Anti-ANG2 antibodies, methods for their preparation 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, specifically to an antibody against ANG2 (angiopoietin-2, human angiogenesis 2), its preparation method and use. [Background technology]

[0002] Antibodies against ANG2 can inhibit the formation and leakage of new blood vessels and reduce the occurrence of inflammatory responses. ANG / Tie2 is an important signal pathway that regulates angiogenesis. ANG1 promotes the phosphorylation of endothelial cell receptor Tie2, which attracts vascular smooth muscle cells, pericytes and other perivascular cells to surround and support endothelial cells, promotes vascular remodeling, maintains vascular integrity and regulates vascular function. ANG1 can protect blood vessels from lesions and maintain vascular morphology, but ANG2 promotes vascular leakage, causing hypotension and abnormal vascular structure. Although there are many ANG2 antibodies in clinical trials under corporate development, no antibody drugs against ANG2 have been launched on the market at present. Summary of the Invention [Problem 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 treating diseases and conditions caused by or related to the angiogenesis process, such as neovascular disease of the fundus, rheumatoid arthritis, and psoriasis. In addition, angiogenesis is very important for tumor growth and maintenance, and a monoclonal antibody that inhibits ANG2 can also act in the treatment of cancer. The present invention further provides a bispecific antibody against ANG2 and VEGF. The antibody of the present invention can bind to human ANG2, rabbit ANG2, and monkey ANG2. [Means for solving the problem]

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

[0005] 1. An anti-ANG2 heavy chain single domain antibody comprising HCDR1, HCDR2 and HCDR3 contained in a heavy chain variable region shown in any one of SEQ ID NOs: 1 to 10, or a mutant thereof, Preferably, the HCDR1, HCDR2 and HCDR3 are selected from the group consisting of the following according to the IMGT numbering system: (1) HCDR1 as set forth in SEQ ID NO:21, HCDR2 as set forth in SEQ ID NO:22, and HCDR3 as set forth in SEQ ID NO:23; (2) HCDR1 as set forth in SEQ ID NO: 34, HCDR2 as set forth in SEQ ID NO: 35, and HCDR3 as set forth in SEQ ID NO: 36; (3) HCDR1 as shown in SEQ ID NO: 37, HCDR2 as shown in SEQ ID NO: 38, and HCDR3 as shown in SEQ ID NO: 39; (4) HCDR1 shown in SEQ ID NO:40, HCDR2 shown in SEQ ID NO:41, and HCDR3 shown in SEQ ID NO:42, or variants of 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 the variants have one or more (preferably one, two or three) conservative 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, respectively, and retain binding affinity to 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 amino acids at positions 5 to 8 and 10 to 11 in CDR1 shown in SEQ ID NO: 40, the amino acids at positions 2 to 5 and 7 in CDR2 shown in SEQ ID NO: 41, and the amino acids at positions 1 and 3 to 8 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 variant of HCDR1 shown in SEQ ID NO:40, the first glycine G may be replaced by an amino acid selected from the group consisting of Ala, Val, Leu and Ile, the second phenylalanine F by Tyr, the third proline P by an amino acid selected from the group consisting of Trp and His, the fourth Ser by Thr, the ninth Ser by Thr and the twelfth Gln by Asn, For the variant of HCDR2 shown in SEQ ID NO:41, the first Ile may be replaced by an amino acid selected from the group consisting of Ala, Val, Leu and Gly, the sixth Leu by Ile and the eighth Lys by Arg; In the variant of HCDR3 shown in SEQ ID NO:42, the second Val may be replaced by an amino acid selected from the group consisting of Ala, Gly, Leu and Ile, and the ninth Asp may be replaced by Glu; (5) HCDR1 as shown in SEQ ID NO: 43, HCDR2 as shown in SEQ ID NO: 44, and HCDR3 as shown in SEQ ID NO: 45; (6) HCDR1 as shown in SEQ ID NO: 46, HCDR2 as shown in SEQ ID NO: 44, and HCDR3 as shown in SEQ ID NO: 48; (7) HCDR1 as shown in SEQ ID NO: 49, HCDR2 as shown in SEQ ID NO: 44, and HCDR3 as shown in SEQ ID NO: 48; (8) HCDR1 as shown in SEQ ID NO: 52, HCDR2 as shown in SEQ ID NO: 44, and HCDR3 as shown in SEQ ID NO: 48; (9) HCDR1 shown in SEQ ID NO:55, HCDR2 shown in SEQ ID NO:56, and HCDR3 shown in SEQ ID NO:57.

[0006] 2. The anti-ANG2 heavy chain single domain antibody according to 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 according to item 1 or 2, comprising a heavy chain variable region, the heavy chain variable region comprising or consisting of a sequence set forth in any one of SEQ ID NOs:1 to 10, or a sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the sequence set forth in any one of SEQ ID NOs:1 to 10 and having ANG2 binding activity.

[0008] 4. A recombinant protein comprising the anti-ANG2 heavy chain single domain antibody according to any one of items 1 to 3, preferably the recombinant protein further comprising a biologically active substance, such as a toxin having enzymatic activity or an active fragment thereof (e.g., abrin, ricin A, Pseudomonas exotoxin or diphtheria toxin), tumor necrosis factor or interferon (e.g., IFN-γ), a biological response regulator (e.g., lymphokine, IL-2, IL-2-6, IL-10, particulate GM-CSF), and / or an Fc fragment (e.g., an Fc region derived from IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM, e.g., an Fc region derived from human, rabbit or monkey IgG, IgA1, IgA2, IgD, IgE or IgM), preferably the anti-ANG2 heavy chain single domain antibody and the Fc fragment are linked by a linking peptide, preferably the linking peptide is represented by the formula: GGGGS(SEQ ID NO: 15), Preferably, the Fc fragment is a human IgG1 heavy chain constant region, more preferably an Fc fragment shown in GenBank No.AK303185.1 or SEQ ID NO:29, and more preferably, the recombinant protein comprises or consists of a sequence shown in SEQ ID NO:11 to 20.

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

[0010] 6. The multispecific antibody according to item 5, wherein VHH is an anti-ANG2 heavy chain single domain antibody according to any one of items 1 to 3, and VH and VL are heavy and light chain variable regions of a second type of antibody, preferably the antigen directed by said second type of antibody is selected from an immune cell surface antigen, a tumor antigen, a virus, a bacterium, an endotoxin, a cytokine, or a combination thereof, more preferably PD-L1, PD-1, VEGFA, IL-10, IL-10R, BCMA. , 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 the antigen directed by the second type of antibody is VEGF, more preferably the second antibody is selected from anti-VEGF antibodies, SEQ ID NO: 1 and a light chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:24 (preferably comprising HCDR1 as shown in SEQ ID NO:50, HCDR2 as shown in SEQ ID NO:51 and HCDR3 as shown in SEQ ID NO:53, according to the IMGT numbering system); and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:27 (preferably comprising LCDR1 as shown in SEQ ID NO:54, LCDR2 as shown in SEQ ID NO:58 and LCDR3 as shown in SEQ ID NO:59, according to the IMGT numbering system). More preferably, the heavy chain variable region of the anti-VEGF antibody comprises the sequence shown in SEQ ID NO:24 or SEQ ID and the light chain variable region of the anti-VEGF antibody comprises or consists of a sequence set forth in SEQ ID NO:27 or a sequence set forth in SEQ ID NO:27 having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the sequence set forth in SEQ ID NO:27.

[0011] 7. A multispecific antibody according to 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 connecting 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, preferably said multispecific antibody comprises (1) a heavy chain having a sequence comprising or consisting of SEQ ID NOs: 24, 25, 26, 29, 30, and 4, and a light chain having a sequence comprising or consisting of SEQ ID NOs: 27 and 28, Y400C; (2) a heavy chain having a heavy chain sequence comprising or consisting of SEQ ID NOs: 24, 25, 26, 29, 47, and 4, and a light chain having a light chain sequence comprising or consisting of SEQ ID NOs: 27 and 28; and Y400E comprising or consisting of the above sequence.

[0012] 8. A polynucleotide encoding the anti-ANG2 heavy chain single domain antibody according to any one of items 1 to 3, the recombinant protein according to item 4, or the multispecific antibody according to any one of items 5 to 7.

[0013] 9. A vector comprising the polynucleotide according to item 8.

[0014] 10. A host cell comprising the vector according to item 9.

[0015] 11. A coupling product comprising an anti-ANG2 heavy chain single domain antibody according to any one of items 1 to 3, a recombinant protein according to item 4, or a multispecific antibody according to any one of items 5 to 7, and a coupling moiety, wherein the coupling moiety is a purification 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 biological toxin, polyethylene glycol, or an enzyme.

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

[0017] 13. A pharmaceutical composition comprising the anti-ANG2 heavy chain single domain antibody according to any one of clauses 1 to 3, the recombinant protein according to clause 4, the multispecific antibody according to any one of clauses 5 to 7, or the coupling product according to clause 11, optionally further comprising a pharma- ceutically acceptable vector and / or excipient, and preferably, the pharmaceutical composition is in a form suitable for administration by subcutaneous, intradermal, intravenous, intramuscular or intralesional 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 coupling product according to item 11 in inhibiting the angiogenic activity of ANG2, and in preventing and / or treating a disease caused by an angiogenic process or a disease associated therewith, such as neovascular disease of the fundus, rheumatoid arthritis and psoriasis, and / or tumors, or in the preparation of a medicament for inhibiting the angiogenic activity of ANG2 and preventing and / or treating a disease caused by an angiogenic process or a disease associated therewith, such as neovascular disease of the fundus, rheumatoid arthritis and psoriasis, and / or tumors.

[0019] It should be understood that within the scope of the present invention, any of the above technical features of the present invention and the technical features specifically described hereinafter (e.g., in the Examples) can be combined with each other to form new or preferred technical aspects, which will not be described individually here due to space limitations.

[0020] The terms of the present invention have the ordinary meanings as understood by those skilled in the art. When a term has two or more definitions, as used and / or accepted within the art, the definition of the term used herein is intended 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 the antigen. If the sequences of the antibody heavy and light chain variable regions are known, there are currently several methods for determining the antibody CDR region, including Kabat, IMGT, Chothia and AbM numbering systems. However, the application of various definitions for the CDR of an antibody or its variants is within the scope of the terms defined and used herein. Given the amino acid sequence of the variable region of the antibody, a person skilled in the art can usually determine a particular 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 a VHH antibody is a variable domain of an 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). A VHH domain specifically binds to an epitope without the need for another antigen-binding domain. A VHH domain is a small, stable and highly efficient antigen recognition unit consisting of a single immunoglobulin domain.

[0023] The heavy chain single domain antibodies of the present invention can be conjugated 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, ricin A, Pseudomonas exotoxin or diphtheria toxin), tumor necrosis factor or interferon (e.g., IFN-γ), biological response regulators (e.g., lymphokines, IL-2, IL-2, IL2-6, IL-10, particulate GM-CSF), Fc fragments. The Fc fragment contained in the recombinant protein of the present invention can enable it to form dimers and at the same time extend the half-life of the recombinant protein in the body. In some embodiments, the Fc fragments usable in the present invention can be derived from different subtypes of immunoglobulins, such as IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM.

[0024] The antibody of the present invention may be (i) a polypeptide in which one or more conservative or non-conserved amino acid residues (preferably conservative amino acid residues) have been substituted, or (ii) a polypeptide in which one or more amino acid residues have a substitution, or (iii) a polypeptide formed by fusing the mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, e.g., polyethylene glycol), or (iv) a polypeptide formed by fusing an additional amino acid sequence to the polypeptide sequence (e.g., a leader sequence, a secretory sequence, a sequence for purifying the polypeptide, a fibrinogen sequence, or a fusion protein formed with a 6His tag). Based on the teachings of this document, these fragments, derivatives, and analogs are within the scope of those skilled in the art.

[0025] Conservative Substitutions A "conservative amino acid substitution" refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, non-essential amino acid residues in immunoglobulin polypeptides are preferably replaced with other amino acid residues from the same side chain family. In some other embodiments, the string of amino acids may be replaced with a string of structurally similar amino acids, the latter differing in order and / or side chain family composition.

[0026] The table below provides non-limiting examples of conservative amino acid substitutions, in which a similarity score of 0 or higher indicates a conservative 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 residue in the following groups (a) to (e) is replaced with another amino acid residue in the same group, the groups being: (a) small aliphatic, non-polar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, non-polar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

[0029] Particularly preferred conservative substitutions are: Ala to Gly or Ser, Arg to Lys, Asn to Gln or His, Asp to Glu, Cys to Ser, Gln to Asn, Glu to Asp, Gly to Ala or Pro, His to Asn or Gln, Ile to Leu or Val, Leu to Ile or Val, Lys to Arg, Gln to Glu, Met to Leu, Tyr or Ile, Phe to Met, Leu or Tyr, Ser to Thr, Thr to Ser, Trp to Tyr, Tyr to Trp, and / or Phe to 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 are all neutral amino acids that contain one amino group and one carboxyl group. Serine S and threonine T both belong to the hydroxyamino acids. Cysteine ​​C and methionine M both belong to the sulfur-containing amino acids. Asparagine N and glutamine Q both belong to the amide group-containing amino acids. Aspartic acid D and glutamic acid E both belong to the 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, and therefore have similar structural features and properties and can be conservatively substituted for each other. Effect of the Invention

[0032] In some embodiments, the antibodies according to the invention can be conjugated to a therapeutic agent (e.g., a chemotherapeutic agent, e.g., cisplatin, carboplatin), a drug precursor, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, a drug, or PEG. The antibodies of the invention can be linked or fused to a therapeutic agent, which can include a detectable label, e.g., a radioactive label, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent, can be a drug or toxin, an ultrasound enhancing agent, a non-radioactive label, combinations thereof, and other such moieties known in the art. [Brief description of the drawings]

[0033] [Figure 1] Biological activity of anti-ANG2 heavy chain single domain antibody-Fc binding to human ANG2. [Diagram 2] Biological activity of anti-ANG2 heavy chain single domain antibody-Fc binding to monkey ANG2. [Diagram 3]Biological activity of anti-ANG2 heavy chain single domain antibody-Fc to inhibit the binding of human ANG2 to its receptor hTIE2. [Figure 4] Biological activity of antibodies inhibiting the binding of human ANG2 to HUVEC. [Diagram 5] Biological activity of antibodies inhibiting the binding of human ANG1 to HUVEC. [Figure 6] Inhibition of human ANG2-dependent AKT phosphorylation in HUVEC cells by antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0034] The present invention will be described in detail below with reference to the following examples. As can be understood by those skilled in the art, the following examples are used for illustrative purposes only. The spirit and scope of the present invention are limited by the claims. The methods used in the following examples are conventional methods unless otherwise specified, and the reagents used are commercially available reagents unless otherwise specified.

[0035] Example 1: Screening of heavy chain single domain antibodies against ANG2 The recombinant human ANG2 protein corresponding to amino acids Lys275-Phe496 of human ANG2 is hereinafter abbreviated as hANG2, the sequence of which is set forth in SEQ ID NO:32, and the monkey ANG2 protein is hereinafter abbreviated as cynoANG2, the sequence of which is set forth in SEQ ID NO:33. The amino acid sequence information of exemplary ANG2 ECD molecules is provided in Table 1.

[0036] [Table 2]

[0037] 1.1 Construction of heavy chain single domain antibody phage library Two alpacas were selected for antigen immunization, and after four immunizations with human ANG2 protein, lymphocytes were extracted from 100 ml of peripheral blood of the alpacas, total RNA was extracted using RNAiso Plus reagent (TAKARA, 9109), and the extracted RNA was reverse transcribed into cDNA using PrimeScript II kit (TAKARA, 6210A). By PCR amplification, a 750 bp long nucleic acid fragment of the heavy chain antibody variable region and constant region CH2 was first amplified, and 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 each enzymatically digested with SfiI, and the target fragment was recovered after overnight at 50°C, and ligated according to the ligation molar ratio of vector:fragment = 1:3. The ligation products were electrotransformed into E. coli competent cells TG1, and 10 electrotransformations were performed for each alpaca ligation product. The library sizes were calculated by plating gradient dilution, and the phage library sizes of the two alpacas were 1.21 × 10 9 and 1×10 9 96 clones were randomly picked from the titration plate and identified, revealing a 100% insertion rate.

[0038] 1.2 Panning of heavy chain single domain antibodies against ANG2 Plates were coated with hANG2 protein at 10 μg / well and incubated overnight at 4° C. The next day, the plates were blocked with 1% BSA at room temperature for 2 hours, and then 100 μl of phages (2×10 9pfu / well, derived from the heavy chain single domain antibody phage library constructed in 1.1) was added and incubated at 37°C for 1 h. Then, unbound phages were washed off by washing five times with PBST (PBS containing 0.05% Tween 20). Finally, phages that specifically bound to hANG2 were eluted using glycine-HCl (200 mM), and infected with logarithmic-phase growing E. coli TG1 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 infected with E. coli TG1 and plated, and monoclones were selected from the plate and sequenced. According to the sequence alignment results, the protein sequences of individual clones were analyzed, and clones with different CDR1, CDR2, and CDR3 sequences were considered as different antibody strains, and finally, a total of 10 different antibody strains were obtained.

[0039] [Table 3]

[0040] Example 2: Preliminary evaluation and identification of heavy chain single domain antibodies against ANG2 1. Expression of heavy chain single domain antibody in host bacteria E. coli A single colony was selected from the 10 strains of heavy chain single domain antibodies obtained and inoculated into the TG1 host bacteria, and cultured overnight. The next day, the overnight culture was transferred to the inoculation and amplified, induced with 0.5 mM IPTG, and cultured overnight at 37°C in a shaker. The next day, the mixture was centrifuged and the supernatant was collected for detection.

[0041] 2. Affinity detection of 10 heavy chain single domain antibodies by ELISA The enzyme-labeled plate was coated with 2 μl / well of hANG2 protein solution and BSA, left at 4°C overnight, the supernatant was discarded, 300 μl of blocking solution (PBS containing 3% BSA) was added to each well, and blocked at 37°C for 2 hours. The supernatant collected by centrifugation was added to the enzyme-labeled plate wells at 200 μl / well, and incubated at room temperature for 2 hours. The supernatant was discarded, and 200 μl / well of PBST (PBS contains 0.1% The plate was washed three times with PBST (containing Tween 20), diluted HRP-labeled anti-VHH secondary antibody (Genscript, A01861) was added, the secondary antibody was used after dilution at 1:10000, the diluent was 1% BSA in PBS, the volume was 100μl / well, incubated at room temperature for 1 hour, washed five times with PBST (200μl / well), TMB color development solution (BD, 55214) was added at 100μl / well, and developed at 37 degrees for 8 minutes. 2M HCl stop solution was added at 100μl / well, and the plate was read at 450nm in a microplate reader within 30 minutes after adding the stop solution. 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 coding DNA sequence corresponding to the monoclonal antibody heavy chain variable region (SEQ ID NO: 1-10) of the PCR clone is linked to the coding DNA corresponding to the human IgG1 heavy chain constant region (GenBank No. AK303185.1) to construct the heavy chain antibody, to obtain the expression plasmid of anti-ANG2 heavy chain single domain antibody-Fc, the vector is generally pcDNA3.1(-) (purchased from Invitrogen) or other eukaryotic expression vector. The variable region and the constant region of the above-mentioned heavy chain antibody protein sequence are connected 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] TIFF2024534964000026.tif204161

[0045] Example 4. Expression and purification of anti-ANG2 heavy chain single domain antibody-Fc Plasmid extraction was performed using an endotoxin-free large-scale extraction plasmid kit (Qiagen, product number 12391), and the specific operation was performed according to the specifications provided by the manufacturer. CHO-S cell culture was cultured in CD CHO medium (Gibco, product number 10743-029) at 37°C in a cell incubator with 5% CO2 according to the instructions provided by the manufacturer, and after the cells were prepared, the plasmid containing the anti-ANG2 heavy chain single domain antibody-Fc sequence was co-transfected into the CHO-S cells to express the antibody-Fc. The day after transfection, the culture temperature was lowered to 32°C, and 3.5% 2xEFC+ (Gibco, product number A2503105) was supplemented every day. After culturing for 14 days, the expression supernatant was harvested by centrifugation at 800xg, and then filtered through a 0.22μm filter membrane. The 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 the extinction coefficients corresponding to various proteins. Purity and homogeneity of antibody-Fc were assessed by SDS-PAGE and SE-HPLC. Secondary purification was performed using ion exchange and SEC on 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 This example evaluated the affinity of 10 anti-ANG2 heavy chain single domain antibodies-Fc using an ELISA detection method.

[0047] ANG2 protein solution was applied to 2 μl / well of enzyme-labeled plate, and left at 4 degrees overnight. The supernatant was discarded, and 300 μl of blocking solution (PBS containing 3% BSA) was added to each well, and blocking was performed at 37 degrees for 2 hours. Anti-ANG2 heavy chain single domain antibody-Fc was gradient-diluted, and the dilution solution was PBS containing 1% BSA. For example, the initial concentration of the dilution was 500 nM, and it was diluted 10-fold, and diluted to 8 concentration gradients. Add diluted anti-ANG2 heavy chain single domain antibody-Fc to the enzyme-labeled plate wells at 200 μl per well, leave to stand at room temperature and incubate for 2 hours, discard the supernatant, wash 3 times with 200 μl / well PBST (PBS contains 0.1% Tween20), add diluted HRP-labeled anti-human Fc secondary antibody (SIGMA, A8667), the secondary antibody was used after dilution at 1:20000, the dilution solution was 1% BSA in PBS, the volume was 100 μl / well, incubate at room temperature for 1 hour, wash 5 times with 200 μl / well PBST, add TMB color development solution (BD, 55214) at 100 μl / well, develop at 37 degrees for 8 minutes. Add 2M HCl stop solution at 100 μl / well, and read at 450 nm on a microplate reader within 30 minutes after adding the stop solution. The data was analyzed using GraphPad Prism 6.0 software, affinity fitting was performed, and EC50 values ​​were obtained, and the results are shown in Figures 1 and 2 and Table 5. B2-E2-Fc had the highest affinity for human ANG2 and monkey ANG2 proteins.

[0048] [Table 6]

[0049] Example 6. Inhibition of binding of hANG2 to its receptor hTIE2 by anti-ANG2 heavy chain single domain antibody-Fc 96-well plates were treated with 100 μL of hTIE2-Fc (Novoprotein, CW98) prepared in PBS at 2 μg / mL and incubated at 4° C. overnight. The plates were then washed three times with PBS wash buffer containing 0.1% Tween-20, followed by blocking for 2 hours with 300 μL of PBS containing 1% BSA. 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, incubated at 37° C. for 1 hour, and then the antigen-antibody mixture was added to the 96-well plate and incubated at 37° C. for 1 hour. After washing with PBS wash 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, the diluent was 1% BSA in PBS, the volume was 100 μl / well, incubated at room temperature for 1 hour, washed 5 times with 200 μl / well PBST, TMB development solution (BD, 55214) was added at 100 μl / well, and developed at 37°C for 8 minutes. 2M HCl stop solution was added at 100 μl / well, and reading was performed at 450 nm on a microplate reader within 30 minutes after adding the stop solution. 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. B2E2-Fc had a significant inhibitory effect on the binding of human ANG2 to its receptor TIE2.

[0050] Example 7: Evaluation of acid and thermal stability of anti-ANG2 heavy chain single domain antibody-Fc B2-E2-Fc was evaluated according to the following methods for evaluating acid stability and thermal stability. During protein A affinity chromatography of anti-ANG2 heavy chain single domain antibody-Fc molecules, 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 was kept in the buffer for a certain period of time, sampled at 30 min, neutralized by adding 1 / 10 volume of 1M Tris-HCl (pH 8.0), and the sample was subjected to HPLC-SEC detection. After treatment at pH 3.5 for 30 min, the antibody molecule showed no aggregation or decomposition phenomenon and the purity change was less than 4%, indicating that it can maintain stability in an acidic environment. At the same time, after incubation in a 40°C incubator for 14 days, the sample was subjected to HPLC-SEC detection, indicating that it can maintain stability in a 40°C environment, showing that no aggregation or decomposition phenomenon was observed and the purity change was less than 4%. As shown in the results in Tables 6 and 7, B2-E2-Fc had good acid stability and heat 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, DNA codes were reverse-translated and DNA fragments were synthesized, which were then constructed into an expression vector pcDNA3.1, and expressed by transient transfection into 293 or CHO cells. The supernatant was harvested and protein purified to obtain bispecific antibodies Y400C and Y400E with a purity of 95% or more. Specific vector construction, transient transfection and protein purification methods were referred to in the above-mentioned Examples. The bispecific antibody structure includes a light chain and a heavy chain, in which the light-heavy chain is paired to form an interchain disulfide bond, and the two heavy chains are paired to form an interchain disulfide bond, in which the heavy chain is (VH)-(CH1)-(hinge region)-(Fc)-(connecting peptide)-(VHH), and the light chain is (VL)-(light chain constant region), where VH and VL are the heavy chain and light chain variable regions of an anti-VEGF antibody, and VHH is the variable region of an anti-ANG2 heavy chain single domain antibody.

[0052] [Table 9]

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

[0054] [Table 10]

[0055] As can be seen from the results, both the dual antibody molecules Y400C and Y400E had good thermal stability.

[0056] (3) Binding of bispecific antibodies to hANG2 The test antibody was captured using a Sensor Chip Protein A chip (GE, product number: 28995056), and the antigen human ANG2 protein (623-AN-025 / CF, R&D) was used as the analyte to detect the kinetics and affinity data of binding to the test substance. The starting concentration for the detection of the binding between the antigen and the test sample is 10nM, and based on this, a 2-fold gradient dilution is performed, that is, the concentrations of the antigen dilution are 10nM, 5nM, 2.5nM, 1.25nM, and 0.625nM, respectively, and the antigen is injected sequentially from low to high concentration. One negative control (i.e., 1×HBS-EP+buffer) and one repeat concentration (generally the lowest concentration is repeated) are set, and the start up (1×HBS-EP+buffer) rinse process is performed at least three times to balance the system before injection, and the binding and dissociation tendency between the antigen and the test sample is detected. After dissociation is completed, the regeneration reagent is injected, and the chip is regenerated. After the chip is regenerated, the next concentration is detected. After the detection is completed, the data is fitted by 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 results of detecting the affinity with human ANG2, the bispecific antibody had strong affinity for human ANG2.

[0059] (4) Inhibition of binding of hANG2 to HUVEC cells by bispecific antibodies HUVEC cells (H-003, Allcells) in good growth condition were prepared into a single cell suspension. After cell counting, 10 5The cells were seeded in a 96-well plate at 10 cells / well, and the final concentration of human ANG2 protein (623-AN-025 / CF, R&D) was fixed at 10 μg / ml. The antibody to be detected was added to each well according to the experimental design. The highest antibody concentration was 1000 nM, and the antibody was diluted 5-fold in sequence to set up a total of seven concentration gradients. At the same time, a blank control and a positive control RG7716 (ATAD00534, Atagenix) were set up, and the cells were incubated at room temperature for 30 min, after which the cells in each well were washed three times and resuspended, and then the fluorescently labeled secondary antibody PE anti-His (362603, Biolegend) was added, and incubated at room temperature in the dark for 30 min. The cells in each well were washed three times and resuspended, and then detected by a flow cytometry instrument. As shown in Figure 4, the bispecific antibodies Y400C and Y400E have better blocking activity against ANG2 and HUVEC cells than the control positive antibody.

[0060] [Table 12]

[0061] (5) The bispecific antibody does not inhibit the binding of hANG1 to HUVEC cells In the biological function related to the signal pathway of ANG1 / ANG2-TIE2, preserving ANG1 activity is beneficial for some anti-angiogenesis treatments.Therefore, the antibody of the present invention specifically binds to ANG2 but not to ANG1, and at the same time, only inhibits the binding of ANG2 to its receptor TIE2 but does not inhibit the binding of ANG1 to its receptor TIE2.The antibody of the present invention is particularly useful in inhibiting the angiogenesis-promoting activity of ANG2 and treating diseases and conditions caused by or related to the angiogenesis process.

[0062] HUVEC cells (H-003, Allcells) in good growth condition were prepared into a single cell suspension. After cell counting, 10 5The cells were seeded in a 96-well plate at 10 cells / well, and the final concentration of human ANG1 protein (623-AN / CF, R&D) was fixed at 10 μg / ml. The antibody to be detected was added to each well according to the experimental design. The highest antibody concentration was 1000 nM, and the antibody was diluted 5-fold to set up a total of seven concentration gradients. At the same time, a blank control and a positive control hTIE2-Fc (Novoprotein, CW98) were set up, and the cells were incubated at room temperature for 30 min, after which the cells in each well were washed three times and resuspended, and then the fluorescently labeled secondary antibody PE anti-His (362603, Biolegend) was added, and incubated at room temperature in the dark for 30 min. The cells in each well were washed three times and resuspended, and then detected by a flow cytometry instrument. As shown in Figure 5, the bispecific antibodies Y400C and Y400E had no binding blocking activity against ANG1 and HUVEC cells.

[0063] (6) Bispecific antibodies inhibit hANG2-dependent AKT phosphorylation in HUVEC cells The receptor for the ANG2 protein is naturally expressed on the surface of HUVEC cells, and the binding of ANG2 to the cell surface receptor activates the phosphorylation of AKT. The Phospho-AKT Assay Kit (64AKSPE1-1, Cisbio) contained two labeled antibodies, one with a donor fluorophore and one with a receptor. The first antibody was selected for its specific binding to a phosphorylation motif on the protein, and the second antibody was selected for its ability to recognize the protein independent of its phosphorylation state. Upon phosphorylation of the protein, the labeled antibody is involved in the formation of an immune complex, and the donor fluorophore is in close proximity to the receptor, resulting in a signal. In this experiment, HUVEC cells were co-incubated with serially diluted samples and a fixed concentration of ANG2. The degree of AKT phosphorylation in HUVEC cells was used to evaluate ANG2 functional activity and the inhibitory effect of the samples on ANG2 functional activity. HUVEC cells (70013502, ATCC) in good growth condition were prepared into a single cell suspension. After cell counting, 10 5The cells were seeded in a 96-well plate at 10 cells / well, and the final concentration of human ANG2 protein (623-AN-025 / CF, R&D) was fixed at 10 μg / ml. The antibody to be detected was added to each well according to the experimental design. The highest antibody concentration was 1000 nM, and the antibody was diluted 5-fold in sequence to set up a total of eight concentration gradients. At the same time, a blank control and a positive control RG7716 (ATAD00534, Atagenix) were set up, and the cells were incubated at 37 degrees for 10 min, after which the supernatant in the well was removed, and the lysate was added, followed by shaking at room temperature and incubation for 30 min. The cell lysate was sucked into a 384-well plate, and the detection reagent was added and incubated in the dark for 4 hours, and detection was performed using a PHERAstar. As shown in Figure 6, the bispecific antibodies Y400C and Y400E have a better inhibitory activity on hANG2-dependent AKT phosphorylation of HUVEC cells than the control positive antibody.

[0064] [Table 13]

[0065] All documents mentioned in this application are incorporated herein by reference as if each document was individually incorporated by reference. It should also be understood that after reading the above teachings of the present invention, one skilled in the art may make various changes or modifications to the present invention, and that equivalent forms thereof are also 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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