Formulations comprising bispecific binding molecules that bind to VEGF and Ang2 and uses thereof

Abstract

Formulations comprising a bispecific binding molecule that binds VEGF and Ang2 are provided, in particular pharmaceutical formulations comprising the bispecific binding molecule, a buffer, a stabilizer, and a surfactant. Also provided are uses of these formulations for the treatment or prevention of diseases.

Description

Formulations comprising bispecific binding molecules that bind VEGF and Ang2 and uses thereof Technical Field

[0001] The present invention relates to the field of preparations. More specifically, the present invention relates to preparations comprising antibodies or antigen-binding fragments thereof directed against vascular endothelial growth factor (VEGF / VEGF-A) and angiopoietin-2 (ANG-2), respectively, or comprising bispecific binding molecules (e.g., antibodies) or antigen-binding fragments thereof directed against both vascular endothelial growth factor (VEGF / VEGF-A) and angiopoietin-2 (ANG-2), in particular stable liquid preparations, as well as methods for preparing the antibody preparations and uses of the antibody preparations.

[0002] Background of the Invention

[0003] Angiogenesis has been implicated in the pathogenesis of various diseases, including solid tumors, diseases associated with intraocular neovascularization, rheumatoid arthritis, and psoriasis.

[0004] VEGF is an effective and ubiquitous angiogenesis 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 primarily in their bioavailability, with VEGF165 being the main isoform. It is believed that VEGF is an important regulator of normal and disease-related angiogenesis.

[0005] In addition to the VEGF family, human angiogenin is also believed to be involved in vascular development and postnatal angiogenesis. Human angiogenin includes the naturally occurring agonist angiopoietin-1 (ANG-1) and the naturally occurring antagonist angiopoietin-2 (ANG-2). The effects of ANG-1 are believed to be conserved in adults, where it is widely and constitutively expressed. In contrast, 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 blood vessels to return to and remain in a plastic state that may be more responsive to sprouting signals.

[0006] In recent years, several bispecific antibodies targeting VEGF-A and ANG-2 have been developed (e.g., WO2012131078 and WO2014009465). However, existing bispecific antibodies have poor blocking properties against VEGF and ANG-2, and their large molecular weight results in low molar concentrations when administered once. In particular, for ocular diseases, smaller molecular weight antibodies are typically used for intravitreal administration, requiring less frequent dosing. Therefore, there remains a need for new bispecific binding molecules targeting VEGF-A and ANG-2, particularly those suitable for ocular diseases.

[0007] There is a need for new specific antibodies that can be used to treat and / or prevent ocular diseases and formulations containing such new antibodies, especially formulations with good stability.

[0008] Summary of the Invention

[0009] The present invention addresses these needs by providing pharmaceutical formulations comprising VHH antibodies targeting VEGF-A or ANG-2, or bispecific binding molecules targeting both VEGF-A and ANG-2. Specifically, compared to known antibodies, the bispecific binding molecules of the present invention have a smaller molecular weight and, at the same mass concentration, a higher molar concentration. They also exhibit potent VEGF-A and ANG-2 blocking activity, capable of completely blocking VEGFA-induced primary cell proliferation. Therefore, the molecules of the present invention possess stronger blocking activity clinically and can achieve higher antibody molar concentrations with a single administration, maintaining the efficacy of a single dose for a longer period, and reducing the frequency of ocular administration (e.g., intravitreal injection). Description of the drawings:

[0010] Figure 1 shows the structure of a bispecific binding molecule.

[0011] FIG2 shows that anti-VEGF A VHH antibodies can block the binding of VEGF A to VEGFR2 as determined by ELISA.

[0012] FIG3 shows the effect of humanized anti-Ang2 VHH antibodies in blocking the binding of Ang2 to Tie2 as determined by ELISA.

[0013] FIG4 shows the effects of anti-Ang2 VHH antibody (A) and humanized anti-Ang2 VHH antibody (B) on inhibiting hAng2-Fc-induced phosphorylation in 293-Tie2 cells as determined by ELISA.

[0014] Figure 5 shows the use of the HEK293-KDR reporter assay to detect the effect of anti-VEGF A VHH in blocking VEGFA activation of the KDR receptor.

[0015] Figure 6 shows the effects of anti-VEGF A VHH antibodies in inhibiting VEGF A-induced survival and proliferation of HUVEC cells as measured by CCK-8.

[0016] FIG7 shows the use of the HEK293-KDR reporter assay to detect the effect of the VEGF A / Ang2 bispecific binding molecule IEX04-012 in blocking VEGF-activated KDR receptor.

[0017] Figure 8 shows the effects of bispecific binding molecules IEX04-008, IEX04-010 and IEX04-012 in inhibiting VEGF-induced HUVEC cell survival and proliferation.

[0018] FIG9 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.

[0019] Figure 10 shows flow cytometric assays to determine whether bispecific binding molecules IEX04-008, IEX04-010, and IEX04-012 block the binding of Ang2-Fc to Tie2.

[0020] FIG11 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.

[0021] FIG12 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.

[0022] FIG13 shows the statistics of the proportion of laser spots of grade 4 (Panel A) and grade 3 or above (Panel B) in the laser-induced choroidal neovascularization model.

[0023] FIG14 shows the retinal thickness statistics in the laser-induced choroidal neovascularization model.

[0024] Figure 15 shows the leakage area statistics in the laser-induced choroidal neovascularization model

[0025] FIG16 shows the H&E staining of fundus tissue in the laser-induced choroidal neovascularization model (A) and the lesion area statistics (B).

[0026] FIG17 shows the CD31 staining images (A) and positive cell statistics (B) of the fundus tissue in the laser-induced choroidal neovascularization model.

[0027] definition

[0028] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methodology, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0029] To interpret this specification, the following definitions will apply, and wherever appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0030] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0031] As used herein, the term "and / or" means any one of the alternatives or two or more or all of the alternatives.

[0032] As used herein, the terms "comprising" or "including" are intended to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise indicated, the context of consisting of the stated elements, integers, or steps is also encompassed. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0033] The term "VEGF" as used herein 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 primarily in their bioavailability, with VEGF165 being the predominant isoform. In some embodiments, the VEGF A of the present invention refers to VEGF A from humans, such as VEGF 165 from humans. In one embodiment, the amino acid sequence of VEGFA of the present invention is the amino acid sequence with accession number P15692 (uniprot database).

[0034] As used herein, the term "ANG2" refers to human angiopoietin-2 (ANG-2) (alternatively abbreviated as ANGPT2 or ANG2), which is described, for example, in Maisonpierre, PC et al., Science 277 (1997) 55-60 and Cheung, AH et al., Genomics 48 (1998) 389-91. Ang1 and Ang2 are discovered as ligands for the Tie family of tyrosine kinases, which are selectively expressed in the vascular endothelium. There are currently four identified members of the angiopoietin family. Angiopoietins-3 and -4 (ANG3 and ANG4) may represent widely diverse counterparts of the same locus in mice and humans. 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, PC et al., Science 277 (1997) 55-60). All known angiopoietins primarily bind to Tie2. In some embodiments, the ANG2 of the present invention refers to human Ang2. In some embodiments, human Ang2 comprises the amino acid sequence with accession number O15123 (uniprot database).

[0035] The term "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. Thus, the molecules according to the present invention comprise specificities for at least two different antigens or targets. The molecules according to the present invention also encompass multispecific molecules comprising multiple target binding regions / binding sites, such as trispecific binding molecules. In some embodiments, the bispecific binding molecules of the present invention are bispecific antibodies.

[0036] As used herein, the term "joint" refers to any molecule that enables the different parts of a bispecific binding molecule to be directly connected. The example of a covalently linked joint between different molecular parts includes a peptide joint and a non-protein polymer, including but not limited to a copolymer of polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene or polyethylene glycol, polypropylene glycol. In some embodiments, the joint is a peptide joint, which refers to an amino acid sequence, wherein the sequence is connected to the amino acid sequence of the first part of the binding molecule to the second part of the binding molecule. For example, a peptide joint can connect the first target binding region of the binding molecule to the second target binding region. For example, a peptide joint can also connect a part of an antibody to another part of the antibody, such as connecting the light chain variable region to the heavy chain variable region. Preferably, the peptide joint has a length that is enough to connect two entities in a manner that allows them to maintain their conformation relative to each other so as not to hinder the desired activity.

[0037] The peptide linker may or may not primarily 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. Preferably, the linker is (GGGGS)n(SEQ ID NO:48), wherein n=1, 2, 3 or 4.

[0038] The term "valence" according to the present invention indicates the presence of a specified number of binding sites in a binding molecule, such as an antibody molecule. Thus, the terms bivalent, trivalent, and tetravalent denote the presence of two, three, or four binding sites (target binding regions) in a binding molecule, respectively. The bispecific binding molecules according to the present invention are at least bivalent and can be multivalent, such as bivalent, trivalent, tetravalent, or hexavalent.

[0039] As used herein, the term "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.

[0040] The term "antibody fragment" includes a portion of an intact antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0041] An "antigen-binding fragment" refers to a molecule, other than an intact antibody, that comprises a portion of an intact antibody and binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; dAb (domain antibody); linear antibodies; single-chain antibodies (e.g., scFv); single-domain antibodies such as VHH; diabodies or fragments thereof; or camelid antibodies.

[0042] "VHH," also known as a single-domain antibody (sdAb), refers to a genetically engineered antibody consisting solely of the variable region of a heavy chain antibody, containing only the three HCDRs of the heavy chain variable region. VHHs possess antigen specificity and high affinity with just three HCDRs, whereas conventional antibodies require six CDRs. Crystal structures have shown that VHHs are composed of two β-sheets forming a scaffold, similar to the traditional antibody VH immunoglobulin fold.

[0043] The term "target" refers to the object to which a binding molecule is directed. A target can be an antigen, a ligand, or a receptor.

[0044] The term "antigen" refers to a molecule that elicits an immune response. This immune response may involve the production of antibodies or the activation of specific immune cells, or both. The skilled artisan will appreciate that any macromolecule, including essentially all proteins or peptides, can serve as an antigen. In addition, antigens can be derived from recombinant or genomic DNA. As used herein, the term "epitope" refers to the portion of an antigen (e.g., VEGF or Ang2) that specifically interacts with an antibody molecule.

[0045] "Complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms structurally determined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, and are numbered sequentially starting from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of 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 antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (on the World Wide Web at imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0046] For example, according to different CDR definition schemes, the residues of each CDR are as follows.

[0047] 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).

[0048] Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above-mentioned ways.

[0049] Unless otherwise indicated, in the present invention, when referring to residue positions in the variable region of an antibody (including heavy chain variable region residues and light chain variable region residues), the numbering refers to the 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)).

[0050] In one embodiment, the CDRs in the VHH of the present invention follow the following rules: HCDR1 is defined according to AbM, and HCDR2 and HCDR3 are defined according to Kabat.

[0051] It should be noted that 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 may be different. Therefore, when referring to antibodies defined by 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).

[0052] Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs (under the same assignment system). However, although CDRs are different between antibodies, only a limited number of amino acid positions in the CDRs are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM, Contact and North methods, 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. As will be appreciated by those skilled in the art, the residues of the remainder 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 provided herein. For example, in a variant of a CDR, 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.

[0053] The term "Fc region" is used herein to define the constant regions 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 causing 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). In some embodiments, the Fc region is mutated to enhance its CDC\ADCC\ADCP effector functions. In some embodiments, the Fc region is mutated to weaken or delete its CDC\ADCC\ADCP effector functions.

[0054] "Humanized" antibody refers to an antibody comprising the amino acid residues from non-human CDR and the amino acid residues from people FR. In some embodiments, a humanized antibody will comprise substantially all of at least one, usually two variable domains, wherein all or substantially all of the CDRs (e.g., CDRs) correspond to those of non-human antibodies, and all or substantially all of the FRs correspond to those of people antibodies. Humanized antibodies optionally can comprise at least a portion of an antibody constant region derived from people antibodies. "Humanized form" of an antibody (e.g., non-human antibody) refers to a humanized antibody. "Humanized antibody" or "fully human antibody" or "fully human 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 a non-human source, and it utilizes people's antibody library or other people's antibody encoding sequences. This definition of people's antibody clearly excludes humanized antibodies comprising non-human antigen binding residues.

[0055] As used herein, the terms "anti," "binding," or "specific binding" mean that the binding is selective for the target or antigen and can be distinguished from unwanted or non-specific interactions. The ability of a binding site to bind 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).

[0056] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny screened or selected for the same function or biological activity as the initially transformed cell are included herein.

[0057] The term "label" as used herein refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and promotes the detection of the reagent to which it is conjugated or fused. The label itself can be detectable (e.g., radioisotope labeling or fluorescent labeling) or can catalyze the chemical alteration of a detectable substrate compound or composition in the case of an enzymatic label. The term is intended to encompass direct labeling of a probe or antibody by coupling a detectable substance (i.e., physical connection) to the probe or antibody and indirect labeling of the probe or antibody by reacting with another reagent of the direct labeling. In some embodiments, the label is hFc or biotin.

[0058] "Subject" includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the subject is a human.

[0059] Calculation of sequence identity between sequences is performed as follows.

[0060] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment or non-homologous sequences can be discarded for comparison purposes). In a preferred embodiment, 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 that position.

[0061] Mathematical algorithms can be used to compare sequences and calculate percent identity between two sequences. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (available at http: / / www.gcg.com), which has been integrated into the GAP program in the GCG software package. The Blossum 62 matrix or the PAM250 matrix is ​​used, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 is used, and a length weight of 1, 2, 3, 4, 5, or 6 is used. In another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http: / / www.gcg.com). 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 or nucleotide sequences can also be determined using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weighted remainder table, a gap length penalty of 12, a gap penalty of 4). Additionally or alternatively, the 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.

[0062] The "ocular diseases" of the present invention encompass ocular diseases involving angiogenesis (eg, diseases occurring in the eye), such as ocular diseases associated with corneal neovascularization.

[0063] As used herein, "treat," ...

[0064] As used herein, "prevention" includes the inhibition of the onset or development of a disease or condition or symptoms of a particular disease or condition.

[0065] The term "vector," as used herein, 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 operatively linked. Such vectors are referred to herein as "expression vectors."

[0066] "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 from fresh, frozen and / or preserved organ or tissue samples or biopsy samples or puncture samples; blood or any blood component; body fluids, such as tears, vitreous humor, cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time during the subject's pregnancy or development. In some embodiments, the tissue sample is ocular tissue, such as vitreous. In some embodiments, 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.

[0067] The antibodies used in the preparations of the present invention are also referred to as the antibodies of the present invention. For example, 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. Specific implementation plan

[0068] In one aspect, the present invention provides a liquid formulation 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 formulation is about 5.0-7.5.

[0069] In some embodiments, the liquid formulation comprises an anti-VEGF A VHH antibody.

[0070] In some embodiments, the liquid formulation comprises an anti-Ang2 VHH antibody.

[0071] In some embodiments, the liquid formulation comprises a bispecific binding molecule that binds to VEGF A and Ang2, preferably a bispecific antibody that binds to VEGF A and Ang2.

[0072] In some embodiments, the anti-VEGF A VHH comprises the following three CDRs, HCDR1, HCDR2 and HCDR3, wherein

[0073] HCDR1 comprises or consists of the sequence shown in SEQ ID NO: 1;

[0074] HCDR2 comprises or consists of the sequence shown in SEQ ID NO: 2;

[0075] HCDR3 comprises or consists of the sequence shown in SEQ ID NO: 3;

[0076] or

[0077] HCDR1 comprises or consists of the sequence shown in SEQ ID NO: 6;

[0078] HCDR2 comprises or consists of the sequence shown in SEQ ID NO: 7 or 10;

[0079] HCDR3 comprises or consists of the sequence shown in SEQ ID NO: 8.

[0080] In some embodiments, 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.

[0081] In some embodiments, 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, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.

[0082] In some preferred embodiments, the mutation is not present in a CDR, such as HCDR1, HCDR2 or HCDR3.

[0083] In some embodiments, the anti-Ang2 VHH comprises the following three CDRs, HCDR1, HCDR2, and HCDR3, wherein

[0084] HCDR1 comprises or consists of the sequence shown in SEQ ID NO: 16;

[0085] HCDR2 comprises or consists of the sequence shown in SEQ ID NO: 17 or 20;

[0086] HCDR3 comprises or consists of the sequence shown in SEQ ID NO: 18.

[0087] In some embodiments, 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.

[0088] In some embodiments, 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, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.

[0089] In some preferred embodiments, the mutation is not present in a CDR, such as HCDR1, HCDR2 or HCDR3.

[0090] In some embodiments, 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 described above.

[0091] In some embodiments, the first target binding region is selected from

[0092] VHH that specifically binds to VEGF A;

[0093] An antigen-binding fragment of an antibody that specifically binds to VEGF A, such as an scFv, e.g., the antibody is a fully human or humanized antibody; or

[0094] VEGF receptor (VEGF R) that specifically binds to VEGF A or its extracellular domain or a fusion protein comprising the extracellular domain, such as a fusion protein of the extracellular domain and Fc.

[0095] In some embodiments, the bispecific binding molecule comprises one or two or three or four first or second target binding regions. In some embodiments, the bispecific binding molecule comprises two, three or four target binding regions. In some embodiments, the bispecific binding molecule is bivalent or trivalent or tetravalent. In some embodiments, the bispecific binding molecule is a bispecific antibody.

[0096] In some embodiments, the bispecific binding molecule, such as a bispecific antibody, has the following structure:

[0097] light chain variable region VL of anti-VEGF antibody - linker - heavy chain variable region VH of anti-VEGF antibody - linker - anti-Ang2 VHH or

[0098] Heavy chain variable region VH of anti-VEGF antibody - linker - light chain variable region VL of anti-VEGF antibody - linker - anti-Ang2 VHH;

[0099] The anti-Ang2 VHH comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 16, or consists of the sequence; 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.

[0100] In some embodiments, the structure of the bispecific binding molecule is as shown in Figure 1A or Figure 1B. In some embodiments, the bispecific binding molecule is composed of one chain. In some embodiments, the bispecific binding molecule is bivalent.

[0101] In some embodiments, 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; and LCDR3 comprises or consists of the sequence shown in SEQ ID NO:33.

[0102] In some embodiments, 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.

[0103] In some embodiments, 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 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: 34, or consists of the amino acids of SEQ ID NO: 34. In some embodiments, the heavy chain variable region VH comprises an amino acid sequence that has one or more (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 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. In some preferred embodiments, the mutations are not present in a CDR, such as HCDR1, HCDR2 or HCDR3.

[0104] In some embodiments, 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 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: 30, or consists of the amino acids of SEQ ID NO: 30. In some embodiments, the light chain variable region (VL) comprises an amino acid sequence that has one or more (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 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. In some preferred embodiments, the mutations are not present in the CDRs, such as LCDR1, LCDR2, or LCDR3.

[0105] In some embodiments, 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. In some embodiments, the VHH comprises an amino acid sequence that has 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. In some preferred embodiments, the mutations are not present in a CDR, such as HCDR1, HCDR2 or HCDR3.

[0106] In some embodiments, the linker comprises or consists of the amino acid sequence of SEQ ID NO: 23. In some embodiments, the linker between the light chain variable region and the heavy chain variable region of the anti-VEGF antibody comprises or consists of the amino acid sequence of SEQ ID NO: 23, where, for example, n = 4. In some embodiments, the linker between the anti-VEGF variable region and the anti-Ang2 VHH comprises or consists of the amino acid sequence of SEQ ID NO: 23, where, for example, n = 2 or 3, such as 3.

[0107] In some embodiments, the anti-VEGF A×ANG2 bispecific binding molecule comprises the amino acid sequence of SEQ ID NO: 28, 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: 28, or consists of the amino acids of SEQ ID NO: 28. In some embodiments, the bispecific binding molecule comprises an amino acid sequence that has one or more (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 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. In some preferred embodiments, the mutations are not present in the variable regions of the anti-VEGF antibody and the CDRs of the anti-Ang2 VHH.

[0108] In some embodiments, the bispecific binding molecule, such as a bispecific antibody, has the following structure:

[0109] First anti-VEGF VHH-linker-second anti-VEGF VHH-linker-anti-Ang2 VHH,

[0110] The anti-Ang2 VHH comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 16, or consists of the sequence; 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.

[0111] In some embodiments, the structure of the bispecific binding molecule is as shown in Figure 1C. In some embodiments, the bispecific binding molecule is composed of a single chain. In some embodiments, the bispecific binding molecule is trivalent. In some embodiments, the first anti-VEGF VHH and the second anti-VEGF VHH are the same or different.

[0112] In some embodiments, the anti-VEGF VHH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises or consists of the sequence set forth in SEQ ID NO: 1; HCDR2 comprises or consists of the sequence set forth in SEQ ID NO: 2; and HCDR3 comprises or consists of the sequence set forth in SEQ ID NO: 3.

[0113] or

[0114] 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.

[0115] In some embodiments, 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 that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence as set forth in SEQ ID NO: 4, 5, 9 or 11, or consists of amino acids as set forth in SEQ ID NO: 4, 5, 9 or 11. In some embodiments, the VHH comprises an amino acid sequence that has one or more (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions, compared to the amino acid sequence as set forth in SEQ ID NO: 4, 5, 9 or 11. In some preferred embodiments, the mutations are not present in a CDR, such as HCDR1, HCDR2 or HCDR3.

[0116] In some embodiments, the anti-VEGF VHH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises or consists of the sequence set forth in SEQ ID NO: 6; HCDR2 comprises or consists of the sequence set forth in SEQ ID NO: 7 or 10; and HCDR3 comprises or consists of the sequence set forth in SEQ ID NO: 8.

[0117] In some embodiments, the anti-VEGF VHH comprises the amino acid sequence of SEQ ID NO: 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: 9 or 11, or consists of the amino acids of SEQ ID NO: 9 or 11. In some embodiments, the VHH comprises an amino acid sequence that has one or more (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions, compared to the amino acid sequence of SEQ ID NO: 9 or 11. In some preferred embodiments, the mutations are not present in a CDR, such as HCDR1, HCDR2 or HCDR3.

[0118] In some embodiments, 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. In some embodiments, the VHH comprises an amino acid sequence that has 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. In some preferred embodiments, the mutations are not present in a CDR, such as HCDR1, HCDR2 or HCDR3.

[0119] In some embodiments, the linker comprises or consists of the amino acid sequence of SEQ ID NO: 23. In some embodiments, the linker between the first anti-VEGF VHH and the second anti-VEGF VHH or the linker between the second anti-VEGF VHH and the anti-Ang2 VHH comprises or consists of the amino acid sequence of SEQ ID NO: 23, e.g., n=2.

[0120] In some embodiments, the anti-VEGF A×ANG2 bispecific binding molecule comprises the amino acid sequence of SEQ ID NO: 22, 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: 22, or consists of the amino acids of SEQ ID NO: 22. In some embodiments, the bispecific binding molecule comprises an amino acid sequence that has one or more (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 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. In some preferred embodiments, the mutations are not present in the CDRs of the anti-VEGF VHH and anti-Ang2 VHH.

[0121] In some embodiments, the bispecific binding molecule comprises one or two of the following chains:

[0122] VEGF R extracellular domain-Fc-linker-anti-Ang2 VHH

[0123] The anti-Ang2 VHH comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 16, or consists of the sequence; 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.

[0124] In some embodiments, the structure of the bispecific binding molecule is shown in Figure ID. In some embodiments, the bispecific binding molecule is composed of two chains. In some embodiments, the bispecific binding molecule is tetravalent.

[0125] In some embodiments, 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. In some embodiments, the VHH comprises an amino acid sequence that has 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. In some preferred embodiments, the mutations are not present in a CDR, such as HCDR1, HCDR2 or HCDR3.

[0126] In some embodiments, the VEGFR extracellular domain is an extracellular domain of VEGFR from a human. In some embodiments, the VEGFR extracellular domain comprises a VEGFR1 second antibody-like domain (e.g., FLT1 domain 2) and a VEGFR2 third antibody-like domain (e.g., KDR domain 3). In some embodiments, the VEGFR extracellular domain comprises a human VEGFR1 second antibody-like domain and a human VEGFR2 third antibody-like domain. In some embodiments, 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 to the amino acid sequence of SEQ ID NO: 26, or consists of the amino acids described in SEQ ID NO: 26. In some embodiments, 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 that of the domain shown in SEQ ID NO: 26 (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0127] In some embodiments, 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. In some embodiments, 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 to the amino acid sequence of SEQ ID NO: 27, or consists of the amino acids of SEQ ID NO: 27.

[0128] In some embodiments, the VEGFR extracellular domain-Fc is a fusion protein of the VEGFR extracellular domain and Fc, such as Aflibercept or a derivative thereof.

[0129] In some embodiments, 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 consists of the amino acids of SEQ ID NO:25. In some embodiments, 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, or 1) mutations compared to the amino acid sequence shown in SEQ ID NO: 25, such as 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 that of the domain shown in SEQ ID NO: 25 (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%).

[0130] In some embodiments, the linker comprises or consists of the amino acid sequence of SEQ ID NO: 23, eg, n=3.

[0131] In some embodiments, 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 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: 24, or consists of the amino acids of SEQ ID NO: 24. In some embodiments, the bispecific binding molecule comprises an amino acid sequence that has one or more (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 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. In some preferred embodiments, the mutations are not present in the CDRs of the anti-Ang2 VHH. In some embodiments, the mutated VEGF R extracellular domain-Fc retains a binding affinity for VEGF similar to that of the domain shown in SEQ ID NO: 25 (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0132] In one embodiment of the invention, the antibodies or binding molecules described herein comprise one or more amino acid mutations. In some embodiments, the amino acid mutations comprise amino acid substitutions, insertions, or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

[0133] In a preferred embodiment, the amino acid mutations described herein occur in regions outside of the CDRs (e.g., in the FRs). In some embodiments, the amino acid mutations described herein occur in the constant region of the heavy chain of an antibody, such as the Fc region. In a preferred embodiment, the amino acid mutations in the Fc region weaken or delete the ADCC and / or CDC effects of the antibody.

[0134] In some embodiments, 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.

[0135] In certain embodiments, 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 cytotoxicity, Fc receptor binding, and / or antibody-dependent cellular cytotoxicity. The Fc region variant can include a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid mutation (e.g., substitution) at one or more amino acid positions.

[0136] In certain embodiments, it may be desirable to mutate the variable region of an antibody to generate disulfide bonds, eg, to generate a scFv comprising a disulfide bond mutation.

[0137] In certain embodiments, the antibodies or binding molecules provided herein may be further modified to contain other non-proteinaceous moieties known in the art and readily available. Suitable moieties for the derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, 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), polyvinyl alcohol, and mixtures thereof.

[0138] In some embodiments, the buffer is selected from one or more of histidine, histidine salts, glutamate, phosphate, acetate, citrate, and tris. The histidine salts and glutamate salts include, but are not limited to, salts formed by the amino acids with hydrochloric acid or sulfuric acid. The phosphates, acetates, and citrates include, but are not limited to, alkali metal salts of the corresponding acids, such as lithium, sodium, or potassium salts. For example, the phosphate may 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. Preferably, the buffer is selected from a combination of histidine and histidine hydrochloride, a phosphate, and histidine.

[0139] In some embodiments, the stabilizer is selected from one or more of saccharides, polyols, amino acids or their salts. Preferably, the saccharides are selected from but not limited to sucrose, dextrose, lactose, maltose, trehalose, cyclodextrin, maltodextrin and dextran; the polyols are selected from but not limited to mannitol, sorbitol and xylitol; the amino acids or their salts are selected from one or more of the following: arginine, glycine, proline, methionine and their salts; the cyclodextrin is preferably hydroxypropyl-β-cyclodextrin.

[0140] In some embodiments, 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-β-cyclodextrin.

[0141] In some embodiments, 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. In some preferred embodiments, the liquid formulation of the present invention comprises polysorbate-80 as a surfactant.

[0142] In some embodiments, the concentration of the antibody or bispecific binding molecule in the liquid formulation of the present invention is about 1-200 mg / ml, for example, 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, about 30-100 mg / ml, about 30-100 mg / ml, about 30-100 mg / ml, about 30-100 mg / ml, about 30-100 mg / ml, about 30-150 mg / ml, about 30-170 mg / ml, about 30-100 mg / ml, about 30-100 mg / ml, about 30-100 mg / ml, about 30-100 mg / ml, about 30-15 ...50 mg / ml, about 30-150 mg / ml, about 30-100 mg / ml, about g / ml, 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 mg / ml, preferably about 10-160 mg / ml, more preferably about 30-150 mg / mL.

[0143] In some embodiments, 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, 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 mM, preferably about 5-50 mM, more preferably about 5-30 mM. When the buffer is a multi-component buffer, 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 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90 or about 100 mM.

[0144] In some embodiments, the concentration of the stabilizer in the liquid formulation of the present invention is about 1-1000 mM, about 10-1000 mM, 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 25 0-280 mM, about 200-400 mM or about 100-200 mM, 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 1000 mM; preferably about 50-800 mM, more preferably about 50-500 mM. When the stabilizer is a plurality of components, the concentration of each component can be selected from the following ranges or values: 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 2 ...400mM, about 100-300mM, about 200-350mM, about 200-300mM, about 250-280mM, about 200-400mM, about 100-300mM, about 100-300mM, about 100-300mM, about 100-300 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.

[0145] In some embodiments, 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.

[0146] In some embodiments, 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, more preferably about 6.3-6.8.

[0147] In some embodiments, 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.

[0148] In some embodiments, the liquid preparation is a pharmaceutical preparation, preferably an injection, more preferably an intravitreal injection, or eye drops.

[0149] In some embodiments, the liquid formulations of the present invention comprise:

[0150] (i) about 10-160 mg / ml, preferably 30-150 mg / ml, of the bispecific binding molecule;

[0151] (ii) about 5-20 mM buffer, wherein the buffer is histidine or a combination of histidine and histidine hydrochloride;

[0152] (iii) about 200-350 mM trehalose; and

[0153] (iv) about 0.1-1 mg / ml polysorbate 80;

[0154] The pH of the liquid preparation is about 6.1-7.0, preferably about 6.5.

[0155] In some embodiments, the liquid formulations of the present invention comprise:

[0156] (i) about 10-160 mg / ml, preferably 30-150 mg / ml, of the bispecific binding molecule;

[0157] (ii) about 8-12 mM histidine;

[0158] (iii) about 200-300 mM trehalose; and

[0159] (iv) about 0.2-0.4 mg / ml polysorbate 80;

[0160] The pH of the liquid preparation is about 6.1-6.6, preferably about 6.5.

[0161] In some embodiments, the liquid formulations of the present invention comprise:

[0162] (i) about 10-160 mg / ml, preferably 30-150 mg / ml, of the bispecific binding molecule;

[0163] (ii) about 1-3 mM histidine and about 7-9 mM histidine hydrochloride;

[0164] (iii) about 250-280 mM trehalose; and

[0165] (iv) about 0.2-0.4 mg / ml polysorbate 80;

[0166] The pH of the liquid preparation is about 6.1-6.6, preferably about 6.5.

[0167] In some embodiments, the liquid formulations of the present invention are the formulations disclosed in Examples 15 to 17. In addition, based on the formulations disclosed in Examples 15 to 17, embodiments resulting from fluctuations in the concentration of the components in these formulations by 50%, 40%, 30%, 20%, 10% or 5%, and fluctuations in the pH value by 5%, 4%, 3%, 2%, 1% or 0.5% are also included in the present application.

[0168] The antibody liquid formulation of the present invention may or may not contain other excipients. For example, the antibody liquid formulation of the present invention may further contain a tonicity adjuster. The tonicity adjuster may be selected from the group consisting of sodium acetate, sodium lactate, sodium chloride, potassium chloride, and calcium chloride.

[0169] These and further known pharmaceutical excipients and / or additives suitable for use in the formulations of the present invention are well known in the art and are listed, for example, in "The Handbook of Pharmaceutical Excipients, 4th edition, Rowe et al., ed., American Pharmaceuticals Association (2003); and Remington: the Science and Practice of Pharmacy, 21st edition, Gennaro, ed., Lippincott Williams & Wilkins (2005)".

[0170] It should be understood that, unless otherwise specified, 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, double distilled water, etc. The liquid preparation of the present invention can be prepared or formulated using pharmaceutically acceptable solvents or solutions known in the art. The pharmaceutically acceptable solvents or solutions include but are not limited to, for example, purified water such as ultrapure water, water for injection, sterile water, double distilled water, normal saline, Ringer's solution, glucose injection, etc.

[0171] In some embodiments, the anti-Ang2 VHH antibody is capable of specifically binding to Ang2, such as human Ang2, eg, with high affinity.

[0172] In some embodiments, the anti-VEGFA VHH antibody is capable of specifically binding to VEGF A, such as human VEGF A, e.g., with high affinity.

[0173] In some embodiments, the bispecific binding molecule is capable of specifically binding to Ang2 and VEGFA, eg, human Ang2 and human VEGFA, eg, with high affinity.

[0174] In some embodiments, the anti-Ang2 antibody or anti-VEGFA antibody or bispecific binding molecule has one or more of the following properties:

[0175] (i) the anti-Ang2 antibody or bispecific binding molecule has an inhibitory effect on Ang2-induced Tie2 phosphorylation;

[0176] (ii) the anti-Ang2 antibody or bispecific binding molecule has a blocking effect on the binding between Ang2 and Tie2;

[0177] (iii) the anti-VEGFA antibody or bispecific binding molecule has a blocking effect on VEGFA-activated receptor signaling pathways, for example, as detected by KDR reporter assay;

[0178] (iv) the anti-VEGFA antibody or bispecific binding molecule has a blocking effect on the binding between VEGFA and VEGFR;

[0179] (v) 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 HUVECs) induced by VEGF A;

[0180] (vi) the bispecific binding molecule has an inhibitory effect on vascular endothelial cell (eg, human umbilical vein endothelial cell, such as HUVEC) leakage;

[0181] (vii) The bispecific binding molecule has an inhibitory effect on neovascularization in vivo or in vitro, such as inhibiting fundus or retinal choroidal neovascularization, such as inhibiting leakage caused by neovascularization, such as protecting vascular integrity.

[0182] On the other hand, 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. In a preferred embodiment, 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. In one embodiment, 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.

[0183] The preparation of the present invention (including solid preparation and liquid preparation) can be stored stably for a long time, for example, at least 24 months or longer. In one embodiment, the preparation of the present invention can be stored at about -80 ℃ to about 45 ℃, for example, -80 ℃, about -30 ℃, about -20 ℃, about 0 ℃, about 5 ℃, about 25 ℃, about 35 ℃, about 38 ℃, about 40 ℃, about 42 ℃ or about 45 ℃, 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, and is stable.

[0184] In one embodiment, after storage, the stability of the liquid formulation of the present invention is checked by visual inspection, wherein the liquid formulation 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, no visible foreign matter is present in the formulation under visual inspection with a clarity detector. In one embodiment, after storage, the stability of the liquid formulation 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 on storage day 0 is no more than 20%, preferably no more than 10%, such as 7-8%, more preferably no more than 5%. In one embodiment, after storage, the stability of the liquid formulation of the present invention is checked by measuring the change in purity of the liquid formulation of the present invention, wherein, by size exclusion high performance liquid chromatography (SEC-HPLC), the change in monomer purity relative to the initial value on 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%. In one embodiment, after storage, the stability of the liquid formulation of the present invention is checked by measuring the change in purity of the liquid formulation of the present invention, wherein the change in monomer purity by non-reduced and / or reduced sodium dodecyl sulfate capillary electrophoresis (CE-SDS) method does not exceed 10%, for example, not more than 5%, 4%, 3%, 2% or 1%. In one embodiment, after storage, the stability of the liquid formulation of the present invention is tested by imaging capillary isoelectric focusing (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%, for example, not more than 20%, not more than 10%, not more than 5%, not more than 2% or not more than 1%.

[0185] In another aspect, the present invention provides a delivery device comprising a liquid or solid formulation of the present invention. In one embodiment, the delivery device of the present invention is provided in the form of a prefilled syringe comprising a liquid or solid formulation of the present invention, for example, for intravitreal injection, intravenous, subcutaneous, intradermal or intramuscular injection, or intravenous infusion.

[0186] In another aspect, the present invention provides a method for preparing the liquid formulation according to the present invention, comprising the following steps:

[0187] i. Providing an isolated and purified antibody or bispecific binding molecule of the present invention, and optionally adding it to an ultrafiltration centrifuge tube for centrifugation and concentration;

[0188] ii. 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 above in the liquid formulation of the present invention,

[0189] iii. ultrafiltration of the antibody from step i into the solution from step ii,

[0190] iv. adjusting the concentration of the replaced protein to the concentration defined in the liquid formulation of the present invention;

[0191] v. adding 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;

[0192] vi. optionally sterile filtering the solution of step v; and

[0193] vii. Optionally, the mixture is dispensed into vials, and capped with rubber stoppers and aluminum-plastic caps to obtain finished products.

[0194] In another aspect, the present invention provides a method for preventing or treating an ocular disease in a subject, comprising administering to the subject a formulation of the present invention.

[0195] In some embodiments, the patient has (eg, elevated levels, eg, nucleic acid or protein levels, of) VEGF, eg, VEGF A, and / or Ang2.

[0196] In some embodiments, the ocular disease includes, but is not limited to, an ocular disease associated with angiogenesis, such as an ocular disease associated with corneal neovascularization.

[0197] In some embodiments, the ocular disease treatment would benefit from inhibition of nucleic acid or protein levels of VEGF, such as VEGF A, and / or Ang2.

[0198] In other aspects, the present 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 the relevant diseases or conditions mentioned herein.

[0199] In some embodiments, the formulations of the invention delay the onset of a disorder and / or symptoms associated with the disorder.

[0200] In some embodiments, the formulations of the present 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.

[0201] The 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.

[0202] These and other aspects and embodiments of the present invention are described in the accompanying drawings (a brief description of the drawings follows) and the following detailed description of the invention and are exemplified in the following examples. Any or all of the features discussed above and throughout this application may be combined in various embodiments of the present invention. The following examples further illustrate the present invention, however, it should be understood that the examples are described in an illustrative and non-limiting manner, and that various modifications may be made by those skilled in the art.

[0203] Example 1. Preparation of phage immune library

[0204] Alpaca immune or synthetic library construction

[0205] 1.1 Two healthy adult alpacas (Apac, Chengdu) were selected and 0.5 mg of recombinant protein antigen VEGFA or Ang2 (Sino-Qiao, Beijing) was mixed with Freund's adjuvant in a 1:1 ratio. The alpacas were immunized by multiple subcutaneous injections at the back for a total of four immunizations, with an interval of 2 weeks between immunizations.

[0206] 1.2 Collect 50 ml of alpaca peripheral blood and separate lymphocytes at a rate of 2.5×10 7 1 mL of Trizol reagent was added to each viable cell, and total RNA was extracted using chloroform / isopropanol precipitation. 10 μg of RNA was used as a template for reverse transcription using the PrimeScript Reverse Transcription Kit (Takara). The cDNA was used as a template for a first-round PCR reaction using the forward primer Alp-VhL and the reverse primer Alp-2b / 2cR to generate the first-round PCR product. The first-round PCR product was used as a template for a second-round PCR reaction using the forward primer Alp-VhF and the reverse primer Alp-JHR-SalI to generate the second-round PCR product. The pC3-HF vector and the second-round PCR product were double-digested with SacI and SalI (Thermo Fisher Scientific), respectively. The digested products were then added to T4 ligase (Thermo Fisher Scientific) and electroporated into TG1 competent cells to construct the VHH antibody library. The bacterial culture was frozen at −80°C.

[0207] The revived bacterial suspension was inoculated into 100 ml of YT-AG medium (Shanghai Sangon Co., Ltd.), and infection was performed with M13KO7 helper phage. The cells were resuspended in 2× YT-AK medium (Shanghai Sangon Co., Ltd.) and incubated overnight at 37°C and 200 rpm. The culture supernatant was collected and recombinant phage was prepared by PEG / NaCl precipitation.

[0208] 1.3 The recombinant phage was subjected to three rounds of panning using the biotinylated antigen VEGFA (ACRO) or Ang2 (Beijing Yiqiao). 50ul of M280 magnetic beads (Thermo) and an appropriate amount of biotinylated antigen were added to each tube. After incubation at room temperature for 30 minutes, 1x10 12 The cfu recombinant phage were incubated at room temperature for 1 hour. The resulting mixture was washed 10 times with 1 ml of PBST, each wash lasting 5 minutes. Finally, 0.5 ml of pH 2.5 glycine buffer was added to elute the antigen-bound recombinant phage, which was then used to infect an overnight TG1 culture to prepare recombinant phage for the next round of panning and identification of positive VHH-positive TG1 bacterial clones.

[0209] 1.4 Binding ELISA assay and clone sequencing.

[0210] VEGF A and Ang2 antigens (Beijing Yiqiao Company) were diluted in PBS buffer to 0.5 μg / ml and coated on a 96-well ELISA plate. Refrigerate overnight at 4°C. Wash the antigen-coated plate three times with PBST, add 300 μl of blocking agent per well, and incubate at room temperature for 1 hour. Wash three times with PBST, then add 80 μl of blocking agent plus 20 μl of the expression supernatant from the TG1 strain expressing the positive VHH identified in 1.3 above, and shake at room temperature for 1 hour.

[0211] Wash three times with PBST, add 100 μl / well of Anti-Flag / HRP secondary antibody (Sigma) diluted in blocking agent, and shake at room temperature for 40 minutes. Wash six times with PBST, add TMB colorimetric solution to 100 μl / well, and develop in the dark for 5-15 minutes. Then add 100 μl / well of stop solution. Read the plate using a microplate reader and measure the absorbance at OD450 nm. Select bacterial clones with a reading greater than 0.5 and send them to Jinweizhi for sequencing. Select TG1 bacterial monoclonal clones containing each corresponding VHH sequence, add glycerol, and freeze in a -80°C freezer.

[0212] Example 2. Production and purification of prokaryotic antibodies, and humanization

[0213] The present invention utilizes molecular biological techniques 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 proteins.

[0214] 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 to an OD600 value of 0.5-0.6. Add 1mM IPTG to the bacterial solution to induce expression, culture it overnight at 28°C 200rpm, 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. Pass the bacterial lysate through a 1ml Ni Sepharose pre-column, add PBS and wash twice, add 0.5M imidazole to elute the target protein, and determine the protein concentration by ultraviolet method. The eluted target protein is determined by ultraviolet method for protein concentration, and the divided tubes are placed in a -40 degree refrigerator for storage. The obtained antibody solution is subsequently referred to as the supernatant.

[0215] The CDRs, VHH amino acid sequences, and sequence numbers of the two anti-VEGF A VHH antibodies (LA42F8 and LA46E11) and one anti-Ang2 VHH antibody (LA24C11) obtained in the present invention are shown in the sequence listing.

[0216] The immune library antibodies LA42F8, LA46E11, and LA24C11 obtained above were then humanized in the following steps:

[0217] ① Determine the CDR loop structure;

[0218] ② Find the closest homologous sequence for each V / J region of the heavy chain in the human germline sequence database;

[0219] ③ Screening for the human germline that best matches the heavy and light chains and the lowest amount of back mutations;

[0220] ④ Constructing the CDR region of the chimeric antibody onto the human framework region;

[0221] ⑤ Using sequence and structural features, determine the amino acid positions in the framework region that maintain CDR function;

[0222] ⑥ Perform back mutation at the sequence position determined to be important (return to the input amino acid type);

[0223] ⑦Optimize amino acids at risk sites.

[0224] The three humanized antibodies obtained in the present invention are humanized VHH antibodies LA42F8.5, LA46E11.8, and LA24C11.10. Please refer to the attached sequence table for the CDRs and amino acid sequences of the heavy chain variable regions.

[0225] The expression of the above-mentioned LA42F8, LA46E11, LA24C11 and humanized antibodies LA42F8.5, LA46E11.8, LA24C11.10 in eukaryotic cells was prepared as follows:

[0226] The humanized antibody sequences obtained above were cloned into pcDNA3.1 (Invitrogen) to obtain plasmids containing the antibody sequences.

[0227] 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 cell density on the day of transfection was approximately 3×10 6 cells / ml. Take 1 / 10 of the final volume of F17 medium (Gibco, A13835-01) as the transfection buffer, add the appropriate plasmid and mix well. Add appropriate polyethyleneimine (PEI) (Polysciences, 23966) to the plasmid (the ratio of plasmid to PEI in 293F cells is 1:3), mix well and incubate at room temperature for 10 minutes to obtain a DNA / PEI mixture. After resuspending the cells with the DNA / PEI mixture, incubate at 36.5°C and 8% CO2. After 24 hours, add 2% of the transfection volume of FEED (Sigma) and culture at 36.5°C, 120rpm, and 8% CO2. Culture continuously until the 6th day or when the cell viability is ≤60%, collect the cell supernatant for purification.

[0228] The gravity column used for purification was treated with 0.5M NaOH overnight. The glass bottles were rinsed with distilled water and then dry-baked at 180°C for 4 hours to obtain the purification column. Prior to purification, the collected cell supernatant was centrifuged at 4500 rpm for 30 minutes and the cells discarded. The supernatant was then filtered through a 0.22 μl filter. A Protein A column (Hitrap MabSelect Sure 5*5ml, GE, 11-0034-95) was equilibrated with 10 ml of binding buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.0). The filtered supernatant was applied to the purification column and re-equilibrated with 15 ml of binding buffer. 5 ml of elution buffer (0.1 M citric acid + sodium citrate, pH 3.5) was added, and the eluate was collected. 80 μl of Tris-HCl was added per 1 ml of eluate. The collected antibody was concentrated by ultrafiltration and exchanged into PBS (Gibco, 70011-044) for concentration determination. Except for the antibodies of the present invention used for detection in Table 3, Figure 2, Figure 4A and Figure 5A, all antibodies used in the examples are expressed and purified antibodies unless otherwise specified for the supernatant.

[0229] Similarly, the coding nucleic acids of negative control IgG, positive control BI-anti-VEGF, BI836880, and Faricimab (sequences are shown in the sequence listing) were cloned into pcDNA3.1, transfected into Expi-293 cells, expressed, and purified using the same method as in Example 2.

[0230] Example 3 Determination of the binding kinetics between the chimeric antibody of the present invention and the antigen using thin-layer biofilm interferometry

[0231] The equilibrium dissociation constant (KD) of the antibody of the present invention binding to human Ang2 was determined using thin-layer biofilm interferometry (ForteBio). ForteBio affinity determination was performed according to existing methods (Estep, P et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013. 5(2): p. 270-8).

[0232] Half an hour before the start of the experiment, according to the number of samples, an appropriate number of AMQ (Pall, 1506091) (for sample detection) or AHQ (Pall, 1502051) (for positive control detection) sensors were taken and immersed in SD buffer (PBS 1×, BSA 0.1%, Tween-20 0.05%).

[0233] Take 100 μl of SD buffer, the VHH antibody prepared in Example 2 above, and antigens [including human Ang2 (Beijing Yiqiao), and human VEGF165 (R&D)] and add them to a 96-well black polystyrene half-volume microplate (Greiner, 675076). Arrange the plate according to the sample position and select the sensor position. The instrument setting parameters are as follows: running steps: Baseline, Loading ~ 1nm, Baseline, Association and Dissociation; the running time of each step depends on the sample binding and dissociation speed, the rotation speed is 400 rpm, and the temperature is 30 ° C. Use ForteBio analysis software to analyze K D value.

[0234] In the experiments described in the above assays, the affinities of the antibodies are shown in Table 1:

[0235] Table 1. Affinity constants (equilibrium dissociation constants) of monovalent antigen-antibody binding detected by ForteBio

[0236] ND means not detected

[0237] Table 2. Affinity constants (equilibrium dissociation constants) of antigen-antibody bivalent binding detected by ForteBio

[0238] * Indicates that the dissociation constant exceeds the detection limit of ForteBio

[0239] Example 4 Anti-VEGF A VHH Antibody ELISA Blocking Experiment

[0240] This example demonstrates 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 Product No. 21125) was diluted to 1 μg / ml, and 100 μl / well was plated on an ELISA plate overnight at 4°C. The plate was washed three times with PBST and blocked with 3% BSA for 1.5 hours. The plate was washed three times with PBST, and 50 ng / ml of biotinylated VEGF A165 (ACRO Product No. VE5-H8210) was added and incubated for 1.5 hours. The supernatant of LA42F8 and LA46E11 prepared in Example 2, as well as negative control IgG and 50 μl of the positive control BI-anti-VEGF (starting at 150 μg / ml, serially diluted three times) were incubated with VEGFR-Fc (Beijing Sino-Bio, Product No. 10012-H02H, final concentration 0.2 μg / ml) for 20 minutes before addition to the plate. Wash three times with PBST, add anti-human Fc HRP antibody (Bethyl Catalog No.: A80-104P) (1:10000) and incubate for 30 minutes. Wash six times with PBST, develop with TMB for 5 minutes, and read the OD at 450 nm after termination.

[0241] The blocking results of the three anti-VEGF VHH antibodies obtained in the present invention are shown in Figure 2. Figure 2 shows that the candidate molecules LA42F8 and LA46E11 antibodies can completely block the binding of VEGF A to VEGFR2.

[0242] Example 5 Anti-Ang2 VHH Antibody ELISA Blocking Experiment

[0243] This example demonstrates the blocking effect of the anti-Ang2 VHH antibody of the present invention on the binding of hAng2-biotin (R&D Catalog No.: BT623B / CF) to Tie2 protein.

[0244] Tie2-Fc (Beijing Sino-Bio Products, Cat. No. 10700-H03H) was diluted to 2 μg / ml and 100 μl / well was plated on an ELISA plate overnight at 4°C. The plate was washed three times with PBST and blocked with 3% BSA for 1.5 hours. The plate was washed three times with PBST. 50 μl each of the purified LA24C11.10 and negative control IgG prepared in Example 2 were incubated with hAng2-biotin (final concentration 0.2 μg / ml) for 20 minutes before addition to the plate. The plate was washed three times with PBST and Avidin HRP (1:2000) was added and incubated for 35 minutes. The plate was washed six times with PBST and developed with TMB for 5 minutes. After termination, the OD at 450 nm was read.

[0245] 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.

[0246] Table 3. Purified antibody single-point blocking ELISA

[0247] 1 The antibody refers to the purified prokaryotic supernatant prepared in Example 2, with an initial concentration of 0.556 mg / ml and subsequent dilutions of 1:10 or 1:100;

[0248] 2 The supernatant refers to the unpurified prokaryotic supernatant.

[0249] Similar to Example 4 (using Ang2-Bio (R&D Catalog No.: BT623B / CF)), the effect of LA24C11.10 in blocking the binding of Ang2 to Tie2 was tested by ELISA blocking assay, and the results are shown in Figure 3. It can be seen that LA24C11.10 has a blocking effect on the binding of Ang2 to the receptor Tie2.

[0250] Example 6 Phosphorylation Experiment of Anti-Ang2 VHH Antibody

[0251] This example verifies the inhibitory effect of the anti-Ang2 VHH antibody of the present invention on hAng2-Fc-induced Tie2 phosphorylation.

[0252] hAng2-induced phosphorylation assay

[0253] In this study, Expi293 cells 293-Tie2, which overexpress Tie2, were co-incubated with antibodies and recombinant hAng2-Fc protein. The phosphorylated Tie2 content in the system was detected to reflect the inhibitory effect of different antibodies on hAng2-fc-induced Tie2 phosphorylation.

[0254] Expi-293 cells (293-Tie2) overexpressing human Tie2 were generated by transfecting Expi-293 cells (Thermo) with the pCHO1.0 vector (Invitrogen) carrying the human Tie2 gene (Beijing Sino-Qio Catalog No. HG10700-M) cloned into the multiple cloning site MCS.

[0255] Take 293-Tie2 cells expressing human Tie2 and dilute to 2*10 6 cells / ml, 100ul per well was added to a 96-well plate, centrifuged at 400g for 5min, and the supernatant was removed.

[0256] Experimental culture medium was prepared using Expi293 medium (Thermo catalog number A1435102): test antibodies (LA24C11 (24C11) and LA24C11.0 (hz24C11.10) prepared in Example 2, as well as negative control IgG, and positive control Nesvacumab (prepared according to CN202010573625.2)) were added at an initial concentration of 60 μg / ml and diluted in a 1:2 ratio; hAng2-Fc (Beijing Yiqiao: 10691-H02H) was added to a final concentration of 2.5 μg / ml.

[0257] Resuspend the cells in 100 μl of experimental culture medium per well and incubate at 37°C for 15 min.

[0258] The culture medium was removed by centrifugation, and 100 μl of NP-40 lysis buffer (Biyuntian, catalog number: P0013F) containing 1% protease (Thermo catalog number: 78442) and phosphatase inhibitor (Thermo catalog number: 78442) was added. The mixture was placed on ice for 30 min and centrifuged at 2000 g. The protein supernatant was collected and stored in a -80 degree refrigerator.

[0259] Detect pTie2 concentrations according to the instructions of the Phospho-Tie2 ELISA Kit (R&D Catalog No. DYC2720E). Coat the plate with the capture antibody provided in the kit at a concentration of 4 μg / ml overnight at 4°C. Wash three times with PBST and block with 5% BSA for 1 hour. Add 100 μl of the freeze-thawed protein supernatant from the previous step and a control pTie2 (R&D Catalog No. DYC2720E) for creating a standard curve and incubate at room temperature for 2 hours. (If the pTie2 concentration in the sample is too high, exceeding the detection range of the ELISA, dilute the resulting mixture 2-3 times.) Wash three times with PBST, then add 100 μl of HRP-conjugated anti-pTyr antibody (R&D Catalog No. DYC2720E) and incubate at room temperature for 2 hours. The cells were washed six times with PBST and developed with 100 μl of TMB (Solarbio Catalog No. PR1200). After 15 minutes, the reaction was terminated with 100 μl of stop buffer (Solarbio Catalog No. C1058). OD450-OD620 values ​​were measured using a SpectraMax i3 multi-function microplate reader. The experimental results are shown in Figure 4A (24C11 represents the purified supernatant of LA24C11 prokaryotic expression prepared in Example 2) and Figure 4B.

[0260] Therefore, 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.

[0261] Example 7 Anti-VEGF A VHH Antibody KDR Reporter Blocking Experiment

[0262] VEGF A can bind to the related receptor VEGFR2 (KDR), activate the VEGFR2 signaling pathway, and induce vascular endothelial cell survival, proliferation, and migration. In this study, the KDR reporter experimental system was used to detect the blocking effect of serially diluted antibodies on the VEGFA-activated related receptor signaling pathway using NFAT-RE-luc2P / KDR HEK293 cells (Promega Cat CS181401).

[0263] The experimental method refers to the supplier (Promega):

[0264] Remove the NFAT-RE-luc2P / KDR HEK293 cells that had been replaced with experimental culture medium (DMEM culture medium containing 10% FBS) 3 days in advance, remove the old culture medium, wash once with PBS, and then digest the cells with 1 ml of Accutase solution (Sigma product number: A6964-500ML) until the cells become round and detach from the wall. Terminate the reaction with 5 ml of dilution culture medium, pipette the cells into a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, discard the culture medium, add 10 ml of dilution culture medium (DMEM culture medium containing 10% FBS) to resuspend the cells, mix well and count, the cell viability should be above 90%. Adjust the cell density to 0.8×10 with dilution culture medium. 6 Cells / ml, 50 μl / well were added into 96-well white cell culture plates according to the experimental layout.

[0265] Prepare a mixture of 100 ng / ml VEGF A and serially diluted test antibodies, let it sit for 30 minutes, then add 50 μl / well of the mixture to a 96-well white cell culture plate containing cells and incubate in a 37°C, 5% CO2 incubator for 6 hours. The test samples are as follows: negative control IgG; positive controls BI-anti-VEGF; LA42F8; LA46E11; LA42F8.5; LA46E11.8; Blank (contains only dilution medium without VEGF A or antibody); VEGF A 100 ng / ml (contains only 100 ng / ml VEGF A).

[0266] Remove the 96-well white cell culture plate from the CO2 incubator after incubation for 6 hours and equilibrate to room temperature for 10-15 minutes. Add 100 μl / well of the Bio-Glo Luciferase Assay System, which had been previously equilibrated to room temperature, to the 96-well white cell culture plate according to the experimental layout. Incubate at room temperature in the dark for 5 minutes.

[0267] Use a multifunctional microplate reader to read the fluorescence value. Select the chemiluminescence mode for plate reading mode, the endpoint method for plate reading type, and the wavelength as full wavelength. Collect fluorescence column by column, and the collection time for each column is 1000ms.

[0268] In the experiments described in the above assay, the test results are shown in FIG5 , and the anti-VEGF VHH antibodies LA42F8, LA42F8.5, LA46E11, and LA46E11.8 can all block VEGF A-induced activation of the KDR signaling pathway.

[0269] Example 8 Anti-VEGF A VHH inhibits VEGF A-induced HUVEC survival and proliferation experiment

[0270] VEGF A can act on VEGFR and other related receptors in vascular endothelial cells, promote endothelial cell survival, proliferation and migration, and then induce neovascularization. This experiment is based on VEGF-induced survival and proliferation of human umbilical vein endothelial cells (HUVEC) to detect the inhibitory effect of antibodies on VEGF A-induced primary cell survival and proliferation.

[0271] In this example, the survival and proliferation of HUVEC were determined by CCK-8. The specific method was as follows: HUVEC cells (Allcells product number: H-001-CN) were treated one day in advance, 2000 cells / well were plated in a 96-well culture plate, and incubated in a 37°C, 5% carbon dioxide incubator for 24 hours.

[0272] After the cells adhered, the experimental culture medium containing VEGF A at a final concentration of 10 ng / ml and serially diluted antibodies (LA42F8 prepared in Example 2, starting at a concentration of 80 μg / ml, diluted 1:3, negative control IgG, positive control BI836880, a group containing only 10 ng / ml VEGF A (VEGFA), and a blank group without VEGF A or antibody) was prepared. The endothelial cell culture medium in the 96-well plate was replaced and the cells were incubated in a 37°C, 5% CO2 incubator for 72 hours.

[0273] 10 μl / well of 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.

[0274] Use a multifunctional microplate reader to measure the absorbance OD 450 -OD 620 Reading value,

[0275] In the experiment described in the above assay, the test results are shown in FIG6 , from which it can be seen that the anti-VEGFA antibody LA42F8 can completely inhibit VEGFA-induced HUVEC cell survival and proliferation.

[0276] Example 9 Anti-VEGF A / Ang2 bispecific antibody HEK293-KDR reporter blocking experiment

[0277] The chains of the bispecific binding molecules of the present invention, IEX04-008, IEX04-010 and IEX04-012 (sequences shown in the sequence listing), were constructed into the pcDNA3.1 vector and expressed and purified in 293 cells as described in Example 2.

[0278] In this example, the HEK293-KDR reporter assay was used to detect the blocking effect of the anti-VEGF A / Ang2 bispecific antibody on VEGF A. The experimental method was similar to that of Example 7.

[0279] The binding molecules or controls used were as follows:

[0280] Blank: no VEGF A, no antibody;

[0281] VEGF A: 100 ng / ml VEGF A;

[0282] Negative control IgG: prepared as described above;

[0283] Positive control Faricimab: Prepare as described above, starting at 13.5 μg / ml, with a 1:3 serial dilution.

[0284] Positive control BI-836880: prepared as described above, starting at 13.5 μg / ml, 1:3 serial dilutions;

[0285] IEX04-012: Prepared as described above, starting at 13.5 μg / ml, and diluted 1:3.

[0286] 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.

[0287] Example 10 Anti-VEGF A / Ang2 bispecific binding molecule HUVEC proliferation inhibition experiment

[0288] In this study, the HEK293-KDR reporter assay was used to detect the inhibitory effect of anti-VEGF A / Ang2 bispecific binding molecules on VEGF A-induced HUVEC cell survival and proliferation.

[0289] The experimental method is the same as Example 8, but the antibodies used are as follows:

[0290] Panel A: IEX04-008 prepared as described above, negative control IgG, positive controls Faricimab and BI836880, starting at 20 μg / ml, diluted 1:3, blank (i.e., no antibody and VEGF A), and VEGF A group (i.e., only 20 ng / ml VEGF A added);

[0291] Panel B: The starting concentration was 80 μg / ml, and the negative control IgG, BI-anti-VEGF, IEX04-010 prepared as described above were diluted 1:3 in equal proportions, as well as the blank group (i.e., without antibody and VEGF A), and the VEGF A group (i.e., only 20 ng / ml VEGF A was added);

[0292] Panel 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 VEGF A added).

[0293] The test results are shown in Figure 8. The bispecific binding molecules IEX04-008, IEX04-010, and IEX04-012 all inhibited VEGF-induced HUVEC cell survival and proliferation. Compared with the control antibodies BI836880 and Faricimab, the IC 50 Lower, with better suppression ability.

[0294] Example 11 Anti-VEGF A / Ang2 bispecific antibody Ang2 blocking experiment

[0295] Ang2 can bind to its natural receptor Tie2. In this study, the blocking effect of anti-VEGF A / Ang2 bispecific binding molecules on the binding between Ang2 and Tie2 was detected by ELISA and FACS.

[0296] (1)ELISA

[0297] The ability of IEX04-008, IEX04-010, and IEX04-012, as well as control antibodies BI-836880 and Faricimab, to block the binding of human Ang2 to hTie2 was tested by ELISA.

[0298] hTie2 protein (Beijing Yiqiao) was resuspended in PBS and dissolved to a concentration of 2 μg / ml. The plate was coated overnight. Blocked with 5% BSA for 1 hour, the biotinylated hAngiopoietin-2 protein (R&D) was diluted to 600 μg / ml and added in 50 μl / well. Antibodies prepared as described above (IEX04-008, IEX04-010, IEX04-012, as well as positive control antibodies BI836880 and Faricimab, and negative control IgG) were diluted 1:2 in a series of 8 or 12 dilutions starting at a maximum concentration of 300 nM. The plates were incubated in PBS on ice for 30 minutes. The final biotinylated concentration was 300 ng / ml. The antigen-antibody mixture obtained above was incubated in an ELISA plate for 90 minutes, washed three times with PBS, and the supernatant discarded. 100 μl of Avidin-HRP (Invitrogen) diluted 1:10,000 was added per well and incubated at room temperature for 30 minutes. The plate was then washed six times with PBS. Development was performed with 100 μl of TMB colorimetric solution (solarbio) for 1 minute, and the plate was terminated with 100 μl of stop solution (Solarbio) per well.

[0299] OD readings were performed on each well using a microplate reader. 450 , OD 620 .

[0300] The experimental results show (see Figure 9) that IEX04-008, IEX04-010, IEX04-012 and the control antibody BI836880 all have complete blocking effects, and the IC values ​​of the bispecific binding molecules of the present invention are 50 were significantly lower than those of the positive control antibody.

[0301] (2) Flow cytometry (FACS)

[0302] The ability of IEX04-008, IEX04-010, IEX04-012, positive control antibodies BI-836880 and Faricimab, and negative control IgG to block the binding of human Ang2-hFc to Tie2 on the cell surface was detected by FACS.

[0303] The antigen hAng2-Fc protein (Beijing Yiqiao, Cat. No.: 10691-H02H) was diluted to 4ug / ml, 50μl / well. The antibodies prepared as described above (IEX04-008, IEX04-010, IEX04-012, as well as the positive control antibodies BI-836880 and Faricimab and the negative control IgG) were diluted 2-fold starting from the highest concentration of 800nM, for a total of 12 dilution gradients, 50μl / well, and incubated on ice with PBS 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, 100μl / well. The cells were centrifuged at 300g for 5 minutes, the supernatant was discarded, and the cells were resuspended in the antigen-antibody mixture. Incubated on ice for 30 minutes, 100μl / well of PBS was added, centrifuged at 300g for 5 minutes, washed once with PBS, 100μl of Goat anti-human IgG-PE (SouthernBiotech) diluted 1:200 was added / well, ice bathed for 20 minutes, 100μl / well of PBS was added, centrifuged at 300g for 5 minutes, and washed once with PBS. Resuspended with 100μl PBS, the cell fluorescence signal value was detected by flow cytometry (BD Biosciences). According to its MFI, the concentration-dependent curve was fitted using GraphPad. The results are shown in Figure 10. The figure shows that the bispecific binding molecules IEX04-008, IEX04-010 and IEX04-012 can effectively block the binding of human Ang2-hFc to Tie2, and the IC 50 Lower than the positive control.

[0304] Example 12 Ang2 phosphorylation inhibition experiment of anti-VEGF A / Ang2 bispecific binding molecules

[0305] This example verifies the inhibitory effect of the bispecific binding molecules of the present invention on hAng2-Fc-induced Tie2 phosphorylation using an hAng2-induced phosphorylation experiment.

[0306] In this study, Expi293 cells overexpressing Tie2 (293-Tie2) were co-incubated with bispecific binding molecules and recombinant hAng2-Fc protein. The phosphorylated Tie2 content in the system was detected to reflect the inhibitory effect of different antibodies on hAng2-Fc-induced Tie2 phosphorylation.

[0307] Take the overexpressed 293-Tie2 cells prepared above and dilute to 2*10 6 cell / ml, 100ul per well was added to a 96-well plate, centrifuged at 400g for 5min, and the supernatant was removed.

[0308] Experimental culture medium was prepared using Expi293 medium (Thermo Catalog No. 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 60 μg / ml, diluted in a 1:2 ratio. The final concentration of hAng2-Fc (Beijing Sino-Bio Products Catalog No.: 10691-H02H) was 2.5 μg / ml.

[0309] Resuspend the cells with 100ul of experimental culture medium per well, incubate at 37 degrees for 15 minutes, remove the culture medium by centrifugation, add 100ul of NP-40 lysis buffer containing 1% protease and phosphatase inhibitors, and place on ice for 30 minutes. Centrifuge at 2000g, collect the protein supernatant, and store in a -80 degree refrigerator.

[0310] pTie2 concentration was determined according to the instructions of the Phospho-Tie2 ELISA Kit (R&D DYC2720E). The capture antibody was coated onto the ELISA plate at a concentration of 4 μg / ml overnight at 4°C. The plate was washed three times with PBST and blocked with 5% BSA for 1 hour. 100 μL of the sample to be tested and a pTie2 control (R&D DYC2720E) for standard curve generation were added and incubated at room temperature for 2 hours. (If the pTie2 concentration in the sample is too high, exceeding the detection range of the ELISA, the resulting mixture can be diluted 2-3 times.) The plate was washed three times with PBST, and 100 μL of HRP-conjugated anti-pTyr antibody (R&D Catalog No. DYC2720E) was added and incubated at room temperature for 2 hours. The plate was washed six times with PBST, and 100 μL of TMB was added for color development. After 15 minutes, the reaction was terminated with 100 μL of stop buffer. The OD450-OD620 values ​​of each well were measured using a spectrophotometer.

[0311] 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 Better than the positive control.

[0312] Example 13 Inhibitory Experiment of Anti-VEGF A / Ang2 Bispecific Binding Molecules on Ang2 Leakage into Vascular Endothelial Cells

[0313] This study used the HUVEC-Tie2 leakage assay to identify the effects and functions of anti-VEGF A / Ang2 bispecific binding molecules on vascular endothelial cell leakage.

[0314] HUVEC cells (Allcells catalog number: H-001-CN) were transfected with lentivirus to obtain HUVEC-Tie2 cells overexpressing Tie2.

[0315] Use 300ul of EGM-2 medium on the bottom layer of a mini-well 96-well culture dish, digest with Accutase (Sigma) to obtain HUVEC-Tie2, and resuspend in EGM-2 medium to 1*10 7 Cells / ml, 100ul / well were plated on the upper layer of the culture dish. The lower chamber culture medium (EGM-2 medium) was replaced every 24 hours. After 24 hours, the lower chamber culture medium was replaced with the experimental medium. The experimental medium composition is as follows:

[0316] Blank: EGM-2 culture medium (Lonza catalog number: CC-5035),

[0317] VEGF Group A: EGM-2 medium + 20ng / ml VEGF (R&D Catalog No.: 293-VE)

[0318] IgG group (VEGF A + IgG): EGM-2 medium + 20 ng / ml VEGF + 10 ug / ml IgG;

[0319] Ang1 group (VEGF A + Ang1): EGM-2 medium + 20 ng / ml VEGF (R&D Catalog No.: 293-VE) + 200 ng / ml Ang1 (R&D Catalog No.: 923-AN);

[0320] IEX04-012 group (VEGF A + IEX04-012): EGM-2 medium + 20 ng / ml VEGF + 10 ug / ml IEX04-012;

[0321] BI-836880 group (VEGF A + BI-836880): EGM-2 medium + 20 ng / ml VEGF + 10 ug / ml BI-836880;

[0322] Faricimab group (VEGF A + Faricimab): EGM-2 medium + 20ng / ml VEGF + 10ug / ml Faricimab.

[0323] The above experimental culture medium was placed at 37°C and 5% CO2 for culture.

[0324] After 24 hours, 1 μl of 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% CO2. After 30 minutes, the chamber culture medium was removed and diluted 1:10 with PBS before detection on a multifunctional microplate reader with an excitation wavelength of 488 nm and an emission wavelength of 535 nm.

[0325] The experimental results are shown in FIG12 , which show that the antibody IEX04-012 of the present invention can effectively reduce VEGF-induced vascular endothelial cell permeability.

[0326] Example 14. Laser-induced choroidal neovascularization efficacy test

[0327] This experiment used a rhesus monkey laser-induced choroidal neovascularization model to determine the anti-neovascularization effect of the bispecific binding molecule IEX04-012 of the present invention.

[0328] Rhesus monkeys:

[0329] Species: Rhesus monkey; Grade: Common; Weight: 3.30-4.20 kg at purchase; 3.35-4.35 kg at model establishment; Source: Sichuan Hengshu Biotechnology Co., Ltd., Production License No.: SCXK(Chuan)2019-029, Laboratory Animal Quality Certificate No.: No.0023356;

[0330] This study used laser photocoagulation around the fovea of ​​the rhesus monkey fundus to induce choroidal neovascularization, establishing an animal model similar to human choroidal neovascularization. Fluorescein fundus angiography was performed before and 20 days after photocoagulation to assess model development. Twenty rhesus monkeys (half male and half female) with successful model development were divided into five groups: a model control group, a low-dose IEX04-012 group, a high-dose IEX04-012 group, an Elyea group, and a Faricimab group, with four monkeys in each group (half male and half female).

[0331] On the 21st day after photocoagulation, the monkeys in each group were given medication according to the doses in the table. IEX04-012, Eylea (Bayer) or Faricimab (all dissolved in 0.9% sodium chloride injection) was injected into the vitreous of both eyes. The model control group was given an equal volume of 0.9% sodium chloride injection. Fundus color photography, fluorescein angiography (leakage spot count and leakage area measurement) (Robin J Goody, Wenzheng Hu, Afshin Shafiee et al., Optimization of laser-induced choroidal neovascularization in African green monkeys. Experimental Eye Research, Exp Eye Res. 2011 92(6):464-72), and optical coherence tomography (OCT, Wang Q, Lin X, Xiang W et al., 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) were performed on the 7th, 14th, 21st, and 28th day after administration to observe the inhibitory effect of the test product on choroidal neovascularization. Both eyes were euthanized 29 days after administration and histological examination was performed by immunohistochemistry (HE) staining.

[0332] Experimental Design Table

[0333] The results are shown in Figures 13-15. 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 of the animals in the IEX04-012 treatment group was significantly reduced, 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 treatment group was significantly reduced, and the effect was better than the positive controls Eylea and Faricimab (Figure 15), indicating that the antibodies of the present invention can significantly inhibit leakage caused by neovascularization. In summary, it is proved that the antibodies of the present invention combined with anti-VEGF inhibitors have a significant inhibitory effect on laser-induced fundus neovascularization, and at the same time have the function of protecting vascular integrity.

[0334] After 29 days of administration, rhesus monkeys were anesthetized with sodium pentobarbital (approximately 30 mg / kg intravenously, the dose can 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.

[0335] Both eyes of some animals were fixed with modified Davidson's fixative, embedded in paraffin and sectioned. The laser modeling area was selected for routine HE staining, CD31 IHC staining and other histopathological examinations.

[0336] In the pathological sections, the antibody group of the present invention significantly reduced the retinal lesion area, alleviated retinal edema, and reduced tissue proliferation in the laser-damaged area compared to anti-VEGF treatment alone. The results showed better retinal morphology improvement (see Figure 16), inhibited retinal choroidal neovascularization, and enhanced vascular integrity function (Figure 17).

[0337] Sequence Listing

[0338] Example 15. Experimental Study on Determining the Bispecific Antibody Formulation of the Present Invention

[0339] This experiment mainly investigated the effects of excipients (sodium dihydrogen phosphate (monohydrate), sodium dihydrogen phosphate (heptahydrate), trehalose, histidine, methionine, proline, glycine, sucrose, hydroxypropyl-β-cyclodextrin, and polysorbate 80) on the stability of IEX04-012 protein. Detailed prescription information is shown in Table 5.

[0340] Experimental Materials:

[0341] The information of the excipients and / or reagents used in the formulation research experiments of the present invention is shown in Table 4 below.

[0342] Table 4. Reagent information

[0343] Note: N / A means not involved. Trehalose in the following prescription refers to trehalose (dihydrate).

[0344] Preparation and composition of the preparation

[0345] Buffer solutions for each formulation were prepared according to the mass concentrations listed in Table 5 using ultrapure water as the solvent (their compositions, excluding polysorbate 80, are shown in Table 5). IEX04-012 protein (prepared according to the method in Example 2 above) was then ultrafiltration-displaced into the respective formulation buffer. Following displacement, the protein content of each formulation was adjusted to 40 mg / ml. Polysorbate 80 was added to a final concentration of 0.3 mg / ml. The solutions were then filtered and dispensed into vials, stoppered, and capped. Stability tests were conducted at 40°C ± 2°C.

[0346] Table 5 Alternative prescription information

[0347] Note: Use dilute hydrochloric acid or 1 M NaOH solution to adjust the pH.

[0348] Stability studies

[0349] Detailed experimental conditions and sampling plan are shown in Table 6.

[0350] Table 6. Stability study plan

[0351] Note: After sampling at the above time points, all samples were first placed in a -70℃ refrigerator for testing and thawed and sent for testing as needed.

[0352] Judgment criteria

[0353] Based on the understanding of the product and the precision of the instrument and method, the judgment criteria for whether the sample test value has not changed compared with the initial value are set, as shown in Table 7.

[0354] Table 7. Criteria for judging if quality has not changed

[0355] Note: “*” indicates that this standard is only applicable to prescription determination experiments.

[0356] Experimental results

[0357] The results of the mandatory stability study are detailed in Table 8. The results showed that after one week of storage at 40°C ± 2°C, the appearance and visible foreign matter content of the samples of the remaining formulations, except for formulation 5, which showed a strong opalescence, were acceptable. There were no significant changes in protein content or pH. The purity (SEC-HPLC method) of the samples of all formulations changed, with formulation 7 showing the least decrease. The acidic components of the charge variants of the samples of all formulations increased, while the main components decreased, with formulation 7 showing the least increase. After two weeks of storage at 40°C ± 2°C, the purity (SEC-HPLC method) of the samples of all formulations changed, with formulation 7 showing the least decrease. The acidic components of the charge variants of the samples of all formulations increased, while the main components decreased, with formulation 7 showing the least decrease. The polysorbate 80 content of all formulations remained unchanged. The biological activities of all formulations were within the acceptable range.

[0358] Based on the results of the formulation confirmation experiments, Formulation 7 was selected as the final formulation for IEX04-012. To avoid the use of hydrochloric acid for pH adjustment during production, the buffer system was adjusted to histidine and histidine hydrochloride. Furthermore, because the formulation osmotic pressure of Formulation F7 was relatively low (255 mOsmol / kg), the trehalose concentration was increased to 100 mg / ml (at this point, the formulation osmotic pressure was 350 mOsmol / kg) to ensure that the formulation buffer osmotic pressure met the requirements of the Chinese Pharmacopoeia. The final formulation of IEX04-012 was: 40.0 mg / ml recombinant anti-vascular endothelial growth factor A (VEGF A) and anti-angiopoietin 2 (Ang2) bispecific antibody, 1.25 mg / ml (8 mM) histidine, 0.40 mg / ml (2 mM) L-histidine hydrochloride, 100 mg / ml (264.3 mM) trehalose, and 0.3 mg / ml polysorbate 80, pH 6.5. Subsequent long-term stability results at 25°C and 2-8°C also showed that the IEX04-012 antibody can remain stable for a long time under this formulation condition.

[0359] The long-term stability test results of the final formulation of Prescription 7 are shown in Table 9

[0360] Example 16. Study on high concentration formulation of the bispecific antibody of the present invention (120 mg / ml)

[0361] In this example, a high-concentration (120 mg / ml) bispecific antibody preparation was prepared, and its stability was studied by continuously detecting mass changes during forced, accelerated, and long-term storage.

[0362] A buffer solution (pH 6.5) containing 1.25 mg / ml histidine, 0.40 mg / ml L-histidine hydrochloride, and 100.00 mg / ml trehalose was prepared. IEX04-012 protein (prepared according to Example 2) was then ultrafiltered into this buffer to adjust the protein content to approximately 120 mg / ml. Polysorbate 80 was added to a final concentration of 0.3 mg / ml. The solution was then filtered and dispensed into vials, stoppered, and capped. Stability tests were conducted on these samples at 40°C ± 2°C, 25°C ± 2°C, and 2-8°C. The specific protocol is shown in Table 10.

[0363] Table 10. Stability study plan

[0364] Note: After sampling at the above time points, all samples were first frozen at -70℃ for testing and thawed and sent for testing as needed.

[0365] Judgment criteria

[0366] Based on the understanding of the product and the precision of the instruments and methods, the judgment criteria for whether the sample test value has not changed compared with the initial value are set, as shown in Table 11.

[0367] Experimental results

[0368] The results of the prescription study are detailed in Table 12. The results showed that after being placed at 40℃±2℃ for 1 week and 2 weeks, the main peak content decreased and the aggregate content 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 4 weeks, obvious opalescence was produced. After being placed at 25℃±2℃ for 1 month, 2 months, and 3 months, the appearance and visible foreign matter of all samples were qualified. In the purity (SEC-HPLC) test of the samples, the main peak content decreased and the aggregate content increased; the charge variant-main component decreased and the acidic component increased; the other inspection items were qualified or unchanged. After being placed at 2-8℃ for 6 months and 12 months, the appearance and visible foreign matter of all samples were qualified, and the other inspection items had no significant changes compared with the initial values.

[0369] As described in Example 15, to avoid the use of hydrochloric acid to adjust the pH during production, the buffer system was adjusted to histidine and histidine hydrochloride. The adjusted high-concentration formulation of IEX04-012 was formulated as follows: 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.

[0370] Example 17. Study on high concentration formulation of the bispecific antibody of the present invention (150 mg / ml)

[0371] In this example, a high-concentration (150 mg / ml) bispecific antibody preparation was prepared and its stability was studied.

[0372] Prepare a buffer containing 1.25 mg / ml histidine, 0.40 mg / ml L-histidine hydrochloride, and 100.00 mg / ml trehalose. After adjusting the pH of the buffer to 6.5, ultrafiltration was performed into the buffer to adjust the protein content to approximately 150 mg / ml. Polysorbate 80 was added to a final concentration of 0.3 mg / ml. Filter and aliquot into vials, stopper, and cap. Stability tests were conducted at 40°C ± 2°C and 25°C ± 2°C.

[0373] Experimental results

[0374] The results of the formulation study are detailed in Table 13. The results showed that after 1 and 2 weeks of storage at 40°C ± 2°C, the main peak decreased in purity testing, while aggregates increased. The charge variant (main component) decreased, while the acidic component increased. All other test items were either qualified or unchanged. After 1 and 2 weeks of storage at 25°C ± 2°C, all samples passed the test in terms of appearance and visible foreign matter. The charge variant (main component) decreased, while the acidic component increased. All other test items were either qualified or unchanged.

[0375] Table 13. Prescription study results

[0376] Note: N / A means not tested.

[0377] As mentioned above, in order to avoid the use of hydrochloric acid to adjust the pH during production, the buffer system was adjusted to histidine and histidine hydrochloride. The formulation of the high-concentration formulation of IEX04-012 is: 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.

[0378] It should be noted that the formulations in 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.

[0379] While the exemplary embodiments of the present invention have been described above, it should be understood by those skilled in the art that these disclosures are merely exemplary and that various other substitutions, adaptations, and modifications may be made within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments listed herein.

Claims

1. A liquid preparation comprising (i) a bispecific binding molecule that specifically binds to VEGF A and Ang2; (ii) a buffer, (iii) a stabilizer, and (iv) a surfactant, The pH of the antibody formulation is about 5.0-7.5, The bispecific binding molecule comprises a first target binding region that specifically binds to VEGF A and a second target binding region that specifically binds to Ang2, wherein the second target binding region is an anti-Ang2 VHH, and the 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 or consists of the sequence shown in SEQ ID NO: 17 or 20; HCDR3 comprises or consists of the sequence shown in SEQ ID NO: 18; Optionally, wherein 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, e.g., the antibody is a fully human or humanized antibody; or VEGF receptor (VEGF R) that specifically binds to VEGF A or its extracellular domain or a fusion protein comprising its extracellular domain, such as a fusion protein of its extracellular domain and Fc.

2. The liquid formulation according to claim 1, wherein the anti-Ang2 VHH (1) comprising the amino acid sequence of SEQ ID NO: 19 or 21, or comprising 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 consisting of the amino acids of SEQ ID NO: 19 or 21; or (2) comprising an amino acid sequence that has 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, For example, the bispecific binding molecule is a bispecific antibody.

3. The liquid formulation according to claim 1 or 2, wherein the bispecific binding molecule is bivalent, trivalent or tetravalent.

4. The liquid formulation according to any one of claims 1 to 3, wherein the bispecific binding molecule has the following structure: light chain variable region VL of anti-VEGF antibody - linker - heavy chain variable region VH of anti-VEGF antibody - linker - anti-Ang2 VHH or Heavy chain variable region VH of anti-VEGF antibody - linker - light chain variable region VL of anti-VEGF antibody - linker - anti-Ang2 VHH.

5. The liquid preparation according to claim 4, wherein: 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; and / or 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.

6. The liquid preparation according to any one of claims 1 to 5, wherein 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 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: 34, or consists of the amino acids of SEQ ID NO: 34; or the heavy chain variable region VH comprises an amino acid sequence that has one or several (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 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 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 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: 30, or consists of the amino acids of SEQ ID NO: 30; or the light chain variable region VL comprises an amino acid sequence that has one or several (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 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.

7. The liquid preparation according to any one of claims 4 to 6, wherein (1) the bispecific binding molecule comprises the amino acid sequence of SEQ ID NO: 28, 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: 28, or consists of the amino acids of SEQ ID NO: 28; or (2) 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, or 1) mutations compared to the amino acid sequence shown in SEQ ID NO: 28, wherein the mutation is, for example, a substitution, deletion, or addition, preferably a substitution, such as a conservative substitution.

8. The liquid formulation according to any one of claims 3 to 7, wherein the bispecific binding molecule has the following structure: First anti-VEGF VHH-linker-second anti-VEGF VHH-linker-anti-Ang2 VHH, The first anti-VEGF VHH and the second anti-VEGF VHH are the same or different.

9. The liquid formulation of claim 8, wherein the first anti-VEGF VHH or the second 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; or 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.

10. The liquid formulation according to claim 8 or 9, wherein the first anti-VEGF VHH or the second anti-VEGF VHH comprises the amino acid sequence of 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 to the amino acid sequence of SEQ ID NO: 4, 5, 9 or 11, or consists of amino acids of SEQ ID NO: 4, 5, 9 or 11; or 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: 4, 5, 9 or 11, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions.

11. The liquid preparation according to any one of claims 8 to 10, wherein (1) the bispecific binding molecule comprises the amino acid sequence of SEQ ID NO: 22, 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: 22, or consists of the amino acids of SEQ ID NO: 22; or (2) 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, or 1) mutations compared to the amino acid sequence shown in SEQ ID NO: 22, wherein the mutation is, for example, a substitution, deletion, or addition, preferably a substitution, such as a conservative substitution.

12. The liquid formulation of claim 1, wherein the bispecific binding molecule comprises one or two of the following chains: VEGF R extracellular domain-Fc-linker-anti-Ang2 VHH.

13. The liquid preparation according to claim 12, wherein the VEGFR extracellular domain is an extracellular domain of human VEGFR; preferably, the VEGFR extracellular domain comprises a VEGFR1 second antibody-like domain and a VEGFR2 third antibody-like domain; more preferably, the VEGFR extracellular domain comprises a human VEGFR1 second antibody-like domain and a human VEGFR2 third antibody-like domain.

14. The liquid formulation of claim 12 or 13, wherein the VEGFR extracellular domain comprises the amino acid sequence of SEQ ID NO: 26, 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: 26, or consists of the amino acids of SEQ ID NO:

26.

15. The liquid formulation according to any one of claims 12 to 14, wherein the Fc is derived from human IgG1, IgG2, IgG3 or IgG4, preferably, the Fc comprises the amino acid sequence of SEQ ID NO: 27, 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: 27, or consists of the amino acids of SEQ ID NO:

27.

16. The liquid formulation of any one of claims 12-15, wherein the VEGFR extracellular domain-Fc is a fusion protein of the VEGFR extracellular domain and Fc, such as Aflibercept or a derivative thereof, such as comprising the amino acid sequence of SEQ ID NO: 25, or comprising 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 consisting of the amino acids of SEQ ID NO:

25.

17. The liquid preparation according to any one of claims 12 to 16, wherein (1) the bispecific binding molecule comprises the amino acid sequence of SEQ ID NO: 24, 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: 24, or consists of the amino acids of SEQ ID NO: 24; or (2) 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, or 1) mutations compared to the amino acid sequence shown in SEQ ID NO: 24, wherein the mutation is, for example, a substitution, deletion, or addition, preferably a substitution, such as a conservative substitution.

18. The liquid formulation according to any one of claims 1 to 17, wherein the linker comprises or consists of the amino acid sequence of SEQ ID NO:

23.

19. The liquid preparation according to any one of claims 1 to 18, wherein the buffer is selected from one or more of histidine, histidine salts, glutamate, phosphate, acetate, citrate and tris(hydroxymethylaminomethane); preferably, the buffer is selected from a combination of histidine and histidine hydrochloride, phosphate and histidine; preferably, the phosphate is a combination of disodium hydrogen phosphate and sodium dihydrogen phosphate.

20. The liquid preparation according to any one of claims 1 to 19, wherein the stabilizer is selected from one or more of saccharides, polyols, amino acids or salts thereof, Preferably, the sugar is selected from the group consisting of sucrose, dextrose, lactose, maltose, trehalose, cyclodextrin, maltodextrin and dextran; the polyol is selected from the group consisting of mannitol, sorbitol and xylitol; the amino acid or its salt is selected from the group consisting of arginine, glycine, proline, methionine, arginine salt and a combination thereof; and the cyclodextrin is preferably hydroxypropyl-β-cyclodextrin. More preferably, the stabilizer is sucrose or trehalose, or a combination of sucrose or trehalose with an additional ingredient selected from one or more of the following: glycine, proline, methionine and hydroxypropyl-β-cyclodextrin.

21. The liquid preparation according to any one of claims 1 to 20, wherein the surfactant is selected from polysorbate 20, polysorbate 80, polysorbate 60, or polysorbate 40; Pluronic, preferably polysorbate 80.

22. The liquid formulation according to any one of claims 1 to 21, wherein the concentration of the antibody or antigen-binding fragment thereof is about 1-200 mg / ml, preferably 30-150 mg / mL; and / or The concentration of the buffer is about 0.5-200 mM, preferably 5-30 mM; and / or The concentration of the stabilizer is about 1-1000 mM, preferably about 50-500 mM; and / or The concentration of the surfactant is about 0.01-10 mg / ml, preferably 0.05-2 mg / ml; and / or The pH of the liquid preparation is about 6.1-7.0, preferably about 6.3-6.

8.

23. The liquid preparation according to any one of claims 1 to 22, wherein the liquid preparation is an injection or eye drops, more preferably an intravitreal injection.

24. The liquid formulation according to any one of claims 1 to 18, comprising: (i) about 10-160 mg / ml of the bispecific binding molecule; (ii) about 5-20 mM buffer, wherein the buffer is histidine or a combination of histidine and histidine hydrochloride; (iii) about 200-350 mM trehalose; and (iv) about 0.1-1 mg / ml polysorbate 80; The pH of the liquid preparation is about 6.1-7.0, preferably about 6.

5.

25. The liquid formulation according to claim 24, comprising: (i) about 30-150 mg / ml of the bispecific binding molecule; (ii) about 8-12 mM histidine; (iii) about 200-300 mM trehalose; and (iv) about 0.2-0.4 mg / ml polysorbate 80; The pH of the liquid preparation is about 6.1-6.6, preferably about 6.

5.

26. The liquid formulation according to claim 24, comprising: (i) about 30-150 mg / ml of the bispecific binding molecule; (ii) about 1-3 mM histidine and about 7-9 mM histidine hydrochloride; (iii) about 250-280 mM trehalose; and (iv) about 0.2-0.4 mg / ml polysorbate 80; The pH of the liquid preparation is about 6.1-6.6, preferably about 6.

5.

27. A solid preparation obtained by solidifying the liquid preparation according to any one of claims 1 to 26; the solid preparation is, for example, in the form of a lyophilized powder injection.

28. A delivery device comprising the liquid formulation of any one of claims 1 to 26 or the solid formulation of claim 27, for example in the form of a pre-filled syringe.

29. Use of the liquid preparation according to any one of claims 1 to 26 or the solid preparation according to claim 27 in the preparation of a medicament or a delivery device for preventing or treating an eye disease associated with angiogenesis, such as an eye disease associated with corneal neovascularization.

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