100] In a sixth embodiment of the present invention, an immunoconjugate is provided, which is: (a) A bispecific antibody according to a first aspect of the present invention; and (b) comprising a coupling moiety selected from the group consisting of a detectable label, drug, toxin, cytokine, radionuclide, or enzyme, gold nanoparticles / nanorods, nanomagnetic particles, virus-coated proteins or VLPs, or a combination thereof.

[0101] In another preferred embodiment, the radionuclide is: (i) Diagnostic isotopes selected from the group consisting of: Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or combinations thereof; and / or (ii) Therapeutic isotopes comprising therapeutic isotopes selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb-177, or combinations thereof.

[0102] In another preferred embodiment, the coupling portion is a drug or toxin.

[0103] In another preferred embodiment, the agent is a cytotoxic agent.

[0104] In other preferred embodiments, the cytotoxic agent is selected from the group consisting of antitubulin agents, DNA minor groove binders, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemotherapeutic sensitizers, topoisomerase inhibitors, vinca alkaloids, or combinations thereof.

[0105] Particularly useful classes of cytotoxic agents include, for example, DNA minor groove binders, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic agents include, for example, auristatin, camptothecin, duocalmycin, etoposide, maytansine and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines, or benzodiazepine-containing agents (e.g., pyrrolo[1,4]benzodiazepine (PBD), indolinobenzodiazepine, and oxazolidinobenzodiazepine), vinca alkaloids, or combinations thereof.

[0106] In another preferred embodiment, the toxin is as follows: The following are selected from the group consisting of auristatins (e.g., auristatin E, auristatin F, MMAE, and MMAF), aureomycin, meitansinoids, lysine, lysine A chain, combretastatin, duocalmycin, dorastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, cortisine, dihydroxyanthracinedione, actinomycin, diphtheria toxin, pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modexin A chain, α-Sertin, geronin, mitogen, retostrictosin, phenomycin, enomycin, chrysin, crotin, calicheamicin, saponaria officinalis inhibitors, glucocorticoids, or combinations thereof.

[0107] In another preferred embodiment, the coupling portion is a detectable label.

[0108] In other preferred embodiments, the conjugate is selected from the group consisting of: fluorescent or luminescent labels, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (e.g., IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), and chemotherapeutic agents (e.g., cisplatin).

[0109] In other preferred embodiments, the immunoconjugate comprises a polyvalent (e.g., bivalent) bispecific antibody as described in the first aspect of the present invention.

[0110] In other preferred embodiments, polyvalent means that the immunoconjugate has an amino acid sequence comprising multiple repeats of the bispecific antibody according to the first aspect of the present invention.

[0111] In a seventh aspect of the present invention, the use of a bispecific antibody according to the first aspect of the present invention or an immunoconjugate according to the sixth aspect of the present invention is provided in the manufacture of a pharmaceutical, reagent, detection plate or kit; wherein the reagent, detection plate or kit is used to detect PD-L1 and / or 4-1BB in a sample; wherein the pharmaceutical is used to treat or prevent a tumor expressing PD-L1 (i.e., PD-L1 positive).

[0112] In another preferred embodiment, the coupling portion of the immunoconjugate is a diagnostic isotope.

[0113] In other preferred embodiments, the reagent is one or more reagents selected from the group consisting of isotope tracers, contrast agents, flow detection reagents, cellular immunofluorescence detection reagents, magnetic nanoparticles, and imaging agents.

[0114] In another preferred embodiment, the reagent for detecting PD-L1 and / or 4-1BB in the sample is a contrast agent for detecting PD-L1 and / or 4-1BB molecules (in vivo).

[0115] In other preferred embodiments, detection is either in vivo or in vitro.

[0116] In other preferred embodiments, detection includes flow detection and cellular immunofluorescence detection.

[0117] In another preferred embodiment, the agent is used to block the interaction between PD-1 and PD-L1 and simultaneously activate immune cells by binding 4-1BB.

[0118] In other preferred embodiments, tumors include, but are not limited to, acute myeloid leukemia, chronic myeloid leukemia, multiple myelopathy, non-Hodgkin lymphoma, colorectal cancer, breast cancer, colorectal cancer, gastric cancer, liver cancer, leukemia, kidney tumors, lung cancer, small intestine cancer, bone cancer, prostate cancer, prostate cancer, cervical cancer, lymphoma, adrenal tumors, bladder tumors, or combinations thereof.

[0119] In an eighth aspect of the present invention, a pharmaceutical composition is provided, which is: (i) a bispecific antibody according to the first aspect of the present invention, or an immunoconjugate according to the sixth aspect of the present invention; and (ii) Contains a pharmaceutically acceptable carrier.

[0120] In other preferred embodiments, the coupling portion of the immunoconjugate is a drug, toxin, and / or therapeutic isotope.

[0121] In another preferred embodiment, the pharmaceutical composition further comprises additional agents, such as cytotoxic agents, for treating tumors.

[0122] In other preferred embodiments, additional agents for treating the tumor include paclitaxel, doxorubicin, cyclophosphamide, axitinib, lenvatinib, and pembrolizumab.

[0123] In another preferred embodiment, the pharmaceutical composition is used to treat tumors that express the PD-L1 protein (i.e., PD-L1 positive).

[0124] In another preferred embodiment, the pharmaceutical composition is in the form of an injectable preparation.

[0125] In another preferred embodiment, the pharmaceutical composition is used in the manufacture of pharmaceuticals for the prevention and treatment of tumors.

[0126] In a ninth aspect of the present invention, the following: (i) Use for detecting human PD-L1 molecules and / or 4-1BB molecules; (ii) Use for flow detection; (iii) Use for cellular immunofluorescence detection; (iv) Use for treating tumors; (v) Use for diagnosing tumors; (vi) Use for blocking the interaction between PD-1 and PD-L1; and (vii) Use for binding to 4-1BB and activating immune cells. The use of one or more bispecific antibodies according to a first aspect of the present invention, selected from the group including the above, is provided.

[0127] In another preferred embodiment, the tumor is a tumor that expresses the PD-L1 protein (i.e., a PD-L1-positive tumor).

[0128] In other preferred embodiments, the use is non-diagnostic and non-therapeutic.

[0129] In a tenth aspect of the present invention, a recombinant protein is provided, which has: (i) a bispecific antibody according to the first aspect of the present invention; and (ii) optionally a tag sequence for aiding expression and / or purification.

[0130] In another preferred embodiment, the tag array includes 6His tags, HA tags, and Fc tags.

[0131] In another preferred embodiment, the recombinant protein specifically binds to PD-L1 and / or 4-1BB.

[0132] An eleventh aspect of the present invention is provided for detecting PD-L1 and / or 4-1BB in a sample, the method comprising: (1) contacting the sample with a bispecific antibody described in the first aspect of the present invention; and (2) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of PD-L1 and / or 4-1BB in the sample.

[0133] In a twelfth aspect of the present invention, a method for treating a disease is provided, which comprises administering a bispecific antibody according to the first aspect of the present invention, or an immunoconjugate according to the sixth aspect of the present invention, or a pharmaceutical composition according to the eighth aspect of the present invention, to a subject in need thereof.

[0134] In other preferred embodiments, the subject includes mammals, preferably humans.

[0135] In a thirteenth aspect of the present invention, a PD-L1 and / or 4-1BB detection reagent is provided, characterized in that the detection reagent comprises an immunoconjugate and a detection-acceptable carrier as described in the sixth aspect of the present invention.

[0136] In another preferred embodiment, the coupling portion of the immunoconjugate is a diagnostic isotope.

[0137] In another preferred embodiment, the detectable carrier is a non-toxic, inert aqueous carrier medium.

[0138] In another preferred embodiment, the detection reagent is one or more reagents selected from the group consisting of isotope tracers, contrast agents, flow detection reagents, cellular immunofluorescence detection reagents, magnetic nanoparticles, and imaging agents.

[0139] In another preferred embodiment, the detection reagent is used for in vivo detection.

[0140] In other preferred embodiments, the dosage form of the detection reagent is a liquid or powder (e.g., aqueous solution, injection, lyophilized powder, tablet, buccal, aerosol).

[0141] In a fourteenth aspect of the present invention, a kit for detecting PD-L1 and / or 4-1BB is provided, the kit comprising an immunoconjugate according to the sixth aspect of the present invention or a detection reagent according to the thirteenth aspect of the present invention, as well as instructions.

[0142] In another preferred embodiment, the instructions state that the kit is used for the non-invasive detection of PD-L1 and / or 4-1BB expression in a subject.

[0143] In another preferred embodiment, the kit is used to detect tumors expressing the PD-L1 protein (i.e., PD-L1 positive).

[0144] term To facilitate understanding of this disclosure, certain terms are defined first. As used in this application, unless otherwise expressly stated herein, the following terms have the meanings set forth below. Other definitions are provided throughout this application.

[0145] As used herein, the term “about” may refer to a value or composition within the tolerance range for a particular value or composition as determined by a person skilled in the art, which depends in part on how that value or composition is measured or determined.

[0146] As used herein, the terms “give” and “administer” are to be used interchangeably. The product of the present invention can be administered intravenously, intramuscularly, subcutaneously, intraperitoneally, via the spinal cord, or through other parenteral administration routes. For example, this refers to physically introducing a substance into a target using any of the various methods and delivery systems known to those skilled in the art, including injection or intravenous infusion.

[0147] bispecific antibody In this specification, the terms "bispecific antibody of the present invention," "bispecific antibody of the present invention," and "anti-PD-L1 / 4-1BB bispecific antibody" have the same meaning and all refer to a bispecific antibody that can specifically recognize and bind to PD-L1 and 4-1BB.

[0148] The present invention provides anti-PD-L1 / 4-1BB bispecific antibodies, which include an anti-PD-L1 single-domain antibody and an anti-4-1BB single-domain antibody.

[0149] Preferably, the bispecific antibody is a dimer composed of peptide chain i and peptide chain ii, and the structures of peptide chain i and peptide chain ii are, respectively, formula I and formula II. A-L1-Fc1-L2-B(Formula I) A-L3-Fc2-L4-B (Formula II) As shown, During the ceremony, A and B are, independently, absent, or anti-PD-L1 single-domain antibodies or anti-4-1BB single-domain antibodies; L1, L2, and L3 are either non-existent or linkers, each independently; L4 is the linker; Fc1 and Fc2 are, independently, Fc regions of human immunoglobulin (preferably LALA variants); and The hyphen "-" represents a peptide bond; The bispecific antibody comprises at least one anti-PD-L1 single-domain antibody and at least one anti-4-1BB single-domain antibody. Polypeptides represented by formula I and those represented by formula II form heterodimers via disulfide bond interactions and knob-in-hole structures.

[0150] In one embodiment of the present invention, the bispecific antibody is either a homodimer or a heterodimer.

[0151] In one embodiment, the Fc region is an LALA variant Fc, and has 85% (preferably 90%, more preferably 95%) or more sequence identity with the amino acid sequence described in SEQ ID NO: 3, or the sequence described in SEQ ID NO: 3.

[0152] In other embodiments, Fc1 and Fc2 have a knob mutation and a hole mutation, respectively. Here, a knob mutation refers to the presence of an S132C mutation at position 132 and a T144W mutation at position 144, based on the amino acid sequence described in SEQ ID NO: 3. A hole mutation refers to the presence of a Y127C mutation at position 127, a T144S mutation at position 144 and an L146A mutation at position 146, based on the amino acid sequence described in SEQ ID NO: 3.

[0153] Preferably, Fc1 has the amino acid sequence described in SEQ ID NO: 8, or has 85% (preferably 90%, more preferably 95%) or more sequence identity with the sequence described in SEQ ID NO: 8; and Fc2 has the amino acid sequence described in SEQ ID NO: 9, or has 85% (preferably 90%, more preferably 95%) or more sequence identity with the sequence described in SEQ ID NO: 9.

[0154] As used herein, the terms "single-domain antibody," "nanobody VHH," and "nanobody" have the same meaning and refer to a nanobody (VHH) consisting of only one heavy chain variable region, constructed by cloning the antibody heavy chain variable region, which is the smallest antigen-binding fragment with full functionality. Typically, after obtaining an antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1), the antibody heavy chain variable region is cloned to construct a nanobody (VHH) consisting of only one heavy chain variable region.

[0155] In this specification, the term “variable” means that certain parts of the antibody variable domain differ in sequence, and this contributes to the binding affinity and specificity of each particular antibody to its particular antigen. However, variability is not uniformly distributed throughout the antibody variable domain. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions of the light and heavy chain variable domains. The more conserved portion of the variable domain is called the framework region (FR). The native heavy and light chain variable domains each contain four FR regions, which are broadly folded and connected by three CDRs that form binding loops, although in some cases they may also form a partially folded structure. The CDRs of each chain are adjacent by FR regions and, together with the CDRs of other chains, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). The constant region does not directly participate in the binding of the antibody to the antigen, but exhibits different effector functions, such as participation in antibody-dependent cell-mediated cytotoxicity.

[0156] As used herein, the term “framework region” (FR) refers to an amino acid sequence inserted between CDRs, i.e., a portion of the immunoglobulin light and heavy chain variable regions that are relatively conserved among different immunoglobulins of a single species. Each immunoglobulin light and heavy chain has four FRs, designated FR1-L, FR2-L, FR3-L, FR4-L, and FR1-H, FR2-H, FR3-H, and FR4-H, respectively. Thus, the light chain variable domain may be designated (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L), and the heavy chain variable domain may be represented as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H). Preferably, the FR of the present invention is a human antibody FR or a derivative thereof, and the human antibody FR derivative is substantially identical to a naturally occurring human antibody FR, i.e., sequence identity reaches 85%, 90%, 95%, or 96%, 97%, 98%, or 99%.

[0157] Knowing the amino acid sequence of the CDR, a person skilled in the art can easily determine the framework regions FR1-L, FR2-L, FR3-L, FR4-L and / or FR1-H, FR2-H, FR3-H, FR4-H.

[0158] As used herein, the term “human framework region” refers to a framework region that is substantially (about 85% or more, specifically 90%, 95%, 97%, 99%, or 100%) identical to a naturally occurring human antibody framework region.

[0159] As used herein, the term "affinity" is theoretically defined by the equilibrium association between an intact antibody and an antigen. The affinity of the bispecific antibody of the present invention is K D The value (dissociation constant) or other measurement methods can be used to evaluate or determine it, for example, by bio-layer interferometry (BLI) using a FortebioRed96 instrument.

[0160] As used herein, the term “linker” refers to one or more amino acid residues inserted into an immunoglobulin domain to provide sufficient mobility for folding into an immunoglobulin having a bivariate region in which the light chain and heavy chain domains are exchanged.

[0161] As is known to those skilled in the art, immunoconjugates and fusion expression products include conjugates formed by conjugating drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules to the antibody or fragment of the present invention. The present invention also includes PD-L1 / 4-1BB bispecific antibodies, or cell surface markers or antigens that bind to fragments thereof.

[0162] As used herein, the terms “variable region” and “complementarity-determining region (CDR)” are used interchangeably.

[0163] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody comprises three complementarity-determining regions, CDR1, CDR2, and CDR3.

[0164] In a preferred embodiment of the present invention, the antibody heavy chain includes the heavy chain variable region and the heavy chain constant region described above.

[0165] In the present invention, the terms “antibody of the present invention,” “protein of the present invention,” or “polypeptide of the present invention” are used interchangeably and all refer to polypeptides that specifically bind to PD-L1 and / or 4-1BB proteins, such as proteins or polypeptides having a heavy chain variable region. These may or may not contain initiation methionine.

[0166] The present invention further provides other proteins or fusion expression products having the antibody of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region, provided that the variable region is identical or at least 90% homologous, preferably at least 95% homologous, to the heavy chain variable region of the antibody of the present invention.

[0167] Generally, the antigen-binding properties of an antibody can be explained by three specific regions located in the heavy chain variable region, which are called CDRs. These regions divide the segment into four framework regions (FRs), the amino acid sequences of the four FRs being relatively conserved and not directly involved in the binding reaction. These CDRs form a ring structure, and the β-folds formed by the FRs between them are spatially close to each other. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. By comparing the amino acid sequences of similar antibodies, it is possible to determine which amino acids constitute the FR region or the CDR region.

[0168] The variable region of the antibody heavy chain of the present invention is of particular interest because at least a portion of it is involved in binding to the antigen. Accordingly, the present invention includes these molecules having an antibody heavy chain variable region having a CDR, insofar as the CDR is more than 90% (preferably more than 95%, most preferably more than 98%) homologous to the CDR identified herein.

[0169] The present invention includes not only intact antibodies, but also antibody fragments having immunological activity, or fusion proteins formed by antibodies with other sequences. Therefore, the present invention further includes antibody fragments, derivatives, and analogs.

[0170] As used herein, the terms “fragment,” “derivative,” and “analog” refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. Polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides having one or more substituted conserved or non-conserved amino acid residues (preferably conserved amino acid residues), such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituents on one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, e.g., polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to a polypeptide sequence (e.g., a leader sequence or secretion sequence, or a sequence or proprotein sequence for purifying the polypeptide, or a fusion protein formed by a 6His tag). In light of the teachings herein, such fragments, derivatives, and analogs are within the scope of those skilled in the art.

[0171] The antibody of the present invention refers to a biantibody having activity to bind to PD-L1 and / or 4-1BB protein. The term also refers to polypeptide variants that contain the same CDR region as the antibody of the present invention and have the same function. Such variant forms include (but are not limited to): deletion, insertion, and / or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids at the C-terminus and / or N-terminus, and addition of one or more (usually within 20, preferably within 10, more preferably within 5) amino acids. For example, in the art, substitution of amino acids having closer or similar properties generally does not alter the function of the protein. As another example, the addition of one or more amino acids at the C-terminus and / or N-terminus usually does not alter the function of the protein. The term further refers to active fragments and active derivatives of the antibody of the present invention.

[0172] Polypeptide variants include homologous sequences, conserved variants, allele variants, native mutants, induced mutants, proteins encoded by DNA that can hybridize with the DNA of the antibody of the present invention under highly or low stringent conditions, or polypeptides or proteins obtained by using antiserum against the antibody of the present invention.

[0173] The present invention further provides fusion proteins comprising other polypeptides, such as single-domain antibodies or fragments thereof. In addition to substantially full-length polypeptides, the present invention further comprises fragments of the single-domain antibody of the present invention. Typically, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0174] In the present invention, "conservative variants of the antibody of the present invention" refers to polypeptides formed by substituting at least 10, preferably at least 8, more preferably at least 5, and most preferably at least 3 amino acids in the amino acid sequence with amino acids having similar or close properties to the amino acid sequence of the antibody of the present invention. These conservative variant polypeptides are preferably produced by the amino acid substitutions listed in Table A.

[0175] [Table 1]

[0176] The present invention further provides polynucleotide molecules encoding the above-mentioned antibodies or fragments thereof or fusion proteins. The polynucleotides of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be either a coding strand or a non-coding strand.

[0177] The polynucleotides encoding mature polypeptides of the present invention include a coding sequence encoding only mature polypeptides; a coding sequence for mature polypeptides and various additional coding sequences; and a coding sequence (and any additional coding sequences) and non-coding sequences for mature polypeptides.

[0178] The term "polynucleotide encoding a polypeptide" may include polynucleotides that encode a polypeptide, or it may include additional coding and / or non-coding sequences.

[0179] The present invention further relates to a polynucleotide that hybridizes to the above sequence and has at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention particularly relates to a polynucleotide that can hybridize to the polynucleotide of the present invention under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at low ionic strength and high temperature, e.g., 0.2 × SSC, 0.1% SDS, 60°C; or (2) hybridization in the presence of a denaturant, e.g., 50% (v / v) formamide, 0.1% bovine serum / 0.1% Ficoll, 42°C; or (3) hybridization that occurs only when the identity between the two sequences is at least 90%, preferably more than 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0180] The full-length nucleotide sequence or fragments of the antibody of the present invention can typically be obtained by PCR amplification, recombination, or artificial synthesis. A feasible method is to synthesize the relevant sequence using artificial synthesis, especially when the fragment length is short. Fragments with very long sequences are usually obtained by synthesizing multiple smaller fragments and ligating them. Furthermore, the heavy chain coding sequence can also be fused with an expression tag (e.g., 6His) to form a fusion protein.

[0181] Once a related sequence is obtained, a large quantity of the related sequence can be obtained using recombination. Typically, after cloning into a vector, cells are transformed, and the related sequence is isolated from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) related to this invention include biomolecules in their isolated forms.

[0182] Currently, the DNA sequence (or fragment thereof, or derivative thereof) encoding the protein of the present invention can be obtained entirely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (e.g., vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present invention by chemical synthesis.

[0183] The present invention also relates to vectors comprising the above-mentioned suitable DNA sequence and a suitable promoter or regulatory sequence. These vectors can be used to transform suitable host cells to express a protein.

[0184] The host cell may be a prokaryotic cell such as a bacterial cell; a lower eukaryotic cell such as a yeast cell; or a higher eukaryotic cell such as a mammalian cell. Typical examples include bacterial cells such as Escherichia coli, Streptomyces, and Salmonella typhimurium; fungal cells such as yeast; insect cells such as Drosophila S2 or Sf9; and animal cells such as CHO, COS7, and 293 cells.

[0185] Transformation of host cells with recombinant DNA can be carried out by conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of taking up DNA can be collected after the exponential growth phase and treated with CaCl2 using procedures well known to those skilled in the art. Another method is to use MgCl2. Transformation can also be carried out by electroporation if desired. When the host is a eukaryote, the following DNA transfection methods can be used: conventional mechanical methods such as calcium phosphate coprecipitation, microinjection, electroporation, and liposome packaging.

[0186] The resulting transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. The culture medium can be selected from various conventional media depending on the host cells used. Culture is carried out under conditions suitable for host cell proliferation. After the host cells have proliferated to an appropriate cell density, the selected promoter is induced by an appropriate method (e.g., temperature shift or chemical induction), and the cells are cultured for a further period.

[0187] In the above method, recombinant polypeptides can be expressed intracellularly or on the cell membrane, or secreted extracellularly. Recombinant proteins can be isolated and purified by various separation methods, utilizing their physical, chemical, and other properties, as desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional regeneration treatments, treatment with protein precipitants (salting-out), centrifugation, osmotic disruption, supernatant treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and other liquid chromatography techniques and combinations thereof.

[0188] The antibodies of the present invention can be used alone, or conjugated with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifier, or any combination thereof.

[0189] Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products.

[0190] Therapeutic agents that can be combined with or conjugated to the antibodies of the present invention include, but are not limited to, the following: 1. Radionuclides; 2. Biological toxins; 3. Cytokines such as IL-2; 4. Gold nanoparticles / nanorods; 5. Viruses; 6. Liposomes; 7. Nanomagnetic particles; 8. Prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenylhydrolase-like protein (BPHL)); 10. Chemotherapy agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0191] Pharmaceutical composition The present invention also provides compositions. Preferably, the compositions are pharmaceutical compositions comprising the antibody or its active fragment or its fusion protein and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, the pH of which is usually about 5 to 8, preferably about 6 to 8, but the pH value may be varied depending on the properties of the substance being formulated and the condition to be treated. The prepared pharmaceutical compositions can be administered by conventional routes including (but not limited to) intratumoral, intraperitoneal, intravenous, or topical administration.

[0192] The pharmaceutical composition of the present invention can be used directly for the binding of PD-L1 and / or 4-1BB protein molecules, and therefore can be used to treat tumors. Furthermore, additional therapeutic agents can be used in combination.

[0193] The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001 to 99 wt%, preferably 0.01 to 90 wt%, more preferably 0.1 to 80 wt%) of the single-domain antibody (or its conjugate) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, physiological saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be adapted to the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injectable by conventional methods, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition, such as an injectable or liquid formulation, is preferably manufactured under sterile conditions. The active ingredient is administered in a therapeutically effective amount, for example, about 10 μg / kg body weight to about 50 mg / kg body weight per day. Furthermore, the polypeptide of the present invention can also be used in combination with additional therapeutic agents.

[0194] When using a pharmaceutical composition, a safe and effective amount of immunoconjugate is administered to a mammal. This safe and effective amount is usually at least about 10 μg / kg body weight, often less than or equal to about 50 mg / kg body weight, and preferably about 10 μg / kg body weight to about 10 mg / kg body weight. Nevertheless, when determining the specific dosage, factors such as the route of administration and the patient's health condition must also be considered, and these are within the scope of the skill of a physician skilled in the art.

[0195] labeled antibody In a preferred embodiment of the present invention, the antibody has a detectable label. More preferably, the label is selected from the group consisting of isotopes, colloidal gold labels, colored labels, or fluorescent labels.

[0196] Colloidal gold labeling can be carried out using methods known to those skilled in the art. In a preferred embodiment of the present invention, a PD-L1 / 4-1BB bispecific antibody can be labeled with colloidal gold to obtain a colloidal gold-labeled antibody.

[0197] Detection method The present invention also relates to a method for detecting PD-L1 and / or 4-1BB proteins. The method broadly comprises the following steps: obtaining a cell and / or tissue sample; lysing the sample in a medium; and detecting the level of PD-L1 and / or 4-1BB proteins in the lysed sample.

[0198] In the detection method of the present invention, the sample used is not particularly limited, and a typical example is a cell-containing sample present in a cell preservation solution.

[0199] kit Furthermore, the present invention provides a kit comprising the antibody (or fragment thereof) or a detection plate of the present invention. In a preferred embodiment of the present invention, the kit further comprises a container, instructions for use, a buffer, and the like.

[0200] Furthermore, the present invention provides a detection kit for detecting the levels of PD-L1 and / or 4-1BB, which comprises an antibody that recognizes the PD-L1 and / or 4-1BB protein, a lysis medium for dissolving the sample, and various general detection reagents and buffers such as detection labels and detection substrates. The detection kit may also be contained within an in vitro diagnostic device.

[0201] use As described above, the single-domain antibody of the present invention has broad biological and clinical value, and its use is relevant to many fields such as the diagnosis and treatment of diseases associated with PD-L1 and / or 4-1BB, basic medical research, and biological research. Its preferred use is for targeted therapy against PD-L1 and / or 4-1BB, such as clinical diagnosis and oncology.

[0202] Beneficial effects of the present invention The present invention achieves one or more of the following technical effects: 1) The nanobodies of the present invention are highly specific to human PD-L1 protein and 4-1BB protein with the correct spatial structure. 2) The bispecific antibody of the present invention has strong affinity. 3) The production of the bispecific antibodies of the present invention is simple and straightforward. 4) The bispecific antibody of the present invention has the function of blocking the binding of PD-L1 to the receptor PD-1. At the same time, the bispecific antibody also binds to 4-1BB on immune cells, activating the activity of immune cells in the tumor microenvironment and more effectively improving the effect of inhibiting tumor development and growth. 5) Bispecific antibodies are highly stable and have a long half-life. 6) It has low hepatotoxicity and good safety. 7) There is a very high possibility of a synergistic effect between the first and second protein functional regions of the bispecific antibody of the present invention; for example, its ability to block the binding of PD-L1 to PD-1 is even better than that of the control antibody; its binding levels to human 4-1BB and human PD-L1 proteins are even better than those of the control antibody; and it is also shown to be superior to the control antibody in inducing IL-2 secretion in mixed lymphocytes. This indicates that the bispecific antibody of the present invention can better activate T cells. [Brief explanation of the drawing]

[0203] [Figure 1] Figures 1A to 1D show schematic diagrams of the structures of the bispecific antibodies Bi-400, Bi-088, Bi-401-091, and Bi-061-091, respectively. The meaning of each module is as follows. [ka] [Figure 2A] Figure 2A shows the detection results of the binding of the bispecific antibody of the present invention to human PD-L1 protein overexpressed on the surface of CHO-S cells. [Figure 2B] Figure 2B shows the detection results of the binding of the bispecific antibody of the present invention to the cynomolgus monkey PD-L1 protein overexpressed on the surface of CHO-S cells. [Figure 2C]Figure 2C shows the detection results of binding of the bispecific antibody of the present invention to human 4-1BB protein overexpressed on the surface of CHO-S cells. [Figure 2D] Figure 2D shows the detection results of the binding of the bispecific antibody of the present invention to the cynomolgus monkey 4-1BB protein overexpressed on the surface of CHO-S cells. [Figure 2E] Figure 2E shows the detection results of the binding of the bispecific antibody of the present invention to CHO-S cells. [Figure 3] Figure 3 shows the detection results of the bispecific antibody of the present invention that blocks the binding of PD-L1 protein to human PD-1 protein overexpressed on the surface of CHO-S cells. [Figure 4A] Figure 4A shows the detection results of the bispecific antibody of the present invention simultaneously bound to CHO-S cells overexpressing human 4-1BB and CHO-S cells overexpressing human PD-L1. [Figure 4B] Figure 4B shows the detection results of the bispecific antibody of the present invention that simultaneously binds to human 4-1BB and human PD-L1 proteins. [Figure 5A] Figure 5A shows the detection results of the fluorescence signal activation activity of the bispecific antibody of the present invention in a co-incubation system of CHO-S cells and cells overexpressing the human 4-1BB Jurkat NF-AT luciferase reporter gene. [Figure 5B] Figure 5B shows the detection results of the fluorescence signal activation of the bispecific antibody of the present invention in a co-incubation system of CHO-S cells overexpressing human PD-L1 and cells overexpressing the human 4-1BB Jurkat NF-AT luciferase reporter gene. [Figure 5C] Figure 5C shows the detection results of the fluorescence signal activation of the bispecific antibody of the present invention in a co-incubation system of CT-26 cells overexpressing human PD-L1 and cells overexpressing the human 4-1BB Jurkat NF-AT luciferase reporter gene. [Figure 6A]Figure 6A shows the detection results of T cell activation by the bispecific antibody of the present invention in a co-incubation system of CHO-S cells and human primary T cells. [Figure 6B] Figure 6B shows the results of detecting IL-2 secretion induced by T cell activation in a co-incubation system of PD-L1 CHO-S cells and human primary T cells using the bispecific antibody of the present invention. [Figure 7] Figure 7 shows the results of detecting T cell activation in a mixed lymphocyte reaction system using the bispecific antibody of the present invention. [Figure 8] Figure 8 shows the detection results of the half-life of the bispecific antibody of the present invention in mice. [Figure 9] Figure 9 shows the results of detecting the pharmaceutical effect of the bispecific antibody of the present invention in a PD-L1 / PD-1 / 4-1BB triple transgenic mouse model inoculated with human PD-L1 CT-26 cells. [Figure 10] Figure 10 shows the results of detecting the pharmaceutical effect of the bispecific antibody of the present invention in a human PD-L1 / 4-1BB double transgenic mouse model inoculated with human PD-L1 MC38 cells. [Figure 11] Figure 11 shows the HE staining results of liver tissue with the bispecific antibody of the present invention in a human 4-1BB mouse toxicity test. Each horizontal group represents the results from four mice. [Figure 12] Figure 12 shows the results of CD8 IHC staining of liver tissue with the bispecific antibody of the present invention in a human 4-1BB mouse toxicity test. Several sequences related to the present invention are shown in Table B below:

[0204] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Examples]

[0205] A specific model for carrying out the present invention The present invention will be further described below in combination with specific embodiments. It should be understood that these embodiments are used solely for illustrative purposes and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify conditions are usually carried out under conventional conditions, for example, according to the conditions described in Sambrook et al., Molecular cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise specified.

[0206] If not specified, the C-terminuses of HZ-L-Yr-13&14-16-01 and C-Ye-18-5 used in Examples 2-12 were directly ligated to IgG1 Fc (LALA mutation).

[0207] Example 1: Cloning and expression of bispecific antibodies 1.1 Structure of the antibody construct In this example, four anti-4-1BB / PD-L1 bispecific antibodies were constructed, namely: It consisted of two identical polypeptide chains of Bi-400:2 (the two peptide chains were linked by two pairs of disulfide bonds), and its structural diagram is shown in Figure 1A. The peptide chain had the amino acid sequence described in SEQ ID NO: 1, which included the amino acid sequence of the anti-4-1BB nanobody HZ-L-Yr-13&14-16-01 (SEQ ID NO: 2), the C-terminus of the anti-4-1BB nanobody amino acid sequence was directly ligated to the amino acid sequence of human IgG1 Fc (introduced with the LALA mutation, SEQ ID NO: 3, named IgG1 Fc(LALA)), and the N-terminus of the anti-PD-L1 nanobody C-Ye-18-5 (Publication No.: Chinese Patent Application Publication No. 112480253A) (SEQ ID NO: 4) was ligated to the C-terminus of IgG1 Fc(LALA) via a linker of 21 amino acid residues (G4S)4 (SEQ ID NO: 5). Bi-088: Composed of two identical polypeptide chains, its schematic structure is shown in Figure 1B, the peptide chain having the amino acid sequence described in SEQ ID NO. 6, which includes the anti-PD-L1 nanobody C-Ye-18-5 (Publication No.: Chinese Patent Application Publication No. 112480253A) (SEQ ID NO. 4), the C-terminus of the nanobody amino acid sequence is directly ligated to the amino acid sequence of human IgG1 Fc (LALA mutation introduced, SEQ ID NO. 3), and the N-terminus of the anti-4-1BB nanobody HZ-L-Yr-13&14-16-01 amino acid sequence (SEQ ID NO. 2) is ligated to the C-terminus of IgG1 Fc (LALA) via a linker of 21 amino acid residues (G4S)4 (SEQ ID NO. 5).

[0208] Bi-401-091: Consists of two polypeptide chains, the structural diagram of which is shown in Figure 1. Peptide chain #1 has the amino acid sequence described in SEQ ID NO. 7, which includes the anti-PD-L1 nanobody C-Ye-18-5 (Publication No.: Chinese Patent Application Publication No. 112480253A) (SEQ ID NO. 4), the C-terminus of which is directly ligated to the human IgG1 Fc amino acid sequence (LALA mutation introduced, knob-in-hole mutation introduced, SEQ ID NO. 8), and the N-terminus of the anti-4-1BB nanobody HZ-L-Yr-13&14-16-01 (SEQ ID NO. 2) is ligated to the C-terminus of Fc via a linker of 21 amino acid residues (G4S)4 (SEQ ID NO. 5); peptide chain #2 has the amino acid sequence described in SEQ ID NO. 9, which was the human IgG1 Fc amino acid sequence (LALA mutation introduced, knob-in-hole mutation introduced, SEQ ID NO. 9).

[0209] Bi-061-091: Consists of two polypeptide chains, the structural diagram of which is shown in Figure 1D, peptide chain #1 having the amino acid sequence described in SEQ ID NO: 7; peptide chain #2 having the amino acid sequence described in SEQ ID NO: 10, which contains the anti-PD-L1 nanobody C-Ye-18-5 (Publication No.: Chinese Patent Application Publication No. 112480253A) (SEQ ID NO: 4), and the C-terminus of the nanobody amino acid sequence is directly ligated to the human IgG1 Fc amino acid sequence (LALA mutation introduced to reduce Fc function, knob-in-hole mutation introduced to form a heterodimer, SEQ ID NO: 9).

[0210] 1.2 Gene cloning and protein preparation Referring to the sequences in Table B, gene fragments encoding the corresponding amino acid sequences were constructed in the pCDNA3.1 vector. For peptide chains #1 and #2 of Bi-401-091 and Bi-061-091, these two sequences were expressed in two different plasmids between transient transfections, and disulfide bonds were automatically formed during the cellular expression process.

[0211] Plasmids were transfected into Expi-CHO cells using the ExpiCHO® Expression System Kit (purchased from Thermo). The transfection method was carried out according to the manufacturer's instructions. After culturing the cells for 5 days, the supernatant was collected and the target protein was purified using a separation method with Protein A magnetic beads (purchased from GenScript). The magnetic beads were resuspended in an appropriate volume of binding buffer (PBS + 0.1% Tween 20, pH 7.4) (1 to 4 times the volume of the magnetic beads), then added to the sample to be purified, and incubated at room temperature for 1 hour with gentle shaking. The sample was placed on a magnetic stand (purchased from Beaver), the supernatant was discarded, and the magnetic beads were washed three times with binding buffer. Elution buffer (0.1 M sodium citrate, pH 3.2) was added to a volume 3 to 5 times that of the magnetic beads. After shaking at room temperature for 5 to 10 minutes, the mixture was placed on a magnetic stand, the elution buffer was collected, and the mixture was transferred to a recovery tube containing neutralizing buffer (1 M Tris, pH 8.54) and thoroughly mixed. The prepared bispecific antibody sample was used in subsequent experiments.

[0212] Example 2: Cell-based conjugation of bispecific antibodies CHO cells overexpressing human or cynomolgus monkey 4-1BB(PD-L1) were generated by transfection with the pCHO1.0 vector (purchased from Invitrogen) encoding the cDNA of human or cynomolgus monkey 4-1BB(PD-L1) cloned into an MCS (Multiple Cloning Site) (theoretically, this method allows for obtaining overexpression cells through transfection and stress screening, which can then be confirmed by flow cytometry). The overexpression cells were subjected to expansion culture, and the cell density reached 2 × 10⁻¹⁰ 6After adjusting the cell / ml concentration, the cells were added to a 96-well flow plate at 100 μl / well and centrifuged for later use. Purified 4-1BB antibody was diluted 3-fold from 400 nM in PBS to obtain a total of 12 samples. The diluted samples were added to the 96-well flow plate containing the cells at 100 μl / well, incubated at 4°C for 30 minutes, and then washed twice with PBS. Goat F(ab')2 anti-human IgG-Fc(PE) (purchased from Abcam), diluted 100-fold in PBS, was added at 100 μl / well, incubated at 4°C for 30 minutes, and then washed twice with PBS. The cells were resuspended by adding 100 μl / well of PBS, detected using a CytoFlex (Bechman) flow cytometer, and the corresponding MFI was calculated. INBRX-105-1B (which has only one peptide chain and whose amino acid sequence is described in SEQ ID NO: 11; see International Publication No. 2017123650A2) was used as a bispecific antibody-positive control (Inhibrx anti-PD-L1 / 4-1BB bispecific antibody); nanobody HZ-L-Yr-13&14-16-01 was ligated to human IgG1 Fc (LALA mutation) (SEQ ID NO: 3) as an anti-4-1BB-positive control; and nanobody C-Ye-18-5 (SEQ ID NO: 5) was ligated to human IgG1 Fc (LALA mutation) (SEQ ID NO: 3) as an anti-PD-L1-positive control. Furthermore, two anti-4-1BB monoclonal antibodies, utomirumab (Patent No.: U.S. Patent Application Publication No. 20120237498) (the heavy and light chain sequences are described in SEQ ID NO: 12 and SEQ ID NO: 13, respectively) and urerumab (International Non-Property Name for the Drug: 104, the heavy and light chain sequences are described in SEQ ID NO: 14 and SEQ ID NO: 15, respectively), were used as anti-4-1BB positive controls.

[0213] The experimental results are shown in Figures 2A to 2E.

[0214] As shown in Figure 2A, bispecific antibodies with different structures showed different binding activity to human PD-L1-overexpressing CHO cells, with the binding activity of some bispecific antibodies being comparable to that of the control antibody; as shown in Figure 2B, bispecific antibodies with different structures showed different binding activity to cynomolgus monkey PD-L1-overexpressing CHO cells, with the binding activity of some bispecific antibodies being comparable to that of the control antibody; as shown in Figure 2C, bispecific antibodies with different structures showed different binding activity to human 4-1BB-overexpressing CHO cells, with the binding activity of some bispecific antibodies being comparable to that of the control antibody; as shown in Figure 2D, bispecific antibodies with different structures showed different binding activity to cynomolgus monkey 4-1BB-overexpressing CHO cells, with the binding activity of some bispecific antibodies being comparable to that of the control antibody; and as shown in Figure 2E, bispecific antibodies with different structures did not show any clear nonspecific binding to CHO-S cells.

[0215] Example 3: Bispecific antibody that blocks the binding of PD-L1 to PD-1 CHO cells In this example, CHO-hPD-1 cells (overexpressing human PD-1) were cultured in large quantities at a cell density of 2 × 10⁶ 6The cells were adjusted to the desired concentration per ml and added to a 96-well flow plate at 100 μl / well, then centrifuged for later use. The purified bispecific antibody was diluted 3-fold from 400 nM with PBS to obtain a total of 12 samples. The diluted samples were added to a 96-well sample dilution plate at 60 μl / well. Biotin-labeled human PD-L1 protein (purchased from AcroBiosystems) was added simultaneously at 60 μl / well to achieve a final concentration of 500 ng / ml, and incubated at 4°C for 30 minutes. The co-incubated samples were added at 100 μl / well to the 96-well flow plate containing the cells, incubated at 4°C for 30 minutes, and washed twice with PBS. APC goat anti-mouse IgG antibody (purchased from Biolegend), diluted 100-fold with PBS, was added at 100 μl / well, incubated at 4°C for 30 minutes, and washed twice with PBS. Cells were resuspended by adding 100 μl of PBS per well, and detection was performed using a CytoFlex (Bechman) flow cytometer. The corresponding MFI was then calculated.

[0216] The results are shown in Figure 3. All of the bispecific antibodies with different structures of the present invention were able to block the binding of PD-L1 and PD-1, with the level of blockage varying depending on the structure. The blocking activity of the Bi-088 molecule was equivalent to or better than that of the control antibody.

[0217] Example 4: Detection of bispecific antibody cell-based cobinding In this example, CHO cells overexpressing human 4-1BB were labeled using CFSE (purchased from Thermo), and CHO cells overexpressing human PD-L1 were labeled using CTV (purchased from Thermo). The cells were resuspended in PBS, and the cell density was set to 4 × 10⁶. 6Each cell suspension was prepared in a cell / ml concentration, and the two cell suspensions were mixed in equal proportions. 50 μl / well of the mixture was added to a 96-well U-bottom plate (purchased from Thermo). Simultaneously, the bispecific antibody or control antibody was gradient diluted threefold from a final concentration of 100 nM with PBS to obtain a total of 12 samples. 50 μl / well of each sample was added, and the mixture was incubated at 37°C for 2 hours. Detection was performed using a CytoFlex (Bechman) flow cytometer, and the percentage of bi-positive cells was calculated.

[0218] The results are shown in Figure 4A. The bispecific antibodies were able to simultaneously bind to human 4-1BB overexpressing CHO cells and human PD-L1 overexpressing CHO cells. The binding activity varied depending on the structure of the bispecific antibody, with the binding activity of Bi-088, Bi-400, and Bi-401-091 molecules being equivalent to or better than that of the control antibody.

[0219] Example 5: Bispecific antibody-protein-based co-binding assay Human 4-1BB protein (purchased from ACRO) was dissolved according to the instructions, diluted to 1 μg / ml with ELISA coating solution (purchased from Shanghai Sangon), coated onto an ELISA plate at a concentration of 100 μl / well, allowed to stand overnight at 4°C, washed three times with PBST, and 200 μl / well of 5% BSA (purchased from Shanghai Sangon) was added. Blocking was performed at room temperature for 1 hour. The blocking solution was discarded, and bispecific antibodies or control antibodies were serially diluted 3-fold from a final concentration of 100 nM with 1% BSA to obtain a total of 12 samples. These samples were added at a concentration of 100 μl / well and incubated at room temperature for 2 hours. Biotin-labeled PD-L1 protein (purchased from Kactus), diluted three times with PBST and 1% BSA, was added at a concentration of 100 μl / well. The diluted protein sample was at a concentration of 1 μg / ml, and incubation was performed at room temperature for 1 hour. Washing was performed three times with PBST, and 100 μl / well of (1:10000) SA-HRP (purchased from abcam) diluted with 1% BSA was added and incubated at room temperature for 0.5 hours. Washing was performed three times with PBST, 100 μl / well of ELISA colorimetric solution (purchased from Solarbio) was added and reacted at room temperature for 3 minutes, 50 μl / well of ELISA stop solution (purchased from Solarbio) was added, and the absorbance value at 450 nm was read.

[0220] The results are shown in Figure 4B. The bispecific antibodies could simultaneously bind to human 4-1BB and human PD-L1 proteins, and their binding activity varied depending on the molecular structure. In particular, the binding activity of the Bi-088 molecule was equivalent to or better than that of the control antibody.

[0221] Example 6: Detection of bispecific antibody activity (luciferase reporter cell-based assay) A plasmid encoding human 4-1BB and a plasmid encoding the NF-κB luciferase reporter gene (purchased from Promega) were co-transfected into Jurkat cells to obtain hu4-1BB Jurkat-NF-κB cells. The hu4-1BB Jurkat-NF-κB cells were subjected to expansion culture, and the cells were resuspended in 1640 complete medium and cultured for 4 × 10⁶ days.6 cells / ml. Human PD-L1 overexpressing CHO-S cells, CHO-S cells, and human PD-L1 overexpressing CT-26 cells were diluted to 4×10 5 cells / ml in 1640 complete medium. The above three types of cell suspensions were individually mixed with hu4-1BB Jurkat-NF-κB cells at a ratio of 1:1, added to a sterile 96-well white-bottom plate (purchased from Nunc) at 50 μl / well, and serially diluted three-fold with 1640 complete medium from a final sample concentration of 200 nM to obtain a total of 12 points. A bispecific antibody sample was added. Incubation was carried out at 37 °C and 5% CO2 for 16 hours, and the luciferase signal was detected.

[0222] The results are shown in Figure 5A, Figure 5B, and Figure 5C. No obvious signal activation was observed in the bispecific antibody samples of the co-incubation system of hu4-1BB Jurkat-NF-κB cells and CHO-S cells. Significant signal activation activity was observed in the co-incubation system of hu4-1BB Jurkat-NF-κB cells and human PD-L1 overexpressing CHO-S cells or CT-26 cells overexpressing human PD-L1. This is due to the cross-linking effect caused by PD-L1, whereby 4-1BB on Jurkat cells binds to the other end of the antibody where they are co-clustered, thereby activating the downstream NF-κB pathway. This indicates that the bispecific antibody can activate the NF-κB pathway of human T cells under the PD-L1 cross-linking effect in the human body, further activate T cells, and kill tumors. Among them, the activation activities of Bi-088, Bi-400, and Bi-401-091 were equal to or higher than those of the control antibody.

[0223] Example 7: Detection of bispecific antibody activity (primary T cell activation assay) Human PD-L1 overexpressing CHO-S or CHO cells were treated with mitomycin for 4 hours, and the cell density was adjusted to 2×10 6It was adjusted to cells / ml. OKT-3 antibody (purchased from Biolegend) was diluted to 1 μg / ml with sterile PBS (purchased from Hyclone), and coated at 100 μl / well in a 96-well cell culture flat-bottom plate (purchased from Thermo), and incubated at 37 °C for 2 hours. Frozen human PBMC (purchased from Shanghai Saili) was revived, isolated by a human T cell isolation and purification kit (purchased from Stemcell) to obtain T cells, the T cells were resuspended using X-VIVO15 medium (purchased from LONZA), and the cell density was adjusted to 2×10 6 cells / ml, and mixed at an equal ratio with mitomycin-treated CHO-S cells or human PD-L1 overexpressing CHO cells. After the antibody coating was completed, the coating solution was discarded, washed twice with PBS, the PBS was discarded, the above cell mixed solution was added at 100 μl / well, and at the same time, a bispecific antibody sample serially diluted with X-VIVO15 was added at 100 μl / well, and HZ-L-Yr-13&14-16-1-IgG1Fc (LALA)+C-Ye-18-5-IgG1Fc (LALA) was added to the final sample concentration or combination group, and the final concentrations were 50, 10, 2 nM. Incubation was carried out at 37 °C and 5% CO2 for 3 days, the supernatant was collected, and the IL-2 content was detected.

[0224] The experimental results are shown in FIGS. 6A and 6B. For the bispecific antibody sample in the co-incubation system of T cells and CHO-S cells, there was no obvious signal activation, but in the co-incubation system of T cells and human PD-L1 overexpressing CHO-S cells, significant T cell activation and IL-2 release were observed. Here, the activities of the Bi-088, Bi-400, and Bi-401-091 molecules were equal to or higher than those of the control antibody.

[0225] Example 8: Activity of Bispecific Antibodies in Mixed Lymphocyte Reaction Assay PBMC (purchased from SAILY BIO, SLB-HPB) was resuscitated, centrifuged, and the PBMC was resuspended in 10 ml of X-VIVO-15 medium (purchased from LONZA), cultured in a cell culture incubator at 37 °C for 2 hours, and the adherent cells were removed by suction. 10 ml of DC medium was added: 10 ng / ml GM-CSF (purchased from R&D) and 20 ng / ml IL-4 (purchased from R&D) were added to the X-VIVO-15 medium, cultured for 3 days, supplemented with 5 ml of DC medium, and cultured until day 6. Then, DC maturation medium was added and cultured for 2 days. Here, the DC maturation medium was prepared by adding 1000 U / ml TNF-α (purchased from R&D), 10 ng / ml IL-6 (purchased from R&D), 5 ng / ml IL-1β (purchased from R&D), and 1 μM PGE2 (purchased from Tocris) to the X-VIVO-15 medium. The mature DC cells were collected and adjusted using X-VIVO-15 medium to a cell density of 2×10 5 cells / ml.

[0226] PBMC from other donors (purchased from SAILY BIO, SLB-HPB) was resuscitated, centrifuged, and the PBMC was resuspended in 10 ml of X-VIVO-15 medium. T cells were enriched using a T cell sorting kit (purchased from Stemcell), the T cells were resuspended in X-VIVO-15, adjusted to a cell density of 2×l0 6 cells / ml, mixed with the mature DC cells collected above at a ratio of 1:1, and added to a 96-well U-bottom plate at 100 μl / well. At the same time, a bispecific antibody sample diluted with X-VIVO-15 medium was added at 100 μl / well, which had final concentrations of 10, 2, 0.4, 0.08, 0.016 nM, cultured for 3 days, the supernatant was collected, and the IL-2 expression level was detected by ELISA (purchased from eBioscience).

[0227] The experimental results are shown in Figure 7. The bispecific antibody samples were able to activate T cells in the mixed lymphocyte reaction system to release IL-2, and the activation levels of Bi-088 and Bi-400 were comparable to those of the control antibody.

[0228] Example 9: Half-life of bispecific antibodies in mice For the experiment, Balb / c mice were used, with an equal number of males and females, six mice at each blood sampling point. They were kept under 12 / 12-hour light / dark cycles, at a temperature of 24±2°C, with humidity between 40% and 70%, and had free access to water and food. On the day of the experiment, the bispecific antibody molecule Bi-088 was injected once into the tail vein of each Balb / c mouse at a dose of 10 mg / kg.

[0229] Blood samples were collected at 5 minutes, 0.5 hours, 2 hours, 6 hours, 24 hours, 48 ​​hours, 96 hours, 168 hours, 336 hours, and 504 hours after administration. Blood was collected from the mouse orbit. Whole blood samples were allowed to stand at 2-8°C for 30 minutes, then centrifuged at 12,000 rpm for 5 minutes to collect the serum. The serum was then centrifuged again at 2-8°C at 12,000 rpm for 5 minutes and stored at -80°C. The content of bispecific antibody molecules in the serum was detected by ELISA, and the average value was calculated. The results are shown in Figure 8. The half-life of the bispecific antibody molecule of the present invention in mice was approximately 174 hours.

[0230] Example 10: Pharmaceutical effects of bispecific antibodies in mice In this experiment, human PD-L1-expressing mouse colorectal cancer CT-26 cells (h-PD-L1 KI CT-26) were transplanted into human 4-1BB / PD-L1 / PD-1 transgenic mice (purchased from GemPharmatech) to determine the antitumor effect of the bispecific antibody. First, a mouse model containing h-PD-L1 KI CT-26 tumors was established by subcutaneous inoculation, and 0.6 × 10⁶ antibodies were administered to each mouse. 6 Individual cells were inoculated and grouped, with an average tumor volume of 100-200 mm². 3 When the target was reached, the mice were divided into groups of 6. Treatment was carried out by intraperitoneal injection of different antibodies at different doses, and changes in tumor volume and body weight in the mice of each group were monitored every 2-3 days for 2-3 weeks. The dosages and models are shown in Table 1.

[0231] [Table 3]

[0232] The results are shown in Figure 9. The results showed that the bispecific antibody significantly inhibited the proliferation of h-PD-L1 KI CT-26 cells, and its inhibitory activity was superior to that of the combination drug group. The inhibitory activity of Bi-088 was equivalent to or greater than that of the control antibody INBRX-105-1B.

[0233] Example 11: Pharmacological model of human PD-L1 / 4-1BB double transgenic mice transplanted with huPD-L1 MC-38 cells. In this experiment, we used MC-38 cells expressing human PD-L1 (huPD-L1 MC-38) to determine the antitumor effect of Bi-088 in human PD-L1 / 4-1BB transgenic mice (purchased from Biocytogen Biotechnology Co., Ltd.). First, a mouse model of huPD-L1 MC-38 tumors was established by subcutaneous inoculation, with each mouse being 1 × 10⁶ 6 Individual cells were inoculated, and the groups were divided based on an average tumor volume of 80-120 mm. 3 The procedure was performed when the tumor reached a certain stage. Six mice in each group were treated with different doses of different antibodies by intraperitoneal injection, and the doses and administration methods are shown in Table 2 below. After the mouse tumors had completely regressed, huPD-L1 MC-38 cells were newly inoculated into the opposite side of the mouse, and changes in tumor volume and body weight in the mice of each group were monitored. Monitoring was performed once every 2-3 days for a total of 8 weeks.

[0234] [Table 4]

[0235] The results are shown in Table 3 and Figure 10. [Table 5]

[0236] As a result, after inoculating huPD-L1 MC-38 cells, the tumor volume of the negative control group continued to increase. The tumors of 6 mice in the Bi-088 group completely regressed after treatment. Even when huPD-L1 MC-38 cells were inoculated again on the opposite side 31 days after the final administration, the tumor cells did not proliferate much.

[0237] Example 12: Hepatotoxicity test in human 4-1BB transgenic mice In this experiment, the hepatotoxicity of Bi-088 was determined in human 4-1BB transgenic mice (purchased from Biocytogen Biotechnology Co., Ltd.), with 4 mice in each group. The Bi-088 molecule and the control molecule were intraperitoneally administered by the method shown in Table 4 below. On the 20th day from the first administration, the mice were euthanized and liver tissues were collected for embedding, fixation, sectioning, HE staining, and CD8 positive cell staining.

[0238]

Table 6

[0239] The results are shown in Figures 11 and 12. From the results, in the mice of the urelumab group, obvious infiltration of mononuclear cells and CD8 positive stained cells were seen in the liver. In the INBRX-105-1B group, obvious infiltration of mononuclear cells and CD8 positive stained cells were seen in some mice in the liver. The mice in the Bi-088 group did not show obvious infiltration of mononuclear cells and CD8 positive stained cells in the mouse liver.

[0240] Although specific embodiments of the present invention have been described in detail, those skilled in the art will be able to make various modifications and substitutions based on all the disclosed teachings regarding their details, and it will be understood that all such changes are within the scope of the present invention. The full scope of the present invention is indicated by the appended claims and their equivalents.

Claims

1. The first protein functional region targeting 4-1BB, and A second protein functional domain targeting PD-L1 or PD-1 A bispecific antibody containing; The first protein functional region is an anti-4-1BB single-domain antibody; The second protein functional region is an anti-PD-L1 antibody, an anti-PD-1 antibody, or an antigen-binding fragment thereof. A bispecific antibody comprising a heavy chain variable region, wherein the anti-4-1BB single-domain antibody includes CDR1 having the amino acid sequence described in SEQ ID NO: 16, CDR2 having the amino acid sequence described in SEQ ID NO: 17, and CDR3 having the amino acid sequence described in SEQ ID NO:

18.

2. The bispecific antibody according to claim 1, wherein the anti-4-1BB single-domain antibody has the amino acid sequence described in SEQ ID NO:

2.

3. The bispecific antibody according to any one of claims 1 to 2, wherein the second protein functional region is an anti-PD-L1 single-domain antibody or an anti-PD-1 single-domain antibody.

4. The bispecific antibody according to claim 3, wherein the anti-PD-L1 single-domain antibody includes a heavy chain variable region, and the heavy chain variable region includes CDR1 having the amino acid sequence described in SEQ ID NO: 19, CDR2 having the amino acid sequence described in SEQ ID NO: 20, and CDR3 having the amino acid sequence described in SEQ ID NO:

21.

5. The bispecific antibody according to claim 4, wherein the anti-PD-L1 single-domain antibody has the amino acid sequence described in SEQ ID NO:

4.

6. The bispecific antibody according to any one of claims 1 to 5, wherein the bispecific antibody further comprises one or more IgG Fc fragments; or further comprises one or more IgG constant regions.

7. The bispecific antibody according to claim 6, characterized by one or more of the following: (1) The Fc fragment of IgG is an Fc fragment of IgG1, IgG2, IgG3, or IgG4; (2) The steady-state region of IgG is the heavy chain steady-state region of IgG1, IgG2, IgG3, or IgG4; (3) The Fc fragment of IgG or the constant region of IgG is located between the first protein functional region and the second protein functional region; (4) The Fc fragment of IgG or the heavy chain constant region of IgG is ligated to the C-terminus of the first protein functional region and / or to the C-terminus of the second protein functional region; (5) The Fc fragment of IgG or the heavy chain constant region of IgG is ligated to the first protein functional region directly or via a linker; and the Fc fragment of IgG or the heavy chain constant region of IgG is ligated to the second protein functional region directly or via a linker; (6) The linker has an amino acid sequence independently selected from SEQ ID NO: 5 and SEQ ID NO: 22; (7) In accordance with the EU numbering system, the Fc fragment of IgG or the heavy chain constant region of IgG contains the L234A mutation and the L235A mutation; or the Fc fragment of IgG or the heavy chain constant region of IgG contains the L234A mutation, the L235A mutation and the G237A mutation; (8) The Fc fragment of IgG is the Fc fragment of IgG1 containing the L234A mutation and the L235A mutation; (9) The Fc fragment of IgG1 has the amino acid sequence described in Sequence ID No. 3; (10) The Fc fragment of IgG further comprises a knob-in-hole mutation; (11) The Fc fragment of IgG is the Fc fragment of IgG1 and contains a knob-in-hole mutation; (12) The Fc fragment of IgG is the Fc fragment of IgG1 and has the amino acid sequence described in SEQ ID NO: 8 or SEQ ID NO:

9.

8. The bispecific antibody is the following first peptide chain: A bispecific antibody according to any one of claims 6 to 7, comprising the first protein functional region, the Fc fragment of IgG or the heavy chain constant region of IgG, a linker, and the second protein functional region in order from the N-terminus to the C-terminus; or comprising a first peptide chain comprising the second protein functional region, the Fc fragment of IgG or the heavy chain constant region of IgG, a linker, and the first protein functional region.

9. The bispecific antibody according to claim 8, wherein the first peptide chain has the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO:

6.

10. The bispecific antibody according to claim 8 or 9, wherein the bispecific antibody is a dimer formed by two first peptide chains.

11. The bispecific antibody according to claim 10, characterized by one or more of the following: (1) The dimer is formed by the first peptide chain described in SEQ ID NO: 1 and / or SEQ ID NO: 6; (2) The two first peptide chains are linked by two or three pairs of disulfide bonds; (3) The two first peptide chains further contain knob-in-hole mutations.

12. The bispecific antibody is the following second peptide chain: The bispecific antibody according to claim 8 or 9, further comprising a second peptide chain containing the Fc fragment of IgG or the heavy chain constant region of IgG; the N-terminus and / or C-terminus of the second peptide chain being ligated directly or via a linker to the first protein functional region and / or the second protein functional region.

13. The bispecific antibody according to claim 12, wherein the second peptide chain has the amino acid sequence described in SEQ ID NO: 9 or SEQ ID NO:

10.

14. The bispecific antibody according to any one of claims 12 to 13, wherein the first peptide chain and the second peptide chain form a dimer.

15. The bispecific antibody according to claim 14, characterized by one or more of the following: (1) The first peptide chain and the second peptide chain are linked by two or three pairs of disulfide bonds; (2) The first peptide chain and the second peptide chain further include knob-in-hole mutations; (3) The first peptide chain has the amino acid sequence described in SEQ ID NO: 7, and the second peptide chain has the amino acid sequence described in SEQ ID NO: 9 or SEQ ID NO:

10.

16. An isolated nucleic acid molecule encoding a bispecific antibody according to any one of claims 1 to 15.

17. A vector comprising an isolated nucleic acid molecule as described in claim 16.

18. A host cell comprising an isolated nucleic acid molecule according to claim 16, or a vector according to claim 17.

19. A method for preparing a bispecific antibody according to any one of claims 1 to 15, comprising the steps of culturing a host cell according to claim 18 under appropriate conditions, and recovering the bispecific antibody from the cell culture.

20. A conjugate comprising a bispecific antibody and a coupling moiety, wherein the bispecific antibody is the bispecific antibody according to any one of claims 1 to 15, the coupling moiety is a detectable label, and the coupling moiety is a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

21. A kit comprising a bispecific antibody according to any one of claims 1 to 15, or a conjugate according to claim 20; The kit further comprises a second antibody capable of specifically binding to the bispecific antibody, wherein the second antibody further comprises a detectable label.

22. Use of a bispecific antibody according to any one of claims 1 to 15 in the preparation of a kit for detecting the presence or level of 4-1BB and / or PD-L1 in a sample.

23. A pharmaceutical composition comprising a bispecific antibody according to any one of claims 1 to 15 or a conjugate according to claim 20, further comprising a pharmaceutically acceptable excipient.

24. Use of a bispecific antibody according to any one of claims 1 to 15 or a conjugate according to claim 20 in the manufacture of a pharmaceutical product for the prevention or treatment of malignant tumors.

25. The use according to claim 24, wherein the malignant tumor is selected from the group consisting of rectal cancer, colon cancer, lung cancer, melanoma, liver cancer, gastric cancer, renal cell carcinoma, ovarian cancer, esophageal cancer, and head and neck cancer.

26. A bispecific antibody according to any one of claims 1 to 15 or a conjugate according to claim 20, used for the treatment or prevention of malignant tumors.

27. The bispecific antibody or conjugate according to claim 26, wherein the malignant tumor is selected from the group consisting of rectal cancer, colon cancer, lung cancer, melanoma, liver cancer, gastric cancer, renal cell carcinoma, ovarian cancer, esophageal cancer, and head and neck cancer.

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