WO2023035226A1 - 抗ang2抗体及其制备方法和应用 - Google Patents

抗ang2抗体及其制备方法和应用 Download PDF

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WO2023035226A1
WO2023035226A1 PCT/CN2021/117697 CN2021117697W WO2023035226A1 WO 2023035226 A1 WO2023035226 A1 WO 2023035226A1 CN 2021117697 W CN2021117697 W CN 2021117697W WO 2023035226 A1 WO2023035226 A1 WO 2023035226A1
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seq
ang2
heavy chain
antibody
single domain
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English (en)
French (fr)
Inventor
石剑
方丽娟
严永祥
张敬
华珊
周鹏飞
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Shenzhen Kangzhe Pharmaceutical Development Co Ltd
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Shenzhen Kangzhe Pharmaceutical Development Co Ltd
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Priority to KR1020247010141A priority Critical patent/KR20240048555A/ko
Priority to IL311318A priority patent/IL311318A/en
Priority to CA3231417A priority patent/CA3231417A1/en
Priority to US18/690,330 priority patent/US20240425575A1/en
Priority to EP21956409.3A priority patent/EP4400512A4/en
Priority to JP2024515391A priority patent/JP2024534964A/ja
Priority to CN202180102284.3A priority patent/CN118251412A/zh
Priority to AU2021463580A priority patent/AU2021463580A1/en
Application filed by Shenzhen Kangzhe Pharmaceutical Development Co Ltd filed Critical Shenzhen Kangzhe Pharmaceutical Development Co Ltd
Priority to PCT/CN2021/117697 priority patent/WO2023035226A1/zh
Priority to MX2024002962A priority patent/MX2024002962A/es
Publication of WO2023035226A1 publication Critical patent/WO2023035226A1/zh
Priority to JOJO/P/2024/0051A priority patent/JOP20240051A1/ar
Anticipated expiration legal-status Critical
Priority to CONC2024/0003519A priority patent/CO2024003519A2/es
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    • C07K16/22—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6845—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a cytokine, e.g. growth factors, VEGF, TNF, a lymphokine or an interferon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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    • C07K2317/522—CH1 domain
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to the field of medicine, in particular to an antibody to ANG2 (angiopoietin-2, human angiogenesis 2) and a preparation method and application thereof.
  • ANG2 angiopoietin-2, human angiogenesis 2
  • Antibodies against ANG2 can inhibit the formation and leakage of new blood vessels, and reduce the occurrence of inflammation.
  • ANG/Tie2 is an important signaling pathway regulating angiogenesis.
  • ANG1 promotes phosphorylation of endothelial cell receptor Tie2, 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 pathological changes , and maintain blood vessel morphology, while ANG2 promotes vascular leakage, leading to hypotension and abnormal vascular structure.
  • ANG2 antibodies developed by many companies are undergoing clinical trials, there is currently no antibody drug against ANG2 on the market.
  • the present invention provides a specific monoclonal antibody against ANG2, which plays a role in inhibiting the pro-angiogenic activity of ANG2 and treating diseases and disorders caused by or related to angiogenic processes, such as fundus neovascular diseases, rheumatoid arthritis and psoriasis.
  • angiogenesis is critical for tumor growth and maintenance, and monoclonal antibodies that inhibit ANG2 may also have a role in the treatment of cancer.
  • the present invention also provides bispecific antibodies against ANG2 and VEGF.
  • the antibodies of the present invention are capable of binding to human ANG2, rabbit ANG2 and monkey ANG2.
  • the present invention relates to the following aspects:
  • An anti-ANG2 heavy chain single domain antibody comprising HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region shown in any one of SEQ ID NO: 1-10, or a variant thereof,
  • HCDR1, HCDR2 and HCDR3 selected from the group consisting of:
  • HCDR1 shown in SEQ ID NO:21 HCDR2 shown in SEQ ID NO:22 and HCDR3 shown in SEQ ID NO:23,
  • Variants of HCDR2 shown in and HCDR3 shown in SEQ ID NO:42, and combinations thereof, said variant being HCDR1 shown in SEQ ID NO:40, HCDR2 shown in SEQ ID NO:41 and SEQ ID NO HCDR3 shown in: 42 has one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferred substitution, insertion or deletion) and retains the amino acid sequence with ANG2 binding affinity compared with HCDR3 respectively,
  • the HCDR1 shown in SEQ ID NO:40, the HCDR2 shown in SEQ ID NO:41 and the HCDR3 shown in SEQ ID NO:42 wherein the 5th-8th positions in the CDR1 shown in SEQ ID NO:40 and The 10th-11th amino acid, the 2nd-5th amino acid in the CDR2 shown in SEQ ID NO:41 and the 7th, the 1st amino acid of CDR3 shown in SEQ ID NO:42, the 3rd-8th amino acid are selected from Amino acid X, said 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 Group,
  • the variant of HCDR1 shown in SEQ ID NO:40 wherein the glycine G at position 1 can be replaced by an amino acid selected from the group consisting of: Ala, Val, Leu and Ile; Amino acid F may be replaced by Tyr; Proline P at position 3 may be replaced by an amino acid selected from the group consisting of: Trp and His; Ser at position 4 may be replaced by Thr; Ser at position 9 may be Replaced by Thr; Gln at position 12 can be replaced by Asn;
  • Ile at position 1 can be replaced by an amino acid selected from the group consisting of: Ala, Val, leu and Gly; Leu at position 6 can be replaced by Ile; Lys at the 8th position can be replaced by Arg;
  • Val at position 2 can be replaced by an amino acid selected from the group consisting of: Ala, Gly, Leu and Ile; Asp at position 9 can be replaced by Glu ,
  • HCDR1 shown in SEQ ID NO:43 HCDR2 shown in SEQ ID NO:44 and HCDR3 shown in SEQ ID NO:45,
  • HCDR1 shown in SEQ ID NO:46 HCDR2 shown in SEQ ID NO:44 and HCDR3 shown in SEQ ID NO:48,
  • HCDR1 shown in SEQ ID NO:49 HCDR2 shown in SEQ ID NO:44 and HCDR3 shown in SEQ ID NO:48,
  • HCDR1 set forth in SEQ ID NO:52 HCDR2 set forth in SEQ ID NO:44
  • HCDR3 set forth in SEQ ID NO:48
  • the anti-ANG2 heavy chain single domain antibody described in item 1 or 2 which comprises a heavy chain variable region, and the heavy chain variable region comprises a sequence shown in any one of SEQ ID NO: 1-10 or with SEQ ID NO:
  • the sequence described in any one of ID NO: 1-10 has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98% or 99% homology and a sequence having ANG2 binding activity, or consists of it.
  • a recombinant protein which comprises the anti-ANG2 heavy chain single domain antibody described in any one of items 1-3, preferably, said recombinant protein further comprises a biologically active substance, such as a toxin with enzymatic activity or an active fragment thereof (such as abrin, ricin A, pseudomonas exotoxin or diphtheria toxin), tumor necrosis factor or interferon (such as IFN- ⁇ ), biological response modifiers (such as lymphokines, IL-2, IL- 2, IL2-6, IL-10, GM-CSF), and/or Fc fragments (such as Fc regions from IgG (such as IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM , such as IgG, IgA1, IgA2, IgD, IgE or IgM Fc region from humans, rabbits or monkeys), preferably, the anti-ANG2 heavy
  • a multispecific antibody preferably a bispecific antibody, comprising the anti-ANG2 heavy chain single domain antibody described in any one of items 1-3, preferably the structure of the bispecific antibody is: comprising a light chain and a heavy chain , where the light-heavy chains pair and form an interchain disulfide bond, and the two heavy chains pair and form an interchain disulfide bond, where the heavy chain is (VH)-(CH1)-(hinge region)-(Fc)-( Linker peptide)-(VHH), light chain is (VL)-(light chain constant region).
  • VHH is the anti-ANG2 single domain antibody described in any one of items 1-3, VH and VL are the heavy chain and light chain variable regions of the second antibody, 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 selected from: PD-L1, PD-1, VEGFA, IL -10, IL-10R, BCMA, VEGF, TGF- ⁇ , 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 second antibody is directed against an antigen of VEGF, more preferably the second antibody is selected from an anti-VEGF antibody comprising SEQ ID NO:24
  • Y400C which comprises a heavy chain and a light chain, or consists of it; wherein the sequence of the heavy chain comprises, or consists of, SEQ ID NO: 24, 25, 26, 29, 30 and 4; the sequence of the light chain comprises SEQ ID NO: 27 and 28, or consist of;
  • (2) Y400E which comprises a heavy chain and a light chain, or consists of it; wherein the sequence of the heavy chain comprises, or consists of, SEQ ID NO: 24, 25, 26, 29, 47 and 4; the sequence of the light chain comprises SEQ ID NO: 27 and 28, or consist thereof.
  • a vector comprising the polynucleotide according to item 8.
  • a host cell comprising the vector according to item 9.
  • a conjugate comprising the anti-ANG2 heavy chain single domain antibody described in any one of items 1-3, the recombinant protein described in item 4, or the multispecific antibody described in any one of items 5-7, And a coupling moiety, wherein the coupling moiety is a purification tag (such as a His tag), a detectable label, a drug, a toxin, a cytokine, an enzyme, or a combination thereof; preferably, the coupling moiety is a radioactive isotope , fluorescent substances, chemiluminescent substances, colored substances, chemotherapeutic agents, biotoxins, polyethylene glycol or enzymes.
  • the coupling moiety is a purification tag (such as a His tag), a detectable label, a drug, a toxin, a cytokine, an enzyme, or a combination thereof; preferably, the coupling moiety is a radioactive isotope , fluorescent substances, chemiluminescent substances, colored substances, chemotherapeutic agents,
  • a kit comprising the anti-ANG2 heavy chain single domain antibody described in any one of items 1-3, the recombinant protein described in item 4, the multispecific antibody or item described in any one of items 5-7
  • the conjugate described in 11 preferably, the kit also includes the anti-ANG2 heavy chain single domain antibody specifically recognizing any one of items 1-3, the recombinant protein described in item 4, and items 5-7
  • the anti-ANG2 heavy chain single domain antibody, The recombinant protein or multispecific antibody also includes a detectable label, such as a radioactive isotope, a fluorescent substance, a chemiluminescent substance, a colored substance or an enzyme; preferably, the kit is used to detect the presence or level of ANG2 in a sample; or
  • the kit comprises (1) the anti-ANG2 heavy chain single domain antibody described in any one of items 1-3, the recombinant protein described in item 4, the multispecific antibody or item described in any one of items 5-7
  • a pharmaceutical composition comprising the anti-ANG2 heavy chain single domain antibody described in any one of items 1-3, the recombinant protein described in item 4, the multispecific antibody or item described in any one of items 5-7
  • the conjugate described in 11; optionally, the pharmaceutical composition also includes a pharmaceutically acceptable carrier and/or excipient; preferably, the pharmaceutical composition is suitable for subcutaneous injection, intradermal injection , intravenous injection, intramuscular injection or intralesional injection administration form.
  • the anti-ANG2 heavy chain single domain antibody described in any one of items 1-3, the recombinant protein described in item 4, the multispecific antibody described in any one of items 5-7 or the conjugate described in item 11 Use of substances in inhibiting the pro-angiogenic activity of ANG2, preventing and/or treating diseases caused by or related to angiogenesis processes, such as fundus neovascular diseases, rheumatoid arthritis and psoriasis, and/or tumors , or in preparation for inhibiting the pro-angiogenic activity of ANG2, preventing and/or treating diseases caused by or associated with angiogenic processes such as fundus neovascular diseases, rheumatoid arthritis and psoriasis and/or tumors use in medicines.
  • the CDR regions of an antibody are responsible for the binding specificity of the antibody to an antigen.
  • the known antibody heavy and light chain variable region sequences there are currently several methods for determining antibody CDR regions, including the Kabat, IMGT, Chothia, and AbM numbering systems.
  • each application of the definition to the CDRs of an antibody or variant thereof will be within the scope of the terms as defined and used herein.
  • the variable region amino acid sequence of the antibody one skilled in the art can generally determine the specific CDRs without reliance on any experimental data other than the sequence itself.
  • heavy chain single domain antibodies also known as VHH domains, VHH antibody fragments, or VHH antibodies
  • VHH domains are variable structures of antigen-binding immunoglobulins known as "heavy chain antibodies” (i.e., antibodies lacking light chains) Domain
  • “heavy chain antibodies” i.e., antibodies lacking light chains
  • Domain 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).
  • VHH domains specifically bind epitopes without the need for additional antigen-binding domains.
  • the VHH domain is a small, stable and efficient antigen recognition unit formed by a single immunoglobulin domain.
  • the heavy chain single domain antibody of the present invention can be conjugated with a desired biologically active substance to form a recombinant protein.
  • the biologically active substance is selected from, for example, a toxin with enzymatic activity or an active fragment thereof (such as abrin, ricin A, pseudomonas exotoxin or diphtheria toxin), tumor necrosis factor Or interferon (such as IFN- ⁇ ), biological response regulators (such as lymphokines, IL-2, IL-2, IL2-6, IL-10, granular GM-CSF), Fc fragment.
  • the Fc fragment contained in the recombinant protein of the present invention can make it form a dimer, and at the same time prolong the half-life of the recombinant protein in vivo.
  • the Fc fragments useful in the present invention may be from different subtypes of immunoglobulins such as IgG (such as IgG1, IgG2, IgG3 or IgG4 subtypes), IgAl, IgA2, IgD, IgE or IgM.
  • Antibodies of the invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) having substitutions in one or more amino acid residues or (iii) a mature polypeptide fused to another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional amino acid sequence fused to the polypeptide sequence to form The polypeptide (such as a leader sequence or a secretory sequence or a sequence or protein sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). Such fragments, derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein.
  • a “conservative amino acid substitution” is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues 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
  • side chains of ⁇ -branches for example, threonine, valine, isoleucine amino acids
  • aromatic side chains eg tyrosine, phenylalanine, tryptophan, histidine.
  • nonessential amino acid residues of an immunoglobulin polypeptide are preferably replaced with other amino acid residues from the same side chain family.
  • a string of amino acids may be replaced by a structurally similar string of amino acids that differ in sequence and/or composition of side chain families.
  • Non-limiting examples of conservative amino acid substitutions are provided in the table below, where a similarity score of 0 or higher indicates a conservative substitution between these two amino acids.
  • the conservative substitution is preferably a substitution wherein one amino acid residue within the following groups (a)-(e) is replaced by another amino acid residue within the same group: (a) small Aliphatic, non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e ) Aromatic residues: Phe, Tyr and Trp.
  • Particularly preferred conservative substitutions are as follows: Ala by Gly or by Ser; Arg by Lys; Asn by Gln or His; Asp by Glu; Cys by Ser; Gln by Asn; Glu by Asp; Gly replaced by Ala or replaced by Pro; His replaced by Asn or replaced by Gln; Ile replaced by Leu or replaced by Val; Leu replaced by Ile or replaced by Val; Lys replaced by Arg, replaced by Gln or replaced by Glu; Met Substitution of Leu, substitution of Tyr or substitution of Ile; substitution of Phe by Met, substitution of Leu or substitution of Tyr; substitution of Ser by Thr; substitution of Thr by Ser; substitution of Trp by Tyr; substitution of Tyr by Trp; and/or substitution of Phe Replace with Val, replace with Ile or replace with Leu.
  • amino acids have similar structural characteristics and properties:
  • glycine G alanine A
  • valine V alanine A
  • leucine L alanine L
  • isoleucine I all belong to neutral amino acids containing one amino-carboxyl group
  • Both serine S and threonine T are hydroxyl amino acids.
  • Both cysteine C and methionine M are sulfur-containing amino acids.
  • Both asparagine N and glutamine Q belong to amido-containing amino acids.
  • Both aspartic acid D and glutamic acid E are acidic amino acids.
  • Lysine K and arginine R are both basic amino acids.
  • Both phenylalanine F and tyrosine Y are aromatic amino acids.
  • Tryptophan W, histidine H and proline P are all heterocyclic amino acids. Therefore, similar structural features and properties can be conservatively substituted with each other.
  • the antibodies of the invention can bind to a therapeutic agent (such as a chemotherapeutic drug, such as cisplatin, carboplatin), a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, a pharmaceutical agent, or PEG.
  • a therapeutic agent such as a chemotherapeutic drug, such as cisplatin, carboplatin
  • a prodrug such as a chemotherapeutic drug, such as cisplatin, carboplatin
  • a prodrug such as a chemotherapeutic drug, such as cisplatin, carboplatin
  • a prodrug such as a chemotherapeutic drug, such as cisplatin, carboplatin
  • a prodrug such as a chemotherapeutic drug, such as cisplatin, carboplatin
  • a prodrug such as a chemotherapeutic drug, such as cisp
  • Figure 1 Biological activity of anti-ANG2 heavy chain single domain antibody-Fc binding to human ANG2.
  • Figure 3 The 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 HUVECs.
  • Figure 5 Biological activity of antibodies inhibiting the binding of human ANG1 to HUVECs.
  • the amino acid sequence information of the specific ANG2 ECD molecule is shown in Table 1.
  • alpacas Two alpacas (Alpaca) were selected for antigen immunization, and after four times of immunization with human ANG2 protein, lymphocytes from the peripheral blood of the alpaca were extracted from 100ml, total RNA was extracted with RNAiso Plus reagent (TAKARA, 9109), and PrimeScript II kit ( TAKARA, 6210A) reverse transcribed the extracted RNA into cDNA.
  • TAKARA RNAiso Plus reagent
  • TAKARA, 6210A PrimeScript II kit reverse transcribed the extracted RNA into cDNA.
  • the ligation product was electrotransformed into Escherichia coli competent cell TG1, and the ligation product of each alpaca was transformed by electroporation 10 times.
  • the size of the library was calculated by gradient dilution plating, and the sizes of the phage libraries of the two alpacas were: 1.21 ⁇ 10 9 and 1 ⁇ 10 9 .
  • 96 clones were randomly selected from the titer plate for identification, and the results showed that the insertion rate was 100%.
  • the phages specifically binding to hANG2 were eluted with glycine-hydrochloric acid (200 mM), and infected with Escherichia coli TG1 in logarithmic phase growth, and the phages were produced and purified for the next round of screening. After the same screening process was repeated for 2 rounds, the obtained phages were infected with Escherichia coli TG1 and plated, and single clones were picked from the plate and sequenced. According to the sequence alignment results, the protein sequences of each clone were analyzed, and the clones with different CDR1, CDR2, and CDR3 sequences were regarded as different antibody strains, and finally 10 different antibody strains were obtained.
  • the hANG2 protein solution and BSA were coated with 2 ⁇ l/well of the microtiter plate, and spent overnight at 4; the supernatant was discarded, and 300 ⁇ l of blocking solution (PBS containing 3% BSA) was added to each well, and blocked for 2 hours at 37 degrees; Add 200 ⁇ l per well of the supernatant to the wells of the microplate, incubate at room temperature for 2 hours, discard the supernatant; wash 3 times with 200 ⁇ l/well PBST (PBS containing 0.1% Tween20); add diluted HRP-labeled antibody VHH secondary antibody (Genscript, A01861), the secondary antibody was used after dilution of 1:10000, the dilution solution was 1% BSA in PBS, the volume was 100 ⁇ l/well, and incubated at room temperature for 1 hour; washed 5 times with 200 ⁇ l/well PBST; added TMB Chromogenic solution (BD, 55214) 100 ⁇ l/well, develop color at 37°
  • chain antibody to obtain the anti-ANG2 heavy chain single domain antibody-Fc expression plasmid the vector generally uses pcDNA3.1(-) (purchased from Invitrogen) or other eukaryotic expression vectors.
  • the endotoxin-free large-scale extraction plasmid kit (Qiagen, product number 12391) was used for plasmid extraction, and the specific operation was performed according to the instructions provided by the manufacturer.
  • CHO-S cells were cultured in CD CHO medium (Gibco, Cat. No. 10743-029) according to the instructions provided by the manufacturer, and placed in a 37°C, 5% CO 2 cell incubator for cultivation. After the cells were prepared, the cells containing anti-ANG2 Plasmids of the heavy chain single domain antibody-Fc sequence were co-transfected into CHO-S cells to express the antibody-Fc. On the second day after transfection, the culture temperature was lowered to 32° C., and 3.5% 2 ⁇ EFC+ (Gibco, Cat.
  • the ELISA detection method was used to evaluate the Fc affinity of 10 anti-ANG2 heavy chain single domain antibodies.
  • Anti-ANG2 heavy chain single domain antibody-Fc inhibits the binding of hANG2 to its receptor hTIE2
  • a 96-well plate was treated with 100 ⁇ L of hTIE2-Fc (Novoprotein, CW98) prepared at 2 ⁇ g/mL in PBS and incubated overnight at 4°C. Plates were then washed three times with PBS wash buffer containing 0.1% Tween-20, followed by blocking with 300 ⁇ L of PBS containing 1% BSA for 2 hours.
  • the anti-ANG2 heavy chain single domain antibody-Fc to be tested was diluted and mixed with hANG2 at a final concentration of 10 ⁇ g/mL. After incubation at 37°C for 1 hour, the antigen-antibody mixture was added to a 96-well plate and incubated at 37°C for 1 hour.
  • B2-E2-Fc was evaluated according to the following acid stability and thermal stability evaluation methods.
  • the anti-ANG2 heavy chain single domain antibody-Fc molecule is subjected to protein A affinity chromatography, the anti-ANG2 heavy chain single domain antibody-Fc eluted in the acid elution step (using pH 3.5 citrate buffer) The solution was not neutralized, and after being kept in the buffer for a period of time, a sample was taken at 30 minutes and 1/10 volume of 1M Tris-HCl (pH 8.0) was added for neutralization, and the HPLC-SEC detection of the sample was carried out.
  • Antibody molecules did not show aggregation or degradation after being treated at pH 3.5 for 30 minutes, and the change in purity was less than 4%, indicating that it can maintain stability in an acidic environment.
  • HPLC-SEC detection of the sample was performed after being incubated in a 40°C incubator for 14 days, no aggregation or degradation occurred, and the change in purity was less than 4%, indicating that it could maintain stability in a 40°C environment.
  • B2-E2-Fc has good acid stability and thermal stability.
  • bispecific antibody Y400C and Y400E According to the amino acid sequence of the bispecific antibody shown in Table 8, reverse translation of the DNA code and synthesis of the DNA fragment were performed, constructed into the expression vector pcDNA3.1, and transiently transfected into 293 or CHO cells for expression. The supernatant was harvested for protein purification to obtain bispecific antibodies Y400C and Y400E with a purity of not less than 95%.
  • transient transfection and protein purification methods please refer to the previous examples.
  • the structure of the bispecific antibody is: comprising a light chain and a heavy chain, wherein the light-heavy chain pairs and forms an interchain disulfide bond, and two heavy chains pair and forms an interchain disulfide bond, wherein the heavy chain is (VH )-(CH1)-(hinge region)-(Fc)-(connecting peptide)-(VHH), light chain is (VL)-(light chain constant region), VH and VL are heavy chain and light chain of anti-VEGF antibody Chain variable region, VHH is the variable region of anti-ANG2 heavy chain single domain antibody.
  • the bispecific antibody was placed in a 40-degree incubator for 14 days or 28 days, and then the HPLC-SEC detection of the sample was performed. It was found that the antibody molecule of the present invention did not aggregate or degrade after being treated at 40°C for 28 days, and the change in purity was less than 4%, indicating that it can maintain stability in an environment of 40°C, as shown in Table 9.
  • the sensor chip Protein A chip (GE, article number: 28995056) was used to capture the antibody to be tested, and the antigen human ANG2 protein (623-AN-025/CF, R&D) was used as an analyte to detect the kinetics and affinity data of its binding to the test product.
  • the initial detection concentration of the combination of antigen and the test product is 10nM.
  • 2-fold serial dilution that is, the concentration of antigen dilution is 10nM, 5nM, 2.5nM, 1.25nM, 0.625nM, and the antigen is sequentially from low concentration to Start sample injection at a high concentration, and set up a negative control (i.e.
  • Bispecific antibody inhibits the binding of hANG2 to HUVEC cells
  • HUVEC cells in good growth state (H-003, Allcells) were used to prepare a single cell suspension. After cell counting, 105 cells/well were plated into a 96-well plate; the final concentration of immobilized human ANG2 protein (623-AN-025/CF, R&D) was 10 ⁇ g/ml, and each well was added with the antibody to be tested according to the experimental design.
  • the highest antibody concentration was 1000nM, which was diluted 5 times sequentially, and a total of 7 concentration gradients were set up, and a blank control and a positive control RG7716 (ATAD00534, Atagenix) were set at the same time; after the cells were incubated at room temperature for 30min, the cells in each well were washed 3 times and resuspended. Afterwards, fluorescently labeled secondary antibody PE anti-His (362603, Biolegend) was added, and incubated at room temperature in the dark for 30 min; cells in each well were washed 3 times, resuspended, and detected by flow cytometry. As shown in Figure 4, the blocking activity of bispecific antibodies Y400C and Y400E on ANG2 and HUVEC cells was better than that of the control positive antibody.
  • Bispecific antibody does not inhibit the binding of hANG1 to HUVEC cells
  • 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 antibodies of the invention are particularly useful in inhibiting the pro-angiogenic activity of ANG2 and in treating diseases and disorders resulting from or associated with the angiogenic process.
  • HUVEC cells in good growth state were used to prepare a single cell suspension. After cell counting, 105 cells/well were plated into 96-well plates; the final concentration of immobilized human ANG1 protein (623-AN/CF, R&D) was 10 ⁇ g/ml, and each well was added with the antibody to be tested according to the experimental design, and the antibody was the highest The concentration was 1000nM, diluted 5-fold sequentially, and a total of 7 concentration gradients were set up, and a blank control and a positive control hTIE2-Fc (Novoprotein, CW98) were set at the same time; after the cells were incubated at room temperature for 30min, the cells in each well were washed 3 times and resuspended.
  • immobilized human ANG1 protein 623-AN/CF, R&D
  • the concentration was 1000nM, diluted 5-fold sequentially, and a total of 7 concentration gradients were set up, and a blank control and a positive control hTIE2-Fc (Novo
  • bispecific antibodies Y400C and Y400E have no binding blocking activity on ANG1 and HUVEC cells.
  • Bispecific antibody inhibits hANG2-dependent AKT phosphorylation in HUVEC cells
  • HUVEC cell surface naturally expresses the receptor of ANG2 protein, when ANG2 binds to the cell surface receptor, it will activate the phosphorylation reaction of AKT.
  • the Phospho-AKT assay kit (64AKSPE1-1, Cisbio) contains two labeled antibodies: one with a donor fluorophore and one with an acceptor. The primary antibody was selected for its specific binding to phosphorylation motifs on the protein, and the secondary antibody was selected for its ability to recognize the protein independent of its phosphorylation state. Phosphorylation of the protein enables the formation of immune complexes involving both the labeled antibody and the close proximity of the donor fluorophore to the acceptor, thereby generating the signal.
  • HUVEC cells were co-incubated with serially diluted samples and a fixed concentration of ANG2.
  • the ANG2 functional activity and the inhibitory effect of the sample on ANG2 functional activity were evaluated by the AKT phosphorylation degree of HUVEC cells.
  • HUVEC cells in good growth state (70013502, ATCC) were prepared into a single cell suspension. After cell counting, 105 cells/well were plated into a 96-well plate; the final concentration of immobilized human ANG2 protein (623-AN-025/CF, R&D) was 10 ⁇ g/ml, and each well was added with the antibody to be tested according to the experimental design.
  • the highest concentration of the antibody was 1000nM, which was diluted 5-fold successively, and a total of 8 concentration gradients were set up, and a blank control and a positive control RG7716 (ATAD00534, Atagenix) were set at the same time; after the cells were incubated at 37°C for 10min, the supernatant in the well was removed, and added After the lysate, shake and incubate at room temperature for 30 minutes, pipette the cell lysate into a 384-well plate, add detection reagents and incubate for 4 hours in the dark, and use PHERAstar for detection. As shown in Figure 6, the AKT phosphorylation inhibitory activity of bispecific antibodies Y400C and Y400E on hANG2-dependent HUVEC cells was superior to that of the control positive antibody.

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Abstract

本发明涉及抗ANG2重链单域抗体，及其应用。

Description

抗ANG2抗体及其制备方法和应用 技术领域
本发明涉及医药领域，具体地涉及ANG2(angiopoietin-2，人血管生成2)的抗体及其制备方法和应用。
背景技术
针对ANG2的抗体能抑制新生血管的生成和渗漏，并减少炎症反应的发生。ANG/Tie2是一种调节血管生成的重要信号通路。ANG1促进内皮细胞受体Tie2磷酸化，吸引血管平滑肌细胞、周细胞等血管周围细胞包围和支持内皮细胞，促进血管重塑、并维持血管的完整性和调节血管功能；ANG1能够保护血管不发生病变，并保持血管形态，而ANG2促进血管渗漏，导致低血压和血管结构异常。尽管有很多企业开发的ANG2抗体在进行临床实验，但是目前尚无针对ANG2的抗体药物上市。
发明内容
本发明提供一种针对ANG2的特异性单克隆抗体，在抑制ANG2的促血管生成活性和治疗由血管生成过程导致的或与其相关的疾病和病症中发挥作用，例如眼底新生血管性疾病、类风湿性关节炎和银屑病。另外，血管生成对肿瘤生长和维持是非常关键的，抑制ANG2的单克隆抗体也能对治疗癌症过程中产生作用。此外，本发明还提供了针对ANG2和VEGF的双特异性抗体。本发明抗体能够与人ANG2、兔ANG2和猴ANG2结合。
具体地，本发明涉及以下方面：
1.抗ANG2重链单域抗体，其包含SEQ ID NO:1-10任一项所示的重链可变区包含的HCDR1，HCDR2和HCDR3，或其变体，
优选地，按照IMGT编号系统，包含选自以下各项组成的组的HCDR1，HCDR2和HCDR3:
(1)SEQ ID NO:21所示的HCDR1，SEQ ID NO:22所示的HCDR2和SEQ ID NO:23所示的HCDR3，
(2)SEQ ID NO:34所示的HCDR1，SEQ ID NO:35所示的HCDR2和SEQ ID NO:36所示的HCDR3，
(3)SEQ ID NO:37所示的HCDR1，SEQ ID NO:38所示的HCDR2和SEQ ID NO:39所示的HCDR3，
(4)SEQ ID NO:40所示的HCDR1，SEQ ID NO:41所示的HCDR2和SEQ ID NO:42所示的HCDR3，或SEQ ID NO:40所示的HCDR1、SEQ ID NO:41所示的HCDR2和SEQ ID NO:42所示的HCDR3的变体，及其组合，所述变体为与SEQ ID NO:40所示的HCDR1，SEQ ID NO:41所示的HCDR2和SEQ ID NO:42所示的HCDR3相比分别具有一个或多个(优选1个、2个或3个)保守氨基酸突变 (优选置换，插入或缺失)并保留与ANG2结合亲和力的氨基酸序列，
优选地，SEQ ID NO:40所示的HCDR1，SEQ ID NO:41所示的HCDR2和SEQ ID NO:42所示的HCDR3，其中SEQ ID NO:40所示的CDR1中第5-8位和第10-11位氨基酸、SEQ ID NO:41所示的CDR2中第2-5位氨基酸和第7位、SEQ ID NO:42所示的CDR3第1位氨基酸、第3-8位氨基酸选自氨基酸X，所述氨基酸X选自Ala，Arg，Asn，Asp，Cys，Gln，Glu，Gly，His，Ile，Leu，Lys，Met，Phe，Pro，Ser，Thr，Trp，Tyr和Val组成的组，
优选地，SEQ ID NO:40所示HCDR1的变体，其中第1位的甘氨酸G可以被选自以下各项组成的组的氨基酸替换：Ala，Val，Leu和Ile；第2位的苯丙氨酸F可以被Tyr替换；第3位的脯氨酸P可以被选自以下各项组成的组的氨基酸替换：Trp和His；第4位的Ser可以被Thr替换；第9位的Ser可以被Thr替换；第12位的Gln可以被Asn替换；
SEQ ID NO:41所示的HCDR2的变体，第1位的Ile可以被选自以下各项组成的组的氨基酸替换：Ala，Val，leu和Gly；第6位的Leu可以被Ile替换；第8位的Lys可以被Arg替换；
SEQ ID NO:42所示的HCDR3的变体，其中第2位的Val可以被选自以下各项组成的组的氨基酸替换：Ala，Gly，Leu和Ile；第9位的Asp可以被Glu替换，
(5)SEQ ID NO:43所示的HCDR1，SEQ ID NO:44所示的HCDR2和SEQ ID NO:45所示的HCDR3，
(6)SEQ ID NO:46所示的HCDR1，SEQ ID NO:44所示的HCDR2和SEQ ID NO:48所示的HCDR3，
(7)SEQ ID NO:49所示的HCDR1，SEQ ID NO:44所示的HCDR2和SEQ ID NO:48所示的HCDR3，
(8)SEQ ID NO:52所示的HCDR1，SEQ ID NO:44所示的HCDR2和SEQ ID NO:48所示的HCDR3，和
(9)SEQ ID NO:55所示的HCDR1，SEQ ID NO:56所示的HCDR2和SEQ ID NO:57所示的HCDR3。
2.项目1所述的抗ANG2重链单域抗体，其中所述ANG2选自人ANG2、兔ANG2、或猴ANG2。
3.项目1或2所述的抗ANG2重链单域抗体，其包含重链可变区，所述重链可变区包含SEQ ID NO:1-10任一项所示的序列或与SEQ ID NO:1-10任一项所述的序列具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同源性且具有ANG2结合活性的序列，或由其组成。
4.重组蛋白，其包含项目1-3任一项所述的抗ANG2重链单域抗体，优选地，所述重组蛋白进一步包含生物活性物质，如具有酶活性的毒素或其活性片段(如相思豆毒蛋白，蓖麻毒蛋白A，假单胞菌外毒素或白喉毒素)，肿瘤坏死因 子或干扰素(如IFN-γ)，生物反应调节物(如淋巴因子，IL-2，IL-2，IL2-6，IL-10，粒GM-CSF)，和/或Fc片段(如来自IgG(例如IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM的Fc区，例如来自人、兔或猴的IgG，IgA1，IgA2，IgD，IgE或IgM的Fc区)，优选地，所述抗ANG2重链单域抗体和所述Fc片段通过连接肽连接，优选地，所述连接肽为(GGGGS)n，n＝1、2或者3，优选地，所述Fc片段为人IgG1重链恒定区，更优选为GenBank No.AK303185.1或SEQ ID NO:29所示的Fc片段，更优选地，所述重组蛋白包含SEQ ID NO:11-20所示的序列或由其组成。
5.多特异性抗体，优选双特异性抗体，其包含项目1-3任一项所述的抗ANG2重链单域抗体，优选所述双特异性抗体的结构为：包含轻链和重链，其中轻-重链配对并形成链间二硫键，两个重链配对并形成链间二硫键，其中重链为(VH)-(CH1)-(铰链区)-(Fc)-(连接肽)-(VHH)，轻链为(VL)-(轻链恒定区)。
6.项目5所述的多特异性抗体，其中VHH为项目1-3任一项所述的抗ANG2单域抗体，VH和VL为第二种抗体的重链和轻链可变区，优选所述第二种抗体针对的抗原选自免疫细胞表面抗原、肿瘤抗原、病毒、细菌、内毒素、细胞因子、或其组合；更优选地选自：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，和/或CD16a，优选第二种抗体针对的抗原为VEGF，更优选第二抗体选自抗VEGF抗体，其包含SEQ ID NO:24所示的重链可变区包含的HCDR1，HCDR2和HCDR3(优选按照IMGT编号系统，包含SEQ ID NO:50所示的HCDR1，SEQ ID NO:51所示的HCDR2和SEQ ID NO:53所示的HCDR3)和SEQ ID NO:27所示的轻链可变区包含的LCDR1，LCDR2和LCDR3(优选按照IMGT编号系统，包含SEQ ID NO:54所示的LCDR1，SEQ ID NO:58所示的LCDR2和SEQ ID NO:59所示的LCDR3)，更优选，所述抗VEGF抗体的重链可变区包含SEQ ID NO:24所示的序列或与SEQ ID NO:24所示的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同源性的序列，或由其组成；所述抗VEGF抗体的轻链可变区包含SEQ ID NO:27所示的序列或与SEQ ID NO:27所示的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同源性的序列，或由其组成。
7.项目5或6所述的多特异性抗体，其中CH1的序列如SEQ ID NO:25所示，铰链区的序列如SEQ ID NO:26所示，Fc恒定区的序列如SEQ ID NO:29所示，连接肽的序列如SEQ ID NO:30或47所示，轻链恒定区的序列如SEQ ID NO:28所示；优选地，所述多特异性抗体选自下组：
(1)Y400C，其包含重链和轻链，或由其组成；其中重链的序列包含SEQ ID NO:24，25，26，29，30和4，或由其组成；轻链的序列包含SEQ ID NO:27和28，或由其组成；
(2)Y400E，其包含重链和轻链，或由其组成；其中重链的序列包含SEQ ID  NO:24，25，26，29，47和4，或由其组成；轻链的序列包含SEQ ID NO:27和28，或由其组成。
8.多核苷酸，其编码项目1-3任一项所述的抗ANG2重链单域抗体、项目4所述的重组蛋白，或项目5-7任一项所述的多特异性抗体。
9.载体，其包含项目8所述的多核苷酸。
10.宿主细胞，其包含项目9所述的载体。
11.偶联物，其包含项目1-3任一项所述的抗ANG2重链单域抗体、项目4所述的重组蛋白，或项目5-7任一项所述的多特异性抗体，以及偶联部分，其中，所述偶联部分为纯化标签(如His标签)、可检测的标记、药物、毒素、细胞因子、酶、或其组合；优选地，所述偶联部分为放射性同位素、荧光物质、化学发光物质、有色物质、化疗剂、生物毒素、聚乙二醇或酶。
12.试剂盒，其包括包含项目1-3任一项所述的抗ANG2重链单域抗体、项目4所述的重组蛋白，项目5-7任一项所述的多特异性抗体或项目11所述的偶联物；优选地，所述试剂盒还包括特异性识别项目1-3任一项所述的抗ANG2重链单域抗体、项目4所述的重组蛋白，项目5-7任一项所述的多特异性抗体或项目11所述的偶联物的抗ANG2重链单域抗体、重组蛋白或多特异性抗体；任选地，所述抗ANG2重链单域抗体、重组蛋白或多特异性抗体还包括可检测的标记，例如放射性同位素、荧光物质、化学发光物质、有色物质或酶；优选地，所述试剂盒用于检测ANG2在样品中的存在或其水平；或者
所述试剂盒包含(1)项目1-3任一项所述的抗ANG2重链单域抗体、项目4所述的重组蛋白，项目5-7任一项所述的多特异性抗体或项目11所述的偶联物，和(2)针对其它抗原的抗体或其抗原结合片段，和/或细胞毒性剂，和/或化疗药，和任选地，使用说明书。
13.药物组合物，其包含项目1-3任一项所述的抗ANG2重链单域抗体、项目4所述的重组蛋白，项目5-7任一项所述的多特异性抗体或项目11所述的偶联物；可选地，所述药物组合物还包括药学上可接受的载体和/或赋形剂；优选地，所述药物组合物为适于通过皮下注射、皮内注射、静脉内注射、肌内注射或病灶内注射施用的形式。
14.项目1-3任一项所述的抗ANG2重链单域抗体、项目4所述的重组蛋白，项目5-7任一项所述的多特异性抗体或项目11所述的偶联物在抑制ANG2的促血管生成活性、预防和/或治疗由血管生成过程导致的或与其相关的疾病例如眼底新生血管性疾病、类风湿性关节炎和银屑病，和/或肿瘤中的用途，或在制备用于抑制ANG2的促血管生成活性、预防和/或治疗由血管生成过程导致的或与其相关的疾病例如眼底新生血管性疾病、类风湿性关节炎和银屑病和/或肿瘤的药物中的用途。
应理解，在本发明范围内，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
在本发明中涉及的术语具备本领域技术人员理解的常规含义。在本技术领域内使用和/或可接受的情况下，一个术语有两个或两个以上定义时，本文使用的术语的定义用于包括所有的含义。
本领域普通技术人员可以理解，抗体的CDR区负责抗体对抗原的结合特异性。在已知抗体重链和轻链可变区序列的情况下，目前有几种确定抗体CDR区的方法，包括Kabat，IMGT，Chothia和AbM编号系统。然而，每种关于抗体或其变体的CDR的定义的应用都将在本文定义和使用的术语的范围内。如果给定该抗体的可变区氨基酸序列，则本领域技术人员通常可确定特定CDR，而不依赖于该序列自身之外的任何实验数据。
在本发明中，重链单域抗体也称为VHH结构域，VHH抗体片段或VHH抗体，是称为“重链抗体”(即缺乏轻链的抗体)的抗原结合免疫球蛋白的可变结构域(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)。VHH结构域特异性结合表位而无需其他抗原结合结构域。VHH结构域为由单一免疫球蛋白结构域形成的小型稳定及高效的抗原识别单元。
本发明的重链单域抗体可以与具有需要的生物活性物质缀合，形成重组蛋白。在一些实施方案中，所述生物活性物质选自例如具有酶活性的毒素或其活性片段(如相思豆毒蛋白，蓖麻毒蛋白A，假单胞菌外毒素或白喉毒素)，肿瘤坏死因子或干扰素(如IFN-γ)，生物反应调节物(如淋巴因子，IL-2，IL-2，IL2-6，IL-10，粒GM-CSF)，Fc片段。本发明重组蛋白包含的Fc片段可以使其形成二聚体，同时延长所述重组蛋白的体内半衰期。在一些实施方案中，可用于本发明的Fc片段可以来自不同亚型的免疫球蛋白如IgG(如IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM。
本发明的抗体可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与6His标签形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
保守替换：
“保守氨基酸替换”是其中氨基酸残基被具有类似侧链的氨基酸残基替换。具有类似侧链的氨基酸残基家族已在本领域中定义，其包括碱性侧链(例如赖氨酸、精氨酸、组氨酸)，酸性侧链(例如天冬氨酸，谷氨酸)，不带电荷的极性侧链(例如，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)，非极性侧链(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、蛋氨酸、色氨酸)，β-支链的侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳族侧链(例如酪氨酸、 苯丙氨酸、色氨酸、组氨酸)。因此，免疫球蛋白多肽的非必需氨基酸残基优选被来自相同侧链家族的其他氨基酸残基替换。在另一些实施方案中，一串氨基酸可被结构上类似的氨基酸串替换，后者在顺序上和/或侧链家族的组成上不同。
在下表中提供了保守性氨基酸替换的非限制性实例，其中相似性得分为0或更高表示在这两个氨基酸之间有保守替换。
  C G P S A T D E N Q H K R V M I L F Y W
W -8 -7 -6 -2 -6 -5 -7 -7 -4 -5 -3 -3 2 -6 -4 -5 -2 0 0 17
Y 0 -5 -5 -3 -3 -3 -4 -4 -2 -4 0 -4 -5 -2 -2 -1 -1 7 10  
F -4 -5 -5 -3 -4 -3 -6 -5 -4 -5 -2 -5 -4 -1 0 1 2 9    
L -6 -4 -3 -3 -2 -2 -4 -3 -3 -2 -2 -3 -3 2 4 2 6      
I -2 -3 -2 -1 -1 0 -2 -2 -2 -2 -2 -2 -2 4 2 5        
M -5 -3 -2 -2 -1 -1 -3 -2 0 -1 -2 0 0 2 6          
V -2 -1 -1 -1 0 0 -2 -2 -2 -2 -2 -2 -2 4            
R -4 -3 0 0 -2 -1 -1 -1 0 1 2 3 6              
K -5 -2 -1 0 -1 0 0 0 1 1 0 5                
H -3 -2 0 -1 -1 -1 1 1 2 3 6                  
Q -5 -1 0 -1 0 -1 2 2 1 4                    
N -4 0 -1 1 0 0 2 1 2                      
E -5 0 -1 0 0 0 3 4                        
D -5 1 -1 0 0 0 4                          
T -2 0 0 1 1 3                            
A -2 1 1 1 2                              
S 0 1 1 1                                
P -3 -1 6                                  
G -3 5                                    
C 12                                      
在一些实施方案中，所述保守替换优选地是这样的替换，即，其中下列组(a)–(e)内的一个氨基酸被同组内的另一氨基酸残基替换：(a)小的脂肪族、非极性或弱极性的残基：Ala,Ser,Thr,Pro和Gly；(b)极性、带负电荷的残基及其(不带电荷的)酰胺：Asp,Asn,Glu和Gln；(c)极性、带正电荷的残基:His,Arg和Lys；(d)大的脂肪族、非极性残基:Met,Leu,Ile,Val和Cys；以及(e)芳族残基:Phe,Tyr和Trp。
特别优选的保守替换如下：Ala替换成Gly或替换成Ser；Arg替换成Lys；Asn替换成Gln或替换成His；Asp替换成Glu；Cys替换成Ser；Gln替换成Asn；Glu替换成Asp；Gly替换成Ala或替换成Pro；His替换成Asn或替换成Gln；Ile替换成Leu或替换成Val；Leu替换成Ile或替换成Val；Lys替换成Arg,替换成Gln或替换成Glu；Met替换成Leu,替换成Tyr或替换成Ile；Phe替换成Met,替换成Leu或替换成Tyr；Ser替换成Thr；Thr替换成Ser；Trp替换成Tyr；Tyr替换成Trp；和/或Phe替换成Val,替换成Ile或替换成Leu。
在一些实施方案中，以下氨基酸具有相似的结构特征和性能：
如甘氨酸G、丙氨酸A、缬氨酸V、亮氨酸L和异亮氨酸I均属于含一氨基一羧基的中性氨基酸，
丝氨酸S和苏氨酸T均属于羟基氨基酸，
半胱氨酸C和甲硫氨酸M均属于含硫氨基酸，
天冬酰胺N和谷氨酰胺Q均属于含酰胺基氨基酸，
天冬氨酸D和谷氨酸E均属于酸性氨基酸，
赖氨酸K和精氨酸R均属于碱性氨基酸，
苯丙氨酸F和酪氨酸Y均属于芳香族氨基酸，
色氨酸W，组氨酸H和脯氨酸P均属于杂环氨基酸。因此相似的结构特征和性能，彼此之间可以进行保守置换。
在一些实施方案中，本发明所述抗体可以结合治疗剂(如化疗药，如顺铂、卡铂)、药物前体、肽、蛋白、酶、病毒、脂质、生物反应调节剂、药剂或PEG。本发明抗体可以连接至或融合至治疗剂上，该治疗剂可包括可检测标记物，如放射性标记物、免疫调节剂、激素、酶、寡核苷酸、光活性治疗剂或诊断剂、细胞毒性剂，其可为药物或毒素，超声增强剂，非放射性标记物，它们的组合和其他这类本领域已知的成分。
附图说明
图1.抗ANG2重链单域抗体-Fc结合人ANG2的生物学活性。
图2.抗ANG2重链单域抗体-Fc结合猴ANG2的生物学活性。
图3.抗ANG2重链单域抗体-Fc抑制人ANG2与其受体hTIE2结合的生物学活性。
图4.抗体抑制人ANG2与HUVEC结合的生物学活性。
图5.抗体抑制人ANG1与HUVEC结合的生物学活性。
图6.抗体抑制人ANG2依赖的HUVEC细胞的AKT磷酸化。
具体实施方式
下面通过实施例详细描述本发明。本领域的普通技术人员可以理解，下述实施例仅是用于举例说明的目的。本发明的精神和范围由权利要求所限定。下述实施例中所用方法如无特别说明均为常规方法，所用的试剂如无特别说明均为可商购的试剂。
实施例1：针对ANG2的重链单域抗体的筛选
对应人ANG2的Lys275-Phe496位氨基酸的重组人ANG2蛋白，后简称hANG2，序列如SEQ ID NO:32所示；猴ANG2蛋白的序列如SEQ ID NO:33所示，后简称cynoANG2。具体ANG2 ECD分子的氨基酸序列信息如表1。
表1.重组人ANG2序列信息
Figure PCTCN2021117697-appb-000001
Figure PCTCN2021117697-appb-000002
1.1重链单域抗体噬菌体文库的构建：
选取两只羊驼(Alpaca)进行抗原免疫，用人ANG2蛋白进行4次免疫后，提取羊驼100ml外周血的淋巴细胞，用RNAiso Plus试剂(TAKARA，9109)提取总RNA，使用PrimeScript II试剂盒(TAKARA，6210A)将提取的RNA反转录成cDNA。用PCR扩增的方法先扩增750bp长的重链抗体可变区和恒定区CH2的核酸片段，然后以上一步中回收的750bp长的核酸片段为模板扩增目的片段，即重链抗体可变区片段。将载体pComb3XSS(NBbiolab，NPL-001,)与目的片段分别用SfiI进行酶切，50℃过夜酶切，回收目的片段，按照连接摩尔比例载体：片段＝1:3的比例连接。将连接产物电转化至大肠杆菌感受态细胞TG1中，每一只羊驼的连接产物进行10次电击转化。通过梯度稀释铺板，计算库容的大小，两只羊驼的噬菌体文库大小分别为：1.21×10 9和1×10 9。随机从测滴度平板上挑取96个克隆进行鉴定，结果表明插入率为100％。
1.2针对ANG2的重链单域抗体淘选：
用hANG2蛋白10μg/孔包被平板，4℃过夜。第二天用1％BSA室温封闭2小时后，加入100μl噬菌体(2×10 9pfu/孔，来自1.1所构建的重链单域抗体噬菌文库)，37℃孵育1小时。之后用PBST(PBS中含有0.05％吐温20)洗5遍，以洗掉不结合的噬菌体。最后用甘氨酸-盐酸(200mM)将与hANG2特异性结合的噬菌体洗脱下来，并感染处于对数期生长的大肠杆菌TG1，产生并纯化噬菌体用于下一轮的筛选。相同筛选过程重复2轮后，将获得的噬菌体感染大肠杆菌TG1并铺板，从平板上挑取单克隆并测序。根据序列比对结果分析各个克隆的蛋白序列，把CDR1、CDR2、CDR3序列不相同的克隆视为不同抗体株，最终共获得10株不同的抗体。
表2.重链单域抗体可变区氨基酸序列信息(Numbering Scheme IMGT)
Figure PCTCN2021117697-appb-000003
Figure PCTCN2021117697-appb-000004
Figure PCTCN2021117697-appb-000005
实施例2：针对ANG2的重链单域抗体的初步评价鉴定
1.重链单域抗体在宿主菌大肠杆菌中表达：
将所获得10株重链单域抗体的TG1宿主菌挑选单菌落接种、培养过夜，第二天将过夜菌种转接扩增，0.5mM IPTG诱导，37℃摇床培养过夜。第二天，离心收集上清检测。
2.ELISA检测10个重链单域抗体亲和力：
将hANG2蛋白溶液和BSA分别以2μl/孔包被酶标板，4度过夜；弃上清，每孔加入300μl封闭液(含3％BSA的PBS)，37度封闭2小时；将离心收集的上清以每孔200μl加入到酶标板孔中，室温下静置孵育2小时，弃上清；用200μl/孔PBST(PBS含0.1％Tween20)洗涤3次；加入稀释好的HRP标记的抗VHH二抗(Genscript，A01861)，二抗以1：10000稀释后使用，稀释液为1％BSA的PBS，体积为100μl/孔，室温孵育1小时；用200μl/孔PBST洗涤5次；加TMB显色液(BD，55214)100μl/孔，37度显色8分钟。加2M HCl终止液100μl/孔，终止液加入后，在30分钟内进行酶标仪450nm读数。结果见下表3。
表3.重链单域抗体与hANG2抗原结合的OD值
克隆号 结合hANG2的OD值 结合BSA的OD值
B1-G8 2.021 0.065
B1-G3 1.572 0.067
B4-D3 1.679 0.059
B2-E2 1.962 0.070
B2-F10 2.061 0.063
B4-C11 1.58 0.071
B1-H9 1.495 0.058
B3-H7 1.82 0.056
B3-C11 1.63 0.071
B2-A6 2.39 0.049
实施例3：抗ANG2重链单域抗体-Fc的质粒制备
通过将PCR克隆的单抗重链可变区(SEQ ID NO：1～10)对应的编码DNA序列与人IgG1重链恒定区(GenBank No.AK303185.1)对应的编码DNA连接，来构建重链抗体，以获得抗ANG2重链单域抗体-Fc表达质粒，载体一般用pcDNA3.1(-)(购自Invitrogen)或其他真核表达载体。所述的重链抗体蛋白序列的可变区与恒定区之间使用连接肽相连，所述连接肽为(GGGGS)n，n＝1、2或者3。
表4.抗ANG2重链单域抗体-Fc氨基酸序列
Figure PCTCN2021117697-appb-000006
Figure PCTCN2021117697-appb-000007
Figure PCTCN2021117697-appb-000008
实施例4.抗ANG2重链单域抗体-Fc的表达与纯化
利用无内毒素大量提取质粒试剂盒(Qiagen，货号12391)进行质粒提取，具体操作按照厂商提供的说明书进行。CHO-S细胞培养根据厂商提供的说明书在CD CHO培养基(Gibco，货号10743-029)中，置于37℃，5％CO 2细胞培养箱中进行培养，准备好细胞后，将含有抗ANG2重链单域抗体-Fc序列的质粒一起共转染到CHO-S细胞中，以表达抗体-Fc。在转染后的第二天，培养温度下调到32℃，并每天补加3.5％2×EFC+(Gibco，货号A2503105)，培养14天后，800×g离心收获表达上清。并用0.22μm滤膜过滤。通过蛋白A亲和层析和阳离子交换层析纯化得到培养上清液中的抗体-Fc。通过在280nm处的UV吸光度以及每种蛋白对应的消光系数测定纯化的抗体-Fc浓度。通过SDS-PAGE和SE-HPLC评估抗体-Fc的纯度和均质性。或者使用离子交换和Superdex 200的SEC进行二次纯化制备高纯度抗体-Fc样品备用。
实施例5.抗ANG2重链单域抗体-Fc结合ANG2的亲和力
本实施例使用ELISA检测方法评估10个抗ANG2重链单域抗体-Fc亲和力。
将ANG2蛋白溶液分别以2μl/孔包被酶标板，4度过夜；弃上清，每孔加入300μl封闭液(含3％BSA的PBS)，37度封闭2小时；梯度稀释抗ANG2重链单域抗体-Fc，稀释液为1％BSA的PBS。比如稀释的初始浓度为500nM，10倍稀释，稀释成8个浓度梯度。将稀释好的抗ANG2重链单域抗体-Fc以每孔200μl加入到酶标板孔中，室温下静置孵育2小时，弃上清；用200μl/孔PBST(PBS含0.1％Tween20)洗涤3次；加入稀释好的HRP标记的抗人Fc二抗(SIGMA，A8667)，二抗以1：20000稀释后使用，稀释液为1％BSA的PBS，体积为100μl/孔，室温孵育1小时；用200μl/孔PBST洗涤5次；加TMB显色液(BD，55214)100μl/孔，37度显色8分钟。加2M HCl终止液100μl/孔，终止液加入后，在30分钟内进行酶标仪450nm读数。用GraphPad Prism6.0软件分析数据，进行亲和力的拟合，得到EC50值，结果见图1、图2和表5。B2-E2-Fc对人ANG2和猴ANG2蛋白的亲和力最高。
表5.重链单域抗体对ANG2蛋白的亲和力
Figure PCTCN2021117697-appb-000009
Figure PCTCN2021117697-appb-000010
实施例6.抗ANG2重链单域抗体-Fc抑制hANG2与其受体hTIE2的结合
用100μL的2μg/mL在PBS中制备的hTIE2-Fc(Novoprotein，CW98)处理96孔板，并在4℃孵育过夜。然后将板用含有0.1％Tween-20的PBS洗涤缓冲液洗涤三次，接着加入300μL含有1％BSA的PBS封闭2小时。将待测抗ANG2重链单域抗体-Fc稀释后与终浓度为10μg/mL的hANG2混合，37℃孵育1小时后，将抗原抗体混合物加入96孔板，并将在37℃孵育1小时。用含有0.1％Tween-20的PBS洗涤缓冲液洗涤后，加入稀释好的HRP标记的抗His二抗(Proteintech，66005-1-Ig)，二抗以1：2000稀释后使用，稀释液为1％BSA的PBS，体积为100μl/孔，室温孵育1小时；用200μl/孔PBST洗涤5次；加TMB显色液(BD，55214)100μl/孔，37度显色8分钟。加2M HCl终止液100μl/孔，终止液加入后，在30分钟内进行酶标仪450nm读数。用GraphPad Prism6.0软件分析数据，进行亲和力的拟合，得到EC50值，结果见图3，B2E2-Fc对人ANG2与其受体TIE2的结合有明显的抑制作用。
实施例7：抗ANG2重链单域抗体-Fc的酸稳定性和热稳定性评估
按照以下酸稳定性和热稳定性评估方法对B2-E2-Fc进行评估。抗ANG2重链单域抗体-Fc分子进行protein A亲和层析时，在酸洗脱步骤中(使用pH3.5的柠檬酸缓冲液)，洗脱下来的抗ANG2重链单域抗体-Fc溶液不进行中和，在该缓冲液中保持一段时间后，在第30min取样加入1/10体积的1M Tris-HCl(pH 8.0)进行中和，并进行该样品的HPLC-SEC检测。抗体分子在经过pH 3.5处理30min后未出现聚集或降解现象，纯度变化小于4％，说明其在酸性环境中能保持稳定性。同时，在经40℃培养箱孵育14天后进行该样品的HPLC-SEC检测，未出现聚集或降解现象，纯度变化小于4％，说明其在40℃环境中能保持稳定性。如表6和表7结果显示，B2-E2-Fc具有良好的酸稳定性和热稳定性。
表6.抗ANG2重链单域抗体-Fc酸稳定性评估结果
Figure PCTCN2021117697-appb-000011
表7.抗ANG2重链单域抗体-Fc热稳定性评估结果
Figure PCTCN2021117697-appb-000012
实施例8.构建双特异性抗体及应用
(1)双特异性抗体构建
按表8所示的双特异性抗体氨基酸序列，进行DNA编码的逆向翻译和DNA片段的合成，构建至表达载体pcDNA3.1中，瞬时转染至293或者CHO细胞中表达。收获上清进行蛋白纯化，获得纯度不低于95％的双特异性抗体Y400C和Y400E。具体载体构建，瞬时转染和蛋白纯化方法参加前述的各实施例。所述双特异性抗体的结构为：包含轻链和重链，其中轻-重链配对并形成链间二硫键，两个重链配对并形成链间二硫键，其中重链为(VH)-(CH1)-(铰链区)-(Fc)-(连接肽)-(VHH)，轻链为(VL)-(轻链恒定区)，VH和VL为抗VEGF抗体的重链和轻链可变区，VHH为抗ANG2重链单域抗体的可变区。
表8：双特异性抗体氨基酸序列(Numbering scheme IMGT)
Figure PCTCN2021117697-appb-000013
Figure PCTCN2021117697-appb-000014
(2)双特异性抗体的热稳定性考察
将双特异性抗体放置在40度培养箱处理14天或者28天后进行该样品的HPLC-SEC检测。结果发现，本发明抗体分子在经40℃处理28天后未出现聚集或降解现象，纯度变化小于4％，说明其在40℃环境中能保持稳定性，如表9所示。
表9：抗体热稳定性评估结果
Figure PCTCN2021117697-appb-000015
结果显示，双抗体分子Y400C和Y400E均具有良好热稳定性。
(3)双特异性抗体与hANG2的结合
用Sensor Chip ProteinA芯片(GE，货号：28995056)捕获供试抗体，抗原人ANG2蛋白(623-AN-025/CF，R&D)作为分析物检测其与供试品结合的动力学和亲和力数据。抗原与供试品结合的检测起始浓度为10nM，在此基础上，2倍梯度稀释，即抗原稀释的浓度分别为10nM、5nM、2.5nM、1.25nM、0.625nM，抗原依次从低浓度到高浓度开始进样，并设置1个阴性对照(即1×HBS-EP+buffer)和1个重复浓度(一般为最低浓度重复)，进样前至少进行3次Start up(1×HBS-EP+buffer)的冲洗流程以平衡系统，检测抗原与供试品的结合和解离趋势，当解离完成后，再生试剂进样，对芯片进行再生，芯片再生完成后，进行下一个浓度的检测。检测完成后，利用数据分析软件(Biacore T200 Evaluation Software)中的1:1Binding拟合方式进行数据拟合试验结果见表10。
表10：抗体对人ANG2亲和力检测结果
Figure PCTCN2021117697-appb-000016
与人ANG2的亲和力检测结果显示，双特异性抗体对人ANG2具有较强的亲和力。
(4)双特异性抗体抑制hANG2与HUVEC细胞的结合
将生长状态良好的HUVEC细胞(H-003，Allcells)用制备成单细胞悬液。细胞计数后，以10 5个细胞/孔铺入96孔板；固定人ANG2蛋白(623-AN-025/CF，R&D)的终浓度10μg/ml，各孔按照实验设计分别加入待测抗体，抗体最高浓度为1000nM，依次5倍稀释，共设置7个浓度梯度，同时设置空白对照和阳性对照RG7716(ATAD00534，Atagenix)；将细胞置于室温孵育30min后，各孔细胞洗3次、重悬后，加入荧光标记二抗PE anti-His(362603，Biolegend)，室温避光孵育30min；各孔细胞洗涤3次、重悬后流式上机检测。如图4所示，双特异性抗体Y400C和Y400E对ANG2与HUVEC细胞的阻断活性优于对照阳性抗体。
表11：抗体抑制人ANG2与HUVEC结合的生物学活性
样品 IC50(nM)
Y400C 19.43
Y400E 30.29
B2-E2-Fc 11.19
RG7716 53.87
(5)双特异性抗体不抑制hANG1与HUVEC细胞的结合
在ANG1/ANG2-TIE2这个信号通路相关的生物学功能中，保存ANG1活性对某些抗血管生成的治疗是有益的。因此本发明中的抗体特异性结合ANG2但不结合ANG1，同时只抑制ANG2和其受体TIE2的结合但不抑制ANG1和其受体TIE2的结合。本发明的抗体在抑制ANG2的促血管生成活性和治疗由血管生成过程导致的或与其相关的疾病和病症中特别有用。
将生长状态良好的HUVEC细胞(H-003，Allcells)用制备成单细胞悬液。细胞计数后，以10 5个细胞/孔铺入96孔板；固定人ANG1蛋白(623-AN/CF，R&D)的终浓度10μg/ml，各孔按照实验设计分别加入待测抗体，抗体最高浓度为1000nM，依次5倍稀释，共设置7个浓度梯度，同时设置空白对照和阳性对照hTIE2-Fc(Novoprotein，CW98)；将细胞置于室温孵育30min后，各孔细胞洗3次、重悬后，加入荧光标记二抗PE anti-His(362603，Biolegend)，室温避光孵育30min；各孔细胞洗涤3次、重悬后流式上机检测。如图5所示，双特异性抗体Y400C和Y400E对ANG1与HUVEC细胞没有结合阻断活性。
(6)双特异性抗体抑制hANG2依赖的HUVEC细胞的AKT磷酸化
HUVEC细胞表面天然表达ANG2蛋白的受体，当ANG2与细胞表面受体结合，会激活AKT磷酸化反应。Phospho-AKT分析试剂盒(64AKSPE1-1，Cisbio)包含两种标记抗体：一种带有供体荧光团，另一种带有受体。选择第一抗体的原因在与其与蛋白质上磷酸化基序的特异性结合，第二抗体是由于其识别蛋白质的能力独立于其磷酸化状态而选择的。蛋白质的磷酸化使免疫复合物的形成既涉及标记的抗体，又使供体的荧光团与受体紧密接近，从而产生信号。在本实验中，HUVEC细胞与连续稀释的样品和固定浓度的ANG2共孵育。通过HUVEC细胞的AKT磷酸化程度，评估ANG2功能活性以及样品对ANG2功能活性的抑制作用。将生长状态良好的HUVEC细胞(70013502，ATCC)制备成单细胞悬液。细胞计数后，以10 5个细胞/孔铺入96孔板；固定人ANG2蛋白(623-AN-025/CF，R&D)的终浓度10μg/ml，各孔按照实验设计分别加入待测抗体，抗体最高浓度为1000nM，依次5倍稀释，共设置8个浓度梯度，同时设置空白对照和阳性对照RG7716(ATAD00534，Atagenix)；将细胞置于37度孵育10min后，移去孔内上清，加入裂解液后，室温震荡孵育30min，吸取细胞裂解液至384孔板，加入检测试剂避光孵育4小时，使用PHERAstar进行检测。如图6所示，双特异性抗体Y400C和Y400E对hANG2依赖的HUVEC细胞的AKT磷酸化抑制活性优于对照阳性抗体。
表12：抗体抑制人ANG2依赖的HUVEC细胞的AKT磷酸化
样品 IC50(nM)
Y400C 1.262
Y400E 2.269
RG7716 27.31
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (14)

  1. 抗ANG2重链单域抗体，其包含SEQ ID NO:1-10任一项所示的重链可变区包含的HCDR1，HCDR2和HCDR3，或其变体，
    优选地，按照IMGT编号系统，包含选自以下各项组成的组的HCDR1，HCDR2和HCDR3:
    (1)SEQ ID NO:21所示的HCDR1，SEQ ID NO:22所示的HCDR2和SEQ ID NO:23所示的HCDR3，
    (2)SEQ ID NO:34所示的HCDR1，SEQ ID NO:35所示的HCDR2和SEQ ID NO:36所示的HCDR3，
    (3)SEQ ID NO:37所示的HCDR1，SEQ ID NO:38所示的HCDR2和SEQ ID NO:39所示的HCDR3，
    (4)SEQ ID NO:40所示的HCDR1，SEQ ID NO:41所示的HCDR2和SEQ ID NO:42所示的HCDR3，或SEQ ID NO:40所示的HCDR1、SEQ ID NO:41所示的HCDR2和SEQ ID NO:42所示的HCDR3的变体，及其组合，所述变体为与SEQ ID NO:40所示的HCDR1，SEQ ID NO:41所示的HCDR2和SEQ ID NO:42所示的HCDR3相比分别具有一个或多个(优选1个、2个或3个)保守氨基酸突变(优选置换，插入或缺失)并保留与ANG2结合亲和力的氨基酸序列，
    优选地，SEQ ID NO:40所示的HCDR1，SEQ ID NO:41所示的HCDR2和SEQ ID NO:42所示的HCDR3，其中SEQ ID NO:40所示的CDR1中第5-8位和第10-11位氨基酸、SEQ ID NO:41所示的CDR2中第2-5位氨基酸和第7位、SEQ ID NO:42所示的CDR3第1位氨基酸、第3-8位氨基酸选自氨基酸X，所述氨基酸X选自Ala，Arg，Asn，Asp，Cys，Gln，Glu，Gly，His，Ile，Leu，Lys，Met，Phe，Pro，Ser，Thr，Trp，Tyr和Val组成的组，
    优选地，SEQ ID NO:40所示HCDR1的变体，其中第1位的甘氨酸G可以被选自以下各项组成的组的氨基酸替换：Ala，Val，Leu和Ile；第2位的苯丙氨酸F可以被Tyr替换；第3位的脯氨酸P可以被选自以下各项组成的组的氨基酸替换：Trp和His；第4位的Ser可以被Thr替换；第9位的Ser可以被Thr替换；第12位的Gln可以被Asn替换；
    SEQ ID NO:41所示的HCDR2的变体，第1位的Ile可以被选自以下各项组成的组的氨基酸替换：Ala，Val，leu和Gly；第6位的Leu可以被Ile替换；第8位的Lys可以被Arg替换；
    SEQ ID NO:42所示的HCDR3的变体，其中第2位的Val可以被选自以下各项组成的组的氨基酸替换：Ala，Gly，Leu和Ile；第9位的Asp可以被Glu替换，
    (5)SEQ ID NO:43所示的HCDR1，SEQ ID NO:44所示的HCDR2和SEQ ID NO:45所示的HCDR3，
    (6)SEQ ID NO:46所示的HCDR1，SEQ ID NO:44所示的HCDR2和SEQ ID  NO:48所示的HCDR3，
    (7)SEQ ID NO:49所示的HCDR1，SEQ ID NO:44所示的HCDR2和SEQ ID NO:48所示的HCDR3，
    (8)SEQ ID NO:52所示的HCDR1，SEQ ID NO:44所示的HCDR2和SEQ ID NO:48所示的HCDR3，和
    (9)SEQ ID NO:55所示的HCDR1，SEQ ID NO:56所示的HCDR2和SEQ ID NO:57所示的HCDR3。
  2. 权利要求1所述的抗ANG2重链单域抗体，其中所述ANG2选自人ANG2、兔ANG2、或猴ANG2。
  3. 权利要求1或2所述的抗ANG2重链单域抗体，其包含重链可变区，所述重链可变区包含SEQ ID NO:1-10任一项所示的序列或与SEQ ID NO:1-10任一项所述的序列具有至少80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同源性且具有ANG2结合活性的序列，或由其组成。
  4. 重组蛋白，其包含权利要求1-3任一项所述的抗ANG2重链单域抗体，优选地，所述重组蛋白进一步包含生物活性物质，如具有酶活性的毒素或其活性片段(如相思豆毒蛋白，蓖麻毒蛋白A，假单胞菌外毒素或白喉毒素)，肿瘤坏死因子或干扰素(如IFN-γ)，生物反应调节物(如淋巴因子，IL-2，IL-2，IL2-6，IL-10，粒GM-CSF)，Fc片段(如来自IgG(例如IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM的Fc区，例如来自人、兔或猴的IgG，IgA1，IgA2，IgD，IgE或IgM的Fc区)，优选地，所述抗ANG2重链单域抗体和所述Fc片段通过连接肽连接，优选地，所述连接肽为(GGGGS)n，n＝1、2或者3，优选地，所述Fc片段为人IgG1重链恒定区，更优选为GenBank No.AK303185.1所示的Fc片段，更优选地，所述重组蛋白包含SEQ ID NO:11-20所示的序列或由其组成。
  5. 多特异性抗体，优选双特异性抗体，其包含权利要求1-3任一项所述的抗ANG2重链单域抗体，优选所述双特异性抗体的结构为：包含轻链和重链，其中轻-重链配对并形成链间二硫键，两个重链配对并形成链间二硫键，其中重链为(VH)-(CH1)-(铰链区)-(Fc)-(连接肽)-(VHH)，轻链为(VL)-(轻链恒定区)。
  6. 权利要求5所述的多特异性抗体，其中VHH为权利要求1-3任一项所述的抗ANG2单域抗体，VH和VL为第二种抗体的重链和轻链可变区，优选所述第二种抗体针对的抗原选自免疫细胞表面抗原、肿瘤抗原、病毒、细菌、内毒素、细胞因子、或其组合；更优选地选自：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，和/或CD16a，优选第二种抗体针对的抗原为VEGF，更优选第二抗体选自抗VEGF抗体，其包含SEQ ID NO:24所示的重链可变区包含的HCDR1，HCDR2和HCDR3(优选按照IMGT编号系统， 包含SEQ ID NO:50所示的HCDR1，SEQ ID NO:51所示的HCDR2和SEQ ID NO:53所示的HCDR3)和SEQ ID NO:27所示的轻链可变区包含的LCDR1，LCDR2和LCDR3(优选按照IMGT编号系统，包含SEQ ID NO:54所示的LCDR1，SEQ ID NO:58所示的LCDR2和SEQ ID NO:59所示的LCDR3)，更优选，所述抗VEGF抗体的重链可变区包含SEQ ID NO:24所示的序列或与SEQ ID NO:24所示的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同源性的序列，或由其组成；所述抗VEGF抗体的轻链可变区包含SEQ ID NO:27所示的序列或与SEQ ID NO:27所示的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％同源性的序列，或由其组成。
  7. 权利要求5或6所述的多特异性抗体，其中CH1的序列如SEQ ID NO:25所示，铰链区的序列如SEQ ID NO:26所示，Fc恒定区的序列如SEQ ID NO:29所示，连接肽的序列如SEQ ID NO:30或47所示，轻链恒定区的序列如SEQ ID NO:28所示。
  8. 多核苷酸，其编码权利要求1-3任一项所述的抗ANG2重链单域抗体、权利要求4所述的重组蛋白，或权利要求5-7任一项所述的多特异性抗体。
  9. 载体，其包含权利要求8所述的多核苷酸。
  10. 宿主细胞，其包含权利要求9所述的载体。
  11. 偶联物，其包含权利要求1-3任一项所述的抗ANG2重链单域抗体、权利要求4所述的重组蛋白，或权利要求5-7任一项所述的多特异性抗体，以及偶联部分，其中，所述偶联部分为纯化标签(如His标签)、可检测的标记、药物、毒素、细胞因子、酶、或其组合；优选地，所述偶联部分为放射性同位素、荧光物质、化学发光物质、有色物质、化疗剂、生物毒素、聚乙二醇或酶。
  12. 试剂盒，其包括包含权利要求1-3任一项所述的抗ANG2重链单域抗体、权利要求4所述的重组蛋白，权利要求5-7任一项所述的多特异性抗体或权利要求11所述的偶联物；优选地，所述试剂盒还包括特异性识别权利要求1-3任一项所述的抗ANG2重链单域抗体、权利要求4所述的重组蛋白，权利要求5-7任一项所述的多特异性抗体或权利要求11所述的偶联物的抗ANG2重链单域抗体、重组蛋白或多特异性抗体；任选地，所述抗ANG2重链单域抗体、重组蛋白或多特异性抗体还包括可检测的标记，例如放射性同位素、荧光物质、化学发光物质、有色物质或酶；优选地，所述试剂盒用于检测ANG2在样品中的存在或其水平；或者
    所述试剂盒包含(1)权利要求1-3任一项所述的抗ANG2重链单域抗体、权利要求4所述的重组蛋白，权利要求5-7任一项所述的多特异性抗体或权利要求11所述的偶联物，和(2)针对其它抗原的抗体或其抗原结合片段，和/或细胞毒性剂，和/或化疗药，和任选地，使用说明书。
  13. 药物组合物，其包含权利要求1-3任一项所述的抗ANG2重链单域抗体、权利要求4所述的重组蛋白，权利要求5-7任一项所述的多特异性抗体或权利要 求11所述的偶联物；可选地，所述药物组合物还包括药学上可接受的载体和/或赋形剂；优选地，所述药物组合物为适于通过皮下注射、皮内注射、静脉内注射、肌内注射或病灶内注射施用的形式。
  14. 权利要求1-3任一项所述的抗ANG2重链单域抗体、权利要求4所述的重组蛋白，权利要求5-7任一项所述的多特异性抗体或权利要求11所述的偶联物在抑制ANG2的促血管生成活性、预防和/或治疗由血管生成过程导致的或与其相关的疾病例如眼底新生血管性疾病、类风湿性关节炎和银屑病，和/或肿瘤中的用途，或在制备用于抑制ANG2的促血管生成活性、预防和/或治疗由血管生成过程导致的或与其相关的疾病例如眼底新生血管性疾病、类风湿性关节炎和银屑病和/或肿瘤的药物中的用途。
PCT/CN2021/117697 2021-09-10 2021-09-10 抗ang2抗体及其制备方法和应用 Ceased WO2023035226A1 (zh)

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US18/690,330 US20240425575A1 (en) 2021-09-10 2021-09-10 Anti-ang2 antibody, preparation method therefor, and application thereof
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KR1020247010141A KR20240048555A (ko) 2021-09-10 2021-09-10 항 ang2 항체 및 그 제조방법과 응용
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JOJO/P/2024/0051A JOP20240051A1 (ar) 2021-09-10 2024-03-10 جسم مضاد لـ ang2، وطريقة تحضيره، واستخداماته
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