Anti-ANG2 antibody, method of preparation thereof, and use

Abstract

The present invention relates to anti-ANG2 heavy chain single domain antibodies and uses thereof.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and specifically relates to an antibody against ANG2 (angiopoietin-2, human angiogenesis 2), a method for preparing the same, and its use.

Background Art

[0002] Antibodies against ANG2 can inhibit the formation and leakage of new blood vessels and reduce the occurrence of inflammatory reactions. ANG / Tie2 is an important signaling pathway for regulating angiogenesis. ANG1 promotes the phosphorylation of endothelial cell receptor Tie2, attracts perivascular cells such as vascular smooth muscle cells and pericytes to surround and support endothelial cells, promotes vascular remodeling, maintains vascular integrity, regulates vascular function, and ANG1 can protect blood vessels from lesions and maintain vascular morphology. However, ANG2 promotes vascular leakage and causes hypotension and abnormal vascular structure. Although there are many ANG2 antibodies in clinical trials developed by enterprises, at present, antibody drugs against ANG2 have not yet entered the market.

Summary of the Invention

Problems to be Solved by the Invention

[0003] The present invention provides a specific monoclonal antibody against ANG2, which acts in inhibiting the angiogenesis-promoting activity of ANG2 and in the treatment of diseases and disorders caused by the angiogenesis process or diseases and disorders related thereto, such as fundus neovascular diseases, rheumatoid arthritis, and psoriasis. In addition, angiogenesis is very important for tumor growth and maintenance, and monoclonal antibodies that inhibit ANG2 can also act in the treatment process of cancer. The present invention further provides a bispecific antibody against ANG2 and VEGF. The antibodies of the present invention can bind to human ANG2, rabbit ANG2, and monkey ANG2.

Means for Solving the Problems

[0004] Specifically, the present invention relates to the following aspects.

[0005] 1. An anti-ANG2 heavy chain single-domain antibody containing HCDR1, HCDR2, and HCDR3, or their variants, which are included in the heavy chain variable region shown in any one of SEQ ID NO: 1 to 10, Preferably, according to the IMGT numbering system, it includes HCDR1, HCDR2, and HCDR3 selected from the group consisting of the following items: (1) HCDR1 shown in SEQ ID NO:21, HCDR2 shown in SEQ ID NO:22, and HCDR3 shown in SEQ ID NO:23. (2) HCDR1 shown in SEQ ID NO:34, HCDR2 shown in SEQ ID NO:35, and HCDR3 shown in SEQ ID NO:36. (3) HCDR1 shown in SEQ ID NO:37, HCDR2 shown in SEQ ID NO:38, and HCDR3 shown in SEQ ID NO:39. (4) Variants of HCDR1 shown in SEQ ID NO:40, HCDR2 shown in SEQ ID NO:41, and HCDR3 shown in SEQ ID NO:42, or HCDR1 shown in SEQ ID NO:40, HCDR2 shown in SEQ ID NO:41, and HCDR3 shown in SEQ ID NO:42, and combinations thereof, wherein each variant has one or more (preferably one, two, or three) conserved amino acid mutations (preferably substitutions, insertions, or deletions) compared to HCDR1 shown in SEQ ID NO:40, HCDR2 shown in SEQ ID NO:41, and HCDR3 shown in SEQ ID NO:42, and has an amino acid sequence that retains binding affinity with ANG2. Preferably, in HCDR1 shown in SEQ ID NO:40, HCDR2 shown in SEQ ID NO:41, and HCDR3 shown in SEQ ID NO:42, the 5th to 8th and 10th to 11th amino acids in CDR1 shown in SEQ ID NO:40, the 2nd to 5th and 7th amino acids in CDR2 shown in SEQ ID NO:41, and the 1st and 3rd to 8th amino acids in CDR3 shown in SEQ ID NO:42 are selected from amino acid X, and the amino acid X is selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val. Preferably, in the HCDR1 variant shown in SEQ ID NO:40, the first glycine G may be substituted with an amino acid selected from the group consisting of Ala, Val, Leu, and Ile; the second phenylalanine F may be substituted with Tyr; the third proline P may be substituted with an amino acid selected from the group consisting of Trp and His; the fourth Ser may be substituted with Thr; the ninth Ser may be substituted with Thr; and the twelfth Gln may be substituted with Asn. In the HCDR2 variant shown in SEQ ID NO:41, the first Ile may be substituted with an amino acid selected from the group consisting of Ala, Val, Leu, and Gly, the sixth Leu may be substituted with Ile, and the eighth Lys may be substituted with Arg. In the HCDR3 variant shown in SEQ ID NO:42, the second Val may be substituted with an amino acid selected from the group consisting of Ala, Gly, Leu, and Ile, and the ninth Asp may be substituted with Glu. (5) HCDR1 shown in SEQ ID NO:43, HCDR2 shown in SEQ ID NO:44, and HCDR3 shown in SEQ ID NO:45. (6) HCDR1 shown in SEQ ID NO:46, HCDR2 shown in SEQ ID NO:44, and HCDR3 shown in SEQ ID NO:48. (7) HCDR1 shown in SEQ ID NO:49, HCDR2 shown in SEQ ID NO:44, and HCDR3 shown in SEQ ID NO:48. (8) HCDR1 shown in SEQ ID NO:52, HCDR2 shown in SEQ ID NO:44, and HCDR3 shown in SEQ ID NO:48. (9) These are HCDR1 shown in SEQ ID NO: 55, HCDR2 shown in SEQ ID NO: 56, and HCDR3 shown in SEQ ID NO: 57.

[0006] 2. An anti-ANG2 heavy chain single-domain antibody as described in item 1, wherein the ANG2 is selected from human ANG2, rabbit ANG2, or monkey ANG2.

[0007] 3. An anti-ANG2 heavy chain single-domain antibody as described in item 1 or 2, comprising a heavy chain variable region, wherein the heavy chain variable region comprises or is composed of a sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with the sequence described in any one of SEQ ID NO: 1 to 10 and having ANG2 binding activity.

[0008] 4. Recombinant protein comprising an anti-ANG2 heavy-chain single-domain antibody as described in any one of items 1 to 3, wherein the recombinant protein further comprises a bioactive substance, such as an enzymatic toxin or an active fragment thereof (e.g., abrin, lysine A, Pseudomonas exotoxin, or diphtheria toxin), tumor necrosis factor, or interferon (e.g., IFN-γ), a bioreaction modifier (e.g., lymphokine, IL-2, IL-2-6, IL-10, granular GM-CSF), and / or an Fc fragment (e.g., an Fc region derived from IgG (e.g., subtypes of IgG1, IgG2, IgG3, or IgG4), IgA1, IgA2, IgD, IgE, or IgM, e.g., an Fc region derived from human, rabbit, or monkey IgG, IgA1, IgA2, IgD, IgE, or IgM), wherein the anti-ANG2 heavy-chain single-domain antibody and the Fc fragment are linked by a linking peptide, wherein the linking peptide is GGGGS(SEQ The sequence is ID NO:30), GGGGSGGGGS (SEQ ID NO:60), or GGGGSGGGGSGGGGS (SEQ ID NO:47), preferably the Fc fragment is the human IgG1 heavy chain constant region, more preferably the Fc fragment shown in GenBank No.AK303185.1 or SEQ ID NO:29, and more preferably the recombinant protein contains or is composed of the sequences shown in SEQ ID NO:11-20.

[0009] 5. A multispecific antibody, preferably a bispecific antibody, comprising an anti-ANG2 heavy chain single-domain antibody as described in any one of items 1 to 3, wherein the structure of the bispecific antibody comprises a light chain and a heavy chain, wherein the light-heavy chains pair to form an interchain disulfide bond, and the two heavy chains pair to form an interchain disulfide bond, wherein the heavy chain is (VH)-(CH1)-(hinge region)-(Fc)-(linked peptide)-(VHH), and the light chain is (VL)-(light chain constant region).

[0010] 6. A multispecific antibody as described in item 5, wherein VHH is an anti-ANG2 heavy-chain single-domain antibody as described in any one of items 1 to 3, and VH and VL are the heavy-chain and light-chain variable regions of the second antibody, and preferably the antigen targeted by the second antibody is selected from immune cell surface antigens, tumor antigens, viruses, bacteria, endotoxins, cytokines, or combinations thereof, more preferably PD-L1, PD-1, VEGFA, IL-10, IL-10R, BCMA The antigen targeted by the second antibody is selected from VEGF, TGF-β, CTLA-4, LAG-3, TIGIT, CEA, CD38, SLAMF7, B7-H3, Her2, EpCAM, CD19, CD20, CD30, CD33, CD47, CD52, CD133, EGFR, GD2, GD3, GM2, RANKL, CD3, and / or CD16a, preferably VEGF, and more preferably the second antibody is selected from anti-VEGF antibodies and SEQ The heavy chain variable region shown in ID NO:24 includes HCDR1, HCDR2, and HCDR3 (preferably including HCDR1 shown in SEQ ID NO:50, HCDR2 shown in SEQ ID NO:51, and HCDR3 shown in SEQ ID NO:53, according to the IMGT numbering system), and the light chain variable region shown in SEQ ID NO:27 includes LCDR1, LCDR2, and LCDR3 (preferably including LCDR1 shown in SEQ ID NO:54, LCDR2 shown in SEQ ID NO:58, and LCDR3 shown in SEQ ID NO:59, according to the IMGT numbering system), and more preferably the heavy chain variable region of the anti-VEGF antibody is the sequence shown in SEQ ID NO:24 or SEQ ID The light chain variable region of the anti-VEGF antibody includes or is composed of sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% homology with the sequence shown in NO:24.

[0011] 7. A multispecific antibody as described in item 5 or 6, wherein the sequence of CH1 is shown in SEQ ID NO:25, the sequence of the hinge region is shown in SEQ ID NO:26, the sequence of the Fc constant region is shown in SEQ ID NO:29, the sequence of the linked peptide is shown in SEQ ID NO:30 or 47, and the sequence of the light chain constant region is shown in SEQ ID NO:28, and preferably, the multispecific antibody is (1) A heavy chain whose sequence includes SEQ ID NO: 24, 25, 26, 29, 30 and 4, or a heavy chain composed of the above sequences, and a light chain whose sequence includes SEQ ID NO: 27 and 28, or a light chain composed of the above sequences, or Y400C composed of the above sequences, (2) Selected from the group consisting of a heavy chain whose heavy chain sequence includes SEQ ID NO: 24, 25, 26, 29, 47 and 4, or a heavy chain composed of the above sequences, and a light chain whose light chain sequence includes SEQ ID NO: 27 and 28, or a light chain composed of the above sequences, or Y400E composed of the above sequences.

[0012] 8. A polynucleotide encoding an anti-ANG double-chain single-domain antibody as described in any one of items 1 to 3, a recombinant protein as described in item 4, or a multispecific antibody as described in any one of items 5 to 7.

[0013] 9. A vector containing the polynucleotides described in Section 8.

[0014] 10. Host cells containing the vector described in Section 9.

[0015] 11. A coupling product comprising an anti-ANG double-chain single-domain antibody as described in any one of items 1 to 3, a recombinant protein as described in item 4, or a multispecific antibody as described in any one of items 5 to 7, and a coupling moiety, wherein the coupling moiety is a purified tag (e.g., a His tag), a detectable label, a drug, a toxin, a cytokine, an enzyme, or a combination thereof, preferably the coupling moiety is a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, a chemotherapeutic agent, a biotoxin, polyethylene glycol, or an enzyme.

[0016] 12. A kit comprising an anti-ANG2 heavy-chain single-domain antibody as described in any one of items 1 to 3, a recombinant protein as described in item 4, a multispecific antibody as described in any one of items 5 to 7, or a coupling product as described in item 11, wherein the kit further comprises an anti-ANG2 heavy-chain single-domain antibody, recombinant protein, or multispecific antibody that specifically recognizes the anti-ANG2 heavy-chain single-domain antibody as described in any one of items 1 to 3, a recombinant protein as described in item 4, a multispecific antibody as described in any one of items 5 to 7, or a coupling product as described in item 11, and optionally the anti-ANG2 heavy-chain single-domain antibody, recombinant protein, or multispecific antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance, a chemiluminescent substance, a colorant, or an enzyme, wherein the kit is used to detect the presence or level of ANG2 in a sample, or The kit comprises (1) an anti-ANG2 heavy-chain single-domain antibody as described in any one of items 1 to 3, a recombinant protein as described in item 4, a multispecific antibody as described in any one of items 5 to 7, or a coupling product as described in item 11, and (2) an antibody against another antigen or its antigen-binding fragment, and / or a cytotoxic agent, and / or a chemotherapeutic agent, and a selectable instruction manual.

[0017] 13. A pharmaceutical composition comprising an anti-ANG double-chain single-domain antibody as described in any one of items 1 to 3, a recombinant protein as described in item 4, a multispecific antibody as described in any one of items 5 to 7, or a coupling product as described in item 11, wherein the pharmaceutical composition is pharmaceutically acceptable Carrier The pharmaceutical composition further comprises and / or excipients, and preferably is in a form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection or intrafocal injection.

[0018] 14. Use of the anti-ANG2 heavy chain single domain antibody according to any one of items 1 to 3, the recombinant protein according to item 4, the multispecific antibody according to any one of items 5 to 7, or the conjugate according to item 11 to suppress the angiogenesis promoting activity of ANG2, for the prevention and / or treatment of diseases caused by or related to the angiogenesis process, such as ocular neovascular diseases, rheumatoid arthritis and psoriasis, and / or tumors, or for the preparation of a pharmaceutical for suppressing the angiogenesis promoting activity of ANG2 and for the prevention and / or treatment of diseases caused by or related to the angiogenesis process, such as ocular neovascular diseases, rheumatoid arthritis and psoriasis and / or tumors.

[0019] It should be understood that within the scope of the present invention, any combination of the above technical features of the present invention and the technical features specifically described in the following text (for example, in the examples) can constitute a novel or preferred technical aspect. For the convenience of the paper, it will not be described individually here.

[0020] The terms according to the present invention have the ordinary meaning understood by those skilled in the art. When a term has two or more definitions within the art and / or is acceptable, the definition of the term used in this manuscript is used to include all meanings.

[0021] As can be understood by those skilled in the art, the CDR region of an antibody is responsible for the binding specificity of the antibody to an antigen. When the sequences of the heavy and light chain variable regions of an antibody are known, there are currently several methods for determining the antibody CDR region, including the Kabat, IMGT, Chothia and AbM numbering systems. However, the application of various definitions regarding the CDR of an antibody or its variant is within the scope of the terms defined and used in this manuscript. Given the amino acid sequence of the variable region of the antibody, those skilled in the art can usually determine the specific CDR without relying on any experimental data other than the sequence itself.

[0022] In the present invention, a heavy-chain single-domain antibody is also called a VHH domain, and a VHH antibody fragment or VHH antibody is a variable domain of antigen-binding immunoglobulin called a "heavy-chain antibody" (i.e., an antibody lacking a light chain) (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, BEndahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains", Nature 363, 446-448, 1993). The VHH domain specifically binds to an epitope without requiring other antigen-binding domains. The VHH domain is a small, stable, and highly efficient antigen recognition unit consisting of a single immunoglobulin domain.

[0023] The heavy-chain single-domain antibody of the present invention can conjugate to a substance having the required biological activity to form a recombinant protein. In some embodiments, the biologically active substance is selected from, for example, toxins or active fragments thereof having enzymatic activity (e.g., abrin, lysine A, Pseudomonas exotoxin, or diphtheria toxin), tumor necrosis factor or interferon (e.g., IFN-γ), biological reaction modifiers (e.g., lymphokines, IL-2, IL-2-6, IL-10, granular GM-CSF), and Fc fragments. The Fc fragment contained in the recombinant protein of the present invention can cause it to form dimers and simultaneously extend the in vivo half-life of the recombinant protein. In some embodiments, the Fc fragments usable in the present invention may be derived from different subtypes of immunoglobulins, such as IgG (e.g., subtypes of IgG1, IgG2, IgG3, or IgG4), IgA1, IgA2, IgD, IgE, or IgM.

[0024] The antibodies of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, (ii) polypeptides having substituents on one or more amino acid residues, (iii) polypeptides formed by fusing a mature polypeptide with one other compound (e.g., a compound that extends the polypeptide half-life, e.g., polyethylene glycol), or (iv) polypeptides formed by fusing an added amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence or a sequence for purifying this polypeptide or a fibrinogen sequence or a fusion protein formed with a 6His tag). As taught in this paper, these fragments, derivatives and analogues are well known to those skilled in the art.

[0025] Conservative substitution "Conservative amino acid substitution" refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. The family of amino acid residues having similar side chains has already been defined in this art and includes 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), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, it is preferable that non-essential amino acid residues in immunoglobulin polypeptides be replaced with other amino acid residues derived from the same side chain family. In some other embodiments, the amino acid chain may be replaced with a structurally similar amino acid chain, the latter differing in order and / or the composition of its side chain family.

[0026] The table below provides unrestricted examples of conserved amino acid substitutions, where a similarity score of 0 or higher indicates the presence of a conserved substitution between the two amino acids.

[0027] [Table 1]

[0028] In some embodiments, the conservative substitution is preferably a substitution in which one amino acid from the following groups (a) to (e) is replaced by another amino acid residue within the same group, the groups being (a) small aliphatic, nonpolar 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, nonpolar residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0029] Particularly preferred conservative substitutions are as follows: replace Ala with Gly or Ser, Arg with Lys, Asn with Gln or His, Asp with Glu, Cys with Ser, Gln with Asn, Glu with Asp, Gly with Ala or Pro, His with Asn or Gln, Ile with Leu or Val, Leu with Ile or Val, Lys with Arg, Gln or Glu, Met with Leu, Tyr or Ile, Phe with Met, Leu or Tyr, Ser with Thr, Thr with Ser, Trp with Tyr, Tyr with Trp, and / or Phe with Val, Ile or Leu.

[0030] In some embodiments, the following amino acids have similar structural characteristics and properties.

[0031] For example, glycine G, alanine A, valine V, leucine L, and isoleucine I all belong to the category of neutral amino acids, each containing one amino group and one carboxyl group. Serine S and threonine T both belong to the hydroxyamino acid group. Cysteine ​​C and methionine M are both sulfur-containing amino acids. Asparagine N and glutamine Q are both amino acids containing an amide group. Aspartic acid D and glutamic acid E are both acidic amino acids. Lysine K and arginine R are both basic amino acids. Phenylalanine F and tyrosine Y are both aromatic amino acids. Tryptophan W, histidine H, and proline P are all heterocyclic amino acids. Therefore, their similar structural characteristics and properties allow for conservative substitution between them. [Effects of the Invention]

[0032] In some embodiments, the antibodies described in the present invention can be conjugated to therapeutic agents (e.g., chemotherapeutic agents, e.g., cisplatin, carboplatin), drug precursors, peptides, proteins, enzymes, viruses, lipids, bioreaction modifiers, drugs, or PEG. The antibodies of the present invention can be linked to or fused to therapeutic agents, which may include detectable labels, e.g., radiolabels, immunomodulators, hormones, enzymes, oligonucleotides, photoactive therapeutic agents or diagnostic agents, cytotoxic agents, drugs or toxins, sonication enhancers, non-radiolabels, combinations thereof, and other such components known in the art. [Brief explanation of the drawing]

[0033] [Figure 1] This describes the biological activity of the anti-ANG2 heavy chain single-domain antibody-Fc in binding to human ANG2. [Figure 2] This describes the biological activity of the anti-ANG2 heavy chain single-domain antibody-Fc in binding to monkey ANG2. [Figure 3]This describes the biological activity of the anti-ANG2 heavy chain single-domain antibody-Fc, which inhibits the binding of human ANG2 to its receptor hTIE2. [Figure 4] This describes the biological activity of antibodies in inhibiting the binding of human ANG2 to HUVEC. [Figure 5] This describes the biological activity of antibodies in inhibiting the binding of human ANG1 to HUVEC. [Figure 6] This involves the suppression of AKT phosphorylation in human ANG2-dependent HUVEC cells by antibodies. [Modes for carrying out the invention] [Examples]

[0034] The present invention will be described in detail below with reference to examples. These examples are provided solely for illustrative purposes, so that those skilled in the art may understand them. The spirit and scope of the present invention are limited by the claims. Unless otherwise stated, the methods used in the following examples are conventional methods, and the reagents used are commercially available reagents unless otherwise stated.

[0035] Example 1: Screening of heavy chain single-domain antibodies against ANG2 The recombinant human ANG2 protein corresponding to the amino acids at positions Lys275-Phe496 of human ANG2 is abbreviated as hANG2 below, and its sequence is shown in SEQ ID NO:32. The monkey ANG2 protein has its sequence shown in SEQ ID NO:33 and is abbreviated as cynoANG2 below. The specific amino acid sequence information for the ANG2 ECD molecule is shown in Table 1.

[0036] [Table 2]

[0037] 1.1 Construction of a heavy-chain single-domain antibody phage library Two alpacas were selected and subjected to antigen immunization. After four immunizations with human ANG2 protein, lymphocytes were extracted from 100 ml of alpaca peripheral blood. Total RNA was extracted using RNAiso Plus reagent (TAKARA, 9109), and the extracted RNA was reverse transcribed into cDNA using the PrimeScript II kit (TAKARA, 6210A). Using PCR amplification, the 750 bp long nucleic acid fragments of the heavy chain antibody variable region and the constant region CH2 were first amplified. Then, the target fragment, i.e., the heavy chain antibody variable region fragment, was amplified using the 750 bp long nucleic acid fragment recovered in the previous step as a template. The vector pComb3XSS (NBbiolab, NPL-001) and the target fragment were enzymatically cleaved with SfiI, incubated overnight at 50°C, and the target fragment was recovered. The fragments were then ligated according to a ligation molar ratio of vector:fragment = 1:3. The ligation products were electrically converted to E. coli competent cells TG1, and each alpaca's ligation product was subjected to 10 electric shock conversions. After plating by gradient dilution, the library size was calculated, and the size of the two alpaca phage libraries was 1.21 × 10⁶ each. 9 and 1 x 10 9 The results showed that 96 clones were randomly selected and identified from the titer measurement plate, and the insertion rate was 100%.

[0038] 1.2 Panning of heavy chain single-domain antibodies against ANG2 Plates were coated with 10 μg / well of hANG2 protein and left overnight at 4°C. The following day, after blocking with 1% BSA at room temperature for 2 hours, 100 μl of phage (2 × 10⁶) was added. 9PFU (pfu / well, derived from the heavy chain single-domain antibody phage library constructed in 1.1) was added and incubated at 37°C for 1 hour. Unbound phages were then washed away by washing five times with PBST (PBS containing 0.05% Tween 20). Finally, phages specifically bound to hANG2 were eluted using glycine hydrochloride (200 mM), and these were used to infect E. coli TG1 in logarithmic phase growth to generate and purify phages for use in the next round of screening. After repeating the same screening process for two rounds, the obtained phages were plated after infecting E. coli TG1, and monoclones were selected from the plates and sequenced. The protein sequences of individual clones were analyzed according to the sequence alignment results, and clones with different CDR1, CDR2, and CDR3 sequences were considered different antibody strains, resulting in a total of 10 different antibody strains.

[0039] [Table 3]

[0040] Example 2: Preliminary evaluation and identification of a heavy chain single-domain antibody against ANG2 1. Expression of heavy chain single-domain antibodies in the host bacterium Escherichia coli Single colonies of the obtained 10 strains of heavy chain single-domain antibodies were selected and inoculated into TG1 host bacteria. The cultures were allowed to mature overnight. The following day, the bacteria that had matured overnight were transferred and amplified. The antibodies were induced with 0.5 mM IPTG and cultured in a shaker at 37°C overnight. The following day, the supernatant was collected by centrifugation and detected.

[0041] 2. Affinity detection of 10 heavy chain single-domain antibodies by ELISA Coat the enzyme-labeled plate with 2 μl each of hANG2 protein solution and BSA per well, incubate overnight at 4°C, discard the supernatant, add 300 μl blocking solution (PBS containing 3% BSA) to each well, block at 37°C for 2 hours, collect the supernatant by centrifugation, add 200 μl of the supernatant to each well of the enzyme-labeled plate, incubate at room temperature for 2 hours, discard the supernatant, and add 200 μl / well of PBST (PBS containing 0.1% BSA) The plates were washed three times with Tween20, and diluted HRP-labeled anti-VHH secondary antibody (Genscript, A01861) was added. The secondary antibody was used after dilution at 1:10000, and the diluent was PBS with 1% BSA, in a volume of 100 μl / well. The plates were incubated at room temperature for 1 hour, washed five times with PBST at 200 μl / well, and 100 μl / well of TMB chromogenic solution (BD, 55214) was added. The plates were incubated at 37°C for 8 minutes. 100 μl / well of 2M HCl stop solution was added, and within 30 minutes after adding the stop solution, the plates were read at 450 nm using a microplate reader. The results are shown in Table 3 below.

[0042] [Table 4]

[0043] Example 3: Preparation of plasmid for anti-ANG2 heavy chain single-domain antibody-Fc The expression plasmid for anti-ANG2 heavy chain single-domain antibody-Fc is obtained by constructing a heavy chain antibody by ligating the coding DNA sequence corresponding to the variable region (SEQ ID NO: 1~10) of the monoclonal antibody heavy chain of a PCR clone to the coding DNA corresponding to the constant region of the human IgG1 heavy chain (GenBank No. AK303185.1). The vector is generally pcDNA3.1(-) (purchased from Invitrogen) or other eukaryotic expression vectors. The variable region and constant region of the heavy chain antibody protein sequence are linked by a linking peptide, which is GGGGS (SEQ ID NO: 30), GGGGSGGGGS (SEQ ID NO: 60), or GGGGSGGGGSGGGGS (SEQ ID NO: 47).

[0044] [Table 5] TIFF0007905428000006.tif204161

[0045] Example 4. Expression and purification of anti-ANG2 heavy chain single-domain antibody-Fc. Plasmid extraction was performed using an endotoxin-free high-volume plasmid extraction kit (Qiagen, product number 12391), and the specific procedure was carried out according to the specifications provided by the manufacturer. CHO-S cells were cultured in CD CHO medium (Gibco, product number 10743-029) at 37°C in a 5% CO2 cell incubator, according to the instructions provided by the manufacturer. After the cells were prepared, antibody-Fc was expressed by co-transfecting the CHO-S cells with a plasmid containing the anti-ANG2 heavy-chain single-domain antibody-Fc sequence. The culture temperature was lowered to 32°C the day after transfection, and 3.5% 2×EFC+ (Gibco, product number A2503105) was added daily. After 14 days of culture, the supernatant was harvested by centrifugation at 800×g. The supernatant was then filtered through a 0.22 μm filter membrane. Antibody-Fc in the culture supernatant was obtained by purification using protein A affinity chromatography and cation exchange chromatography. The concentration of purified antibody-Fc was measured by UV absorbance at 280 nm and extinction coefficients corresponding to various proteins. The purity and homogeneity of antibody-Fc were evaluated by SDS-PAGE and SE-HPLC. Alternatively, secondary purification was performed using ion exchange and SEC on a Superdex 200 to prepare high-purity antibody-Fc samples for use.

[0046] Example 5. Binding affinity of anti-ANG2 heavy chain single-domain antibody-Fc to ANG2 In this example, the affinity of 10 anti-ANG2 double-chain single-domain antibodies-Fc was evaluated using an ELISA detection method.

[0047] Enzyme-labeled plates were coated with 2 μl / well of ANG2 protein solution, incubated overnight at 4°C, the supernatant was discarded, 300 μl of blocking solution (PBS containing 3% BSA) was added to each well, and the mixture was blocked at 37°C for 2 hours. Anti-ANG2 double-chain single-domain antibody-Fc was then gradient diluted, with the diluent being PBS containing 1% BSA. For example, the initial concentration of the dilution was 500 nM, which was then diluted 10-fold to create eight concentration gradients. Diluted anti-ANG double-chain single-domain antibody-Fc was added to each well of an enzyme-labeled plate at a rate of 200 μl. The wells were incubated at room temperature for 2 hours, the supernatant was discarded, and the wells were washed three times with 200 μl / well of PBST (PBS containing 0.1% Tween20). Diluted HRP-labeled anti-human Fc secondary antibody (SIGMA, A8667) was added. The secondary antibody was used after dilution at a ratio of 1:20000. The diluent was PBS with 1% BSA, in a volume of 100 μl / well. The wells were incubated at room temperature for 1 hour, washed five times with 200 μl / well of PBST, and 100 μl / well of TMB chromogenic solution (BD, 55214) was added. The wells were incubated at 37°C for 8 minutes. 100 μl / well of 2M HCl stop solution was added, and within 30 minutes after adding the stop solution, the wells were read at 450 nm using a microplate reader. The data was analyzed using GraphPad Prism 6.0 software, affinity fitting was performed, and EC50 values ​​were obtained. The results are shown in Figures 1 and 2 and Table 5. B2-E2-Fc showed the highest affinity for human ANG2 and monkey ANG2 proteins.

[0048] [Table 6]

[0049] Example 6. Inhibition of hANG2 binding to its receptor hTIE2 by anti-ANG2 heavy chain single-domain antibody-Fc. A 96-well plate was treated with 100 μL of hTIE2-Fc (Novoprotein, CW98) prepared in 2 μg / mL PBS and incubated overnight at 4°C. The plate was then washed three times with PBS washing buffer containing 0.1% Tween-20, followed by 300 μL of PBS containing 1% BSA and blocking for 2 hours. The anti-ANG2 heavy chain single-domain antibody-Fc to be detected was diluted and mixed with hANG2 at a final concentration of 10 μg / mL. After incubation at 37°C for 1 hour, the antigen-antibody mixture was added to the 96-well plate and incubated at 37°C for 1 hour. After washing with PBS washing buffer containing 0.1% Tween-20, diluted HRP-labeled anti-His secondary antibody (Proteintech, 66005-1-Ig) was added. The secondary antibody was used after dilution at 1:2000, with PBS containing 1% BSA in a volume of 100 μl / well. The plates were incubated at room temperature for 1 hour, washed five times with PBST at 200 μl / well, and 100 μl / well of TMB chromogenic solution (BD, 55214) was added. The plates were developed at 37°C for 8 minutes. 100 μl / well of 2M HCl stop solution was added, and within 30 minutes after adding the stop solution, the plates were read at 450 nm using a microplate reader. The data was analyzed using GraphPad Prism 6.0 software, affinity fitting was performed, and EC50 values ​​were obtained. The results are shown in Figure 3, and B2E2-Fc had a significant inhibitory effect on the binding of human ANG2 to its receptor TIE2.

[0050] Example 7: Evaluation of the acid and thermal stability of anti-ANG2 double-chain single-domain antibody-Fc B2-E2-Fc was evaluated according to the following methods for evaluating acid and thermal stability. During protein A affinity chromatography of the anti-ANG2 heavy chain single-domain antibody-Fc molecule, in the acid elution step (using citrate buffer at pH 3.5), the eluted anti-ANG2 heavy chain single-domain antibody-Fc solution was not neutralized but held in the buffer for a certain period of time. After sampling at 30 min, 1 / 10 volume of 1 M Tris-HCl (pH 8.0) was added for neutralization, and the sample was subjected to HPLC-SEC detection. After treatment at pH 3.5 for 30 min, no aggregation or degradation was observed in the antibody molecule, and the change in purity was less than 4%, indicating that it can maintain stability in an acidic environment. Simultaneously, after incubation in a 40°C incubator for 14 days, HPLC-SEC detection of the sample was performed, and no aggregation or degradation was observed, and the change in purity was less than 4%, indicating that it can maintain stability in a 40°C environment. As shown in the results in Tables 6 and 7, B2-E2-Fc exhibited good acid and thermal stability.

[0051] [Table 7] [Table 8] Example 8. Construction and use of bispecific antibodies (1) Construction of bispecific antibodies According to the bispecific antibody amino acid sequences shown in Table 8, the DNA code was reverse-translated and DNA fragments were synthesized, constructed in the expression vector pcDNA3.1, and expressed by transient transfecting 293 or CHO cells. The supernatant was harvested and protein purified to obtain bispecific antibodies Y400C and Y400E with a purity of 95% or higher. The specific vector construction, transient transfection, and protein purification methods were described in the examples above. The structure of the bispecific antibody included a light chain and a heavy chain, of which the light-heavy chain paired to form an interchain disulfide bond, and the two heavy chains paired to form an interchain disulfide bond. The heavy chain was (VH)-(CH1)-(hinge region)-(Fc)-(linked peptide)-(VHH), and the light chain was (VL)-(light chain constant region). VH and VL were the heavy chain and light chain variable region of the anti-VEGF antibody, and VHH was the variable region of the anti-ANG2 heavy chain single-domain antibody.

[0052] [Table 9]

[0053] (2) Consideration of the thermal stability of bispecific antibodies The bispecific antibodies were treated in a 40°C incubator for 14 or 28 days, after which the samples were subjected to HPLC-SEC detection. The results showed that the antibody molecules of the present invention did not exhibit aggregation or degradation after 28 days of treatment at 40°C, and the change in purity was less than 4%, indicating that they can maintain stability in a 40°C environment, as shown in Table 9.

[0054] [Table 10]

[0055] As the results show, both the biantibody molecules Y400C and Y400E exhibited good thermal stability.

[0056] (3) Binding of bispecific antibody to hANG2 The test antibody is captured using the Sensor Chip Protein A chip (GE, product number: 28995056), and the antigen human ANG2 protein (623-AN-025 / CF, R&D) is detected as an analyte, with kinetic and affinity data for its binding to the test sample being obtained. The detection starting concentration for antigen-test binding was 10 nM. Based on this, a 2x gradient dilution was performed, resulting in antigen dilution concentrations of 10 nM, 5 nM, 2.5 nM, 1.25 nM, and 0.625 nM, respectively. Antigen injection was started sequentially from low to high concentrations, with one negative control (i.e., 1 × HBS-EP + buffer) and one repeat concentration (generally the lowest concentration being repeated). A start-up (1 × HBS-EP + buffer) rinse was performed at least three times to balance the system before injection. The binding and dissociation tendencies between the antigen and test were detected. After dissociation was complete, a regeneration reagent was injected to regenerate the tip, and detection of the next concentration was performed after tip regeneration was complete. After detection was complete, data fitting was performed using the 1:1 Binding fitting method in the data analysis software (Biacore T200 Evaluation Software), and the test results are shown in Table 10.

[0057] [Table 11]

[0058] As can be seen from the detection results of affinity with human ANG2, the bispecific antibody showed strong affinity for human ANG2.

[0059] (4) Inhibition of hANG2 binding to HUVEC cells by bispecific antibodies HUVEC cells (H-003, Allcells) in good growth condition were prepared as a single-cell suspension. After counting the cells, 10 5Cells were seeded in 96-well plates at a concentration of 10 μg / ml per well. The final concentration of human ANG2 protein (623-AN-025 / CF, R&D) was fixed at 10 μg / ml. Each well was then fitted with the antibody to be detected according to the experimental design. The maximum antibody concentration was 1000 nM, and seven concentration gradients were established by sequential 5-fold dilutions. Simultaneously, a blank control and a positive control RG7716 (ATAD00534, Atagenix) were prepared. After incubating the cells at room temperature for 30 minutes, the cells in each well were washed three times and resuspended. Fluorescently labeled secondary antibody PE anti-His (362603, Biolegend) was added, and the cells were incubated at room temperature in the dark for 30 minutes. The cells in each well were then washed three times, resuspended, and detected using a flow cytometry instrument. As shown in Figure 4, bispecific antibodies Y400C and Y400E exhibited superior blocking activity against ANG2 and HUVEC cells compared to the control-positive antibody.

[0060] [Table 12]

[0061] (5) Bispecific antibodies do not inhibit the binding of hANG1 to HUVEC cells. In the biological functions related to the ANG1 / ANG2-TIE2 signaling pathway, preserving ANG1 activity is beneficial for some anti-angiogenic therapies. Therefore, the antibody of the present invention specifically binds to ANG2 but not to ANG1, and simultaneously inhibits the binding of ANG2 to its receptor TIE2 but not to ANG1 to its receptor TIE2. The antibody of the present invention is particularly useful in inhibiting the angiogenic activity of ANG2 and in treating diseases and conditions caused by or related to the angiogenic process.

[0062] HUVEC cells (H-003, Allcells) in good growth condition were prepared as single-cell suspensions. After cell counting, 10 5Cells were seeded in 96-well plates at a concentration of 10 μg / ml per well. The final concentration of human ANG1 protein (623-AN / CF, R&D) was fixed at 10 μg / ml. Each well was then mixed with the antibody to be detected according to the experimental design. The maximum antibody concentration was 1000 nM, and seven concentration gradients were established by sequential 5-fold dilutions. A blank control and a positive control hTIE2-Fc (Novoprotein, CW98) were also prepared. After incubating the cells at room temperature for 30 minutes, the cells in each well were washed three times and resuspended. Fluorescently labeled secondary antibody PE anti-His (362603, Biolegend) was added, and the cells were incubated at room temperature in the dark for 30 minutes. The cells in each well were then washed three times, resuspended, and detected using a flow cytometry instrument. As shown in Figure 5, the bispecific antibodies Y400C and Y400E did not exhibit binding blocking activity against ANG1 and HUVEC cells.

[0063] (6) Inhibition of hANG2-dependent AKT phosphorylation in HUVEC cells by bispecific antibodies HUVEC cells spontaneously express the ANG2 protein receptor on their surface, and when ANG2 binds to the receptor on the cell surface, the phosphorylation reaction of AKT is activated. The Phospho-AKT analysis kit (64AKSPE1-1, Cisbio) contained two types of labeled antibodies: one with a donor fluorophore and one with a receptor. The first antibody was selected for its specific binding to the phosphorylation motif on the protein, and the second antibody was selected because its ability to recognize the protein is independent of its phosphorylation state. Protein phosphorylation involves the labeling antibody in the formation of an immune complex, and the donor fluorophore comes into close proximity to the receptor, producing a signal. In this experiment, HUVEC cells were co-incubated with sequentially diluted samples and fixed concentrations of ANG2. The degree of AKT phosphorylation in HUVEC cells was used to evaluate ANG2 functional activity and the inhibitory effect of the sample on ANG2 functional activity. Well-developed HUVEC cells (70013502, ATCC) were prepared as single-cell suspensions. After cell counting, 10 5Cells were seeded in 96-well plates at a concentration of 10 μg / ml per well. The final concentration of human ANG2 protein (623-AN-025 / CF, R&D) was fixed at 10 μg / ml. Each well was then mixed with the antibody to be detected according to the experimental design. The maximum antibody concentration was 1000 nM, and eight concentration gradients were established by sequential 5-fold dilutions. A blank control and a positive control RG7716 (ATAD00534, Atagenix) were also prepared. After incubating the cells at 37°C for 10 minutes, the supernatant from the wells was removed, lysate was added, and the cells were incubated with shaking at room temperature for 30 minutes. The cell lysates were then aspirated into a 384-well plate, detection reagents were added, and the cells were incubated in the dark for 4 hours. Detection was performed using PHERAstar. As shown in Figure 6, the bispecific antibodies Y400C and Y400E exhibited superior inhibitory activity against AKT phosphorylation in hANG2-dependent HUVEC cells compared to the control-positive antibody.

[0064] [Table 13]

[0065] All documents referenced in this invention are cited by reference in this application, just as each document is cited independently by reference. Furthermore, after reviewing the above teachings of this invention, those skilled in the art should understand that various changes or modifications can be made to the invention, and that these equivalent forms remain within the scope of the claims of this application.

Claims

1. An anti-ANG2 heavy chain single-domain antibody, wherein the heavy chain variable region includes HCDR1 shown in SEQ ID NO: 40, HCDR2 shown in SEQ ID NO: 41, and HCDR3 shown in SEQ ID NO:

42.

2. The anti-ANG2 heavy chain single-domain antibody according to claim 1, wherein the ANG2 is selected from human ANG2 or monkey ANG2.

3. The anti-ANG2 heavy chain single-domain antibody according to claim 1, wherein the heavy chain variable region includes the sequence shown in SEQ ID NO: 4, or is composed of the above sequence.

4. A recombinant protein comprising an anti-ANG2 heavy chain single-domain antibody and an Fc fragment as described in claim 1, wherein the anti-ANG2 heavy chain single-domain antibody and the Fc fragment are linked by a linking peptide, the linking peptide is GGGGGSGGGGGGGS (SEQ ID NO: 47), and the Fc fragment is a human IgG1 heavy chain constant region.

5. The recombinant protein according to claim 4, wherein the Fc fragment is the Fc fragment shown in SEQ ID NO:

61.

6. The recombinant protein according to claim 4, wherein the recombinant protein includes the sequence shown in SEQ ID NO: 14, or is composed of the above sequence.

7. A multispecific antibody comprising the anti-ANG double-chain single-domain antibody described in claim 1.

8. A bispecific antibody, wherein the structure of the bispecific antibody comprises a light chain and a heavy chain, wherein the light-heavy chain pairs to form an interchain disulfide bond, and the two heavy chains pair to form an interchain disulfide bond, wherein the heavy chain is (VH)-(CH1)-(hinge region)-(Fc)-(linked peptide)-(VHH), the light chain is (VL)-(light chain constant region), VHH is the anti-ANG2 heavy chain single-domain antibody, VH and VL are the heavy chain and light chain variable region of the second antibody, and the antigen targeted by the second antibody is selected from immune cell surface antigens, tumor antigens, viruses, bacteria, endotoxins, cytokines, or combinations thereof, as described in claim 7.

9. The multispecific antibody according to claim 8, wherein the second antibody is an anti-VEGF antibody and, according to the IMGT numbering system, comprises HCDR1 shown in SEQ ID NO: 50, HCDR2 shown in SEQ ID NO: 51, and HCDR3 shown in SEQ ID NO: 53, and also comprises LCDR1 shown in SEQ ID NO: 54, LCDR2 shown in SEQ ID NO: 58, and LCDR3 shown in SEQ ID NO:

59.

10. The multispecific antibody according to claim 9, wherein the heavy chain variable region of the anti-VEGF antibody includes or is composed of the sequence shown in SEQ ID NO: 24, and the light chain variable region of the anti-VEGF antibody includes or is composed of the sequence shown in SEQ ID NO:

27.

11. The multispecific antibody according to any one of claims 8 to 10, wherein the sequence of CH1 is shown in SEQ ID NO: 25, the sequence of the hinge region is shown in SEQ ID NO: 26, the sequence of Fc is shown in SEQ ID NO: 29, the sequence of the linked peptide is shown in SEQ ID NO: 30 or 47, and the sequence of the light chain constant region is shown in SEQ ID NO:

28.

12. A polynucleotide encoding an anti-ANG double-chain single-domain antibody according to any one of claims 1 to 3, a recombinant protein according to claim 4, or a multispecific antibody according to any one of claims 7 to 11.

13. A vector comprising the polynucleotide described in claim 12.

14. A host cell comprising the vector according to claim 13.

15. A coupling product comprising an anti-ANG2 heavy chain single-domain antibody, recombinant protein, or multispecific antibody, and a coupling moiety, wherein the coupling moiety is a purified tag, a detectable label, a drug, a toxin, a cytokine, an enzyme, or a combination thereof, wherein the recombinant protein comprises the anti-ANG2 heavy chain single-domain antibody described in claim 1, and the multispecific antibody is a bispecific antibody comprising the anti-ANG2 heavy chain single-domain antibody described in claim 1. A coupling story.

16. The coupling product according to claim 15, wherein the coupling portion is a radioactive isotope, a fluorescent substance, a chemiluminescent substance, a colored substance, a chemotherapeutic agent, a biotoxin, polyethylene glycol, or an enzyme.

17. A kit comprising (1) an anti-ANG2 heavy chain single-domain antibody according to any one of claims 1 to 3, a recombinant protein according to claim 4, a multispecific antibody according to any one of claims 7 to 11, or a coupling product according to claim 15, and (2) an antibody against another antigen or an antigen-binding fragment thereof, and / or a cytotoxic agent, and / or a chemotherapeutic agent, and a selectable instruction manual.

18. A pharmaceutical composition comprising an anti-ANG2 heavy-chain single-domain antibody according to any one of claims 1 to 3, a recombinant protein according to claim 4, a multispecific antibody according to any one of claims 7 to 11, or a coupling product according to claim 15, wherein the pharmaceutical composition further optionally comprises a pharmaceutically acceptable carrier and / or excipient, and the pharmaceutical composition is in a form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection, or intrafocal injection.

19. Use of the anti-ANG2 heavy chain single-domain antibody according to any one of claims 1 to 3, the recombinant protein according to claim 4, the multispecific antibody according to any one of claims 7 to 11, or the coupling product according to claim 15 in the preparation of a pharmaceutical product for inhibiting the angiogenesis-promoting activity of ANG2 and for preventing and / or treating diseases caused by or related to the angiogenesis process.

20. The use according to claim 19, wherein the disease caused by or related to the angiogenesis process is retinal neovascular disease, rheumatoid arthritis, and psoriasis.

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