CN112794904A

CN112794904A - Application of humanized antibody for resisting 4-1BB

Info

Publication number: CN112794904A
Application number: CN201911105605.6A
Authority: CN
Inventors: 程联胜; 刘雯婷; 赵群; 张大艳
Original assignee: Hefei Hankemab Biotechnology Co ltd
Current assignee: Hefei Hankemab Biotechnology Co ltd
Priority date: 2019-11-13
Filing date: 2019-11-13
Publication date: 2021-05-14
Anticipated expiration: 2039-11-13
Also published as: CN112794904B

Abstract

The invention discloses an application of a humanized antibody for resisting 4-1 BB. The antibody can be used for detecting 4-1BB, blocking a 4-1BB/4-1BBL signal channel, stimulating T cell activation, promoting the secretion of IFN-gamma by T cells, inhibiting colon cancer tumors and cell growth, and treating and/or preventing colon cancer; the HCDR1, HCDR2 and HCDR3 of the antibody are sequentially shown as 31-35, 50-64 and 98-106 of SEQ ID No.1, and the LCDR1, LCDR2 and LHCDR3 are sequentially shown as 24-34, 50-56 and 89-97 of SEQ ID No. 2. The antibodies of the invention have high affinity for human 4-1BB, are effective in enhancing T cell responses, and can be used to modulate T cell and antibody-mediated immune responses, as immunomodulators in the treatment of diseases such as cancer, autoimmune diseases, inflammatory diseases, and infectious diseases.

Description

Application of humanized antibody for resisting 4-1BB

Technical Field

The invention relates to the technical field of biology, in particular to application of a humanized antibody for resisting 4-1 BB.

Background

4-1BB, also known as CD137, TNFRSF9, etc., is a transmembrane protein of the Tumor Necrosis Factor Receptor Superfamily (TNFRS), which was first discovered by Kwon and Weissman during the activated murine T cell clonal differentiation screen. Schwarzh et al isolated human 4-1BB (hu4-1BB) having homology to murine 4-1BB from human activated T cells and designated CD 137. 4-1BB is a type I membrane glycoprotein, which is activation-dependent unlike expression of PD-1/PD-L, 4-1BB, which mediates costimulatory signals for T cell activation, and is present on the surface of immune cells including activated NK cells, activated T cells, Dendritic Cells (DCs), mast cells, monocytes, and neutrophils. Human 4-1BB, which consists of 255 amino acid residues, is expressed on the cell surface in monomeric and dimeric forms, and readily forms trimers with ligands, thus opening signal transduction.

Studies have shown that 4-1BB mediated costimulatory signals can enhance T cell function, improve T cell surveillance on tumor cells and enhance immune defense against viral infections. Some 4-1BB agonist antibodies are capable of increasing costimulatory molecule expression and significantly enhance T cell immune responses, leading to anti-tumor efficacy. The ligand 4-1BBL or the activated 4-1BB monoclonal antibody is used for activating 4-1BB, so that the T cells and the antigen presenting cells can be stimulated to proliferate and secrete cytokines, and the anti-tumor immune response level of an organism is improved. Further, 4-1BB monotherapy and combination therapy tumor models have identified a persistent anti-tumor protective T cell memory response. At the same time, 4-1BB agonists have also been shown to suppress autoimmune responses in a number of art-recognized autoimmune models. 4-1BB signaling pathway induces CD4 in vivo⁺T cell mediated immune tolerance, preventing the occurrence and development of autoimmune diseasesAnd (6) unfolding. The dual activity of 4-1BB provides anti-tumor activity while reducing autoimmune side effects.

Currently, no agonist drugs are on the market for 4-1BB, are at the fastest stage of clinical development, and are few, failing to meet the needs for immunotherapeutic approaches for various diseases, including cancer. Aiming at a 4-1BB target point, an 'accelerator' medicament is developed, the existing cancer medicament (such as Rituxan of Roche) is utilized to help activate an immune system, a novel tumor immunotherapy is developed, the market demand is met, the drug resistance disadvantage of PD-1/PD-L1 is avoided, and the application prospect is good.

Disclosure of Invention

The object of the present invention is to provide a use of a humanized antibody against 4-1 BB.

In a first aspect, the invention claims the use of an antibody or antigen binding fragment thereof or a nucleic acid molecule or expression cassette, recombinant vector or recombinant cell or pharmaceutical composition in any one of:

(A1) preparing a product for testing 4-1BB, or testing 4-1 BB.

In practical applications, the antibodies or antigen binding fragments thereof can be used as a diagnostic tool to detect 4-1BB in the blood or tissue of patients with cancer, autoimmune or other diseases.

Wherein the 4-1BB may be human 4-1BB or monkey 4-1 BB.

(A2) Preparing a product for blocking the 4-1BB/4-1BBL signaling pathway, or blocking the 4-1BB/4-1BBL signaling pathway.

In a specific embodiment of the invention, the 4-1BB/4-1BBL signaling pathway is specifically the human 4-1BB/4-1BBL signaling pathway.

(A3) Preparing a product for stimulating T cell activation, or stimulating T cell activation.

Further, the T cell activation is dependent on Fc γ R | | a and/or Fc γ R | | B.

(A4) Preparing a product for promoting the secretion of IFN-gamma by the T cells, or promoting the secretion of IFN-gamma by the T cells.

(A5) Preparing a product for inhibiting the growth of colon cancer cells, or inhibiting the growth of colon cancer cells.

In a particular embodiment of the invention, the colon cancer cell is specifically a MC38 cell.

(A6) Preparing a product for inhibiting the growth of colon cancer tumors or inhibiting the growth of colon cancer tumors.

In a specific embodiment of the invention, the colon cancer tumor is a colon cancer tumor caused by MC38 cells.

(A7) Preparing a product for treating and/or preventing colon cancer, or treating and/or preventing colon cancer.

The amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of the antibody are shown as 31-35 th, 50-64 th and 98-106 th positions from the N end of SEQ ID No.1 in sequence; the amino acid sequences of LCDR1, LCDR2 and LHCDR3 in the light chain variable region are shown as 24-34 th, 50-56 th and 89-97 th positions from the N end of SEQ ID No. 2.

The nucleic acid molecule is a nucleic acid molecule encoding the antibody or the antigen-binding fragment.

The expression cassette is an expression cassette comprising the nucleic acid molecule.

The recombinant vector is a recombinant vector containing the nucleic acid molecule.

The recombinant cell is a recombinant cell containing the nucleic acid molecule.

The pharmaceutical composition comprises: (a1) the antibody or the antigen-binding fragment; (a2) a pharmaceutically acceptable excipient, diluent or carrier.

In a second aspect, the invention claims the use of an antibody or antigen-binding fragment thereof or a pharmaceutical composition for any one of:

(B1) as an immunopotentiator, or in the preparation of an immunopotentiator.

Wherein the immune enhancer is either an enhancer of an anti-tumor immune response or an enhancer of an anti-viral immune response.

(B2) As an immunomodulator, or in the preparation of an immunomodulator.

Wherein the immunomodulator can be specifically an immunomodulator of a T cell mediated autoimmune disease.

The amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of the antibody are shown as 31-35 th, 50-64 th and 98-106 th positions from the N end of SEQ ID No.1 in sequence; the amino acid sequences of LCDR1, LCDR2 and LHCDR3 in the light chain variable region of the antibody are shown as 24-34 th, 50-56 th and 89-97 th positions from the N end of SEQ ID No.2 in sequence.

In a third aspect, the invention claims the use of an antibody or antigen binding fragment thereof or a nucleic acid molecule or expression cassette, recombinant vector or recombinant cell or pharmaceutical composition in any one of:

(C1) preparing a product for the treatment and/or prevention and/or diagnosis of a disease characterized by a deregulated 4-1BB expression, or the treatment and/or prevention and/or diagnosis of a disease characterized by a deregulated 4-1BB expression.

(C2) Preparing a product for treating and/or preventing and/or diagnosing cancer, or treating and/or preventing and/or diagnosing cancer.

Further, the cancer may be a cancer in which 4-1BB expression is deregulated.

(C3) Preparing a product for treating and/or preventing and/or diagnosing an autoimmune disease, or treating and/or preventing and/or diagnosing an autoimmune disease.

Further, the autoimmune disease can be an autoimmune disease with deregulated 4-1BB expression.

(C4) Preparing a product for treating and/or preventing and/or diagnosing inflammatory diseases, or treating and/or preventing and/or diagnosing inflammatory diseases.

Further, the inflammatory disease may be an inflammatory disease in which 4-1BB expression is deregulated.

(C5) Preparing a product for treating and/or preventing and/or diagnosing infectious diseases, or treating and/or preventing and/or diagnosing infectious diseases.

Further, the infectious disease may be an infectious disease with deregulated 4-1BB expression.

Further, the amino acid sequence of the heavy chain variable region is at positions 1-117 of SEQ ID No.1 from the N-terminus, or has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identity (the disparity is preferably in the Framework Region (FR)) with positions 1-117 of SEQ ID No.1 from the N-terminus. The amino acid sequence of the light chain variable region is 1-107 th from the N terminal of SEQ ID No.2, or has more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% of consistency with 1-107 th from the N terminal of SEQ ID No.2 (the inconsistency is preferably in a Framework Region (FR)).

The heavy chain constant region of the antibody is a human IgG4 constant region, and the specific amino acid sequence is shown as the 118-444 th position from the N end of SEQ ID No. 1. The light chain constant region of the antibody is a human kappa light chain constant region, and the specific amino acid sequence is shown as 108-214 from the N end of SEQ ID No. 2.

Further, the heavy chain has the amino acid sequence of SEQ ID No.1, or has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identity with SEQ ID No.1 (the discordance is preferably in the Framework Region (FR)). The amino acid sequence of the light chain is SEQ ID No.2, or has more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% of consistency with SEQ ID No.2 (the inconsistency is preferably in a Framework Region (FR)).

The antibodies of the invention may be of any class, such as IgG, IgM, IgE, IgA or IgD. Preferably the 4-1BB antibody is of the IgG class, such as the IgG1, IgG2, IgG3 or IgG4 subtype, more preferably the IgG4 subtype.

The light chain type of the antibody of the present invention may be a Kappa chain or a lambda chain, preferably a Kappa chain.

In a particular embodiment of the invention, the antibody is specifically the humanized antibody Hanke10F4 directed against human 4-1 BB. The Hanke10F4 mainly binds to Domain1 and Domain 2A in the extracellular region of human 4-1 BB.

The antibodies provided by the invention have one or more of the following properties or characteristics:

1) specifically binds to human 4-1 BB;

2) binds to human and cynomolgus monkey 4-1 BB;

3) binds to human or cynomolgus monkey 4-1 BB;

4) to some extent, it blocks the binding of human 4-1BBL (4-1BB ligand) to human 4-1 BB;

5) is an IgG, such as IgG1, IgG2, IgG3 or IgG 4;

6) is a human antibody, or a humanized antibody.

The antibodies or antigen binding fragments thereof of the invention have agonist activity against human 4-1BB and stimulate CD4⁺TCD8⁺T cell proliferation, obviously increase IFN-gamma expression, to a certain extent show that the antibody or antigen binding portion thereof can regulate and control immune system by regulating immune cell activity, and can be applied to immune enhancement of immune enhancer anti-tumor or anti-virus immune reactionOr an immunomodulator in a T cell mediated autoimmune disease.

Antibodies of the invention can be prepared by methods known in the art, including conventional monoclonal antibody techniques. Can be prepared by expression in transfected cells such as immortalized eukaryotic cells such as myeloma or hybridoma cells. Antibodies can be converted from one class or subtype to another by methods known in the art. Furthermore, the antibody provided by the present invention may be a monoclonal antibody or a polyclonal antibody, preferably a monoclonal antibody.

The antibodies of the invention may be produced by techniques known in the art, including conventional monoclonal antibody techniques, such as standard somatic hybridization techniques, viral or oncogenic transformation of B lymphocytes, or recombinant antibody techniques, as described in detail below.

Hybridoma production is a common method, and is known in the art. First, a Balb/c mouse was immunized by transient expression of human 4-1BB protein. The amino acid sequence of the human 4-1BB antigen is shown in SEQ ID No.5 (wherein the 24 th to 184 th positions are extracellular regions), and the immunizing antigen is isolated or purified human 4-1BB, and can be a human 4-1BB fragment, such as an extracellular domain of human 4-1 BB. Animal immunization, which can be performed by any method known in the art, is performed on non-human animals such as mice, rats, sheep, and the like. Following immunization of an animal with human 4-1BB antigen, antibody-producing immortalized cell lines are prepared from cells isolated from the immunized animal. After immunization, the animals are sacrificed to obtain immortalized lymph nodes and/or splenic B cells, cell fusion is carried out by a conventional method, positive cell clones, namely cells producing anti-4-1 BB antibody, are screened by an ELISA method, and then are repeatedly subcloned and further screened, and hybridomas with good growth state, high antibody yield and 100% positive culture supernatant are selected. Hybridomas can be expanded in vivo or in vitro in cell culture in syngeneic animals, animals lacking the immune system, and nude mice. Methods for selecting, cloning and amplifying hybridomas are well known to those skilled in the art. The immunoglobulin subtype secreted by the hybridoma cells was detected as IgG1 using a biphasic agar diffusion assay. Antibodies of the invention may also be prepared using phage display methods. Such phage display methods for isolating human antibodies are well established in the art.

After expression of the antibody, including antibody, dimer, single light, heavy chain, or other immunoglobulin forms, may be purified according to standard methods in the art, such as ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, and the like.

Wherein the antigen-binding fragment may comprise one or more of: (1) the light chain of the antibody described hereinbefore; (2) the heavy chain of the antibody described hereinbefore; (3) the light chain variable region of the antibody described above; (4) the heavy chain variable region of the antibody described above; (5) one or more CDR regions of the antibody described above.

Further, the antigen binding fragment may be any one of: (1) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (2) a F (ab') 2 fragment which is a bivalent fragment comprising two Fab fragments having a hinge region linked by a disulfide bond; (3) an Fd fragment consisting of the VH and CH1 domains; (4) an FV fragment consisting of the VL and VH domains of a single arm of an antibody; (5) isolated CDRs (e.g., CDRs from a light chain and/or CDRs from a heavy chain); (6) and the single-chain antibody can be in an scFv structure, and has an additional Linker structure compared with an FV fragment, and the single-chain antibody can be provided with different tags, such as scFv-His, scFv-Fc and the like.

In a specific embodiment of the invention, the antigen-binding fragment provided by the invention is a single chain antibody (scFv). Single chain antibodies (scFv) consist of a single polypeptide chain comprising a VL domain linked to a VH domain, wherein the VL domain and VH domain are paired to form a monovalent molecule. Single chain antibodies can be prepared according to methods known in the art.

Wherein the nucleic acid molecule may be DNA or RNA, and may or may not comprise an intron sequence. Preferably, the nucleic acid molecule is a cDNA molecule.

Further, in the nucleic acid molecule, the nucleotide sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region encoding the antibody are shown as 91-105, 148-192 and 292-318 of SEQ ID No.3 from the 5' end in sequence; the nucleotide sequences of LCDR1, LCDR2 and LHCDR3 in the light chain variable region encoding the antibody are shown as 70-102 th, 148-168 th and 265-291 th positions from the 5' end of SEQ ID No.4 in sequence.

Further, in the nucleic acid molecule, the nucleotide sequence encoding the heavy chain variable region of the antibody has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identity (the place of inconsistency is preferably in the Framework Region (FR)) with position 1 to position 351 from the 5 'end of SEQ ID No.3 or with position 1 to position 351 from the 5' end of SEQ ID No. 3. The nucleotide sequence of the light chain variable region of the antibody is the 1 st-321 th site from the 5 'end of SEQ ID No.4 or has more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% of consistency with the 1 st-321 th site from the 5' end of SEQ ID No.4 (the inconsistency is preferably in a Framework Region (FR)).

In the nucleic acid molecule, the nucleotide sequence encoding the heavy chain constant region variable region of the antibody is shown as position 352-1332 from the 5' end in SEQ ID No. 3. The nucleotide sequence of the light chain constant region of the antibody is the 322 nd-642 nd position from the 5' end of SEQ ID No. 4.

More specifically, in the nucleic acid molecule, the nucleotide sequence encoding the heavy chain of the antibody is SEQ ID No.3 or has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identity with SEQ ID No.3 (the place of inconsistency is preferably in the Framework Region (FR)). The nucleotide sequence of the light chain encoding the antibody is SEQ ID No.4 or has more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% identity with SEQ ID No.4 (the place of inconsistency is preferably in the Framework Region (FR)).

The nucleic acid molecules of the invention can be obtained using suitable molecular biological techniques. For antibodies expressed by a hybridoma, the cdnas encoding the light and heavy chains of the antibody prepared from the hybridoma can be obtained by PCR amplification or cDNA cloning techniques.

The nucleic acid molecule of the invention may encode the amino acid sequence of a portion of a light or heavy chain, a full-length light or heavy chain, or an antibody derivative or antigen-binding fragment thereof.

Isolated DNA encoding VH can be converted to a full-length heavy chain gene by linking the DNA encoding VH to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH 3). Human heavy chain constant region gene sequences are known in the art and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region. Preferred are the IgG4 constant regions.

An isolated DNA encoding a VL region can be converted into a full-length light chain gene by ligating the DNA encoding VL to another DNA molecule encoding a light chain constant region CL. Human light chain constant region gene sequences are known in the art, and DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region can be a Kappa or Lambda constant region.

Wherein the vector is an expression vector useful for expression of a binding molecule, such as an antibody or antigen-binding fragment thereof.

To express the antibodies of the invention, DNA encoding partial or full length light and heavy chains is inserted into an expression vector, and transcription and translation of the DNA molecule is initiated. The antibody light chain gene and the antibody heavy chain gene may be inserted into separate vectors, or into the same expression vector. The insertion method may be any suitable method known, such as linkage of complementary restriction sites on the antibody gene fragment to the vector. The light and heavy chain variable regions of the antibodies described herein can be used to generate full-length antibody genes of any antibody type and subtype.

The invention further provides a host cell (i.e.the recombinant cell) comprising a nucleic acid molecule provided by the invention. The host cell may be any cell available for expression vectors. For example, higher eukaryotic host cells, such as mammalian cells, include, for example, Chinese Hamster Ovary (CHO) cells, lower eukaryotic host cells, such as yeast cells, and may be prokaryotic cells, such as bacterial cells, E.coli, and the like. Transfection methods for introducing the recombinant nucleic acid construct into a host cell include, for example, electroporation, calcium phosphate transfection, DEAE-dextran, lipofection, phage infection, and the like. When an expression vector encoding a gene for an antibody is introduced into a host cell by a known method, the antibody is produced by culturing the host cell sufficiently to allow the binding molecule to be expressed in the host cell.

The vector may be a plasmid, cosmid, phage or viral vector. The plasmids may be pCDNA3.4 and pCDNA3.4-L or others.

The nucleotide sequence of the anti-4-1 BB antibody of the present invention can be easily mutated by a person of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identity to the nucleotide sequence of the anti-4-1 BB antibody of the present invention are derived from the nucleotide sequence of the present invention and are identical to the sequence of the present invention as long as they encode the anti-4-1 BB antibody and have the activity of the anti-4-1 BB antibody.

In the specific embodiment of the invention, the DNA fragment (coding gene of antibody heavy chain) shown in SEQ ID No.3 is cloned between the enzyme cutting site Xba I and Hand III of pcDNA3.4 vector to obtain the recombinant expression vector (named pcDNA3.4-H) for expressing the heavy chain of the antibody; the DNA fragment shown in SEQ ID No.4 (coding gene of antibody light chain) is cloned between enzyme cutting sites Xba I and Hand III of pcDNA3.4 vector to obtain recombinant expression vector (named pcDNA3.4-L) for expressing the light chain of the antibody. In order to be more beneficial to protein expression, when the recombinant expression vector is constructed, a kozak co-recognition sequence (5 ' -GCCACC-3 ') and a coding sequence (5'-ATGGAGTTTGGGCTGAGTTGGGTCTTTCTGGTCGCAATTCTGCTGAAGGGAGTGCAGTGC-3') of a signal peptide are introduced at the upstream of the DNA fragments (coding genes of the heavy chain and the light chain of the antibody) shown in SEQ ID No.3 and SEQ ID No.4, and the coding sequence of the signal peptide is connected with the 5 ' end of the DNA fragments shown in SEQ ID No.3 and SEQ ID No. 4. The recombinant cell is obtained by co-transfecting HEK293 cells with the two recombinant expression vectors (pcDNA3.4-H and pcDNA3.4-L) for respectively expressing the heavy chain and the light chain of the antibody. The recombinant cells express the antibody Hanke10F4 described above.

Experiments prove that the antibody provided by the invention can be combined with human 4-1BB, shows high affinity to the human 4-1BB, and effectively enhances T cell response. On the one hand, the antibodies compete to some extent with human 4-1BBL for binding to human 4-1BB, and on the other hand, the antibodies induce the production of INF- γ in a co-stimulatory assay. The antibodies of the invention are useful for modulating T cell and antibody-mediated immune responses.

Binding of the antibodies of the invention to human 4-1BB results in an enhanced immune response. The antibodies and antigen binding fragments thereof provided by the invention are useful as immune enhancers, or immune modulators of T cell mediated autoimmune diseases. The antibodies and antigen binding fragments thereof may also be used as diagnostic tools to detect 4-1BB in the blood or tissue of patients with cancer, autoimmune or other diseases.

The antibodies and antigen binding fragments thereof provided by the present invention have a wide range of therapeutic uses as immunomodulators, such as cancer, autoimmune diseases, inflammatory diseases, infectious diseases, and the like.

Drawings

FIG. 1 is a hybridoma antibody SDS-PAGE.

FIG. 2 is a diagram showing the structure of an antibody.

FIG. 3 shows that the hybridoma secretes antibody recognizing human 4-1 BB.

FIG. 4 shows that the hybridoma-secreting antibody recognizes monkey 4-1 BB.

FIG. 5 is a graph showing that the antigen-binding fragment recognizes human 4-1 BB.

FIG. 6 shows that the antigen binding fragment recognizes monkey 4-1 BB.

FIG. 7 shows a comparison of the affinities of Hankering10F4 and HKB 6.

FIG. 8 is a graph showing that antibody Hanke10F4 blocks the binding of 4-1BBL to 4-1 BB.

Fig. 9 shows that antibody Hanke10F4 competes with the control antibody epitope.

Fig. 10 shows Hanke10F4 and HKB6 epitope competition.

FIG. 11 is a luciferase activity assay.

FIG. 12 is the antibody Hanke10F4 activating CD4⁺And (4) detecting T cell effect. In the figure, each group of three column charts are respectively CD4 under the conditions of high, medium and low (2 mu g/ml, 0.4 mu g/ml and 0.08 mu g/ml) concentration of the antibody from left to right⁺Activation effects of T cells.

FIG. 13 shows that antibody Hanke10F4 activates CD8⁺And (4) detecting T cell effect. Each set of three bar graphsThe activation effect of the T cells under the conditions of high, medium and low (3 mug/ml, 0.5 mug/ml, 0.08 mug/ml) concentrations of the antibody is respectively arranged from left to right.

Fig. 14 shows that antibody Hanke10F4 inhibits tumor growth.

Fig. 15 is a toxicological assay for the antibody Hanke10F 4.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In the quantitative tests in the following examples, three replicates were set up and the results averaged.

In the following examples, positive control antibodies were Utomillumab (PF-05082566, patent No.: US 8337850B 2) from Pfizer and Urelumab (BMS-663513, patent No.: US 7288638B 2) from Bristol Myers Squibb, both of which were anti-human 4-1BB antibodies. The negative control antibody was human IgG (product of jinsburg).

In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA.

The pCDNA3.4 vector (Invitrogen, Cat: A14697) in the following examples.

The conventional equipment and reagents were as follows:

1. a 96-well microplate (Nunc Corp.);

2. plating buffer solution: phosphate buffer at pH 7.0;

3. washing liquid: phosphate buffer at pH 7.0 containing only 0.05% by volume of Tween 20;

4. sealing liquid: washing solution containing only 10g/L BSA.

5. Horse radish peroxidase labeled avidin;

6. chromogenic substrate: tetramethyl benzidine;

7. stopping liquid: 1M sulfuric acid.

Wherein the solvent of the phosphate buffer solution with the pH of 7.0 is water, and the solutes are sodium chloride, potassium dihydrogen phosphate and disodium hydrogen phosphate; the concentration of the sodium chloride in the phosphate buffer solution with the pH of 7.0 is 135mM, the concentration of the potassium chloride in the phosphate buffer solution with the pH of 7.0 is 2.7mM, the concentration of the potassium dihydrogen phosphate in the phosphate buffer solution with the pH of 7.0 is 1.5mM, and the concentration of the disodium hydrogen phosphate in the phosphate buffer solution with the pH of 7.0 is 8 mM.

Example 1 preparation of hybridoma antibodies

1.1 immunization of mice

3 Balb/C mice and 3C 57b1/6 mice with the age of 4-6 weeks are selected, and the mice are immunized by taking a human 4-1BB extracellular domain (24-186 th site of SEQ ID No.5) as an antigen for 1 time every 2 weeks and 3 times in total. Collecting blood from tail vein after 3 rd immunization, detecting antibody production by indirect ELISA, selecting appropriate immunized mouse, taking eyeball, removing neck, killing, aseptically taking mouse spleen cell, and performing cell fusion by conventional method to prepare anti-human 4-1BB monoclonal antibody hybridoma cell strain.

1.2 hybridoma selection

Anti-human 4-1BB monoclonal antibody hybridoma cells were screened by ELISA. ELISA plates were coated with 10. mu.g/ml human 4-1BB (SEQ ID No.5), blocked overnight at 4 ℃. The cell culture supernatant to be tested was added in order, incubated at 37 ℃ for 1 hour, then washed with PBST 3 times, added with 4000-fold diluted horseradish peroxidase-labeled goat anti-mouse (ThermoFisher Co.) and incubated at 37 ℃ for 45min, after washing with PBST 3 times, developed with Tetramethylbenzidine (TMB) (ThermoFisher Co.) substrate and then measured for OD at 450nm wavelength.

Positive cell clones were screened by ELISA and subcloned repeatedly until all hybridoma cell culture supernatants were 100% positive. The hybridoma cells were cloned by limiting dilution. The plates were incubated at 37 ℃ in 5% CO₂The cells were cultured in an incubator and after about 5 days, cell clones were observed under a microscope. And (3) replacing the liquid at a proper time, detecting, and carrying out amplification culture on the positive and well-grown monoclonal cell strain to obtain the anti-human 4-1BB monoclonal antibody hybridoma cell strain, and freezing the cell strain in time.

1.3 hybridoma antibody identification

And (3) carrying out step-by-step amplification culture on the positive and well-grown anti-human 4-1BB antibody hybridoma cell strain, centrifuging (10000rpm for 10 minutes), taking the supernatant, and purifying the obtained supernatant by using a Protein G affinity chromatography column. The specific operation is as follows: the method comprises the steps of balancing a Protein G column (GE company) with PBS, culturing supernatant, passing through the column, pre-eluting 5 column volumes with a solution A (a formula: solvent is water, solute and concentration are 20mM sodium phosphate and 500mM NaCl, and pH is 5.0), eluting 5 column volumes with a solution B (a formula: solvent is water, solute and concentration are 20mM sodium acetate and 150mM NaCl, and pH is 3.5), collecting elution peaks, and concentrating a 30KDa concentration centrifuge tube to obtain the antibody.

The immunization resulted in 15 hybridoma antibody cell lines, and the screening resulted in 6 candidate hybridoma cell lines, and the details of the cell lines and the respective corresponding antibodies are shown in table 1 after subtype identification and primary screening.

TABLE 1 anti-human 4-1BB hybridoma cell lines

Serial number	Numbering	Subtypes of antibodies	Fused cells
				1	37G10F4	IgG1,κ	Sp2/0
2	23C2E9	IgG1,κ	Sp2/0
				3	2C2F2	IgG1,κ	Sp2/0
4	32C2G6	IgG2b,κ	Sp2/0
				5	13F8H8	IgM,κ	Sp2/0
6	37G11E2B3	IgM,κ	Sp2/0

The two candidates 13F8H8 and 37G11E2B3 are eliminated, cell supernatants are collected, purified and concentrated respectively, and SDS-PAGE electrophoresis is carried out on the samples, and the electrophoresis detection result is shown in figure 1.

Sequencing of the obtained anti-human 4-1BB antibody revealed that the obtained anti-human 4-1BB antibody had a molecular weight of 150Kb, comprised of a heavy chain and a light chain, and was a typical whole antibody. The antibody structure is shown in FIG. 2.

1.4 hybridoma secretory antibody recognition of human 4-1BB

The affinity of 4 hybridomas secreting antibodies to recognize human 4-1BB was assessed by ELISA. A96-well plate (96-well microplate, Nunc Co., Ltd.) was coated with human 4-1BB (SEQ ID No.5) at a concentration of 1. mu.g/ml, 100. mu.l/well, overnight at 4 ℃. The plate was washed three times, 300. mu.l of blocking solution (formula see above) was added to each well, and blocked at 37 ℃ for 1 hour. The plates were washed three times with washing solution (recipe see above), and the antibodies were diluted to 4000ng/ml with sample diluent (PBS-T plus 1% bovine serum albumin) and then diluted 4-fold into a gradient with 7 Ep tubes, two replicate wells per concentration, 100 μ Ι/well, incubated for 1 hour. The plate was washed three times with a washing solution, 100. mu.l of a 8000-fold diluted horseradish peroxidase-labeled goat anti-mouse (ThermoFisher Co.) was added to each well, and shaken for 0.5 hour. The plate was washed three times, and 100. mu.l of Tetramethylbenzidine (TMB) (ThermoFisher Co.) was added to each well. The reaction was stopped by adding 1M sulfuric acid. OD was measured at 450nm using a BIO-TEK ELX-800 microplate reader.

Through the above steps, the hybridoma secreting antibody can recognize and bind to human 4-1BB, the ELISA result OD450 value is shown in Table 2, and the analysis chart is shown in FIG. 3.

TABLE 2 hybridoma secretory antibody recognition of human 4-1BB

1.5 hybridoma secretion antibody recognition of monkey 4-1BB

The affinity of the hybridoma-secreting antibodies for recognition of monkey 4-1BB was assessed by ELISA. Specifically, the monkey 4-1BB (Cy4-1BB) (SEQ ID No.6) antigen was diluted to 1. mu.g/ml with plating buffer (formulation see above), and 100. mu.l of plate was added to each well, overnight at 4 ℃. Hybridoma secretory antibody and positive control antibody Utomillumab were diluted to 1000ng/ml with sample diluent, and then diluted 10-fold with 7 Ep tubes to form a gradient solution, two multiple wells were set for each concentration, 100. mu.l of each well was added to the plate, and the plate was shaken for 1 hour. After washing the plate again, 100. mu.l of a 8000-fold diluted horseradish peroxidase-labeled goat anti-mouse (ThermoFisher Co.) was added to each well, and the mixture was shaken for 0.5 hour. After washing, 100. mu.l of Tetramethylbenzidine (TMB) (ThermoFisher Co.) was added for color development.

Through the steps, experiments prove that the hybridoma secreting antibodies can all recognize Cy4-1BB molecules, the ELISA result OD450 values are shown in Table 3, and the analysis chart is shown in FIG. 4.

TABLE 3 hybridoma secretion antibody recognition of monkey 4-1BB

Example 2 antibody humanization and characterization of scFv-Fc antigen binding fragment Properties

Secreted antibody 37G10F4 was selected from panning as described in the present invention. Analyzing the gene sequence of the antibody, then determining a humanized mutation site through homologous modeling and optimization of an antibody Fab, scanning the surface, simulating virtual mutation and molecular dynamics, and determining key amino acids and the like. Through humanization, 12 antigen-binding fragments in the form of candidate scFv-Fc (human IgG4) were obtained for further characterization as described in this example. Candidate scFv-Fc molecules were expressed and purified and then identified in ELISA (including identification of their ability to recognize human and monkey 4-1BB, see example 1 for the relevant steps), as shown in table 4, and as shown in fig. 5 and 6 for ELISA results.

TABLE 4 scFv-Fc molecules recognize human 4-1BB

Note: the CDR regions of the heavy and light chains of the 12 candidate scFv-Fc were identical and there was an amino acid mutation in the Framework Region (FR).

Example 3 IgG humanized antibody

The exemplary antibody identified in this example, Hanke10F4, was humanized engineered from hybridoma-secreting antibody 37G10F4, i.e., transformed from scFvB60103 in table 4. This example details expression and purification of Hanke10F4 and subsequent identification in ELISA, FACS, luciferase reporter assay, T cell activation system.

3.1 antibody expression and purification

According to the gene sequence obtained by sequencing, a full-length gene sequence is synthesized, the heavy chain coding gene of the Hanke10F4 antibody is shown as SEQ ID No.3, and the light chain coding gene of the Hanke10F4 antibody is shown as SEQ ID No. 4.

The 1 st to 351 st positions of SEQ ID No.3 are encoding genes of the VH of the heavy chain variable region, wherein the encoding sequences of the CDR1, the CDR2 and the CDR3 are respectively shown as the 91 st to 105 th positions, the 148 th and the 192 th positions and the 292 nd and the 318 th positions of SEQ ID No. 3.

The 1-321 positions of SEQ ID No.4 are the genes encoding the light chain variable region VL, wherein the coding sequences of CDR1, CDR2 and CDR3 are shown as the 70-102 positions, 148-168 positions and 265-291 positions of SEQ ID No.4, respectively.

The 5 'ends of SEQ ID No.3 and SEQ ID No.4 are respectively and sequentially added with a recognition sequence of XbaI (5' -TCTAGA-3 '), a kozak consensus recognition sequence (5' -GCCACC-3 ') and a coding sequence of a signal peptide (5'-ATGGAGTTTGGGCTGAGTTGGGTCTTTCTGGTCGCAATTCTGCTGAAGGGAGTGCAGTGC-3'), the coding sequence of the signal peptide is connected with the 5' ends of SEQ ID No.3 and SEQ ID No.4, then the 3 'ends of SEQ ID No.3 and SEQ ID No.4 are respectively and sequentially added with a Terminator (TGA) and a recognition sequence of HindIII (5' -AAGCTT-3 '), the TGA is connected with the 3' ends of SEQ ID No.3 and SEQ ID No.4, and the obtained new DNA sequences are respectively marked as heavy chain gene A and light chain gene A. Artificially synthesizing a heavy chain gene A, carrying out enzyme digestion on the heavy chain gene A by XbaI and HindIII, connecting the enzyme digested heavy chain gene A with a vector framework obtained by carrying out enzyme digestion on a pCDNA3.4 vector by XbaI and HindIII to obtain a recombinant vector, and naming the recombinant vector which is verified to be correct by sequencing as pCDNA3.4-H; the light chain gene A is cut by XbaI and HindIII and then connected with a vector framework obtained by cutting a pCDNA3.4 vector by XbaI and HindIII to obtain a recombinant vector, and the recombinant vector verified to be correct by sequencing is named as pCDNA3.4-L.

The pCDNA3.4-H and pCDNA3.4-L were introduced into the human embryonic kidney cell line HEK293F (ATCC U.S. cell bank) to obtain recombinant cells, which were then placed at 37 ℃ with 5% CO₂The culture was carried out in a shaking incubator at 120 rpm.

The antibody Protein was purified from the culture supernatant using a Protein A affinity column. The specific operation is as follows: the method comprises the steps of balancing a Protein A column (GE company) by PBS, culturing supernatant, passing through the column, pre-eluting 5 column volumes by adopting a solution A (a formula: solvent is water, solute and concentration are 20mM sodium phosphate and 500mM NaCl, and pH5.0), eluting 5 column volumes by adopting a solution B (a formula: solvent is water, solute and concentration are 20mM sodium acetate and 150mM NaCl, and pH3.5), collecting elution peaks, and concentrating a 30KDa concentration centrifuge tube to obtain the antibody, namely the antihuman 4-1BB antibody.

Sequencing of the anti-human 4-1BB antibody revealed that the obtained anti-human 4-1BB antibody was an intact antibody, i.e., the antibody was Hanke10F 4. The antibody consists of a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID No.1, and the amino acid sequence of the light chain is shown as SEQ ID No. 2. The heavy chain type of the Hanke10F4 antibody is IgG4 and the light chain type is kappa chain.

Positions 1-117 of SEQ ID No.1 are the heavy chain variable region VH, wherein the sequences of CDR1, CDR2 and CDR3 are shown as positions 31-35, 50-64 and 98-106 of SEQ ID No.1, respectively.

The sequences of CDR1, CDR2 and CDR3 in the light chain variable region VL of SEQ ID No. 1-107 are shown at positions 24-34, 50-56 and 89-97, respectively, of SEQ ID No. 2.

3.2 identification of affinity for human or monkey 4-1BB

(1) ELISA detection

See step 1.4 in example 1, the 96-well plate was directly coated with human 4-1BB or monkey 4-1BB, respectively, antibody samples (Hanke10F4 and Utomillumab) as primary antibodies, and horseradish peroxidase-labeled goat anti-mouse as secondary antibodies. Tetramethylbenzidine (TMB) is used as a color reagent, and the color development is stopped by sulfuric acid. OD was measured at 450nm using a BIO-TEKELX-800 microplate reader.

Table 5 shows the results of ELISA using the antibody Hanke10F4 in the form of IgG4, which was recognized by both human 4-1BB and monkey 4-1 BB.

TABLE 5 binding of Hanke10F4 in ELISA assay

(2) FACS detection

The reagents used included:

a FACS buffer solution obtained by adding BSA and sodium azide to PBS, wherein the mass percent concentration of BSA in the buffer solution is 2% and the mass percent concentration of sodium azide is 0.02%; goat anti-human FITC secondary antibody (Sigma, protected from light).

The cells used in the experiment are CHO-K1 cells (CHO-K1-hu4-1BB) with high cell membrane surface expression of human 4-1BB and CHO-K1 cells (CHO-K1-cy4-1BB) with high cell membrane surface expression of monkey 4-1BB, which are constructed by Hefeijiagaku Mebo, and wild type CHO-K1 (from Shanghai cell bank of Zhongkou institute) without 4-1BB expression.

Human 4-1BB gene sequence (SEQ ID No.7) and monkey 4-1BB gene sequence (SEQ ID No.8) were synthesized by hand, and then inserted into pCDNA3.4 vectors according to a method commonly used in the art, followed by introduction into wild-type CHO-K1 cells using Lipofectamine 3000 transfection reagent (Invitrogen). Then G418 (purchased from Biotechnology engineering (Shanghai) Co., Ltd.) is added for pressure screening, and CHO-K1 cells (CHO-K1-hu4-1BB) with high cell membrane expression of human 4-1BB and CHO-K1 cells (CHO-K1-cy4-1BB) with high cell membrane expression of monkey 4-1BB are finally obtained.

The binding activity of the Hanke10F4 monoclonal antibody to human 4-1BB was tested using CHO-K1-hu4-1 BB. Culturing the test cells to 80% full using T75 flask, trypsinizing, centrifuging at 1000rpm for 5min, and collecting cells (10 cells per flask)⁶About one), washing with about 1ml of buffer solution, centrifuging, and resuspending the cells to obtain a cell suspension having a cell concentration of 1X 10⁷Individual cells/ml. Mu.l of each dilution gradient of Hanke10F4 antibody dilution was prepared, 25. mu.l of the cell suspension was added to each tube containing different concentrations of antibody, and the final antibody concentration was as shown in Table 6, and the tube containing 0. mu.g/ml antibody dilution was used as a blank. After incubation for 30min, 1ml was usedAfter washing twice with FACS buffer, adding goat anti-human FITC secondary antibody, pipetting, resuspending, and incubating for 30min in dark place. The tube was washed twice with 1ml FACS buffer, then resuspended with 500. mu.l FACS buffer per tube, placed on ice and protected from light, and tested on the machine. Antibody Utomilumab (feverfew pharmaceuticals, ltd.) was used as a positive control.

According to the above procedure, the binding activity of the Hanke10F4 monoclonal antibody to monkey 4-1BB was examined using CHO-K1-cy4-1 BB.

Through the above steps, the experiment proves that the Hanke10F4 monoclonal antibody has high binding activity with human 4-1BB and monkey 4-1BB molecules, as shown in Table 6.

TABLE 6 FACS detection of 4-1BB antibody affinity detection results

(3) FACS comparison of antibody affinities

The CHO-K1-hu4-1BB assay was used to compare the binding activity of the Hanke10F4, HKB6 monoclonal antibodies to human 4-1 BB. Culturing the test cells to 80% full using T75 flask, trypsinizing, centrifuging at 1000rpm for 5min, and collecting cells (10 cells per flask)⁶About one), washing with about 1ml of buffer solution, centrifuging, and resuspending the cells to obtain a cell suspension having a cell concentration of 1X 10⁷Individual cells/ml. Each dilution gradient of 25. mu.l of Hanke10F4 antibody dilution or 25. mu.l of HKB6 antibody dilution was prepared, 25. mu.l of the cell suspension was added to each of the centrifuge tubes to which antibodies of different concentrations were added and mixed, and the final antibody concentration was as shown in Table 7, and the centrifuge tube to which the antibody dilution of 0. mu.g/ml antibody was added was used as a blank control. After incubation for 30min, the cells were washed twice with 1ml FACS buffer, and then goat anti-human FITC secondary antibody was added, resuspended by pipetting, and incubated for 30min in the dark. The tube was washed twice with 1ml FACS buffer, then resuspended with 500. mu.l FACS buffer per tube, placed on ice and protected from light, and tested on the machine.

HKB6 is the HKB6 antibody described in patent application No. 201811541545.8.

Through the above steps, as shown in Table 7 and FIG. 7, Hanke10F4 binds human 4-1BB with significantly higher affinity than HKB 6.

TABLE 7 comparison of the affinities of Hankering10F4 and HKB6

3.3 ligand Competition

CHO-K1-hu4-1BB was used to compare competitive binding activity between the Hanke10F4 antibody and human 4-1 BBL. The secondary antibody was SA-FITC (Biolegend), and other procedures (except for the following) and dilutions were performed in the same manner as for FACS detection in step 3.2. The amino acid sequence of human 4-1BBL is shown in SEQ ID No. 9.

The Hanke10F4 was labeled with biotin as Bio-Hanke10F4 in accordance with conventional labeling methods for proteins in the art. Bio-Hanke10F4 was diluted with sample diluent to 1.5. mu.g/ml solution A. Meanwhile, a dilution B of human 4-1BBL-hIgG (or Hanke10F4) is prepared, the concentration of the fixed solution A is unchanged, and the concentration of the solution B is a certain multiple of the concentration of A (the specific multiple is shown in Table 8). And uniformly mixing the solution A and the solution B in equal proportion, and incubating with CHO-K1-hu4-1 BB.

The reverse competition was also tested by labelling human 4-1BBL with biotin as Bio-4-1BBL and diluting solution A at a concentration of 2. mu.g/ml with sample diluent. Meanwhile, a Hanke10F4 (or human 4-1BBL-hIgG) diluent B is prepared, the concentration of the fixed solution A is unchanged, and the concentration of the solution B is a certain multiple of the concentration of A (the specific multiple is shown in Table 8). The solutions A and B were mixed in equal proportions and incubated with CHO-K1-hu4-1 BB.

Through the steps, the experiment proves that the Hanke10F4 monoclonal antibody has competition relation with the binding site of the human 4-1BBL, and the specific result is shown in the table 8 and the figure 8, namely, the monoclonal antibody can block the signaling pathway of the human 4-1BB/4-1BBL to a certain extent along with the dosage of the medicine.

TABLE 8 FACS detection of ligand blockade

3.4 epitope Competition

3.4.1 Hanke10F4 competes with the positive control antibody antigen-binding epitope

CHO-K1-hu4-1BB was used to compare epitope competition between the Hanke10F4 antibody and the control antibodies Utomillumab and Urelumab. The secondary antibody was SA-FITC (Biolegend), and other procedures (except for the following) and dilutions were performed in the same manner as for FACS detection in step 3.2.

Hanke10F4 was labeled Bio-Hanke10F4 with biotin and solution A was diluted to a concentration of 1.5. mu.g/ml with sample diluent. And simultaneously preparing Utomillumab, Urelumab and Hanke10F4 antibody diluent B, wherein the concentration of the fixed solution A is unchanged, and the concentration of the solution B is a certain multiple of A (the specific multiple is shown in Table 9). The solutions A and B were mixed in equal proportions and incubated with CHO-K1-hu4-1 BB.

Through the steps, repeated experiments prove that the Hanke10F4 monoclonal antibody has a certain degree of competition relationship with antigen binding sites of Utomillumab and Urelumab, and the competition with Utomillumab is stronger than that with Urelumab. Specific results are shown in table 9 and fig. 9.

TABLE 9 FACS detection of epitope Competition

3.4.2 Hanke10F4 antigen binding epitope analysis with positive control antibody

The extracellular region of human 4-1BB antigen is replaced by the corresponding extracellular region of mouse 4-1BB, different target genes are synthesized, the target genes are respectively inserted into pCDNA3.4 vectors to obtain DNA plasmids of different 4-1BB antigens, and cell strains expressing different 4-1BB antigens on the cell surface are obtained by transient transformation, which is shown in Table 10. FACS analysis of the binding activity of antibodies to different 4-1BB antigens was performed, and the binding of antibodies to specific epitopes of the extracellular region of human 4-1BB was analyzed.

The transient operation specifically uses an Expi293 expression system (Thermo Fisher) product, and operates the flow according to the product specification. Respectively adding an Expi Fectamine transfection reagent and DNA plasmids into OptiMEM to obtain solutions A and B, and then uniformly mixing the solution A and the solution B to obtain a solution C. The solution C is added into Expi293 cells (Thermo Fisher) and incubated overnight to obtain cell strains with high expression of 4-1BB antigen on the cell surfaces.

Wherein, the full-length amino acid sequence of the human 4-1BB antigen is shown as SEQ ID No.5 (the corresponding coding gene is shown as SEQ ID No.7), and the extracellular region is 24-186 (the corresponding coding gene is shown as 70-558 of SEQ ID No. 7). The full-length amino acid sequence of the mouse 4-1BB antigen is shown as SEQ ID No.10 (the corresponding coding gene is shown as SEQ ID No. 11), and the extracellular region is 24-187 (the corresponding coding gene is shown as 70-561 of SEQ ID No. 11).

The cell strain obtained by transient transformation is added with an antibody sample (Utomillumab, Hanke10F4 or Urelumab) as a primary antibody to be incubated with the cells according to the FACS detection step in the step 3.2, then sheep anti-human FITC is added to be incubated as a secondary antibody, the cells are washed twice by FACS buffer solution, then 500 mu l of FACS buffer solution is added into each tube to be resuspended, protected from light and detected on a machine.

According to the analysis of patent and literature reports, the Utomilumab mainly binds to Domain3 and Domain4 of the extracellular region of human 4-1BB, and the Urelumab mainly binds to Domain1 of the extracellular region of human 4-1 BB. Through the above operation, it can be known that the trend of the fluorescence intensity of Urelumab decreases in two groups of cells a and F, and Utomilumab decreases in three groups of cells B, C and D, and the results are in line with the research report. While the trend of the fluorescence intensity of Hanke10F4 decreased in the two groups of cells A and F, and decreased greatly in the group of cells A, which was much lower than that of Urelumab, and because the fluorescence intensity of Hanke10F4 did not decrease and increase in the group of cells B, it was demonstrated that Domain1 and 2A are critical for the affinity of Hanke10F4 to human 4-1BB, while Domain2B,3A and 3B have little influence, Hanke10F4 mainly binds to the Domain1 and Domain 2A regions in the extracellular region of human 4-1BB, and is different from the antigen binding epitopes of the control antibodies Utomimab and Urelumab (Table 10). Hanke10F4 has a degree of competition with the positive control antibody, which may be due to the spatial epitope of the antigen antibody.

TABLE 10 analysis of mean fluorescence intensity of antibody binding to human 4-1BB epitope

Note: the specific structure of the antigen in cell A, "human 4-1BB + murine 4-1BB domain1,2A,2B (aa 24-86)" means that the amino acids at positions 24-86 in the full length of the human 4-1BB antigen shown in SEQ ID No.5 are replaced with the amino acids at positions 24-86 in the full length of the murine 4-1BB antigen shown in SEQ ID No. 10. The specific structure of the antigen in cell B, "human 4-1BB + murine 4-1BB domain2B,3A,3B (aa 64-118)" refers to the replacement of amino acids 64-118 of the full length of the human 4-1BB antigen shown in SEQ ID No.5 with amino acids 64-118 of SEQ ID No. 10. The specific structure of the antigen in cell C, "human 4-1BB + murine 4-1BB domain3A,3B,4A (aa 87-133)", refers to the substitution of the amino acids at positions 87-133 of the full length of the human 4-1BB antigen shown in SEQ ID No.5 with the amino acids at positions 87-133 of SEQ ID No. 10. The specific structure of the antigen in cell D, "human 4-1BB + murine 4-1BB domain3B,4A,4B (aa 97-159)" refers to the replacement of the amino acids at positions 97-159 of the full length of the human 4-1BB antigen shown in SEQ ID No.5 with the amino acids at positions 97-159 of SEQ ID No. 10. The specific structure of the antigen in cell E, "human 4-1BB + murine 4-1BB domain4A,4B, NF (aa 119-186)" refers to the replacement of the amino acid at position 119-186 in the full length of the human 4-1BB antigen shown in SEQ ID No.5 with the amino acid at position 119-186 in SEQ ID No. 10. The specific structure of the antigen in cell F, "human 4-1BB + murine 4-1BB domain1,4B, NF (aa24-46, aa 139-186)" refers to the replacement of the amino acids at positions 24-46 and 139-186 of the full length of the human 4-1BB antigen shown in SEQ ID No.5 with the amino acids at positions 24-46 and 139-186 of SEQ ID No. 10.

3.4.3 Hanke10F4 competes with HKB6 antigen binding epitope

CHO-K1-hu4-1BB was used to compare the antigen binding epitope competition relationship between the Hanke10F4 antibody and HKB 6. The secondary antibody was SA-FITC (Biolegend), and other procedures (except for the following) and dilutions were performed in the same manner as for FACS detection in step 3.2.

Hanke10F4 was labeled Bio-Hanke10F4 with biotin and solution A was diluted to a concentration of 1.5. mu.g/ml with sample diluent. And preparing HKB6 and Hanke10F4 antibody diluent B at the same time, wherein the concentration of the fixed solution A is unchanged, and the concentration of the solution B is a certain multiple of A (the specific multiple is shown in Table 11). The solutions A and B were mixed in equal proportions and incubated with CHO-K1-hu4-1 BB.

Through the above steps, the experiment proves that the Hanke10F4 monoclonal antibody has no competitive relationship with the HKB6, namely, the antigen binding epitopes of the two antibodies are different, and the specific results are shown in Table 11 and FIG. 10.

TABLE 11 Hanke10F4 and HKB6 epitope competition

3.5 luciferase reporter genes

HEK293 cells expressing human 4-1BB and stably integrated NF κ B luciferase reporter were prepared. Human 4-1BB sequence (SEQ ID No.7) as the target gene was inserted into pCDNA3.4 vector to obtain plasmid A, and NF-. kappa.B element sequence (SEQ ID No.12) and luciferase gene (SEQ ID No.13) as the target gene were inserted into different sites of pGL4.10 vector (Youbao organism) according to the general method in the art to obtain plasmid B. Then, A, B two plasmids were introduced together into HEK293 cells (Shanghai cell Bank of China) using Lipofectamine 3000 transfection reagent (Invitrogen). Then G418 (purchased from Biotechnology engineering (Shanghai) Co., Ltd.) is added for pressurized screening, and HEK293 cells expressing human 4-1BB and stably integrated NF kappa B luciferase reporter genes are finally obtained. Cells were cultured and harvested, washed and resuspended in phenol red free complete medium (DMEM, containing 10% fetal bovine serum), and 40. mu.l of cells plated in black 96-well plates (Corning, 3603) at 5X 10⁴Per well. Detection antibody was added to each well in the presence of the 2:1 ratio cross-linking antibody Fab' goat anti-human IgG Fc (Jackson, 109-. The plate was incubated in a cell incubator for 6 hours, 40. mu.l of ONE-Glo Luciferase assay sysytem reagent (Promega) was added and incubated for 10min, and the chemiluminescence value was measured.

Through the steps, experiments prove that both the Hanke10F4 monoclonal antibody and the Utomilumab can activate a reporter gene expression signal, and specific results are shown in a figure 11. This shows that Hanke10F4 has similar functions to Utomillumab, and can activate the T cell activation signal pathway, thereby promoting the secretion of cytokines by T cells and starting the T cell immune function.

3.6 in vitro drug action

3.6.1 CD4⁺T cell activation effect assay

An anti-CD3 antibody (Biolegend, cat # 317325) was diluted to 1. mu.g/ml with PBS to obtain an anti-CD3 antibody solution; the antibody Hanke10F4 was diluted with PBS at three concentrations, high, medium and low, to give solutions of Hanke10F4 antibody at 4. mu.g/ml, 0.4. mu.g/ml and 0.04. mu.g/ml, respectively. The anti-CD3 antibody solution is respectively and uniformly mixed with three concentrations of Hanke10F4 antibody solution according to the volume ratio of 1:1, then added into a 96-well plate for plate wrapping, 50 mu l/well and provided with 3 multiple wells, and the 96-well plate is stood at 37 ℃ for incubation for 1 hour. Three washes with PBS were performed the following day before use. The positive control antibody was designated Utomillumab and the negative control antibody was designated IgG (Biolegend, cat. 403601).

Human CD4⁺T cell isolation: whole blood was collected from healthy persons, PBMC cells were isolated using lymphocyte separation medium (Sigma Co.) according to the instructions thereof, and human CD4 was used⁺T cell magnetic beads (BD Co., Ltd., cat # 557767), isolated and purified according to the instructions to obtain human CD4⁺T cells for use.

Will CD4⁺T cells were added to 96 cell culture plates incubated at 37 ℃ and plated at a density of 1X 10⁵One/well, put 96-well plate at 37 deg.C, 5% CO₂Culturing in an incubator for 3 days, collecting cell supernatant, and detecting the secretion amount of the cell factor INF-gamma by ELISA.

Through the steps, experiments prove that the Hanke10F4 antibody is similar to Utomilumab and can stimulate CD4+ T cells to activate and secrete cytokine INF-gamma. The specific results are shown in FIG. 12.

3.6.2 CD8⁺T cell activation effect assay

The anti-CD3 antibody was diluted to 0.5. mu.g/ml with PBS, added to a 96-well plate (Corning), 60. mu.l/well, and incubated at 37 ℃ for 1 hour in a resting state. Washed with PBS and then stably transformed into fine CHO-K1The cell strain (CHO-K1-CD32A or CHO-K1-CD32B or CHO-K1) was added to a 96-well plate at 100. mu.l/well at 1X 10⁴Per well. The 96-well plate was then placed in a cell incubator overnight.

The cells used in the experiment are CHO-K1 cells (CHO-K1-CD32A) with the cell membrane surface highly expressing human Fc gamma R | | A (CD32A) and CHO-K1 cells (CHO-K1-CD32B) and CHO-K1 cells with the cell membrane surface highly expressing human Fc gamma R | | B (CD32B) constructed by Hefeijiake Mibo.

The gene sequence encoding human Fc γ R | | A (SEQ ID No.14) and the gene sequence encoding human Fc γ R | | B (SEQ ID No.15) were synthesized by hand, and then inserted into pCDNA3.4 vector according to a method commonly used in the art, respectively, and then introduced into wild-type CHO-K1 cells using Lipofectamine 3000 transfection reagent (Invitrogen, Inc.), respectively. Then G418 (purchased from Biotechnology engineering (Shanghai) Co., Ltd.) is added for pressurized screening, and CHO-K1 cells (CHO-K1-CD32A) with high cell membrane expression of human Fc gamma R I A and CHO-K1 cells (CHO-K1-CD32B) with high cell membrane expression of human Fc gamma R I B are finally obtained.

Human CD8⁺T cell isolation: whole blood was collected from healthy persons, PBMC cells were isolated using lymphocyte separation medium (Sigma Co.) according to the instructions thereof, and human CD8 was used⁺T cell magnetic beads (BD Co., Ltd., cat # 557766), isolated and purified according to the instructions to obtain human CD8⁺T cells for use.

After overnight incubation in 96-well plates, cell supernatants were aspirated and 100. mu.l of human CD8 was added per well⁺T cell, 1X 10⁵Per well. The antibody was then diluted with medium to the target concentration and added to a 96-well plate at 100. mu.l/well, with the final concentrations: 3. mu.g/ml, 0.5. mu.g/ml, 0.08. mu.g/ml, 3 duplicate wells per concentration were set. The 96-well plate is put into a cell culture box for incubation for 3 days, and the secretion amount of the cell factor INF-gamma in the cell culture supernatant is detected by an ELISA method.

Through the steps, experiments prove that the Hanke10F4 antibody can stimulate the activation of T cells similarly to Utomillumab and Urelumab, and the activation of the T cells depends on Fc gamma R | | A, Fc gamma R | | B. The specific results are shown in FIG. 13.

3.7 in vivo drug action

Experimental selection B-hCD137(4-1BB) mice were tested for anti-tumor efficacy in Hanke10F 4. The B-hCD137(4-1BB) mouse model was a genetically engineered mouse, in which a human h4-1BB gene was chimeric to the genome of a C57BL/6 mouse, which was derived from a Baiosai map.

Culturing MC38 cells (mouse colon cancer cells) (ATCC) to 80% full, digesting with pancreatin, centrifuging at 1000rpm for 5min, collecting cells, cleaning, centrifuging, and resuspending cells to obtain cell suspension with cell viability of 95% or more.

The MC38 cell line was inoculated subcutaneously on the dorsal (shaved) side of the test mice (2X 10 cells per mouse)⁶Cells, 100 μ l). When the average tumor volume of the tumor-bearing mice reaches 100-³At the time, the mice were randomly divided into 4 groups of 6 mice per experimental design. The concentration of Hanke10F4 and Utomillumab was set to 1mg/kg per administration and the concentration of Urelumab was set to 0.3mg/kg per administration, and the administration was performed 2 times per week for 3 weeks. The antibody was diluted with physiological saline as required, stored in a refrigerator at 4 ℃ and administered by subcutaneous injection 2 times per week. Animals were examined twice weekly for survival and activity following tumor inoculation. The method comprises the following steps: tumor growth, motility, diet, weight and other abnormal behaviors.

Through the steps, experiments prove that the Hanke10F4 antibody, similar to the Utomilumab and Urelumab, can inhibit the growth of MC38 tumor without influencing the mobility and the weight of a mouse, and the anti-tumor effects of the Hanke10F4 antibody and the Utomilumab antibody are almost the same. The specific results are shown in FIG. 14.

3.8 toxicology in vivo

The test mice were randomly divided into 4 groups, and the physiological saline, Hanke10F4, Utomilumab and Urelumab were administered 2 times per week for 3 weeks at a concentration of 30mg/kg each time. After the administration, the tail is cut off and blood is taken, and the content of AST (aspartate aminotransferase) and ALT (alanine aminotransferase) in the peripheral blood of the mice is detected. As shown in fig. 15, Urelumab showed significantly higher AST content compared to saline group, indicating that the antibody is at risk of hepatotoxicity, while Hanke10F4 showed no significant difference in AST and ALT content from Utomilumab, indicating that Hanke10F4 also has low toxicity as does Utomilumab.

Sequence listing

<110> Sakuaimaibo Biotechnology Limited

<120> use of a humanized antibody against 4-1BB

<130> GNCLN192309

<141> 2019-11-13

<160> 15

<170> SIPOSequenceListing 1.0

<210> 1

<211> 444

<212> PRT

<213> Artificial sequence

<400> 1

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Ile Thr Cys Thr Val Ser Gly Ser Ser Leu Thr Ser Tyr

20 25 30

Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Gly Leu

35 40 45

Gly Val Ile Trp Pro Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Met

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu

65 70 75 80

Lys Met Ser Ser Leu Thr Ala Ala Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Arg Val Thr Gly Thr Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210> 2

<211> 214

<212> PRT

<213> Artificial sequence

<400> 2

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 3

<211> 1332

<212> DNA

<213> Artificial sequence

<400> 3

caggtgcagc tgcaggagtc tggcccaggc ctggtgaagc cctctgagac cctgagcatc 60

acctgtacag tgagcggcag ctccctgaca tcctacggag tgcactgggt gcggcagcca 120

cctggcaagg gcctggaggg actgggcgtg atctggccag gaggcagcac caactataat 180

tccgccctga tgagccggct gacaatctct aaggacaaca gcaagtccca ggtgagcctg 240

aagatgtcta gcctgaccgc cgccgacaca gccatgtact attgcgcccg ggtgaccggc 300

acatggtact tcgacgtgtg gggccagggc accacagtga ccgtgtcctc tgctagcacc 360

aagggcccat ccgtcttccc cctggcgccc tgctccagga gcacctccga gagcacagcc 420

gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 480

ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 540

tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacgaagac ctacacctgc 600

aacgtagatc acaagcccag caacaccaag gtggacaaga gagttgagtc caaatatggt 660

cccccatgcc caccatgccc agcacctgag ttccttgggg gaccatcagt cttcctgttc 720

cccccaaaac ccaaggacac tctcatgatc tcccggaccc ctgaggtcac gtgcgtggtg 780

gtggacgtga gccaggaaga ccccgaggtc cagttcaact ggtacgtgga tggcgtggag 840

gtgcataatg ccaagacaaa gccgcgggag gagcagttca acagcacgta ccgtgtggtc 900

agcgtcctca ccgtcctgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtc 960

tccaacaaag gcctcccgtc ctccatcgag aaaaccatct ccaaagccaa agggcagccc 1020

cgagagccac aggtgtacac cctgccccca tcccaggagg agatgaccaa gaaccaggtc 1080

agcctgacct gcctggtcaa aggcttctac cccagcgaca tcgccgtgga gtgggagagc 1140

aatgggcagc cggagaacaa ctacaagacc acgcctcccg tgctggactc cgacggctcc 1200

ttcttcctct acagcagact aaccgtggac aagagcaggt ggcaggaggg gaatgtcttc 1260

tcatgctccg tgatgcatga ggctctgcac aaccactaca cacagaagag cctctccctg 1320

tctctgggta aa 1332

<210> 4

<211> 642

<212> DNA

<213> Artificial sequence

<400> 4

gacatccaga tgacccagtc cccaagctcc ctgtccgcct ctctgggcga tagggtgaca 60

atcagctgct ccgcctctca gggcatctcc aactacctga attggtatca gcagaagcca 120

gacggcaccg tgaagctgct gatctactat accagcacac tgcactccgg agtgccaagc 180

cggttcagcg gctccggctc tggcaccgac tttaccctga caatctctag cctgcagccc 240

gaggatgtgg ccacatacta ttgtcagcag tactctaagc tgccttggac cttcggcggc 300

ggcacaaagc tggagatcaa gcgtacggtg gctgcaccat ctgtcttcat cttcccgcca 360

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420

cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480

gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540

ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600

ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gc 642

<210> 5

<211> 255

<212> PRT

<213> Artificial sequence

<400> 5

Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu

1 5 10 15

Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro

20 25 30

Ala Gly Thr Phe Cys Asp Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys

35 40 45

Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile

50 55 60

Cys Arg Gln Cys Lys Gly Val Phe Arg Thr Arg Lys Glu Cys Ser Ser

65 70 75 80

Thr Ser Asn Ala Glu Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly

85 90 95

Ala Gly Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu

100 105 110

Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln

115 120 125

Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys

130 135 140

Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro

145 150 155 160

Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala

165 170 175

Pro Ala Arg Glu Pro Gly His Ser Pro Gln Ile Ile Ser Phe Phe Leu

180 185 190

Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu

195 200 205

Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

210 215 220

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

225 230 235 240

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

245 250 255

<210> 6

<211> 254

<212> PRT

<213> Artificial sequence

<400> 6

Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu

1 5 10 15

Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Leu Cys Ser Asn Cys Pro

20 25 30

Ala Gly Thr Phe Cys Asp Asn Asn Arg Ser Gln Ile Cys Ser Pro Cys

35 40 45

Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile

50 55 60

Cys Arg Gln Cys Lys Gly Val Phe Lys Thr Arg Lys Glu Cys Ser Ser

65 70 75 80

Thr Ser Asn Ala Glu Cys Asp Cys Ile Ser Gly Tyr His Cys Leu Gly

85 90 95

Ala Glu Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu

100 105 110

Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln

115 120 125

Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys

130 135 140

Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro

145 150 155 160

Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Ala Thr Pro Pro Ala

165 170 175

Pro Ala Arg Glu Pro Gly His Ser Pro Gln Ile Ile Phe Phe Leu Ala

180 185 190

Leu Thr Ser Thr Val Val Leu Phe Leu Leu Phe Phe Leu Val Leu Arg

195 200 205

Phe Ser Val Val Lys Arg Ser Arg Lys Lys Leu Leu Tyr Ile Phe Lys

210 215 220

Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys

225 230 235 240

Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

245 250

<210> 7

<211> 765

<212> DNA

<213> Artificial sequence

<400> 7

atgggcaact cctgctacaa catcgtggcc accctgctgc tggtgctgaa cttcgagcgc 60

acccgctccc tgcaggaccc atgctccaat tgtcccgccg gcaccttctg cgataacaat 120

aggaaccaga tctgctctcc ctgtccccct aatagcttta gctccgccgg aggacagagg 180

acatgcgaca tctgtagaca gtgcaagggc gtgttcagga cccgcaagga gtgttctagc 240

acaagcaacg ccgagtgcga ctgtacccct ggatttcact gcctgggagc aggatgttcc 300

atgtgcgagc aggattgtaa gcagggccag gagctgacca agaagggctg caaggactgc 360

tgtttcggca ccttcaacga tcagaagagg ggcatctgtc gcccttggac caactgcagc 420

ctggatggca agtccgtgct ggtgaatggc acaaaggaga gggacgtggt gtgcggacct 480

tctccagccg atctgagccc aggcgcctcc tctgtgaccc caccagcacc agcaagagag 540

cctggacact ccccacagat catctccttc tttctggccc tgacctctac agccctgctg 600

ttcctgctgt tctttctgac cctgaggttt tccgtggtga agaggggccg caagaagctg 660

ctgtacatct tcaagcagcc cttcatgaga cccgtgcaga ccacacagga ggaggatggc 720

tgctcttgta ggtttccaga ggaggaggag ggaggatgtg agctg 765

<210> 8

<211> 762

<212> DNA

<213> Artificial sequence

<400> 8

atgggcaact cctgttacaa tatcgtggcc accctgctgc tggtgctgaa cttcgagcgg 60

acaagaagcc tgcaggacct gtgctccaat tgtcctgccg gcaccttctg cgataacaat 120

cggagccaga tctgctcccc atgtccccct aactctttta gctccgccgg aggacagagg 180

acatgcgaca tctgtagaca gtgcaagggc gtgtttaaga cccggaagga gtgttctagc 240

acatctaatg ccgagtgcga ctgtatcagc ggctatcact gcctgggcgc cgagtgtagc 300

atgtgcgagc aggattgtaa gcagggccag gagctgacca agaagggctg caaggactgc 360

tgtttcggca ccttcaacga tcagaagagg ggcatctgtc gcccctggac caactgctct 420

ctggatggca agagcgtgct ggtgaatggc acaaaggaga gagacgtggt gtgcggccct 480

tccccagcag atctgtctcc tggcgcctcc tctgccaccc cacccgcccc agccagggag 540

cccggccact ccccccagat catcttcttt ctggccctga ccagcacagt ggtgctgttc 600

ctgctgttct ttctggtgct gcggtttagc gtggtgaagc ggtccagaaa gaagctgctg 660

tacatcttca agcagccttt tatgaggcca gtgcagacca cacaggagga ggacggctgc 720

tcctgtaggt tcccagagga ggaggaggga ggatgcgagc tg 762

<210> 9

<211> 254

<212> PRT

<213> Artificial sequence

<400> 9

Met Glu Tyr Ala Ser Asp Ala Ser Leu Asp Pro Glu Ala Pro Trp Pro

1 5 10 15

Pro Ala Pro Arg Ala Arg Ala Cys Arg Val Leu Pro Trp Ala Leu Val

20 25 30

Ala Gly Leu Leu Leu Leu Leu Leu Leu Ala Ala Ala Cys Ala Val Phe

35 40 45

Leu Ala Cys Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser

50 55 60

Ala Ala Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp

65 70 75 80

Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val

85 90 95

Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp

100 105 110

Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu

115 120 125

Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe

130 135 140

Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser

145 150 155 160

Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala

165 170 175

Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala

180 185 190

Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala

195 200 205

Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His

210 215 220

Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val

225 230 235 240

Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu

245 250

<210> 10

<211> 256

<212> PRT

<213> Artificial sequence

<400> 10

Met Gly Asn Asn Cys Tyr Asn Val Val Val Ile Val Leu Leu Leu Val

1 5 10 15

Gly Cys Glu Lys Val Gly Ala Val Gln Asn Ser Cys Asp Asn Cys Gln

20 25 30

Pro Gly Thr Phe Cys Arg Lys Tyr Asn Pro Val Cys Lys Ser Cys Pro

35 40 45

Pro Ser Thr Phe Ser Ser Ile Gly Gly Gln Pro Asn Cys Asn Ile Cys

50 55 60

Arg Val Cys Ala Gly Tyr Phe Arg Phe Lys Lys Phe Cys Ser Ser Thr

65 70 75 80

His Asn Ala Glu Cys Glu Cys Ile Glu Gly Phe His Cys Leu Gly Pro

85 90 95

Gln Cys Thr Arg Cys Glu Lys Asp Cys Arg Pro Gly Gln Glu Leu Thr

100 105 110

Lys Gln Gly Cys Lys Thr Cys Ser Leu Gly Thr Phe Asn Asp Gln Asn

115 120 125

Gly Thr Gly Val Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Arg

130 135 140

Ser Val Leu Lys Thr Gly Thr Thr Glu Lys Asp Val Val Cys Gly Pro

145 150 155 160

Pro Val Val Ser Phe Ser Pro Ser Thr Thr Ile Ser Val Thr Pro Glu

165 170 175

Gly Gly Pro Gly Gly His Ser Leu Gln Val Leu Thr Leu Phe Leu Ala

180 185 190

Leu Thr Ser Ala Leu Leu Leu Ala Leu Ile Phe Ile Thr Leu Leu Phe

195 200 205

Ser Val Leu Lys Trp Ile Arg Lys Lys Phe Pro His Ile Phe Lys Gln

210 215 220

Pro Phe Lys Lys Thr Thr Gly Ala Ala Gln Glu Glu Asp Ala Cys Ser

225 230 235 240

Cys Arg Cys Pro Gln Glu Glu Glu Gly Gly Gly Gly Gly Tyr Glu Leu

245 250 255

<210> 11

<211> 768

<212> DNA

<213> Artificial sequence

<400> 11

atgggcaaca attgttacaa cgtggtggtc atcgtgctgc tgctggtggg atgcgagaaa 60

gtgggagccg tgcagaactc ctgcgacaat tgtcagcccg gcaccttctg taggaagtac 120

aatcccgtgt gcaagtcctg tccccctagc accttcagct ccatcggcgg ccagcccaac 180

tgcaatatct gtagggtgtg cgccggctat ttccgcttta agaagttctg ctctagcacc 240

cacaacgccg agtgcgagtg tatcgagggc tttcactgtc tgggacccca gtgtaccagg 300

tgcgagaagg actgcagacc aggacaggag ctgacaaagc agggctgtaa gacctgctct 360

ctgggcacat tcaacgatca gaatggcacc ggcgtgtgcc ggccttggac aaattgcagc 420

ctggacggca gatccgtgct gaagaccggc accacagaga aggatgtggt gtgcggccca 480

cccgtggtgt ccttttctcc tagcaccaca atctctgtga cacctgaggg cggcccagga 540

ggacacagcc tgcaggtgct gaccctgttt ctggccctga caagcgccct gctgctggcc 600

ctgatcttta tcaccctgct gttctccgtg ctgaagtgga tcaggaagaa gttcccacac 660

atctttaagc agcccttcaa gaagaccaca ggagcagcac aggaggagga cgcatgctcc 720

tgtaggtgcc ctcaggaaga agagggagga ggaggaggct acgagctg 768

<210> 12

<211> 14

<212> DNA

<213> Artificial sequence

<400> 12

tggggacttt ccgc 14

<210> 13

<211> 1650

<212> DNA

<213> Artificial sequence

<400> 13

atggaagatg ccaaaaacat taagaagggc ccagcgccat tctacccact cgaagacggg 60

accgccggcg agcagctgca caaagccatg aagcgctacg ccctggtgcc cggcaccatc 120

gcctttaccg acgcacatat cgaggtggac attacctacg ccgagtactt cgagatgagc 180

gttcggctgg cagaagctat gaagcgctat gggctgaata caaaccatcg gatcgtggtg 240

tgcagcgaga atagcttgca gttcttcatg cccgtgttgg gtgccctgtt catcggtgtg 300

gctgtggccc cagctaacga catctacaac gagcgcgagc tgctgaacag catgggcatc 360

agccagccca ccgtcgtatt cgtgagcaag aaagggctgc aaaagatcct caacgtgcaa 420

aagaagctac cgatcataca aaagatcatc atcatggata gcaagaccga ctaccagggc 480

ttccaaagca tgtacacctt cgtgacttcc catttgccac ccggcttcaa cgagtacgac 540

ttcgtgcccg agagcttcga ccgggacaaa accatcgccc tgatcatgaa cagtagtggc 600

agtaccggat tgcccaaggg cgtagcccta ccgcaccgca ccgcttgtgt ccgattcagt 660

catgcccgcg accccatctt cggcaaccag atcatccccg acaccgctat cctcagcgtg 720

gtgccatttc accacggctt cggcatgttc accacgctgg gctacttgat ctgcggcttt 780

cgggtcgtgc tcatgtaccg cttcgaggag gagctattct tgcgcagctt gcaagactat 840

aagattcaat ctgccctgct ggtgcccaca ctatttagct tcttcgctaa gagcactctc 900

atcgacaagt acgacctaag caacttgcac gagatcgcca gcggcggggc gccgctcagc 960

aaggaggtag gtgaggccgt ggccaaacgc ttccacctac caggcatccg ccagggctac 1020

ggcctgacag aaacaaccag cgccattctg atcacccccg aaggggacga caagcctggc 1080

gcagtaggca aggtggtgcc cttcttcgag gctaaggtgg tggacttgga caccggtaag 1140

acactgggtg tgaaccagcg cggcgagctg tgcgtccgtg gccccatgat catgagcggc 1200

tacgttaaca accccgaggc tacaaacgct ctcatcgaca aggacggctg gctgcacagc 1260

ggcgacatcg cctactggga cgaggacgag cacttcttca tcgtggaccg gctgaagagc 1320

ctgatcaaat acaagggcta ccaggtagcc ccagccgaac tggagagcat cctgctgcaa 1380

caccccaaca tcttcgacgc cggggtcgcc ggcctgcccg acgacgatgc cggcgagctg 1440

cccgccgcag tcgtcgtgct ggaacacggt aaaaccatga ccgagaagga gatcgtggac 1500

tatgtggcca gccaggttac aaccgccaag aagctgcgcg gtggtgttgt gttcgtggac 1560

gaggtgccta aaggactgac cggcaagttg gacgcccgca agatccgcga gattctcatt 1620

aaggccaaga agggcggcaa gatcgccgtg 1650

<210> 14

<211> 951

<212> DNA

<213> Artificial sequence

<400> 14

atgaccatgg agacacagat gtcccagaac gtgtgccctc ggaacctgtg gctgctgcag 60

ccactgaccg tgctgctgct gctggcctct gccgatagcc aggcagcagc accacctaag 120

gccgtgctga agctggagcc accctggatc aatgtgctgc aggaggattc tgtgaccctg 180

acatgtcagg gagcacggag ccctgagtct gacagcatcc agtggttcca caatggcaac 240

ctgatcccta cccacacaca gccatcctac cggttcaagg ccaacaataa cgattctggc 300

gagtatacct gccagacagg ccagacctcc ctgtctgacc ccgtgcacct gacagtgctg 360

agcgagtggc tggtgctgca gacccctcac ctggagttcc aggagggcga gacaatcatg 420

ctgcggtgtc actcctggaa ggacaagcca ctggtgaagg tgaccttctt tcagaatggc 480

aagagccaga agttctccca cctggacccc accttcagca tccctcaggc caatcacagc 540

cactccggcg actaccactg cacaggcaac atcggctata ccctgtttag ctccaagccc 600

gtgaccatca cagtgcaggt gccaagcatg ggctctagct cccccatggg catcatcgtg 660

gccgtggtca tcgcaaccgc agtggcagca atcgtggcag ccgtggtggc cctgatctac 720

tgtaggaaga agcgcatctc cgccaactct acagatcccg tgaaggcagc acagttcgag 780

cctccaggca ggcagatgat cgccatccgg aagagacagc tggaggagac aaataacgat 840

tacgagaccg ccgacggcgg ctatatgaca ctgaatccac gcgcccccac cgacgatgac 900

aagaacatct atctgaccct gccccctaat gaccacgtga acagcaataa c 951

<210> 15

<211> 930

<212> DNA

<213> Artificial sequence

<400> 15

atgggcatcc tgtccttcct gcccgtgctg gcaaccgagt ccgactgggc cgattgcaag 60

tctccacagc catggggaca catgctgctg tggaccgccg tgctgtttct ggcaccagtg 120

gcaggaacac ctgcagcacc acctaaggcc gtgctgaagc tggagcctca gtggatcaat 180

gtgctgcagg aggacagcgt gaccctgaca tgtcggggca cacactcccc agagtctgat 240

agcatccagt ggttccacaa tggcaacctg atcccaaccc acacacagcc ctcttaccgg 300

ttcaaggcca acaataacga cagcggcgag tatacctgcc agacaggcca gacctccctg 360

tctgatccag tgcacctgac agtgctgagc gagtggctgg tgctgcagac cccacacctg 420

gagttccagg agggcgagac aatcgtgctg aggtgtcact cctggaagga caagcccctg 480

gtgaaggtga ccttctttca gaatggcaag tccaagaagt tcagccgctc cgaccctaat 540

ttttctatcc cacaggccaa ccactctcac agcggcgatt accactgcac aggcaacatc 600

ggctacaccc tgtatagctc caagcctgtg accatcacag tgcaggcccc ttctagctcc 660

ccaatgggca tcatcgtggc agtggtgacc ggaatcgcag tggcagccat cgtggcagca 720

gtggtggccc tgatctactg ccggaagaag agaatctctg ccctgcctgg ctatccagag 780

tgtagggaga tgggcgagac actgcccgag aagcctgcca atccaaccaa cccagacgag 840

gcagataaag tgggagcaga gaataccatc acatatagcc tgctgatgca ccccgatgcc 900

ctggaggagc ctgacgatca gaacagaatc 930

Claims

1. The application is characterized in that: the use is of an antibody or antigen-binding fragment thereof or a nucleic acid molecule or expression cassette, a recombinant vector or a recombinant cell or a pharmaceutical composition in any one of:

(A1) preparing a product for detecting 4-1BB, or detecting 4-1 BB;

(A2) preparing a product for blocking a 4-1BB/4-1BBL signaling pathway, or blocking a 4-1BB/4-1BBL signaling pathway;

(A3) preparing a product for stimulating T cell activation, or stimulating T cell activation;

(A4) preparing a product for promoting the secretion of IFN-gamma by the T cells, or promoting the secretion of IFN-gamma by the T cells;

(A5) preparing a product for inhibiting the growth of colon cancer cells, or inhibiting the growth of colon cancer cells;

(A6) preparing a product for inhibiting the growth of colon cancer tumors, or inhibiting the growth of colon cancer tumors;

(A7) preparing a product for treating and/or preventing colon cancer, or treating and/or preventing colon cancer;

the amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of the antibody are shown as 31-35 th, 50-64 th and 98-106 th positions from the N end of SEQ ID No.1 in sequence; and/or the amino acid sequences of LCDR1, LCDR2 and LHCDR3 in the light chain variable region of the antibody are shown as 24-34, 50-56 and 89-97 of SEQ ID No.2 from the N end;

the nucleic acid molecule is a nucleic acid molecule encoding the antibody or the antigen-binding fragment;

the expression cassette is an expression cassette containing the nucleic acid molecule;

the recombinant vector is a recombinant vector containing the nucleic acid molecule;

the recombinant cell is a recombinant cell containing the nucleic acid molecule;

2. The application is characterized in that: the use is of an antibody or antigen binding fragment thereof or a pharmaceutical composition in any one of:

(B1) as an immunopotentiator, or for the preparation of an immunopotentiator;

(B2) as an immunomodulator, or in the preparation of an immunomodulator;

3. The application is characterized in that: the use is of an antibody or antigen-binding fragment thereof or a nucleic acid molecule or expression cassette, a recombinant vector or a recombinant cell or a pharmaceutical composition in any one of:

(C1) preparing a product for the treatment and/or prevention and/or diagnosis of a disease characterized by a deregulated 4-1BB expression, or for the treatment and/or prevention and/or diagnosis of a disease characterized by a deregulated 4-1BB expression;

(C2) preparing a product for the treatment and/or prevention and/or diagnosis of cancer, or treating and/or preventing and/or diagnosing cancer;

(C3) preparing a product for treating and/or preventing and/or diagnosing an autoimmune disease, or treating and/or preventing and/or diagnosing an autoimmune disease;

(C4) preparing a product for treating and/or preventing and/or diagnosing an inflammatory disease, or treating and/or preventing and/or diagnosing an inflammatory disease;

(C5) preparing a product for treating and/or preventing and/or diagnosing infectious diseases, or treating and/or preventing and/or diagnosing infectious diseases;

4. Use according to any one of claims 1 to 3, characterized in that: the amino acid sequence of the heavy chain variable region is 1-117 th sites from the N end of SEQ ID No.1, or has more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% of consistency with the 1-117 th sites from the N end of SEQ ID No. 1; and/or

The amino acid sequence of the light chain variable region is 1-107 th from the N end of SEQ ID No.2, or has more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% of consistency with 1-107 th from the N end of SEQ ID No. 2.

5. Use according to any one of claims 1 to 4, characterized in that: the antibody is IgG;

further, the antibody is IgG 4.

6. Use according to any one of claims 1 to 5, characterized in that: the light chain type of the antibody is Kappa chain.

7. Use according to any one of claims 1 to 6, characterized in that: the amino acid sequence of the heavy chain of the antibody is SEQ ID No.1, or the heavy chain has more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% of consistency with the SEQ ID No. 1; and/or

The amino acid sequence of the light chain of the antibody is SEQ ID No.2 or has more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% of consistency with SEQ ID No. 2.

8. Use according to any one of claims 1 to 7, characterized in that: in the nucleic acid molecule, the nucleotide sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region encoding the antibody are shown as 91-105 th, 148-192 th and 292-318 th in sequence from the 5' end of SEQ ID No. 3; and/or

In the nucleic acid molecule, the nucleotide sequences of the LCDR1, LCDR2 and LHCDR3 in the light chain variable region encoding the antibody are shown as 70-102 th, 148-168 th and 265-291 th sites from the 5' end of SEQ ID No.4 in sequence.

9. Use according to any one of claims 1 to 8, wherein: in the nucleic acid molecule, the nucleotide sequence of the heavy chain variable region encoding the antibody is 1-351 from the 5 'end of SEQ ID No.3 or has more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% identity with the 1-351 from the 5' end of SEQ ID No. 3; and/or

In the nucleic acid molecule, the nucleotide sequence of the light chain variable region of the encoding antibody is 1-321 th from 5 'end of SEQ ID No.4 or has more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% of consistency with 1-321 th from 5' end of SEQ ID No. 4.

10. Use according to any one of claims 1 to 9, characterized in that: in the nucleic acid molecule, the nucleotide sequence of the heavy chain of the encoding antibody is SEQ ID No.3 or has more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% of consistency with SEQ ID No. 3; and/or

In the nucleic acid molecule, the nucleotide sequence of the light chain of the encoding antibody is SEQ ID No.4 or has more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% of consistency with SEQ ID No. 4.