Formulations Comprising Bispecific Binding Molecules that Bind VEGF and ANG2 and Uses Thereof

Abstract

The present invention relates to formulations comprising bispecific binding molecules that bind to VEGF and Ang2, and uses thereof, particularly to pharmaceutical formulations comprising the bispecific binding molecules, a buffer, a stabilizer, and a surfactant, and further to therapeutic or prophylactic uses of these formulations.

Description

[Technical Field]

[0001] The present invention relates to the field of formulations. More specifically, the present invention relates to a formulation comprising, separately, an antibody or antigen-binding fragment thereof against vascular endothelial growth factor (VEGF / VEGF-A) and an antibody or antigen-binding fragment thereof against angiopoietin-2 (ANG-2), or a formulation comprising specific antibodies (e.g., antibodies) or antigen-binding fragments thereof against both vascular endothelial growth factor (VEGF / VEGF-A) and angiopoietin-2 (ANG-2), and a formulation comprising antigen-binding fragments against vascular endothelial growth factor (VEGF / VEGF) or both vascular endothelial growth factor (VEGF / VEGF-A) and angiopoietin-2 (ANG-2), particularly stable liquid formulations, as well as methods for producing antibody formulations and uses of the antibody formulations. [Background technology]

[0002] Angiogenesis is involved in the pathogenesis of a variety of diseases, including solid tumors, diseases associated with intraocular neovascularization, rheumatoid arthritis, and psoriasis.

[0003] VEGF is a potent, ubiquitous angiogenic factor. The VEGF family includes VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, placental growth factor (PIGF), and endocrine-derived VEGF (EG-VEGF). Active forms of VEGF are synthesized as homodimers or heterodimers with other VEGF family members. VEGF-A exists in six isotypes generated by alternative splicing: VEGF121, VEGF145, VEGF165, VEGF183, VEGF189, and VEGF206. These isotypes are primarily differentiated by differences in bioavailability, with VEGF165 being the predominant isotype. VEGF is believed to be a key regulator of normal and disease-related angiogenesis.

[0004] In addition to the VEGF family, human angiopoietins are also thought to be involved in vascular development and postnatal angiogenesis. Human angiopoietins include the natural agonist angiopoietin-1 (ANG-1) and the natural antagonist angiopoietin-2 (ANG-2). The role of ANG-1 is widely and constitutively expressed in adults and is thought to be conserved. In contrast, ANG-2 expression is primarily restricted to sites of vascular remodeling and is thought to inhibit the constitutive stabilization or maturation function of ANG-1, thereby restoring and maintaining a plastic state in which blood vessels are more responsive to sprouting signals.

[0005] In recent years, some bispecific antibodies targeting VEGF-A and ANG-2 have been developed (e.g., WO2012131078 and WO2014009465). However, existing bispecific antibodies have low inhibitory potency against VEGF and Ang-2, and their large molecular weights result in low molar concentrations upon single administration. Particularly in ocular diseases, antibodies administered intravitreally generally have small molecular weights, which necessitates less frequent administration. Therefore, there remains a need for novel bispecific binding molecules targeting VEGF-A and ANG-2 that are particularly suitable for ocular diseases.

[0006] There is a need for novel specific antibodies that can be used to treat and / or prevent ocular diseases, as well as formulations containing such novel antibodies, particularly formulations with excellent stability. Summary of the Invention

[0007] The present invention fulfills these needs by providing pharmaceutical formulations containing VHH antibodies targeting VEGF-A or ANG-2, or bispecific binding molecules targeting both VEGF-A and ANG2. In particular, the bispecific binding molecules of the present invention have a smaller molecular weight and a higher molar concentration at the same mass concentration compared to known antibodies. Furthermore, they have high blocking activity against VEGF-A and Ang2, and can completely inhibit VEGFA-induced primary cell proliferation. Therefore, the molecules of the present invention have stronger blocking activity in clinical settings, allow for a higher molar concentration of antibody in a single administration, maintain the efficacy of a single administration for a longer period, and reduce the frequency of ocular administration (e.g., intravitreal injection). [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 shows the structure of a bispecific binding molecule. [Figure 2] FIG. 2 shows that anti-VEGF A VHH antibodies inhibit the binding of VEGF A to VEGFR2 by ELISA. [Figure 3] FIG. 3 shows the inhibitory effect of humanized anti-Ang2 VHH antibodies on the binding of Ang2 to Tie2 by ELISA. [Figure 4] FIG. 4 shows the effect of anti-Ang2 VHH antibodies (A) and humanized anti-Ang2 VHH antibodies (B) on inhibiting phosphorylation of 293-Tie2 cells by hAng2-Fc, as measured by ELISA. [Figure 5] FIG. 5 shows the effect of anti-VEGF A VHHs in inhibiting VEGFA activation of the KDR receptor as measured by the HEK293-KDR reporter assay. [Figure 6] FIG. 6 shows the inhibitory effect of anti-VEGF A VHH antibodies on VEGF A-induced survival and proliferation of HUVEC cells as measured by CCK-8. [Figure 7]FIG. 7 shows the efficacy of the VEGF A / Ang2 bispecific binding molecule IEX04-012 in inhibiting VEGF activation of the KDR receptor as measured by the HEK293-KDR reporter assay. [Figure 8] FIG. 8 shows the effect of bispecific binding molecules IEX04-008, IEX04-010 and IEX04-012 on the inhibition of HUVEC cell survival and proliferation by VEGF. [Figure 9] FIG. 9 shows by ELISA that the bispecific binding molecules IEX04-008, IEX04-010, and IEX04-012 inhibit the binding of human Ang2 to Tie2. [Figure 10] FIG. 10 shows that bispecific binding molecules IEX04-008, IEX04-010, and IEX04-012 inhibit the binding of Ang2-Fc to Tie2 by flow cytometry assay. [Figure 11] FIG. 11 shows that the bispecific binding molecule of the invention, IEX04-012, effectively inhibits hAng2-Fc-induced phosphorylation of 293-Tie2 in vitro by flow cytometry assay. [Figure 12] FIG. 12 shows that the bispecific binding molecule IEX04-012 of the invention reduces VEGF-induced permeability of vascular endothelial cells, ie, inhibits VEGF-induced leakage of HUVEC cells. [Figure 13] FIG. 13 shows the laser spot ratio statistics for grade 4 (Panel A) and grade 3 or higher (Panel B) in the laser-induced choroidal neovascularization model. [Figure 14] FIG. 14 shows the statistics of retinal thickness in the laser-induced choroidal neovascularization model. [Figure 15] FIG. 15 shows the statistics of leakage area in the laser-induced choroidal neovascularization model. [Figure 16] FIG. 16 shows the H&E staining pattern of fundus tissue (A) and the statistics of the lesion area (B) in the laser-induced choroidal neovascularization model. [Figure 17] FIG. 17 shows the CD31 staining pattern of fundus tissue (A) and the statistics of positive cells (B) in a laser-induced choroidal neovascularization model. DETAILED DESCRIPTION OF THE INVENTION

[0009] definition Before describing the present invention in detail below, it should be understood that this invention is not limited to the particular methodology, protocols, and reagents described herein. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0010] For the purpose of describing this specification, the following definitions will be used, and where appropriate, terms used in the singular will also include the plural and vice versa. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0011] The term "about" when used in conjunction with a numerical value is intended to include numerical values ​​ranging from a lower limit of 5% less than the stated numerical value to an upper limit of 5% more than the stated numerical value.

[0012] As used herein, the term "and / or" refers to one, more than one, or all of the alternatives.

[0013] As used herein, the term "comprising" means that the recited elements, integers, or steps are included, but not that other elements, integers, or steps are excluded. As used herein, the term "comprising" also includes cases where the recited elements, integers, or steps are entirely comprised, unless otherwise specified. For example, when referring to an antibody variable region "comprising" a particular sequence, it is intended to encompass an antibody variable region consisting of that particular sequence.

[0014] The term "VEGF" as used herein refers to an angiogenic factor. The VEGF family includes VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, placental growth factor (PIGF), and endocrine-derived VEGF (EG-VEGF). Active forms of VEGF are synthesized as homodimers or heterodimers with other VEGF family members. VEGF-A exists in six isotypes generated by alternative splicing: VEGF121, VEGF145, VEGF165, VEGF183, VEGF189, and VEGF206. These isotypes are primarily distinguished by differences in bioavailability, with VEGF165 being the predominant isotype. In some embodiments, the VEGF A of the present invention refers to VEGF A of human origin, e.g., VEGF165 of human origin. In one embodiment, the amino acid sequence of the VEG A of the present invention is the amino acid sequence of accession number P15692 (Uniproto database).

[0015] The term "ANG2" as used herein refers to human angiopoietin-2 (humanized), as 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 were discovered as ligands for the tyrosine kinase family Tie, which is selectively expressed in vascular endothelium. Currently, four members of the angiopoietin family have been defined. Angiopoietin-3 and -4 (ANG3 and ANG4) are various counterparts of the same gene locus in mice and humans. Ang1 and Ang2 were first 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, ANG2 of the present invention refers to Ang2 derived from humans. In some embodiments, human Ang2 comprises the amino acid sequence of accession number O15123 (UniProto database).

[0016] The term "multispecific binding molecule" refers to a multispecific binding molecule that is at least bispecific, i.e., comprises at least a first target binding region and a second target binding region, where the first target binding region binds to one target or antigen and the second target binding region binds to another antigen or target. Thus, molecules according to the invention comprise specificities for at least two different antigens or targets. Molecules according to the invention also include multispecific molecules comprising multiple target binding regions / sites, such as trispecific binding molecules. In some embodiments, bispecific binding molecules of the invention are bispecific antibodies.

[0017] The term "linker," as used herein, refers to any molecule that allows for the direct linking of different portions of a bispecific binding molecule. Examples of linkers for establishing a covalent bond between different portions of a molecule include peptide linkers and non-protein polymers, including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyalkylene oxide, or copolymers of polyethylene glycol and polypropylene glycol. In some embodiments, the linker is a peptide linker, which refers to an amino acid sequence that connects the amino acid sequence of a first portion of a binding molecule to the second portion of the binding molecule. For example, a peptide linker can connect a first target-binding region of a binding molecule to a second target-binding region. For example, a peptide linker can also connect one portion of an antibody to another portion of the antibody, such as a light chain variable region and a heavy chain variable region. Preferably, the peptide linker is long enough to link the two entities while maintaining their relative conformations with each other without interfering with the desired activity.

[0018] Peptide linkers may, but need not, primarily contain 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), where n is an integer equal to or greater than 1 (preferably 2, 3, 4, 5, 6, 7, 8, 9, or 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), where n = 1, 2, 3, or 4.

[0019] The term "valency" as used herein refers to the presence of a specific number of binding sites in a binding molecule, such as an antibody molecule. Thus, the terms bivalent, trivalent, and tetravalent refer to the presence of two, three, or four binding sites (target binding regions) in a binding molecule, respectively. Bispecific binding molecules of the invention are at least bivalent and may be multivalent (e.g., bivalent, trivalent, tetravalent, or hexavalent).

[0020] As used herein, the term "target binding region" refers to any portion of a multispecific binding molecule, e.g., a bispecific binding molecule, that binds to a specific target or antigen. A 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 rest of the BsAB and may or may not bind to the target as a separate entity. A target binding region can be a receptor or ligand, or a domain of a receptor that can bind to a ligand.

[0021] The term "antibody fragment" includes a portion of an intact antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0022] "Antigen-binding fragment" refers to a molecule distinct from an intact antibody, which contains a portion of the 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, domain antibodies (dAb), linear antibodies, single-chain antibodies (e.g., scFv), single-domain antibodies (e.g., VHH), bivalent antibodies or fragments thereof, and camelid antibodies.

[0023] "VHH," also known as single-domain antibody (sdAb), refers to a genetically engineered antibody composed solely of the variable region of a heavy chain antibody, including only the three HCDRs of the heavy chain variable region. While a typical antibody requires six CDRs, VHH has high specificity and high affinity for antigens with only three HCDRs. Crystal structures have shown that VHH forms a scaffold consisting of two β-sheets, similar to the folding of conventional antibody VH immunoglobulins.

[0024] The term "target" refers to the substance to which a binding molecule binds. A target can be an antigen, a ligand, a receptor, etc.

[0025] The term "antigen" refers to a molecule that elicits an immune response. Such an immune response may include the production of antibodies, the activation of specific immune cells, or both. Those skilled in the art will appreciate that essentially any macromolecule, including any protein or peptide, can be used as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. As used herein, the term "epitope" refers to a fragment of an antigen (e.g., VEGF or Ang2) that specifically interacts with an antibody molecule.

[0026] "Complementarity-determining regions" or "CDR regions" or "CDRs" are regions in the variable region of an antibody that are highly variable in sequence, form structurally defined loops ("hypervariable loops"), and / or contain residues that contact the antigen ("antigen contact sites"). CDRs are primarily responsible for binding to an antigen epitope. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus. The CDRs in an antibody heavy chain variable region are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs in an antibody light chain variable region are referred to as LCDR1, LCDR2, and LCDR3. For a given amino acid sequence of a light chain variable region or a heavy chain variable region, the exact amino acid sequence boundaries of each CDR can be determined, for example, by Chothia (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)), based on the three-dimensional structure of an antibody and the topology of the CDR loops, Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), or the International ImmunoGeneTics database (IMGT) (imgt.cines.fr / on the World Wide Web), based on antibody sequence variability. These can be determined using any one or a combination of many well-known antibody CDR assignment systems, including the North CDR definition based on affinity propagation clustering using multiple crystal structures.

[0027] For example, according to different CDR determination schemes, the residues of each CDR are described as follows: [Table 1]

[0028] Unless otherwise specified, the term "CDR" or "CDR sequence" as used herein includes CDR sequences determined by any of the above methods.

[0029] Unless otherwise specified, residue positions in antibody variable regions (including heavy chain variable region residues and light chain variable region residues) are numbered 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)).

[0030] In one embodiment, the CDRs of a VHH of the invention are determined according to the following rules: HCDR1 is determined according to AbM, and HCDR2 and HCDR3 are determined according to Kabat.

[0031] It should be noted that the CDR boundaries of the variable regions of the same antibody determined by different assignment systems may differ. That is, the CDR sequences of the variable regions of the same antibody defined by different assignment systems will be different. Thus, when defining an antibody having a specific CDR sequence as defined herein, the scope of that antibody also includes antibodies whose variable region sequences contain the specified CDR sequences, but whose CDR boundaries differ from the specific CDR boundaries defined herein due to the applied protocol (e.g., the rules of different assignment systems, or a combination thereof).

[0032] Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs (even under the same assignment system). However, although CDRs differ from antibody to antibody, the amino acid positions within the CDRs that are directly involved in antigen binding are limited. The minimum overlapping region can be determined using at least two of the Kabat, Chothia, AbM, Contact, and North methods, thereby obtaining a "minimum binding unit" for antigen binding. The minimum binding unit may also be a subregion of the CDR. As will be apparent to those skilled in the art, the remaining residues of the CDR sequence can be determined by the antibody structure and protein folding. Therefore, variants of the CDRs described herein are also contemplated. For example, in a CDR variant, the amino acid residues of the minimum binding unit may remain unchanged, while the remaining CDR residues defined by Kabat or Chothia may be replaced with conservative amino acid residues.

[0033] As used herein, the term "Fc region" is used to define the CH2 and CH3 constant regions of an immunoglobulin heavy chain and includes native-sequence Fc regions and variant Fc regions. Native or wild-type Fc regions bind to various Fc receptors on the surface of immune cells, thereby triggering CDC, ADCC, or ADCP effector function. Such effector function typically requires that the Fc region be associated with a binding domain (e.g., an antibody variable region). In some embodiments, the Fc region is mutated to enhance its CDC, ADCC, or ADCP effector function. In some embodiments, the Fc region is mutated to weaken or eliminate its CDC, ADCC, or ADCP effector function.

[0034] A "humanized" antibody refers to an antibody that contains amino acid residues derived from non-human CDRs and human FRs. In some embodiments, a humanized antibody contains at least one, and typically two, substantially all variable domains, in which all or substantially all CDRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has been humanized. The terms "human antibody," "fully human antibody," and "fully humanized antibody" are used interchangeably and refer to an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or obtained from a non-human source utilizing a human antibody library or other human antibody-encoding sequences. The definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0035] As used herein, the terms "anti," "binding," or "specific binding" refer to a binding effect that is selective for a target or antigen and can be distinguished from unwanted or nonspecific interactions. The ability of a binding moiety to bind to a specific target or antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or known binding assays (e.g., radioimmunoassay (RIA), biolayer interferometry, MSD assay, or surface plasmon resonance (SPR)).

[0036] The terms "host cell," "host cell line," and "host cell culture" are used synonymously 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 the progeny derived therefrom, regardless of the number of transfers. The progeny may not be exactly identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny screened or selected from the originally transformed cell that have the same function or biological activity are also included herein.

[0037] As used herein, the term "label" refers to a compound or composition that is attached directly or indirectly as a conjugate or fusion to an agent, such as a polynucleotide probe or antibody, to facilitate detection of the conjugated or fused agent. The label itself is detectable (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, is capable of catalyzing a chemical change to a detectable substrate compound or composition. The term includes direct labeling of a probe or antibody by attaching (i.e., physically linking) a detectable substance to the probe or antibody, and indirect labeling of a probe or antibody by reacting with another directly labeled reagent. In some embodiments, the label is hFc or biotin.

[0038] A "subject" includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, goats, cats, dogs, 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.

[0039] Calculation of sequence identity between sequences is performed as follows.

[0040] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., for optimal alignment, gaps can be introduced in either or both of the first amino acid or nucleic acid sequence or the second amino acid or nucleic acid sequence, and non-homologous sequences can be discarded for comparison). In a preferred embodiment, the length of the aligned reference sequence for comparison is at least 30%, preferably at least 40%, more preferably at least 50% or 60%, and even more preferably at least 70%, 80%, 90%, or 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. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at this position.

[0041] A mathematical algorithm can be used to compare two sequences and calculate the percent identity between sequences. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needlema and Wunsch algorithm ((1970) J. Mol. Biol., 48:444-453; available at http: / / www.gcg.com) incorporated into the GAP program of the GCG software package, using a Blossom 62 matrix or a PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6. In another preferred embodiment, the percent identity between two nucleotide acid sequences is determined using the GAP program of the GCG software package (available at http: / / www.gcg.com) using a NWSgapdna.CMP matrix, a gap weight of 40, 50, 60, 70, or 80, and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the set that should be used unless otherwise specified) is a Blossum62 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 algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17), as incorporated into the ALIGN program (version 2.0), using a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, or alternatively, the nucleic acid and protein sequences described herein can further be used as "query sequences" to conduct searches against public databases, for example, to identify other family member or related sequences.

[0042] "Ocular diseases" as described herein include ocular diseases associated with neovascularization (eg, diseases occurring in the eye), such as ocular diseases associated with corneal neovascularization.

[0043] As used herein, "treatment" (or "treating" or "treating") refers to slowing, interrupting, halting, alleviating, halting, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.

[0044] As used herein, "prevention" (or "preventing" or "preventing") includes inhibiting the onset or progression of symptoms of a disease or disorder, or inhibiting the onset or progression of a particular disease or disorder.

[0045] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are used as self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are 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."

[0046] "Subject / patient sample" refers to a collection of cells or fluids obtained from a patient or subject. Sources of tissue or cell samples include, for example, fresh, frozen, and / or preserved organ or tissue samples, solid tissues such as biopsy samples and paracentesis samples, blood or blood components, bodily fluids such as tears, vitreous humor, cerebrospinal fluid, amniotic fluid, peritoneal fluid, interstitial fluid, and cells obtained from pregnant or developing subjects. In some embodiments, the tissue sample is ocular tissue such as the vitreous. In some embodiments, the sample is tears or vitreous humor. Tissue samples may contain compounds that are not naturally mixed with tissue, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0047] The antibody used in the formulation of the present invention may also be referred to as the antibody of the present invention. For example, the anti-VEGF A VHH antibody contained in the formulation of the present invention may also be referred to as the anti-VEGF A VHH antibody of the present invention.

[0048] Detailed Description In one embodiment, the present invention provides a liquid formulation comprising: (i) an anti-VEGF A VHH antibody or an anti-Ang2 VHH antibody, or a bispecific binding molecule that binds VEGF A and Ang2, (ii) a buffering agent, (iii) a stabilizer, and (iv) a surfactant, wherein the liquid formulation has a pH of about 5.0 to 7.5.

[0049] In some embodiments, the liquid formulation comprises an anti-VEGF A VHH antibody.

[0050] In some embodiments, the liquid formulation comprises an anti-Ang2 VHH antibody.

[0051] In some embodiments, the liquid formulation comprises a bispecific binding molecule to VEGF A and Ang2, preferably a bispecific antibody that binds to VEGF A and Ang2.

[0052] In some embodiments, the anti-VEGF A VHH comprises three CDRs: 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.

[0053] HCDR3 comprises or consists of the sequence set forth in SEQ ID NO:2.

[0054] or HCDR1 comprises or consists of the sequence set forth in SEQ ID NO:6.

[0055] HCDR2 comprises or consists of the sequence set forth in SEQ ID NO: 7 or 10.

[0056] HCDR3 comprises or consists of the sequence set forth in SEQ ID NO:8.

[0057] In some embodiments, the anti-VEGF VHH comprises or consists of the amino acid sequence 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 set forth in SEQ ID NO: 4, 5, 9, or 11.

[0058] In some embodiments, the anti-VEGF VHH comprises an amino acid sequence having 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 set forth in SEQ ID NO: 4, 5, 9, or 11, wherein the mutations are, for example, substitutions, deletions, or additions, preferably substitutions such as conservative substitutions.

[0059] In some preferred embodiments, the mutation is not in a CDR, such as HCDR1, HCDR2, or HCDR3.

[0060] In some embodiments, the anti-Ang2 VHH comprises three CDRs: HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises or consists of the sequence set forth in SEQ ID NO:16.

[0061] HCDR1 comprises or consists of the sequence set forth in SEQ ID NO:16.

[0062] HCDR2 comprises or consists of the sequence set forth in SEQ ID NO: 17 or 20.

[0063] HCDR3 comprises or consists of the sequence set forth in SEQ ID NO:18.

[0064] In some embodiments, the anti-Ang2 VHH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19 or 21, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 19 or 21.

[0065] In some embodiments, the anti-Ang2 VHH comprises an amino acid sequence having 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 set forth in SEQ ID NO: 19 or 21, wherein the mutations are, for example, substitutions, deletions, or additions, preferably substitutions such as conservative substitutions.

[0066] In some preferred embodiments, the mutation is not in a CDR, such as HCDR1, HCDR2, or HCDR3.

[0067] In some embodiments, a 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, where the second target binding region is an anti-Ang2 VHH, such as an anti-Ang2 VHH described above.

[0068] In some embodiments, the first target binding region is selected from the following:

[0069] VHH that specifically binds to VEGF A.

[0070] an antigen-binding fragment of an antibody that specifically binds to VEGF A, e.g., an scFv, e.g., the antibody is a fully human antibody or a humanized antibody; or The VEGF receptor (VEGF R) specifically binds to VEGF A or its extracellular domain, or a fusion protein containing the extracellular domain, for example, a fusion protein of the extracellular domain and Fc.

[0071] In some embodiments, the bispecific binding molecule comprises one, two, 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, trivalent, or tetravalent. In some embodiments, the bispecific binding molecule is a bispecific antibody.

[0072] In some embodiments, the bispecific binding molecule, e.g., bispecific antibody, has the following structure:

[0073] anti-VEGF antibody light chain variable region VL-linker-anti-VEGF antibody heavy chain variable region VH-linker-anti-Ang2 VHH, or Anti-VEGF antibody heavy chain variable region VH-linker-anti-VEGF antibody light chain variable region VL-linker-anti-Ang2 VHH.

[0074] wherein the anti-Ang2 VHH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises or consists of the sequence set forth in SEQ ID NO: 16. HCDR2 comprises or consists of the sequence set forth in SEQ ID NO: 17 or 20. HCDR3 comprises or consists of the sequence set forth in SEQ ID NO: 18.

[0075] In some embodiments, the structure of the bispecific binding molecule is shown in Figure 1A or Figure 1B. In some embodiments, the bispecific binding molecule consists of one chain. In some embodiments, the bispecific binding molecule is bivalent.

[0076] 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 set forth in SEQ ID NO: 31, LCDR2 comprises or consists of the sequence set forth in SEQ ID NO: 32, and LCDR3 comprises or consists of the sequence set forth in SEQ ID NO: 33.

[0077] 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 set forth in SEQ ID NO: 35, HCDR2 comprises or consists of the sequence set forth in SEQ ID NO: 36, and HCDR3 comprises or consists of the sequence set forth in SEQ ID NO: 37.

[0078] In some embodiments, the heavy chain variable region VH of the anti-VEGF antibody comprises or consists of the amino acid sequence set forth in SEQ ID NO: 34, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 34. In some embodiments, the heavy chain variable region VH comprises an amino acid sequence having one or more (preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or less) mutations compared to the amino acid sequence set forth in SEQ ID NO: 34, where the mutations are, for example, substitutions, deletions, or additions, and are preferably substitutions such as conservative substitutions. In some preferred embodiments, the mutations are not present in a CDR, such as HCDR1, HCDR2, or HCDR3.

[0079] In some embodiments, the light chain variable region VL of the anti-VEGF antibody comprises or consists of the amino acid sequence set forth in SEQ ID NO: 30, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 30. In some embodiments, the light chain variable region VL comprises an amino acid sequence having one or more (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) mutations compared to the amino acid sequence set forth in SEQ ID NO: 30, wherein the mutations are, for example, substitutions, deletions, or additions, preferably substitutions such as conservative substitutions. In some preferred embodiments, the mutations are not present in a CDR, such as LCDR1, LCDR2, or LCDR3.

[0080] In some embodiments, the anti-Ang2 VHH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19 or 21, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 19 or 21. In some embodiments, the VHH comprises an amino acid sequence having 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 set forth in SEQ ID NO: 19 or 21, where the mutations are, for example, 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. In some embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23. In some embodiments, the linker between the light chain variable region and the heavy chain variable region of an anti-VEGF antibody comprises or consists of the amino acid sequence set forth in 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 set forth in SEQ ID NO: 23, where, for example, n=2 or 3, e.g., 3.

[0081] In some embodiments, the anti-VEGF A x ANG2 bispecific binding molecule comprises or consists of the amino acid sequence set forth in SEQ ID NO: 28, or comprises an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, the bispecific binding molecule comprises an amino acid sequence having one or more (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) mutations compared to the amino acid sequence set forth in SEQ ID NO: 28, where the mutations are, for example, substitutions, deletions, or additions, preferably substitutions such as conservative substitutions.

[0082] In some embodiments, the bispecific binding molecule, e.g., bispecific antibody, has the following structure:

[0083] first anti-VEGF VHH-linker-second anti-VEGF VHH-linker-anti-Ang2 VHH, wherein the anti-Ang2 VHH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises or consists of the sequence set forth in SEQ ID NO: 16, HCDR2 comprises or consists of the sequence set forth in SEQ ID NO: 17 or 20, and HCDR3 comprises or consists of the sequence set forth in SEQ ID NO: 18.

[0084] In some embodiments, the structure of the bispecific binding molecule is shown in Figure 1C. In some embodiments, the bispecific binding molecule consists of one chain. In some embodiments, the bispecific binding molecule is trivalent. In some embodiments, the first anti-VEGF VHH is the same as or different from the second anti-VEGF VHH.

[0085] 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. HCDR3 comprises or consists of the sequence set forth in SEQ ID NO: 3. Alternatively, 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. HCDR3 comprises or consists of the sequence set forth in SEQ ID NO: 8.

[0086] In some embodiments, the anti-VEGF VHH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4, 5, 9, or 11, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 4, 5, 9, or 11. In some embodiments, the VHH comprises an amino acid sequence having 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 set forth in SEQ ID NO: 19 or 21, wherein the mutations are, for example, 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.

[0087] 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. HCDR3 comprises or consists of the sequence set forth in SEQ ID NO: 8.

[0088] In some embodiments, the anti-VEGF VHH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 9 or 11, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 9 or 11. In some embodiments, the VHH comprises an amino acid sequence having 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 set forth in SEQ ID NO: 9 or 11, wherein the mutations are, for example, 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.

[0089] In some embodiments, the anti-Ang2 VHH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19 or 21, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 19 or 21. In some embodiments, the VHH comprises an amino acid sequence having 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 set forth in SEQ ID NO: 19 or 21, where the mutations are, for example, 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.

[0090] In some embodiments, the linker comprises or consists of the amino acid sequence set forth in 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 set forth in SEQ ID NO: 23. For example, n=2.

[0091] In some embodiments, the anti-VEGF A x ANG2 bispecific binding molecule comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22, or comprises an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, the bispecific binding molecule comprises an amino acid sequence having one or more (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) mutations compared to the amino acid sequence set forth in SEQ ID NO: 22, where the mutations are, for example, 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 the anti-Ang2 VHH.

[0092] In some embodiments, the bispecific binding molecule comprises one or two of the following chains:

[0093] VEGF R extracellular domain-Fc-linker-anti-Ang2 VHH, wherein the anti-Ang2 VHH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises or consists of the sequence set forth in SEQ ID NO: 16, HCDR2 comprises or consists of the sequence set forth in SEQ ID NO: 17 or 20, and HCDR3 comprises or consists of the sequence set forth in SEQ ID NO: 18.

[0094] In some embodiments, the structure of the bispecific binding molecule is shown in Figure ID. In some embodiments, the bispecific binding molecule consists of two chains. In some embodiments, the bispecific binding molecule is tetravalent.

[0095] In some embodiments, the anti-Ang2 VHH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19 or 21, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 19 or 21. In some embodiments, the VHH comprises an amino acid sequence having 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 set forth in SEQ ID NO: 19 or 21, where the mutations are, for example, 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.

[0096] In some embodiments, the VEGFR extracellular domain is an extracellular domain of a VEGFR derived from a human. In some embodiments, the VEGFR extracellular domain comprises a second antibody-like domain of VEGFR1 (e.g., FLT1 domain 2) and a third antibody-like domain of VEGFR2 (e.g., KDR domain 3). In some embodiments, the VEGFR extracellular domain comprises a second antibody-like domain of human VEGFR1 and a third antibody-like domain of human VEGFR2. In some embodiments, the VEGFR extracellular domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:26, or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:26. In some embodiments, the VEGFR extracellular domain comprises an amino acid sequence having one or more (preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or less) mutations compared to the amino acid sequence set forth in SEQ ID NO: 26. The mutations are, for example, substitutions, deletions, or additions, preferably substitutions such as conservative substitutions. Preferably, the VEGFR extracellular domain has a binding affinity for VEGF similar to that of the domain set forth in SEQ ID NO: 26 (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similarity).

[0097] 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 or consists of the amino acid sequence set forth in SEQ ID NO: 27. Alternatively, the Fc comprises an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 27.

[0098] In some embodiments, the VEGF R extracellular domain-Fc is a fusion protein of the VEGFR extracellular domain and Fc, for example, aflibercept or a derivative thereof.

[0099] In some embodiments, the VEGF R extracellular domain-Fc comprises or consists of the amino acid sequence set forth in SEQ ID NO: 25. Alternatively, it comprises an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 25. In some embodiments, the VEGF R extracellular domain-Fc comprises an amino acid sequence having 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 set forth in SEQ ID NO: 25, wherein the mutations are, for example, substitutions, deletions, or additions, preferably substitutions such as conservative substitutions. Preferably, the VEGF R extracellular domain-Fc has a binding affinity for VEGF similar to that of the domain set forth in SEQ ID NO:25 (e.g., having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% similarity).

[0100] In some embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23. For example, n=3.

[0101] In some embodiments, the anti-VEGF A x ANG2 bispecific binding molecule comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24, or comprises an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, the bispecific binding molecule comprises an amino acid sequence having one or more (preferably no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) mutations compared to the amino acid sequence set forth in SEQ ID NO: 24, where the mutations are, for example, 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 has a binding affinity for VEGF similar to that of the domain set forth in SEQ ID NO:25 (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% similarity).

[0102] In one embodiment of the present invention, the antibodies or binding molecules described herein contain one or more amino acid mutations. In some embodiments, the amino acid mutations include amino acid substitutions, insertions, or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

[0103] In preferred embodiments, the amino acid mutations described herein occur in regions other than the CDRs (e.g., FRs). In some embodiments, the amino acid mutations described herein occur in the antibody heavy chain constant region, such as the Fc region. In preferred embodiments, the amino acid mutations in the Fc region reduce or eliminate the ADCC and / or CDC effects of the antibody.

[0104] In some embodiments, the substitution is a conservative substitution, which refers to the substitution of an amino acid for another amino acid of the same class, for example, an acidic amino acid for another acidic amino acid, a basic amino acid for another basic amino acid, or a neutral amino acid for another neutral amino acid.

[0105] In certain embodiments, one or more amino acid mutations are introduced into the Fc region of an antibody provided herein, thereby creating an Fc region variant that alters one or more functional properties of the antibody, such as serum half-life, complement fixation, complement-dependent cytotoxicity, Fc receptor binding, and / or antibody-dependent cellular cytotoxicity. The Fc region variant may comprise a human Fc region sequence (such as a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid mutation (e.g., substitution) at one or more amino acid positions.

[0106] In certain embodiments, it may be necessary to mutate the variable region of an antibody, for example, to produce an scFv that contains disulfide bond mutations.

[0107] In certain embodiments, the antibodies or binding molecules provided herein can be further modified to contain other non-proteinaceous moieties known in the art and readily available. Moieties suitable for derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, glucan, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), glucan or poly(n-vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0108] In some embodiments, the buffering agent is selected from one or more of histidine, histidine salts, glutamic acid, phosphoric acid, acetic acid, citric acid, and tris(hydroxymethyl)aminomethane. Histidine salts and glutamic acid include, but are not limited to, salts formed with amino acids and hydrochloric acid or sulfuric acid. Phosphoric acid, acetic acid, and citric acid include, but are not limited to, alkali metal salts of the corresponding acids (such as lithium, sodium, or potassium salts). For example, the phosphate salts may be in hydrated or non-hydrated form, including, but not limited to, disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, dilithium hydrogen phosphate, lithium dihydrogen phosphate, or combinations thereof. Preferably, the histidine salt is histidine hydrochloride, and the phosphate salt is a combination of disodium hydrogen phosphate and sodium dihydrogen phosphate. Preferably, the buffering agent is selected from a combination of histidine and histidine hydrochloride, a phosphate salt, and histidine.

[0109] In some embodiments, the stabilizer is selected from one or more of sugars, polyols, and amino acids or salts thereof. Preferably, the sugars are selected from, but not limited to, sucrose, dextrose, lactose, maltose, trehalose, cyclodextrin, maltodextrin, and glucan. The polyols are selected from, but not limited to, mannitol, sorbitol, and xylitol. The amino acids or salts thereof are selected from one or more of arginine, glycine, proline, methionine, and salts thereof. The cyclodextrin is preferably hydroxypropyl-β-cyclodextrin.

[0110] In some embodiments, the stabilizer is sucrose or trehalose, hi some embodiments, the stabilizer is a combination of sucrose or trehalose with an additional component selected from one or more of glycine, proline, methionine, and hydroxypropyl-β-cyclodextrin.

[0111] In some embodiments, the surfactant is a non-ionic surfactant such as an alkyl poly(ethylene oxide), a polysorbate (such as polysorbate 20, polysorbate 80, polysorbate 60, or polysorbate 40), Pluronic®, etc. In some preferred embodiments, the liquid formulations of the present invention comprise polysorbate-80 as a surfactant.

[0112] In some embodiments, the concentration of the antibody or bispecific binding molecule in the liquid formulation of the invention is, e.g., about 1-200 mg / mL, e.g., 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. 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.

[0113] 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, or about 8-12 mM, e.g., 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 contains multiple components, 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, e.g., 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.

[0114] 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 250-280 mM, about 200-400 mM, or about 100-200 mM, e.g., 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 comprises multiple components, the concentration of each component can be selected from the following ranges or values:

[0115] About 20-800 mM, about 30-700 mM, about 50-800 mM, about 50-500 mM, about 100-400 mM, about 100-300 mM, about 200-350 mM, about 200-300 mM, about 250-280 mM, about 200-400 mM 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, e.g., 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.

[0116] In some embodiments, the concentration of surfactant in the liquid formulations 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.

[0117] In some embodiments, the pH of the liquid formulations 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, e.g., 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.

[0118] In some embodiments, the liquid formulations of the present invention have an osmolality of 200-400 mOsmol / kg or 250-350 mOsmol / kg, for example, an osmolality of 350 mOsmol / kg.

[0119] In some embodiments, the liquid formulation is a pharmaceutical formulation, preferably an injectable formulation, more preferably an intravitreal injectable formulation, or an eye drop formulation.

[0120] In some embodiments, the liquid formulation of the present invention comprises:

[0121] (i) about 10-160 mg / mL, preferably 30-150 mg / mL, of the bispecific binding molecule. (ii) about 5-20 mM buffer, the buffer being histidine or a combination of histidine and histidine hydrochloride;

[0122] (iii) about 200-350 mM trehalose, and (iv) about 0.1-1 mg / mL of polysorbate 80, wherein The pH of the liquid formulation is about 6.1-7.0, preferably about 6.5.

[0123] In some embodiments, the liquid formulation of the present invention comprises:

[0124] (i) about 10-160 mg / mL, preferably 30-150 mg / mL, of the bispecific binding molecule. (iii) about 8-12 mM histidine, and (iii) about 200-300 mM trehalose, and (iv) about 0.2-0.4 mg / mL of polysorbate 80, wherein The pH of the liquid formulation is about 6.1-6.6, preferably about 6.5.

[0125] In some embodiments, the liquid formulation of the present invention comprises:

[0126] (i) about 10-160 mg / mL, preferably 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 of polysorbate 80, wherein The pH of the liquid formulation is about 6.1-6.6, preferably about 6.5.

[0127] In some embodiments, the liquid formulations of the present invention are those disclosed in Examples 15 through 17. Additionally, embodiments resulting from a 50%, 40%, 30%, 20%, 10%, or 5% variation in the concentration of ingredients in such formulations, and a 5%, 4%, 3%, 2%, 1%, or 0.5% variation in pH value, based on the formulations disclosed in Examples 15 through 17, are also encompassed by the present invention.

[0128] 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.

[0129] These and other known pharmaceutical excipients and / or additives suitable for use in the formulations of the present invention are well known in the art, as described, for example, in The Handbook of Pharmaceutical Excipients, 4th edition, edited by Rowe, American Pharmaceuticals Association (2003); and Remington: The Science and Practice of Pharmacy, 21st edition, edited by Gennaro, Lippincott Williams & Wilkins (2005).

[0130] Unless otherwise specified, it should be understood that the liquid formulations of the present invention further comprise a vehicle, including, but not limited to, purified water such as ultrapure water, water for injection, sterilized water, and double-distilled water. The liquid formulations of the present invention can be prepared or formulated using pharmaceutically acceptable solvents or solutions known in the art. Such pharmaceutically acceptable solvents or solutions include, but are not limited to, purified water such as ultrapure water, water for injection, sterilized water, double-distilled water, saline, Ringer's solution, and glucose injection.

[0131] In some embodiments, the anti-Ang2 VHH antibody can specifically bind to Ang2, eg, human Ang2, eg, with high affinity.

[0132] In some embodiments, the anti-VEGF A VHH antibody can specifically bind to VEGF A, eg, human VEGF A, eg, with high affinity.

[0133] In some embodiments, the bispecific binding molecule can specifically bind to Ang2 and VEGFA, eg, human Ang2 and human VEGFA, eg, with high affinity.

[0134] In some embodiments, the anti-Ang2 antibody or anti-VEGFA antibody or bispecific binding molecule has one or more of the following properties:

[0135] (i) The anti-Ang2 antibody or bispecific binding molecule has the effect of inhibiting the phosphorylation of Tie2 by Ang2.

[0136] (ii) The anti-Ang2 antibody or bispecific binding molecule has the effect of blocking the binding of Ang2 to Tie2.

[0137] (iii) The anti-VEGFA antibody or bispecific binding molecule has the effect of blocking a VEGFA activation-associated receptor signaling pathway (e.g., as detected by a KDR reporter).

[0138] (iv) The anti-VEGFA antibody or bivalent binding molecule has the effect of inhibiting the binding of VEGFA to VEGFR.

[0139] (v) The anti-VEGFA antibody or bispecific binding molecule has the effect of inhibiting VEGF A-induced cell (e.g., vascular endothelial cells, e.g., human umbilical vein endothelial cells, e.g., HUVEC) survival and proliferation.

[0140] (vi) The bispecific binding molecule has the effect of inhibiting leakage of vascular endothelial cells (eg, human umbilical vein endothelial cells, e.g., HUVEC).

[0141] (vii) The bispecific binding molecule has the effect of inhibiting neovascularization in vivo or in vitro, e.g., inhibiting neovascularization of the fundus or retina and choroid, e.g., inhibiting leakage due to neovascularization, e.g., preserving the integrity of blood vessels.

[0142] In another aspect, the present invention provides a solid formulation obtained by solidifying the liquid formulation of the present invention. The solidification process can be carried out, for example, by crystallization, spray drying, or freeze-drying. In one preferred embodiment, the solid formulation is in the form of, for example, a freeze-dried powder for injection. The solid formulation can be reconstituted with an appropriate solvent before use to form a reconstituted formulation of the present invention. The reconstituted formulation is also a liquid formulation of the present invention. In one embodiment, the appropriate solvent is selected from ultrapure water, water for injection, organic solvent for injection (including, but not limited to, oil for injection, ethanol, propylene glycol, etc.), and combinations thereof.

[0143] The formulations of the present invention (including solid and liquid formulations) can be stably stored for long periods, for example, at least 24 months or longer. In one embodiment, the formulations of the present invention can be stably stored at about -80°C to about 45°C, e.g., -80°C, about -30°C, about -20°C, about 0°C, about 5°C, about 25°C, about 35°C, about 38°C, about 40°C, about 42°C, or about 45°C, for 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.

[0144] In one embodiment, the stability of a liquid formulation of the present invention is visually inspected after storage, and the liquid formulation of the present invention remains a clear to slightly opalescent, colorless to pale yellow, particle-free liquid in appearance. In one embodiment, visual inspection with a transparency detector reveals that no visible particles are present in the formulation. In one embodiment, the stability of a liquid formulation of the present invention is tested by measuring the change in protein content after storage, where the rate of change in protein content, as measured, for example, by ultraviolet spectrophotometry (UV), is 20% or less, preferably 10% or less, e.g., 7-8%, more preferably 5% or less, relative to the initial value on day 0 of storage. In one embodiment, the stability of a liquid formulation of the present invention is tested by measuring the change in purity of a liquid formulation of the present invention after storage, where the change in monomer purity, as measured by size-exclusion high-performance liquid chromatography (SEC-HPLC), is 10% or less, e.g., 5% or less, 4% or 3% or less, e.g., 1-2%, preferably 1% or less, relative to the initial value on day 0 of storage. In one embodiment, the stability of a liquid formulation of the invention is tested by measuring the change in purity of the formulation after storage, where the change in monomer purity is reduced to 10% or less, e.g., 5%, 4%, 3%, 2%, or 1% or less, as measured by non-reducing and / or reducing sodium dodecyl sulfate capillary electrophoresis (CE-SDS). In one embodiment, the stability of a liquid formulation of the invention is tested by imaging capillary isoelectric focusing (iCIEF) after storage, where the total change in charge variants (major, acidic, and basic species) is 30% or less, e.g., 20% or less, 10% or less, 5% or less, 2% or less, or 1% or less, relative to the initial value on day 0 of storage.

[0145] In another aspect, the present invention provides a delivery device comprising the 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 pre-filled syringe containing the liquid or solid formulation of the present invention, for example, for use in intravitreal, intravenous, subcutaneous, intradermal or intramuscular injection, or intravenous infusion.

[0146] In another aspect, the present invention provides a method for preparing the liquid formulation of the present invention, comprising the steps of:

[0147] i. An isolated and purified antibody or bispecific binding molecule of the invention is provided and, if desired, placed in an ultrafiltration centrifuge tube and concentrated by centrifugation.

[0148] ii. A solution of buffers and stabilizers, preferably aqueous, is prepared, wherein the identity and concentration of the buffers and stabilizers, and the pH of the solution are preferably as defined above for the liquid formulations of the present invention.

[0149] iii. Ultrafiltering the antibody from step i into the solution from step ii.

[0150] iv. The resulting protein solution is adjusted to the concentration specified for the liquid formulation of the present invention.

[0151] v. A surfactant or a solution thereof, preferably an aqueous solution, is added to adjust the final concentration of the surfactant to the concentration specified in the liquid formulation of the present invention.

[0152] vi. If necessary, sterile filter the solution from step v.

[0153] vii. If necessary, aliquot the solution into vials, seal the vials with rubber stoppers and aluminum-plastic caps, and use the product.

[0154] In another aspect, the invention provides a method for preventing or treating an eye disease in a subject, comprising administering to the subject a formulation of the invention.

[0155] In some embodiments, the patient has VEGF, eg, VEGF A, and / or Ang2 (eg, high levels, eg, nucleic acid or protein levels).

[0156] In some embodiments, the ocular disease includes, but is not limited to, angiogenesis-associated ocular diseases, such as corneal neovascularization-associated ocular diseases.

[0157] In some embodiments, treatment of ocular diseases is beneficial by inhibition of VEGF, eg, VEGF A and / or Ang2, at the nucleic acid or protein level.

[0158] In another aspect, the invention provides the use of a formulation of the invention in the manufacture or preparation of a medicament or delivery device for a use described herein, e.g., for the prevention or treatment of a related disease or condition described herein.

[0159] In some embodiments, the formulations of the present invention delay the onset of the condition and / or symptoms associated with the condition.

[0160] In some embodiments, the formulations of the present invention can also be administered in combination with one or more other therapies, e.g., therapies and / or other therapeutic agents, e.g., for the uses described herein, e.g., for the prevention or treatment of a related disease or related condition described herein.

[0161] The formulations of the present invention can be administered by known methods, for example, by topical administration, such as intraocular administration or conjunctival administration. In some embodiments, administration is by injection or eye drops.

[0162] These and other aspects and embodiments are illustrated in the drawings (a brief description of the drawings follows) and the detailed description below, and are illustrated in the examples below. All or part of the features described above and throughout the invention can be combined in various embodiments of the invention. The following examples further illustrate the invention. However, it should be understood that these examples are illustrative and not limiting. Various modifications may be made by those skilled in the art. [Example]

[0163] Example 1: Preparation of a phage immune library Construction of alpaca immune or synthetic libraries 1.1 Two healthy adult alpacas (Chengdu NBbiolab, Co. Ltd.) were selected and immunized four times with 0.5 mg of recombinant protein antigen VEGFA or Ang2 (Beijing Sino Biological) homogeneously mixed with Freund's adjuvant at a 1:1 ratio via subcutaneous injection at multiple sites on the back (with a 2-week interval between immunizations).

[0164] 1.2 Peripheral blood (50 mL) was collected from alpacas, lymphocytes were isolated, and total RNA was extracted using chloroform / isopropanol precipitation with 1 mL of Trizol reagent added per 2.5 × 10 viable cells. Reverse transcription was performed using the PrimeScript Reverse Transcription Kit (Takara) with 10 μg of RNA as a template. The first round of PCR was performed using the cDNA as a template and forward primer Alp-VhL and reverse primer Alp-2b / 2cR to obtain the first-round PCR product. The first-round PCR product was used as a template for a second round of PCR using forward primer Alp-VhF and reverse primer Alp-JHR-SalI to obtain the second-round PCR product. The pC3-HF vector and the second PCR product were digested twice with SacI and SalI, respectively (Thermo). The digested products were ligated with T4 ligase (Thermo) and electrotransformed into TG1-sensitive cells to construct a VHH antibody library. The bacterial solution was stored frozen at -80°C.

[0165] The thawed bacterial suspension was inoculated into 100 mL of YT-AG medium (Shanghai Sangon Biotech) and infected with M13KO7 helper phage. The bacterial cell pellet was resuspended in 2x YT-AK medium (Shanghai Sangon Biotech) and cultured overnight at 37°C and 200 rpm. The culture supernatant was collected, and recombinant phage was prepared using the PEG / NaCl precipitation method.

[0166] 1.3 Recombinant phages were subjected to three rounds of panning experiments using biotin-labeled antigens VEGFA (ACRO) or Ang2 (Beijing Sino Biological). 50 μL of M280 magnetic beads (Thermo) and the appropriate amount of biotin-labeled antigen were added to each tube and incubated at room temperature for 30 minutes. 1 × 1012 cfu of recombinant phages were added and incubated at room temperature for 1 hour. The resulting mixture was washed ten times with 1 mL of PBST for 5 minutes each. Finally, 0.5 mL of glycine buffer, pH 2.5, was added to elute the antigen-bound recombinant phages, which were then infected with TG1 cells and cultured overnight. Recombinant phages were prepared for the next panning experiment, and TG1 bacterial clones positive for the VHH were identified.

[0167] 1.4 Binding activity was detected by binding ELISA and clones were sequenced.

[0168] VEGF A and Ang2 antigens (Beijing Sino Biological) were prepared in advance and diluted to 0.5 μg / mL in PBS buffer. They were coated onto a 96-well ELISA plate and stored overnight in a refrigerator at 4°C. The antigen-coated plate was washed three times with PBST, and 300 μL of blocking reagent was added to each well and left at room temperature for 1 hour. The plate was then washed three times with PBST, and 80 μL of blocking reagent and 20 μL of the expression supernatant of the TG1 strain, which was positive for the VHH identified in 1.3 above, were added and shaken at room temperature for 1 hour.

[0169] The plate was washed three times with PBST, and 100 μL / well of anti-Flag / HRP secondary antibody (Sigma) diluted in blocking reagent was added and shaken at room temperature for 40 minutes. The plate was washed six times with PBST, and 100 μL / well of TMB color development solution was added. The color development reaction was allowed to proceed in the dark for 5-15 minutes. 100 μL / well of stop solution was then added. The plate was read using a microplate reader to measure absorbance at OD450 nm. Bacterial clones with a reading of 0.5 or higher were selected and sent to Genewiz for sequencing. A single clone of the TG1 strain containing the corresponding VHH sequence was selected and glycerol was added. The mixture was frozen and stored in a -80°C freezer.

[0170] Example 2: Prokaryotic antibody production, purification and humanization According to the present invention, an antibody sequence was obtained from an anti-VEGFA or Ang2-positive phage using molecular biological techniques, and the obtained VHH-containing TG1 monoclone was used for expression and purification to obtain a VHH antibody protein.

[0171] The TG1 strain containing the VHH expression plasmid identified in Example 1 was inoculated into 800 mL of LB-Amp medium and cultured at 37°C and 200 rpm until an OD600 value of 0.5-0.6 was reached. Expression was induced by adding 1 mM IPTG to the bacterial suspension, and the cells were cultured overnight at 28°C and 200 rpm. The culture supernatant was collected. After centrifugation, 15 mL of PB and 1 mg / mL polymyxin were added to resuspend the bacteria. The mixture was centrifuged again and filtered through a 0.22 μm filter. The bacterial lysate was passed through a 1 mL Ni Sepharose precolumn, and the column was washed twice with PBS. The target protein was eluted with 0.5 M imidazole, and the protein concentration was measured by UV light. The eluted target protein was measured by UV light, dispensed into multiple tubes, and stored in a refrigerator at -40°C. The resulting antibody solution is hereafter referred to as the "supernatant." The amino acid sequences of the CDRs and VHHs of two anti-VEGF A VHH antibodies (LA42F8 and LA46E11) and one anti-Ang2 VHH antibody (LA24C11) obtained according to the present invention, as well as SEQ ID NOs, are shown in the sequence listing.

[0172] Next, the immune librarian LA42F8, LA46E11, and LA24C11 obtained above were humanized as follows.

[0173] (1) Determine the CDR loop structure.

[0174] (2) For each V / J region of the heavy chain, the closest homologous sequence is searched for in the human geralin sequence database.

[0175] (3) Screening the human genome for the most highly identical heavy and light chains with minimal back mutations.

[0176] (4) Constructing the CDRs of a chimeric antibody on the framework of a human antibody.

[0177] (5) Based on the sequence and structural features, amino acid positions that maintain CDR function in the framework are determined.

[0178] (6) Add back mutations (back to the input amino acids) at identified critical positions.

[0179] (7) Optimize the amino acids at risk sites.

[0180] The amino acid sequences of the CDRs and heavy chain variable regions of three humanized antibodies obtained according to the present invention, namely humanized VHH antibodies LA42F8.5, LA46E11.8 and LA24C11.10, are shown in the attached sequence listing.

[0181] The expression and preparation of the above-mentioned LA42F8, LA46E11, LA24C11, and humanized antibodies LA42F8.5, LA46E11.8, and LA24C11.10 in eukaryotic cells are as follows.

[0182] The humanized antibody sequences obtained above were cloned into pcDNA3.1 (Invitrogen) to obtain plasmids containing the antibody sequences.

[0183] Expi-293 cells (Invitrogen) were passaged according to the desired transfection amount. The day before transfection, the cell density was adjusted to 1.5 × 10 6 The cell density on the day of transfection was adjusted to approximately 3 × 10 cells / mL. 6 The transfection buffer was 1 / 10 of the final volume of F17 medium (Gibco, A13835-01). The appropriate amount of plasmid was added and mixed uniformly. The appropriate amount of polyethylenimine (PEI) (Polysciences, 23966) was added to the plasmid (for 293F cells, the ratio of plasmid to PEI was 1:3), mixed, and incubated at room temperature for 10 minutes to prepare a DNA / PEI mixture. After resuspending the DNA / PEI mixture, the cells were transfected at 36.5°C and 8% CO2. After 24 hours, 2% of the transfection amount of FEED (Sigma) was added, and the cells were cultured at 36.5°C, 8% CO2, and 120 rpm. On the 6th day of passage, or when viability fell below 60%, the cell supernatant was collected and purified.

[0184] A gravity column for purification was treated with 0.5 M NaOH overnight. The glassware was washed with distilled water and dried at 180 °C for 4 hours to prepare a purification column. Prior to purification, the collected cell supernatant was centrifuged at 4500 rpm for 30 minutes, and the cells were discarded. The supernatant was filtered through a 0.22 μL filter. A Protein A column (Hitrap Mabselect Sure5 × 5 mL, 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 loaded onto the purification column and re-equilibrated with 15 mL of binding buffer. Five mL of elution buffer (citric acid + 0.1 M sodium citrate, pH 3.5) was added. The eluate was collected, and 80 μL of Tris-HCl was added per mL of eluate. The recovered antibodies were buffer exchanged with PBS (Gibco, 70011-044) by ultrafiltration / dialysis, and their concentrations were measured. In Table 3, Figure 2, Figure 4A, and Figure 5A, except for the antibodies of the present invention used for detection, all antibodies used in the examples were expressed and purified by these methods unless otherwise specified for the supernatants described below.

[0185] Similarly, the coding nucleic acids of negative control IgG, positive control BI-anti-VEGF, BI836880, and faricimab (sequences shown in the sequence listing) were cloned into pcDNA3.1, transfected into Expi-293 cells, expressed, and purified in the same manner as in Example 2.

[0186] Example 3: Binding kinetics of the chimeric antibody of the present invention to an antigen (measured by biolayer interferometry) The equilibrium dissociation constant (KD) of the antibodies of the present invention binding to human Ang2 was measured by biolayer interferometry (ForteBio). A prior art ForteBio affinity assay was performed (Estep, P., et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning, MAbs, 2013.5(2):270-8).

[0187] Thirty minutes before the experiment, depending on the number of samples, an appropriate number of AMQ (Pall, 1506091) (for sample detection) or AHQ (Pall, 1502051) (for positive control detection) sensors were immersed in SD buffer (PBS 1x, BSA 0.1%, Tween®-20 0.05%).

[0188] 100 μL of SD buffer, the VHH antibodies prepared in Example 2 above, and antigens (including human Ang2 (Beijing Sino Biological) and human VEGF165 (R&D)) were added to a 96-well black polystyrene half-area microplate (Greiner, 675076). The sensors were positioned according to the sample location. The instrument settings were baseline, load -1 nm, baseline, association, and dissociation in this order, with the duration of each step depending on the association and dissociation rates. The rotation speed was 400 rpm and the temperature was 30°C. D The values ​​were analyzed using ForteBio analysis software.

[0189] In experiments performed with the above assays, the affinities of the antibodies are shown in Table 1: [Table 2] [Table 3]

[0190] Example 4: ELISA inhibition assay (anti-VEGF A VHH antibodies) This example demonstrates the inhibitory effect of anti-VEGF A VHHs of the present invention on the binding of hVEGF A to its receptor KDR. SA (Thermo, Cat. No. 21125) was diluted to 1 μg / mL and 100 μL / well was plated onto a microplate 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 biotin-labeled VEGF A165 (ACRO, Cat. No. VE5-H8210) was added, followed by incubation of the plate for 1.5 hours. The LA42F8 supernatant, LA46E11 supernatant, negative control IgG, and positive control BI-anti-VEGF (50 μL) of the antibodies prepared in Example 2 (initial concentration 150 μg / mL, 3-fold dilution) were pre-reacted with VEGFR-Fc (Beijing Sino Biological, catalog number 10012-H02H, final concentration 0.2 μg / mL) for 20 minutes, and then the mixture was added to the plate. The plate was washed three times with PBST, and anti-human Fc HRP antibody (Bethyl, catalog number A80-104P) (1:10000) was added and incubated for 30 minutes. The plate was washed six times with PBST, and the color reaction was developed with TMB for 5 minutes. After stopping, the OD450 nm was measured.

[0191] The inhibition 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 completely inhibit the binding of VEGF A to VEGFR2.

[0192] Example 5: ELISA inhibition assay (anti-Ang2 VHH antibodies) This example demonstrates the inhibitory effect of the anti-Ang2 VHH antibody of the present invention on the binding of hAng2-biotin (R&D, catalog number BT623B / CF) to Tie2 protein.

[0193] Tie2-Fc (Beijing Sino Biological, catalog number 10700-H03H) was diluted to 2 μg / mL and added to a microplate at 100 μL / well. The plate was then incubated overnight at 4°C. The plate was washed three times with PBST and blocked with 3% BSA for 1.5 hours. The purified LA24C11.10 and negative control IgG (50 μL each) prepared in Example 2 were pre-incubated with hAng2-biotin (final concentration 0.2 μg / mL) for 20 minutes, and the resulting mixture was added 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. The color reaction was developed with TMB for 5 minutes, stopped, and the OD450 nm was measured.

[0194] The inhibitory results of the anti-Ang2 VHH antibodies obtained in the present invention are shown in Table 3, and it was found that the candidate molecule LA24C11 has the effect of inhibiting the binding of Ang2 to its receptor Tie2. [Table 4]

[0195] Similar to Example 4 (using Ang2-Bio (R&D, catalog number BT623B / CF)), the inhibitory effect of LA24C11.10 on the binding of Ang2 to Tie2 was detected by ELISA inhibition experiments. The results are shown in Figure 3. LA24C11.10 was found to have an inhibitory effect on the binding of Ang2 to its receptor, Tie2.

[0196] Example 6: Phosphorylation assay of anti-Ang2 VHH antibodies This example demonstrates the inhibitory effect of the anti-Ang2 VHH antibodies of the present invention on hAng2-Fc-induced Tie2 phosphorylation.

[0197] The hAng2 phosphorylation assay was carried out as follows.

[0198] In this study, Tie2-overexpressing Expi293 cells (293-Tie2) were co-cultured with antibodies and recombinant hAng2-Fc protein, and the content of phosphorylated Tie2 in the system was detected to reflect the inhibitory effects of different antibodies on hAng2-fc-induced Tie2 phosphorylation.

[0199] The pCHO1.0 vector (Invitrogen) carrying the human Tie2 gene (Beijing Sino Biological, catalog no. HG10700-M) cloned into the multiple cloning site (MCS) was transfected into Expi-293 cells (Thermo) to generate Expi-293 cells overexpressing human Tie2, i.e., 293-Tie2 cells.

[0200] 2 × 10 293-Tie2 cells overexpressing human Tie2 6 cells / mL, added to a 96-well plate at 100 μL / well, centrifuged at 400 g for 5 min, and the supernatant was discarded.

[0201] Experimental media were prepared using Expi293 medium (Thermo, catalog number A1435102): test antibodies (LA24C11 (24C11) and LA24C11.0 (hz24C11.10) prepared in Example 2, negative control IgG, and positive control nesbacumab (prepared based on CN202010573625.2)) were added at an initial concentration of 60 μg / mL and serially diluted at a ratio of 1:2. The final concentration of hAng2-Fc (Beijing Sino Biological: 10691-H02H) was 2.5 μg / mL.

[0202] Cells were resuspended in 100 μL of experimental medium per well and incubated at 37°C for 15 min.

[0203] The medium was removed by centrifugation, and 100 μL of NP-40 lysis buffer (Beyotime, Cat. No. P0013F) containing 1% protease (Thermo, Cat. No. 78442) and phosphatase inhibitors (Thermo, Cat. No. 78442) was added. The plates were left on ice for 30 minutes, centrifuged at 2000 g, and the protein supernatant was collected and stored in a refrigerator at -80°C.

[0204] The concentration of pTie2 was measured according to the instructions of the Phosphorylated Tie2 ELISA Kit (R&D, Catalog No. DYC2720E). The capture antibody included in the kit was coated onto a microplate at a concentration of 4 μg / mL and incubated overnight at 4°C. The plate was washed three times with PBST and blocked with 5% BSA for 1 hour. A standard curve was prepared by adding 100 μL of the frozen-thawed protein supernatant obtained in the last step to control pTie2 (R&D, Catalog No. DYC2720E). This mixture was then incubated at room temperature for 2 hours. (If the pTie2 concentration in the sample is too high beyond the ELISA detection range, the resulting mixture can be diluted 2-3 times and administered to the subject.) The plate was washed three times with PBST, and 100 μL of HRP-conjugated anti-pTyr antibody (R&D, Catalog No. DYC2720E) was added. The mixture was then incubated at room temperature for 2 hours. The plate was washed six times with PBST, and 100 μL of TMB (Solarbio, Cat. No. PR1200) was added to develop the color. After 15 minutes, 100 μL of stop buffer (Solarbio, Cat. No. C1058) was added to stop the reaction. OD450-OD620 was measured using a SpectraMax i3 multimode microplate reader. The results are shown in Figure 4A (24C11 is the purified LA24C11 prokaryotic expression supernatant prepared in Example 2) and Figure 4B.

[0205] Therefore, the anti-Ang2 VHH antibodies LA24C11 and LA24C11.10 of the present invention can effectively inhibit hAng2-Fc-induced phosphorylation of 293-Tie2 cells in vitro.

[0206] Example 7: KDR reporter blocking assay of anti-VEGF A VHH antibodies VEGF-A binds to its related receptor, VEGFR2 (KDR), activating the VEGFR2 signaling pathway, which induces endothelial cell survival, proliferation, and migration. In this study, we used a KDR reporter experimental system to detect the blocking effect of gradient-diluted antibodies on the VEGFA-related receptor signaling pathway using NFAT-RE-luc2P / KDR HEK293 cells (Promega, Cat. No. CS181401).

[0207] The experimental procedures followed the instructions of the supplier (Promega). NFAT-RE-luc2P / KDR HEK293 cells were cultured in experimental medium (DMEM medium containing 10% FBS) three days prior. The old medium was removed by pipetting. After washing the medium once with PBS, the cells were digested with 1 mL of accutase solution (Sigma, catalog no. A6964-500ML) until the cells became round and detached from the cell wall. The reaction was stopped with 5 mL of dilution medium. The cells were transferred to a centrifuge tube by pipetting and centrifuged at 1000 rpm for 5 minutes. The medium was discarded, and 10 mL of dilution medium (DMEM medium containing 10% FBS) was added to resuspend the cells. The cells were mixed uniformly and counted. Cell viability should be above 90%. The cell density in dilution medium was adjusted to 0.8 × 10 6 cells / mL and added 50 μL / well to a 96-well white cell culture plate according to the experimental layout.

[0208] A mixture of 100 ng / mL VEGF A and serially diluted antibody was prepared and allowed to stand for 30 minutes before adding 50 μL / well to a 96-well white cell culture plate containing cells. The plate was placed in a 37°C incubator with 5% carbon dioxide and incubated for 6 hours. The samples tested were as follows: negative control IgG, positive control BI-anti-VEGF, LA42F8, LA46E11, LA42F8.5, and LA46E11.8. Blank: contained diluted medium only, no VEGF A or antibody; VEGF A 100 ng / mL: contained 100 ng / mL VEGF A only.

[0209] After 6 hours of incubation, the 96-well white cell culture plate was removed from the carbon dioxide incubator and equilibrated at room temperature for 10-15 minutes. The Bio-Glo Luciferase Assay System, previously equilibrated at room temperature, was added to the 96-well white cell culture plate at 100 μL / well according to the experimental layout, and the plate was incubated at room temperature in the dark for 5 minutes.

[0210] Fluorescence values ​​were measured using a multimode microplate reader with the plate reading mode set to chemiluminescence, the plate reading type set to endpoint, and the wavelength set to full wavelength. Fluorescence was collected row by row, with a collection time of 1000 ms for each row.

[0211] In the experiments performed using the above test method, the detection results in Figure 5 showed that the anti-VEGF VHH antibodies LA42F8, LA42F8.5, LA46E11, and LA46E11.8 all inhibited the activation of the KDR signaling pathway by VEGF A.

[0212] Example 8: Inhibition assay of VEGF A-induced HUVEC survival and proliferation by anti-VEGF A VHHs VEGF A may induce angiogenesis by acting on VEGFR and other related receptors on vascular endothelial cells, promoting their survival, proliferation, and migration. This example is based on the inhibitory effects of antibodies on VEGF-induced human umbilical vein endothelial cell (HUVEC) survival and proliferation, as well as on VEGF A-induced primary cell survival and proliferation.

[0213] In this example, HUVEC survival and proliferation were measured using CCK-8. The specific method was as follows: HUVECs (Allcells, catalog number H-001-CN) were treated one day in advance, seeded at 2000 cells / well in a 96-well culture plate, and cultured in a 37°C incubator containing 5% carbon dioxide for 24 hours.

[0214] After the cells bound to the wall, VEGF A at a final concentration of 10 ng / mL and serially diluted antibodies (LA42F8 prepared in Example 2, initial concentration 80 μg / mL, serially diluted at a 1:3 ratio, negative control IgG, positive control BI836880, a group containing only 10 ng / mL VEGF A (VEGFA), and a blank containing neither VEGF A nor antibody) were prepared, the endothelial cell medium in the 96-well plate was replaced with experimental medium, and the plate was placed in an incubator at 37°C with 5% carbon dioxide and cultured for 72 hours.

[0215] CCK-8 detection solution (Dojindo, catalog number CK04) was added at 10 μL / well, and the plate was placed in a 5% carbon dioxide-containing incubator at 37°C and cultured for 12 to 24 hours.

[0216] Using a multimode microplate reader, measure the absorbance OD 450 -OD 620 The values ​​were measured.

[0217] The results of the experiments performed using the above assay are shown in Figure 6. The results demonstrate that the anti-VEGFA antibody LA42F8 completely inhibits VEGFA-induced survival and proliferation of HUVEC cells.

[0218] Example 9: HEK293-KDR reporter blocking assay of anti-VEGF A / Ang2 bispecific antibodies The chains of the bispecific binding molecules of the invention IEX04-008, IEX04-010 and IEX04-012 (sequences shown in the sequence listing) were constructed in the pcDNA3.1 vector and the bispecific binding molecules were expressed in 293 cells and purified as in Example 2.

[0219] In this example, the HEK293-KDR reporter assay was used to detect that the anti-VEGF A / Ang2 bivalent antibody has an inhibitory effect on VEGF A. The experimental procedures were as described in Example 7.

[0220] The binding molecules or controls used were as follows: Blank: no VEGF A and antibody; VEGF A: 100 ng / mL VEGF A.

[0221] Negative control IgG: same as above Positive control faricimab: Prepared as above with an initial concentration of 13.5 μg / mL and serially diluted at a ratio of 1:3.

[0222] Positive control BI-836880: Prepared as above, with an initial concentration of 13.5 μg / mL and serially diluted at a ratio of 1:3.

[0223] IEX04-012: Prepared as above, with an initial concentration of 13.5 μg / mL and serially diluted at a ratio of 1:3.

[0224] The detection results in Figure 7 demonstrate that the bispecific binding molecule IEX04-012 inhibits the activation of the KDR signaling pathway by VEGF, and has superior inhibitory activity compared to the control antibody BI836880.

[0225] Example 10: HUVEC proliferation inhibition assay of anti-VEGF A / Ang2 bispecific binding molecules In this study, the inhibitory effect of anti-VEGF A / Ang2 bispecific binding molecules on VEGF A-induced survival and proliferation of HUVEC cells was detected by HEK293-KDR reporter assay.

[0226] The experimental procedure was the same as in Example 8, and the antibodies used were as follows:

[0227] Panel A: IEX04-008, negative control IgG, positive controls faricimab and BI836880, blank (i.e., no antibody and VEGF A), and VEGF A group (i.e., VEGFA only at 20 ng / mL) were prepared as above, with an initial concentration of 20 μg / mL and serially diluted at a ratio of 1:3.

[0228] Panel B: Negative control IgG, BI-anti-VEGF, IEX04-010, blank (i.e., no antibody and VEGF A), and VEGF A group (i.e., only 20 ng / mL VEGFA added) were prepared as above, with an initial concentration of 80 μg / mL and serially diluted at a ratio of 1:3.

[0229] Panel C: IEX04-012, negative control IgG, positive controls faricimab and BI836880, blank (i.e., no antibody and VEGF A), and VEGF A group (i.e., 20 ng / mL VEGFA only) were prepared as above, with an initial concentration of 20 nM and serially diluted at a ratio of 1:3.

[0230] The detection results in Figure 8 showed that the bispecific binding molecules IEX04-008, IEX04-010, and IEX04-012 all inhibited VEGF-induced HUVEC cell survival and proliferation, and IEX04-012 had a lower IC50 and stronger inhibitory potency than the control antibodies BI836880 and faricimab.

[0231] Example 11: Ang2 blocking assay of anti-VEGF A / Ang2 bispecific antibodies Ang2 can bind to its natural receptor Tie2, and in this study, we detected the blocking effect of anti-VEGF A / Ang2 bispecific binding molecules on the binding between Ang2 and Tie2 by ELISA assay and FACS assay.

[0232] (1) ELISA The ability of IEX04-008, IEX04-010, IEX04-012, and the control antibodies BI-836880 and faricimab to inhibit the binding of human Ang2 to hTie2 was measured by ELISA.

[0233] hTie2 protein (Beijing Sino Biological) was resuspended and dissolved in PBS at a concentration of 2 μg / mL, coated onto a microplate, and incubated overnight. The plate was blocked with 5% BSA for 1 hour, and biotinylated antigen (recombinant biotinylated hAngiopoietin-2 protein (R&D)) was diluted to 600 μg / mL and added to the plate at 50 μL / well. Antibodies prepared as described above (IEX04-008, IEX04-010, IEX04-012, positive control antibody BI836880, and faricimab, negative control IgG) were serially diluted in 8 or 12 steps at a 1:2 ratio from a maximum concentration of 300 nM. 50 μL of diluted antibody was added per well and incubated on ice in PBS for 30 minutes, resulting in a final biotinylated antigen concentration of 300 ng / mL. The antigen-antibody mixture obtained above was incubated in a microplate for 90 minutes, washed three times with PBS, and the supernatant was discarded. Avidin-HRP (Invitrogen) was added to each well at a 1:10,000 dilution (100 μL), incubated at room temperature for 30 minutes, and washed six times with PBS. TMB dye solution (Solarbio) was added to each well at a rate of 100 μL per well, allowed to develop for 1 minute, and the reaction was stopped by adding 100 μL of stop solution (Solarbio) per well.

[0234] Measure the OD of the plate using a microplate reader. 450 and O.D. 620 I read it in.

[0235] The results (see Figure 9) show that IEX04-008, IEX04-010, IEX04-012, and the control antibody BI836880 all exhibited complete inhibitory effects, and the IC50 of the bispecific binding molecules of the invention was significantly lower than the IC50 of the positive control antibody.

[0236] (2) Flow cytometry assay (FACS) The ability of IEX04-008, IEX04-010, IEX04-012, the positive control antibodies BI-836880 and faricimab, and the negative control IgG to inhibit the binding of human Ang2-hFc to cell surface Tie2 was detected by FACS.

[0237] Antigen hAng2-Fc protein (Beijing Sino Biological, Catalog No. 10691-H02H) was diluted to 4 μg / mL and added at 50 μL per well. Antibodies prepared as described above (IEX04-008, IEX04-010, IEX04-012, positive control antibody BI-836880, and faricimab, negative control IgG) were serially diluted in two-fold increments starting from a maximum concentration of 800 nM, creating a total of 12 dilution gradients. The diluted antibodies were added at 50 μL per well and incubated on ice in PBS for 30 minutes. The final concentration of antigen hAng2-Fc protein was 2 μg / mL, and the maximum final concentration of each antibody was 400 nM. 293-Tie2 cells prepared as described above were plated at 2 × 10 5The cells / well ratio was adjusted and added at 100 μL per well. The cells were centrifuged at 300 g for 5 minutes. The supernatant was discarded. Next, the cells were resuspended in the antigen-antibody mixture. The mixture was incubated on ice for 30 minutes. 100 μL of PBS was added per well. After centrifugation at 300 g for 5 minutes, the mixture was washed once with PBS. 100 μL of 1:200 diluted goat anti-human IgG-PE (SouthernBiotech) was added to each well. After a 20-minute ice bath, 100 μL of PBS was added per well. The mixture was centrifuged at 300 g for 5 minutes and washed once with PBS. The cells were resuspended in 100 μL of PBS. The fluorescence signal of the cells was measured using a flow cytometer (BD Biosciences). A concentration-dependent curve was fitted using GraphPad according to the MFI. The results are shown in Figure 10. The bispecific binding molecules IEX04-008, IEX04-010, and IEX04-012 all effectively inhibited the binding of human Ang2-hFc to Tie2, demonstrating lower IC50 values ​​than the positive control.

[0238] Example 12: Ang2 phosphorylation inhibition assay of anti-VEGF A / Ang2 bispecific binding molecules In this example, it is shown that the bispecific binding molecules of the present invention exhibit an inhibitory effect on hAng2-Fc-induced Tie2 phosphorylation using an hAng2-induced phosphorylation assay.

[0239] In this study, Expi293 cells overexpressing Tie2 (293-Tie2) were co-cultured with bispecific binding molecules and recombinant hAng2-Fc protein, and the content of phosphorylated Tie2 in the system was detected to reflect the inhibitory effects of different antibodies on the phosphorylation of Tie2 by hAng2-Fc.

[0240] 2 × 10 overexpressing 293-Tie2 cells prepared above were cultured in a 2 × 10 6 cells / mL, added to a 96-well plate at 100 μL / well, centrifuged at 400 g for 5 min, and the supernatant was discarded.

[0241] Experimental medium was prepared using Expi293 medium (Thermo, catalog no. A1435102) and serially diluted with test antibodies (IEX04-012, positive control BI-836880 and faricimab, and negative control IgG) at a final concentration of 60 μg / mL at a ratio of 1:2. hAng2-Fc (Beijing Sino Biological, catalog no. 10691-H02H) was added to the medium at a final concentration of 2.5 μg / mL.

[0242] Cells were resuspended in 100 μL of experimental medium per well and incubated at 37°C for 15 minutes. The medium was removed by centrifugation. 100 μL of NP-40 lysis buffer containing 1% protease and phosphatase inhibitors was added, and the plate was placed on ice for 30 minutes. The plate was centrifuged at 2000 g, and the protein supernatant was collected and stored in a refrigerator at -80°C.

[0243] The concentration of pTie2 was measured according to the instructions of the phosphorylated Tie2 ELISA kit (R&D, DYC2720E). The capture antibody was coated onto a microplate at a concentration of 4 μg / mL and incubated overnight at 4°C. The plate was washed three times with PBST and blocked with 5% BSA for 1 hour. A standard curve was prepared by adding 100 μL of the target sample and control pTie2 (R&D, DYC2720E). The mixture was incubated at room temperature for 2 hours. (If the pTie2 concentration in the sample is too high beyond the ELISA detection range, the resulting mixture can be diluted 2-3 times and administered to the subject.) The plate was washed three times with PBST, and 100 μL of anti-pTyr antibody conjugated with HRP (R&D, catalog no. DYC2720E) was added. The mixture was incubated at room temperature for 2 hours. The plate was washed six times with PBST, and 100 μL of TMB was added to develop the color. After 15 minutes, 100 μL of stop buffer was added to stop the reaction. The OD450-OD620 of each well was measured using a spectrophotometer.

[0244] The results are shown in Figure 11. The antibody IEX04-012 of the present invention effectively inhibited the phosphorylation of 293-Tie2 by hAng2-Fc in vitro, with an IC50 superior to that of the positive control.

[0245] Example 13: Inhibition assay of Ang2 vascular endothelial cell leakage by anti-VEGF A / Ang2 bispecific binding molecules In this study, the effect and function of anti-VEGF A / Ang2 bispecific binding molecules on vascular endothelial cell leakage were identified by HUVEC-Tie2 leakage assay.

[0246] HUVEC-Tie2 cells overexpressing Tie2 were obtained by transfecting HUVEC cells (Allcells, catalog number H-001-CN) with lentivirus.

[0247] 300 μL of EGM-2 medium was plated on the bottom of a mini-well 96-well insert culture dish, and the cells were digested with actinase (Sigma) to obtain HUVEC-Tie2. 1 × 10 cells were cultured in EGM-2 medium. 7 The cells were resuspended at 0.05% ethanol / mL and seeded at 100 μL / well in the upper chamber of a culture dish. The medium in the lower chamber (EGM-2 medium) was replaced every 24 hours, and after 24 hours, the medium in the lower chamber was replaced with experimental medium. The experimental medium contained the following components:

[0248] Blank: EGM-2 medium (Lonza, Cat. No. CC-5035)

[0249] VEGF Group A: EGM-2 medium + 20 ng / mL VEGF (R&D, Catalog No. 293-VE).

[0250] IgG group (VEGF A + IgG): EGM-2 medium + 20 ng / mL VEGF + 10 μg / mL IgG.

[0251] 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).

[0252] IEX04-012 group (VEGF A + IEX04-012): EGM-2 medium + 20 ng / mL VEGF + 10 μg / mL IEX04-012.

[0253] BI-836880 group (VEGF A + BI-836880): EGM-2 medium + 20 ng / mL VEGF + 10 μg / mL BI-836880, and

[0254] Faricimab group (VEGF A + faricimab): EGM-2 medium + 20 ng / mL VEGF + 10 μg / mL faricimab.

[0255] The above experimental medium was cultured under conditions of 37°C and 5% CO2.

[0256] After 24 hours, 1 μL of FITC-dextran (Sigma, Cat. No. FD2000S-1G) (4 mg / mL) was added to the experimental medium in each well. The mixture was incubated at 37°C in 5% CO2. After 30 minutes, the lower chamber medium was removed and diluted 1:10 with PBS. The results were analyzed using a multimode microplate reader with an excitation wavelength of 488 nm and an emission wavelength of 535 nm.

[0257] The results are shown in Figure 12. As a result, it was found that the antibody IEX04-012 of the present invention effectively reduces VEGF-induced permeability of vascular endothelial cells.

[0258] Example 14: Pharmacokinetic study of laser-induced choroidal neovascularization In this study, a laser-induced choroidal neovascularization model using rhesus monkeys was employed to evaluate the resistance of the bispecific binding molecule IEX04-012 of the present invention to choroidal neovascularization.

[0259] Rhesus monkeys: Species: Rhesus monkey; Grade: Standard grade; Weight: Purchase weight 3.30-4.20 kg, Formed weight 3.35-4.35 kg; Source: Sichuan Hengshu Bio-Technology; Manufacturing permit number: SCXK(Sichuan)2019-029; Laboratory animal quality certification number: No. 0023356 In this study, we performed laser photocoagulation on the perifoveal area of ​​the macular retina of rhesus monkeys to induce choroidal neovascularization in the fundus, establishing an animal model similar to human choroidal neovascularization. Fundus fluorescein angiography was performed before and 20 days after photocoagulation to assess the status of neovascularization. Twenty rhesus monkeys (half male and half female) that successfully underwent neovascularization were divided into five groups: model control group, low-dose IEX04-012 group, high-dose IEX04-012 group, Elyea group, and faricimab group. Each group consisted of four monkeys, half male and half female.

[0260] On day 21 after photocoagulation, monkeys in each group were individually administered the doses listed in the table. IEX04-012, Eylea (Bayer), or faricimab (all dissolved in 0.9% sodium chloride injection) was injected intravitreally into both eyes, and the model control group received the same volume of 0.9% sodium chloride injection. Color fundus photography, fundus fluorescein angiography (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 monkeys by spectral-domain optical coherence tomography. British Journal of Ophthalmology, 2015, 99 (1) :119-24) were performed on animals in each group on days 7, 14, 21, and 28 after administration to observe the inhibitory effect of the test sample on choroidal neovascularization. On day 29 after administration, the animals were euthanized, and both eyes were enucleated for histological examination using immunohistochemistry (HE) staining.

[0261] Experimental Design Table [Table 5]

[0262] The results in Figures 13-15 demonstrate that the bispecific binding molecules of the present invention exhibited significant antiangiogenic effects 28 days after administration. Furthermore, the statistics for the number of grade 4 leakage spots (Figure 13A) and grade 3-4 leakage spots (Figure 13B) showed that the number of high leakage spots was significantly lower in animals treated with IEX04-012 than in animals treated with the control and positive control groups. Optical coherence tomography (OCT) revealed a significant decrease in retinal thickness and reduced retinal edema in animals treated with IEX04-012, suggesting a superior effect compared to the control group (Figure 14). Fundus fluorescence angiography revealed a significant decrease in the area of ​​fundus leakage in the IEX04-012 group, an effect superior to that of the positive controls Eylea and faricimab (Figure 15). These results demonstrate that the antibodies of the present invention significantly inhibit leakage caused by neovascularization. In conclusion, it was confirmed that the antibody of the present invention, when combined with an anti-VEGF inhibitor, has a significant inhibitory effect on laser-induced ocular fundus neovascularization and has the function of protecting the integrity of blood vessels.

[0263] On the 29th day after administration, the animals were anesthetized with sodium pentobarbital (approximately 30 mg / kg intravenously, depending on their body weight; the dose could be adjusted depending on the animal's health condition) and euthanized by bleeding from the abdominal aorta or femoral artery. The animals were observed roughly and both eyes were removed.

[0264] The eyes of some animals were fixed in modified Davidson's fixative and embedded in paraffin, and laser-formed areas were selected for routine HE staining, including CD31 IHC staining, and histopathological examination was performed.

[0265] Pathological sections of the antibody group of the present invention showed a significant reduction in the area of ​​retinopathy, retinal edema, and tissue hyperplasia in the laser-damaged area compared to anti-VEGF monotherapy, demonstrating improved retinal morphology (see Figure 16), suppression of retinal-choroidal neovascularization, and enhanced vascular integrity (Figure 17).

[0266] Array List [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] Example 15: Formulation determination test of bispecific antibodies of the present invention In this experiment, the effects of additives (sodium dihydrogen phosphate (monohydrate), disodium hydrogen phosphate (heptahydrate), trehalose, histidine, methionine, proline, glycine, sucrose, hydroxypropyl-β-cyclodextrin, polysorbate 80) on the stability of IEX04-012 protein were investigated, and the detailed formula information is shown in Table 5.

[0267] material: Information regarding the excipients and / or reagents used in the formulation studies of the present invention is provided in Table 4 below.

[0268] [Table 7]

[0269] Preparation and compounding of formulations The buffer for each formulation (see Table 5 for composition excluding polysorbate 80) was prepared using ultrapure water as the solvent at the mass concentration shown in Table 5, and IEX04-012 protein (prepared by the method described in Example 2 above) was ultrafiltered into each buffer. The protein content of each formulation was adjusted to approximately 40 mg / mL after exchange, and then polysorbate 80 was added to a final concentration of 0.3 mg / mL. The solutions were filtered, dispensed into vials, and sealed with stoppers and caps. The stability of the above samples was investigated at 40 ± 2°C.

[0270] [Table 8]

[0271] Stability testing The experimental conditions and sampling schedule are detailed in Table 6.

[0272] [Table 9]

[0273] standard Based on product knowledge, equipment, and method accuracy, the criteria for determining the absence of a quality change in measurements compared to the initial value were as detailed in Table 1.

[0274] [Table 10]

[0275] result The results of the forced stability test are shown in Table 8. After storage at 40 ± 2°C for one week, the other formulations met the acceptance criteria for appearance and visible particles better than Formulation 5. Formulation 5 exhibited severe cloudiness. No significant changes were observed in protein content and pH. All formulation samples showed changes in purity (SEC-HPLC), with the slightest decrease in purity (SEC-HPLC) observed in Formulation 7. For the charge variants, all formulation samples showed an increase in acidic substance content and a decrease in major substance content, with Formulation 7 showing the least significant increase in acidic substance content. After storage at 40 ± 2°C for two weeks, all formulation samples showed changes in purity (SEC-HPLC), with Formulation 7 showing the least significant decrease in purity (SEC-HPLC). For the charge variants, all formulation samples showed an increase in acidic substance content and a decrease in major component content, with Formulation 7 showing the least significant decrease in major component content. No change in polysorbate 80 content was observed in any of the formulations. All formulations were within acceptable bioactivity ranges. [Table 11-1] [Table 11-2] [Table 11-3]

[0276] Based on the results of the formula determination experiment, Formula 7 was selected as the formula of choice for IEX04-012. To avoid the need to add hydrochloric acid for pH adjustment during manufacturing, the buffer was changed to histidine / histidine hydrochloride, and because the osmolality of Formula F7 was low at 255 mOsmol / kg, the trehalose concentration was increased to 100 mg / mL (equivalent to an osmolality of 350 mOsmol / kg). As a result, the osmolality of the formulation buffer met the requirements of the Chinese Pharmacopoeia. Therefore, the final formula for IEX04-012 was recombinant anti-vascular endothelial growth factor A (VEGF A) / anti-angiopoietin 2 (Ang2) bispecific antibody at 40.0 mg / mL, histidine (8 mM) at 1.25 mg / mL, L-histidine hydrochloride (2 mM) at 0.40 mg / mL, trehalose (264.3 mM) at 100 mg / mL, and polysorbate 80, pH 6.5 at 0.3 mg / mL. Subsequent long-term stability studies at 25°C and 2-8°C confirmed that the IEX04-012 antibody was stable for extended periods under these formulation conditions.

[0277] The results of the long-term stability study of final formulation 7 are shown in Table 9. [Table 12]

[0278] Example 16: Study of a high dose formulation (120 mg / mL) of a bispecific antibody of the invention In this example, a high-dose (120 mg / mL) bispecific antibody formulation was prepared and its stability was investigated by monitoring its quality under forced or accelerated conditions and long-term storage.

[0279] A buffer (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 (prepared by the method in Example 2) was transferred to the buffer by ultrafiltration, the protein content was adjusted to approximately 120 mg / mL, polysorbate 80 was added to a final concentration of 0.3 mg / mL, the mixture was filtered, and the mixture was dispensed into vials, which were then sealed with stoppers and caps. The stability of the above samples was examined at 40±2°C, 25±2°C, and 2-8°C, with the specific schedule shown in Table 10.

[0280] [Table 13]

[0281] standard Based on product knowledge, equipment, and method accuracy, the criteria for determining the absence of a quality change in measurements compared to the initial value were as detailed in Table 1. [Table 14]

[0282] result The results of the formulation study are detailed in Table 12. After 1 or 2 weeks of storage at 40 ± 2°C, purity testing showed a decrease in the main peak content and an increase in aggregate content. For the charge variants, the main species decreased and the acidic species increased; all other parameters were within acceptable limits or unchanged. Significant milkiness was observed at week 4. After 1, 2, and 3 months of storage at 25 ± 2°C, all samples were rated as pass for appearance and visible particles. Purity testing (SEC-HPLC) showed a decrease in the main peak content and an increase in aggregate content. For the charge variants, the main species decreased but the acidic species increased. All other parameters were rated as within acceptable limits or unchanged. After 6 and 12 months of storage at 2-8°C, all samples met the pass criteria for appearance and visible particles; no significant changes were observed compared to initial values. [Table 15]

[0283] As described in Example 15, the buffer was changed to histidine / histidine hydrochloride to eliminate the need for adding hydrochloric acid for pH adjustment during manufacturing. The modified IEX04-012 high-dose formulation contained the following: recombinant vascular endothelial growth factor A (VEGF A) / angiogenesis inhibitor 2 (Ang2) bispecific antibody 120.0 mg / mL, histidine 1.25 mg / mL, L-histidine hydrochloride 0.40 mg / mL, trehalose 100 mg / mL, and polysorbate 80 0.3 mg / mL.

[0284] Example 17: Study of a high dose formulation (150 mg / mL) of a bispecific antibody of the invention In this example, a high-dose (150 mg / mL) bispecific antibody formulation was prepared and its stability was investigated.

[0285] A buffer containing 1.25 mg / mL histidine, 0.40 mg / mL L-histidine hydrochloride, and 100.00 mg / mL trehalose was prepared and adjusted to pH 6.5. IEX04-012 was transferred to the buffer by ultrafiltration, the protein content adjusted to approximately 150 mg / mL, and polysorbate 80 was added to a final concentration of 0.3 mg / mL. The sample was filtered, dispensed into vials, and sealed with stoppers and caps. The stability of the sample was investigated at 40±2°C and 25±2°C.

[0286] result The results of the formulation study are shown in Table 13. After storage at 40±2°C for one or two weeks, the purity test showed a decrease in the main peak content and an increase in aggregate content, and the charge variant showed a decrease in the main species and an increase in the acidic species, but all other parameters were within acceptable limits or showed no change. After storage at 25±2°C for one or two weeks, all samples were rated as pass for appearance and visible particles. For the charge variant, the main species decreased but the acidic species increased. All other parameters were within acceptable limits or showed no change. [Table 16]

[0287] As explained above, the buffer was changed to histidine / histidine hydrochloride to eliminate the need for adding hydrochloric acid for pH adjustment during manufacturing. The IEX04-012 high-dose formulation contained the following: recombinant vascular endothelial growth factor A (VEGF A) / angiogenesis inhibitor 2 (Ang2) bispecific antibody 150.0 mg / mL, histidine 1.25 mg / mL, L-histidine hydrochloride 0.40 mg / mL, trehalose 100 mg / mL, and polysorbate 80 0.3 mg / mL.

[0288] It should be noted that the exemplary formulations of the present application are also applicable to other specific binding molecules of the present application, particularly other bispecific binding molecules exemplified (see sequence listing above), and comparable results can be achieved.

[0289] Although exemplary embodiments of the present invention have been described above, those skilled in the art will recognize that these are merely exemplary and that various other substitutions, adaptations, and modifications are possible within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments described herein.

Claims

1. below: (i) a bispecific binding molecule that specifically binds to VEGF A and Ang2; (ii) a buffering agent; (iii) a stabilizer, and (iv) surfactants, wherein the antibody formulation has a pH of about 5.0-7.5; the bispecific binding molecule comprises a first target binding region that specifically binds VEGF A and a second target binding region that specifically binds Ang2, the second target binding region being an Ang VHH comprising the following three CDRs: HCDR1, HCDR2, and HCDR3; said HCDR1 comprises or consists of the sequence set forth in SEQ ID NO: 16; said HCDR2 comprises or consists of the sequence set forth in SEQ ID NO: 17 or 20; said HCDR3 comprises or consists of the sequence set forth in SEQ ID NO: 18; Optionally, the first target binding region comprises: VHHs that specifically bind to VEGF A, an antigen-binding fragment (e.g., scFv) of an antibody that specifically binds to VEGF A, e.g., the antibody is a fully human antibody or a humanized antibody, or a VEGF receptor (VEGF R) that specifically binds to VEGF A, or its extracellular domain, or a fusion protein containing the extracellular domain (e.g., a fusion protein of the extracellular domain and Fc); The liquid formulation is selected from the group consisting of:

2. the anti-Ang2 VHH (1) comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 19 or 21, or comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 19 or 21, or (2) An amino acid sequence having one or more (preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or less) mutations compared to the amino acid sequence set forth in SEQ ID NO: 19 or 21, wherein the mutations are, for example, substitutions, deletions, or additions, preferably substitutions such as conservative substitutions; For example, the bispecific binding molecule is a bispecific antibody. The liquid formulation of claim 1.

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

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

5. the light chain variable region VL of the anti-VEGF antibody comprises LCDR1, LCDR2, and LCDR3; said LCDR1 comprises or consists of the sequence set forth in SEQ ID NO: 31; said LCDR2 comprises or consists of the sequence set forth in SEQ ID NO: 32; said LCDR3 comprises or consists of the sequence set forth in SEQ ID NO: 33; and / or the heavy chain variable region VH of the anti-VEGF antibody comprises HCDR1, HCDR2, and HCDR3; said HCDR1 comprises or consists of the sequence set forth in SEQ ID NO: 35; said HCDR2 comprises or consists of the sequence set forth in SEQ ID NO: 36; The HCDR3 comprises or consists of the sequence set forth in SEQ ID NO: 37; 5. The liquid formulation of claim 4.

6. The heavy chain variable region VH of the anti-VEGF antibody comprises or consists of the amino acid sequence set forth in SEQ ID NO: 34, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 34, or the heavy chain variable region VH comprises an amino acid sequence having one or more (preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or less) mutations compared to the amino acid sequence set forth in SEQ ID NO: 34, the mutations being, for example, substitutions, deletions, or additions, preferably substitutions such as conservative substitutions; The light chain variable region VL of the anti-VEGF antibody comprises or consists of the amino acid sequence set forth in SEQ ID NO: 30, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 30, or the light chain variable region VL comprises an amino acid sequence having one or more (preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or less) mutations compared to the amino acid sequence set forth in SEQ ID NO: 30, the mutations being, for example, substitutions, deletions, or additions, preferably substitutions such as conservative substitutions. A liquid formulation according to any one of claims 1 to 5.

7. (1) the bispecific binding molecule comprises or consists of the amino acid sequence set forth in SEQ ID NO:28, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:28; or (2) The bispecific binding molecule comprises an amino acid sequence having one or more (preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or less) mutations compared to the amino acid sequence set forth in SEQ ID NO: 28, wherein the mutations are, for example, substitutions, deletions, or additions, preferably substitutions such as conservative substitutions. The liquid formulation according to any one of claims 4 to 6.

8. The bispecific binding molecule has the following structure: a first anti-VEGF VHH-linker-second anti-VEGF VHH-linker-anti-Ang2 VHH, wherein the first anti-VEGF VHH is the same as or different from the second anti-VEGF VHH; A liquid formulation according to any one of claims 3 to 7.

9. the first anti-VEGF VHH or the second anti-VEGF VHH comprises HCDR1, HCDR2, and HCDR3; said HCDR1 comprises or consists of the sequence set forth in SEQ ID NO: 1; said HCDR2 comprises or consists of the sequence set forth in SEQ ID NO:2; The HCDR3 comprises or consists of the sequence set forth in SEQ ID NO: 3; or said HCDR1 comprises or consists of the sequence set forth in SEQ ID NO:6; said HCDR2 comprises or consists of the sequence set forth in SEQ ID NO: 7 or 10; The HCDR3 comprises or consists of the sequence set forth in SEQ ID NO: 8; 9. The liquid formulation of claim 8.

10. The first or second anti-VEGF VHH comprises: comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 4, 5, 9, or 11, or comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 4, 5, 9, or 11; or The VHH comprises an amino acid sequence having one or more (preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or less) mutations compared to the amino acid sequence set forth in SEQ ID NO: 4, 5, 9, or 11, wherein the mutations are, for example, substitutions, deletions, or additions, and preferably substitutions such as conservative substitutions.

10. The liquid formulation of claim 8 or 9.

11. (1) the bispecific binding molecule comprises or consists of the amino acid sequence set forth in SEQ ID NO:22, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:22; or (2) The bispecific binding molecule comprises an amino acid sequence having one or more (preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or less) mutations compared to the amino acid sequence set forth in SEQ ID NO: 22, wherein the mutations are, for example, substitutions, deletions, or additions, preferably substitutions such as conservative substitutions. A liquid formulation according to any one of claims 8 to 10.

12. The bispecific binding molecule comprises the following chain: VEGF R extracellular domain-Fc-linker-anti-Ang2 VHH 2. The liquid formulation of claim 1, comprising one or two of the following:

13. The VEGFR extracellular domain is an extracellular domain of a VEGFR derived from a human, and preferably, the VEGFR extracellular domain comprises a second antibody-like domain of VEGFR1 and a third antibody-like domain of VEGFR2, and more preferably, the VEGFR extracellular domain comprises a second antibody-like domain of human VEGFR1 and a third antibody-like domain of human VEGFR2.

13. The liquid formulation of claim 12.

14. The VEGFR extracellular domain comprises or consists of the amino acid sequence set forth in 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 set forth in SEQ ID NO:

26.

14. The liquid formulation of claim 12 or 13.

15. The Fc is derived from human IgG1, IgG2, IgG3, or IgG4, and preferably the Fc comprises or consists of the amino acid sequence set forth in 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 set forth in SEQ ID NO:

27. A liquid formulation according to any one of claims 12 to 14.

16. The VEGF R extracellular domain-Fc is a fusion protein of a VEGFR extracellular domain and Fc, such as aflibercept or a derivative thereof, for example, the VEGF R extracellular domain-Fc comprises or consists of the amino acid sequence set forth in SEQ ID NO: 25, 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 set forth in SEQ ID NO: 25; A liquid formulation according to any one of claims 12 to 15.

17. (1) The bispecific binding molecule comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 24, or (2) The bispecific binding molecule comprises an amino acid sequence having one or more (preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or less) mutations compared to the amino acid sequence set forth in SEQ ID NO: 24, wherein the mutations are, for example, substitutions, deletions, or additions, preferably substitutions such as conservative substitutions. A liquid formulation according to any one of claims 12 to 16.

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

23.

19. The buffering agent is selected from one or more of histidine, histidine salts, glutamic acid, phosphoric acid, acetic acid, citric acid, and tris(hydroxymethyl)aminomethane; preferably, the buffering agent is selected from a combination of histidine and histidine hydrochloride, phosphoric acid, and histidine; preferably, the phosphoric acid is a combination of disodium phosphate and sodium dihydrogen phosphate; A liquid formulation according to any one of claims 1 to 18.

20. the stabilizer is selected from one or more of a sugar, a polyol, and an amino acid or a salt thereof; Preferably, the sugars are selected from sucrose, dextrose, lactose, maltose, trehalose, cyclodextrins, maltodextrins, and glucans; the polyol is selected from mannitol, sorbitol, and xylitol; the amino acid or salt thereof is selected from arginine, glycine, proline, methionine, a salt of arginine, and combinations thereof; The cyclodextrin is preferably hydroxypropyl-β-cyclodextrin, More preferably, the stabilizer is sucrose, trehalose, or a combination of sucrose or alginate with an additional component; the additional component is selected from one or more of glycine, proline, methionine, and hydroxypropyl-β-cyclodextrin; A liquid formulation according to any one of claims 1 to 19.

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

22. 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 5-500 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 formulation is about 6.1-7.0, preferably about 6.3-6.

8. A liquid formulation according to any one of claims 1 to 21.

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

24. below: (i) about 10-160 mg / mL of the bispecific binding molecule; (ii) about 5-20 mM of a buffering agent, which is histidine or a combination of histidine and histidine hydrochloride; (iii) about 200-350 mM trehalose; and (iv) about 0.1-1 mg / mL of polysorbate 80; 19. The liquid formulation according to any one of claims 1 to 18, wherein the liquid formulation has a pH of about 6.1-7.0, preferably about 6.

5.

25. below: (i) about 30-150 mg / mL of the bispecific binding molecule; (iii) about 8-12 mM histidine; (iii) about 200-300 mM trehalose; and (iv) about 0.2-0.4 mg / mL polysorbate 80; A liquid formulation comprising:

25. The liquid formulation of claim 24, wherein the pH of the liquid formulation is about 6.1-6.6, preferably about 6.

5.

26. below: (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; A liquid formulation comprising:

25. The liquid formulation of claim 24, wherein the pH of the liquid formulation is about 6.1-6.6, preferably about 6.

5.

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

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

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

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