WO2024114746A1 - 包含结合VEGF和Ang2的双特异性结合分子的制剂以及其用途 - Google Patents

包含结合VEGF和Ang2的双特异性结合分子的制剂以及其用途 Download PDF

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WO2024114746A1
WO2024114746A1 PCT/CN2023/135525 CN2023135525W WO2024114746A1 WO 2024114746 A1 WO2024114746 A1 WO 2024114746A1 CN 2023135525 W CN2023135525 W CN 2023135525W WO 2024114746 A1 WO2024114746 A1 WO 2024114746A1
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amino acid
acid sequence
vegf
antibody
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French (fr)
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刘懿
何冰
马一冬
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信达生物制药(苏州)有限公司
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    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • the present invention relates to the field of preparations. More specifically, the present invention relates to a preparation comprising antibodies or antigen-binding fragments thereof directed against vascular endothelial growth factor (VEGF/VEGF-A) and against angiopoietin-2 (ANG-2), respectively, or comprising a bispecific binding molecule (e.g., antibody) or antigen-binding fragment thereof directed against both vascular endothelial growth factor (VEGF/VEGF-A) and angiopoietin-2 (ANG-2), especially a stable liquid preparation, as well as a method for preparing the antibody preparation and the use of the antibody preparation.
  • VEGF/VEGF-A vascular endothelial growth factor
  • ANG-2 angiopoietin-2
  • Angiogenesis is involved in the pathogenesis of various diseases, including solid tumors, diseases associated with intraocular neovascularization, rheumatoid arthritis, and psoriasis.
  • VEGF is an effective and ubiquitous angiogenic 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 are mainly different because of their bioavailability, and VEGF165 is the main isoform. It is believed that VEGF is an important regulator of normal and disease-related angiogenesis.
  • human angiogenins are also believed to be involved in vascular development and postnatal angiogenesis.
  • Human angiogenins include the naturally occurring agonist angiopoietin-1 (ANG-1) and the naturally occurring antagonist angiopoietin-2 (ANG-2).
  • ANG-1 the naturally occurring agonist angiopoietin-1
  • ANG-2 the naturally occurring antagonist angiopoietin-2
  • the effects of ANG-1 are believed to be conserved in adults, where it is widely and constitutively expressed.
  • ANG-2 expression is primarily restricted to sites of vascular remodeling, where it is believed to block the constitutive stabilizing or maturation function of ANG-1, thereby allowing the blood vessels to return to and remain in a plastic state that may be more responsive to sprouting signals.
  • bispecific antibodies targeting VEGF-A and ANG-2 have been developed (e.g., WO2012131078 and WO2014009465).
  • the existing bispecific antibodies have poor blocking properties against VEGF and Ang2, and due to their large molecular weight, the molar concentration is low when administered at a single time.
  • antibodies with smaller molecular weights that are applied intravitreally are usually used, and less frequent administration is required. Therefore, there is still a need for new bispecific binding molecules targeting VEGF-A and ANG-2, especially for ocular diseases.
  • the present invention satisfies the above needs by providing a pharmaceutical preparation comprising a VHH antibody targeting VEGF-A or ANG-2 or a bispecific binding molecule targeting both VEGF-A and ANG2.
  • the bispecific binding molecule of the present invention has a smaller molecular weight and a higher molar concentration at the same mass concentration than known antibodies; and has a stronger VEGF A and Ang2 blocking activity, can completely block VEGFA-induced primary cell proliferation. Therefore, the molecule of the present invention has stronger blocking activity clinically, and can make the antibody molar concentration higher during single administration, maintain the single administration effect longer, and reduce the frequency of ocular administration (such as intravitreal injection).
  • Figure 1 shows the structure of a bispecific binding molecule.
  • Figure 2 shows that the anti-VEGF A VHH antibody measured by ELISA can block the binding of VEGF A to VEGFR2.
  • Figure 3 shows the effect of humanized anti-Ang2 VHH antibody in blocking the binding of Ang2 to Tie2 as measured by ELISA.
  • Figure 4 shows the effects of anti-Ang2 VHH antibody (A) and humanized anti-Ang2 VHH antibody (B) in inhibiting hAng2-Fc-induced phosphorylation in 293-Tie2 cells as measured by ELISA.
  • Figure 5 shows the application of the HEK293-KDR reporter method to detect the effect of anti-VEGF A VHH in blocking VEGFA from activating the KDR receptor.
  • Figure 6 shows the effect of anti-VEGF A VHH antibody in inhibiting VEGF A-induced survival and proliferation of HUVEC cells as measured by CCK-8.
  • Figure 7 shows the application of the HEK293-KDR reporter method to detect the effect of the VEGF A/Ang2 bispecific binding molecule IEX04-012 in blocking VEGF activation of the KDR receptor.
  • FIG8 shows the effects of bispecific binding molecules IEX04-008, IEX04-010 and IEX04-012 in inhibiting VEGF-induced HUVEC cell survival and proliferation.
  • FIG. 9 shows the blocking effect of the bispecific binding molecules IEX04-008, IEX04-010 and IEX04-012 on the binding of human Ang2 to Tie2 as determined by ELISA.
  • FIG. 10 shows flow cytometry assay to determine whether bispecific binding molecules IEX04-008, IEX04-010 and IEX04-012 block the binding of Ang2-Fc to Tie2.
  • FIG. 11 shows the application of flow cytometry to determine that the bispecific binding molecule IEX04-012 of the present invention effectively inhibits hAng2-Fc-induced 293-Tie2 phosphorylation in vitro.
  • FIG. 12 shows that the bispecific binding molecule IEX04-012 of the present invention reduces VEGF-induced vascular endothelial cell permeability, ie, inhibits VEGF-induced HUVEC cell leakage.
  • FIG13 shows the statistics of the proportion of laser spots of grade 4 ( Figure A) and grade 3 or above ( Figure B) in the laser-induced choroidal neovascularization model.
  • FIG. 14 shows retinal thickness statistics in the laser-induced choroidal neovascularization model.
  • Figure 15 shows the leakage area statistics in the laser-induced choroidal neovascularization model
  • FIG16 shows the H&E staining of fundus tissue in the laser-induced choroidal neovascularization model (A) and lesion area statistics (B).
  • FIG. 17 shows the CD31 staining image (A) and positive cell statistics (B) of the fundus tissue in the laser-induced choroidal neovascularization model.
  • the term “comprising” or “including” means including the stated elements, integers or steps, but does not exclude any other elements, integers or steps.
  • the term “comprising” or “including” when used, unless otherwise indicated, it also covers the situation consisting of the stated elements, integers or steps.
  • an antibody variable region “comprising” a specific sequence when referring to an antibody variable region “comprising” a specific sequence, it is also intended to cover the antibody variable region consisting of the specific 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 in their bioavailability, with VEGF165 being the main isoform.
  • VEGF A of the present invention refers to VEGF A from humans, such as VEGF 165 from humans.
  • the amino acid sequence of 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 as: ANGPT2 or ANG2), which is described, for example, in Maisonpierre, P.C. et al., Science 277 (1997) 55-60 and Cheung, A.H. et al., Genomics 48 (1998) 389-91.
  • Ang1 and Ang2 were discovered as ligands of the tyrosine kinase family Tie, which is selectively expressed in the vascular endothelium. There are currently 4 defined members of the angiopoietin family.
  • Angiopoietin-3 and -4 may represent widely diverse counterparts of the same locus in mouse and man.
  • Ang1 and Ang2 were originally identified as agonists and antagonists, respectively, in tissue culture experiments (for ANG1, see Davis, S. et al., Cell 87 (1996) 1161-69; for ANG2, see Maisonpierre, P.C. et al., Science 277 (1997) 55-60). All known angiopoietins bind primarily to Tie2.
  • ANG2 of the present invention refers to Ang2 from humans.
  • human Ang2 comprises an amino acid sequence with accession number O15123 (uniprot database).
  • multispecific binding molecule refers to a multispecific binding molecule that is at least bispecific, e.g., a bispecific binding molecule, i.e., the molecule comprises at least a first target binding region and a second target binding region, wherein the first target binding region binds one target or antigen and the second target binding region binds another antigen or target.
  • the molecule according to the invention comprises specificity for at least two different antigens or targets.
  • the molecule according to the invention also encompasses multispecific molecules comprising multiple target binding regions/binding sites, such as trispecific binding molecules.
  • the bispecific binding molecule of the invention is a bispecific antibody.
  • linker refers to any moiety that enables direct connection of the different parts of a bispecific binding molecule.
  • the example of the covalently linked joint between different molecular parts includes a peptide joint and a non-protein polymer, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene or a copolymer of polyethylene glycol and polypropylene glycol.
  • the joint is a peptide joint, which refers to a sequence of amino acids, wherein the sequence connects the amino acid sequence of the first part of the binding molecule to the second part of the binding molecule.
  • a peptide joint can connect the first target binding region of the binding molecule to the second target binding region.
  • a peptide joint can also connect a part of an antibody to another part of the antibody, such as connecting a light chain variable region to a heavy chain variable region.
  • the peptide joint has a length that is sufficient to connect two entities in a manner that allows them to maintain their conformations relative to each other so as not to interfere with the desired activity.
  • the peptide linker may or may not mainly include the following amino acid residues: Gly, Ser, Ala or Thr.
  • Useful linkers include glycine-serine polymers, including, for example, (GS)n(SEQ ID NO:43), (GSGGS)n(SEQ ID NO:44), (GGGGS)n(SEQ ID NO:45), (GGGS)n(SEQ ID NO:46) and (GGGGS)nG(SEQ ID NO:47), wherein n is an integer of at least 1 (and preferably 2, 3, 4, 5, 6, 7, 8, 9, 10).
  • Useful linkers also include glycine-alanine polymers, alanine-serine polymers and other flexible linkers.
  • valence means that there are a specified number of binding sites in a binding molecule, such as an antibody molecule. Therefore, the terms divalent, trivalent, tetravalent respectively mean that there are two, three or four binding sites (target binding regions) in a binding molecule.
  • the bispecific binding molecules according to the present invention are at least divalent and can be multivalent, such as divalent, 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 specific 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 to its target as a separate entity.
  • the target binding region can also be a receptor or a ligand, or a domain of a receptor that is capable of binding a ligand.
  • antibody fragment includes a portion of an intact antibody.
  • the antibody fragment is an antigen-binding fragment.
  • Antigen-binding fragment refers to a molecule different from an intact antibody, which comprises a portion of an intact antibody and binds to 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 antibody; single-chain antibody (e.g., scFv); single-domain antibody such as VHH; bivalent antibody or fragment thereof; or camelid antibody.
  • VHH also known as single domain antibody (sdAb) refers to a genetically engineered antibody consisting only of the variable region of the heavy chain antibody, which contains only three HCDRs of the heavy chain variable region. VHH has antigen specificity and high affinity with only three HCDRs, while ordinary antibodies require six CDRs.
  • the crystal structure shows that VHH is composed of two ⁇ sheets as a scaffold, similar to the traditional antibody VH immunoglobulin fold.
  • target refers to the object to which a binding molecule is directed.
  • the target can be an antigen, a ligand or a receptor.
  • antigen refers to a molecule that triggers 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.
  • epitopope refers to a portion of an antigen (e.g., VEGF or Ang2) that specifically interacts with an antibody molecule.
  • CDR region is a region of an antibody variable domain that is highly variable in sequence and forms structurally defined loops ("hypervariable loops") and/or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigen epitopes.
  • the CDRs of the heavy and light chains are usually referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus.
  • the CDRs located in the antibody heavy chain variable domain are called HCDR1, HCDR2 and HCDR3, while the CDRs located in the antibody light chain variable domain are called LCDR1, LCDR2 and LCDR3.
  • each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example, Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al.
  • the residues of each CDR are as follows.
  • a CDR can also be identified based on having the same Kabat numbering position as a reference CDR sequence (eg, any of the exemplary CDRs of the invention).
  • the residue positions in the antibody variable region refers to the numbering position according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
  • the CDRs in the VHH of the present invention follow the following rules: wherein HCDR1 is determined according to AbM, and HCDR2 and HCDR3 are determined according to Kabat.
  • the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems are different. Therefore, when it comes to defining antibodies using specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined in the present invention due to the application of different schemes (e.g., different assignment system rules or combinations).
  • Antibodies with different specificities have different CDRs (under the same assignment system).
  • CDRs are different between antibodies, only a limited number of amino acid positions in CDRs are directly involved in antigen binding.
  • the minimum overlapping region can be determined, thereby providing a "minimum binding unit" for antigen binding.
  • the minimum binding unit can be a sub-portion of a CDR.
  • the residues of the rest of the CDR sequence can be determined by the structure and protein folding of the antibody. Therefore, the present invention also contemplates variants of any CDR given herein.
  • the amino acid residues of the minimum binding unit can remain unchanged, while the remaining CDR residues defined according to Kabat or Chothia can be replaced by conservative amino acid residues.
  • Fc region is used herein to define the constant region of CH2 and CH3 of an immunoglobulin heavy chain, and the term includes native sequence Fc regions and variant Fc regions.
  • the native or wild-type Fc region can bind to different Fc receptors on the surface of immune cells, thereby being able to cause CDC ⁇ ADCC ⁇ ADCP effector functions. Such effector functions generally require that the Fc region be combined with a binding domain (e.g., an antibody variable region).
  • the Fc region is mutated to enhance its CDC ⁇ ADCC ⁇ ADCP effector functions.
  • the Fc region is mutated to weaken or delete its CDC ⁇ ADCC ⁇ ADCP effector functions.
  • Humanized antibody refers to an antibody comprising amino acid residues from non-human CDR and amino acid residues from human FR.
  • humanized antibody will comprise substantially all at least one, usually two variable domains, wherein all or substantially all CDRs (e.g., CDRs) correspond to those of non-human antibodies, and all or substantially all FRs correspond to those of human antibodies.
  • Humanized antibody optionally can comprise at least a portion of antibody constant regions derived from human antibodies.
  • “Humanized form" of antibody (e.g., non-human antibody) refers to humanized antibodies.
  • Humanized antibody or “full human antibody” or “full human source antibody” can be used interchangeably, and it refers to an antibody with such an amino acid sequence, and the amino acid sequence corresponds to the amino acid sequence of following antibodies, and the antibody is generated by people or human cells or is derived from non-human sources, and it utilizes human antibody library or other human antibody coding sequences. This definition of human antibody clearly excludes humanized antibodies comprising non-human antigen binding residues.
  • the terms "anti,” “binding,” or “specific binding” mean that the binding is selective for a target or antigen and can be distinguished from unwanted or non-specific interactions.
  • the ability of a binding site to bind to a specific 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 thin-layer interferometry or MSD assays or surface plasmon resonance (SPR).
  • host cell refers to cells into which exogenous nucleic acids are introduced, including progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include primary transformed cells and progeny derived therefrom, without regard to the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Included herein are mutant progeny screened or selected for the same function or biological activity as in the initially transformed cell.
  • label refers to a compound or composition that is conjugated or fused directly or indirectly to an agent (such as a polynucleotide probe or an antibody) and facilitates detection of the agent to which it is conjugated or fused.
  • the label itself can be detectable (e.g., a radioisotope label or a fluorescent label) or can catalyze a chemical alteration of a detectable substrate compound or composition in the case of an enzymatic label.
  • the term is intended to encompass direct labeling by coupling (i.e., physically linking) a detectable substance to a probe or antibody.
  • the label is hFc or biotin.
  • Subjects include mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, 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 subject is a human.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences may be discarded for comparison purposes).
  • the length of the reference sequence being aligned 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 length of the reference sequence.
  • 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 at the corresponding position in the second sequence, then the molecules are identical at this position.
  • Mathematical algorithms can be used to compare sequences and calculate percent identity between two sequences.
  • the Needlema and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm (available at http://www.gcg.com) that has been integrated into the GAP program of the GCG software package is used, using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences.
  • the GAP program in the GCG software package is used (available at http://www.gcg.com) using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70 or 80 and a length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two nucleotide sequences.
  • a particularly preferred parameter set (and one that should be used unless otherwise specified) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • the percent identity between two amino acid sequences or nucleotide sequences can also be determined using the E. Meyers and W.
  • nucleic acid sequences and protein sequences described herein can be further used as "query sequences" to perform searches against public databases to, for example, identify other family member sequences or related sequences.
  • ocular diseases encompass ocular diseases involving angiogenesis (eg, diseases occurring in the eye), such as ocular diseases associated with corneal neovascularization.
  • prevention includes the inhibition of the onset or development of a disease or condition or symptoms of a particular disease or condition.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated 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.”
  • Subject/patient sample refers to a collection of cells or fluids obtained from a patient or subject.
  • the source of the tissue or cell sample can be solid tissue, such as an organ or tissue sample or a biopsy sample or a puncture sample from fresh, frozen and/or preserved; 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 the subject's pregnancy or development.
  • the tissue sample is ocular tissue, such as vitreous.
  • the sample is tears or vitreous humor.
  • the tissue sample may contain compounds that are not naturally mixed with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.
  • the antibodies used in the preparations of the present invention are also referred to as antibodies of the present invention.
  • the anti-VEGF A VHH antibody in the preparations of the present invention is sometimes also referred to as the anti-VEGF A VHH antibody of the present invention.
  • the present invention provides a liquid preparation comprising (i) an anti-VEGF A VHH antibody, an anti-Ang2 VHH antibody, or a bispecific binding molecule that binds to VEGF A and Ang2; (ii) a buffer, (iii) a stabilizer, and (iv) a surfactant, wherein the pH of the liquid preparation is about 5.0-7.5.
  • the liquid formulation comprises an anti-VEGF A VHH antibody.
  • the liquid formulation comprises an anti-Ang2 VHH antibody.
  • the liquid preparation comprises a bispecific binding molecule that binds to VEGF A and Ang2, preferably a bispecific antibody that binds to VEGF A and Ang2.
  • the anti-VEGF A VHH comprises the following three CDRs, HCDR1, HCDR2 and HCDR3, wherein
  • HCDR1 comprises the sequence shown in SEQ ID NO:1, or consists of the sequence;
  • HCDR2 comprises the sequence shown in SEQ ID NO:2, or consists of the sequence;
  • HCDR3 comprises the sequence shown in SEQ ID NO:3, or consists of the sequence;
  • HCDR1 comprises the sequence shown in SEQ ID NO:6, or consists of the sequence;
  • HCDR2 comprises the sequence shown in SEQ ID NO:7 or 10, or consists of the sequence;
  • HCDR3 comprises the sequence shown in SEQ ID NO:8, or consists of the sequence.
  • the anti-VEGF VHH comprises the amino acid sequence of SEQ ID NO:4, 5, 9 or 11, or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:4, 5, 9 or 11, or consists of the amino acids of SEQ ID NO:4, 5, 9 or 11.
  • the anti-VEGF VHH comprises an amino acid sequence having one or several (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence shown in SEQ ID NO: 4, 5, 9 or 11, the mutations being, for example, substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.
  • the mutation is not present in a CDR, such as HCDR1, HCDR2 or HCDR3.
  • the anti-Ang2 VHH comprises the following three CDRs, HCDR1, HCDR2 and HCDR3, wherein
  • HCDR1 comprises or consists of the sequence shown in SEQ ID NO: 16;
  • HCDR2 comprises the sequence shown in SEQ ID NO: 17 or 20, or consists of the sequence;
  • HCDR3 comprises the sequence shown in SEQ ID NO:18, or consists of the sequence.
  • the anti-Ang2 VHH comprises the amino acid sequence of SEQ ID NO: 19 or 21, or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 19 or 21, or consists of the amino acids of SEQ ID NO: 19 or 21.
  • the anti-Ang2 VHH comprises an amino acid sequence having one or several (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence shown in SEQ ID NO: 19 or 21, the mutations being, for example, substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.
  • the mutation is not present in a CDR, such as HCDR1, HCDR2 or HCDR3.
  • the bispecific binding molecule that binds VEGF A and Ang2 comprises a first target binding region that specifically binds VEGF A and a second target binding region that specifically binds Ang2, wherein the second target binding region is an anti-Ang2 VHH, such as the anti-Ang2 VHH as described above.
  • the first target binding region is selected from
  • VHH that specifically binds to VEGF A
  • an antigen-binding fragment of an antibody that specifically binds to VEGF A such as an scFv, for example, the antibody is a fully human antibody or a humanized antibody; or
  • VEGF receptor that specifically binds to VEGF A or its extracellular domain or a fusion protein comprising its extracellular domain, such as a fusion protein of the extracellular domain and Fc.
  • the bispecific binding molecule comprises 1 or 2 or 3 or 4 first or second target binding regions. In some embodiments, the bispecific binding molecule comprises 2, 3 or 4 target binding regions. In some embodiments, the bispecific binding molecule is divalent or trivalent or tetravalent. In some embodiments, the bispecific binding molecule is a bispecific antibody.
  • the bispecific binding molecule such as a bispecific antibody, 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 the said sequence; HCDR2 comprises the sequence shown in SEQ ID NO:17 or 20, or consists of the said sequence; HCDR3 comprises the sequence shown in SEQ ID NO:18, or consists of the said sequence.
  • the structure of the bispecific binding molecule is as shown in Figure 1A or Figure 1B.
  • the bispecific binding molecule is composed of one chain.
  • the bispecific binding molecule is bivalent.
  • the light chain variable region VL of the anti-VEGF antibody comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises or consists of the sequence shown in SEQ ID NO:31; LCDR2 comprises or consists of the sequence shown in SEQ ID NO:32; LCDR3 comprises or consists of the sequence shown in SEQ ID NO:33.
  • 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 or consists of the sequence shown in SEQ ID NO:36; and HCDR3 comprises or consists of the sequence shown in SEQ ID NO:37.
  • the heavy chain variable region VH of the anti-VEGF antibody comprises the amino acid sequence of SEQ ID NO: 34, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 34, or consists of the amino acids of SEQ ID NO: 34.
  • the heavy chain variable region VH comprises an amino acid sequence having one or more (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence of SEQ ID NO: 34, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.
  • the mutations are not present in CDRs, such as HCDR1, HCDR2 or HCDR3.
  • the light chain variable region VL of the anti-VEGF antibody comprises the amino acid sequence of SEQ ID NO: 30, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 30, or consists of the amino acids of SEQ ID NO: 30.
  • the light chain variable region VL comprises an amino acid sequence having one or more (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence of SEQ ID NO: 30, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.
  • the mutations are not present in CDRs, such as LCDR1, LCDR2 or LCDR3.
  • the anti-Ang2 VHH comprises an amino acid sequence of SEQ ID NO: 19 or 21, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 19 or 21, or consists of the amino acids of SEQ ID NO: 19 or 21.
  • the VHH comprises an amino acid sequence having one or more (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence of SEQ ID NO: 19 or 21, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.
  • the mutations are not present in CDRs, 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 comprises the amino acid sequence of SEQ ID NO: 28, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 28, or consists of the amino acids of SEQ ID NO: 28.
  • the bispecific binding molecule comprises an amino acid sequence having one or several (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence of SEQ ID NO: 28, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.
  • the mutations are not present in the CDRs of the anti-VEGF antibody variable region and the anti-Ang2 VHH.
  • the bispecific binding molecule such as a bispecific antibody, 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 the said sequence; HCDR2 comprises the sequence shown in SEQ ID NO:17 or 20, or consists of the said sequence; HCDR3 comprises the sequence shown in SEQ ID NO:18, or consists of the said sequence.
  • the structure of the bispecific binding molecule is as shown in Figure 1C.
  • the bispecific binding molecule consists of one chain.
  • the bispecific binding molecule is trivalent.
  • the first anti-VEGF VHH is the same or different from the second anti-VEGF VHH.
  • the anti-VEGF VHH comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the sequence shown in SEQ ID NO:1; HCDR2 comprises or consists of the sequence shown in SEQ ID NO:2; HCDR3 comprises or consists of the sequence shown in SEQ ID NO:3;
  • HCDR1 comprises the sequence shown in SEQ ID NO:6, or consists of the said sequence
  • HCDR2 comprises the sequence shown in SEQ ID NO:7 or 10, or consists of the said sequence
  • HCDR3 comprises the sequence shown in SEQ ID NO:8, or consists of the said sequence.
  • the anti-VEGF VHH comprises an amino acid sequence as set forth in SEQ ID NO: 4, 5, 9 or 11, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or consists of the amino acids set forth in SEQ ID NO: 4, 5, 9 or 11.
  • the VHH comprises an amino acid sequence having one or several (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence set forth in SEQ ID NO: 4, 5, 9 or 11, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.
  • the mutations are not present in a CDR, such as HCDR1, HCDR2 or HCDR3.
  • the anti-VEGF VHH comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the sequence shown in SEQ ID NO:6; HCDR2 comprises or consists of the sequence shown in SEQ ID NO:7 or 10; and HCDR3 comprises or consists of the sequence shown in SEQ ID NO:8.
  • the anti-VEGF VHH comprises an amino acid sequence as set forth in SEQ ID NO: 9 or 11, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or consists of the amino acids set forth in SEQ ID NO: 9 or 11.
  • the VHH comprises an amino acid sequence having one or more (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence set forth in SEQ ID NO: 9 or 11, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.
  • the mutations are not present in a CDR, such as HCDR1, HCDR2 or HCDR3.
  • the anti-Ang2 VHH comprises the amino acid sequence of SEQ ID NO: 19 or 21, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 19 or 21, or consists of the amino acids of SEQ ID NO: 19 or 21.
  • the VHH comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 19 or 21.
  • mutations are not present in CDRs, 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 comprises the amino acid sequence of SEQ ID NO: 22, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 22, or consists of the amino acids of SEQ ID NO: 22.
  • the bispecific binding molecule comprises an amino acid sequence having one or more (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence of SEQ ID NO: 22, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.
  • the mutations are not present in the CDRs of the anti-VEGF VHH and the anti-Ang2 VHH.
  • the bispecific binding molecule comprises one or two 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 the said sequence; HCDR2 comprises the sequence shown in SEQ ID NO:17 or 20, or consists of the said sequence; HCDR3 comprises the sequence shown in SEQ ID NO:18, or consists of the said sequence.
  • the structure of the bispecific binding molecule is shown in Figure ID.
  • the bispecific binding molecule is composed of two chains.
  • the bispecific binding molecule is tetravalent.
  • the anti-Ang2 VHH comprises an amino acid sequence of SEQ ID NO: 19 or 21, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 19 or 21, or consists of the amino acids of SEQ ID NO: 19 or 21.
  • the VHH comprises an amino acid sequence having one or several (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence of SEQ ID NO: 19 or 21, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.
  • the mutations are not present in CDRs, 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 second antibody-like domain of VEGFR1 (e.g., FLT1 domain 2) and a third antibody-like domain of VEGFR2 (e.g., KDR domain 3).
  • 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 an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence described in SEQ ID NO: 26, or consists of the amino acids described in SEQ ID NO: 26.
  • the VEGFR extracellular domain comprises an amino acid sequence having one or several (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence shown in SEQ ID NO: 26, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions, and preferably the VEGFR extracellular domain retains a binding affinity to VEGF similar to the domain shown in SEQ ID NO: 26 (e.g., having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).
  • 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 an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 27, or consists of the amino acids of SEQ ID NO: 27.
  • the VEGF R extracellular domain-Fc is a fusion protein of the VEGFR extracellular domain and Fc, such as Aflibercept or its derivatives.
  • the VEGF R extracellular domain-Fc comprises the amino acid sequence of SEQ ID NO:25, or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:25, or is composed of the amino acids described in SEQ ID NO:25.
  • the VEGF R extracellular domain-Fc comprises an amino acid sequence having one or several (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence shown in SEQ ID NO:25, the mutations being, for example, substitutions, deletions or additions, preferably substitutions, such as conservative substitutions, and preferably the VEGF R extracellular domain-Fc retains a binding affinity to VEGF similar to the domain shown in SEQ ID NO:25 (for example, having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).
  • one chain of the anti-VEGF A ⁇ ANG2 bispecific binding molecule comprises the amino acid sequence of SEQ ID NO: 24, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 24, or consists of the amino acids of SEQ ID NO: 24.
  • the bispecific binding molecule comprises an amino acid sequence having one or more (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations compared to the amino acid sequence of SEQ ID NO: 24, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.
  • the mutations are not present in the CDRs of the anti-Ang2 VHH.
  • the mutated VEGF R extracellular domain-Fc retains a binding affinity to VEGF similar to the domain shown in SEQ ID NO:25 (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).
  • the antibodies or binding molecules described herein comprise one or more amino acid mutations.
  • the amino acid mutations comprise replacement, insertion or deletion of an amino acid.
  • the amino acid changes described herein are amino acid replacements, preferably conservative replacements.
  • the amino acid mutations described in the present invention occur in regions outside of CDR (e.g., in FR). In some embodiments, the amino acid mutations described in the present invention occur in the constant region of the heavy chain of an antibody, such as the Fc region, and in a preferred embodiment, the amino acid mutations in the Fc region weaken or delete the ADCC and/or CDC effects of the antibody.
  • the substitution is a conservative substitution.
  • a conservative substitution refers to the substitution of one amino acid by another amino acid within the same class, such as an acidic amino acid by another acidic amino acid, a basic amino acid by another basic amino acid, or a neutral amino acid by another neutral amino acid.
  • one or more amino acid mutations can be introduced into the Fc region of an antibody provided herein to generate an Fc region variant to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, complement dependent cellular toxicity, Fc receptor binding and/or antibody dependent cellular toxicity.
  • Fc region variants can include a human Fc region sequence (e.g., human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid mutation (e.g., substitution) at one or more amino acid positions.
  • variable region of an antibody may be desirable to mutate the variable region of an antibody to generate disulfide bonds, for example to generate a scFv comprising a disulfide bond mutation.
  • the antibodies or binding molecules provided herein may be further modified to contain other non-protein moieties known in the art and readily available.
  • Suitable moieties for the derivatization include, but are not limited to, water-soluble polymers.
  • water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinylene glycol homopol
  • the buffer is selected from one or more of histidine, histidine salt, glutamate, phosphate, acetate, citrate and tris(hydroxymethyl)aminomethane.
  • the histidine salt and glutamate include but are not limited to salts formed by the amino acid and hydrochloric acid or sulfuric acid.
  • the phosphate, acetate and citrate include but are not limited to alkali metal salts of the corresponding acid such as lithium, sodium or potassium salts.
  • the phosphate can be in the form of a hydrate or a non-hydrate, including but not limited to disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, dilithium hydrogen phosphate, lithium dihydrogen phosphate or a combination thereof; preferably, the histidine salt is histidine hydrochloride, and the phosphate is a combination of disodium hydrogen phosphate and sodium dihydrogen phosphate.
  • the buffer is selected from a combination of histidine and histidine hydrochloride, phosphate and histidine.
  • the stabilizer is selected from one or more of sugars, polyols, amino acids or their salts.
  • the sugar is selected from but not limited to: sucrose, dextrose, lactose, maltose, trehalose, cyclodextrin, maltodextrin and dextran;
  • the polyol is selected from but not limited to: mannitol, sorbitol and xylitol;
  • the amino acid or its salt is selected from one or more of the following: arginine, glycine, proline, methionine and their salts;
  • the cyclodextrin is preferably hydroxypropyl- ⁇ -cyclodextrin.
  • the stabilizer is sucrose or trehalose. In some embodiments, the stabilizer is a combination of sucrose or trehalose with an additional ingredient selected from one or more of the following: glycine, proline, methionine, and hydroxypropyl- ⁇ -cyclodextrin.
  • the surfactant is a nonionic surfactant, including but not limited to alkyl poly (ethylene oxide); polysorbate, such as polysorbate-20, polysorbate-80, polysorbate-60, or polysorbate-40; Pluronic, etc.
  • the liquid formulation of the present invention comprises polysorbate-80 as a surfactant.
  • the concentration of the antibody or bispecific binding molecule in the liquid formulation of the invention is about 1-200 mg/ml, such as about 1-150 mg/ml, about 10-190 mg/ml, about 20-180 mg/ml, about 30-170 mg/ml, about 30-150 mg/ml, about 30-170 mg/ml, about 30-100 mg/ml, about 30-100 mg/ml, such as about 5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190 or about 200 mg/ml, preferably about 10-160 mg/ml, more preferably about 30-150 mg/mL.
  • the concentration of the buffer in the liquid formulation of the present invention is about 0.5-200 mM, about 1-100 mM, about 5-50 mM, about 5-30 mM, about 5-20 mM, about 5-15 mM, about 8-12 mM, for example, about 5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190 or about 200 mM, preferably about 5-50 mM, more preferably about 5-30 mM.
  • the concentration of each component can be selected from the following ranges or values: 0.2-100 mM, about 0.5-50 mM, about 2-25 mM, about 2-15 mM, about 1-10 mM, about 2-8 mM, about 1-3 mM, 0.5-4 mM, about 7-9 mM, about 6-10 mM, about 5-10 mM, for example, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 51, about 52, about 53, about 54, about 55 About 8, about 9, about 10, about 15, about 20, about 25, about 30, about 40
  • the concentration of the stabilizer in the liquid formulation of the present invention is about 1-1000mM, about 10-1000mM, about 20-800mM, about 30-700mM, about 50-800mM, about 50-500mM, about 100-400mM, about 100-300mM, about 200-350mM, about 200-300mM, about 250-280mM, about 200-400mM or about 100-200mM, for example, about 10, about 20, about 50, about 80, about 100, about 150, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900 or about 1000mM; preferably about 50-800mM, more preferably about 50-500mM.
  • the concentration of each component can be selected from the following ranges or values: about 20-800 mM, about 30-700 mM, about 50-800 mM, about 50-500 mM, about 100-400 mM, about 100-300 mM, about 200-350 mM, about 200-300 mM, about 250-280 mM, about 2 ...400 mM, about 100-300 mM, about 200-350 mM, about 200-300 M or about 100-200 mM, about 0.5-100 mM, about 1-50 mM, about 2-50 mM, about 5-20 mM, about 8-12 mM, for example, about 1, about 2, about 3, about 4, about 5, about 10, about 20, about 30, about 50, about 80, about 100, about 150, about 200, about 300, about 400, about 500 or about 600 mM.
  • the concentration of the surfactant in the liquid formulation of the present invention is about 0.01-10 mg/ml, for example, about 0.05-5, about 0.05-2, about 0.1-1, about 0.1-5, about 0.2-2, about 0.3-1, about 0.2-0.4 mg/ml about 0.4-0.8 or about 0.5-0.6 mg/ml, for example, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7 or about 0.8 mg/ml, preferably about 0.05-5 mg/ml, more preferably 0.05-2 mg/ml.
  • the pH of the liquid formulation of the present invention is about 5.5-7.2, 6.0-7.0, 6.1-7.0, 6.2-7.0, 6.2-6.6 or 6.3-6.8, for example, about 5.0, about 5.2, about 5.4, about 5.6, about 5.8, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1 or about 7.2, preferably about 6.1-7.0, and more preferably about 6.3-6.8.
  • the liquid formulation of the present invention has an osmotic pressure of 200-400 mOsmol/kg, 250-350 mOsmol/kg, for example, an osmotic pressure of 350 mOsmol/kg.
  • the liquid preparation is a pharmaceutical preparation, preferably an injection, more preferably an intravitreal injection, or eye drops.
  • the liquid formulation of the present invention comprises:
  • the pH of the liquid preparation is about 6.1-7.0, preferably about 6.5.
  • the liquid formulation of the present invention comprises:
  • the pH of the liquid preparation is about 6.1-6.6, preferably about 6.5.
  • the liquid formulation of the present invention comprises:
  • the pH of the liquid preparation is about 6.1-6.6, preferably about 6.5.
  • the liquid formulation of the present invention is the formulation disclosed in Examples 15 to 17.
  • embodiments resulting from fluctuations of 50%, 40%, 30%, 20%, 10% or 5% in the concentration values of the components in these formulations, and fluctuations of 5%, 4%, 3%, 2%, 1% or 0.5% in the pH value are also included in the present application.
  • the antibody liquid preparation of the present invention may or may not contain other excipients.
  • the antibody liquid preparation of the present invention may also contain a tension regulator.
  • the tension regulator may be selected from the group consisting of sodium acetate, sodium lactate, sodium chloride, potassium chloride and calcium chloride.
  • the liquid preparation of the present invention further comprises a solvent, including but not limited to, for example, purified water such as ultrapure water, water for injection, sterile water, distilled water, etc.
  • a solvent including but not limited to, for example, purified water such as ultrapure water, water for injection, sterile water, distilled water, etc.
  • the liquid preparation of the present invention can be prepared or formulated using a pharmaceutically acceptable solvent or solution known in the art.
  • the pharmaceutically acceptable solvent or solution includes but is not limited to, for example, purified water such as ultrapure water, water for injection, sterile water, distilled water, normal saline, Ringer's solution, glucose injection, etc.
  • the anti-Ang2 VHH antibody is capable of specifically binding to Ang2, such as human Ang2, e.g., with high affinity.
  • the anti-VEGFA VHH antibody is capable of specifically binding to VEGF A, such as human VEGF A, e.g., with high affinity.
  • the bispecific binding molecule is capable of specifically binding to Ang2 and VEGFA, eg, human Ang2 and human VEGFA, eg, with high affinity.
  • the anti-Ang2 antibody or anti-VEGFA antibody or bispecific binding molecule has one or more of the following properties:
  • the anti-Ang2 antibody or bispecific binding molecule has an inhibitory effect on Ang2-induced Tie2 phosphorylation
  • the anti-Ang2 antibody or bispecific binding molecule has a blocking effect on the binding between Ang2 and Tie2;
  • Anti-VEGFA antibodies or bispecific binding molecules have the ability to block VEGFA-activated receptor signaling pathways Effects, such as through KDR reporter detection;
  • the anti-VEGFA antibody or 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 cells (e.g., primary cells, such as vascular endothelial cells, such as human umbilical vein endothelial cells, such as HUVEC) induced by VEGF A;
  • cells e.g., primary cells, such as vascular endothelial cells, such as human umbilical vein endothelial cells, such as HUVEC
  • the bispecific binding molecule has an inhibitory effect on leakage of vascular endothelial cells (eg, human umbilical vein endothelial cells, such as HUVEC);
  • vascular endothelial cells eg, 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 choroid neovascularization, such as inhibiting leakage caused by neovascularization, such as protecting vascular integrity.
  • the present invention provides a solid preparation obtained by subjecting the liquid preparation of the present invention to a solidification treatment.
  • the solidification treatment is implemented by, for example, a crystallization method, a spray drying method, or a freeze drying method.
  • the solid preparation is, for example, in the form of a lyophilized powder injection.
  • the solid preparation can be reconstituted in an appropriate solvent before use to form a reconstituted preparation of the present invention.
  • the reconstituted preparation is also a liquid preparation of the present invention.
  • the appropriate solvent is selected from purified water such as ultrapure water, water for injection, an organic solvent for injection, including but not limited to oil for injection, ethanol, propylene glycol, etc., or a combination thereof.
  • the preparation of the present invention can be stored stably for a long time, for example, at least 24 months or longer.
  • the preparation of the present invention can be stored at least 10 days, at least 20 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or longer under the conditions of about -80 ° C to about 45 ° C, for example, -80 ° C, about -30 ° C, about -20 ° C, about 0 ° C, about 5 ° C, about 25 ° C, about 35 ° C, about 38 ° C, about 40 ° C, about 42 ° C or about 45 ° C, and be stable.
  • the stability of the liquid preparation of the present invention is visually inspected, wherein the liquid preparation of the present invention remains clear to slightly opalescent in appearance, is a colorless to light yellow liquid, and is free of foreign matter. In one embodiment, visual inspection under a clarity detector shows that no visible foreign matter is present in the preparation. In one embodiment, after storage, the stability of the liquid preparation of the present invention is checked by measuring the change in protein content, wherein, for example, by ultraviolet spectrophotometry (UV), the rate of change in protein content relative to the initial value of the storage day 0 is no more than 20%, preferably no more than 10%, such as 7-8%, and more preferably no more than 5%.
  • UV ultraviolet spectrophotometry
  • the stability of the liquid preparation of the present invention is checked by measuring the change in purity of the liquid preparation of the present invention, wherein, by size exclusion high performance liquid chromatography (SEC-HPLC), the change in monomer purity relative to the initial value of the storage day 0 is no more than 10%, such as no more than 5%, 4%, 3%, such as a change of no more than 1-2%, preferably no more than 1%.
  • SEC-HPLC size exclusion high performance liquid chromatography
  • the stability of the liquid preparation of the present invention is checked by measuring the change in purity of the liquid preparation of the present invention, wherein the change value of the monomer purity decreases by no more than 10%, such as no more than 5%, 4%, 3%, 2% or 1% by non-reduced and/or reduced sodium dodecyl sulfate capillary electrophoresis (CE-SDS).
  • CE-SDS non-reduced and/or reduced sodium dodecyl sulfate capillary electrophoresis
  • the stability of the liquid preparation of the present invention is detected by imaging capillary isoelectric focusing electrophoresis (iCIEF), wherein the sum of the change values of the charge variants (main component, acidic component and basic component) of the antibody relative to the initial value on storage day 0 does not exceed 30%, such as no more than 20%, no more than 10%, no more than 5%, no more than 2% or no more than 1%.
  • iCIEF capillary isoelectric focusing electrophoresis
  • the present invention provides a delivery device comprising a liquid formulation or a solid formulation of the present invention.
  • the delivery device of the present invention is in the form of a prefilled syringe comprising a liquid formulation or a solid formulation of the present invention.
  • the composition can be provided in the form of an intravitreal injection, intravenous, subcutaneous, intradermal or intramuscular injection, or intravenous infusion.
  • the present invention provides a method for preparing a liquid formulation according to the present invention, comprising the following steps:
  • the solution of the buffer and stabilizer is preferably an aqueous solution, preferably, the type, concentration and pH of the buffer and stabilizer are as defined in the liquid preparation of the present invention above,
  • a surfactant or a solution thereof preferably an aqueous solution, to a final concentration of the surfactant to a concentration defined in the liquid formulation of the present invention
  • step v optionally sterile filtering the solution of step v;
  • the mixture is dispensed into vials, and covered with rubber stoppers and aluminum-plastic caps to obtain finished products.
  • the present invention provides a method for preventing or treating an ocular disease in a subject, comprising administering the formulation of the present invention to the subject.
  • the patient has (e.g., elevated levels, such as nucleic acid or protein levels) VEGF, such as VEGF A, and/or Ang2.
  • VEGF vascular endothelial growth factor
  • Ang2 vascular endothelial growth factor A
  • 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 will benefit from inhibition of nucleic acid or protein levels of VEGF, such as VEGF A, and/or Ang2.
  • VEGF such as VEGF A, and/or Ang2.
  • the invention provides the use of a formulation of the invention in the manufacture or preparation of a medicament or a delivery device for the uses described herein, such as for preventing or treating a disease or condition as mentioned herein.
  • the formulations of the invention delay the onset of a disorder and/or symptoms associated with the disorder.
  • the formulations of the invention can also be administered in combination with one or more other therapies, such as treatment modalities and/or other therapeutic agents, for the uses described herein, such as for preventing and/or treating the relevant diseases or conditions mentioned herein.
  • therapies such as treatment modalities and/or other therapeutic agents, for the uses described herein, such as for preventing and/or treating the relevant diseases or conditions mentioned herein.
  • administration route of the preparation of the present invention is according to known methods, for example, topical administration, such as intraocular administration, ocular surface administration. In some embodiments, administration is by injection or instillation.
  • RNA RNA
  • PrimeScript reverse transcription kit Takara
  • Use cDNA as a template for the first round of PCR reaction, use forward primer Alp-VhL and reverse primer Alp-2b/2cR to obtain the first round of PCR products.
  • the pC3-HF vector and the second round of PCR products 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 electroporated to construct the VHH antibody library, and the bacterial solution was frozen at -80°C.
  • the revived bacterial liquid was inoculated into 100 ml YT-AG medium (Shanghai Bioengineering Co., Ltd.), and M13KO7 helper phage was added for infection.
  • the bacteria were resuspended in 2 ⁇ YT-AK medium (Shanghai Bioengineering Co., Ltd.), and cultured overnight at 37°C 200rpm.
  • the culture supernatant was collected and the recombinant phage was prepared by PEG/NaCl precipitation method.
  • Recombinant phages were subjected to three rounds of panning experiments using biotin-labeled antigens VEGFA (ACRO) or Ang2 (Beijing Yiqiao). 50ul M280 magnetic beads (Thermo) and an appropriate amount of biotin-labeled antigen were added to each tube. After incubation at room temperature for 30 minutes, 1x10 12 cfu of recombinant phages were added and incubated at room temperature for 1 hour. The obtained mixture was added with 1ml PBST and washed 10 times, with each washing time of 5 minutes.
  • VEGF A and Ang2 antigens (Beijing Yiqiao Company) in advance and dilute them to 0.5ug/ml with PBS buffer to coat 96-well ELISA plates, and keep them in a refrigerator at 4°C overnight. Wash the antigen-coated plates 3 times with PBST, add blocking agent to 300ul/well, and stand at room temperature for 1 hour. Wash 3 times with PBST, add 80ul blocking agent + 20ul expression supernatant of TG1 bacteria with positive VHH identified in 1.3 above, and shake at room temperature for 1 hour.
  • the present invention utilizes molecular biological technology to obtain antibody sequences in anti-VEGFA or Ang2 positive phages, and utilizes the above-obtained TG1 monoclonal expression and purification containing positive VHH to obtain VHH antibody protein.
  • TG1 bacteria containing the VHH expression plasmid identified in Example 1 Take the TG1 bacteria containing the VHH expression plasmid identified in Example 1, inoculate it into 800ml LB-Amp medium, and culture it at 37°C 200rpm until the OD600 value is 0.5-0.6. Add 1mM IPTG to the bacterial solution to induce expression, culture it at 28°C 200rpm overnight, collect the culture supernatant, add 15ml PB+1mg/ml polymyxin to resuspend the bacteria after centrifugation, centrifuge again, and filter it with a 0.22um filter membrane.
  • the immune library antibodies LA42F8, LA46E11, and LA24C11 obtained above were humanized, and the steps were as follows:
  • the three humanized antibodies obtained in the present invention respectively obtained the CDRs of the humanized VHH antibodies LA42F8.5, LA46E11.8, and LA24C11.10, and the amino acid sequences of the heavy chain variable regions are shown in the attached sequence table.
  • LA42F8, LA46E11, LA24C11 and humanized antibodies LA42F8.5, LA46E11.8, LA24C11.10 in eukaryotic cells was prepared as follows:
  • the humanized antibody sequences obtained above were cloned into pcDNA3.1 (Invitrogen) to obtain plasmids containing the antibody sequences.
  • Expi-293 cells (Invitrogen) were passaged according to the required transfection volume, and the cell density was adjusted to 1.5 ⁇ 10 6 cells/ml the day before transfection. The cell density on the day of transfection was about 3 ⁇ 10 6 cells/ml. 1/10 of the final volume of F17 medium (Gibco, A13835-01) was taken as transfection buffer, and the appropriate plasmid was added and mixed. Appropriate polyethyleneimine (PEI) (Polysciences, 23966) was added to the plasmid (the ratio of plasmid to PEI in 293F cells was 1:3), mixed and incubated at room temperature for 10 minutes to obtain a DNA/PEI mixture.
  • PEI polyethyleneimine
  • the gravity column used for purification was treated with 0.5M NaOH overnight, and the glass bottles were washed with distilled water and then dry-baked at 180°C for 4h to obtain the purification column. Before purification, the collected cell supernatant was centrifuged at 4500rpm for 30min and the cells were discarded. The supernatant was then filtered using a 0.22 ⁇ l filter. Protein A column (Hitrap Mabselect Sure 5*5ml, GE, 11-0034-95) was equilibrated with 10ml binding buffer (sodium phosphate 20mM. NaCl 150mM, PH7.0). The filtered supernatant was added to the purification column and re-equilibrated with 15ml binding buffer.
  • 10ml binding buffer sodium phosphate 20mM. NaCl 150mM, PH7.0
  • coding nucleic acids of negative control IgG, positive control BI-anti-VEGF, BI836880, and Faricimab were cloned into pcDNA3.1, transfected into Expi-293 cells, expressed, and purified using the same method as in Example 2.
  • Example 3 Determination of the binding kinetics between the chimeric antibody of the present invention and the antigen using biofilm thin layer interferometry
  • the equilibrium dissociation constant (KD) of the antibody of the present invention binding to human Ang2 was determined by using the thin-layer biofilm interferometry technique (ForteBio).
  • the ForteBio affinity determination was performed 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.
  • SA Thermo catalog number 21125
  • SA was diluted to 1ug/ml, and 100ul/well was spread on the ELISA plate at 4°C overnight. Wash 3 times with PBST and add 3% BSA to block for 1.5h.
  • VEGF A165 ACRO catalog number VE5-H8210
  • LA42F8 supernatant LA46E11 supernatant and negative control IgG prepared in Example 2 of the antibody (initial concentration of 150ug/ml, 3-fold serial dilution), and 50ul of positive control BI-anti-VEGF) with VEGFR-Fc (Beijing Yiqiao, catalog number: 10012-H02H final concentration 0.2ug/ml) and incubate for 20min before adding to the plate.
  • FIG. 2 shows that the candidate molecules LA42F8 and LA46E11 antibodies can completely block the binding of VEGF A to VEGFR2.
  • Tie2-Fc (Beijing Yiqiao Product No.: 10700-H03H) was diluted to 2ug/ml, and 100ul/well was spread on the ELISA plate at 4°C overnight. Wash 3 times with PBST, and add 3% BSA to block for 1.5h. Wash 3 times with PBST, and incubate 50ul of the purified LA24C11.10 and negative control IgG prepared in Example 2 with hAng2-biotin (final concentration 0.2ug/ml) for 20min in advance and then add to the plate. Wash 3 times with PBST, and add Avidin HRP (1:2000) and incubate for 35min. Wash 6 times with PBST, develop with TMB for 5min, and read OD450nm after termination.
  • the blocking results of the anti-Ang2 VHH antibody 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, and subsequently diluted 1:10 or 1:100;
  • 2Supernatant refers to the unpurified prokaryotic supernatant.
  • This example verifies the inhibitory effect of the anti-Ang2 VHH antibody of the present invention on Tie2 phosphorylation induced by hAng2-Fc.
  • Expi293 cells 293-Tie2 overexpressing Tie2 were co-incubated with antibodies and recombinant hAng2-Fc protein, and the content of phosphorylated Tie2 in the system was detected to reflect the inhibitory effect of different antibodies on hAng2-fc-induced Tie2 phosphorylation.
  • Expi-293 cells overexpressing human Tie2, 293-Tie2 cells were generated by transfecting Expi-293 cells (Thermo) with a pCHO1.0 vector (Invitrogen) carrying the human Tie2 gene (Sino Biologics, Inc., Cat. No. HG10700-M) cloned into the multiple cloning site MCS.
  • the experimental culture medium was prepared using Expi293 culture medium (Thermo catalog number A1435102): the test antibodies (LA24C11 (24C11) and LA24C11.0 (hz24C11.10) prepared in Example 2, as well as the negative control IgG, and the positive control Nesvacumab (prepared according to CN202010573625.2)) were added thereto, with an initial concentration of 60ug/ml and then diluted in a 1:2 ratio; the final concentration of hAng2-Fc (Beijing Yiqiao: 10691-H02H) was 2.5ug/ml.
  • Expi293 culture medium Thermo catalog number A143510293 culture medium (Thermo catalog number A1435102): the test antibodies (LA24C11 (24C11) and LA24C11.0 (hz24C11.10) prepared in Example 2, as well as the negative control IgG, and the positive control Nesvacumab (prepared according to CN202010573625.2)) were added thereto,
  • the culture medium was removed by centrifugation, and 100 ul of NP-40 lysis buffer (Biyuntian, Catalog No.: P0013F) containing 1% protease (Thermo Catalog No.: 78442) and phosphatase inhibitor (Thermo Catalog No.: 78442) was added, and the mixture was placed on ice for 30 min, centrifuged at 2000 g, and the protein supernatant was collected and stored in a -80 degree refrigerator.
  • NP-40 lysis buffer Boyuntian, Catalog No.: P0013F
  • protease Thermo Catalog No.: 78442
  • phosphatase inhibitor Thermo Catalog No.: 78442
  • the concentration of pTie2 was detected.
  • the capture antibody in the kit was coated on the ELISA plate at a concentration of 4ug/ml at 4°C overnight. Wash three times with PBST and block with 5% BSA for 1h. Add 100ul of the protein supernatant obtained in the previous step and the control pTie2 (R&D catalog number: DYC2720E) for making a standard curve and incubate at room temperature for 2h (if the concentration of pTie2 in the sample is too high and exceeds the detection range of ELISA, the obtained mixture can be diluted 2-3 times).
  • the anti-Ang2 VHH antibodies LA24C11 and LA24C11.10 of the present invention can effectively inhibit hAng2-Fc-induced 293-Tie2 cell phosphorylation in vitro.
  • VEGF A can bind to the related receptor VEGFR2 (KDR), activate the VEGFR2 signaling pathway, and induce vascular endothelial cell survival, proliferation, and migration.
  • KDR related receptor VEGFR2
  • the KDR reporter experimental system was used to detect the expression of graded dilutions of antibodies against VEGFA using NFAT-RE-luc2P/KDR HEK293 cells (Promega Cat CS181401). Blockade of activation-related receptor signaling pathways.
  • the experimental method refers to the supplier (Promega):
  • DMEM culture medium containing 10% FBS the experimental culture medium
  • the experimental culture medium DMEM culture medium containing 10% FBS
  • 1ml Accutase solution Sigma catalog number: A6964-500ML
  • 5ml dilution culture medium pipette the cells into a centrifuge tube, centrifuge at 1000rpm for 5min, discard the culture medium, add 10ml dilution culture medium (DMEM culture medium containing 10% FBS) to resuspend the cells, mix and count, the cell viability should be above 90%.
  • Use dilution culture medium to adjust the cell density to 0.8 ⁇ 10 6 cells/ml, and add 50 ⁇ l/well to a 96-well white cell culture plate according to the experimental layout.
  • VEGF A a mixture of 100ng/ml VEGF A and gradiently diluted antibodies to be tested, let it stand for 30 minutes, and place 50 ⁇ l/well of a 96-well white cell culture plate containing cells in a 37°C, 5% carbon dioxide incubator for 6 hours.
  • the samples to be tested are as follows: negative control IgG; positive control BI-anti-VEGF; LA42F8; LA46E11; LA42F8.5; LA46E11.8; Blank: only contains dilution medium, no VEGF A, no antibody; VEGF A 100ng/ml: only contains 100ng/ml VEGF A.
  • test results are shown in Figure 5, and the anti-VEGF VHH antibodies LA42F8, LA42F8.5, LA46E11, and LA46E11.8 can all block VEGF A-induced KDR signaling pathway activation.
  • 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 was based on VEGF-induced survival and proliferation of human umbilical vein endothelial cells (HUVEC), and detected the inhibitory effect of antibodies on VEGF A-induced primary cell survival and proliferation.
  • VVEEC human umbilical vein endothelial cells
  • HUVEC cells Allcells catalog number: H-001-CN
  • H-001-CN Allcells catalog number: H-001-CN
  • 2000 cells/well were plated in a 96-well culture plate, and incubated in a 37°C, 5% carbon dioxide incubator for 24 hours.
  • the experimental culture medium containing VEGF A at a final concentration of 10 ng/ml and gradiently diluted antibodies (LA42F8 prepared in Example 2, starting concentration 80 ug/ml 1:3 equal dilution, negative control IgG, positive control BI836880, a group containing only 10 ng/ml VEGF A (VEGFA), and Blank without VEGF A and antibody addition) was prepared, and the endothelial cell culture medium in the 96-well plate was replaced, and the plate was placed in a 37°C, 5% carbon dioxide incubator for incubation for 72 hours.
  • LA42F8 prepared in Example 2, starting concentration 80 ug/ml 1:3 equal dilution, negative control IgG, positive control BI836880, a group containing only 10 ng/ml VEGF A (VEGFA), and Blank without VEGF A and antibody addition
  • CCK-8 detection solution (Tongren Chemical Product No.: CK04) was added and incubated in a 37°C, 5% carbon dioxide incubator for 12 to 24 hours.
  • the chains of the bispecific binding molecules of the present invention IEX04-008, IEX04-010 and IEX04-012 were constructed into the pcDNA3.1 vector, and the bispecific binding molecules were expressed and purified in 293 cells as described in Example 2.
  • Example 7 the blocking effect of anti-VEGF A/Ang2 bispecific antibody on VEGF A was detected by HEK293-KDR reporter experiment.
  • the experimental method is referred to Example 7.
  • binding molecules or controls used were as follows:
  • VEGF A 100ng/ml VEGF A;
  • Negative control IgG prepared as described above;
  • Positive control Faricimab prepared as described above, starting concentration 13.5ug/ml, 1:3 serial dilution;
  • Positive control BI-836880 prepared as described above, starting concentration 13.5ug/ml, 1:3 serial dilution;
  • IEX04-012 Prepared as described above, starting concentration 13.5ug/ml, 1:3 serial dilutions.
  • 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 HEK293-KDR reporter experiment was used to detect the inhibitory effect of anti-VEGF A/Ang2 bispecific binding molecules on VEGF A-induced HUVEC cell survival and proliferation.
  • the experimental method is the same as in Example 8, but the antibodies used are as follows:
  • Figure A IEX04-008 prepared as described above, negative control IgG, positive control Faricimab and BI836880, with a starting concentration of 20ug/ml, 1:3 isocratic dilution, Blank (i.e., no antibody and VEGF A), and VEGF A group (i.e., only 20ng/ml VEGFA was added);
  • Figure B The starting concentration was 80ug/ml, the negative control IgG, BI-anti-VEGF, IEX04-010 prepared as described above were diluted 1:3, the blank was Blank (i.e., no antibody and VEGF A), and the VEGF A group (i.e., only 20ng/ml VEGFA was added);
  • Figure C IEX04-012 prepared as described above, negative control IgG, positive controls Faricimab and BI836880 with a starting concentration of 20 nM, 1:3 isocratic dilution, Blank (i.e., no antibody and VEGF A), and VEGF A group (i.e., only 20 ng/ml VEGFA added).
  • the test results are shown in FIG8 .
  • the bispecific binding molecules IEX04-008, IEX04-010, and IEX04-012 all inhibited VEGF-induced HUVEC cell survival and proliferation.
  • IEX04-012 had a lower IC 50 and had better inhibitory ability.
  • Ang2 can bind to its natural receptor Tie2. This study used ELISA and FACS methods to detect the blocking effect of anti-VEGF A/Ang2 bispecific binding molecules on the binding between Ang2 and Tie2.
  • hTie2 protein (Beijing Yiqiao) was resuspended and dissolved in PBS to a concentration of 2ug/ml, and coated on the ELISA plate overnight. Use 5% BSA to block for 1h, dilute the biotin antigen Recombinant Biotinylated hAngiopoietin-2 protein (R&D) to 600ug/mlM, 50 ⁇ l/well.
  • the antibodies prepared as described above were diluted 1:2 from the highest concentration of 300nM, a total of 8 or 12 dilution gradients, 50 ⁇ l/well, incubated in PBS on ice for 30min, and the final concentration of biotin antigen was 300ng/ml.
  • the antigen-antibody mixture obtained as described above was incubated on the ELISA plate for 90 minutes, washed three times with PBS, the supernatant was discarded, 100 ⁇ l Avidin-HRP (Invitrogen) diluted 1:10000 was added per well, incubated at room temperature for 30 minutes, and washed six times with PBS. 100 ul/well TMB colorimetric solution (solarbio) was used for color development for 1 minute, and 100 ul/well of stop solution (Solarbio) was used for termination.
  • the OD 450 and OD 620 of each well were read using a microplate reader.
  • the antigen hAng2-Fc protein (Beijing Yiqiao, Cat. No.: 10691-H02H) was diluted to 4ug/ml, 50 ⁇ l/well.
  • the antibodies prepared as described above (IEX04-008, IEX04-010, IEX04-012, positive control antibodies BI-836880 and Faricimab, and negative control IgG) were diluted 2-fold from the highest concentration of 800nM, with a total of 12 dilution gradients, 50 ⁇ l/well, and incubated on PBS ice for 30min.
  • the final concentration of the antigen hAng2-Fc protein was 2ug/ml, and the highest final concentration of each antibody was 400nM.
  • the 293-Tie2 cells prepared as described above were adjusted to 2 ⁇ 10 5 cells/well, 100 ⁇ l/well.
  • the cells were centrifuged at 300g for 5min, the supernatant was discarded, and the cells were resuspended in the antigen-antibody mixture.
  • This example verifies the inhibitory effect of the bispecific binding molecules of the present invention on hAng2-Fc-induced Tie2 phosphorylation, and uses an hAng2-induced phosphorylation experiment.
  • the experimental culture medium was prepared using Expi293 culture medium (Thermo catalog number A1435102), to which the test antibodies (IEX04-012 prepared as above, positive controls BI-836880 and Faricimab, and negative control IgG) were added at a maximum final concentration of 60ug/ml, and diluted in a 1:2 ratio in sequence.
  • the final concentration of hAng2-Fc (Beijing Yiqiao catalog number: 10691-H02H) was 2.5ug/ml.
  • the concentration of pTie2 was detected, and the capture antibody was coated on the ELISA plate at a concentration of 4ug/ml overnight at 4°C. Wash three times with PBST and block with 5% BSA for 1h. Add 100ul of the sample to be tested and the control pTie2 (R&D DYC2720E) for making a standard curve and incubate at room temperature for 2h (if the concentration of sample pTie2 is too high and exceeds the detection range of ELISA, 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 hAng2-Fc-induced 293-Tie2 phosphorylation 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 molecules on Ang2 vascular endothelial cell leakage
  • HUVEC-Tie2 leakage assay was used to identify the effect and function of anti-VEGF A/Ang2 bispecific binding molecules on vascular endothelial cell leakage.
  • HUVEC cells (Allcells catalog number: H-001-CN) were transfected with lentivirus to obtain HUVEC-Tie2 cells overexpressing Tie2.
  • EGM-2 medium Use a mini-well 96-well insert culture dish to spread 300ul of EGM-2 medium on the lower layer, digest with Accutase (Sigma) to obtain HUVEC-Tie2, resuspend with EGM-2 medium to 1*10 7 cells/ml, and spread 100ul/well on the upper layer of the culture dish. Replace the lower chamber medium (EGM-2 medium) every 24h, and replace the lower chamber medium with the experimental medium after 24 hours.
  • the experimental medium composition is as follows:
  • VEGF Group A EGM-2 medium + 20ng/ml VEGF (R&D catalog number: 293-VE)
  • IgG group (VEGF A + IgG): EGM-2 medium + 20ng/ml VEGF + 10ug/ml IgG;
  • Ang1 group (VEGF A + Ang1): EGM-2 medium + 20 ng/ml VEGF (R&D catalog number: 293-VE) + 200 ng/ml Ang1 (R&D catalog 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 culture medium was placed at 37°C and 5% CO 2 for culture.
  • FITC-Dextran (Sigma catalog number: FD2000S-1G) (4 mg/ml) was added to each well of the upper chamber experimental culture medium and placed at 37°C, 5% CO 2 . After 30 minutes, the lower chamber culture medium was removed and diluted 1:10 with PBS and detected on a multifunctional microplate reader with an excitation wavelength of 488 nm and an emission wavelength of 535 nm.
  • FIG12 The experimental results are shown in FIG12 , which show that the antibody IEX04-012 of the present invention can effectively reduce the permeability of vascular endothelial cells induced by VEGF.
  • the rhesus monkey laser-induced choroidal neovascularization model was used to determine the anti-neovascularization effect of the bispecific binding molecule IEX04-012 of the present invention.
  • This experiment used laser photocoagulation around the fovea of the rhesus monkey fundus to induce fundus choroidal angiogenesis and establish an animal model similar to human choroidal neovascularization.
  • Fluorescein fundus angiography was performed before and 20 days after photocoagulation to determine the modeling status, and 20 rhesus monkeys (half male and half female) with successful modeling were selected and divided into 5 groups, namely model control group, IEX04-012 low-dose group, IEX04-012 high-dose group, Elyea group, and Faricimab group, with 4 monkeys in each group, half male and half female.
  • OCT optical coherence tomography
  • the bispecific binding molecules of the present invention showed significant anti-neovascularization effects 28 days after administration.
  • the number of fourth-order leakage spots (Figure 13A) and third to fourth-order leakage spots (Figure 13B) showed that the number of high leakage spots in the IEX04-012 group of treated animals was significantly less than that in the control and positive control groups.
  • OCT results showed that the retinal thickness was significantly reduced in the IEX04-012 treated animals, indicating that the degree of retinal edema was reduced, and the effect was better than the control ( Figure 14).
  • Fluorescein fundus angiography results showed that the fundus leakage area in the IEX04-012 treated group was significantly reduced, and the effect was better than the positive controls Eylea and Faricimab ( Figure 15), indicating that the antibody of the present invention can significantly inhibit the leakage caused by neovascularization.
  • the antibody of the present invention combined with the anti-VEGF inhibitor has a significant inhibitory effect on laser-induced fundus neovascularization, and at the same time has the function of protecting vascular integrity.
  • rhesus monkeys were anesthetized with sodium pentobarbital (approximately 30 mg/kg injected intravenously, and the dose could be adjusted according to the health status of the animals) according to their body weight, and euthanized by bleeding from the abdominal aorta or femoral artery. Gross observation was performed, and bilateral eyeballs were removed.
  • the antibody group of the present invention showed a significant reduction in retinal lesion area, reduced retinal edema, and reduced tissue proliferation in the laser damaged area compared to anti-VEGF treatment alone.
  • the results showed better improvement in retinal morphology (see Figure 16), inhibition of retinal choroidal neovascularization, and enhanced vascular integrity function (Figure 17).
  • This experiment mainly investigated the effects of excipients (sodium dihydrogen phosphate (monohydrate), sodium dihydrogen phosphate (heptahydrate), trehalose, histidine, methionine, proline, glycine, sucrose, hydroxypropyl- ⁇ -cyclodextrin and polysorbate 80) on the stability of IEX04-012 protein.
  • excipients sodium dihydrogen phosphate (monohydrate), sodium dihydrogen phosphate (heptahydrate), trehalose, histidine, methionine, proline, glycine, sucrose, hydroxypropyl- ⁇ -cyclodextrin and polysorbate 80
  • N/A means not involved.
  • the trehalose in the following prescription refers to trehalose (dihydrate).
  • ultrapure water was used as the solvent to prepare the buffer of each prescription according to the table (except for polysorbate 80, its composition is shown in Table 5), and the IEX04-012 protein (prepared according to the method in Example 2 above) was ultrafiltered and replaced into the respective prescription buffer. After the replacement was completed, the protein content of each prescription was adjusted to 40 mg/ml; polysorbate 80 was added to a final concentration of 0.3 mg/ml; filtered and dispensed into vials, stoppered and capped. The above samples were subjected to stability investigation at 40°C ⁇ 2°C.
  • results of the mandatory stability study are detailed in Table 8.
  • the results show that after being placed at 40°C ⁇ 2°C for 1 week, except for prescription 5 which showed a heavier opalescence, the appearance and visible foreign matter of the samples of the other prescriptions were qualified; the protein content and pH value did not change significantly; the purity (SEC-HPLC method) of the samples of all prescriptions changed, and the purity (SEC-HPLC method) of the sample of prescription 7 decreased the least; the acidic components of the charge variants of all prescription samples increased, the main components decreased, and the acidic components of the sample of prescription 7 increased the least.
  • the purity (SEC-HPLC method) of the samples of all prescriptions changed, and the purity (SEC-HPLC method) of the sample of prescription 7 decreased the least; the acidic components of the charge variants of all prescription samples increased, the main components decreased, and the main components of the sample of prescription 7 decreased the least.
  • the polysorbate 80 content of all prescriptions did not change. The biological activity of all prescriptions was within the acceptable range.
  • formulation 7 was determined as the final formulation of IEX04-012.
  • the buffer system was adjusted to histidine and histidine hydrochloride.
  • the trehalose concentration was increased to 100mg/ml (the formulation osmotic pressure was 350mOsmol/kg at this time) to make the formulation buffer osmotic pressure meet the requirements of the Chinese Pharmacopoeia, that is, the final formulation of IEX04-012 was: 40.0mg/ml recombinant anti-vascular endothelial growth factor A (VEGF A) and anti-angiopoietin 2 (Ang2) bispecific antibody, 1.25mg/ml (8mM) histidine, 0.40mg/ml (2mM) L-histidine hydrochloride, 100mg/ml
  • VEGF A vascular endothelial growth factor A
  • Ang2 anti-angiopoietin 2
  • a high-concentration (120 mg/ml) bispecific antibody preparation was prepared, and its stability was studied by continuously detecting the mass change during forced, accelerated and long-term storage.
  • the buffer system was adjusted to histidine and histidine hydrochloride, and the adjusted IEX04-012 high-concentration formulation formulation was: 120.0 mg/ml recombinant anti-vascular endothelial growth factor A (VEGF A) and anti-angiopoietin 2 (Ang2) bispecific antibody, 1.25 mg/ml histidine, 0.40 mg/ml L-histidine hydrochloride, 100 mg/ml trehalose and 0.3 mg/ml polysorbate 80.
  • VEGF A vascular endothelial growth factor A
  • Ang2 anti-angiopoietin 2
  • the results of the prescription study are detailed in Table 13.
  • the results show that after being placed at 40°C ⁇ 2°C for 1 week and 2 weeks, the main peak content decreased and the aggregate increased in the purity test; the charge variant-main component decreased and the acidic component increased; the other inspection items were qualified or unchanged. After being placed at 25°C ⁇ 2°C for 1 week and 2 weeks. All samples were qualified in appearance and visible foreign matter. The charge variant-main component decreased and the acidic component increased; the other inspection items were qualified or unchanged.
  • N/A means not tested.
  • the buffer system was adjusted to histidine and histidine hydrochloride, and the prescription of the high-concentration preparation of IEX04-012 was: 150.0 mg/ml recombinant anti-vascular endothelial growth factor A (VEGF A) and anti-angiopoietin 2 (Ang2) bispecific antibody, 1.25 mg/ml histidine, 0.40 mg/ml L-histidine hydrochloride, 100 mg/ml trehalose and 0.3 mg/ml polysorbate 80.
  • VEGF A vascular endothelial growth factor A
  • Ang2 anti-angiopoietin 2
  • formulations of the examples of the present application are also applicable to other specific binding molecules of the present invention, especially other bispecific binding molecules in the examples (see the sequence table above), and achieve comparable effects.

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Abstract

提供了包含结合VEGF和Ang2的双特异性结合分子的制剂，特别地提供了包含该双特异性结合分子、缓冲剂、稳定剂和表面活性剂的药物制剂。还提供了这些制剂的疾病治疗或预防用途。

Description

包含结合VEGF和Ang2的双特异性结合分子的制剂以及其用途 技术领域
本发明涉及制剂领域。更具有而言，本发明涉及制剂，其包含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)的抗体或其抗原结合片段，或者包含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)或其抗原结合片段，尤其是稳定的液体制剂，以及用于制备所述抗体制剂的方法，以及所述抗体制剂的用途。
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。
需要新的能够用来治疗和/或预防眼部疾病的特异性抗体及包含这类新抗体的制剂，尤其是具有良好稳定性的制剂。
发明内容
本发明通过提供包含靶向VEGF-A或ANG-2的VHH抗体或者同时针对VEGF-A和ANG2的双特异性结合分子的药物制剂来满足上述需求。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VEGF 165。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,Science 277(1997)55-60和Cheung,A.H.等人,Genomics48(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,Cell 87(1996)1161-69；关于ANG2，参见Maisonpierre,P.C.等人,Science 277(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(SEQ ID NO:43)、(GSGGS)n(SEQ ID NO:44)、(GGGGS)n(SEQ ID NO:45)、(GGGS)n(SEQ ID NO:46)和(GGGGS)nG(SEQ ID NO:47)，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。优选的，所述接头是(GGGGS)n(SEQ ID NO:48)，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)Nature 342:877-883，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 of Health(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。
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“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。
“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，受试者是人。
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Blossum 62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Blossum 62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。
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。
本发明制剂中使用的抗体也称为本发明的抗体。例如本发明制剂中的抗VEGF A的VHH抗体有时也称为本发明的抗VEGF A的VHH抗体。
具体实施方案
在一个方面，本发明提供了一种液体制剂，其包含(i)抗VEGF A的VHH抗体、抗Ang2的VHH抗体或者结合VEGF A和Ang2的双特异性结合分子；(ii)缓冲剂，(iii)稳定剂，和(iv)表面活性剂，所述液体制剂的pH为约5.0-7.5。
在一些实施方案中，所述液体制剂包含抗VEGF A的VHH抗体。
在一些实施方案中，所述液体制剂包含抗Ang2的VHH抗体。
在一些实施方案中，所述液体制剂包含结合VEGF A和Ang2的双特异性结合分子，优选为结合VEGF A和Ang2的双特性抗体。
在一些实施方案中，所述抗VEGF A的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，所述抗VEGFVHH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，所述抗VEGF 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，所述抗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，所述抗Ang2 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，所述的结合VEGF A和Ang2的双特异性结合分子包含特异性结合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，所述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，所述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，所述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，所述双特异性结合分子为双特异性抗体。
在一些实施方案中，所述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，所述的抗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，所述的抗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，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，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，所述的抗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。
在一些实施方案中，所述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，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，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，所述的抗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。
在一些实施方案中，所述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 domain 2)和VEGFR2第三抗体样结构域(例如KDR domain 3)。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，所述的抗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IgG4Fc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。
在一些实施方案中，所述的缓冲剂选自组氨酸、组氨酸盐、谷氨酸盐、磷酸盐、乙酸盐、柠檬酸盐和三羟甲基氨基甲烷中的一种或多种。所述组氨酸盐、谷氨酸盐包括但不限于所述氨基酸与盐酸或硫酸形成的盐。所述磷酸盐、乙酸盐、柠檬酸盐包括但不限于相应酸的碱金属盐例如锂、钠或钾盐。例如，磷酸盐可以为水合物形式或非水合物形式的，包括但不限于磷酸氢二钠、磷酸二氢钠、磷酸氢二钾、磷酸二氢钾、磷酸氢二锂、磷酸二氢锂或其组合；优选地，组氨酸盐为盐酸组氨酸，磷酸盐为磷酸氢二钠和磷酸二氢钠的组合。优选地，所述的缓冲剂选自组氨酸和盐酸组氨酸的组合、磷酸盐和组氨酸。
在一些实施方案中，所述稳定剂选自糖类、多元醇、氨基酸或其盐中的一种或多种，优选地，所述糖类选自但不限于：蔗糖、右旋糖、乳糖、麦芽糖、海藻糖、环糊精、麦芽糖糊精和葡聚糖，所述多元醇选自但不限于：甘露醇、山梨醇和木糖醇，所述氨基酸或其盐选自以下的一种或多种：精氨酸、甘氨酸、脯氨酸、甲硫氨酸以及它们的盐；所述环糊精优选为羟丙基-β-环糊精。
在一些实施方案中，所述稳定剂是蔗糖或海藻糖。在一些实施方案中，所述稳定剂是蔗糖或海藻糖与另外的成分的组合，所述另外的成分选自以下的一种或多种：甘氨酸、脯氨酸、甲硫氨酸和羟丙基-β-环糊精。
在一些实施方案中，所述的表面活性剂是非离子型表面活性剂，包括但不限于，烷基聚(环氧乙烯)；聚山梨酯，诸如聚山梨酯-20、聚山梨酯-80、聚山梨酯-60、或聚山梨酯-40；普洛尼克等。在一些优选实施方案中，本发明的液体制剂中包含聚山梨酯-80作为表面活性剂。
在一些实施方案中，本发明的液体制剂中的所述抗体或双特异性结合分子的浓度为约1-200mg/ml，例如约1-150mg/ml、约10-190mg/ml、约20-180mg/ml、约30-170mg/ml、约30-150mg/ml、约30-170mg/ml、约30-100mg/ml、约30-100mg/ml，例如约5、约10、约15、约20、约25、约30、约40、约50、约60、约70、约80、约90、约100、约110、约120、约130、约140、约150、约160、约170、约180、约190或约200mg/ml，优选地为约10-160mg/ml，更优选地为约30-150mg/mL。
在一些实施方案中，本发明的液体制剂中的缓冲剂的浓度为约0.5-200mM、约1-100mM、约5-50mM、约5-30mM、约5-20mM、约5-15mM、约8-12mM，例如约5、约10、约15、约20、约25、约30、约40、约50、约60、约70、约80、约90、约100、约110、约120、约130、约140、约150、约160、约170、约180、约190或约200mM，优选地为约5-50mM，更优选地为约5-30mM。当缓冲剂为多种成分时，各成分的浓度可选自如下范围或值：0.2-100mM、约0.5-50mM、约2-25mM、约2-15mM、约1-10mM、约2-8mM，约1-3mM、0.5-4mM、约7-9mM、约6-10mM、约5-10mM，例如约1、约2、约3、约4、约5、约6、约7、 约8、约9、约10、约15、约20、约25、约30、约40、约50、约60、约70、约80、约90或约100mM。
在一些实施方案中，本发明的液体制剂中的稳定剂的浓度为约1-1000mM、约10-1000mM、约20-800mM、约30-700mM、约50-800mM、约50-500mM、约100-400mM、约100-300mM、约200-350mM、约200-300mM、约250-280mM、约200-400mM或约100-200mM，例如，约10、约20、约50、约80、约100、约150、约200、约300、约400、约500、约600、约700、约800、约900或约1000mM；优选地约50-800mM，更优选地约50-500mM。当稳定剂为多种成分时，各成分的浓度可选自如下范围或值：约20-800mM、约30-700mM、约50-800mM、约50-500mM、约100-400mM、约100-300mM、约200-350mM、约200-300mM、约250-280mM、约200-400mM或约100-200mM、约0.5-100mM、约1-50mM、约2-50mM、约5-20mM、约8-12mM，例如，约1、约2、约3、约4、约5、约10、约20、约30、约50、约80、约100、约150、约200、约300、约400、约500或约600mM。
在一些实施方案中，本发明的液体制剂中的表面活性剂的浓度为约0.01-10mg/ml，例如约0.05-5、约0.05-2、约0.1-1、约0.1-5、约0.2-2、约0.3-1、约0.2-0.4mg/ml约0.4-0.8或约0.5-0.6mg/ml，例如约0.2、约0.3、约0.4、约0.5、约0.6、约0.7或约0.8mg/ml，优选地约0.05-5mg/ml，更优选地0.05-2mg/ml。
在一些实施方案中，本发明的液体制剂的pH为约5.5-7.2、6.0-7.0、6.1-7.0、6.2-7.0、6.2-6.6或6.3-6.8，例如约5.0、约5.2、约5.4、约5.6、约5.8、约6.0、约6.1、约6.2、约6.3、约6.4、约6.5、约6.6、约6.7、约6.8、约6.9、约7.0、约7.1或约7.2，优选地为约6.1-7.0，更优选地为约6.3-6.8。
在一些实施方案中，本发明的液体制剂具有200-400mOsmol/kg、250-350mOsmol/kg的渗透压，例如350mOsmol/kg的渗透压。
在一些实施方案中，所述液体制剂为药物制剂，优选为注射剂，更优选为玻璃体腔注射剂，或滴眼液。
在一些实施方案中，本发明的液体制剂包含：
(i)约10-160mg/ml优选30-150mg/ml的所述双特异性结合分子；
(ii)约5-20mM缓冲剂，所述缓冲剂为组氨酸或者为组氨酸和组氨酸盐酸盐的组合；
(iii)约200-350mM海藻糖；和
(iv)约0.1-1mg/ml聚山梨醇酯80；
其中所述液体制剂的pH为约6.1-7.0，优选地约6.5。
在一些实施方案中，本发明的液体制剂包含：
(i)约10-160mg/ml优选30-150mg/ml的所述双特异性结合分子；
(ii)约8-12mM组氨酸；
(iii)约200-300mM海藻糖；和
(iv)约0.2-0.4mg/ml聚山梨醇酯80；
其中所述液体制剂的pH为约6.1-6.6，优选地约6.5。
在一些实施方案中，本发明的液体制剂包含：
(i)约10-160mg/ml优选30-150mg/ml的所述双特异性结合分子；
(ii)约1-3mM组氨酸和约7-9mM组氨酸盐酸盐；
(iii)约250-280mM海藻糖；和
(iv)约0.2-0.4mg/ml聚山梨醇酯80；
其中所述液体制剂的pH为约6.1-6.6，优选地约6.5。
在一些实施方案中，本发明的液体制剂是实施例15-17中所公开的制剂。此外，在实施例15-17中所公开的制剂的基础上，由这些制剂中各成分的浓度值上下浮动50％、40％、30％、20％、10％或5％，以及pH值上下浮动5％、4％、3％、2％、1％或0.5％所产生的实施方案也包括在本申请中。
本发明的抗体液体制剂中可以包含或不包含其它赋形剂。例如，本发明的抗体液体制剂还可包含张力调节剂。张力调节剂可以选自下组：醋酸钠、乳酸钠、氯化钠、氯化钾和氯化钙。
这些和另外已知的药物赋形剂和/或适用于本发明制剂的添加剂是本领域公知的，例如，列出于“The Handbook of Pharmaceutical Excipients，第4版，Rowe等人编，American Pharmaceuticals Association(2003)；和Remington:the Science and Practice of Pharmacy，第21版，Gennaro编，Lippincott Williams&Wilkins(2005)”。
应当理解，如无特别说明，本发明的液体制剂还包含溶媒，包括但不限于例如纯化水如超纯水、注射用水、无菌水、双蒸水等。本发明的液体制剂可使用本领域已知的可药用的溶剂或溶液来制备或配制。所述可药用的溶剂或溶液包括但不限于例如纯化水如超纯水、注射用水、无菌水、双蒸水、生理盐水、林格氏溶液、葡萄糖注射液等。
在一些实施方案中，所述的抗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，所述的抗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，所述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，所述的抗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。
另一方面，本发明提供了一种固体制剂，其是通过将本发明的液体制剂经固化处理而获得的。所述固化处理是通过例如结晶法、喷雾干燥法、冷冻干燥法实施的。在一个优选的实施方案中，所述固体制剂例如是冻干粉针剂形式。固体制剂可在使用前，通过重构于适当的溶媒中，形成本发明的重构制剂。所述重构制剂也是一种本发明的液体制剂。在一个实施方案中，所述适当的溶媒选自纯化水如超纯水、注射用水、注射用有机溶剂，包括但不限于注射用油、乙醇、丙二醇等，或其组合。
本发明的制剂(包括固体制剂和液体制剂)可以长期稳定储存，例如至少24个月或更长时间。在一个实施方案中，本发明的制剂可以在约-80℃至约45℃，例如-80℃、约-30℃、约-20℃、约0℃、约5℃、约25℃、约35℃、约38℃、约40℃、约42℃或约45℃的条件下，储存至少10天、至少20天、至少1个月、至少2个月、至少3个月、至少4个月、至少5个月、至少6个月、至少7个月、至少8个月、至少9个月、至少10个月、至少11个月、至少12个月、至少18个月、至少24个月，至少36个月，或更长时间，且是稳定的。
在一个实施方案中，在储存后，通过目视检查本发明液体制剂的稳定性，其中本发明液体制剂在外观上保持为澄明至微乳光，为无色至淡黄色液体，且无异物。在一个实施方案中，在澄明度检测仪下目视检查，制剂中无可见异物存在。在一个实施方案中，在储存后，通过测定蛋白含量变化，检查本发明液体制剂的稳定性，其中例如通过紫外分光光度(UV)法，相对于储存第0天的初始值，蛋白含量变化率不超过20％，优选不超过10％，例如7-8％，更优选不超过5％。在一个实施方案中，在储存后，通过测定本发明液体制剂的纯度变化，检查本发明液体制剂的稳定性，其中通过体积排阻高效液相色谱法(SEC-HPLC)，相对于储存第0天的初始值，单体纯度的变化值不超过10％，例如不超过5％、4％、3％、例如变化值不超过1-2％，优选不超过1％。在一个实施方案中，在储存后，通过测定本发明液体制剂的纯度变化，检查本发明液体制剂的稳定性，其中通过非还原型和/或还原型十二烷基硫酸钠毛细管电泳(CE-SDS)法，单体纯度的变化值下降不超过10％，例如不超过5％、4％、3％、2％或1％。在一个实施方案中，在储存后，通过成像毛细管等电聚焦电泳(iCIEF)检测本发明液体制剂的稳定性，其中相对于储存第0天的初始值，抗体的电荷变异体(主成分、酸性组分和碱性组分)的变化值总和不超过不超过30％，例如不超过20％、不超过10％、不超过5％、不超过2％或不超过1％。
在另一个方面，本发明提供了一种递送装置，其包含本发明的液体制剂或固体制剂。在一个实施方案中，本发明的递送装置以包含本发明的液体制剂或固体制剂的预填充注射器形 式提供，例如用于玻璃体腔注射、静脉内、皮下、皮内或者肌内注射、静脉内输注。
在另一方面，本发明提供根据本发明的液体制剂的制备方法，其包括以下步骤：
i.提供分离纯化的本发明的抗体或双特异性结合分子，并任选地将其加入到超滤离心管中离心浓缩；
ii.配置缓冲剂、稳定剂的溶液优选水溶液，优选地，缓冲剂、稳定剂的种类、浓度和溶液的pH如上面本发明的液体制剂中所定义，
iii将步骤i的抗体超滤置换至步骤ii的溶液中，
iv.调整置换后的蛋白质的浓度到本发明的液体制剂所定义的浓度；
v.加入表面活性剂或其溶液优选水溶液，使表面活性剂的终浓度至对本发明的液体制剂中所定义的浓度；
vi.任选地步骤v的溶液无菌过滤；和
vii.任选地分装至西林瓶中，加盖橡胶塞和铝塑盖，获得成品。
另一方面，本发明提供了在受试者中预防或治疗眼部疾病的方法，包括向受试者施用本发明的制剂。
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制剂在生产或制备药物或递送装置中的用途，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。
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2.5×107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1x1012cfu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1.5×106个细胞/ml。转染当天细胞密度约为3×106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CO2。24h后补加转染体积2％的FEED(Sigma)，于36.5℃，120rpm，8％的CO2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.NaCl 150mM,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(PBS 1×，BSA 0.1％，Tween-20 0.05％)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KD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)
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)
*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
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*106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Tie2 ELISA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0.8×106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 A 100ng/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/ml 1:3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OD450-OD620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IC50更低，具有更优的抑制能力。
实施例11抗VEGF A/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300nM 1：2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OD450，OD620。
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IC50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2×105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PBS 100μl/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IC50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*106cell/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IC50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*107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％CO2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％CO2.30min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-induced 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 Ophthalmology,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
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，CD31IHC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




实施例15.本发明的双特异性抗体处方确定实验
本实验主要考察辅料(磷酸二氢钠(一水合物)、磷酸氢二钠(七水合物)、海藻糖、组氨酸、甲硫氨酸、脯氨酸、甘氨酸、蔗糖、羟丙基-β-环糊精和聚山梨酯80)对IEX04-012蛋白稳定性的影响，详细处方信息见表5。
实验材料：
本发明的处方研究实验种的所用的辅料和/或试剂信息参见下面的表4。
表4.试剂信息

注：N/A表示不涉及。下文处方中的海藻糖是指海藻糖(二水合物)。
制剂的制备和组成
按照表5中的质量浓度使用超纯水作为溶剂按表格配制各个处方的缓冲液(除了聚山梨酯80外，其组成如表5所示)，将IEX04-012蛋白(根据上面实施例2中的方法制备)超滤置换至各自的处方缓冲液中。置换完成后，调节各处方蛋白含量至40mg/ml；加入聚山梨酯80，使其终浓度为0.3mg/ml；过滤分装至西林瓶，加塞、轧盖。上述样品于40℃±2℃条件下进行稳定性考察。
表5备选处方信息表

注：用稀盐酸或1M NaOH溶液调节pH。
稳定性研究
详细实验条件及取样计划见表6。
表6.稳定性考察方案
注：上述时间点取样后均先放入-70℃冰箱中冻存待检，按需化冻送检。
判断标准
根据对产品的认识以及仪器和方法的精密度，设定了样品检测值与初始值相比质量未发生变化的判断标准，具体见表7。
表7.质量未发生变化的判断标准
注：“*”表示该标准仅适用于处方确定实验。
实验结果
强制稳定性研究结果详见表8。结果表明，40℃±2℃条件下放置1周，除处方5显示出较重乳光，其余处方的样品外观、可见异物均合格；蛋白含量和pH值均未发生明显变化；所有处方的样品的纯度(SEC-HPLC法)均发生变化，处方7样品纯度(SEC-HPLC法)下降最少；所有处方样品电荷变异体的酸性组分均上升，主成分均下降，处方7样品酸性组分上升最少。40℃±2℃条件下放置2周，所有处方的样品的纯度(SEC-HPLC法)均发生变化，处方7样品纯度(SEC-HPLC法)下降最少；所有处方样品电荷变异体的酸性组分均上升，主成分均下降，处方7样品主成分下降最少。所有处方的聚山梨酯80含量未发生变化。所有处方的生物学活性都在接受范围。


基于处方确定实验结果，确定处方7作为IEX04-012最后选定的制剂处方。为生产时避免使用盐酸调节pH，将缓冲体系调整为组氨酸和盐酸组氨酸，同时因处方F7的制剂渗透压偏低(255mOsmol/kg)，因此将海藻糖浓度提高至100mg/ml(此时制剂渗透压为350mOsmol/kg)，使其制剂缓冲液渗透压符合中国药典要求，即IEX04-012最终制剂处方为：40.0mg/ml重组抗血管内皮生长因子A(VEGF A)和抗血管生成素2(Ang2)双特异性抗体，1.25mg/ml(8mM)组氨酸，0.40mg/ml(2mM)L-组氨酸盐酸盐，100mg/ml(264.3mM)海藻糖和0.3mg/ml聚山梨酯80，pH 6.5。后续长时间的25℃和2～8℃稳定性的结果也表明，IEX04-012抗体在该处方条件下能够长时间维持稳定。
处方7最终制剂处方长期稳定性实验结果见表9
实施例16.本发明的双特异性抗体的高浓度处方研究(120mg/ml)
本实施例制备了高浓度(120mg/ml)的双特异性抗体制剂，并通过持续检测强制、加速和长期保存时的质量变化，研究了其稳定性。
配制含1.25mg/ml组氨酸、0.40mg/ml L-组氨酸盐酸盐、100.00mg/ml海藻糖的缓冲液(pH 6.5)，后将IEX04-012(按照实施例2方法制备)蛋白超滤置换至该缓冲液中，调节蛋白含量至约120mg/ml；加入聚山梨酯80使其终浓度为0.3mg/ml；过滤分装至西林瓶，加塞、轧盖。上述样品于40℃±2℃，25℃±2℃和2～8℃条件下进行稳定性考察，具体方案见表10。
表10.稳定性考察方案
注：上述时间点取样后均先放入-70℃冻存待检，按需化冻送检。
判断标准
根据对产品的认识以及仪器和方法的精密度，设定了样品检测值与初始值相比质量未发生变化的判断标准，具体见表11。
实验结果
处方研究结果详见表12。结果表明，40℃±2℃条件下放置1周，2周，纯度检测中，主峰含量下降，聚合体增加；电荷变异体-主成分减少，酸性组分增加；其他检项均合格或无变化。放置4周，产生明显乳光。25℃±2℃条件下放置1月，2月，和3月。所有样品外观、可见异物均合格。样品的纯度(SEC-HPLC)检测中，主峰含量下降，聚合体含量上升；电荷变异体-主成分减少，酸性组分增加；其他检项均合格或无变化。2～8℃条件下放置6月和12月。所有样品外观、可见异物均合格，其他检测项与初始值相比无明显变化。
如实施例15中所述，为生产时避免使用盐酸调节pH，将缓冲体系调整为组氨酸和盐酸组氨酸，调整后的IEX04-012高浓度规格制剂处方为：120.0mg/ml重组抗血管内皮生长因子A(VEGF A)和抗血管生成素2(Ang2)双特异性抗体，1.25mg/ml组氨酸，0.40mg/ml L-组氨酸盐酸盐，100mg/ml海藻糖和0.3mg/ml聚山梨酯80。
实施例17.本发明的双特异性抗体的高浓度处方研究(150mg/ml)
本实施例制备了高浓度(150mg/ml)的双特异性抗体制剂，并研究了其稳定性。
配制含1.25mg/ml组氨酸、0.40mg/ml L-组氨酸盐酸盐、100.00mg/ml海藻糖的缓冲液，将缓冲液pH调节至6.5后，将IEX04-012蛋白超滤置换至该缓冲液中，调节蛋白含量至约150mg/ml；加入聚山梨酯80使其终浓度为0.3mg/ml；过滤分装至西林瓶，加塞、轧盖。上述样品于40℃±2℃和25℃±2℃条件下进行稳定性考察。
实验结果
处方研究结果详见表13。结果表明，40℃±2℃条件下放置1周，2周，纯度检测中，主峰含量下降，聚合体增加；电荷变异体-主成分减少，酸性组分增加；其他检项均合格或无变化。25℃±2℃条件下放置1周和2周。所有样品外观、可见异物均合格。电荷变异体-主成分减少，酸性组分增加；其他检项均合格或无变化。
表13.处方研究结果
注：N/A表示未检测。
如前所述，为生产时避免使用盐酸调节pH，将缓冲体系调整为组氨酸和盐酸组氨酸，IEX04-012高浓度规格制剂处方为：150.0mg/ml重组抗血管内皮生长因子A(VEGF A)和抗血管生成素2(Ang2)双特异性抗体，1.25mg/ml组氨酸，0.40mg/ml L-组氨酸盐酸盐，100mg/ml海藻糖和0.3mg/ml聚山梨酯80。
应当注意的是，本申请的实施例制剂处方同样适用于本发明的其他特异性结合分子尤其是实施例中的其他双特异性结合分子(可参见上面的序列表)，并取得相当的效果。
以上描述了本发明的示例性实施方案，本领域技术人员应当理解的是，这些公开内容仅是示例性的，在本发明的范围内可以进行各种其它替换、适应和修改。因此，本发明不限于文中列举的具体实施方案。

Claims (29)

  1. 一种液体制剂，其包含
    (i)特异性结合VEGF A和Ang2的双特异性结合分子；
    (ii)缓冲剂，
    (iii)稳定剂，和
    (iv)表面活性剂，
    所述抗体制剂的pH为约5.0-7.5，
    所述双特异性结合分子包含特异性结合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抗Ang2的VHH，该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；
    任选地，其中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，例如其胞外结构域与Fc的融合蛋白。
  2. 根据权利要求1所述的液体制剂，其中所述抗Ang2的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，
    例如，所述双特异性结合分子为双特异性抗体。
  3. 根据权利要求1或2所述的液体制剂，其中所述双特异性结合分子为二价、三价或四价的，。
  4. 根据权利要求1-3中任何一项所述的液体制剂，其中所述双特异性结合分子具有以下结构：
    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。
  5. 根据权利要求4所述的液体制剂，其中：
    所述抗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
    所述抗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。
  6. 根据权利要求1-5中任何一项所述的液体制剂，其中
    所述抗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；
    所述抗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。
  7. 根据权利要求4-6中任何一项所述的液体制剂，其中
    (1)所述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；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。
  8. 根据权利要求3-7中任何一项所述的液体制剂，其中所述双特异性结合分子具有以下结构：
    第一抗VEGF VHH-接头-第二抗VEGF VHH-接头-抗Ang2VHH，
    其中第一抗VEGF VHH与第二抗VEGF VHH相同或不同。
  9. 根据权利要求8所述的液体制剂，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。
  10. 根据权利要求8或9所述的液体制剂，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。
  11. 根据权利要求8-10中任何一项所述的液体制剂，其中
    (1)所述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；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。
  12. 根据权利要求1所述的液体制剂，其中所述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Ang2VHH。
  13. 根据权利要求12所述的液体制剂，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。
  14. 根据权利要求12或13所述的液体制剂，其中所述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。
  15. 根据权利要求12-14中任何一项所述的液体制剂，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。
  16. 根据权利要求12-15中任何一项所述的液体制剂，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。
  17. 根据权利要求12-16中任何一项所述的液体制剂，其中
    (1)所述双特异性结合分子包含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。
  18. 根据权利要求1-17中任何一项所述的液体制剂，其中所述接头包含SEQ ID NO:23的氨基酸序列，或由其组成。
  19. 根据权利要求1-18中任何一项所述的液体制剂，其中所述的缓冲剂选自组氨酸、组氨酸盐、谷氨酸盐、磷酸盐、乙酸盐、柠檬酸盐和三羟甲基氨基甲烷中的一种或多种；优选地，所述的缓冲剂选自组氨酸和盐酸组氨酸的组合、磷酸盐和组氨酸；优选地，所述磷酸盐为磷酸氢二钠和磷酸二氢钠的组合。
  20. 根据权利要求1-19中任何一项所述的液体制剂，其中所述稳定剂选自糖类、多元醇、氨基酸或其盐中的一种或多种，
    优选地，所述糖类选自：蔗糖、右旋糖、乳糖、麦芽糖、海藻糖、环糊精、麦芽糖糊精和葡聚糖，所述多元醇选自：甘露醇、山梨醇和木糖醇，所述氨基酸或其盐选自精氨酸、甘氨酸、脯氨酸、甲硫氨酸、精氨酸盐和其组合；所述环糊精优选为羟丙基-β-环糊精。
    更优选地，所述稳定剂是蔗糖或海藻糖，或者是蔗糖或海藻糖与另外的成分的组合，所述另外的成分选自以下的一种或多种：甘氨酸、脯氨酸、甲硫氨酸和羟丙基-β-环糊精。
  21. 根据权利要求1-20中任何一项所述的液体制剂，其中所述表面活性剂选自聚山梨酯-20、聚山梨酯-80、聚山梨酯-60、或聚山梨酯-40；普洛尼克，优选为聚山梨酯-80。
  22. 根据权利要求1-21中任何一项所述的液体制剂，其中所述抗体或其抗原结合片段的浓度为约1-200mg/ml，优选30-150mg/mL；和/或
    所述缓冲剂的浓度为约0.5-200mM，优选5-30mM；和/或
    所述稳定剂的浓度为约1-1000mM，优选约50-500mM；和/或
    所述表面活性剂的浓度为约0.01-10mg/ml，优选0.05-2mg/ml；和/或
    所述液体制剂的pH为约6.1-7.0，优选约6.3-6.8。
  23. 根据权利要求1-22中任何一项所述的液体制剂，其中所述液体制剂为注射剂或滴眼液，更优选为玻璃体腔注射剂。
  24. 根据权利要求1-18中任何一项所述的液体制剂，其包含：
    (i)约10-160mg/ml的所述双特异性结合分子；
    (ii)约5-20mM缓冲剂，所述缓冲剂为组氨酸或者为组氨酸和组氨酸盐酸盐的组合；
    (iii)约200-350mM海藻糖；和
    (iv)约0.1-1mg/ml聚山梨醇酯80；
    其中所述液体制剂的pH为约6.1-7.0，优选地约6.5。
  25. 根据权利要求24所述的液体制剂，其包含：
    (i)约30-150mg/ml的所述双特异性结合分子；
    (ii)约8-12mM组氨酸；
    (iii)约200-300mM海藻糖；和
    (iv)约0.2-0.4mg/ml聚山梨醇酯80；
    其中所述液体制剂的pH为约6.1-6.6，优选地约6.5。
  26. 根据权利要求24所述的液体制剂，其包含：
    (i)约30-150mg/ml的所述双特异性结合分子；
    (ii)约1-3mM组氨酸和约7-9mM组氨酸盐酸盐；
    (iii)约250-280mM海藻糖；和
    (iv)约0.2-0.4mg/ml聚山梨醇酯80；
    其中所述液体制剂的pH为约6.1-6.6，优选地约6.5。
  27. ー种固体制剂，其是通过将权利要求1-26中任何一项所述的液体制剂经固化处理而获得的；所述固体制剂例如是冻干粉针剂形式。
  28. 一种递送装置，其包含权利要求1-26中任何一项所述的液体制剂或权利要求27所述的固体制剂，例如预填充注射器形式的。
  29. 权利要求1-26中任何一项所述的液体制剂或权利要求27所述的固体制剂在制备用于预防或治疗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的药物或递送装置中的用途。
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