4-1BB binding proteins

Abstract

The invention discloses a 4-1BB binding protein which can be specifically bound with 4-1BB, does not block the binding of 4-1BB and 4-1BBL, and can effectively activate an NF-kappa B signal and effectively activate T cells under a crosslinking condition.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to 4-1BB binding proteins, such as single domain antibodies, heavy chain antibodies and / or fusion proteins binding to 4-1BB and applications thereof. Background Art

[0002] 4-1BB, also known as CD137, belongs to the tumor necrosis factor receptor superfamily and plays an important role in T cell activation-driven immune response, mainly promoting T cell proliferation, acquiring effector function, promoting immune memory, and inhibiting activation-induced cell death. The earliest agonistic antibodies targeting 4-1BB to enter the clinic include Urelumab from Bristol-Myers Squibb (BMS) and Utomilumab from Pfizer, which have shown potential as monotherapy or combination therapy in mouse tumor models, but in clinical practice, due to toxicity or lack of efficacy (Chester C, Sanmamed MF, Wang J, Melero I. Immunotherapy targeting 4-1BB: mechanistic rationale, clinical results, and future strategies. Blood. 2018; 131(1): 49-57.), there is a great clinical demand for safe and effective new therapies targeting 4-1BB. Summary of the invention

[0003] The present invention provides a 4-1BB binding protein, which can specifically bind to 4-1BB, does not block the binding of 4-1BB and 4-1BBL (4-1BB ligand), can effectively activate NF-κB signals under cross-linking conditions, and effectively activates T cells.

[0004] On the one hand, the present invention provides a 4-1BB binding protein comprising a heavy chain variable region, wherein the heavy chain variable region comprises one or more of the following complementarity determining regions (CDRs): HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in any one of SEQ ID NOs:8-13 or SEQ ID NOs:31-36, and HCDR3 as shown in any one of SEQ ID NOs:14-19 or SEQ ID NOs:37-42.

[0005] In some embodiments, the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in SEQ ID NO:8-13, and HCDR3 as shown in any one of SEQ ID NO:14-19.

[0006] In some embodiments, the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in SEQ ID NO:8, and HCDR3 as shown in any one of SEQ ID NOs:14-17.

[0007] In some embodiments, the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in SEQ ID NO:9, and HCDR3 as shown in SEQ ID NO:14 or 19.

[0008] In some embodiments, the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:14 or 16.

[0009] In some embodiments, the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in SEQ ID NO:11 or 12, and HCDR3 as shown in SEQ ID NO:16.

[0010] In some embodiments, the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in SEQ ID NO:13, and HCDR3 as shown in SEQ ID NO:18 or 19.

[0011] In some embodiments, the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in SEQ ID NO:31-36, and HCDR3 as shown in any one of SEQ ID NO:37-42.

[0012] In some embodiments, the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in SEQ ID NO:31, and HCDR3 as shown in any one of SEQ ID NOs:37-40.

[0013] In some embodiments, the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in SEQ ID NO:32, and HCDR3 as shown in SEQ ID NO:37 or 42.

[0014] In some embodiments, the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in SEQ ID NO:33, and HCDR3 as shown in SEQ ID NO:37 or 39.

[0015] In some embodiments, the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in SEQ ID NO:34 or 35, and HCDR3 as shown in SEQ ID NO:39.

[0016] In some embodiments, the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in SEQ ID NO:36, and HCDR3 as shown in SEQ ID NO:41 or 42.

[0017] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1-6, 29-30, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity compared to the amino acid sequence shown in any one of SEQ ID NOs: 1-6, 29-30, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 1-6, 29-30.

[0018] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1-6, 29-30.

[0019] In some embodiments, the 4-1BB binding protein is an antibody or an antigen binding fragment. In some embodiments, the 4-1BB binding protein is in the form of a single domain antibody (such as a nanobody), a heavy chain antibody, a bispecific antibody, a multispecific antibody, Fab, Fab', F(ab')2, Fv, or scFv.

[0020] In some embodiments, the 4-1BB binding protein is a fusion protein, further comprising an Fc region. In some embodiments, the Fc is an Fc of an immunoglobulin. In some embodiments, the Fc is an Fc of an IgG. In some embodiments, the Fc is an Fc of IgG1, IgG2, IgG3, or IgG4, or a variant thereof. In some embodiments, the amino acid sequence of the Fc of the IgG1 is shown in SEQ ID NO: 21.

[0021] In some embodiments, the Fc is a variant Fc region. In some embodiments, relative to the parent Fc region, the variant Fc region has one or more amino acid modifications, such as substitutions, deletions or insertions. In some embodiments, relative to the parent Fc region activity, the amino acid modifications in the Fc region change the effector function activity. In some embodiments, the variant Fc region may have altered (i.e., increased or decreased) antibody-dependent cellular cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), phagocytosis, opsonization or cell binding activity. In some embodiments, relative to the parent Fc region, the amino acid modifications in the Fc region may change the affinity for FcγR (Fcγ receptor). In some embodiments, the Fc region is derived from IgG1 or IgG4. In some embodiments, the Fc mutation comprises one or more of N297A, L234A / F234A, L235A, L235E and G237A mutations (Eu numbering). In some embodiments, the Fc region mutation is E345R or S440Y (Eu numbering). In some embodiments, the IgG1 Fc comprises a N297A mutation (Eu numbering).

[0022] In some embodiments, a peptide linker is further connected between the C-terminus of the heavy chain variable region of the fusion protein and the N-terminus of the Fc region. In some embodiments, the sequence of the peptide linker is (G m S) n , wherein each m is independently 2, 3, 4 or 5, and n is independently 1, 2, 3, 4, 5 or 6. In some embodiments, the sequence of the linker is (GGGGS) n , said n is independently 1, 2, 3, 4, 5 or 6. In some embodiments, the amino acid sequence of said peptide linker 1 is as shown in SEQ ID NO:8.

[0023] In some embodiments, the fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 22-27, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity compared to the amino acid sequence shown in any one of SEQ ID NOs: 22-27, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 22-27.

[0024] In some embodiments, the fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 22-27.

[0025] In some embodiments, the fusion protein comprises two identical amino acid sequences.

[0026] Table 1: CDR amino acid sequences

[0027]

[0028] Table 2: Amino acid sequence

[0029]

[0030]

[0031]

[0032] In one aspect, the present invention provides a biomaterial comprising:

[0033] 1) A nucleic acid molecule encoding the 4-1BB binding protein of the present invention or a portion thereof;

[0034] 2) an expression vector comprising the nucleic acid molecule of the present invention;

[0035] 3) A host cell comprising the nucleic acid molecule or expression vector of the present invention.

[0036] In one aspect, the present invention provides a pharmaceutical composition comprising the 4-1BB binding protein of the present invention and a pharmaceutically acceptable carrier.

[0037] In one aspect, the present invention provides use of the above-mentioned 4-1BB binding protein, biomaterial or pharmaceutical composition in the preparation of a drug for treating or preventing a disease.

[0038] On the one hand, the present invention provides the use of the above-mentioned 4-1BB binding protein, biomaterial or pharmaceutical composition in the treatment or prevention of disease. On the one hand, the present invention provides a method for treating or preventing a disease, comprising administering an effective dose of the above-mentioned 4-1BB binding protein, biomaterial or pharmaceutical composition to a patient in need. In some embodiments, the disease is a tumor, a viral infection, an autoimmune disease or an inflammatory disease. In some embodiments, the tumor includes melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, liver cancer, colon cancer, prostate cancer, gastric cancer, kidney cancer, bladder cancer, pancreatic cancer, breast cancer, ovarian cancer, endometrial cancer, esophageal cancer, soft tissue sarcoma, bile duct cancer, thyroid cancer, hepatocellular carcinoma, mesothelioma, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 : Binding experiment results of 4-1BB-VHH-Fc fusion protein and cells expressing 4-1BB.

[0040] Figure 2 and3 : Competition experiment between 4-1BB-VHH-Fc fusion protein and 4-1BB ligand binding.

[0041] Figure 4 : Comparison of activation of the 293T-41BB reporter system by different fusion proteins in the presence of cross-linking antibodies, where the cross-linking antibody is anti-human IgG (Fc specific).

[0042] Figure 5 : Comparison of reporter system activation after different fusion proteins were fixed and cross-linked in 96-well plates. DETAILED DESCRIPTION

[0043] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0044] the term

[0045] It should be noted that the term "a" entity refers to one or more of that entity, e.g., "an antibody" should be understood as one or more antibodies, and thus, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein.

[0046] As used herein, the terms "comprising" or "including" mean that the antibody, composition, method, etc. include the listed elements, such as components or steps, but do not exclude others. "Essentially consisting of" means that the antibody, composition, method, etc. exclude other elements that have a fundamental effect on the characteristics of the combination, but do not exclude elements that have no essential effect on the antibody, composition, method, etc. "Consisting of" means excluding elements not specifically listed.

[0047] The term "polypeptide" is intended to encompass the singular "polypeptide" as well as the plural "polypeptide", and refers to a molecule composed of amino acid monomers linearly linked by amide bonds (also called peptide bonds). The term "polypeptide" refers to any single chain or multiple chains of two or more amino acids, and does not refer to the specific length of the product. Therefore, the definition of "polypeptide" includes peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains" or any other terms used to refer to two or more amino acid chains, and "polypeptides" can be used to replace any of the above terms, or used interchangeably with any of the above terms. The term "polypeptide" is also intended to refer to products modified after the expression of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage or non-natural amino acid modifications. The polypeptide can be derived from a natural biological source or produced by recombinant technology, but it is not necessarily translated from a specified nucleic acid sequence, and it may be produced in any manner including chemical synthesis.

[0048] In the present invention, the 4-1BB antigen may be derived from a mammal, such as a human, a rat, a mouse, or a monkey.

[0049] Those of ordinary skill in the art will appreciate that the CDR region of an antibody is responsible for the binding specificity of the antibody to an antigen. In the case of known antibody heavy chain and light chain variable region sequences, there are currently several methods for determining the CDR region of an antibody, including Kabat, IMGT, Chothia and AbM numbering systems. However, the application of each definition of the CDR of an antibody or its variants will be within the scope of the terms defined and used herein. If the variable region amino acid sequence of the antibody is given, those skilled in the art can usually determine which residues are included in a specific CDR, without relying on any experimental data outside the sequence itself.

[0050] "Antibody", "antigen-binding fragment" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody and any antigen-binding fragment thereof or a single chain thereof. Therefore, the term "antibody" includes any protein or peptide that contains at least a portion of an immunoglobulin molecule that has biological activity of binding to an antigen. Antibodies and antigen-binding fragments include but are not limited to the complementarity determining region (CDR) of a heavy chain or light chain or its ligand-binding portion, a heavy chain variable region (VH), a light chain variable region (VL), a heavy chain constant region (CH), a light chain constant region (CL), a framework region (FR) or any portion thereof, or at least a portion of a binding protein. The CDR region includes the CDR region (LCDR1-3) of the light chain and the CDR region (HCDR1-3) of the heavy chain. The heavy chain constant region (CH) includes the CH1 domain, the hinge (e.g., the upper, middle and / or lower hinge region) domain, the CH2 domain, and the CH3 domain; the crystallizable segment (Fc region) is equivalent to the CH2 and CH3 domains. Antibodies and antigen-binding fragments can specifically recognize and bind to polypeptides or polypeptide complexes of one or more (such as two) antigens. Antibodies or antigen-binding fragments that specifically recognize and bind to multiple (such as two) antigens can be referred to as multispecific (such as bispecific) antibodies or antigen-binding fragments.

[0051] A complete antibody includes a heavy chain or and a light chain. The categories of heavy chains include gamma, mu, alpha, delta or epsilon (γ, μ, α, δ, ε), among which there are some subclasses (e.g., γ1-γ4). The nature of the chain determines the "type" of the antibody, which is IgG, IgM, IgA, IgD or IgE, respectively. Antibody subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, etc., have been fully characterized and the functional specificity conferred is also known. All antibody types are within the scope of protection disclosed in the present invention. In some embodiments, the antibody includes two heavy chains or and two light chains, and the four chains are connected in a "Y" configuration by disulfide bonds, wherein the light chain starts from the "Y" mouth and continues through the variable region to surround the heavy chain.

[0052] The term "antibody fragment" or "antigen binding fragment" refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of its structure, an antibody fragment binds to the same antigen that is recognized by the intact antibody. The term "antibody fragment" includes aptamers, spiegelmers, and diabodies. The term "antigen binding fragment" also includes any synthetic or genetically engineered protein that acts as an antibody by binding to a specific antigen to form a complex.

[0053] The term "Fab" generally refers to the part of a conventional antibody (e.g., IgG) that binds to an antigen, including the heavy chain variable region VH, light chain variable region VL, and heavy chain constant region domain CH1 and light chain constant region CL of the antibody. In conventional antibodies, the C-terminus of VH is linked to the N-terminus of CH1 to form a heavy chain Fd fragment, the C-terminus of VL is linked to the N-terminus of CL to form a light chain, and the C-terminus of CH1 is further linked to the hinge region and other constant region domains of the heavy chain to form a heavy chain. In some embodiments, "Fab" also refers to a variant structure of Fab. For example, in some embodiments, the C-terminus of VH is linked to the N-terminus of CL to form a polypeptide chain, and the C-terminus of VL is linked to the N-terminus of CH1 to form another polypeptide chain, forming a Fab (cross VH / VL) structure; in some embodiments, the CH1 of Fab is not linked to the hinge region, but the C-terminus of CL is linked to the hinge region of the heavy chain, forming a Fab (cross Fd / LC) structure.

[0054] "Single-chain antibody", "single-chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin. In some aspects, these regions are connected to a short linker peptide of 10 to about 25 amino acids. The linker can be rich in glycine to increase flexibility, as well as rich in serine or threonine to increase solubility, and can connect the N-terminus of VH and the C-terminus of VL, or vice versa. Although the protein has been stripped of the constant region and a linker has been introduced, it retains the specificity of the original immunoglobulin. ScFv molecules are generally known in the art, for example, as described in U.S. Patent No. 5,892,019.

[0055] The term "single domain antibody" or "sdAb" refers to an antigen-binding fragment that contains only a single antibody variable region. A single sdAb can bind to an antigen without pairing with a corresponding CDR-containing polypeptide. In some cases, sdAb is engineered from camelid HcAb, and its heavy chain variable domain is referred to herein as "VHH" (heavy chain variable domain of a heavy chain antibody). Some VHHs are also referred to as nanobodies. VHH has the following structure from N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In this article, a VHH that can bind to 4-1BB may be referred to as 4-1BB-VHH.

[0056] The term "heavy chain antibody" or "HcAb" refers to a functional antibody comprising heavy chains (VHH, CH2 and CH3) but lacking the light chains normally present in antibodies. Camelids (eg, camels, llamas or alpacas) are known to produce HcAbs.

[0057] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology or identity can be determined by comparing the positions in each sequence that can be aligned. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous or identical at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.

[0058] A polynucleotide or polynucleotide sequence (or polypeptide or antibody sequence) having a certain percentage (e.g., 90%, 95%, 98% or 99%) of "identity or sequence identity" to another sequence means that when the sequences are aligned, the percentage of bases (or amino acids) in the two sequences being compared are the same. The alignment and identity percentage or sequence identity can be determined using visual inspection or software programs known in the art, such as the software programs described in Ausubel et al. eds. (2007) in Current Protocols in Molecular Biology. Preferably, the alignment is performed using the default parameters. One such alignment program is BLAST using default parameters, such as BLASTN and BLASTP, both using the following default parameters: Geneticcode=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sortby=HIGHSCORE; Databases=non-redundant; GenBank+EMBL+DDBJ+PDB+GenBankCDStranslations+SwissProtein+SPupdate+PIR. Biologically equivalent polynucleotides are polynucleotides that have the above specified percentage identities and encode polypeptides having the same or similar biological activity.

[0059] "A conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, non-essential amino acid residues of immunoglobulin polypeptides are preferably replaced with other amino acid residues from the same side chain family. In other embodiments, a string of amino acids may be substituted with a string of structurally similar amino acids that differ in order and / or in the composition of the side chain family.

[0060] In some embodiments, the conservative substitution is preferably a substitution in which an amino acid within the following groups (a)-(e) is substituted by another amino acid residue within the same group: (a) small aliphatic, non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, non-polar residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0061] Particularly preferred conservative substitutions are as follows: Ala is replaced by Gly or Ser; Arg is replaced by Lys; Asn is replaced by Gln or His; Asp is replaced by Glu; Cys is replaced by Ser; Gln is replaced by Asn; Glu is replaced by Asp; Gly is replaced by Ala or Pro; His is replaced by Asn or Gln; Ile is replaced by Leu or Val; Leu is replaced by Ile or Val; Lys is replaced by Arg, Gln or Glu; Met is replaced by Leu, Tyr or Ile; Phe is replaced by Met, Leu or Tyr; Ser is replaced by Thr; Thr is replaced by Ser; Trp is replaced by Tyr; Tyr is replaced by Trp; and / or Phe is replaced by Val, Ile or Leu.

[0062] The 4-1BB binding protein disclosed in the present invention includes modified derivatives, i.e., modified by the covalent connection of any type of molecule to the 4-1BB binding protein, wherein the covalent connection does not prevent the 4-1BB binding protein from binding to the epitope. The 4-1BB binding protein can be glycosylated, acetylated, pegylated, phosphorylated, amidated, derivatized by known protection / blocking groups, proteolytic cleavage, connected to cell ligands or other proteins, etc. Any of the numerous chemical modifications can be carried out by prior art, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc.

[0063] In some embodiments, the 4-1BB binding protein can be conjugated to a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, a pharmaceutical agent, or PEG.

[0064] "Pharmaceutically acceptable" refers to materials listed in the pharmacopoeia for use in animals, particularly for human medicine. In addition, "pharmaceutically acceptable carriers and / or excipients" will generally be any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid.

[0065] The term "carrier" refers to a diluent, adjuvant, excipient or carrier that can be applied to a patient together with the active ingredient. Such pharmaceutical carriers can be sterile liquids, such as water and oil, including oils of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and aqueous glucose solutions and glycerol solutions can also be used as liquid carriers, particularly for injection solutions. Suitable pharmaceutical excipients include starch, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If necessary, the composition can also contain a small amount of wetting agent or emulsifier, or pH buffer. Antibacterial agents such as benzyl alcohol or methyl paraben, antioxidants such as ascorbic acid or sodium bisulfite are also foreseeable. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release preparations, etc. The composition can be formulated into a suppository with traditional adhesives and carriers such as triglycerides. Oral formulations can include standard carriers, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by EW Martin, which is incorporated herein by reference. Such compositions will contain clinically effective doses of antibodies or antigen-binding fragments, preferably in purified form, together with an appropriate number of carriers and / or excipients, to provide a dosage form suitable for the patient. The preparation should be suitable for administration mode. The parent preparation can be packaged in an ampoule, a disposable syringe, or a multi-dose vial made of glass or plastic.

[0066] The 4-1BB binding protein of the present invention (including antibodies, antigen binding fragments or fusion proteins, etc.) includes neutral or salt forms. Pharmaceutically acceptable salts include, but are not limited to, salts formed with anions derived from, for example, hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed with cations derived from, for example, sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. . The 4-1BB binding protein described herein can be neutral, i.e., substantially free of net charge, such as when the pH of the 4-1BB binding protein composition is the isoelectric point of the protein, the protein is electrically neutral. The protein described herein can exist in the form of a positive ion, such as when the pH is below the isoelectric point, the protein molecule as a whole displays a positive charge. The protein described herein can exist in the form of a negative ion, such as when the pH is above the isoelectric point, the protein molecule as a whole displays a negative charge.

[0067] The effective dose and treatment regimen for treating a specific patient will depend on various factors, including the specific 4-1BB binding protein or derivative used, the patient's age and weight, general health, sex and diet, as well as the administration time, excretion frequency, drug combination, and the severity of the specific disease treated. These factors are judged by medical care personnel included in the scope of ordinary technicians in the art. The dosage used can be determined by pharmacology and pharmacokinetic principles well known in the art. In some embodiments, the dosage of the 4-1BB binding protein of the present invention applied to the patient is 0.01mg / kg to 100mg / kg patient weight each time. In some embodiments, it is administered once a week, every two weeks, every three weeks, or every four weeks.

[0068] "About" refers to the normal error range of the corresponding numerical value that is easily known to those skilled in the relevant technical field. In some embodiments, "about" mentioned herein refers to the described numerical value and its ±10%, ±5% or ±1% range.

[0069] Preparation of antibodies or fusion proteins

[0070] Various methods for preparing antibodies or fusion proteins are known in the art, such as hybridoma technology, recombinant DNA technology, transgenic mouse technology, and phage display library methods for preparing antibodies.

[0071] Antibodies or fusion proteins can be prepared using conventional recombinant DNA techniques. Vectors and cell lines that produce antibodies or fusion proteins can be selected, constructed and cultured using techniques known to those skilled in the art. These techniques are described in various laboratory manuals and major publications, such as Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells, DL Hacker, FMWurm, in Reference Module in Life Sciences, 2017, the entire contents of which, including supplementary content, are incorporated herein by reference.

[0072] In some embodiments, the DNA encoding the antibody or fusion protein can be designed and synthesized according to the amino acid sequence of the antibody or fusion protein described herein in a conventional manner, placed in an expression vector, and then transfected into a host cell, and the transfected host cell is cultured in a culture medium to produce a monoclonal antibody or fusion protein. In some embodiments, the expression vector includes at least one promoter element, a protein coding sequence, a transcription termination signal, and a polyA tail. Other elements include enhancers, Kozak sequences, and donor and acceptor sites for RNA splicing on both sides of the insertion sequence. Efficient transcription can be obtained by the early and late promoters of SV40, the long terminal repeats from retroviruses such as RSV, HTLV1, HIVI, and the early promoters of cytomegalovirus, and promoters of other cells such as the actin promoter can also be used. Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, PLXSN, pLNCX, pcDNA3.1 (+ / -), pcDNA / Zeo (+ / -), pcDNA3.1 / Hygro (+ / -), PSVL, PMSG, pRSVcat, pSV2dhfr, pBC12MI and pCS2, etc. Commonly used mammalian host cells include HEK293 cells, Cos1 cells, Cos7 cells, CV1 cells, mouse L cells and CHO cells, etc.

[0073] In some embodiments, the inserted gene fragment needs to contain a screening marker, and common screening markers include dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance and other screening genes, so as to facilitate the screening and separation of successfully transfected cells. The constructed plasmid is transfected into host cells without the above genes, and after culturing in a selective medium, the successfully transfected cells grow in large quantities and produce the desired target protein. The obtained antibody or fusion protein can be separated or purified by conventional technical means, such as protein A-agarose gel, ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis or affinity chromatography.

[0074] Example 1: Enrichment and screening of anti-4-1BB VHH positive clones

[0075] The biopanning method was used to enrich 4-1BB-positive VHH clones in the phage display library using biotin-labeled 4-1BB antigen, and a total of 192 monoclonal clones were identified. Then, VHHs with binding activity to human 4-1BB antigen were screened by ELISA. The positive clones were sequenced and their CDR region sequences were compared and analyzed. The sequences and CDRs included are shown in Table 3. The amino acid sequences are shown in Tables 1 and 2.

[0076] Table 3: Composition of 4-1BB VHH

[0077]

[0078] Example 2: Construction, expression and biophysical characterization of 4-1BB-VHH-Fc fusion protein

[0079] This example describes the construction and expression of an exemplary 4-1BB-VHH-Fc fusion protein. Seven fusion proteins were designed and expressed, each of which contained a polypeptide chain of the following structure from the N-terminus to the C-terminus: VHH domain of anti-4-1BB single domain antibody (sdAb) (sequences are shown in SEQ ID NO: 1 to 6, respectively) - peptide linker (linker 1, sequence is shown in SEQ ID NO: 20) - Fc domain of IgG1 (sequence is shown in SEQ ID NO: 21). The specific names and corresponding sequences are shown in Table 4.

[0080] Table 4: Structure and corresponding sequence of 4-1BB-VHH-Fc fusion protein

[0081]

[0082] The positive control antibodies were Urelumab and Utomilumab.

[0083] Plasmids expressing the above fusion proteins and two positive control antibodies were prepared and transiently expressed in 293F cells. Purification was performed by protein A. Sequencing analysis showed that the sequences of the eight proteins were consistent with the designs.

[0084] Example 3: Determination of binding of 4-1BB-VHH-Fc fusion protein to target

[0085] The ability of the fusion protein to bind 4-1BB can be determined using surface plasmon resonance (eg, Biacore), fluorescence assisted cell sorting (FACS), or a combination thereof, and the analysis can be performed on activated T cells.

[0086] The binding kinetics of different fusion proteins to 4-1BB were determined using a surface plasmon resonance (SPR) biosensor BIACORE T200 (GE Healthcare). Starting from 100nM, 4-1BB-VHH-Fc fusion protein samples of different concentrations were prepared with 3-fold serial dilutions in BIAcore's HEPES-buffered saline buffer, for a total of 7 gradients. Each sample was fixed on the sensor chip by Fc capture. The antigen 4-1BB (Sino-Bio, Cat. No. 10041-H08H) was used as the analyte. Dissociation (kd) and binding (ka) rate constants were obtained using T200 evaluation software (Biacore, GE Healthcare). The apparent equilibrium dissociation constant (K D ) by K d Ka As shown in Table 5, each fusion protein exhibited 4-1BB binding kinetics comparable to that of the positive control antibody.

[0087] Table 5: Affinity test results of 4-1BB-VHH-Fc fusion protein

[0088] ka(1 / Ms) kd(1 / s) KD(M) VHH1-Fc 2.93E+04 7.33E-03 2.5E-07 VHH2-Fc 3.02E+04 2.32E-03 7.71E-08 VHH6-Fc 5.57E+04 8.54E-03 1.53E-07 VHH8-Fc 4.86E+04 7.35E-03 1.51E-07 VHH13-Fc 5.05E+04 4.87E-03 9.64E-08 Urolumab 6.84E+05 6.89E-03 1.01E-08 Urecitumab 4.96E+05 1.59E-03 3.22E-09

[0089] Example 4: Analysis of the binding of 4-1BB-VHH-Fc fusion protein to cells expressing 4-1BB by FACS

[0090] The binding of 4-1BB-VHH-Fc fusion protein to HEK-293T (293T-4-1BB) cells expressing 4-1BB was evaluated using a fluorescence activated cell sorting (FACS)-based assay. Different fusion protein samples (starting from 100nM, 2-fold serial dilution, a total of 11 concentrations) were prepared as the primary antibody for FACS analysis. 293T-4-1BB cells were dissociated from adherent culture flasks and mixed with fusion protein samples of different concentrations (all in 96-well plates). Ureruzumab and Utoluzumab were used as anti-4-1BB antibody positive controls. The mixture was balanced at 4 ° C for 60 minutes and washed three times with PBS buffer. Then phycoerythrin (PE)-conjugated sheep anti-human IgG Fc antibody (Invitrogen) used as a secondary antibody was added and balanced at 4 ° C for 30 minutes in the dark. The cells were washed again with PBS buffer and analyzed by flow cytometry. Using nonlinear regression, data were analyzed using GraphPadPRISM 8 (GraphPad Software, San Diego, CA). The results are as follows Figure 1 As shown, FACS binding assays demonstrated that each fusion protein was able to bind to cells expressing 4-1BB.

[0091] The preparation method of HEK-293T (293T-4-1BB) cells is as follows: HEK-293T cells are transfected with a luciferase reporter gene vector containing an NF-κB promoter, and a stable cell line is obtained by screening with hygromycin resistance (100 μg / ml), and on this basis, a vector expressing h4-1BB is transfected, and puromycin (0.3 μg / ml) is added to screen to obtain a 4-1BB / NF-κB reporter gene-HEK-293T stable cell line. The amino acid sequence of h4-1BB is shown in SEQ ID NO: 28.

[0092] Example 5: FACS analysis of the inhibition of the binding of 4-1BB and its ligand 4-1BBL by 4-1BB-VHH-Fc fusion protein

[0093] In order to evaluate the inhibitory ability of the fusion protein to bind 4-1BB and its ligand 4-1BBL (competitive test), 4-1BB-VHH-Fc fusion protein was prepared (starting from 100nM, 2-fold serial dilution, a total of 12 concentrations). 293T cells expressing human 4-1BB (ie, HEK-293T (293T-4-1BB), preparation method see Example 4) were dissociated from adherent culture flasks and mixed with different concentrations of each fusion protein and 1nM h4-1BBL protein with biotin labeling (Sino-Bio, Cat. No. 15693-H42H-B). Utolu and Urerumab, which have similar functions, were used as positive controls for anti-4-1BB antibodies. The mixture was balanced at 4°C for 60 minutes and washed three times with PBS buffer. Then PE-streptavidin secondary antibody (Invitrogen) was added to the mixture and balanced at 4°C in the dark for 30 minutes. Subsequently, the cells were washed with PBS buffer and analyzed by flow cytometry. Data were analyzed using GraphPad PRISM 8 (GraphPad Software, San Diego, CA) using nonlinear regression. The competition assay demonstrated the ability of Urecitumab to effectively inhibit the 4-1BB / 4-1BBL interaction at low concentrations (0.1-10 nM / ml), while different fusion protein samples and Urecitumab were unable to inhibit the 4-1BB / 4-1BBL interaction at high concentrations (10-100 nM / ml). Figure 2 and Figure 3 The binding data in indicate that neither 4-1BB-VHH-Fc fusion protein nor Ureluzumab blocks the binding of 4-1BB and 4-1BBL.

[0094] Example 6: In vitro functional assay of 4-1BB-VHH-Fc fusion protein

[0095] Activation of the 4-1BB pathway by 4-1BB-VHH-Fc samples can be studied using a variety of bioassays that monitor T cell proliferation, IFN-γ release, IL-2 secretion, or expression of reporter genes driven by signaling in the 4-1BB pathway.

[0096] Six different 4-1BB-VHH-Fc fusion proteins were selected for in vitro bioactivity evaluation. The bioactivity of anti-4-1BB agonistic antibodies in a 4-1BB cell-based assay was characterized as follows using 293T-4-1BB cells. Figure 4The specific experiment is as follows: Different fusion proteins were mixed with anti-human IgG (Fc specific) (goat antibody, Sigma-Aldrich, catalog number I2136) at a ratio of 1:1, or without anti-human IgG (Fc specific), 293T-4-1BB cells were mixed with serially diluted fusion protein samples (starting from 500nM, 3-fold serial dilution, with 10 concentrations) and placed in a cell culture incubator for culture. After about 6 hours of stimulation, Bio-Lite TM Luciferase Assay System luciferase reagent (Novagen) was added to cells to measure NF-κB activity. Data were analyzed using nonlinear regression using GraphPad PRISM 8 (GraphPad Software, San Diego, CA), and EC was calculated. 50 Anti-human IgG (Fc specific) (goat anti, polyclonal antibody) can specifically bind to the Fc of at least two 4-1BB-VHH-Fc fusion proteins, produce an aggregation effect of the fusion protein, and then activate the 4-1BB activation activity of the fusion protein. Figure 4 The reporter gene assay showed that each fusion protein could effectively activate the NF-κB signal in the presence of anti-human IgG (Fc specific), but did not activate the NF-κB signal when present alone. The control antibody urerulumab could effectively activate the NF-κB signal alone, indicating that the fusion protein of the present invention requires cross-linking to be activated and has higher safety.

[0097] Another situation can also reflect that different fusion proteins can effectively activate NF-κB signaling under cross-linking. In this case, fusion protein samples were prepared separately (starting from 100nM, 2-fold serial dilution, with 10 concentrations), and the above samples were placed on ELISA plates at 4°C overnight to coat the fusion proteins on the ELISA plates. After cross-linking the fusion proteins on the ELISA plates, 293T-4-1BB cells were added. After stimulation for about 6 hours, Bio-Lite TM Luciferase Assay System luciferase reagent (Novagen) was added to cells to measure NF-κB activity. Data were analyzed using nonlinear regression using GraphPad PRISM 8 (GraphPad Software, San Diego, CA), and EC was calculated. 50 value. Figure 5 Reporter gene assays also demonstrated that all fusion proteins could effectively activate NF-κB signaling after cross-linking.

Claims

1. A 4-1BB binding protein, comprising a heavy chain variable region, wherein the heavy chain variable region comprises one or more of the following complementarity determining regions (CDRs): HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in any one of SEQ ID NOs:8-13 or SEQ ID NOs:31-36, and HCDR3 as shown in any one of SEQ ID NOs:14-19 or SEQ ID NOs:37-42.

2. The 4-1BB binding protein according to claim 1, The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in SEQ ID NO:8-13, and HCDR3 as shown in any one of SEQ ID NO:14-19; or, The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in SEQ ID NO:8, and HCDR3 as shown in any one of SEQ ID NOs:14-17; or, The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in SEQ ID NO:9, and HCDR3 as shown in SEQ ID NO:14 or 19; or, The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:14 or 16; or, The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in SEQ ID NO:11 or 12, and HCDR3 as shown in SEQ ID NO:16; or, The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in SEQ ID NO:13, and HCDR3 as shown in SEQ ID NO:18 or 19; or, The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in SEQ ID NO:31-36, and HCDR3 as shown in any one of SEQ ID NO:37-42; or, The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in SEQ ID NO:31, and HCDR3 as shown in any one of SEQ ID NOs:37-40; or, The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 7, HCDR2 as shown in SEQ ID NO: 32, and HCDR3 as shown in SEQ ID NO: 37 or 42; or, The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in SEQ ID NO:33, and HCDR3 as shown in SEQ ID NO:37 or 39; or, The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in SEQ ID NO:34 or 35, and HCDR3 as shown in SEQ ID NO:39; or, The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:7, HCDR2 as shown in SEQ ID NO:36, and HCDR3 as shown in SEQ ID NO:41 or 42.

3. The 4-1BB binding protein according to claim 1 or 2, wherein the heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1-6, 29-30, or an amino acid sequence having at least 80% identity with the amino acid sequence as shown in any one of SEQ ID NOs: 1-6, 29-30, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence as shown in any one of SEQ ID NOs: 1-6, 29-30.

4. A 4-1BB binding protein comprising the amino acid sequence shown in any one of SEQ ID NOs: 1-6, 29-30.

5. The 4-1BB binding protein according to any one of claims 1 to 4 is a single domain antibody, a heavy chain antibody, a bispecific antibody, a multispecific antibody, Fab, Fab', F(ab')2, Fv, or scFv.

6. The 4-1BB binding protein according to any one of claims 1 to 4 is a fusion protein, further comprising an Fc region; preferably, the Fc is an Fc of an immunoglobulin, such as IgG1 Fc; or, the Fc is a variant Fc having one or more amino acid modifications, such as substitutions, deletions or insertions.

7. The binding protein according to claim 6, wherein a peptide linker is further connected between the C-terminus of the heavy chain variable region of the fusion protein and the N-terminus of the Fc region.

8. The binding protein as claimed in claim 7, wherein the sequence of the peptide linker is (G m S) n , wherein each m is independently 2, 3, 4 or 5, and n is independently 1, 2, 3, 4, 5 or 6.

9. A 4-1BB binding protein comprising an amino acid sequence as shown in any one of SEQ ID NOs: 22-27.

10. Biomaterials, for 1) A nucleic acid molecule encoding the 4-1BB binding protein according to any one of claims 1 to 9; 2) an expression vector comprising a nucleic acid molecule encoding the 4-1BB binding protein according to any one of claims 1 to 9; or 3) A host cell comprising a nucleic acid molecule or an expression vector encoding the 4-1BB binding protein according to any one of claims 1 to 9.

11. A pharmaceutical composition comprising the 4-1BB binding protein according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier.

12. Use of the 4-1BB binding protein according to any one of claims 1 to 9, the biomaterial according to claim 10 or the pharmaceutical composition according to claim 11 in the preparation of a medicament for treating or preventing a disease; preferably, the disease is a tumor, an autoimmune disease or an inflammatory disease.

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