WO2022253314A1 - 结合VEGF和Ang2的双特异性结合分子以及其用途 - Google Patents

结合VEGF和Ang2的双特异性结合分子以及其用途 Download PDF

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WO2022253314A1
WO2022253314A1 PCT/CN2022/096848 CN2022096848W WO2022253314A1 WO 2022253314 A1 WO2022253314 A1 WO 2022253314A1 CN 2022096848 W CN2022096848 W CN 2022096848W WO 2022253314 A1 WO2022253314 A1 WO 2022253314A1
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seq
amino acid
acid sequence
antibody
vegf
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English (en)
French (fr)
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黎一鸣
胡思怡
荆华
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信达生物制药(苏州)有限公司
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Priority to CA3221192A priority Critical patent/CA3221192A1/en
Priority to CN202280039743.2A priority patent/CN117500828A/zh
Priority to AU2022287100A priority patent/AU2022287100A1/en
Priority to EP22815354.0A priority patent/EP4349861A1/en
Priority to KR1020247000079A priority patent/KR20240023095A/ko
Priority to IL309035A priority patent/IL309035A/en
Publication of WO2022253314A1 publication Critical patent/WO2022253314A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to antibodies against vascular endothelial growth factor (VEGF/VEGF-A) and against angiopoietin-2 (ANG-2), respectively, and simultaneously against vascular endothelial growth factor (VEGF/VEGF-A) and angiopoietin-2 Bispecific binding molecules (such as antibodies) to (ANG-2), methods for their preparation, pharmaceutical compositions comprising said antibodies or molecules, and uses thereof.
  • VEGF/VEGF-A vascular endothelial growth factor
  • ANG-2 angiopoietin-2 Bispecific binding molecules
  • Angiogenesis is involved in the pathogenesis of various diseases including solid tumors, diseases associated with intraocular neovascularization, rheumatoid arthritis and psoriasis.
  • VEGF is a potent and ubiquitous vascular growth factor.
  • VEGF family members include VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, placental growth factor (PIGF), and endocrine gland-derived VEGF (EG-VEGF).
  • the active form of VEGF is synthesized as a homodimer or heterodimer with other VEGF family members.
  • VEGF-A exists in six isoforms generated by alternative splicing: VEGF121, VEGF145, VEGF165, VEGF183, VEGF189 and VEGF206. These isoforms differ mainly by their bioavailability, with VEGF165 being the predominant isoform.
  • VEGF is believed to be an important regulator of normal and disease-associated angiogenesis.
  • human angiogenin is also thought to be involved in vascular development and postnatal angiogenesis.
  • Human angiopoietins include the naturally occurring agonist Angiopoietin-1 (ANG-1 ) and the naturally occurring antagonist Angiopoietin-2 (ANG-2).
  • ANG-1 is thought to be conserved in adults, where it is ubiquitously and constitutively expressed.
  • ANG-2 expression is largely restricted to sites of vascular remodeling, where it is thought to block the constitutive stabilization or maturation function of ANG-1, thereby allowing vessels to return to plasticity that may be more responsive to sprouting signals state and remain in this plastic state.
  • bispecific antibodies targeting VEGF-A and ANG-2 have been developed (eg WO2012131078 and WO2014009465).
  • the existing bispecific antibodies have poor blocking properties against VEGF and Ang2, and because the molecular weight is too large, the molar concentration in a single administration is low.
  • smaller molecular weight antibodies applied intravitreally are often used and require less frequent dosing. Therefore, there is still a need for new bispecific binding molecules targeting VEGF-A and ANG-2, especially suitable for ocular diseases.
  • the present invention develops novel VHH antibodies targeting VEGF-A or ANG-2, as well as bispecific binding molecules targeting both VEGF-A and ANG2.
  • the bispecific binding molecule of the present invention has a smaller molecular weight, and at the same mass concentration, the molar concentration is higher; and it has stronger VEGF A and Ang2 blocking activity, and can completely block Breakdown of VEGFA-induced primary cell proliferation. Therefore, the molecule of the present invention has stronger blocking activity clinically, and can make the molar concentration of the antibody higher in a single administration, maintain the drug effect of a single administration for a longer time, reduce ocular application (such as glass body cavity injection) frequency.
  • Figure 1 shows the structure of a bispecific binding molecule.
  • Figure 2 shows the ability of anti-VEGF A VHH antibodies to block the binding of VEGF A to VEGFR2 as determined by ELISA.
  • Figure 3 shows the effect of humanized anti-Ang2 VHH antibody blocking Ang2 binding to Tie2 determined by ELISA method.
  • Figure 4 shows the effect of anti-Ang2 VHH antibody (A) and humanized anti-Ang2 VHH antibody (B) on inhibiting hAng2-Fc-induced phosphorylation of 293-Tie2 cells by ELISA method.
  • Figure 5 shows the application of HEK293-KDR reporter method to detect the effect of anti-VEGF A VHH on blocking VEGFA activation of KDR receptor.
  • Figure 6 shows the effect of anti-VEGF A VHH antibodies determined by CCK-8 on inhibiting VEGF A-induced survival and proliferation of HUVEC cells.
  • Figure 7 shows the application of the HEK293-KDR reporter method to detect the effect of VEGF A/Ang2 bispecific binding molecule IEX04-012 on blocking VEGF activation of KDR receptors.
  • Figure 8 shows the effect of bispecific binding molecules IEX04-008, IEX04-010 and IEX04-012 in inhibiting VEGF-induced survival and proliferation of HUVEC cells.
  • Figure 9 shows that the bispecific binding molecules IEX04-008, IEX04-010 and IEX04-012 block the binding of human Ang2 to Tie2 as determined by ELISA.
  • Figure 10 shows flow cytometric assays to determine that bispecific binding molecules IEX04-008, IEX04-010 and IEX04-012 block the binding of Ang2-Fc to Tie2.
  • Figure 11 shows that the bispecific binding molecule IEX04-012 of the present invention effectively inhibits hAng2-Fc-induced phosphorylation of 293-Tie2 in vitro as determined by flow cytometry.
  • Figure 12 shows that the bispecific binding molecule IEX04-012 of the present invention reduces VEGF-induced permeability of vascular endothelial cells, that is, inhibits VEGF-induced HUVEC cell leakage.
  • Figure 13 shows the statistics of the proportion of laser spots of grade 4 (figure A) and above grade 3 (figure B) in the laser-induced choroidal neovascularization model.
  • Figure 14 shows retinal thickness statistics in a laser-induced choroidal neovascularization model.
  • Figure 15 shows the leakage area statistics in the laser-induced choroidal neovascularization model
  • Figure 16 shows the H&E staining image (A) and statistics of the lesion area (B) of the fundus tissue of the laser-induced choroidal neovascularization model.
  • Figure 17 shows the CD31 staining pattern (A) and positive cell statistics (B) of the fundus tissue of the laser-induced choroidal neovascularization model.
  • the term “comprising” or “comprising” means including stated elements, integers or steps, but not excluding any other elements, integers or steps.
  • the term “comprising” or “comprises” is used, unless otherwise specified, it also covers the situation consisting of the mentioned elements, integers or steps.
  • an antibody variable region that "comprises” a particular sequence it is also intended to encompass an antibody variable region that consists of that particular sequence.
  • VEGF refers to vascular growth factor.
  • VEGF family members include VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, placental growth factor (PIGF), and endocrine gland-derived VEGF (EG-VEGF).
  • the active form of VEGF is synthesized as a homodimer or heterodimer with other VEGF family members.
  • VEGF-A exists in six isoforms generated by alternative splicing: VEGF121, VEGF145, VEGF165, VEGF183, VEGF189 and VEGF206. These isoforms differ mainly by their bioavailability, with VEGF165 being the predominant isoform.
  • the VEGF A of the present invention refers to VEGF A from humans, such as VEGF 165 from humans.
  • the amino acid sequence of the VEGFA of the present invention is the amino acid sequence with accession number P15692 (uniprot database).
  • ANG2 refers to human angiopoietin-2 (ANG-2) (alternatively abbreviated: ANGPT2 or ANG2), which is described, for example, in Maisonpierre, P.C. et al., Science 277 (1997) 55-60 and Cheung , described in A.H. et al., Genomics 48 (1998) 389-91.
  • Ang1 and Ang2 were discovered as ligands of Tie, a family of tyrosine kinases selectively expressed in the vascular endothelium. There are currently four identified members of the angiopoietin family.
  • Angiopoietin-3 and -4 may represent widely diverse counterparts of the same locus in mouse and human.
  • Ang1 and Ang2 were initially identified as agonists and antagonists, respectively, in tissue culture experiments (for ANG1, see Davis, S. et al., Cell87 (1996) 1161-69; for ANG2, see Maisonpierre, P.C. et al., Science277 (1997 )55-60). All known angiopoietins bind primarily to Tie2.
  • ANG2 of the present invention refers to Ang2 from human.
  • human Ang2 comprises the amino acid sequence with accession number 015123 (uniprot database).
  • multispecific binding molecule refers to a multispecific binding molecule that is at least bispecific, such as a bispecific binding molecule, i.e. the molecule comprises at least a first target binding region and a second target binding region, wherein the The first target binding region binds one target or antigen and the second target binding region binds another antigen or target.
  • the molecules according to the invention comprise specificities for at least two different antigens or targets.
  • Molecules according to the invention also encompass multispecific molecules comprising multiple target binding regions/binding sites, such as trispecific binding molecules.
  • bispecific binding molecules of the invention are bispecific antibodies.
  • linker refers to any molecule that enables the direct linking of different parts of a bispecific binding molecule.
  • linkers that establish covalent linkages between different molecular moieties include peptide linkers and non-proteinaceous polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene or polyethylene glycol, polypropylene glycol copolymer.
  • the linker is a peptide linker, which refers to a sequence of amino acids, wherein the sequence joins the amino acid sequence of the first part of the binding molecule to the second part of the binding molecule.
  • a peptide linker can link a first target-binding region of a binding molecule to a second target-binding region.
  • a peptide linker can also join one part of an antibody to another part of an antibody, such as joining a light chain variable region to a heavy chain variable region.
  • the peptide linker is of a length sufficient to link the two entities in such a way that they maintain their conformation relative to each other so as not to interfere with the desired activity.
  • the peptide linker may or may not consist essentially of the following amino acid residues: Gly, Ser, Ala or Thr.
  • Useful linkers include glycine-serine polymers including, for example, (GS) n , (GSGGS) n , (GGGGS) n , (GGGS) n, and (GGGGS) nG , where n is at least 1 (and preferably 2, 3, 4, 5, 6, 7, 8, 9, 10) integers.
  • Useful linkers also include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers.
  • Useful linkers also include glycine monomer polymerizations, such as (G) n , where n is an integer of at least 4 (and, for example, 4-20, such as 4, 5, 6, 7, 8, 9, 10 and more).
  • valence means that there is a specified number of binding sites in a binding molecule, eg an antibody molecule.
  • bivalent, trivalent, tetravalent indicate the presence, respectively, of two, three or four binding sites (target binding regions) in the binding molecule.
  • Bispecific binding molecules according to the invention are at least bivalent and may be multivalent, eg bivalent, trivalent, tetravalent or hexavalent.
  • target binding region refers to any portion of a multispecific binding molecule, such as a bispecific binding molecule, that binds a particular target or antigen.
  • the target binding region can be, for example, an antibody or immunoglobulin itself or an antibody fragment. Such a target binding region may or may not have a tertiary structure independent of the remainder of the BsAB, and may or may not bind its target as a separate entity.
  • the target binding region can also be a receptor or a ligand, or a domain of a receptor capable of binding a ligand.
  • antibody fragment includes a portion of an intact antibody.
  • antibody fragments are antigen-binding fragments.
  • Antigen-binding fragment refers to a molecule, distinct from an intact antibody, that comprises a portion of an intact antibody and that binds the antigen to which the intact antibody binds.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; dAb (domain antibody); linear antibodies; single chain antibodies (e.g. scFv); ; a diabody or a fragment thereof; or a camelid antibody.
  • VHH also known as single domain antibody (single domain antibody, sdAb) refers to a genetically engineered antibody consisting only of the variable region of the heavy chain antibody (Variable region), which only contains the 3 HCDRs of the variable region of the heavy chain .
  • VHH has antigen specificity and high affinity with only 3 HCDRs, while ordinary antibodies need 6 CDRs.
  • Target refers to a bound substance to which a binding molecule is directed.
  • Targets can be antigens, ligands or receptors.
  • antigen refers to a molecule that elicits an immune response. This immune response may involve antibody production or activation of specific immune cells, or both.
  • antigens can be derived from recombinant or genomic DNA.
  • epipe refers to the portion of an antigen (eg, VEGF or Ang2) that specifically interacts with an antibody molecule.
  • a “complementarity determining region” or “CDR region” or “CDR” is an antibody variable domain that is hypervariable in sequence and forms a structurally defined loop ("hypervariable loop") and/or contains antigen-contacting residues ( "antigen contact point”).
  • the CDRs are primarily responsible for binding to antigenic epitopes.
  • the CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially starting from the N-terminus.
  • the CDRs located within the variable domain of an antibody heavy chain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the variable domain of an antibody light chain are referred to as LCDR1, LCDR2, and LCDR3.
  • each CDR can be determined using any one or combination of a number of well-known antibody CDR assignment systems, including For example: Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al, "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology , 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, U.S.
  • the residues of each CDR are as follows.
  • a CDR can also be determined based on having the same Kabat numbering position as a reference CDR sequence (eg, any of the exemplary CDRs of the invention).
  • the CDRs in the VHH of the present invention follow the rules: HCDR1 is determined according to AbM, and HCDR2 and HCDR3 are determined according to Kabat.
  • the boundaries of the CDRs of the variable region of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable region of the same antibody defined under different assignment systems are different.
  • the scope of said antibody also covers antibodies whose variable region sequences comprise said particular CDR sequence, but due to the application of a different protocol (e.g. Different assignment system rules or combinations) cause the claimed CDR boundary to be different from the specific CDR boundary defined in the present invention.
  • Antibodies with different specificities have different binding sites for different antigens
  • CDRs vary from antibody to antibody, only a limited number of amino acid positions within a CDR are directly involved in antigen binding.
  • a minimal binding unit may be a subsection of a CDR.
  • the residues of the remainder of the CDR sequences can be determined from the structure and protein folding of the antibody. Accordingly, the invention also contemplates variations of any of the CDRs presented herein. For example, in a variant of a CDR, the amino acid residues of the smallest binding unit can remain unchanged, while the remaining CDR residues defined according to Kabat or Chothia can be replaced by conserved amino acid residues.
  • Fc region is used herein to define the CH2 and CH3 constant regions of an immunoglobulin heavy chain and the term includes native sequence Fc regions and variant Fc regions.
  • the natural or wild-type Fc region can bind to different Fc receptors on the surface of immune cells, thereby causing CDC ⁇ ADCC ⁇ ADCP effector functions.
  • Such effector functions generally require the association of an Fc region with a binding domain (eg, an antibody variable region).
  • the Fc region is mutated to enhance its CDC ⁇ ADCC ⁇ ADCP effector function.
  • the Fc region is mutated to attenuate or delete its CDC ⁇ ADCC ⁇ ADCP effector function.
  • a “humanized” antibody is one that comprises amino acid residues from non-human CDRs and amino acid residues from human FRs.
  • a humanized antibody will comprise substantially all of at least one, usually two, variable domains, wherein all or substantially all of the CDRs (e.g., CDRs) correspond to those of a non-human antibody, and all Or substantially all of the FRs correspond to those of a human antibody.
  • a humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody.
  • a "humanized form" of an antibody (eg, a non-human antibody) refers to an antibody that has been humanized.
  • Human antibody or “fully human antibody” or “fully human antibody” are used interchangeably and refer to an antibody having an amino acid sequence corresponding to that of an antibody derived from a human Or human cells are produced or derived from non-human sources that utilize human antibody repertoires or other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.
  • the term "anti”, “bind” or “specifically binds” means that the binding is selective for the target or antigen and can be distinguished from unwanted or non-specific interactions.
  • the ability of a binding site to bind a particular target or antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art such as by radioimmunoassay (RIA) or biofilm thin layer interferometry or MSD assay or surface plasmon resonance (SPR) assay.
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • MSD assay biofilm thin layer interferometry
  • SPR surface plasmon resonance
  • an effective amount refers to such an amount or dose of the antibody or fragment or composition or combination of the present invention, which produces the desired effect in a patient in need of treatment or prevention after being administered to the patient in single or multiple doses.
  • a “therapeutically effective amount” refers to an amount effective, at dosages required, and for periods of time required, to achieve the desired therapeutic result.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody fragment or composition or combination are outweighed by the therapeutically beneficial effects.
  • a “therapeutically effective amount” preferably inhibits a measurable parameter or improves a measurable parameter by at least about 40%, even more preferably at least about 50%, 55%, 60%, 65%, 70%, 75%, relative to an untreated subject %, 80%, 85%, 90% or even 100%.
  • prophylactically effective amount refers to an amount effective, at dosages required, and for periods of time required, to achieve the desired prophylactic result. Typically, a prophylactically effective amount will be less than a therapeutically effective amount because the prophylactic dose is administered in the subject before or at an earlier stage of the disease.
  • host cell refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parental cell, but may contain mutations. Mutant progeny screened or selected for the same function or biological activity in originally transformed cells are included herein.
  • label refers to a compound or composition that is directly or indirectly conjugated or fused to an agent, such as a polynucleotide probe or antibody, and facilitates detection of the agent to which it is conjugated or fused.
  • Labels can themselves be detectable (eg, radioisotopic or fluorescent labels) or, in the case of enzymatic labels, can catalyze the chemical alteration of a detectable substrate compound or composition.
  • the term is intended to encompass both direct labeling of a probe or antibody by conjugating (ie, physically linking) a detectable substance to the probe or antibody as well as indirect labeling of a probe or antibody by reacting with another reagent that is directly labeled.
  • the label is hFc or biotin.
  • “Individual” or “subject” includes mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., , mice and rats). In some embodiments, the individual or subject is a human.
  • an “isolated” antibody or molecule is one that has been separated from a component of its natural environment.
  • the antibody or molecule is purified to greater than 95% or 99% purity, such as by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or determined by reverse phase HPLC).
  • electrophoresis e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatography e.g., ion exchange or determined by reverse phase HPLC.
  • the sequences are aligned for optimal comparison purposes (e.g., a first and second amino acid sequence or nucleic acid sequence may be placed between a first and a second amino acid sequence or nucleic acid sequence for optimal alignment). Gaps may be introduced in one or both or non-homologous sequences may be discarded for comparison purposes).
  • the length of the reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80% , 90%, 100% of the reference sequence length.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the comparison of sequences and the calculation of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm available at http://www.gcg.com available)
  • the identity between two amino acid sequences was determined using the Blossum62 matrix or the PAM250 matrix with gap weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6. sex percentage.
  • using the GAP program in the GCG software package (available at http://www.gcg.com), using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70 or 80 and Length weights of 1, 2, 3, 4, 5 or 6 determine the percent identity between two nucleotide sequences.
  • a particularly preferred parameter set (and one that should be used unless otherwise stated) is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. It is also possible to use the PAM120 weighted remainder table, gap length penalty 12, gap penalty 4), utilize the E. Meyers and W.
  • nucleic acid sequences and protein sequences described herein can further be used as "query sequences" to perform searches against public databases, eg, to identify other family member sequences or related sequences.
  • eye disease in the present invention encompasses eye diseases involving angiogenesis (eg, diseases occurring in the eye), such as eye diseases associated with corneal neovascularization.
  • pharmaceutical excipient refers to a diluent, adjuvant (such as Freund's adjuvant (complete and incomplete)), excipient, carrier or stabilizer, etc., which are administered together with the active substance.
  • adjuvant such as Freund's adjuvant (complete and incomplete)
  • excipient carrier or stabilizer, etc.
  • composition refers to a composition that is present in a form that permits the biological activity of the active ingredients contained therein to be effective and that does not contain additional substances that are unacceptably toxic to the subject to which the composition is administered. ingredients.
  • treating means slowing, interrupting, arresting, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.
  • prevention includes the inhibition of the occurrence or development of a disease or disorder or a symptom of a particular disease or disorder.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it has been linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors.”
  • tissue sample refers to a collection of cells or fluid obtained from a patient or subject.
  • the source of tissue or cell samples can be solid tissue like from fresh, frozen and/or preserved organ or tissue samples or biopsy samples or puncture samples; blood or any blood component; body fluids such as tears, vitreous humor, cerebrospinal fluid , amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time during pregnancy or development of a subject.
  • the tissue sample is ocular tissue, such as the vitreous.
  • the sample is tear fluid or vitreous humor.
  • Tissue samples may contain compounds that are not naturally intermingled with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.
  • the present invention relates to VHH antibodies against VEGF A.
  • the anti-VEGF VHH of the present invention comprises the following 3 CDRs, HCDR1, HCDR2 and HCDR3, wherein
  • HCDR1 comprises the sequence shown in SEQ ID NO: 1, or consists of said sequence;
  • HCDR2 comprises the sequence shown in SEQ ID NO:2, or consists of said sequence;
  • HCDR3 comprises the sequence shown in SEQ ID NO:3, or consists of said sequence;
  • HCDR1 comprises the sequence shown in SEQ ID NO:6, or consists of said sequence;
  • HCDR2 comprises the sequence shown in SEQ ID NO:7 or 10, or consists of said sequence;
  • HCDR3 comprises the sequence shown in SEQ ID NO: 8, or consists of said sequence.
  • the VHH comprises the amino acid sequence described in SEQ ID NO: 4, 5, 9 or 11, or comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, or described by SEQ ID NO: 4, 5, 9 or 11 amino acid composition.
  • the VHH comprises one or more (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) Amino acid sequences mutated, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.
  • the mutation is absent in a CDR, such as HCDR1, HCDR2 or HCDR3.
  • the present invention relates to VHH antibodies against Ang2.
  • the anti-Ang2 VHH of the present invention comprises the following 3 CDRs, HCDR1, HCDR2 and HCDR3, wherein
  • HCDR1 comprises the sequence shown in SEQ ID NO: 16, or consists of said sequence;
  • HCDR2 comprises the sequence shown in SEQ ID NO: 17 or 20, or consists of said sequence;
  • HCDR3 comprises the sequence shown in SEQ ID NO: 18, or consists of said sequence.
  • the VHH comprises the amino acid sequence described in SEQ ID NO: 19 or 21, or comprises at least 90%, 91%, 92% of the amino acid sequence described in SEQ ID NO: 19 or 21 , 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, or consists of amino acids described in SEQ ID NO: 19 or 21.
  • the VHH comprises one or more (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2. 1) Amino acid sequences mutated, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.
  • the mutation is absent in a CDR, such as HCDR1, HCDR2 or HCDR3.
  • the invention relates to a bispecific binding molecule that binds VEGF A and Ang2, comprising a first target binding region that specifically binds VEGF A and a second target binding region that specifically binds Ang2, wherein the second The target binding region is an anti-Ang2 VHH, such as an anti-Ang2 VHH of the invention as described above.
  • the first target binding region is selected from
  • an antigen-binding fragment such as a scFv, of an antibody that specifically binds VEGF A, for example, the antibody is a fully human antibody or a humanized antibody; or
  • a VEGF receptor specifically binding to VEGF A or its extracellular domain or a fusion protein comprising its extracellular domain, such as a fusion protein of an extracellular domain and Fc.
  • a bispecific binding molecule of the invention comprises 1 or 2 or 3 or 4 first or second target binding domains. In some embodiments, the bispecific binding molecules of the invention comprise 2, 3 or 4 target binding regions. In some embodiments, bispecific binding molecules of the invention are bivalent or trivalent or tetravalent. In some embodiments, bispecific binding molecules of the invention are bispecific antibodies.
  • a bispecific binding molecule such as a bispecific antibody, of the invention has the following structure:
  • the anti-Ang2 VHH comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 16, or consists of said sequence; HCDR2 comprises the sequence shown in SEQ ID NO: 17 or 20, or consists of said sequence Composition; HCDR3 comprises the sequence shown in SEQ ID NO: 18, or consists of said sequence.
  • the structure of the above-mentioned bispecific binding molecule is as shown in Figure 1A or Figure 1B.
  • the bispecific binding molecule described above consists of one chain.
  • the bispecific binding molecules described above are bivalent.
  • the light chain variable region VL of the anti-VEGF antibody comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the sequence shown in SEQ ID NO:31 or consists of it; LCDR2 comprises the sequence shown in SEQ ID NO:32 Sequence or is formed by it; LCDR3 comprises the sequence shown in SEQ ID NO:33 or is formed by it.
  • the heavy chain variable region VH of the anti-VEGF antibody comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the sequence shown in SEQ ID NO:35; HCDR2 comprises the sequence shown in SEQ ID NO:36 The sequence or consists of it; HCDR3 comprises or consists of the sequence shown in SEQ ID NO:37.
  • the heavy chain variable region VH of the anti-VEGF antibody of the present invention comprises the amino acid sequence described in SEQ ID NO: 34, or comprises an amino acid sequence described in SEQ ID NO: 34 at least 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, or consists of amino acids described in SEQ ID NO:34.
  • the heavy chain variable region VH comprises one or more (preferably no more than 10, 9, 8, 7, 6, 5, 4 , 3, 2, 1) mutated amino acid sequences, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.
  • the mutation is absent in a CDR, such as HCDR1, HCDR2 or HCDR3.
  • the light chain variable region VL of the anti-VEGF antibody of the present invention comprises the amino acid sequence described in SEQ ID NO: 30, or comprises at least 90% of the amino acid sequence described in SEQ ID NO: 30 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, or consists of amino acids described in SEQ ID NO:30.
  • the light chain variable region VL comprises one or more (preferably no more than 10, 9, 8, 7, 6, 5, 4 , 3, 2, 1) mutated amino acid sequences, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.
  • the mutation is absent in a CDR, such as LCDR1, LCDR2 or LCDR3.
  • the anti-Ang2 VHH of the present invention comprises the amino acid sequence described in SEQ ID NO: 19 or 21, or comprises at least 90%, 91% of the amino acid sequence described in SEQ ID NO: 19 or 21 , 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, or consists of amino acids described in SEQ ID NO: 19 or 21.
  • the VHH comprises one or more (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2. 1) Amino acid sequences mutated, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.
  • the mutation is absent in a CDR, such as HCDR1, HCDR2 or HCDR3.
  • the linker comprises, or consists of, the amino acid sequence of SEQ ID NO: 23.
  • the anti-VEGF A ⁇ ANG2 bispecific binding molecule of the present invention comprises the amino acid sequence of SEQ ID NO:28, or comprises at least 90% of the amino acid sequence described in said SEQ ID NO:28, An amino acid sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of the amino acids set forth in SEQ ID NO:28.
  • the bispecific binding molecule comprises one or more (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) Amino acid sequences mutated, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.
  • the mutations are absent in the CDRs of the variable region of the anti-VEGF antibody and the anti-Ang2 VHH.
  • a bispecific binding molecule such as a bispecific antibody, of the invention has the following structure:
  • the anti-Ang2 VHH comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 16, or consists of said sequence; HCDR2 comprises the sequence shown in SEQ ID NO: 17 or 20, or consists of said sequence Composition; HCDR3 comprises the sequence shown in SEQ ID NO: 18, or consists of said sequence.
  • the structure of the bispecific binding molecule described above is shown in Figure 1C.
  • the bispecific binding molecule described above consists of one chain.
  • the bispecific binding molecules described above are trivalent.
  • the first anti-VEGF VHH is the same or different than the second anti-VEGF VHH.
  • the anti-VEGF VHH comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO:1, or consists of said sequence; HCDR2 comprises the sequence shown in SEQ ID NO:2, or consists of Said sequence is composed; HCDR3 comprises the sequence shown in SEQ ID NO:3, or is made up of said sequence;
  • HCDR1 comprises the sequence shown in SEQ ID NO:6, or consists of said sequence
  • HCDR2 comprises the sequence shown in SEQ ID NO:7 or 10, or consists of said sequence
  • HCDR3 comprises the sequence shown in SEQ ID NO:8 A sequence, or consisting of said sequence.
  • the anti-VEGF VHH comprises the amino acid sequence described in SEQ ID NO: 4, 5, 9 or 11, or comprises the amino acid sequence described in SEQ ID NO: 4, 5, 9 or 11 An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or an amino acid sequence derived from SEQ ID NO: 4, 5, 9 or 11 The amino acid composition described.
  • the VHH comprises one or more (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) Amino acid sequences mutated, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.
  • the mutation is absent in a CDR, such as HCDR1, HCDR2 or HCDR3.
  • the anti-VEGF VHH comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO:6, or consists of said sequence; HCDR2 comprises the sequence shown in SEQ ID NO:7 or 10, Or be made up of said sequence; HCDR3 comprises the sequence shown in SEQ ID NO:8, or be made up of said sequence.
  • the anti-VEGF VHH comprises the amino acid sequence set forth in SEQ ID NO:9 or 11, or comprises at least 90%, 91%, An amino acid sequence having 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of the amino acids set forth in SEQ ID NO:9 or 11.
  • the VHH comprises one or more (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2. 1) Amino acid sequences mutated, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.
  • the mutation is absent in a CDR, such as HCDR1, HCDR2 or HCDR3.
  • the anti-Ang2 VHH of the present invention comprises the amino acid sequence described in SEQ ID NO: 19 or 21, or comprises at least 90%, 91% of the amino acid sequence described in SEQ ID NO: 19 or 21 , 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, or consists of amino acids described in SEQ ID NO: 19 or 21.
  • the VHH comprises one or more (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2. 1) Amino acid sequences mutated, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.
  • the mutation is absent in a CDR, such as HCDR1, HCDR2 or HCDR3.
  • the linker comprises, or consists of, the amino acid sequence of SEQ ID NO: 23.
  • the anti-VEGF A ⁇ ANG2 bispecific binding molecule of the present invention comprises the amino acid sequence of SEQ ID NO:22, or comprises at least 90% of the amino acid sequence described in said SEQ ID NO:22, An amino acid sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of the amino acids set forth in SEQ ID NO:22.
  • the bispecific binding molecule comprises one or more (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) Amino acid sequences mutated, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.
  • the mutation is absent in the CDRs of the anti-VEGF VHH and anti-Ang2 VHH.
  • the bispecific binding molecule of the invention comprises one or both of the following chains:
  • the anti-Ang2 VHH comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 16, or consists of said sequence; HCDR2 comprises the sequence shown in SEQ ID NO: 17 or 20, or consists of said sequence Composition; HCDR3 comprises the sequence shown in SEQ ID NO: 18, or consists of said sequence.
  • the structure of the bispecific binding molecule described above is shown in Figure 1D.
  • the bispecific binding molecule described above consists of two chains.
  • the bispecific binding molecules described above are tetravalent.
  • the anti-Ang2 VHH comprises the amino acid sequence described in SEQ ID NO: 19 or 21, or comprises at least 90%, 91% of the amino acid sequence described in SEQ ID NO: 19 or 21 , 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, or consists of amino acids described in SEQ ID NO: 19 or 21.
  • the VHH comprises one or more (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2. 1) Amino acid sequences mutated, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.
  • the mutation is absent in a CDR, such as HCDR1, HCDR2 or HCDR3.
  • the VEGFR extracellular domain is an extracellular domain of VEGFR from a human.
  • the VEGFR extracellular domain comprises a VEGFR1 second antibody-like domain (eg, FLT1 domain2) and a VEGFR2 third antibody-like domain (eg, KDR domain3).
  • the VEGFR extracellular domain comprises a second antibody-like domain of human VEGFR1 and a third antibody-like domain of human VEGFR2.
  • the VEGFR extracellular domain comprises the amino acid sequence of SEQ ID NO: 26, or comprises at least 90%, 91%, 92%, 93% of the amino acid sequence described in SEQ ID NO: 26 %, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, or consists of amino acids described in SEQ ID NO:26.
  • the VEGFR extracellular domain comprises one or more (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutated amino acid sequence, said mutation such as substitution, deletion or addition, preferably substitution, such as conservative substitution, preferably said VEGFR extracellular domain retains the structure shown in SEQ ID NO:26 Domains are similar (eg, have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) binding affinity to VEGF.
  • the Fc is an Fc derived from human IgG1, IgG2, IgG3 or IgG4, such as a wild-type Fc, or an Fc variant known in the art.
  • the Fc comprises the amino acid sequence of SEQ ID NO: 27, or comprises at least 90%, 91%, 92%, 93%, 94% of the amino acid sequence described in SEQ ID NO: 27 , 95%, 96%, 97%, 98% or 99% identical amino acid sequence, or consists of amino acids described in SEQ ID NO:27.
  • the VEGFR extracellular domain-Fc is a fusion protein of VEGFR extracellular domain and Fc, such as Aflibercept or its derivatives.
  • the VEGFR extracellular domain-Fc comprises the amino acid sequence of SEQ ID NO: 25, or comprises at least 90%, 91%, 92% of the amino acid sequence described in SEQ ID NO: 25 %, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, or consists of amino acids described in SEQ ID NO:25.
  • the VEGFR extracellular domain-Fc comprises one or more (preferably no more than 10, 9, 8, 7, 6, 5 , 4, 3, 2, 1) mutated amino acid sequences, said mutations such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions, preferably said VEGFR extracellular domain-Fc retention and SEQ ID NO
  • the domains shown in :25 have similar (eg, have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) binding affinity to VEGF.
  • one chain of the anti-VEGF A ⁇ ANG2 bispecific binding molecule of the present invention comprises the amino acid sequence of SEQ ID NO: 24, or comprises at least one chain of the amino acid sequence described in SEQ ID NO: 24 An amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting of the amino acids set forth in SEQ ID NO:24.
  • the bispecific binding molecule comprises one or more (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) Amino acid sequences mutated, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.
  • the mutation is absent in the CDRs of the anti-Ang2 VHH.
  • the mutated VEGFR extracellular domain-Fc remains similar to the domain shown in SEQ ID NO: 25 (e.g., has at least 70%, 75%, 80%, 85%, 90% , 95%, 96%, 97%, 98%, 99%) binding affinity to VEGF.
  • the antibodies or binding molecules described herein comprise one or more amino acid mutations.
  • amino acid mutations include amino acid substitutions, insertions or deletions.
  • amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.
  • the amino acid mutations described in the present invention occur in regions outside the CDRs (eg in FRs). In some embodiments, the amino acid mutations described in the present invention occur in the heavy chain constant region of the antibody, such as the Fc region. In a preferred embodiment, the amino acid mutations in the Fc region weaken or delete the ADCC and/or Or CDC function.
  • substitutions are conservative substitutions.
  • a conservative substitution is one in which an amino acid is replaced by another within the same class, such as one acidic amino acid by another acidic amino acid, one basic amino acid by another basic amino acid, or one neutral amino acid by another neutral amino acid replacement.
  • one or more amino acid mutations can be introduced into the Fc region of the antibodies provided herein to generate Fc region variants to alter one or more functional properties of the antibody, such as serum half-life, Complement fixation, complement dependent cytotoxicity, Fc receptor binding and/or antibody dependent cytotoxicity.
  • Fc region variants may include human Fc region sequences (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc regions) comprising amino acid mutations (e.g., substitutions) at one or more amino acid positions.
  • variable region of an antibody may be desirable to mutate the variable region of an antibody to create disulfide bonds, eg, to generate scFvs comprising mutations in disulfide bonds.
  • the antibodies or binding molecules provided herein can be further modified to contain other non-proteinaceous moieties known and readily available in the art.
  • Moieties suitable for such derivatization include, but are not limited to, water soluble polymers.
  • Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl -1,3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), and dextran or poly(n-ethylene pyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyols (such as glycerin), polyvinyl alcohol
  • an anti-Ang2 VHH antibody of the invention is capable of specifically binding Ang2, e.g., human Ang2, e.g., with high affinity.
  • an anti-VEGFA VHH antibody of the invention is capable of specifically binding VEGF A, such as human VEGF A, e.g., with high affinity.
  • the bispecific binding molecules of the invention are capable of specifically binding Ang2 and VEGFA, eg human Ang2 and human VEGFA, eg with high affinity.
  • an anti-Ang2 antibody or anti-VEGFA antibody or bispecific binding molecule of the invention has one or more of the following properties:
  • the anti-Ang2 antibody or the bispecific binding molecule has an inhibitory effect on Ang2-induced Tie2 phosphorylation
  • the anti-Ang2 antibody or the bispecific binding molecule has a blocking effect on the binding between Ang2 and Tie2;
  • the anti-VEGFA antibody or bispecific binding molecule has a blocking effect on the VEGFA activation-related receptor signaling pathway, for example, detected by KDR reporter;
  • the anti-VEGFA antibody or the bispecific binding molecule has a blocking effect on the binding between VEGFA and VEGFR;
  • the anti-VEGFA antibody or bispecific binding molecule has an inhibitory effect on the survival and proliferation of VEGF A-induced cells (e.g. primary cells, e.g. vascular endothelial cells, e.g. human umbilical vein endothelial cells, e.g. HUVEC);
  • VEGF A-induced cells e.g. primary cells, e.g. vascular endothelial cells, e.g. human umbilical vein endothelial cells, e.g. HUVEC
  • the bispecific binding molecule has an inhibitory effect on the leakage of vascular endothelial cells (such as human umbilical vein endothelial cells, such as HUVEC);
  • the bispecific binding molecule has an inhibitory effect on neovascularization in vivo or in vitro, such as inhibiting fundus or retinal choroidal neovascularization, such as inhibiting leakage caused by neovascularization, such as protecting the integrity of blood vessels.
  • the invention provides a nucleic acid encoding any of the above VHH or bispecific binding molecules or any chain thereof.
  • a vector comprising said nucleic acid is provided.
  • the vector is an expression vector, such as a eukaryotic expression vector.
  • Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs).
  • the vector is eg pcDNA3.1.
  • a host cell comprising said nucleic acid or said vector is provided. In one embodiment, the host cell is eukaryotic.
  • the host cell is selected from yeast cells, mammalian cells such as CHO cells (eg CHO-S) or 293 cells (eg 293F or HEK293 cells) or other cells suitable for the production of antibodies or fragments thereof.
  • the host cell is prokaryotic, such as E. coli, such as TG1.
  • the nucleic acid of the present invention comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NO: 4, 5, 9, 11, 19, 21, 22, 24, 28, or encoding an amino acid sequence selected from any one of SEQ ID NO: :
  • the amino acid sequence shown in any one of 4, 5, 9, 11, 19, 21, 22, 24, 28 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%,
  • a host cell comprising said vector.
  • Suitable host cells for cloning or expression of vectors encoding VHH or bispecific binding molecules include prokaryotic or eukaryotic cells as described herein.
  • VHHs can be produced in bacteria. After expression, the VHH is present in the culture supernatant and can be further purified.
  • the host cell is prokaryotic, such as a bacterium, such as E. coli, such as TG1.
  • the host cell is eukaryotic.
  • the host cell is selected from yeast cells, mammalian cells, or other cells suitable for the production of antibodies or fragments thereof.
  • eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors.
  • fungal and yeast strains in which glycosylation pathways have been "humanized” result in the production of antibodies with partially or fully human glycosylation patterns.
  • Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts.
  • mammalian cell lines adapted for growth in suspension can be used.
  • useful mammalian host cell lines are the monkey kidney CV1 line transformed with SV40 (COS-7); the human embryonic kidney line (HEK293, 293F or 293T cells), and the like.
  • Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells, CHO-S cells, ExpiCHO, etc.; and myeloma cell lines such as YO, NSO and Sp2/0. Suitable mammalian host cell lines for antibody production are known in the art.
  • a method for preparing a VHH or bispecific binding molecule of the invention comprises, under conditions suitable for expression of the VHH or bispecific binding molecule or chain thereof, culturing or a nucleic acid of a bispecific binding molecule (eg, any polypeptide chain and/or multiple polypeptide chains) or a host cell comprising an expression vector of said nucleic acid, as provided above, and optionally obtained from said host cell ( or host cell culture medium) to recover the VHH or bispecific binding molecule.
  • a nucleic acid of a bispecific binding molecule eg, any polypeptide chain and/or multiple polypeptide chains
  • a host cell comprising an expression vector of said nucleic acid, as provided above, and optionally obtained from said host cell ( or host cell culture medium) to recover the VHH or bispecific binding molecule.
  • a nucleic acid encoding a VHH or bispecific binding molecule of the invention e.g., a molecule as described above, e.g., any polypeptide chain and/or multiple polypeptide chains
  • a nucleic acid encoding a VHH or bispecific binding molecule of the invention is isolated and inserted into one or Multiple vectors for further cloning and/or expression in host cells.
  • Such nucleic acids are readily isolated and sequenced using routine procedures.
  • VHHs or bispecific binding molecules prepared as described herein can be purified by known art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like.
  • the actual conditions used to purify a particular protein will also depend on such factors as net charge, hydrophobicity, hydrophilicity, and will be apparent to those skilled in the art.
  • the purity of antibody molecules of the invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.
  • VHHs or bispecific binding molecules provided herein can be identified, screened for, or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art.
  • the VHH or bispecific binding molecule of the invention is tested for its target (eg antigen) binding activity, eg by known methods such as biofilm thin layer interferometry, ELISA, and the like. Binding to VEGF A and/or Ang2 can be assayed using methods known in the art, exemplary methods are disclosed herein.
  • radioimmunoassay (RIA) or biofilm thin layer interferometry or MSD assay or surface plasmon resonance (SPR) or flow cytometry is used to measure.
  • the invention also provides assays for identifying biologically active VHHs or bispecific binding molecules.
  • Biological activity is selected from for example
  • VEGF A-induced cells e.g. primary cells, e.g. vascular endothelial cells, e.g. human umbilical vein endothelial cells, e.g. HUVEC;
  • vascular endothelial cells eg, human umbilical vein endothelial cells, such as HUVEC
  • the inhibitory effect of the bispecific binding molecule on neovascularization in vivo or in vitro such as inhibiting fundus or retinal choroidal neovascularization, such as inhibiting leakage caused by neovascularization, such as protecting the integrity of blood vessels.
  • Cells for use in any of the above in vitro assays are primary cells or cell lines, including cells that naturally express or overexpress Ang2 receptors (e.g. Tie2) or VEGFR (e.g. VEGFR2, i.e. KDR), e.g. 293 overexpressing Tie2 or KDR cells, such as HEK293 or Expi293; or vascular endothelial cells, such as human umbilical vein endothelial cells, such as HUVEC.
  • Ang2 receptors e.g. Tie2
  • VEGFR2 e.g. VEGFR2, i.e. KDR
  • 293 overexpressing Tie2 or KDR cells such as HEK293 or Expi293
  • vascular endothelial cells such as human umbilical vein endothelial cells, such as HUVEC.
  • assays can be performed using labels such as biotin or hFc.
  • the invention provides immunoconjugates comprising any of the VHHs or bispecific binding molecules described herein.
  • the immunoconjugate comprises one or more additional therapeutic agents or markers.
  • the invention provides a composition or medicament or formulation comprising any of the VHH or bispecific binding molecules described herein, preferably the composition is a pharmaceutical composition.
  • the composition further comprises pharmaceutical excipients.
  • a composition eg, a pharmaceutical composition, comprises a VHH or bispecific binding molecule of the invention in combination with one or more other therapeutic agents.
  • compositions including pharmaceutical compositions
  • medicaments or formulations comprising a VHH or bispecific binding molecule of the invention.
  • suitable pharmaceutical excipients such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • a composition or medicament or formulation of the invention may be in a variety of forms. These forms include, for example, liquid, semisolid, and solid dosage forms, such as liquid solutions (eg, injections or eye drops), powders or suspensions, liposomes, and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.
  • the composition or medicament or formulation of the present invention may be eye drops.
  • a medicament or formulation comprising a VHH or bispecific binding molecule described herein may be prepared by mixing a VHH or bispecific binding molecule of the invention having the desired purity with one or more optional pharmaceutical excipients, Preferably in the form of a lyophilized formulation or an aqueous solution.
  • compositions or medicaments or formulations of the invention may also contain more than one active ingredient as required for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to also provide other therapeutic agents.
  • sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody in the form of shaped articles such as films or microcapsules.
  • the invention also provides a pharmaceutical combination or pharmaceutical combination comprising a VHH or bispecific binding molecule of the invention, and one or more other therapeutic agents.
  • the present invention also provides a complete kit containing the drug combination, for example, the complete kit contains in the same package:
  • One aspect of the present invention provides a method for preventing or treating eye diseases in a subject, comprising administering to the subject an effective amount of the anti-VEGF VHH of the present invention, the anti-Ang2 VHH of the present invention or the bispecific binding compound of the present invention molecule, or a composition or drug or formulation comprising it.
  • the patient has (e.g., elevated levels, e.g., at the nucleic acid or protein level) VEGF, e.g., VEGFA, and/or Ang2 in the patient.
  • VEGF e.g., VEGFA
  • Ang2 Ang2
  • the ocular disease includes, but is not limited to, an ocular disease associated with angiogenesis, such as an ocular disease associated with corneal neovascularization.
  • the ocular disease treatment would benefit from inhibition of VEGF, e.g., VEGFA, and/or Ang2, at the nucleic acid or protein level.
  • VEGF e.g., VEGFA, and/or Ang2
  • the invention provides the use of an anti-VEGF VHH of the invention, an anti-Ang2 VHH of the invention, or a bispecific binding molecule of the invention, or a composition comprising the same, for the manufacture or preparation of a medicament for use herein
  • the use is, for example, for preventing or treating the related diseases or conditions mentioned herein.
  • an anti-VEGF VHH of the invention delays resolution of the disorder and/or symptoms associated with the disorder. attack.
  • an anti-VEGF VHH of the invention can also be combined with one or more other therapies, such as therapeutic modalities and/or other therapeutic agents for the uses described herein, for example for the prevention and/or treatment of the related diseases or conditions mentioned herein.
  • therapies such as therapeutic modalities and/or other therapeutic agents for the uses described herein, for example for the prevention and/or treatment of the related diseases or conditions mentioned herein.
  • the route of administration of the anti-VEGF VHH of the invention, the anti-Ang2 VHH of the invention or the bispecific binding molecule of the invention or a composition or medicament or formulation comprising the same is according to known methods, e.g., topical administration, e.g. intraocular administration , Ocular surface application. In some embodiments, administration is by injection or instillation.
  • the anti-Ang2 VHH antibodies provided herein can be used to detect the presence of Ang2 in a biological sample.
  • the anti-VEGFA VHH antibodies provided herein can be used to detect the presence of VEGFA in a biological sample.
  • the anti-bispecific binding molecules provided herein can be used to detect the presence of Ang2 and/or VEGFA in a biological sample.
  • the term "detection" as used herein includes quantitative or qualitative detection, and exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays methods, PCR-techniques (eg, RT-PCR).
  • the biological sample is a bodily fluid or ocular tissue, such as vitreous humor.
  • the method comprises contacting a biological sample with a VHH or bispecific binding molecule as described herein under conditions permissive for its binding to Ang2 or VEGFA, and detecting the presence of the VHH or bispecific binding molecule in the VHH or bispecific binding molecule. Whether a complex is formed between the molecule and Ang2 or VEGFA. Complex formation indicates the presence of Ang2 or VEGFA.
  • the method can be an in vitro or in vivo method.
  • an antibody of the invention is used to select a subject suitable for treatment with a VHH or bispecific binding molecule of the invention, eg wherein Ang2 or VEGFA is the biomarker used to select said subject.
  • a labeled VHH or bispecific binding molecule is provided.
  • Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as moieties that are detected indirectly, such as enzymes or ligands, for example, Through enzymatic reactions or molecular interactions.
  • the label is, for example, a label such as biotin or hFc.
  • a sample is obtained prior to treatment with a VHH or bispecific binding molecule of the invention. In some embodiments, the sample is obtained prior to other therapy. In some embodiments, the sample is obtained during or after treatment with the other therapy.
  • Ang2 and/or VEGFA are detected prior to treatment, e.g., prior to initiation of treatment or prior to a treatment after a treatment interval.
  • a method of treating a disease of the present invention comprising: testing the presence of Ang2 and/or VEGF A on a subject (e.g., a sample) (e.g., a sample from a subject), thereby Determining the Ang2 and/or VEGF A value, comparing the Ang2 and/or VEGF A value to a control value (e.g., a value in a normal individual), and administering treatment to the subject if the Ang2 and/or VEGF A value is greater than the control value
  • a control value e.g., a value in a normal individual
  • RNA 1.2 Collect 50ml of alpaca peripheral blood, separate lymphocytes, add 1mL Trizol reagent per 2.5 ⁇ 10 7 living cells, and extract total RNA by chloroform/isopropanol precipitation method. Take 10ug RNA as a template, and use PrimeScript Reverse Transcription Kit (Takara Company) for reverse transcription. The cDNA was used as a template for the first-round PCR reaction, and the forward primer Alp-VhL and the reverse primer Alp-2b/2cR were used to obtain the first-round PCR product.
  • the first-round PCR product was used as a template for the second-round PCR reaction, and the forward primer Alp-VhF and the reverse primer Alp-JHR-SalI were used to obtain the second-round PCR product.
  • the pC3-HF vector and the second-round PCR product were double-digested with SacI and SalI (Thermo Company), respectively, and the digested products were added to T4 ligase (Thermo Company) for reaction, and TG1 competent cells were electrotransformed to construct a VHH antibody library. Freeze the bacterial solution at -80°C.
  • Ang2 antigen (Beijing Yiqiao Company) diluted with PBS buffer to 0.5ug/ml coated 96-well ELISA plate, refrigerated overnight at 4°C.
  • the antigen-coated plate was washed 3 times with PBST, and the blocking agent was added to 300ul/well, and left at room temperature to block for 1 hour. Wash 3 times with PBST, add 80ul blocking agent + 20ul expression supernatant of the positive VHH TG1 bacteria identified in 1.3 above, shake at room temperature for 1 hour.
  • the present invention utilizes molecular biology techniques to obtain antibody sequences in anti-VEGFA or Ang2 positive phages, and utilizes the TG1 monoclonal expression and purification obtained above to obtain VHH antibody proteins containing positive VHH.
  • TG1 bacterium containing the VHH expression plasmid identified in Example 1 Take the TG1 bacterium containing the VHH expression plasmid identified in Example 1, inoculate it into 800ml LB-Amp medium, and cultivate it at 200rpm at 37°C until the OD600 value is 0.5-0.6. Add 1mM IPTG to the bacterial solution to induce expression, culture overnight at 28°C and 200rpm, collect the culture supernatant, add 15ml PB+1mg/ml polymyxin to resuspend the bacteria after centrifugation, centrifuge again, and filter through a 0.22um filter membrane.
  • amino acid sequences of the CDRs and heavy chain variable regions of humanized VHH antibodies LA42F8.5, LA46E11.8, and LA24C11.10 obtained from the three humanized antibodies obtained in the present invention please refer to the attached sequence list.
  • LA42F8, LA46E11, LA24C11 and humanized antibodies LA42F8.5, LA46E11.8, LA24C11.10 in eukaryotic cells is as follows:
  • the humanized antibody sequences obtained above were cloned into pcDNA3.1 (Invitrogen) to obtain plasmids containing the antibody sequences, respectively.
  • Expi-293 cells (Invitrogen) were passaged according to the desired transfection volume, and the cell density was adjusted to 1.5 ⁇ 10 6 cells/ml the day before transfection. On the day of transfection, the cell density was about 3 ⁇ 10 6 cells/ml.
  • Take F17 medium Gibco, A13835-01
  • F17 medium Gibco, A13835-01
  • Add appropriate plasmid and mix well.
  • Add appropriate polyethyleneimine (PEI) Polysciences, 23966) to the plasmid (the ratio of plasmid to PEI is 1:3 in 293F cells), mix well and incubate at room temperature for 10 min to obtain a DNA/PEI mixture.
  • PEI polyethyleneimine
  • the gravity column used for purification was treated overnight with 0.5M NaOH, the glass bottle was washed with distilled water, and then dry-baked at 180°C for 4 hours to obtain a purification column. Before purification, the cell supernatant collected above was centrifuged at 4500 rpm for 30 min, and the cells were discarded. The supernatant was then filtered using a 0.22 ⁇ l filter. A protein A column (Hitrap Mabselect Sure 5*5ml, GE, 11-0034-95) was equilibrated with 10ml of binding buffer (sodium phosphate 20mM. NaCl 150mM, pH7.0).
  • the coding nucleic acids of the negative control IgG, positive control BI-anti-VEGF, BI836880, and Faricimab were cloned into pcDNA3.1, transfected into Expi-293 cells, expressed and purified, and the method was the same as the implementation Example 2.
  • Example 3 Determination of binding kinetics of chimeric antibody and antigen of the present invention by biofilm thin layer interference technique
  • the equilibrium dissociation constant (KD) of the antibody of the present invention binding to human Ang2 was determined by biofilm thin layer interferometry technique (ForteBio). ForteBio affinity determination was carried out according to the existing method (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5 (2): p. 270-8).
  • ND means not detected
  • This example verifies the blocking effect of the anti-VEGF A VHH of the present invention on the binding of hVEGF A to the receptor KDR.
  • Dilute SA Thermo Product No. 21125
  • PBST wash 3 times with PBST, add 3% BSA to block for 1.5h.
  • FIG. 2 shows that the candidate molecules LA42F8 and LA46E11 antibodies can completely block the combination of VEGF A and VEGFR2.
  • the anti-Ang2 VHH antibody blocking results obtained in the present invention are shown in Table 3.
  • the candidate molecule LA24C11 has a blocking effect on the binding of Ang2 to the receptor Tie2.
  • Antibody refers to the purified prokaryotic supernatant prepared in Example 2, with an initial concentration of 0.556 mg/ml, followed by 1:10 or 1:100 dilution;
  • Supernatant refers to unpurified prokaryotic supernatant.
  • Example 4 Similar to Example 4 (using Ang2-Bio (R&D product number: BT623B/CF)), the effect of LA24C11.10 on blocking the binding of Ang2 and Tie2 was detected by ELISA blocking experiment, and the results are shown in FIG. 3 . It can be seen that LA24C11.10 has a blocking effect on the binding of Ang2 to the receptor Tie2.
  • This example verifies the inhibitory effect of the anti-Ang2 VHH antibody of the present invention on Tie2 phosphorylation induced by hAng2-Fc.
  • Expi-293 overexpressing human Tie2 was generated by transfecting Expi-293 cells (Thermo) with the pCHO1.0 vector (Invitrogen) carrying the human Tie2 gene cloned into the MCS of the multi-cloning site (Beijing Yiqiao Cat. No.: HG10700-M) Cells 293-Tie2 cells.
  • Use Expi293 medium (Thermo product number A1435102) to configure the experimental medium: add the test antibody (LA24C11 (24C11) and LA24C11.0 (hz24C11.10) prepared in Example 2), and negative control IgG, and positive control Nesvacumab (according to CN202010573625.2)), the initial concentration is 60ug/ml, followed by 1:2 equal dilution; the final concentration of hAng2-Fc (Beijing Yiqiao: 10691-H02H) is 2.5ug/ml.
  • NP-40 lysate (Beiyuntian, catalog number: P0013F) containing 1% protease (Thermo catalog number: 78442) and phosphatase inhibitor (Thermo catalog number: 78442), place on ice for 30min, and centrifuge at 2000g , collect the protein supernatant and store it in a -80°C refrigerator.
  • the concentration of pTie2 was detected according to the instructions of the phosphorylated Tie2 ELISA kit (R&D product number: DYC2720E), and the capture antibody in the kit was coated on the microtiter plate at a concentration of 4 ug/ml overnight at 4°C. Washed three times with PBST, blocked with 5% BSA for 1h.
  • both the anti-Ang2 VHH antibodies LA24C11 and LA24C11.10 of the present invention can effectively inhibit hAng2-Fc-induced phosphorylation of 293-Tie2 cells in vitro.
  • VEGF A can bind to the related receptor VEGFR2 (KDR), activate the VEGFR2 signaling pathway, and induce the survival, proliferation and migration of vascular endothelial cells.
  • KDR related receptor VEGFR2
  • This study uses the KDR reporter experimental system, using NFAT-RE-luc2P/KDR HEK293 cells (Promega Cat CS181401), to detect the blocking effect of serially diluted antibodies on VEGFA activation-related receptor signaling pathways.
  • the experimental method refers to the description of the supplier (Promega):
  • the antibody mixture to be tested with a concentration of 100ng/ml VEGF A and gradient dilution, let it stand for 30min, put 50 ⁇ l/well in a 96-well white cell culture plate containing cells, and incubate in a 37°C, 5% carbon dioxide incubator for 6h.
  • the samples to be tested are as follows: negative control IgG; positive control BI-anti-VEGF; LA42F8; LA46E11; LA42F8.5; : Contains only 100ng/ml VEGF A.
  • VEGF A can act on VEGFR and other related receptors in vascular endothelial cells, promote the survival, proliferation and migration of vascular endothelial cells, and then induce neovascularization. This experiment is based on VEGF-induced survival and proliferation of human umbilical vein endothelial cells (HUVEC). Antibody Inhibition of VEGF A-Induced Survival and Proliferation of Primary Cells.
  • HUVEC cells Allcells product number: H-001-CN
  • 2000 cells/well were spread in a 96-well culture plate, and placed at 37°C. , 5% carbon dioxide incubator for 24 hours,
  • CCK-8 detection solution (Tongren Chemical Product Number: CK04), and incubate in a 37°C, 5% carbon dioxide incubator for 12 to 24 hours.
  • the chains of IEX04-008, IEX04-010 and IEX04-012 (sequence shown in the sequence listing) of the bispecific binding molecule of the present invention were constructed into the pcDNA3.1 vector, and expressed in 293 cells as described in Example 2 and purifying said bispecific binding molecule.
  • the blocking effect of anti-VEGF A/Ang2 bispecific antibody on VEGF A was detected by HEK293-KDR reporter experiment.
  • the experimental method refers to Example 7.
  • binding molecules or controls applied were as follows:
  • VEGF A 100ng/ml VEGF A;
  • Negative control IgG prepared as described above;
  • Positive control Faricimab prepared as above, the initial concentration is 13.5ug/ml, 1:3 serial dilution;
  • Positive control BI-836880 prepared as described above, the initial concentration is 13.5ug/ml, 1:3 serial dilution;
  • IEX04-012 Prepared as above, the initial concentration is 13.5ug/ml, 1:3 serial dilution.
  • the test results are shown in Figure 7.
  • the bispecific binding molecule IEX04-012 inhibits VEGF-induced KDR signaling pathway activation, and has better inhibitory ability than the control antibody BI836880.
  • the experimental method is as in Example 8, but the antibodies used are as follows:
  • Figure A The initial concentration is 20ug/ml, IEX04-008 prepared as above, negative control IgG, positive control Faricimab and BI836880 prepared as described above in 1:3 equal ratio dilution, the blank is Blank (that is, no antibody and VEGF A), and VEGF A group (that is, only add 20ng/ml VEGFA);
  • Figure B The negative control IgG, BI-anti-VEGF, IEX04-010 prepared as described above with an initial concentration of 80ug/ml, 1:3 equal ratio dilution, the blank is Blank (i.e. no antibody and VEGF A), and VEGF A group (that is, only add 20ng/ml VEGFA);
  • Figure C IEX04-012 prepared as above, negative control IgG, positive control Faricimab and BI836880 prepared as described above with an initial concentration of 20nM, 1:3 equal dilution, the blank is Blank (ie no antibody and VEGF A), and VEGF A group (i.e. only added 20ng/ml VEGFA).
  • the test results are shown in Figure 8.
  • the bispecific binding molecules IEX04-008, IEX04-010, and IEX04-012 all inhibited the VEGF-induced HUVEC cell survival and proliferation.
  • the IEX04-012 IC 50 is lower and has better inhibitory ability.
  • Ang2 can bind to its natural receptor Tie2.
  • ELISA and FACS methods were used to detect the blocking of the binding between Ang2 and Tie2 by anti-VEGF A/Ang2 bispecific binding molecules.
  • the hTie2 protein (Beijing Yiqiao) was resuspended and dissolved in PBS to a concentration of 2ug/ml, and coated onto an ELISA plate overnight. 5% BSA was used to block for 1h, and the biotin antigen Recombinant Biotinylated hAngiopoietin-2 protein (R&D) was diluted to 600ug/mlM, 50 ⁇ l/well.
  • the antigen hAng2-Fc protein (Beijing Yiqiao, product number: 10691-H02H) was diluted to 4ug/ml, 50 ⁇ l/well.
  • the antibodies prepared as described above (IEX04-008, IEX04-010, IEX04-012 and positive control antibodies BI-836880 and Faricimab and negative control IgG) were serially diluted 2 times starting from the highest concentration of 800nM, a total of 12 dilution gradients, 50 ⁇ l/well, incubate in PBS on ice for 30 minutes, the final concentration of antigen hAng2-Fc protein is 2ug/ml, and the highest final concentration of each antibody is 400nM.
  • the cells were resuspended in 100 ⁇ l PBS, and the fluorescence signal value of the cells was detected by a flow cytometer (BD Biosciences). According to its MFI, the concentration-dependent curve was fitted with GraphPad. The results are shown in Figure 10. The figure shows that the bispecific binding molecules IEX04-008, IEX04-010 and IEX04-012 can effectively block the binding of human Ang2-hFc to Tie2, and the IC 50 is lower than that of the positive control.
  • This example verifies the inhibitory effect of the bispecific binding molecule of the present invention on the phosphorylation of Tie2 induced by hAng2-Fc, which uses the phosphorylation experiment induced by hAng2.
  • Tie2-overexpressing Expi293 cells, 293-Tie2 were co-incubated with bispecific binding molecules and recombinant hAng2-Fc protein, and the phosphorylation of Tie2 induced by hAng2-Fc by different antibodies was detected by detecting the content of phosphorylated Tie2 in the system. chemical inhibition.
  • the concentration of pTie2 was detected according to the instructions of the phosphorylated Tie2 ELISA kit (R&D DYC2720E), and the capture antibody was coated on the microplate at a concentration of 4ug/ml for 4 nights. Washed three times with PBST, blocked with 5% BSA for 1h. Add 100ul of the sample to be tested, and the control pTie2 (R&D DYC2720E) to make a standard curve and incubate at room temperature for 2h (if the concentration of the sample pTie2 is too high and exceeds the ELISA detection range, the obtained mixture can be diluted 2-3 times).
  • the experimental results are shown in Figure 11.
  • the antibody IEX04-012 of the present invention can effectively inhibit the phosphorylation of 293-Tie2 induced by hAng2-Fc in vitro, and the IC 50 is better than that of the positive control.
  • Example 13 Inhibition experiment of anti-VEGF A/Ang2 bispecific binding molecule on Ang2 vascular endothelial cell leakage
  • HUVEC-Tie2 cells overexpressing Tie2 were obtained by transfecting HUVEC cells with lentivirus (Product No. H-001-CN of Allcells).
  • EGM-2 medium Spread 300ul of EGM-2 medium on the lower layer of a mini-well 96-well insert culture dish, digest with Accutase (Sigma) to obtain HUVEC-Tie2, and resuspend to 1*10 7 cells/ml in EGM-2 medium, 100ul/ The wells were laid on the upper layer of the Petri dish. Every 24h, replace the culture medium in the lower chamber (EGM-2 medium), and replace the culture medium in the lower chamber after 24 hours as the experimental medium, and the composition of the experimental medium is as follows:
  • VEGF group A EGM-2 medium + 20ng/ml VEGF (R&D product number: 293-VE)
  • IgG group EGM-2 medium+20ng/ml VEGF+10ug/ml IgG;
  • VEGF A+Ang1 EGM-2 medium + 20ng/ml VEGF (R&D product number: 293-VE) + 200ng/ml Ang1 (R&D product number: 923-AN);
  • IEX04-012 group (VEGF A+IEX04-012): EGM-2 medium+20ng/ml VEGF+10ug/ml IEX04-012;
  • BI-836880 group (VEGF A+BI-836880): EGM-2 medium+20ng/ml VEGF+10ug/ml BI-836880;
  • Faricimab group (VEGF A+Faricimab): EGM-2 medium+20ng/ml VEGF+10ug/ml Faricimab.
  • the above experimental medium was placed at 37°C, 5% CO 2 for cultivation.
  • FITC-Dextran Sigma product number: FD2000S-1G (4mg/ml) (4mg/ml) to each well of the experimental medium in the upper chamber, place it at 37°C, 5% CO 2 for 30min, remove the medium in the upper chamber, and dilute it with PBS After dilution of 1:10, it was detected on a multi-functional microplate reader, the wavelength of excitation light was 488nm, and the wavelength of emission light was 535nm.
  • Example 14 Drug efficacy test of laser-induced choroidal neovascularization
  • the rhesus monkey After 29 days of administration, the rhesus monkey was anesthetized with pentobarbital sodium according to body weight (about 30 mg/kg intravenously, and the dose can be adjusted according to the animal's health status), and euthanized by bleeding from the abdominal aorta or femoral artery. side eyeball.
  • the eyes of some animals were fixed with modified Davidson’s fixative solution, paraffin-embedded and sectioned, and the laser modeling area was selected for routine HE staining, CD31 IHC staining and other histopathological examinations.
  • the antibody group of the present invention compared with anti-VEGF treatment alone, significantly reduced the retinopathy area, reduced retinal edema, and reduced tissue hyperplasia in the laser-damaged area.
  • the results showed better improvement in retinal morphology (see Figure 16), and inhibited Retinochoroidal neovascularization enhanced vascular integrity function (Fig. 17).

Abstract

提供分别针对血管内皮生长因子(VEGF/VEGF-A)和针对血管生成素-2(ANG-2)的抗体，同时针对血管内皮生长因子(VEGF/VEGF-A)和血管生成素-2(ANG-2)的双特异性结合分子(例如抗体)，以及其制备方法，包含所述抗体或分子的药物组合物，及其应用。

Description

结合VEGF和Ang2的双特异性结合分子以及其用途
本发明涉及分别针对血管内皮生长因子(VEGF/VEGF-A)和针对血管生成素-2(ANG-2)的抗体，同时针对血管内皮生长因子(VEGF/VEGF-A)和血管生成素-2(ANG-2)的双特异性结合分子(例如抗体)，以及其制备方法，包含所述抗体或分子的药物组合物，及其应用。
发明背景
血管发生参与各种疾病的发病机理，所述疾病包括实体瘤、眼内新生血管化相关的疾病、类风湿性关节炎(rheumatoid arthritis)和银屑病。
VEGF是有效且普遍存在的血管生长因子。VEGF家族成员包括VEGF-A、VEGF-B、VEGF-C、VEGF-D、VEGF-E、胎盘生长因子(PIGF)和内分泌腺衍生的VEGF(EG-VEGF)。VEGF的活性形式与其他VEGF家族成员合成为同型二聚体或异型二聚体。VEGF-A以通过选择性剪接生成的六种同种型存在：VEGF121、VEGF145、VEGF165、VEGF183、VEGF189和VEGF206。这些同种型主要因其生物可利用性而不同，其中VEGF165是主要同种型。据信，VEGF是正常和疾病相关血管发生的重要调节剂。
除VEGF家族之外，还认为人血管生成素参与了血管发育和出生后的血管发生。人血管生成素包括天然存在的激动剂血管生成素-1(ANG-1)以及天然存在的拮抗剂血管生成素-2(ANG-2)。认为ANG-1的作用在成人中是保守的，它在成人中广泛地且组成型地表达。相比之下，ANG-2表达主要限于血管重塑的部位，其中认为它阻断ANG-1的组成型稳定化或成熟化功能，从而允许血管恢复到可能对芽生信号更具响应性的塑性状态并保持在该塑性状态下。
近年来，已经开发了一些靶向VEGF-A和ANG-2的双特异性抗体(例如WO2012131078和WO2014009465)。然而，现有的双特异性抗体针对VEGF和Ang2的阻断性较差，且由于分子量太大，导致单次给药时摩尔浓度较低。特别地，对于眼部疾病，通常使用通过玻璃体内应用的较小分子量的抗体，且需要较少的给药频率。因此，仍然需要新的，特别是适用于眼部疾病的靶向VEGF-A和ANG-2的双特异性结合分子。
发明内容
本发明开发了新的靶向VEGF-A或ANG-2的VHH抗体，以及同时针对VEGF-A和ANG2的双特异性结合分子。特别地，本发明的双特异性结合分子相比已知的抗体，具有较小的分子量，相同质量浓度下，摩尔浓度更高；且具有较强的VEGF A和Ang2阻断活性，能够完全阻断VEGFA诱导的原代细胞增殖。因此，本发明的分子在临床上具有更强的阻断活性，且能够在单次给药时使得抗体摩尔浓度更高，维持单次给药药效时间更久，降低眼部施用(例如玻璃体腔注射)频率。
附图说明：
图1显示了双特异性结合分子的结构。
图2显示了应用ELISA测定的抗VEGF A VHH抗体能够阻断VEGF A与VEGFR2的结合。
图3显示了应用ELISA法测定的人源化anti-Ang2 VHH抗体阻断Ang2与Tie2结合的作用。
图4显示了应用ELISA法测定的抗Ang2 VHH抗体(A)和人源化抗Ang2 VHH抗体(B)抑制hAng2-Fc诱导的293-Tie2细胞磷酸化的作用。
图5显示了应用HEK293-KDR reporter法检测抗VEGF A VHH阻断VEGFA激活KDR受体的作用。
图6显示了CCK-8测定的抗VEGF A VHH抗体抑制VEGF A诱导HUVEC细胞的存活和增殖的作用。
图7显示了应用HEK293-KDR reporter法检测VEGF A/Ang2双特异性结合分子IEX04-012阻断VEGF激活KDR受体的作用。
图8显示了双特异性结合分子IEX04-008、IEX04-010和IEX04-012抑制VEGF诱导的HUVEC细胞存活和增殖的作用。
图9显示了ELISA法测定的双特异性结合分子IEX04-008、IEX04-010和IEX04-012阻断人Ang2与Tie2的结合。
图10显示了流式细胞检测法测定双特异性结合分子IEX04-008、IEX04-010和IEX04-012阻断Ang2-Fc与Tie2的结合。
图11显示了应用流式细胞检测法测定本发明的双特异性结合分子IEX04-012在体外有效抑制hAng2-Fc诱导的293-Tie2磷酸化。
图12显示了本发明的双特异性结合分子IEX04-012降低VEGF诱导的血管内皮细胞通透性，即抑制VEGF诱导的HUVEC细胞渗漏。
图13显示了激光诱导的脉络膜新生血管化模型中4级(图A)和3级以上(图B)激光斑比例统计。
图14显示了激光诱导的脉络膜新生血管化模型中视网膜厚度统计。
图15显示了激光诱导的脉络膜新生血管化模型中渗漏面积统计
图16显示了激光诱导的脉络膜新生血管化模型眼底组织H&E染色图(A)及病变面积统计(B)。
图17显示了激光诱导的脉络膜新生血管化模型眼底组织CD31染色图(A)及阳性细胞统计(B)。
定义
在下文详细描述本发明前，应理解本发明不限于本文中描述的特定方法学、方案和试剂，因为这些可以变化。还应理解本文中使用的术语仅为了描述具体实施方案，而并不意图限制本发明的范围，其仅会由所附权利要求书限制。除非另外定义，本文中使用的所有技术和科学术语与本发明所属领域中普通技术人员通常的理解具有相同的含义。
为了解释本说明书，将使用以下定义，并且只要适当，以单数形式使用的术语也可以包括复数，并且反之亦然。要理解，本文所用的术语仅是为了描述具体的实施方案，并且不意欲是限制性的。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。
如本文所用，术语“和/或”意指可选项中的任一项或可选项的两项或多项或全部。
如本文所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。
本文所用的术语“VEGF”是指血管生长因子。VEGF家族成员包括VEGF-A、VEGF-B、VEGF-C、VEGF-D、VEGF-E、胎盘生长因子(PIGF)和内分泌腺衍生的VEGF(EG-VEGF)。VEGF的活性形式与其他VEGF家族成员合成为同型二聚体或异型二聚体。VEGF-A以通过选择性剪接生成的六种同种型存在：VEGF121、VEGF145、VEGF165、VEGF183、VEGF189和VEGF206。这些同种型主要因其生物可利用性而不同，其中VEGF165是主要同种型。在一些实施方案中，本发明的VEGF A是指来自人的VEGF A，例如来自人的VEGF165。在一个实施方案中，本发明的VEGFA的氨基酸序列是登录号为P15692(uniprot数据库)的氨基酸序列。
如本文所用，术语“ANG2”指人血管生成素-2(ANG-2)(备选地缩写为：ANGPT2或ANG2)，其例如在Maisonpierre,P.C.等人,Science277(1997)55-60和Cheung,A.H.等人,Genomics 48(1998)389-91中描述。Ang1和Ang2作为血管内皮中选择性表达的酪氨酸激酶家族Tie的配体被发现。目前存在4个确定的血管生成素家族成员。血管生成素-3和-4(ANG3和ANG4)可以代表小鼠和人中相同基因座的广泛多样的对应物。Ang1和Ang2最初在组织培养实验中分别鉴定为激动剂和拮抗剂(关于ANG1，参见Davis,S.等人,Cell87(1996)1161-69；关于ANG2，参见Maisonpierre,P.C.等人,Science277(1997)55-60)。全部已知的血管生成素主要与Tie2结合。在一些实施方案中，本发明的ANG2是指来自人的Ang2。在一些实施方案中，人Ang2包含登录号为O15123(uniprot数据库)的氨基酸序列。
术语“多特异性结合分子”是指至少是双特异性的多特异性结合分子，例如双特异性结合分子，即所述分子包含至少第一靶标结合区和第二靶标结合区，其中所述第一靶标结合区结合一种靶标或抗原且所述第二靶标结合区结合另一抗原或靶标。因此，根据本发明的分子包含对于至少两种不同的抗原或靶标的特异性。根据本发明的分子也涵盖包含多个靶标结合区/结合位点的多特异性分子，诸如三特异性结合分子。在一些实施方案中，本发明的双特异性结合分子是双特异性抗体。
如本文所用的术语“接头”是指使得能够直接连接双特异性结合分子的不同部分的任何分子。在不同分子部分之间建立共价连接的接头的实例包括肽接头和非蛋白质聚合物，包括但不限于聚乙二醇(PEG)、聚丙二醇、聚氧化烯或聚乙二醇、聚丙二醇的共聚物。在一些实施方案中，接头是肽接头，其是指氨基酸的序列，其中所述序列将结合分子的第一部分的氨基酸序列连接至结合分子的第二部分。例如，肽接头可以将结合分子的第一靶标结合区连接至第二靶标结合区。例如，肽接头也可以将抗体的一部分连接至抗体的另一部分，诸如将轻链可变区连接至重链可变区。优选地，所述肽接头具有这样的长度，其足以连接两个实体，其方式使得它们维持它们相对于彼此的构象，使得不妨碍期望的活性。
肽接头可以主要包括或可以不主要包括以下氨基酸残基：Gly、Ser、Ala或Thr。有用的接头包括甘氨酸-丝氨酸聚合物，包括例如(GS) n、(GSGGS) n、(GGGGS) n、(GGGS) n和(GGGGS) nG，其中n是至少1(且优选2、3、4、5、6、7、8、9、10)的整数。有用的接头还 包括甘氨酸-丙氨酸聚合物、丙氨酸-丝氨酸聚合物和其他柔性接头。有用的接头还包括甘氨酸单体聚合，例如(G) n，其中n是至少4(且例如为4-20，例如4、5、6、7、8、9、10以及更多)的整数。优选的，所述接头是(GGGGS) n，其中n＝1、2、3或4。
根据本发明的术语“价”表示在结合分子，例如抗体分子中存在指定数目的结合位点。因此，术语二价、三价、四价分别表示在结合分子中存在两个、三个或四个结合位点(靶标结合区)。根据本发明的双特异性结合分子是至少二价的并且可以是多价的，例如二价、三价、四价或六价的。
如本文所用的术语“靶标结合区”是指多特异性结合分子，例如双特异性结合分子的结合特定靶标或抗原的任何部分。靶标结合区可以是例如抗体或免疫球蛋白本身或抗体片段。这种靶标结合区可以具有或可以不具有独立于BsAB的剩余部分的三级结构，并且可以作为单独实体结合或不结合其靶标。靶标结合区还可以是受体或配体，或受体的能够结合配体的结构域。
术语“抗体片段”包括完整抗体的一部分。在优选的实施方案中，抗体片段为抗原结合片段。
“抗原结合片段”指与完整抗体不同的分子，其包含完整抗体的一部分且结合完整抗体所结合的抗原。抗体片段的例子包括但不限于Fv，Fab，Fab’，Fab’-SH，F(ab’) 2；dAb(domain antibody)；线性抗体；单链抗体(例如scFv)；单结构域抗体例如VHH；双价抗体或其片段；或骆驼科抗体。
“VHH”，又称为单域抗体(single domain antibody，sdAb)，是指仅由重链抗体可变区(Variable region)组成的基因工程抗体，其仅包含重链可变区的3个HCDR。VHH靠仅有的3个HCDR就具备抗原的特异性和高亲和力，而普通抗体则需要6个CDR。晶体结构表明，VHH由2个β片层组成支架，类似于传统抗体VH免疫球蛋白折叠。
术语“靶标”是指结合分子所针对的被结合物。靶标可以是抗原，也可以是配体或受体。
术语“抗原”是指引发免疫应答的分子。这种免疫应答可能涉及抗体产生或特异性免疫细胞的活化，或两者兼有。技术人员将理解，任何大分子，包括基本上所有的蛋白质或肽，都可以用作抗原。此外，抗原可以衍生自重组或基因组DNA。如本文所用，术语“表位”指抗原(例如，VEGF或Ang2)中与抗体分子特异性相互作用的部分。
“互补决定区”或“CDR区”或“CDR”是抗体可变结构域中在序列上高变并且形成在结构上确定的环(“超变环”)和/或含有抗原接触残基(“抗原接触点”)的区域。CDR主要负责与抗原表位结合。重链和轻链的CDR通常被称作CDR1、CDR2和CDR3，从N-端开始顺序编号。位于抗体重链可变结构域内的CDR被称作HCDR1、HCDR2和HCDR3，而位于抗体轻链可变结构域内的CDR被称作LCDR1、LCDR2和LCDR3。在一个给定的轻链可变区或重链可变区氨基酸序列中，各CDR的精确氨基酸序列边界可以使用许多公知的抗体CDR指派系统的任一种或其组合确定，所述指派系统包括例如：基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature342:877-883，Al-Lazikani等人,“Standard conformations for the canonical structures of immunoglobulins”,Journal of Molecular Biology,273,927-948(1997))，基于抗体序列可变性的Kabat(Kabat等人,Sequences of Proteins of Immunological Interest,第4版,U.S.Department of Health and Human Services,National Institutes ofHealth(1987)),AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(在万维网上imgt.cines.fr/上)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。
例如，根据不同的CDR确定方案，每一个CDR的残基如下所述。
Figure PCTCN2022096848-appb-000001
CDR也可以基于与参考CDR序列(例如本发明示例性CDR之任一)具有相同的Kabat编号位置而确定。
除非另有说明，否则在本发明中，术语“CDR”或“CDR序列”涵盖以上述任一种方式确定的CDR序列。
除非另有说明，否则在本发明中，当提及抗体可变区中的残基位置(包括重链可变区残基和轻链可变区残基)时，是指根据Kabat编号系统(Kabat等人，Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.(1991))的编号位置。
在一本实施方案中，本发明的VHH中的CDR按照以下规则：其中HCDR1按照AbM确定，HCDR2和HCDR3按照Kabat确定。
应该注意，基于不同的指派系统获得的同一抗体的可变区的CDR的边界可能有所差异。即不同指派系统下定义的同一抗体可变区的CDR序列有所不同。因此，在涉及用本发明定义的具体CDR序列限定抗体时，所述抗体的范围还涵盖了这样的抗体，其可变区序列包含所述的具体CDR序列，但是由于应用了不同的方案(例如不同的指派系统规则或组合)而导致其所声称的CDR边界与本发明所定义的具体CDR边界不同。
具有不同特异性(即，针对不同抗原的不同结合位点)的抗体具有不同的CDR(在同一指派系统下)。然而，尽管CDR在抗体与抗体之间是不同的，但是CDR内只有有限数量的氨基酸位置直接参与抗原结合。使用Kabat,Chothia,AbM、Contact和North方法中的至少两种，可以确定最小重叠区域，从而提供用于抗原结合的“最小结合单位”。最小结合单位可以是CDR的一个子部分。正如本领域技术人员明了，通过抗体的结构和蛋白折叠，可以确定CDR序列其余部分的残基。因此，本发明也考虑本文所给出的任何CDR的变体。例如，在一个CDR的变体中，最小结合单位的氨基酸残基可以保持不变，而根据Kabat或Chothia定义的其余CDR残基可以被保守氨基酸残基替代。
术语“Fc区”在本文中用于定义免疫球蛋白重链的CH2和CH3的恒定区域，该术语包括天然序列Fc区和变体Fc区。天然或野生型Fc区域能够和免疫细胞表面的不同的Fc受体结合，从而能够引起CDC\ADCC\ADCP效应功能。此类效应器功能一般要求Fc区与结合结构域(例如抗体可变区)联合。在一些实施方案中，Fc区被突变以增强其CDC\ADCC\ADCP效应功能。在一些实施方案中，Fc区被突变以削弱或删除其CDC\ADCC\ADCP效应功能。
“人源化”抗体是指包含来自非人CDR的氨基酸残基和来自人FR的氨基酸残基的抗体。在一些实施方案中，人源化抗体将包含基本上所有的至少一个、通常两个可变结构域，其中所有或基本上所有的CDR(例如，CDR)对应于非人抗体的那些，并且所有或基本上所有的FR对应于人抗体的那些。人源化抗体任选可以包含至少一部分的来源于人抗体的抗体恒定区。抗体(例如非人抗体)的“人源化形式”是指已经进行了人源化的抗体。“人抗体”或“全人抗体”或“全人源抗体”可以互换使用，其指具有这样的氨基酸序列的抗体，所述氨基酸序列对应于下述抗体的氨基酸序列，所述抗体由人或人细胞生成或来源于非人来源，其利用人抗体库或其它人抗体编码序列。人抗体的这种定义明确排除包含非人抗原结合残基的人源化抗体。
如本文所用，术语“抗”、“结合”或“特异性结合”意指结合作用对靶标或抗原是选择性的并且可以与不想要的或非特异的相互作用区别。结合位点与特定靶标或抗原结合的能力可以通过酶联免疫吸附测定法(ELISA)或本领域已知的常规结合测定法如通过放射性免疫测定(RIA)或生物膜薄层干涉测定法或MSD测定法或表面等离子体共振法(SPR)测定。
术语“有效量”指本发明的抗体或片段或组合物或组合的这样的量或剂量，其以单一或多次剂量施用患者后，在需要治疗或预防的患者中产生预期效果。
“治疗有效量”指以需要的剂量并持续需要的时间段，有效实现所需治疗结果的量。治疗有效量也是这样的一个量，其中抗体或抗体片段或组合物或组合的任何有毒或有害作用不及治疗有益作用。相对于未治疗的对象，“治疗有效量”优选地抑制可度量参数或改善可度量参数至少约40％、甚至更优选地至少约50％、55％、60％、65％、70％、75％、80％、85％、90％甚至100％。
“预防有效量”指以需要的剂量并持续需要的时间段，有效实现所需预防结果的量。通常，由于预防性剂量在对象中在疾病较早阶段之前或在疾病较早阶段使用，故预防有效量将小于治疗有效量。
术语“宿主细胞”、“宿主细胞系”和“宿主细胞培养物”可交换地使用且是指其中引入外源核酸的细胞，包括这种细胞的后代。宿主细胞包括“转化体”和“转化的细胞”，其包括初级转化的细胞和来源于其的后代，而不考虑传代的数目。后代在核酸内容上可能与亲本细胞不完全相同，而是可以包含突变。本文中包括在最初转化的细胞中筛选或选择的具有相同功能或生物学活性的突变体后代。
本文所使用的术语“标记”是指被直接或间接缀合或融合至试剂(诸如多核苷酸探针或抗体)并且促进其所缀合或融合的试剂的检测的化合物或组合物。标记本身可以是可检测的(例如，放射性同位素标记或荧光标记)或在酶促标记的情况下可以催化可检测的底物化合物或组合物的化学改变。术语旨在涵盖通过将可检测物质偶联(即，物理连接)至探针或抗体来直接标记探针或抗体以及通过与直接标记的另一种试剂反应来间接标记探针或抗体。在一些实施方案中，标记是hFc或生物素。
“个体”或“受试者”包括哺乳动物。哺乳动物包括但不限于，家养动物(例如，牛，羊，猫，狗和马)，灵长类动物(例如，人和非人灵长类动物如猴)，兔，以及啮齿类动物(例如，小鼠和大鼠)。在一些实施方案中，个体或受试者是人。
“分离的”抗体或分子是这样的抗体或分子，其已经与其天然环境的组分分离。在一些实施 方案中，将抗体或分子纯化至超过95％或99％纯度，如通过例如电泳(例如，SDS-PAGE，等电聚焦(IEF)，毛细管电泳)或层析(例如，离子交换或反相HPLC)确定的。
如下进行序列之间序列同一性的计算。
为确定两个氨基酸序列或两个核酸序列的同一性百分数，将所述序列出于最佳比较目的比对(例如，可以为了最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。在一个优选实施方案中，为比较目的，所比对的参考序列的长度是至少30％、优选地至少40％、更优选地至少50％、60％和甚至更优选地至少70％、80％、90％、100％的参考序列长度。随后比较在对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是相同的。
可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。在一个优选实施方案中，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48:444-453)算法(在http://www.gcg.com可获得)，使用Blossum62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。在又一个优选的实施方案中，使用GCG软件包中的GAP程序(在http://www.gcg.com可获得)，使用NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum62评分矩阵。还可以使用PAM120加权余数表、空位长度罚分12，空位罚分4)，利用已经并入ALIGN程序(2.0版)的E.Meyers和W.Miller算法,((1989)CABIOS,4:11-17)确定两个氨基酸序列或核苷酸序列之间的同一性百分数。额外地或备选地，可以进一步使用本文所述的核酸序列和蛋白质序列作为“查询序列”以针对公共数据库执行检索，以例如鉴定其他家族成员序列或相关序列。
本发明所述的“眼部疾病”涵盖涉及血管发生的眼部疾病(例如发生在眼内的疾病)，例如与角膜新生血管化相关的眼部疾病。
术语“药用辅料”指与活性物质一起施用的稀释剂、佐剂(例如弗氏佐剂(完全和不完全的))、赋形剂、载体或稳定剂等。
术语“药物组合物”指这样的组合物，其以允许包含在其中的活性成分的生物学活性有效的形式存在，并且不包含对施用所述组合物的受试者具有不可接受的毒性的另外的成分。
用于本文时，“治疗”指减缓、中断、阻滞、缓解、停止、降低、或逆转已存在的症状、病症、病况或疾病的进展或严重性。
用于本文时，“预防”包括对疾病或病症或特定疾病或病症的症状的发生或发展的抑制。
术语“载体”当在本文中使用时是指能够增殖与其相连的另一个核酸的核酸分子。该术语包括作为自我复制核酸结构的载体以及结合到已经引入其的宿主细胞的基因组中的载体。一些载体能够指导与其可操作相连的核酸的表达。这样的载体在本文中被称为“表达载体”。
“受试者/患者/个体样品”指从患者或受试者得到的细胞或流体的集合。组织或细胞样品的来源可以是实体组织，像来自新鲜的、冷冻的和/或保存的器官或组织样品或活检样品或穿刺样品；血液或任何血液组分；体液，诸如泪液、玻璃体液、脑脊液、羊膜液(羊水)、腹膜液(腹水)、或间隙液；来自受试者的妊娠或发育任何时间的细胞。在一些实施方案中，组织样品是眼部组织，例如玻璃体。在一些实施方案中，样品是泪液或玻璃体液。组织样品可能包含在自然界中天然不与组织混杂的化合物，诸如防腐剂、抗凝剂、缓冲剂、固定剂、营养物、抗生素、等等。
具体实施方案
I.抗VEGF A的VHH抗体
本发明涉及针对VEGF A的VHH抗体。在一些实施方案中，本发明的抗VEGF VHH包含如下的3个CDR，HCDR1，HCDR2和HCDR3，其中
HCDR1包含SEQ ID NO:1所示的序列，或由所述序列组成；
HCDR2包含SEQ ID NO:2所示的序列，或由所述序列组成；
HCDR3包含SEQ ID NO:3所示的序列，或由所述序列组成；
或者
HCDR1包含SEQ ID NO:6所示的序列，或由所述序列组成；
HCDR2包含SEQ ID NO:7或10所示的序列，或由所述序列组成；
HCDR3包含SEQ ID NO:8所示的序列，或由所述序列组成。
在一些实施方案中，所述VHH包含SEQ ID NO:4、5、9或11所述的氨基酸序列，或包含与所述SEQ ID NO:4、5、9或11所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:4、5、9或11所述的氨基酸组成。
在一些实施方案中，所述VHH包含与SEQ ID NO:4、5、9或11所示的氨基酸序列相比具有一个或几个(优选不超过10、9、8、7、6、5、4、3、2、1个)突变的氨基酸序列，所述突变例如置换、缺失或添加，优选地置换，例如保守置换。
在一些优选的实施方案中，所述突变不存在于CDR，例如HCDR1、HCDR2或HCDR3中。
II.抗Ang2的VHH抗体
本发明涉及针对Ang2的VHH抗体。在一些实施方案中，本发明的抗Ang2 VHH包含如下的3个CDR，HCDR1，HCDR2和HCDR3，其中
HCDR1包含SEQ ID NO:16所示的序列，或由所述序列组成；
HCDR2包含SEQ ID NO:17或20所示的序列，或由所述序列组成；
HCDR3包含SEQ ID NO:18所示的序列，或由所述序列组成。
在一些实施方案中，所述VHH包含SEQ ID NO:19或21所述的氨基酸序列，或包含与所述SEQ ID NO:19或21所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:19或21所述的氨基酸组成。
在一些实施方案中，所述VHH包含与SEQ ID NO:19或21所示的氨基酸序列相比具有一个或几个(优选不超过10、9、8、7、6、5、4、3、2、1个)突变的氨基酸序列，所述突变例如置换、缺失或添加，优选地置换，例如保守置换。
在一些优选的实施方案中，所述突变不存在于CDR，例如HCDR1、HCDR2或HCDR3中。
III.抗VEGF A×ANG2的双特异性结合分子
在本发明的一个方面，本发明涉及结合VEGF A和Ang2的双特异性结合分子，其包含特异性结合VEGF A的第一靶标结合区和特异性结合Ang2的第二靶标结合区，其中第二靶标结合区是抗Ang2的VHH，例如本发明的如上所述的抗Ang2 VHH。
在一些实施方案中，所述第一靶标结合区选自
特异性结合VEGF A的VHH；
特异性结合VEGF A的抗体的抗原结合片段，例如scFv，例如所述抗体为全人抗体或人源化抗体；或
特异性结合VEGF A的VEGF受体(VEGF R)或其胞外结构域或包含其胞外结构域的融合蛋白，例如胞外结构域与Fc的融合蛋白。
在一些实施方案中，本发明的双特异性结合分子包含1个或2个或3个或4个第一或第二靶标结合区。在一些实施方案中，本发明的双特异性结合分子包含2个、3个或4个靶标结合区。在一些实施方案中，本发明的双特异性结合分子是二价的或3价的或4价的。在一些实施方案中，本发明的双特异性结合分子为双特异性抗体。
在本发明的一个方面，本发明的双特异性结合分子，例如双特异性抗体具有以下结构：
抗VEGF抗体的轻链可变区VL-接头-抗VEGF抗体的重链可变区VH-接头-抗Ang2 VHH或
抗VEGF抗体的重链可变区VH-接头-抗VEGF抗体的轻链可变区VL-接头-抗Ang2 VHH；
其中抗Ang2 VHH包含HCDR1，HCDR2和HCDR3，其中HCDR1包含SEQ ID NO:16所示的序列，或由所述序列组成；HCDR2包含SEQ ID NO:17或20所示的序列，或由所述序列组成；HCDR3包含SEQ ID NO:18所示的序列，或由所述序列组成。
在一些实施方案中，上述双特异性结合分子的结构如图1A或图1B所示。在一些实施方案中，上述双特异性结合分子由一条链组成。在一些实施方案中，上述双特异性结合分子为二价的。
在一些实施方案中，抗VEGF抗体的轻链可变区VL包含LCDR1，LCDR2和LCDR3，其中LCDR1包含SEQ ID NO:31所示的序列或由其组成；LCDR2包含SEQ ID NO:32所示的序列或由其组成；LCDR3包含SEQ ID NO:33所示的序列或由其组成。
在一些实施方案中，抗VEGF抗体的重链可变区VH包含HCDR1，HCDR2和HCDR3，其中HCDR1包含SEQ ID NO:35所示的序列或由其组成；HCDR2包含SEQ ID NO:36所示的序列或由其组成；HCDR3包含SEQ ID NO:37所示的序列或由其组成。
在一些实施方案中，本发明的抗VEGF抗体的重链可变区VH包含SEQ ID NO:34所述的氨基酸序列，或包含与所述SEQ ID NO:34所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:34所述的氨基酸组成。在一些实施方案中，所述重链可变区VH包含与SEQ ID NO:34所示的氨基酸序列相比具有一个或几个(优选不超过10、9、8、7、6、5、4、3、2、1个)突变的氨基酸序列，所述突变例如置换、缺失或添加，优选地置换，例如保守置换。在一些优选的实施方案中，所述突变不存在于CDR，例如HCDR1、HCDR2或HCDR3中。
在一些实施方案中，本发明的抗VEGF抗体的轻链可变区VL包含SEQ ID NO:30所述的氨基酸序列，或包含与所述SEQ ID NO:30所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:30所述的氨基酸组成。在一些实施方案中，所述轻链可变区VL包含与SEQ ID NO:30所示的氨基酸序列相比具有一个或几个(优选不超过10、9、8、7、6、5、4、3、2、1个)突变的氨基酸序列，所述突变例如置换、缺失或添加，优选地置换，例如保守置换。在一些优选的实施方案中，所述突变不存在于CDR，例如LCDR1、LCDR2或LCDR3中。
在一些实施方案中，本发明的抗Ang2 VHH包含SEQ ID NO:19或21所述的氨基酸序列，或包含与所述SEQ ID NO:19或21所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:19或21所述的氨基酸组成。在一些实施方案中，所述VHH包含与SEQ ID NO:19或21所示的氨基酸序列相比具有一个或几个(优选不超过10、9、8、7、6、5、4、3、2、1个)突变的氨基酸序列，所述突变例如置换、缺失或添加，优选地置换，例如保守置换。在一些优选的实施方案中，所述突变不存在于CDR，例如HCDR1、HCDR2或HCDR3中。
在一些实施方案中，所述接头包含SEQ ID NO:23的氨基酸序列，或由其组成。在一些实施方案中，在抗VEGF抗体的轻链可变区和重链可变区之间的接头包含SEQ ID NO:23的氨基酸序列，或由其组成，其中例如n＝4。在一些实施方案中，抗VEGF可变区与抗Ang2 VHH之间的接头包含SEQ ID NO:23的氨基酸序列，或由其组成，其中例如n＝2或3，例如3。
在一些实施方案中，本发明的抗VEGF A×ANG2的双特异性结合分子包含SEQ ID NO:28的氨基酸序列，或包含与所述SEQ ID NO:28所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:28所述的氨基酸组成。在一些实施方案中，所述双特异性结合分子包含与SEQ ID NO:28所示的氨基酸序列相比具有一个或几个(优选不超过10、9、8、7、6、5、4、3、2、1个)突变的氨基酸序列，所述突变例如置换、缺失或添加，优选地置换，例如保守置换。在一些优选的实施方案中，所述突变不存在于抗VEGF抗体可变区和抗Ang2 VHH的CDR中。
在本发明的另一个方面，本发明的双特异性结合分子，例如双特异性抗体具有以下结构：
第一抗VEGF VHH-接头-第二抗VEGF VHH-接头-抗Ang2 VHH,
其中抗Ang2 VHH包含HCDR1，HCDR2和HCDR3，其中HCDR1包含SEQ ID NO:16所示的序列，或由所述序列组成；HCDR2包含SEQ ID NO:17或20所示的序列，或由所述序列组成；HCDR3包含SEQ ID NO:18所示的序列，或由所述序列组成。
在一些实施方案中，上述双特异性结合分子的结构如图1C所示。在一些实施方案中，上述双特异性结合分子由一条链组成。在一些实施方案中，上述双特异性结合分子为三价的。在一些实施方案中，第一抗VEGF VHH与第二抗VEGF VHH相同或不同。
在一些实施方案中，抗VEGF VHH包含HCDR1，HCDR2和HCDR3，其中HCDR1包含SEQ ID NO:1所示的序列，或由所述序列组成；HCDR2包含SEQ ID NO:2所示的序列，或由所述序列组成；HCDR3包含SEQ ID NO:3所示的序列，或由所述序列组成；
或者
HCDR1包含SEQ ID NO:6所示的序列，或由所述序列组成；HCDR2包含SEQ ID NO:7或10所示的序列，或由所述序列组成；HCDR3包含SEQ ID NO:8所示的序列，或由所述序列组成。
在一些实施方案中，所述抗VEGF VHH包含SEQ ID NO:4、5、9或11所述的氨基酸序列，或包含与所述SEQ ID NO:4、5、9或11所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:4、5、9或11所述的氨基酸组成。在一些实施方案中，所述VHH包含与SEQ ID NO:4、5、9或11所示的氨基酸序列相比具有一个或几个(优选不超过10、9、8、7、6、5、4、3、2、1个)突变的氨基酸序列，所述突变例如置换、缺失或添加，优选地置换，例如保守置换。在一些优选的实施方案中，所述突变不存在于CDR，例如HCDR1、HCDR2或HCDR3中。
在一些实施方案中，抗VEGF VHH包含HCDR1，HCDR2和HCDR3，其中HCDR1包含SEQ ID NO:6所示的序列，或由所述序列组成；HCDR2包含SEQ ID NO:7或10所示的序列，或由所述序列组成；HCDR3包含SEQ ID NO:8所示的序列，或由所述序列组成。
在一些实施方案中，所述抗VEGF VHH包含SEQ ID NO:9或11所述的氨基酸序列，或包含与所述SEQ ID NO:9或11所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:9或11所述的氨基酸组成。在一些实施方案中，所述VHH包含与SEQ ID NO:9或11所示的氨基酸序列相比具有一个或几个(优选不超过10、9、8、7、6、5、4、3、2、1个)突变的氨基酸序列，所述突变例如置换、缺失或添加，优选地置换，例如保守置换。在一些优选的实施方案中，所述突变不存在于CDR，例如HCDR1、HCDR2或HCDR3中。
在一些实施方案中，本发明的抗Ang2 VHH包含SEQ ID NO:19或21所述的氨基酸序列，或包含与所述SEQ ID NO:19或21所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:19或21所述的氨基酸组成。在一些实施方案中，所述VHH包含与SEQ ID NO:19或21所示的氨基酸序列相比具有一个或几个(优选不超过10、9、8、7、6、5、4、3、2、1个)突变的氨基酸序列，所述突变例如置换、缺失或添加，优选地置换，例如保守置换。在一些优选的实施方案中，所述突变不存在于CDR，例如HCDR1、HCDR2或HCDR3中。
在一些实施方案中，所述接头包含SEQ ID NO:23的氨基酸序列，或由其组成。在一些实施方案中，第一抗VEGF VHH和第二抗VEGF VHH之间的接头或第二抗VEGF VHH与抗Ang2 VHH之间的接头包含SEQ ID NO:23的氨基酸序列，或由其组成，例如n＝2。
在一些实施方案中，本发明的抗VEGF A×ANG2的双特异性结合分子包含SEQ ID NO:22的氨基酸序列，或包含与所述SEQ ID NO:22所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:22所述的氨基酸组成。在一些实施方案中，所述双特异性结合分子包含与SEQ ID NO:22所示的氨基酸序列相比具有一个或几个(优选不超过10、9、8、7、6、5、4、3、2、1个)突变的氨基酸序列，所述突变例如置换、缺失或添加，优选地置换，例如保守置换。在一些优选的实施方案中，所述突变不存在于抗VEGF VHH和抗Ang2 VHH的CDR中。
在本发明的另一个方面，本发明的双特异性结合分子包含一条或两条以下链：
VEGF R胞外结构域-Fc-接头-抗Ang2 VHH
其中抗Ang2 VHH包含HCDR1，HCDR2和HCDR3，其中HCDR1包含SEQ ID NO:16所示的序列，或由所述序列组成；HCDR2包含SEQ ID NO:17或20所示的序列，或由所述序列组成；HCDR3包含SEQ ID NO:18所示的序列，或由所述序列组成。
在一些实施方案中，上述双特异性结合分子的结构如图1D所示。在一些实施方案中，上述双特异性结合分子由两条链组成。在一些实施方案中，上述双特异性结合分子为四价的。
在一些实施方案中，所述的抗Ang2 VHH包含SEQ ID NO:19或21所述的氨基酸序列，或包含与所述SEQ ID NO:19或21所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:19或21所述的氨基酸组成。在一些实施方案中，所述VHH包含与SEQ ID NO:19或21所示的氨基酸序列相比具有一个或几个(优选不超过10、9、8、7、6、5、4、3、2、1个)突变的氨基酸序列，所述突变例如置换、缺失或添加，优选地置换，例如保守置换。在一些优选的实施方案中，所述突变不存在于CDR，例如HCDR1、HCDR2或HCDR3中。
在一些实施方案中，VEGFR胞外结构域为来自人的VEGFR的胞外结构域。在一些实施方案中，所述VEGFR胞外结构域包含VEGFR1第二抗体样结构域(例如FLT1 domain2)和VEGFR2第三抗体样结构域(例如KDR domain3)。在一些实施方案中，所述VEGFR胞外结构域包含人VEGFR1第二抗体样结构域和人VEGFR2第三抗体样结构域。在一些实施方案中，所述VEGFR胞外结构域包含SEQ ID NO:26的氨基酸序列，或包含与所述SEQ ID NO:26所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:26所述的氨基酸组成。在一些实施方案中，所述VEGFR胞外结构域包含与SEQ ID NO:26所示的氨基酸序列相比具有一个或几个(优选不超过10、9、8、7、6、5、4、3、2、1个)突变的氨基酸序列，所述突变例如置换、缺失或添加，优选地置换，例如保守置换，优选地所述VEGFR胞外结构域保留与SEQ ID NO:26所示的结构域相似(例如具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％、99％)的与VEGF的结合亲和力。
在一些实施方案中，所述Fc为来源于人IgG1、IgG2、IgG3或IgG4的Fc，例如野生型Fc，或本领域已知的Fc变体。在一些实施方案中，所述Fc包含SEQ ID NO:27的氨基酸序列，或包含与所述SEQ ID NO:27所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:27所述的氨基酸组成。
在一些实施方案中，所述VEGF R胞外结构域-Fc为VEGFR胞外结构域与Fc的融合蛋白，例如Aflibercept或其衍生物。
在一些实施方案中，所述VEGF R胞外结构域-Fc包含SEQ ID NO:25的氨基酸序列，或包含与所述SEQ ID NO:25所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:25所述的氨基酸组成。在一些实施方案中，所述VEGF R胞外结构域-Fc包含与SEQ ID NO:25所示的氨基酸序列相比具有一个或几个(优选不超过10、9、8、7、6、5、4、3、2、1个)突变的氨基酸序列，所述突变例如置换、缺失或添加，优选地置换，例如保守置换，优选地所述VEGF R胞外结构域-Fc保留与SEQ ID NO:25所示的结构域相似(例如具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％、99％)的与VEGF的结合亲和力。
在一些实施方案中，所述接头包含SEQ ID NO:23的氨基酸序列，或由其组成，例如n＝3。
在一些实施方案中，本发明的抗VEGF A×ANG2的双特异性结合分子的一条链包含SEQ ID NO:24的氨基酸序列，或包含与所述SEQ ID NO:24所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:24所述的氨基酸组成。在一些实施方案中，所述双特异性结合分子包含与SEQ ID NO:24所示的氨基酸序列相比具有一个或几个(优选不超过10、9、8、7、6、5、4、3、2、1个)突变的氨基酸序列，所述突变例如置换、缺失或添加，优选地置换，例如保守置换。在一些优选的实施方案中，所述突变不存在于抗Ang2 VHH的CDR中。在一些实施方案中，所述具有突变的VEGF R胞外结构域-Fc保留与SEQ ID NO:25所示的结构域相似(例如具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％、99％)的与VEGF的结合亲和力。
在本发明的一个实施方案中，本文所述的抗体或结合分子包含一个或多个氨基酸突变。在一些实施方案中，氨基酸突变包括氨基酸的置换、插入或缺失。优选的，本文所述的氨基酸改变为氨基酸置换，优选地保守置换。
在优选的实施方案中，本发明所述的氨基酸突变发生在CDR外的区域(例如在FR中)。在一些实施方案中，本发明所述的氨基酸突变发生在抗体重链恒定区，例如Fc区上，在优选的实施方案中，所述Fc区上的氨基酸突变削弱或删除了抗体的ADCC和/或CDC作用。
在一些实施方案中，置换为保守性置换。保守置换是指一个氨基酸经相同类别内的另一氨基酸置换，例如一个酸性氨基酸经另一酸性氨基酸置换，一个碱性氨基酸经另一碱性氨基酸置换，或一个中性氨基酸经另一中性氨基酸置换。
在某些实施方案中，可在本文中所提供抗体的Fc区中引入一个或多个氨基酸突变，以此产生Fc区变体，以改变抗体的一种或多种功能特性，例如血清半衰期、补体结合、补体依赖性细胞毒性、Fc受体结合和/或抗体依赖性细胞毒性。Fc区变体可包括在一或多个氨基酸位置处包含氨基酸突变(例如置换)的人Fc区序列(例如人IgGl、IgG2、IgG3或IgG4 Fc区)。
在某些实施方案中，可能需要对抗体的可变区进行突变以产生二硫键，例如产生包含二硫键突变的scFv。
在某些实施方案中，本文中所提供的抗体或结合分子可进一步经修饰为含有本领域中已知且轻易获得的其他非蛋白质部分。适合所述衍生作用的部分包括，但不限于，水溶性聚合物。水溶性聚合物的非限制性实例包括，但不限于，聚乙二醇(PEG)、乙二醇/丙二醇共聚物、羧甲基纤维素、葡聚糖、聚乙烯醇、聚乙烯吡咯烷酮、聚-1,3-二烷、聚-1,3,6-三烷、乙烯/马来酸酐共聚物、聚氨基酸(均聚物或无规共聚物)、及葡聚糖或聚(n-乙烯基吡咯烷酮)聚乙二醇、丙二醇均聚物、聚环氧丙烷/氧化乙烯共聚物、聚氧乙基化多元醇(例如甘油)、聚乙烯醇、及其混合物。
IV.本发明VHH抗体或双特异性结合分子的性质
在一些实施方案中，本发明抗Ang2 VHH抗体能够特异性结合Ang2，例如人Ang2，例如以高亲和力。
在一些实施方案中，本发明的抗VEGFA VHH抗体能够特异性结合VEGF A，例如人VEGF A，例如以高亲和力。
在一些实施方案中，本发明的双特异性结合分子能够特异性结合Ang2和VEGFA，例如人Ang2和人VEGFA，例如以高亲和力。
在一些实施方案中，本发明的抗Ang2抗体或抗VEGFA抗体或双特异性结合分子具有一种或多种以下性质：
(i)抗Ang2抗体或双特异性结合分子对于Ang2诱导的Tie2磷酸化具有抑制作用；
(ii)抗Ang2抗体或双特异性结合分子对于Ang2与Tie2之间结合具有阻断作用；
(iii)抗VEGFA抗体或双特异性结合分子对于VEGFA激活相关受体信号通路具有阻断作用，例如通过KDR reporter检测；
(iv)抗VEGFA抗体或双特异性结合分子对于VEGFA与VEGFR之间结合具有阻断作用；
(v)抗VEGFA抗体或双特异性结合分子对于VEGF A诱导的细胞(例如原代细胞，例如血管内皮细胞，例如人脐静脉内皮细胞，例如HUVEC)的存活和增殖具有抑制作用；
(vi)双特异性结合分子对血管内皮细胞(例如人脐静脉内皮细胞，例如HUVEC)渗漏具有抑制作用；
(vii)双特异性结合分子在体内或体外对新生血管化具有抑制作用，例如抑制眼底或视网膜脉络膜新生血管化，例如抑制新生血管引起的渗漏，例如保护血管完整性。
V.本发明的核酸以及包含其的宿主细胞
在一方面，本发明提供了编码以上任何VHH或双特异性结合分子或其任一条链的核酸。在一个实施方案中，提供包含所述核酸的载体。在一个实施方案中，载体是表达载体，例如真核表达载体。载体包括但不限于病毒、质粒、粘粒、λ噬菌体或酵母人工染色体(YAC)。在一个实施方案中，载体是例如pcDNA3.1。在一个实施方案中，提供包含所述核酸或所述载体的宿主细胞。在一个实施方案中，宿主细胞是真核的。在另一个实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞(例如CHO细胞(例如CHO-S)或293细胞(例如293F或HEK293细胞))或适用于制备抗体或其片段的其它细胞。在另一个实施方案中，宿主细胞是原核的，例如大肠杆菌，例如TG1。
例如，本发明的核酸包含编码选自SEQ ID NO:4、5、9、11、19、21、22、24、28中任一项所示氨基酸序列的核酸，或编码与选自SEQ ID NO:4、5、9、11、19、21、22、24、28中任一项所示的氨基酸序列具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的氨基酸序列的核酸。
在一个实施方案中，提供包含所述载体的宿主细胞。用于克隆或表达编码VHH或双特异性结合分子的载体的适当宿主细胞包括本文描述的原核或真核细胞。例如，VHH可在细菌中产生。在表达后，VHH存在于在培养物上清中，并且可以进一步纯化。
在一个实施方案中，宿主细胞是原核的，例如是细菌，例如大肠杆菌，例如TG1。
在一个实施方案中，宿主细胞是真核的。在另一个实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞或适用于制备抗体或其片段的其它细胞。例如，真核微生物诸如丝状真菌或酵母是关于编码抗体的载体的合适克隆或表达宿主。例如，糖基化途径已经进行“人源化”的真菌和酵母菌株导致产生具有部分或完全人糖基化模式的抗体。适于表达糖基化抗体的宿主细胞也衍生自多细胞生物体(无脊椎动物和脊椎动物)。也可以将脊椎动物细胞用作宿主。例如，可以使用被改造以适合于悬浮生长的哺乳动物细胞系。有用的哺乳动物宿主细胞系的其它实例是用SV40转化的猴肾CV1系(COS-7)；人胚肾系(HEK293、293F或293T细胞)等。其它有用的哺乳动物宿主细胞系包括中国仓鼠卵巢(CHO)细胞，包括DHFR-CHO细胞、CHO-S细胞、ExpiCHO等；以及骨髓瘤细胞系如Y0，NS0和Sp2/0。本领域已知适合产生抗体的哺乳动物宿主细胞系。
VI.本发明的VHH或双特异性结合分子的生产和纯化
在一个实施方案中，提供了制备本发明VHH或双特异性结合分子的方法，其中所述方法包括，在适合VHH或双特异性结合分子或其链表达的条件下，培养包含编码所述VHH或双特异性结合分子(例如任意一条多肽链和/或多条多肽链)的核酸或包含所述核酸的表达载体的宿主细胞，如上文所提供的，和任选地从所述宿主细胞(或宿主细胞培养基)回收所述VHH或双特异性结合分子。
为了重组产生本发明的分子，分离编码本发明VHH或双特异性结合分子(例如上文所描述的分子，例如任意一条多肽链和/或多条多肽链)的核酸，并将其插入一个或多个载体，用于在宿主细胞中进一步克隆和/或表达。此类核酸易于使用常规规程分离和测序。
如本文所述制备的VHH或双特异性结合分子可以通过已知的现有技术如高效液相色谱、离子交换层析、凝胶电泳、亲和层析、大小排阻层析等纯化。用来纯化特定蛋白质的实际条件还取决于净电荷、疏水性、亲水性等因素，并且这些对本领域技术人员是显而易见的。可以通过多种熟知分析方法中的任一种方法确定本发明的抗体分子的纯度，所述熟知分析方法包括尺寸排阻层析、凝胶电泳、高效液相色谱等。
VII.测定法
可以通过本领域中已知的多种测定法对本文中提供的VHH或双特异性结合分子进行鉴定，筛选，或表征其物理/化学特性和/或生物学活性。一方面，对本发明的VHH或双特异性结合分子测试其靶标(例如抗原)结合活性，例如通过已知的方法诸如生物膜薄层干涉技术、ELISA，等来进行。可使用本领域已知方法来测定对VEGF A和/或Ang2的结合，本文中公开了例示性方法。在一些实施方案中，使用放射性免疫测定(RIA)或生物膜薄层干涉测定法或MSD测定法或表面等离子体共振法(SPR)或流式细胞术测量。
本发明还提供了用于鉴定具有生物学活性的VHH或双特异性结合分子的测定法。生物学活性选自例如
(i)抗Ang2抗体或双特异性结合分子对于Ang2诱导的Tie2磷酸化的抑制作用；
(ii)抗Ang2抗体或双特异性结合分子对于Ang2与Tie2之间结合的阻断作用；
(iii)抗VEGFA抗体或双特异性结合分子对于VEGFA激活相关受体信号通路的阻断作用，例如通过KDR reporter检测；
(iv)抗VEGFA抗体或双特异性结合分子对于VEGFA与VEGFR之间结合的阻断作用；
(iv)抗VEGFA抗体或双特异性结合分子对于VEGF A诱导的细胞(例如原代细胞，例如血管内皮细胞，例如人脐静脉内皮细胞，例如HUVEC)的存活和增殖的抑制作用；
(vi)双特异性结合分子对血管内皮细胞(例如人脐静脉内皮细胞，例如HUVEC)渗漏的抑制作用；
(vii)双特异性结合分子在体内或体外对新生血管化的抑制作用，例如抑制眼底或视网膜脉络膜新生血管化，例如抑制新生血管引起的渗漏，例如保护血管完整性。
供任何上述体外测定法使用的细胞为原代细胞或细胞系，包括天然表达或过表达Ang2受体(例如Tie2)或VEGFR(例如VEGFR2，即KDR)的细胞，例如过表达Tie2或KDR的293细胞，例如HEK293或Expi293；或血管内皮细胞，例如人脐静脉内皮细胞，例如HUVEC。
在一些实施方案中，测定可以应用生物素或hFc等标记物进行。
可以理解的是，能够使用本发明的抗体和别的活性剂的组合来进行任何上述测定法。
VIII.免疫缀合物和药物组合物
在一些实施方案，本发明提供了包含本文所述的任何VHH或双特异性结合分子的免疫缀合物。优选地，所述免疫缀合物包含一种或多种其他治疗剂或标记物。
在一些实施方案中，本发明提供包含本文所述的任何VHH或双特异性结合分子的组合物或药物或制剂，优选地组合物为药物组合物。在一个实施方案中，所述组合物还包含药用辅料。在一个实施方案中，组合物，例如，药物组合物，包含本发明的VHH或双特异性结合分子，以及一种或多种其它治疗剂的组合。
本发明还包括包含本发明的VHH或双特异性结合分子的组合物(包括药物组合物)或药物或制剂。这些组合物或药物或制剂还可以包含合适的药用辅料，如本领域中已知的药用载体、药用赋形剂，包括缓冲剂。
如本文所用，“药用载体”包括生理上相容的任何和全部溶剂、分散介质、等渗剂和吸收延迟剂等。
对于药用辅料的使用及其用途，亦参见“Handbook of Pharmaceutical Excipients”,第八版,R.C.Rowe,P.J.Seskey和S.C.Owen,Pharmaceutical Press,London,Chicago。
本发明的组合物或药物或制剂可以处于多种形式。这些形式例如包括液体、半固体和固体剂型，如液态溶液剂(例如，注射液或滴眼液)、散剂或混悬剂、脂质体剂和栓剂。优选的形式取决于预期的施用模式和治疗用途。例如，本发明的组合物或药物或制剂可以是滴眼液。
可以通过将具有所需纯度的本发明的VHH或双特异性结合分子与一种或多种任选的药用辅料混合来制备包含本文所述的VHH或双特异性结合分子的药物或制剂，优选地以冻干制剂或水溶液的形式。
本发明的组合物或药物或制剂还可以包含超过一种活性成分，所述活性成分是被治疗的特定适应证所需的，优选具有不会不利地彼此影响的互补活性的那些活性成分。例如，理想的是还提供其它治疗剂。
可制备持续释放制剂。持续释放制剂的合适实例包括含有抗体的固体疏水聚合物的半渗透基质，所述基质呈成形物品，例如薄膜或微囊形式。
本发明还提供了药物组合或药物组合产品，其包含本发明的VHH或双特异性结合分子，以及一种或多种其它治疗剂。本发明还提供了包含所述药物组合的成套药盒，例如所述成套药盒在同一包装内包含：
-含有包含本发明的VHH或双特异性结合分子的药物组合物的第一容器；
-含有包含其它治疗剂的药物组合物的第二容器。
IX.用途和方法
本发明一方面提供了在受试者中预防或治疗眼部疾病的方法，包括向受试者施用有效量的本发明的抗VEGF VHH、本发明的抗Ang2 VHH或本发明的双特异性结合分子，或包含其的组合物或药物或制剂。
在一些实施方案中，所述患者中具有(例如升高水平的，例如核酸或蛋白质水平的)VEGF，例如VEGF A，和/或Ang2。
在一些实施方案中，所述眼部疾病包括但不限于与血管发生相关的眼部疾病，例如与角膜新生血管化相关的眼部疾病。
在一些实施方案中，所述眼部疾病治疗将受益于抑制核酸或蛋白质水平的VEGF，例如VEGF A，和/或Ang2。
在其他方面，本发明提供本发明的抗VEGF VHH、本发明的抗Ang2 VHH或本发明的双特异性结合分子或包含其的组合物在生产或制备药物中的用途，所述药物用于本文所述的用途，例如用于预防或治疗本文提及的相关疾病或病症。
在一些实施方案中，本发明的抗VEGF VHH、本发明的抗Ang2 VHH或本发明的双特异性结合分子或包含其的组合物或药物或制剂会延迟病症和/或与病症相关的症状的发作。
在一些实施方案中，本发明的抗VEGF VHH、本发明的抗Ang2 VHH或本发明的双特异性结合分子或包含其的组合物或药物或制剂还能与一种或多种其它疗法例如治疗方式和/或其它治疗剂组合施用，用于本文所述的用途，例如用于预防和/或治疗本文提及的相关疾病或病症。
本发明的抗VEGF VHH、本发明的抗Ang2 VHH或本发明的双特异性结合分子或包含其的组合物或药物或制剂的施用途径是根据已知方法，例如，局部施用，例如眼内施用、眼表施用。在一些实施方案，通过注射或滴入施用。
X.用于诊断和检测的方法和组合物
在某些实施方案中，本文中提供的抗Ang2 VHH抗体可以用于检测Ang2在生物样品中 的存在。在某些实施方案中，本文中提供的抗VEGFA VHH抗体可以用于检测VEGFA在生物样品中的存在。在某些实施方案中，本文中提供的抗双特异性结合分子可以用于检测Ang2和/或VEGFA在生物样品中的存在。
术语“检测”用于本文中时，包括定量或定性检测，示例性的检测方法可以涉及免疫组织化学、免疫细胞化学、流式细胞术(例如，FACS)、抗体分子复合的磁珠、ELISA测定法、PCR-技术(例如，RT-PCR)。在某些实施方案中，生物样品是体液或眼部组织，例如玻璃体液。
在某些实施方案中，所述方法包括将生物样品与如本文所述的VHH或双特异性结合分子在允许其与Ang2或VEGFA结合的条件下接触，并检测在该VHH或双特异性结合分子和Ang2或VEGFA之间是否形成复合物。复合物的形成表示存在Ang2或VEGFA。该方法可以是体外或体内方法。在一个实施方案中，本发明的抗体用于选择适合利用本发明的VHH或双特异性结合分子治疗的受试者，例如其中Ang2或VEGFA是用于选择所述受试者的生物标记物。
在某些实施方案中，提供标记的VHH或双特异性结合分子。标记包括但不限于，被直接检测的标记或部分(如荧光标记、发色团标记、电子致密标记、化学发光标记和放射性标记)，以及被间接检测的部分，如酶或配体，例如，通过酶促反应或分子相互作用。在一些实施方案中，所述标记例如生物素或hFc等标记物。
在本文中提供的一些实施方案中，样品是在用本发明的VHH或双特异性结合分子治疗之前获得的。在一些实施方案中，样品是在用其他疗法之前获得的。在一些实施方案中，样品是在用其他疗法治疗过程中，或者用其他疗法治疗后获得的。
在一些实施方案中，在治疗之前，例如，在起始治疗之前或在治疗间隔后的某次治疗之前检测Ang2和/或VEGF A。
在一些实施方案中，提供了一种治疗本发明疾病的方法，所述方法包括：对受试者(例如，样品)(例如，受试者样品)检验Ang2和/或VEGF A的存在，因而确定Ang2和/或VEGF A值，将Ang2和/或VEGF A值与对照值(例如正常个体中的值)比较，并且如果Ang2和/或VEGF A值大于对照值，则向受试者施用治疗有效量的任选与一种或多种其他疗法组合的本发明所述的VHH或双特异性结合分子，因而治疗所述疾病。
本发明的这些以及其它方面和实施方案在附图(附图简述紧随其后)和以下的发明详述中得到描述并且示例于以下实施例中。上文以及整个本申请中所论述的任何或所有特征可以在本发明的各种实施方案中组合。以下实施例进一步说明本发明，然而，应理解实施例以说明而非限定的方式来描述，并且本领域技术人员可以进行多种修改。
实施例1.噬菌体免疫库的制备
羊驼免疫或合成库构建
1.1选取健康成年羊驼(成都阿帕克公司)2只，将0.5mg重组蛋白抗原VEGFA或Ang2(北京义翘公司)与弗氏佐剂按1:1比例混匀，背部皮下多点注射的方式免疫羊驼，共免疫四次，免疫间隔为2周。
1.2采集50ml羊驼外周血，分离淋巴细胞，按照每2.5×10 7个活细胞加入1mL Trizol试剂，采用氯仿/异丙醇沉淀方法抽提总RNA。取10ug RNA作为模板，使用PrimeScript逆转录试剂盒(Takara公司)进行反转录。将cDNA作为模版进行第一轮PCR反应，使用正向引物Alp-VhL和反向引物Alp-2b/2cR，获得第一轮PCR产物。将第一轮PCR产物作为模板进行第二轮PCR反应，使用正向引物Alp-VhF和反向引物Alp-JHR-SalI，获得第二轮PCR产物。将pC3-HF载体和第二轮PCR产物分别使用SacI和SalI(Thermo公司)进行双酶切，酶切产物加入T4连接酶(Thermo公司)反应，电转化TG1感受态细胞，构建VHH抗体文库，将菌液在-80℃冻存。
取复苏的菌液接种至100ml YT-AG培养基(上海生工公司)中，加入M13KO7辅助噬菌体进行感染，用2×YT-AK培养基(上海生工公司)重悬菌体，37℃200rpm培养过夜。收集培养上清，采用PEG/NaCl沉淀方法制备重组噬菌体。
1.3重组噬菌体使用生物素标记抗原VEGFA(ACRO公司)或Ang2(北京义翘公司)进行3轮淘洗实验。每管加入50ul M280磁珠(Thermo公司)和适量生物素标记抗原，室温孵育30分钟后，加入1x10 12cfu重组噬菌体室温孵育1小时。将获得的混合物加入1ml PBST洗涤10次，每次洗涤时间5分钟。最后加入0.5ml pH2.5甘氨酸缓冲液，洗脱抗原结合的重组噬菌体，侵染TG1过夜培养，制备重组噬菌体，用于下一轮淘洗实验并且鉴定阳性VHH的TG1细菌克隆。
1.4 Binding ELISA检测结合活性和克隆测序。
提前取VEGF A，Ang2抗原(北京义翘公司)用PBS缓冲液稀释成0.5ug/ml包被96孔ELISA板，4℃冰箱过夜。抗原包被板用PBST洗3遍，加入封闭剂至300ul/孔，室温静置封闭1小时。PBST洗3遍，加入80ul封闭剂+20ul上述1.3鉴定的阳性VHH的TG1菌的表达上清，室温振荡1小时。
PBST洗3遍，加入封闭剂稀释的Anti-Flag/HRP二抗(Sigma公司)100ul/孔，室温震荡40分钟。PBST洗6遍，加入TMB显色液至100ul/孔，避光显色5-15分钟。再加入100ul/孔终止液。酶标仪读数，测定OD450nm吸光值，选取读值大于0.5的细菌克隆送金唯智测序，选取含有各个对应VHH序列的TG1菌单克隆加入甘油，冻于-80℃冰箱。
实施例2.原核抗体的生产和纯化，及人源化
本发明利用分子生物学技术，获得抗VEGFA或Ang2阳性噬菌体中的抗体序列，并利用上述获得的含有阳性VHH的TG1单克隆表达纯化获得VHH抗体蛋白。
取实施例1鉴定的含有VHH表达质粒的TG1菌，接种到800ml LB-Amp培养基中，37℃200rpm培养至OD600值0.5-0.6。菌液加入1mM IPTG诱导表达，28℃200rpm培养过夜，收集培养上清，离心后加入15ml PB+1mg/ml多粘菌素重悬细菌，再次离心，0.22um滤膜过滤。将细菌裂解液流过1ml Ni Sepharose预先柱，加入PBS洗涤2次，加入0.5M咪唑洗脱目标蛋白，用紫外法测定蛋白浓度。洗脱的目标蛋白用紫外法测定蛋白浓度，分装多管置于-40度冰箱保存。获得的抗体溶液后续称为上清。
本发明获得的2个抗VEGF A VHH抗体(LA42F8和LA46E11)和1个抗Ang2 VHH抗体(LA24C11)的CDR、和VHH的氨基酸序列，以及序列编号请参见序列表。
之后将上述获得的免疫库抗体LA42F8，LA46E11，LA24C11抗体人源化，步骤如下：
①确定CDR环结构；
②在人种系序列数据库为重链的每个V/J区域找到最接近的同源序列；
③筛选与重链轻链最匹配的人种系以及最低量的回复突变；
④将嵌合抗体的CDR区构建至人的骨架区上；
⑤使用序列和结构特征，确定骨架区中起到维持CDR功能的氨基酸位置；
⑥在确定为重要的序列位置进行回复突变(返回到输入氨基酸类型)；
⑦优化风险位点的氨基酸。
本发明获得的3个人源化抗体分别获得人源化VHH抗体LA42F8.5，LA46E11.8，LA24C11.10的CDR、和重链可变区的氨基酸序列请参见所附序列表。
上述LA42F8，LA46E11，LA24C11和人源化抗体LA42F8.5，LA46E11.8，LA24C11.10在真核细胞中的表达制备如下：
将从上文获得的人源化抗体序列克隆到pcDNA3.1(Invitrogen)，分别获得包含抗体序列的质粒。
根据所需转染体积传代Expi-293细胞(Invitrogen)，转染前一天将细胞密度调整至1.5×10 6个细胞/ml。转染当天细胞密度约为3×10 6个细胞/ml。取终体积1/10的F17培养基(Gibco，A13835-01)作为转染缓冲液，加入适当的所述质粒，混匀。加合适的聚乙烯亚胺(PEI)(Polysciences，23966)到质粒中(质粒与PEI的比例在293F细胞中为1:3)，混匀后室温孵育10min，获得DNA/PEI混合物。用DNA/PEI混合物重悬细胞后，36.5℃，8％的CO 2。24h后补加转染体积2％的FEED(Sigma)，于36.5℃，120rpm，8％的CO 2条件下培养。连续培养至第6天或者细胞活力≤60％时，收集细胞上清进行纯化。
将纯化使用的重力柱使用0.5M NaOH过夜处理，玻璃瓶等用蒸馏水洗净后在180℃干烤4h，获得纯化柱。纯化前将上述收集的细胞上清4500rpm离心30min，弃掉细胞。再将上清使用0.22μl的滤器过滤。使用10ml结合缓冲液(磷酸钠20mM.NaCl150mM,PH7.0)平衡Protein A柱(Hitrap Mabselect Sure 5*5ml，GE，11-0034-95)。将过滤后的上清加入纯化柱后使用15ml结合缓冲液再平衡。加5ml洗脱缓冲液(柠檬酸+柠檬酸钠0.1M,pH3.5)，收集洗脱液，每1ml的洗脱液加入80μl Tris-HCl。将收集的抗体超滤浓缩交换到PBS(Gibco，70011-044)中，并检测浓度。除表3，图2，图4A和图5A中的用于检测的本发明抗体，后续如无特殊提及上清，实施例中所用的抗体均为此表达纯化的抗体。
类似地，阴性对照IgG、阳性对照BI-anti-VEGF、BI836880、Faricimab(序列见序列表)的编码核酸克隆到pcDNA3.1后，转染Expi-293细胞后，表达并纯化获得，方法同实施例2。
实施例3生物膜薄层干涉技术测定本发明的嵌合抗体与抗原的结合动力学
采用生物膜薄层干涉测定技术(ForteBio)测定本发明抗体结合人Ang2的平衡解离常数(KD)。ForteBio亲和力测定按照现有的方法(Estep,P等人，High throughput solution Based measurement of antibody-antigen affinity and epitope binning.MAbs,2013.5(2):第270-8页)进行。
实验开始前半个小时，根据样品数量，取合适数量的AMQ(Pall，1506091)(用于样品检测)或AHQ(Pall，1502051)(用于阳性对照检测)传感器浸泡于SD buffer(PBS1×，BSA  0.1％，Tween-200.05％)中。
取100μl的SD缓冲液、上文实施例2中制备的VHH抗体、抗原[包括人Ang2(北京义翘)、及人VEGF165(R&D)]，分别加入到96孔黑色聚苯乙烯半量微孔板(Greiner，675076)中。根据样品位置布板，选择传感器位置。仪器设置参数如下：运行步骤：Baseline、Loading～1nm、Baseline、Association和Dissociation；各个步骤运行时间取决于样品结合和解离速度，转速为400rpm，温度为30℃。使用ForteBio分析软件分析K D值。
在以上测定法所述的实验中，抗体的亲和力如表1所示：
表1.ForteBio检测抗原抗体单价结合的亲和力常数(平衡解离常数)
Figure PCTCN2022096848-appb-000002
ND表示未检测
表2.ForteBio检测抗原抗体双价结合的亲和力常数(平衡解离常数)
Figure PCTCN2022096848-appb-000003
*代表解离常数超过ForteBio检测极限
实施例4抗VEGF A VHH抗体ELISA阻断实验
本实施例验证了本发明抗VEGF A VHH对于hVEGF A与受体KDR结合的阻断作用。将SA(Thermo货号21125)稀释成1ug/ml，100ul/孔铺于酶标版中4℃过夜。PBST洗3遍，加入3％BSA封闭1.5h。PBST洗3遍，加入50ng/ml生物素标记的VEGF A165(ACRO货号VE5-H8210)，孵育1.5h，提前将抗体(起始浓度为150ug/ml，3倍连续稀释)的实施例2中制备的LA42F8上清、LA46E11上清和阴性对照IgG，以及阳性对照BI-anti-VEGF)50ul与VEGFR-Fc(北京义翘，货号：10012-H02H终浓度0.2ug/ml)孵育20min后加入板中。PBST洗3遍，加入抗human Fc HRP抗体(Bethyl货号：A80-104P)(1:10000)孵育30min。PBST洗6遍，TMB显色5min，终止后OD450nm读数。
本发明获得的3个抗VEGF VHH抗体阻断结果如图2。图2显示了候选分子LA42F8,LA46E11抗体能够完全阻断VEGF A与VEGFR2结合。
实施例5抗Ang2 VHH抗体ELISA阻断实验
本实施例验证了本发明抗体抗Ang2 VHH对于hAng2-biotin(R&D货号：BT623B/CF)与Tie2蛋白结合的阻断作用
将Tie2-Fc(北京义翘货号：10700-H03H)稀释成2ug/ml，100ul/孔铺于酶标版中4℃过夜。PBST洗3遍，加入3％BSA封闭1.5h。PBST洗3遍，提前将实施例2中制备的纯化的LA24C11.10与阴性对照IgG各50ul与hAng2-biotin(终浓度0.2ug/ml)孵育20min后加入板中。PBST洗3遍，加入Avidin HRP(1:2000)孵育35min。PBST洗6遍，TMB显色5min，终止后OD450nm读数。
本发明获得的抗Ang2 VHH抗体阻断结果如表3，候选分子LA24C11对Ang2于受体Tie2结合有阻断作用。
表3.纯化抗体单点blocking ELISA
Figure PCTCN2022096848-appb-000004
1抗体是指实施例2制备的纯化原核上清，起始浓度为0.556mg/ml，后续经1:10或1:100稀释；
2上清是指未经纯化的原核上清。
与实施例4类似的(应用Ang2-Bio(R&D货号：BT623B/CF))，通过ELISA阻断实验检测了LA24C11.10阻断Ang2与Tie2的结合的作用，结果如图3所示。可见，LA24C11.10对Ang2于受体Tie2结合有阻断作用。
实施例6抗Ang2 VHH抗体的磷酸化实验
本实施例验证了本发明的抗体抗Ang2 VHH抗体对于hAng2-Fc诱导的Tie2磷酸化抑制作用。
hAng2诱导的磷酸化实验
本研究将抗体和重组hAng2-Fc蛋白共同孵育过表达Tie2的Expi293细胞293-Tie2，通过检测体系中磷酸化Tie2的含量，从而反应出不同抗体对hAng2-fc诱导的Tie2磷酸化抑制作用。
通过向Expi-293细胞(Thermo)转染携带克隆至多克隆位点MCS的人Tie2基因(北京义翘货号：HG10700-M)的pCHO1.0载体(Invitrogen)，产生过表达人Tie2的Expi-293细胞293-Tie2细胞。
取过表达人Tie2的293-Tie2细胞，稀释至2*10 6细胞/ml，每孔100ul加入96孔板，400g离心5min，去上清。
使用Expi293培养基(Thermo货号A1435102)配置实验培养基：其中加入测试抗体(实施例2中制备的LA24C11(24C11)和LA24C11.0(hz24C11.10)，以及阴性对照IgG，和阳性对照Nesvacumab(依据CN202010573625.2制备))，起始终浓度为60ug/ml，依次1:2等比稀释；hAng2-Fc(北京义翘：10691-H02H)终浓度为2.5ug/ml。
用每孔100ul实验培养基重悬细胞，37℃孵育15min，
离心去除培养基，加入100ul含1％蛋白酶(Thermo货号：78442)及磷酸酶抑制剂(Thermo货号：78442)的NP-40裂解液(碧云天，货号：P0013F)，冰上放置30min，2000g离心，收集蛋白上清，放于-80度冰箱存储。
按照磷酸化Tie2ELISA试剂盒(R&D货号：DYC2720E)的说明检测pTie2浓度，将试剂盒中的捕获抗体以4ug/ml浓度包被至酶标板上4℃过夜。PBST洗涤三次，5％BSA封闭1h。加入冻融的上一步获得的蛋白上清100ul，及对照pTie2(R&D货号：DYC2720E)用于制作标准曲线室温孵育2h(若样品pTie2浓度过高，超过ELISA检测范围的时候，可对获得的混合物进行2～3倍稀释)。PBST洗涤三次，加入100ul缀合有HRP的抗pTyr抗体(R&D货号：DYC2720E),室温孵育2h。PBST洗涤6次，加入100ulTMB(Solarbio货号：PR1200)显色，15min后加入100ul终止缓冲液(Solarbio货号：C1058)终止反应。应用多功能酶标仪SpectraMax i3测定OD450-OD620。实验结果参见图4A(24C11为实施例2中制备的LA24C11原核表达纯化上清)和图4B。
因此，本发明的抗Ang2 VHH抗体LA24C11和LA24C11.10均可以在体外有效抑制hAng2-Fc诱导的293-Tie2细胞磷酸化。
实施例7抗VEGF A VHH抗体KDR reporter阻断实验
VEGF A可以与相关受体VEGFR2(KDR)结合，激活VEGFR2信号通路，诱导血管内皮细胞存活，增殖和迁移等作用，本研究利用KDR reporter实验体系，使用NFAT-RE-luc2P/KDR HEK293细胞(Promega Cat CS181401)，检测梯度稀释的抗体对VEGFA激活相关受体信号通路的阻断作用。
实验方法参考供应商(Promega)说明：
将提前3天换成实验培养基(含10％FBS的DMEM培养基)的NFAT-RE-luc2P/KDR HEK293细胞取出，吸除旧培养基，用PBS洗涤一次，之后用1ml Accutase solution(Sigma货号：A6964-500ML)消化细胞，直至细胞变圆脱壁，用5ml稀释培养基终止反应，吸取细胞至离心管中，1000rpm离心5min，弃去培养基，加入10ml稀释培养基(含10％FBS的DMEM培养基)重悬细胞，混匀后计数，细胞活率应在90％以上。用稀释培养基调整细胞密度至0.8×10 6个/ml，50μl/孔按照实验布局加入96孔白色细胞培养板中，
配置浓度为100ng/ml VEGF A和梯度稀释的待测抗体混合液，静置30min，50μl/孔含有细胞的96孔白色细胞培养板中，放入37℃、5％二氧化碳培养箱中孵育6h。其中，待测样品如下：阴性对照IgG；阳性对照BI-anti-VEGF；LA42F8；LA46E11；LA42F8.5；LA46E11.8；Blank：仅含稀释培养基，无VEGF A，无抗体；VEGF A100ng/ml：仅含100ng/ml VEGF A。
从二氧化碳培养箱中取出已孵育6h的96孔白色细胞培养板，平衡10～15min至室温。将提前拿出平衡至室温的Bio-Glo Luciferase Assay System按照实验布局100μl/孔加入96孔白色细胞培养板中，室温避光孵育5min。
使用多功能酶标仪进行荧光读值，读板模式选择化学发光模式、读板类型选择终点法、波长设定为全波长，逐列收集荧光，每列收集时间为1000ms。
在以上测定法所述的实验中，检测结果如图5显示，抗VEGF VHH抗体LA42F8，LA42F8.5，LA46E11，LA46E11.8均可阻断VEGF A诱导的KDR信号通路激活。
实施例8抗VEGF A VHH抑制VEGF A诱导的HUVEC存活增殖实验
VEGF A可作用于血管内皮细胞中VEGFR等相关的受体，促进血管内皮细胞存活，增殖和迁移，进而诱导新生血管化，本实验基于VEGF诱导人脐静脉内皮细胞(HUVEC)存活和增殖，检测抗体对VEGF A诱导的原代细胞存活和增殖的抑制作用。
本实施例通过CCK-8测定HUVEC的存活和增殖，具体方法如下：提前一天处理细胞HUVEC(Allcells货号：H-001-CN)，2000细胞/孔铺于96孔培养板中，放入37℃、5％二氧化碳培养箱中孵育24小时，
待细胞贴壁后，配置含终浓度为10ng/ml VEGF A和梯度稀释的抗体(实施例2中制备的LA42F8，起始浓度80ug/ml1:3等比稀释，阴性对照IgG，阳性对照BI836880，仅含10ng/ml VEGF A的组(VEGFA)，以及无VEGF A和抗体添加的Blank)实验培养基，置换96孔板中内皮细胞培养基，放入37℃、5％二氧化碳培养箱中孵育72小时，
10μl/孔加入CCK-8检测液(同仁化学货号：CK04)，放入37℃、5％二氧化碳培养箱中孵育12～24小时，
使用多功能酶标仪进行吸光度OD 450-OD 620读值，
在以上测定法所述的实验中，检测结果如图6所示，从图中可以看出，抗VEGFA抗体LA42F8能够完全抑制VEGFA诱导的HUVEC细胞存活和增殖。
实施例9抗VEGF A/Ang2双特异性抗体HEK293-KDR reporter阻断实验
将本发明的双特异性结合分子的链IEX04-008、IEX04-010和IEX04-012(序列如序列表所示)的链构建到pcDNA3.1载体，如实施例2所述在293细胞中表达和纯化所述双特异性结合分子。
本实施例通过HEK293-KDR reporter实验检测了抗VEGF A/Ang2双特异性抗体对VEGF A的阻断作用。实验方法参照实施例7。
应用的结合分子或对照如下：
Blank：无VEGF A，无抗体；
VEGF A：100ng/ml VEGF A；
阴性对照IgG：如上所述制备；
阳性对照Faricimab：如上所述制备，起始浓度为13.5ug/ml,1:3梯度稀释；
阳性对照BI-836880：如上所述所述制备，起始浓度为13.5ug/ml,1:3梯度稀释；
IEX04-012：如上所述制备，起始浓度为13.5ug/ml,1:3梯度稀释。
检测结果如图7所示，双特异性结合分子IEX04-012抑制VEGF诱导KDR信号通路激活，与对照抗体BI836880相比，具有更优的抑制能力。
实施例10抗VEGF A/Ang2双特异性结合分子HUVEC增殖抑制实验
本研究通过HEK293-KDR reporter实验检测了抗VEGF A/Ang2双特异性结合分子对VEGF A诱导的HUVEC细胞存活和增殖的抑制作用。
实验方法如实施例8，但是应用的抗体如下：
图A:起始浓度为20ug/ml,1:3等比稀释的如上所述制备的IEX04-008、阴性对照IgG，阳性对照Faricimab和BI836880，空白为Blank(即无抗体和VEGF A)，以及VEGF A组(即仅添加20ng/ml VEGFA)；
图B：起始浓度为80ug/ml，1:3等比稀释的如上所述制备的阴性对照IgG、BI-anti-VEGF、IEX04-010、空白为Blank(即无抗体和VEGF A)，以及VEGF A组(即仅添加20ng/ml VEGFA)；
图C:起始浓度为20nM，1:3等比稀释的如上所述制备的IEX04-012、阴性对照IgG，阳性对照Faricimab和BI836880，空白为Blank(即无抗体和VEGF A)，以及VEGF A组(即仅添加20ng/ml VEGFA)。
检测结果如图8所示在，双特异性结合分子IEX04-008，IEX04-010，IEX04-012均抑制了VEGF诱导的HUVEC细胞存活和增殖，与对照抗体BI836880和Faricimab相比，IEX04-012的IC 50更低，具有更优的抑制能力。
实施例11抗VEGFA/Ang2双特异性抗体Ang2阻断实验
Ang2可与其天然受体Tie2结合，本研究通过ELISA法和FACS法检测了抗VEGF A/Ang2双特异性结合分子对Ang2与Tie2之间结合的阻断。
(1)ELISA
通过ELISA检测了IEX04-008，IEX04-010和IEX04-012以及对照抗体BI-836880和Faricimab阻断人Ang2与hTie2结合的能力。
将hTie2蛋白(北京义翘)使用PBS重悬并溶解至2ug/ml浓度，包被至酶标板过夜。使用5％BSA封闭1h，将生物素抗原Recombinant Biotinylated hAngiopoietin-2蛋白(R&D)稀释至600ug/mlM，50μl/孔。将如上所述制备的抗体(IEX04-008，IEX04-010、IEX04-012以及阳性对照抗体BI836880和Faricimab和阴性对照IgG)从最高浓度300nM1：2等比稀释，共8个或12稀释梯度，50μl/孔，于PBS冰上孵育30min，生物素抗原终浓度300ng/ml。将如上所述获得的抗原抗体混合液孵育至酶标板90min，PBS洗三次，弃上清，加1:10000稀释的100μl Avidin-HRP(Invitrogen)/孔，常温30min，加PBS洗六次。使用100ul/孔TMB 显色液(solarbio)显色1min，并用终止液(Solarbio)100ul/孔终止。
应用酶标仪对每孔读数OD 450，OD 620。
实验结果表明(参见图9)，IEX04-008，IEX04-010，IEX04-012以及对照抗体BI836880都具有完全阻断效果，且本发明的双特异性结合分子的IC 50均显著小于阳性对照抗体。
(2)流式细胞检测(FACS)
通过FACS检测了IEX04-008，IEX04-010，IEX04-012以及阳性对照抗体BI-836880和Faricimab和阴性对照IgG阻断人Ang2-hFc与细胞表面的Tie2结合的能力。
将抗原hAng2-Fc蛋白(北京义翘，货号：10691-H02H)稀释至4ug/ml，50μl/孔。将如上所述制备的抗体(IEX04-008，IEX04-010、IEX04-012以及阳性对照抗体BI-836880和Faricimab和阴性对照IgG)从最高浓度800nM开始进行2倍梯度稀释，共12个稀释梯度，50μl/孔，于PBS冰上孵育30min，抗原hAng2-Fc蛋白终浓度2ug/ml，每个抗体最高终浓度400nM。将如上所述制得的293-Tie2细胞调节至2×10 5细胞/孔，100μl/孔。细胞于300g离心5min，弃上清，重悬于抗原抗体混合液。冰上孵育30min，加PBS100μl/孔，300g离心5min，PBS清洗1次，加1:200稀释的100μl Goat anti-human IgG-PE(SouthernBiotech)/孔，冰浴20min，加PBS 100μl/孔，300g离心5min，PBS清洗1次。用100μl PBS重悬，细胞流式仪(BD Biosciences)检测细胞荧光信号值。根据其MFI，用GraphPad拟合浓度依赖的曲线。结果如图10所示。图中显示，双特异性结合分子IEX04-008、IEX04-010和IEX04-012都能有效阻断人Ang2-hFc与Tie2的结合，且IC 50低于阳性对照。
实施例12抗VEGF A/Ang2双特异性结合分子的Ang2磷酸化抑制实验
本实施例验证了本发明双特异性结合分子对于hAng2-Fc诱导的Tie2磷酸化的抑制作用，其应用了hAng2诱导的磷酸化实验。
本研究将双特异性结合分子和重组hAng2-Fc蛋白共同孵育过表达Tie2的Expi293细胞，293-Tie2，通过检测体系中磷酸化Tie2的含量，从而反应出不同抗体对hAng2-Fc诱导的Tie2磷酸化抑制作用。
取如上制备的过表达293-Tie2细胞，稀释至2*10 6cell/ml，每孔100ul加入96孔板，400g离心5min，去上清。
使用Expi293培养基(Thermo货号A1435102)配置实验培养基，其中分别加入了测试抗体(如上制备的IEX04-012、阳性对照BI-836880和Faricimab、阴性对照IgG)最高终浓度为60ug/ml，依次1:2等比稀释，hAng2-Fc(北京义翘货号：10691-H02H)终浓度为2.5ug/ml。
用每孔100ul实验培养基重悬细胞，37度孵育15min，离心去除培养基，加入100ul含1％蛋白酶及磷酸酶抑制剂的NP-40裂解液，冰上放置30min。2000g离心，收集蛋白上清，放于-80度冰箱存储，
按照磷酸化Tie2 ELISA试剂盒(R&D DYC2720E)说明检测pTie2浓度，将捕获抗体以4ug/ml浓度包被至酶标板上4度过夜。PBST洗涤三次，5％BSA封闭1h。加入待测样品100ul，及对照pTie2(R&D DYC2720E)用于制作标准曲线室温孵育2h(若样品pTie2浓度过高，超过ELISA检测范围的时候，可对获得的混合物进行2～3倍稀释)。PBST洗涤三次，加入100ul缀合有HRP的抗pTyr抗体(R&D货号：DYC2720E),室温孵育2h。PBST洗涤6次，加入100ul TMB显色，15min后加入100ul终止缓冲液终止反应。应用分光光度计测 定每孔的OD450-OD620。
实验结果如图11所示。本发明的抗体IEX04-012可以在体外有效抑制hAng2-Fc诱导的293-Tie2磷酸化，且IC 50优于阳性对照。
实施例13抗VEGF A/Ang2双特异性结合分子对Ang2血管内皮细胞渗漏的抑制实验
本研究通过HUVEC-Tie2渗漏实验鉴定抗VEGF A/Ang2双特异性结合分子对血管内皮细胞渗漏的作用和功能。
使用慢病毒转染HUVEC细胞(Allcells货号：H-001-CN)获得过表达Tie2的HUVEC-Tie2细胞。
使用mini-well 96孔插入式培养皿下层铺EGM-2培养基300ul，用Accutase(Sigma)消化并获得HUVEC-Tie2，使用EGM-2培养基重悬至1*10 7细胞/ml,100ul/孔铺于培养皿上层。每隔24h置换下室培养基(EGM-2培养基)，24小时后更换下室培养基为实验培养基，所述实验培养基组成如下：
Blank：EGM-2培养基(Lonza货号：CC-5035)，
VEGF A组：EGM-2培养基+20ng/ml VEGF(R&D货号：293-VE)
IgG组(VEGF A+IgG)：EGM-2培养基+20ng/ml VEGF+10ug/ml IgG；
Ang1组(VEGF A+Ang1)：EGM-2培养基+20ng/ml VEGF(R&D货号：293-VE)+200ng/ml Ang1(R&D货号：923-AN)；
IEX04-012组(VEGF A+IEX04-012)：EGM-2培养基+20ng/ml VEGF+10ug/ml IEX04-012；
BI-836880组(VEGF A+BI-836880)：EGM-2培养基+20ng/ml VEGF+10ug/ml BI-836880；
Faricimab组(VEGF A+Faricimab)：EGM-2培养基+20ng/ml VEGF+10ug/ml Faricimab。
将上述实验培养基放置于37℃、5％CO 2培养。
24小时后上室实验培养基每孔加入1ul FITC-Dextran(Sigma货号：FD2000S-1G)(4mg/ml),放置于37℃、5％CO 2.30min后取下室培养基，使用PBS稀释1:10稀释后于多功能酶标仪检测，激发光波长488nm，发射光波长535nm。
实验结果如图12，其显示本发明抗体IEX04-012能够有效降低VEGF诱导的血管内皮细胞通透性。
实施例14.激光诱导的脉络膜新生血管化药效试验
本实验采用恒河猴激光诱导的脉络膜新生血管化模型测定本发明的双特异性结合分子IEX04-012的抗新生血管化作用。
恒河猴：
种属：恒河猴；等级：普通级；体重：购入时体重3.30～4.20kg；造模时体重3.35～4.35kg；来源：四川横竖生物科技股份有限公司，生产许可证号：SCXK(川)2019-029，实验动物质量合格证编号：No.0023356；
本试验采用激光围绕恒河猴眼底黄斑中心凹光凝，诱导眼底脉络膜血管新生，建立与人类脉络膜新生血管类似的动物模型。光凝前及光凝后20天进行荧光素眼底血管造影判定造模情况，选择造模成功的20只恒河猴(雌雄各半)分5组，分别为模型对照组、IEX04-012低剂量组、IEX04-012高剂量组、Elyea组、Faricimab组5组，每组4只猴，雌雄各半。
光凝后第21天，对各组猴分别按表中剂量进行给药，双眼玻璃体注射给予IEX04-012、Eylea(Bayer)或Faricimab(均溶于0.9％氯化钠注射液)，模型对照组给与等体积的0.9％氯化钠注射液。各组动物分别于给药后7、14、21、28天进行眼底彩色照相、荧光素眼底血管造影(渗漏斑统计和渗漏面积测量)(Robin J Goody,Wenzheng Hu,Afshin Shafiee等人，Optimization of laser-ind uced choroidal neovascularization in African green monkeys.Experimental Eye Research,Exp Eye Res.2011 92(6):464-72)，光学相干断层扫描检查(OCT，Wang Q,Lin X,Xiang W等人，Assessment of laser induction of Bruch's membrane disruption in monkey by spectral-domain optical coherence tomography.British Journal of Ophthal mology,2015,99(1):119-24)，观察供试品对脉络膜新生血管的抑制情况。于给药后29天实施安乐死后取双眼进行免疫组织化学(HE)染色的组织学检查。
实验设计表
Figure PCTCN2022096848-appb-000005
结果如图13-15显示，本发明双特异性结合分子在给药28天后即显示出显著的抗新生血管化作用，四级渗漏光斑(图13A)和三至四级渗漏光斑(图13B)数量统计显示，IEX04-012组治疗动物，高渗漏光斑数量显著少于对照和阳性对照给药组。OCT结果显示，IEX04-012治疗组动物中，视网膜厚度显著降低，提示视网膜水肿程度减轻，且效果优于对照(图14)。荧光素眼底血管照影结果显示，IEX04-012治疗组眼底渗漏面积显著降低，且效果优于阳性对照Eylea和Faricimab(图15)，说明本发明抗体能显著抑制新生血管引起的渗漏。综上所述，证明本发明抗体本发明抗体联合抗VEGF抑制剂对激光诱导的眼底新生血管化有明显的抑制效果，同时具有保护血管完整性功能。
恒河猴给药29天后根据体重以戊巴比妥钠麻醉(静脉注射约30mg/kg，可根据动物健康状况调整剂量)，腹主动脉或股动脉放血安乐死，进行大体观察，并摘取双侧眼球。
部分动物双眼以改良的Davidson’s固定液固定，石蜡包埋切片，并选取激光造模区域进行常规HE染色，CD31 IHC染色等组织病理学检查。
本发明抗体组在病理切片中，相对抗VEGF单独治疗，视网膜病变区域显著减少，视网膜水肿减轻，激光损伤区域组织增生减少，结果显示了更好的视网膜形态学改善(参见图16)，抑制了视网膜脉络膜新生血管化，增强了血管完整性功能(图17)。
序列表
Figure PCTCN2022096848-appb-000006
Figure PCTCN2022096848-appb-000007
Figure PCTCN2022096848-appb-000008
Figure PCTCN2022096848-appb-000009
Figure PCTCN2022096848-appb-000010

Claims (32)

  1. 抗Ang2的VHH抗体，其包含如下的3个CDR，HCDR1，HCDR2和HCDR3，其中
    HCDR1包含SEQ ID NO:16所示的序列，或由所述序列组成；
    HCDR2包含SEQ ID NO:17或20所示的序列，或由所述序列组成；
    HCDR3包含SEQ ID NO:18所示的序列，或由所述序列组成。
  2. 权利要求1的VHH抗体，其中所述VHH
    (1)包含SEQ ID NO:19或21所述的氨基酸序列，或包含与所述SEQ ID NO:19或21所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:19或21所述的氨基酸组成；或
    (2)包含与SEQ ID NO:19或21所示的氨基酸序列相比具有一个或几个(优选不超过10、9、8、7、6、5、4、3、2、1个)突变的氨基酸序列，所述突变例如置换、缺失或添加，优选地置换，例如保守置换。
  3. 结合VEGFA和Ang2的双特异性结合分子，其包含特异性结合VEGF A的第一靶标结合区和特异性结合Ang2的第二靶标结合区，其中第二靶标结合区为权利要求1或2所述的VHH抗体，
    任选地，其中第一靶标结合区选自：
    特异性结合VEGFA的VHH；
    特异性结合VEGFA的抗体的抗原结合片段，例如scFv，例如所述抗体为全人抗体或人源化抗体；或
    特异性结合VEGF A的VEGF受体(VEGF R)或其胞外结构域或包含其胞外结构域的融合蛋白，例如其胞外结构域与Fc的融合蛋白。
  4. 权利要求3的双特异性结合分子，其为双特异性抗体。
  5. 权利要求3或4的双特异性结合分子，其为二价、三价或四价的。
  6. 权利要求4或5的双特异性结合分子，其具有以下结构：
    抗VEGF抗体的轻链可变区VL-接头-抗VEGF抗体的重链可变区VH-接头-抗Ang2 VHH或
    抗VEGF抗体的重链可变区VH-接头-抗VEGF抗体的轻链可变区VL-接头-抗Ang2 VHH。
  7. 权利要求6的双特异性结合分子，其中：
    抗VEGF抗体的轻链可变区VL包含LCDR1，LCDR2和LCDR3，其中LCDR1包含SEQ ID NO:31所示的序列或由其组成；LCDR2包含SEQ ID NO:32所示的序列或由其组成；LCDR3包含SEQ ID NO:33所示的序列或由其组成；和/或
    抗VEGF抗体的重链可变区VH包含HCDR1，HCDR2和HCDR3，其中HCDR1包含SEQ ID NO:35所示的序列或由其组成；HCDR2包含SEQ ID NO:36所示的序列或由其组成； HCDR3包含SEQ ID NO:37所示的序列或由其组成。
  8. 权利要求6或7的双特异性结合分子，其中
    抗VEGF抗体的重链可变区VH包含SEQ ID NO:34所述的氨基酸序列，或包含与所述SEQ ID NO:34所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:34所述的氨基酸组成；或所述重链可变区VH包含与SEQ ID NO:34所示的氨基酸序列相比具有一个或几个(优选不超过10、9、8、7、6、5、4、3、2、1个)突变的氨基酸序列，所述突变例如置换、缺失或添加，优选地置换，例如保守置换；
    抗VEGF抗体的轻链可变区VL包含SEQ ID NO:30所述的氨基酸序列，或包含与所述SEQ ID NO:30所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:30所述的氨基酸组成；或所述轻链可变区VL包含与SEQ ID NO:30所示的氨基酸序列相比具有一个或几个(优选不超过10、9、8、7、6、5、4、3、2、1个)突变的氨基酸序列，所述突变例如置换、缺失或添加，优选地置换，例如保守置换。
  9. 权利要求6-8中任一项所述的双特异性结合分子，其中
    (1)所述结合分子包含SEQ ID NO:28的氨基酸序列，或包含与所述SEQ ID NO:28所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:28所述的氨基酸组成；或
    (2)所述双特异性结合分子包含与SEQ ID NO:28所示的氨基酸序列相比具有一个或几个(优选不超过10、9、8、7、6、5、4、3、2、1个)突变的氨基酸序列，所述突变例如置换、缺失或添加，优选地置换，例如保守置换。
  10. 权利要求4或5的双特异性结合分子，其具有以下结构：
    第一抗VEGF VHH-接头-第二抗VEGF VHH-接头-抗Ang2 VHH，
    其中第一抗VEGF VHH与第二抗VEGF VHH相同或不同。
  11. 权利要求10的双特异性结合分子，其中第一抗VEGF VHH或第二抗VEGF VHH包含HCDR1，HCDR2和HCDR3，其中
    HCDR1包含SEQ ID NO:1所示的序列，或由所述序列组成；HCDR2包含SEQ ID NO:2所示的序列，或由所述序列组成；HCDR3包含SEQ ID NO:3所示的序列，或由所述序列组成；
    或者
    HCDR1包含SEQ ID NO:6所示的序列，或由所述序列组成；HCDR2包含SEQ ID NO:7或10所示的序列，或由所述序列组成；HCDR3包含SEQ ID NO:8所示的序列，或由所述序列组成。
  12. 权利要求10或11的双特异性结合分子，其中第一抗VEGF VHH或第二抗VEGF VHH包含SEQ ID NO:4、5、9或11所述的氨基酸序列，或包含与所述SEQ ID NO:4、5、9或11所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:4、5、9或11所述的氨基酸组成；或所述VHH包含 与SEQ ID NO:4、5、9或11所示的氨基酸序列相比具有一个或几个(优选不超过10、9、8、7、6、5、4、3、2、1个)突变的氨基酸序列，所述突变例如置换、缺失或添加，优选地置换，例如保守置换。
  13. 权利要求10-12中任一项的双特异性结合分子，其中
    (1)所述结合分子包含SEQ ID NO:22的氨基酸序列，或包含与所述SEQ ID NO:22所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:22所述的氨基酸组成；或
    (2)所述双特异性结合分子包含与SEQ ID NO:22所示的氨基酸序列相比具有一个或几个(优选不超过10、9、8、7、6、5、4、3、2、1个)突变的氨基酸序列，所述突变例如置换、缺失或添加，优选地置换，例如保守置换。
  14. 权利要求3的双特异性结合分子，其包含一条或两条以下链：
    VEGF R胞外结构域-Fc-接头-抗Ang2 VHH。
  15. 权利要求14的双特异性结合分子，其中所述VEGFR胞外结构域为来自人的VEGFR的胞外结构域；优选地，所述VEGFR胞外结构域包含VEGFR1第二抗体样结构域和VEGFR2第三抗体样结构域；更优先地，所述VEGFR胞外结构域包含人VEGFR1第二抗体样结构域和人VEGFR2第三抗体样结构域。
  16. 权利要求14或15的双特异性结合分子，所述VEGFR胞外结构域包含SEQ ID NO:26的氨基酸序列，或包含与所述SEQ ID NO:26所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:26所述的氨基酸组成。
  17. 权利要求14-16中任一项的双特异性结合分子，其中所述Fc为来源人IgG1、IgG2、IgG3或IgG4的Fc，优选地，所述Fc包含SEQ ID NO:27的氨基酸序列，或包含与所述SEQ ID NO:27所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:27所述的氨基酸组成。
  18. 权利要求14-17中任一项的双特异性结合分子，其中所述VEGF R胞外结构域-Fc为VEGFR胞外结构域与Fc的融合蛋白，例如Aflibercept或其衍生物，例如包含SEQ ID NO:25的氨基酸序列，或包含与所述SEQ ID NO:25所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:25所述的氨基酸组成。
  19. 权利要求14-18中任一项的双特异性结合分子，其中
    (1)所述结合分子包含SEQ ID NO:24的氨基酸序列，或包含与所述SEQ ID NO:24所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:24所述的氨基酸组成；或
    (2)所述双特异性结合分子包含与SEQ ID NO:24所示的氨基酸序列相比具有一个或几个(优选不超过10、9、8、7、6、5、4、3、2、1个)突变的氨基酸序列，所述突变例如置换、缺失或添加，优选地置换，例如保守置换。
  20. 权利要求3-19中任一项的双特异性结合分子，其中所述接头包含SEQ ID NO:23的氨基酸序列，或由其组成。
  21. 抗VEGF A的VHH抗体，其包含如下的3个CDR，HCDR1，HCDR2和HCDR3，其中
    HCDR1包含SEQ ID NO:1所示的序列，或由所述序列组成；
    HCDR2包含SEQ ID NO:2所示的序列，或由所述序列组成；
    HCDR3包含SEQ ID NO:3所示的序列，或由所述序列组成；
    或者
    HCDR1包含SEQ ID NO:6所示的序列，或由所述序列组成；
    HCDR2包含SEQ ID NO:7或10所示的序列，或由所述序列组成；
    HCDR3包含SEQ ID NO:8所示的序列，或由所述序列组成。
  22. 权利要求21的VHH抗体，其
    (1)包含SEQ ID NO:4、5、9或11所述的氨基酸序列，或包含与所述SEQ ID NO:4、5、9或11所述的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或由SEQ ID NO:4、5、9或11所述的氨基酸组成；或
    (2)包含与SEQ ID NO:4、5、9或11所示的氨基酸序列相比具有一个或几个(优选不超过10、9、8、7、6、5、4、3、2、1个)突变的氨基酸序列，所述突变例如置换、缺失或添加，优选地置换，例如保守置换。
  23. 核酸分子，其编码权利要求1或2或21或22中任一项的VHH抗体，或权利要求3-20中任一项的双特异性结合分子或所述结合分子的链。
  24. 表达载体，其包含权利要求23的核酸分子，优选地，所述表达载体为pcDNA3.1。
  25. 宿主细胞，其包含权利要求23所述的核酸分子或权利要求24所述的表达载体，优选地，所述宿主细胞是原核的或真核的，例如细菌如大肠杆菌细胞，例如TG1；或293细胞，例如293F或Expi-293细胞。
  26. 制备权利要求1或2或21或22中任一项的VHH抗体，或权利要求3-20中任一项的双特异性结合分子的方法，所述方法包括，在适合所述VHH抗体或或双特异性结合分子或其链表达的条件下，培养权利要求25的宿主细胞，和任选地从所述宿主细胞(或宿主细胞培养基)回收所述VHH或双特异性结合分子。
  27. 免疫缀合物，其包含权利要求1或2或21或22中任一项的VHH抗体，或权利要求3-20中任一项的双特异性结合分子
  28. 药物组合物或制剂，其包含权利要求1或2或21或22中任一项的VHH抗体，或权利要求3-20中任一项的双特异性结合分子，以及任选地一种或多种其他治疗剂，以及任选地药用辅料。
  29. 在受试者中预防或治疗眼部疾病的方法，包括向受试者施用有效量的权利要求1或2或21或22中任一项的VHH抗体，或权利要求3-20中任一项的双特异性结合分子，权利要求27的免疫缀合物或权利要求28的药物组合物或制剂。
  30. 权利要求29的方法，其中所述眼部疾病选自与血管发生相关的眼部疾病，例如与角 膜新生血管化相关的眼部疾病。
  31. 权利要求29或30的方法，其中所述患者具有(例如升高水平的，例如核酸或蛋白质水平的)VEGF，例如VEGF A，和/或Ang2。
  32. 权利要求29-31中任一项的方法，其中所述方法还包括向患者施用一种或多种疗法，例如治疗方式和/或其它治疗剂。
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