Bispecific binding molecules that bind VEGF and Ang2 and uses thereof

Abstract

The present invention relates to antibodies against vascular endothelial growth factor (VEGF / VEGF-A) and angiopoietin-2 (ANG-2), respectively, and bispecific binding molecules (e.g., antibodies) against vascular endothelial growth factor (VEGF / VEGF-A) and angiopoietin-2 (ANG-2) simultaneously, as well as methods for their production, and pharmaceutical compositions comprising said antibodies or molecules and uses thereof.

Description

[Technical field]

[0001] The present invention relates to antibodies against vascular endothelial growth factor (VEGF / VEGF-A) and angiopoietin-2 (ANG-2), respectively, and bispecific binding molecules (e.g., antibodies) against vascular endothelial growth factor (VEGF / VEGF-A) and angiopoietin-2 (ANG-2) simultaneously, as well as methods for their production, and pharmaceutical compositions comprising said antibodies or molecules and uses thereof. [Background technology]

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

[0003] VEGF is a potent and ubiquitous vascular growth factor. VEGF family members include VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, placental growth factor (PIGF), and endocrine-derived VEGF (EG-VEGF). The active form of VEGF is synthesized as a homodimer or heterodimer with other VEGF family members. VEGF-A exists in six isoforms, VEGF121, VEGF145, VEGF165, VEGF183, VEGF189, and VEGF206, generated by alternative splicing. These isoforms differ mainly by their bioavailability, with VEGF165 being the predominant isoform. VEGF is considered to be a key regulator of normal and disease-associated 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 naturally occurring agonist angiopoietin-1 (ANG-1) and the naturally occurring antagonist angiopoietin-2 (ANG-2). The role of ANG-1 is conserved in adults and is thought to be expressed broadly and constitutively in adults. In contrast, expression of ANG-2 is primarily restricted to sites of vascular remodeling and is thought to block the constitutive stabilizing or maturation function of ANG-1, thereby allowing blood vessels to revert to and maintain a plastic state in which they may be more responsive to sprouting signals.

[0005] In recent years, some bispecific antibodies targeting VEGF-A and ANG-2 have already been developed (e.g., WO2012131078 and WO2014009465). However, existing bispecific antibodies have relatively weak blocking properties against VEGF and Ang2, and their molecular weight is too large, resulting in low molar concentrations when administered in a single dose. In particular, for ocular diseases, a method of intravitreal administration of lower molecular weight antibodies is usually used, and less frequent administration is required. Therefore, there is still a need for new bispecific binding molecules that are particularly applicable to ocular diseases and target VEGF-A and ANG-2. Summary of the Invention

[0006] The present invention develops new VHH antibodies targeting VEGF-A or ANG-2, and bispecific binding molecules against VEGF-A and ANG2 at the same time. In particular, the bispecific binding molecules of the present invention have lower molecular weights, higher molar concentrations at the same mass concentration, and relatively strong VEGF A and Ang2 blocking activity, and can completely block VEGFA-induced primary cell proliferation, compared with known antibodies. Thus, the molecules of the present invention have clinically stronger blocking activity, and can provide a higher antibody molar concentration at a single dose, maintain the efficacy of a single dose for a longer period, and reduce the frequency of intraocular administration (e.g., intravitreal injection). [Brief description of the drawings]

[0007] [Figure 1] 1 shows the structure of a bispecific binding molecule. [Diagram 2] Figure 2 shows that anti-VEGF A VHH antibodies are capable of blocking the binding of VEGF A to VEGFR2 as measured by ELISA. [Diagram 3] The effect of humanized anti-Ang2 VHH antibodies to block the binding of Ang2 to Tie2 was measured by ELISA. [Figure 4] 1 shows the inhibitory effect of anti-Ang2 VHH antibody (A) and humanized anti-Ang2 VHH antibody (B) on Ang2-Fc-induced phosphorylation of 293-Tie2 cells, as measured by ELISA. [Diagram 5] 1 shows that the HEK293-KDR reporter assay detects the effect of anti-VEGF A VHH in blocking activation of the KDR receptor by VEGFA. [Figure 6] Figure 2 shows the inhibitory effect of anti-VEGF A VHH antibodies on VEGF A-induced HUVEC cell survival and proliferation as measured by CCK-8. [Figure 7] This shows that the HEK293-KDR reporter assay detects the blocking effect of the VEGF A / Ang2 bispecific binding molecule IEX04-012 on the activation of the KDR receptor by VEGF. [Figure 8] FIG. 1 shows that the bispecific binding molecules IEX04-008, IEX04-010 and IEX04-012 detect the inhibitory effect of VEGF-induced HUVEC cell survival and proliferation. [Figure 9] FIG. 1 shows that the bispecific binding molecules IEX04-008, IEX04-010 and IEX04-012 block the binding of human Ang2 to Tie2 as measured by ELISA. [Figure 10]FIG. 1 shows that bispecific binding molecules IEX04-008, IEX04-010 and IEX04-012 block the binding of Ang2-Fc to Tie2 as measured by flow cytometry assay. [Figure 11] FIG. 1 shows that the bispecific binding molecule IEX04-012 of the present invention effectively inhibits hAng2-Fc-induced 293-Tie2 phosphorylation in vitro as measured by flow cytometry assay. [Figure 12] FIG. 1 shows that the bispecific binding molecule IEX04-012 of the invention reduces VEGF-induced vascular endothelial cell permeability, i.e. inhibits VEGF-induced HUVEC cell leakage. [Figure 13] Statistics of the ratio of laser spots of grade 4 (Figure A) and grade 3 or higher (Figure B) in a laser-induced choroidal neovascularization model are shown. [Figure 14] 1 shows retinal thickness statistics in a laser-induced choroidal neovascularization model. [Figure 15] 1 shows statistics of leakage area in a laser-induced choroidal neovascularization model. [Figure 16] FIG. 1 shows H&E staining of fundus tissue from a laser-induced choroidal neovascularization model (A) and lesion area statistics (B). [Figure 17] FIG. 1 shows a CD31 staining diagram (A) and positive cell statistics (B) of fundus tissue from a laser-induced choroidal neovascularization model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] definition Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methodology, forms, or reagents described herein, as they may be modified. It should also be understood that the terms used herein are merely for the purpose of describing specific embodiments, and are not intended to limit the scope of the present invention, which is limited only by the claims. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art.

[0009] The following definitions will be used to interpret the specification, and where appropriate, terms used in the singular may 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.

[0010] The term "about," when used in conjunction with a number or numerical value, is meant to cover a range of numbers or numerical values ​​from 5% less than the number or numerical value specified as the lower limit to 5% more than the number or numerical value specified as the upper limit.

[0011] As used herein, the term "and / or" means any one of available options or two or more or all of available options.

[0012] As used herein, the term "containing" or "comprises" means including the element, integer or step, but not excluding any other element, integer or step. When the term "comprises" or "comprises" is used herein, unless otherwise specified, it also includes the case where the element, integer or step is mentioned. For example, when an antibody variable region "comprising" a certain sequence is mentioned, it is also intended to include an antibody variable region consisting of this specific sequence.

[0013] The term "VEGF" as used herein refers to vascular growth factor. VEGF family members include VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, placental growth factor (PIGF), and endocrine-derived VEGF (EG-VEGF). The active form of VEGF is synthesized as a homodimer or heterodimer with other VEGF family members. VEGF-A exists in six isoforms, VEGF121, VEGF145, VEGF165, VEGF183, VEGF189, and VEGF206, which are generated by alternative splicing. These isoforms differ mainly by their bioavailability, with VEGF165 being the major isoform. In some embodiments, the VEGF A of the present invention refers to VEGF A of human origin, such as VEGF165 of human origin. In one embodiment, the amino acid sequence of the VEGFA of the present invention is the amino acid sequence of accession number P15692 (uniprot database).

[0014] As used herein, the term "ANG2" refers to human angiopoietin-2 (ANG-2) (alternatively abbreviated as ANGPT2 or ANG2), e.g., as described in Maisonpierre, PC et al., Science 277 (1997) 55-60, and Cheung, AH et al., Genomics 48 (1998) 389-91. Ang1 and Ang2 are discovered as ligands for Tie, a family of tyrosine kinases selectively expressed in vascular endothelium. There are currently four defined angiopoietin family members. Angiopoietin-3 and -4 (ANG3 and ANG4) may represent widely divergent counterparts of the same genetic locus in mouse and human. 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, P. C. et al., Science 277 (1997) 55-60). All known angiopoietins bind primarily to Tie2. In some embodiments, ANG2 of the present invention refers to Ang2 of human origin. In some embodiments, human Ang2 comprises the amino acid sequence of accession number 015123 (uniprot database).

[0015] The term "multispecific binding molecule" refers to a multispecific binding molecule of at least two specificities, e.g., a bispecific binding molecule, i.e., the molecule 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, the molecule according to the invention comprises specificity for at least two different antigens or targets. The molecule according to the invention also encompasses multispecific molecules comprising multiple target binding regions / binding sites, e.g., trispecific binding molecules. In some embodiments, the bispecific binding molecule of the invention is a bispecific antibody.

[0016] The term "linker" as used herein refers to any molecule that can be directly linked to different portions of a bispecific binding molecule. Examples of linkers that establish a covalent bond between different molecular portions include peptide linkers and non-protein polymers, including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. In some embodiments, the linker is a peptide linker, where the amino acid sequence of a first portion of a binding molecule is linked to a second portion of the binding molecule. For example, a peptide linker can link a first target binding region of a binding molecule to a second target binding region. For example, a peptide linker can also link a portion of an antibody to another portion of an antibody, such as a light chain variable region to a heavy chain variable region. Preferably, the peptide linker has a length sufficient to link two entities such that they maintain a conformation relative to each other without interfering with the desired activity.

[0017] The peptide linker may comprise predominantly, but not predominantly, the amino acid residues Gly, Ser, Ala, or Thr. Useful linkers include glycine-serine polymers, including (GS)n, (GSGGS)n, (GGGGS)n, (GGGS)n, and (GGGGS)nG, where n is an integer of at least 1 (and preferably 2, 3, 4, 5, 6, 7, 8, 9, or 10). Useful linkers further include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Useful linkers also include glycine monomer polymers, such as (G)n, where n is an integer of at least 4 (and preferably 4-20, such as 4, 5, 6, 7, 8, 9, 10, and more). Preferably, the linker is (GGGGS)n, where n=1, 2, 3, or 4.

[0018] The term "valent" according to the present invention refers to the presence of a certain number of binding sites in an antibody molecule, e.g., a binding molecule. Thus, the terms bivalent, trivalent, tetravalent refer to the presence of two, three, or four binding sites (target binding regions) in a binding molecule, respectively. Bispecific binding molecules according to the present invention are at least bivalent and may be multivalent, e.g., bivalent, trivalent, tetravalent, or hexavalent.

[0019] The term "target binding region" as used herein refers to any portion of a multispecific binding molecule, e.g., a bispecific binding molecule, that binds to a specific target or antigen. The target binding region may 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 its target as a separate entity. The target binding region may also be a receptor or ligand, or a domain of a receptor that can bind to a ligand.

[0020] The term "antibody fragment" includes a portion of an intact antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0021] "Antigen-binding fragment" refers to a molecule that includes a portion of an intact antibody and binds to an antigen bound by the intact antibody, but is distinct from the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, dAb (domain antibody), linear antibody, single chain antibody (e.g., scFv), single domain antibody (e.g., VHH), bivalent antibody or fragment thereof, or camelid antibody.

[0022] A "VHH", also called a single domain antibody (sdAb), is a genetically engineered antibody that is composed only of a heavy chain antibody variable region, including only the three HCDRs of the heavy chain variable region. VHHs rely on only three HCDRs to have antigen specificity and high affinity, whereas a normal antibody requires six CDRs. Crystal structures have shown that VHHs are scaffolded by two β-sheets, similar to the folding of a conventional antibody VH immunoglobulin.

[0023] The term "target" refers to an entity to which a binding molecule binds. A target may be an antigen or a ligand or receptor.

[0024] The term "antigen" refers to a molecule that elicits an immune response. Such immune response may involve the production of antibodies or the activation of specific immune cells, or both. Those skilled in the art will appreciate that any large molecule, including almost any protein or peptide, can serve as an antigen. Antigens may also be derived from recombinant or genomic DNA. As used herein, the term "epitope" refers to the portion of an antigen (e.g., VEGF or Ang2) that specifically interacts with an antibody molecule.

[0025] "Complementarity determining regions", "CDR regions" or "CDRs" are the regions in an antibody variable domain that are hypervariable in sequence and structurally determined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to an antigen epitope. The CDRs of the heavy and light chains are usually called CDR1, CDR2 and CDR3 and are numbered sequentially from the N-terminus. The CDRs in an antibody heavy chain variable domain are called HCDR1, HCDR2 and HCDR3, and the CDRs in an antibody light chain variable domain are called LCDR1, LCDR2 and LCDR3. For a given light or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined by any one or a combination of a number of known antibody CDR assignment systems, including, for example, the Chothia system, which is based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883; Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), which is based on the variability of antibody sequences (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., USDepartment of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics (International The database (IMGT) (available on the World Wide Web at www.imgt.cines.fr / ) and a North CDR definition based on affinity propagation clustering utilizing a large number of crystal structures.

[0026] For example, according to different CDR determination methods, the residues of each CDR are as follows: [Table 1]

[0027] A CDR may be determined by having the same Kabat numbering position as the sequence of a reference CDR (eg, any one of the exemplary CDRs of the invention).

[0028] Unless otherwise specified, in the present invention, the term "CDR" or "CDR sequence" includes a CDR sequence determined by any one of the above methods.

[0029] Unless otherwise specified, in the present invention, references to residue positions in antibody variable regions (including heavy chain variable region residues and light chain variable region residues) refer to numbered positions based on 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 in a VHH of the invention comply with the following rules: where HCDR1 is determined according to AbM, and HCDR2 and HCDR3 are determined according to Kabat.

[0031] It should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained by different allocation systems may differ. That is, there are differences in the CDR sequences of the variable regions of the same antibody defined by different allocation systems. Thus, when an antibody is defined by a specific CDR sequence defined in the present invention, the scope of the antibody also includes antibodies whose variable region sequences contain the specific CDR sequences, but where a different approach (e.g., rules or combinations of different allocation systems) is used and such CDR boundaries differ from the specific CDR boundaries defined in the present invention.

[0032] Antibodies with different specificities (i.e., directed to different binding sites of different antigens) have different CDRs (in the same allocation system). However, even though CDRs differ between antibodies, there are a limited number of amino acid positions in the CDRs that are directly involved in binding to the antigen. The minimum overlapping region can be determined to provide a "minimal binding unit" for antigen binding by at least two of the Kabat, Chothia, AbM, Contact and North methods. The minimal binding unit may be a subpart of one of the CDRs. As known to those skilled in the art, the structure of the antibody and protein folding can determine the residues of the remaining part of the CDR sequence. Thus, the present invention contemplates variants of any of the CDRs provided herein. For example, in a variant of one CDR, the amino acid residues of the minimal binding unit are kept unchanged, but the remaining CDR residues defined according to Kabat or Chothia may be replaced with conservative amino acid residues.

[0033] The term "Fc region" is used herein to define the constant regions of CH2 and CH3 of an immunoglobulin heavy chain, and includes native sequence Fc regions and variant Fc regions. Native or wild-type Fc regions can bind to different Fc receptors on the surface of immune cells, thereby triggering CDC / ADCC / ADCP effector functions. Such effector functions generally require the association of an Fc region with a binding domain (e.g., an antibody variable region). In some embodiments, an Fc region is mutated to enhance its CDC / ADCC / ADCP effector function. In some embodiments, an Fc region is mutated to reduce or eliminate its CDC / ADCC / ADCP effector function.

[0034] A "humanized" antibody refers to an antibody that contains amino acid residues from non-human CDRs and amino acid residues from human FRs. In some embodiments, a humanized antibody contains at least one, typically two, of substantially all of the variable domains, in which all or substantially all of the CDRs (e.g., CDRs) correspond to portions derived from a non-human antibody, and all or substantially all of the FRs correspond to portions derived from a human antibody. A humanized antibody optionally contains 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. "Human antibody" or "fully humanized antibody" or "fully human antibody" may be used interchangeably and refer to an antibody that is generated from a human or human cell, or is derived from a non-human source, and has an amino acid sequence that corresponds to the amino acid sequence of an antibody whose sequence is encoded in a human antibody library or other human antibody. This 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 "specifically binding" mean that the binding action is selective for a target or antigen and can be distinguished from unwanted or non-specific interactions. The ability of a binding site to bind to a particular target or antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other conventional binding assays known in the art, such as radioimmunoassay (RIA), biolayer interferometry, MSD assay, or surface plasmon resonance (SPR).

[0036] The term "effective amount" refers to an amount or dosage that provides the desired effect in a patient in need of treatment or prevention after administration of one or more doses of an antibody or fragment or composition or combination of the invention to the patient.

[0037] A "therapeutically effective amount" refers to an amount that effectively achieves a desired therapeutic result at the required dosage for the required period of time. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the antibody or antibody fragment or composition or combination are not offset by the beneficial effects of the treatment. A "therapeutically effective amount" preferably inhibits or improves a measurable parameter by at least about 40%, more preferably by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 100%, compared to an untreated subject.

[0038] A "prophylactically effective amount" refers to an amount to effectively achieve a desired prophylactic result at a necessary dosage for a necessary period of time. Generally, the prophylactically effective amount will be less than the therapeutically effective amount, since a prophylactic dose is administered prior to or at an earlier stage of disease in a subject.

[0039] The terms "host cell," "host cell line," and "host cell culture" may be used interchangeably and refer to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," and include the primary transformed cell and its progeny regardless of the number of passages. The progeny may not be exactly identical to the parent cell in nucleic acid content and may contain mutations. As used herein, includes mutant progeny that have the same function or biological activity as screened or selected from the primary transformed cell.

[0040] The term "label" as used herein refers to a compound or composition that is directly or indirectly bound or fused to a reagent (e.g., a polynucleotide probe or an antibody) and facilitates detection by the reagent to which it is bound or fused. The label can be detectable itself (e.g., a radioisotope label or a fluorescent label) or, when labeled by enzyme catalysis, can catalyze the chemical modification of a detectable substrate compound or composition. The term is intended to include direct labeling of a probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, and indirect labeling of a probe or antibody by reaction with another reagent that is directly labeled. In some embodiments, the label is hFc or biotin.

[0041] An "individual" or "subject" includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, goats, cats, dogs, and horses), primates (e.g., humans, non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0042] An "isolated" antibody or molecule is one that is separated from a component of the natural environment it is found in. In some embodiments, the antibody or molecule is purified to greater than 95% or 99% purity, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC).

[0043] The sequence identity between sequences is calculated as follows. To determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison). In a preferred embodiment, the length of the reference sequence aligned for comparison is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at this position.

[0044] A mathematical algorithm can realize the sequence comparison between two sequences and the calculation of the identity percentage. In one preferred embodiment, the identity percentage between two amino acid sequences is determined by the Needlema and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm integrated in the GAP program of the GCG software package (available at http: / / www.gcg.com) using a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6. In another preferred embodiment, the identity percentage between two nucleotide sequences is determined by the GAP program of the GCG software package (available at http: / / www.gcg.com) using a NWSgapdna.CMP matrix, and a gap weight of 40, 50, 60, 70 or 80 and a length weight of 1, 2, 3, 4, 5 or 6. A particularly preferred set of parameters (and the one that should be used unless otherwise noted) employs a Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. Percent identity between two amino acid or nucleotide sequences may also be determined using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4:11-17) as integrated into the ALIGN program (version 2.0) using a PAM120 weighted remainder 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 be used as "query sequences" to perform searches against public databases to, for example, identify other family member sequences or related sequences.

[0045] "Ocular diseases" as used herein include eye diseases associated with angiogenesis (eg, diseases occurring within the eye), such as eye diseases associated with corneal neovascularization.

[0046] The term "auxiliary pharmaceutical material" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete or incomplete)), excipient, vector, stabilizer, etc., administered with an active substance.

[0047] The term "pharmaceutical composition" refers to a composition that contains an active ingredient in a form that is effective for the biological activity of the active ingredient, and that does not contain additional ingredients that are unacceptably toxic to a subject to which the composition is administered.

[0048] As used herein, "treatment" refers to alleviating, interrupting, slowing, ameliorating, arresting, reducing, or reversing the progression or severity of an existing symptom, condition, pathology, or disease.

[0049] As used herein, "prevention" includes the inhibition of the onset or progression of a disease, condition, or symptom associated with a particular disease or condition.

[0050] The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes carriers that are self-replicating nucleic acid structures and carriers that are integrated into the genome of a host cell into which they are introduced. Some vectors are capable of directing the expression of a nucleic acid that is operably linked to them. Such carriers are referred to herein as "expression carriers."

[0051] "Subject / patient / individual sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample may be solid tissue, such as fresh, frozen and / or preserved organ or tissue samples, biopsy or puncture samples, blood or any blood component, bodily fluids, such as tears, vitreous fluid, cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites) or interstitial fluid, or cells from any stage of pregnancy or development in a subject. In some embodiments, the tissue sample is ocular tissue, such as the vitreous. In some embodiments, the sample is tears or vitreous fluid. The tissue sample may contain compounds that are not naturally mixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0052] I. Anti-VEGF A VHH antibodies The present invention relates to VHH antibodies against VEGF A. In some embodiments, the anti-VEGF VHH of the invention comprises three CDRs, HCDR1, HCDR2 and HCDR3, wherein: HCDR1 comprises or consists of the sequence shown in SEQ ID NO:1, HCDR2 comprises or consists of the sequence shown in SEQ ID NO:2, HCDR3 comprises or consists of the sequence shown in SEQ ID NO:3, or, HCDR1 comprises or consists of the sequence shown in SEQ ID NO:6, HCDR2 comprises or consists of the sequence shown in SEQ ID NO: 7 or SEQ ID NO: 10, HCDR3 comprises or consists of the sequence shown in SEQ ID NO:8.

[0053] In some embodiments, the VHH comprises an amino acid sequence as set forth in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:9 or SEQ ID NO:11, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence as set forth in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:9 or SEQ ID NO:11, or consists of the amino acids as set forth in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:9 or SEQ ID NO:11.

[0054] In some embodiments, the VHH comprises an amino acid sequence having one or more (preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less) mutations, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions, compared to the amino acid sequence shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:9 or SEQ ID NO:11.

[0055] In some preferred embodiments, the mutation is not present in a CDR, such as HCDR1, HCDR2, or HCDR3.

[0056] II. Anti-Ang2 VHH antibodies The present invention relates to a VHH antibody against Ang2. In some embodiments, the anti-Ang2 VHH of the present invention comprises three CDRs, HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises or consists of the sequence shown in SEQ ID NO: 16, HCDR2 comprises or consists of the sequence shown in SEQ ID NO: 17 or SEQ ID NO: 20, HCDR3 comprises or consists of the sequence shown in SEQ ID NO:18.

[0057] In some embodiments, the VHH comprises an amino acid sequence as set forth in SEQ ID NO:19 or SEQ ID NO:21, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence as set forth in SEQ ID NO:19 or SEQ ID NO:21, or consists of the amino acids as set forth in SEQ ID NO:19 or SEQ ID NO:21.

[0058] In some embodiments, the VHH comprises an amino acid sequence having one or more (preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less) mutations, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions, compared to the amino acid sequence shown in SEQ ID NO: 19 or SEQ ID NO: 21.

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

[0060] III. Anti-VEGF A x ANG2 Bispecific Binding Molecules In one aspect of the invention, the invention relates to a bispecific binding molecule that binds to VEGF A and Ang2 comprising a first target binding region that specifically binds to VEGF A and a second target binding region that specifically binds to Ang2, wherein the second target binding region is an anti-Ang2 VHH, such as an anti-Ang2 VHH of the invention as described above.

[0061] In some embodiments, the first target binding region comprises: VHH that specifically binds to VEGF A, 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 antibody is selected from a VEGF receptor (VEGF R) that specifically binds to VEGF A, or an extracellular domain thereof, or a fusion protein comprising the extracellular domain thereof, for example a fusion protein of the extracellular domain and Fc.

[0062] In some embodiments, the bispecific binding molecules of the invention comprise one or two or three or four first or second target binding regions. In some embodiments, the bispecific binding molecules of the invention comprise two, three or four target binding regions. In some embodiments, the bispecific binding molecules of the invention are bivalent, trivalent or tetravalent. In some embodiments, the bispecific binding molecules of the invention are bispecific antibodies.

[0063] In one embodiment of the invention, a bispecific binding molecule, such as a bispecific antibody, of the invention 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 The antibody has a heavy chain variable region VH-linker of an anti-VEGF antibody, a light chain variable region VL-linker of an anti-VEGF antibody, and an anti-Ang2 VHH. Here, the anti-Ang2 VHH comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the sequence shown in SEQ ID NO: 16, HCDR2 comprises or consists of the sequence shown in SEQ ID NO: 17 or SEQ ID NO: 20, and HCDR3 comprises or consists of the sequence shown in SEQ ID NO: 18.

[0064] In some embodiments, the structure of the bispecific binding molecule is as shown in Figure 1A or Figure 1B. In some embodiments, the bispecific binding molecule consists of a single chain. In some embodiments, the bispecific binding molecule is bivalent.

[0065] 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.

[0066] 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.

[0067] In some embodiments, the heavy chain variable region VH of the anti-VEGF antibody of the present invention comprises an amino acid sequence as 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 with the amino acid sequence as set forth in SEQ ID NO: 34, or consists of the amino acids as 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, 1 or less) mutations, e.g., substitutions, deletions or additions, preferably substitutions, e.g., conservative substitutions, compared to the amino acid sequence as set forth in SEQ ID NO: 34. In some preferred embodiments, the mutations are not present in CDRs, e.g., HCDR1, HCDR2, or HCDR3.

[0068] In some embodiments, the light chain variable region VL of the anti-VEGF antibody of the present invention comprises an amino acid sequence as 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 with the amino acid sequence as set forth in SEQ ID NO: 30, or consists of the amino acids as 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 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less) mutations, e.g., substitutions, deletions or additions, preferably substitutions, e.g., conservative substitutions, compared to the amino acid sequence as set forth in SEQ ID NO: 30. In some preferred embodiments, the mutations are not present in the CDRs, e.g., LCDR1, LCDR2, or LCDR3.

[0069] In some embodiments, the anti-Ang2 VHH of the invention comprises an amino acid sequence as set forth in SEQ ID NO: 19 or SEQ ID NO: 21, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with said amino acid sequence as set forth in SEQ ID NO: 19 or SEQ ID NO: 21, or consists of the amino acids as set forth in SEQ ID NO: 19 or SEQ ID NO: 21. In some embodiments, said VHH comprises an amino acid sequence having one or more (preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less) mutations, e.g. substitutions, deletions or additions, preferably substitutions, e.g. conservative substitutions, compared to the amino acid sequence as set forth in SEQ ID NO: 19 or SEQ ID NO: 21. In some preferred embodiments, said mutations are not present in the CDRs, e.g. HCDR1, HCDR2 or HCDR3.

[0070] In some embodiments, the linker comprises or consists of the amino acid sequence of SEQ ID NO: 23. In some embodiments, the linker between the light chain variable region and the heavy chain variable region of an anti-VEGF antibody comprises or consists of the amino acid sequence of SEQ ID NO: 23, for example, where n=4. In some embodiments, the linker between the anti-VEGF variable region and an anti-Ang2 VHH comprises or consists of the amino acid sequence of SEQ ID NO: 23, for example, where n=2 or 3, for example, 3.

[0071] In some embodiments, the anti-VEGF A×ANG2 bispecific binding molecule of the invention comprises an amino acid sequence as 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 with said amino acid sequence as set forth in SEQ ID NO: 28, or consists of the amino acids as set forth in SEQ ID NO: 28. In some embodiments, said bispecific binding molecule comprises an amino acid sequence having one or more (preferably not more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations, e.g. substitutions, deletions or additions, preferably substitutions, e.g. conservative substitutions, compared to the amino acid sequence as set forth in SEQ ID NO: 28. In some preferred embodiments, said mutations are not present in the CDRs of the anti-VEGF antibody variable region and the anti-Ang2 VHH.

[0072] In another embodiment of the invention, a bispecific binding molecule, such as a bispecific antibody, of the invention has the following structure: a first anti-VEGF VHH-linker-a second anti-VEGF VHH-linker-an anti-Ang2 VHH; Here, the anti-Ang2 VHH comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the sequence shown in SEQ ID NO: 16, HCDR2 comprises or consists of the sequence shown in SEQ ID NO: 17 or SEQ ID NO: 20, and HCDR3 comprises or consists of the sequence shown in SEQ ID NO: 18.

[0073] In some embodiments, the structure of the bispecific binding molecule is as shown in FIG. 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 and the second anti-VEGF VHH are the same or different.

[0074] In some embodiments, the anti-VEGF VHH comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the sequence shown in SEQ ID NO:1, HCDR2 comprises or consists of the sequence shown in SEQ ID NO:2, and HCDR3 comprises or consists of the sequence shown in SEQ ID NO:3; or, HCDR1 comprises or consists of the sequence shown in SEQ ID NO:6, HCDR2 comprises or consists of the sequence shown in SEQ ID NO:7 or SEQ ID NO:10, and HCDR3 comprises or consists of the sequence shown in SEQ ID NO:8.

[0075] In some embodiments, said anti-VEGF VHH comprises an amino acid sequence as set forth in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:9 or SEQ ID NO:11, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with said amino acid sequence as set forth in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:9 or SEQ ID NO:11, or consists of the amino acids as set forth in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:9 or SEQ ID NO:11. In some embodiments, said VHH comprises an amino acid sequence having one or more (preferably not more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations, e.g. substitutions, deletions or additions, preferably substitutions, e.g. conservative substitutions, compared to the amino acid sequence as set forth in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:9 or SEQ ID NO:11. In some preferred embodiments, said mutations are not present in the CDRs, e.g. HCDR1, HCDR2 or HCDR3.

[0076] 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 SEQ ID NO:10, and HCDR3 comprises or consists of the sequence set forth in SEQ ID NO:8.

[0077] In some embodiments, said anti-VEGF VHH comprises an amino acid sequence as set forth in SEQ ID NO:9 or SEQ ID NO:11, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with said amino acid sequence as set forth in SEQ ID NO:9 or SEQ ID NO:11, or consists of the amino acids as set forth in SEQ ID NO:9 or SEQ ID NO:11. In some embodiments, said VHH comprises an amino acid sequence having one or more (preferably not more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations, e.g. substitutions, deletions or additions, preferably substitutions, e.g. conservative substitutions, compared to the amino acid sequence as set forth in SEQ ID NO:9 or SEQ ID NO:11. In some preferred embodiments, said mutations are not present in the CDRs, e.g. HCDR1, HCDR2 or HCDR3.

[0078] In some embodiments, the anti-Ang2 VHH of the invention comprises an amino acid sequence as set forth in SEQ ID NO: 19 or SEQ ID NO: 21, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with said amino acid sequence as set forth in SEQ ID NO: 19 or SEQ ID NO: 21, or consists of the amino acids as set forth in SEQ ID NO: 19 or SEQ ID NO: 21. In some embodiments, said VHH comprises an amino acid sequence having one or more (preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less) mutations, e.g. substitutions, deletions or additions, preferably substitutions, e.g. conservative substitutions, compared to the amino acid sequence as set forth in SEQ ID NO: 19 or SEQ ID NO: 21. In some preferred embodiments, said mutations are not present in the CDRs, e.g. HCDR1, HCDR2 or HCDR3.

[0079] In some embodiments, the linker comprises or consists of the amino acid sequence of SEQ ID NO: 23. In some embodiments, the linker between a first anti-VEGF VHH and a second anti-VEGF VHH, or the linker between a second anti-VEGF VHH and an anti-Ang2 VHH comprises or consists of the amino acid sequence of SEQ ID NO: 23, e.g., n=2.

[0080] In some embodiments, the anti-VEGF A×ANG2 bispecific binding molecule of the invention comprises an amino acid sequence as 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 with said amino acid sequence as set forth in SEQ ID NO: 22, or consists of the amino acids as set forth in SEQ ID NO: 22. In some embodiments, said bispecific binding molecule comprises an amino acid sequence having one or more (preferably not more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations, e.g. substitutions, deletions or additions, preferably substitutions, e.g. conservative substitutions, compared to the amino acid sequence as set forth in SEQ ID NO: 22. In some preferred embodiments, said mutations are not present in the CDRs of the anti-VEGF VHH and the anti-Ang2 VHH.

[0081] In another embodiment of the invention, the bispecific binding molecule of the invention comprises the following chain: VEGF R extracellular domain-Fc-linker-anti-Ang2 VHH, Here, the anti-Ang2 VHH comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the sequence shown in SEQ ID NO: 16, HCDR2 comprises or consists of the sequence shown in SEQ ID NO: 17 or SEQ ID NO: 20, and HCDR3 comprises or consists of the sequence shown in SEQ ID NO: 18.

[0082] In some embodiments, the structure of the bispecific binding molecule is as shown in Figure ID. In some embodiments, the bispecific binding molecule consists of two chains. In some embodiments, the bispecific binding molecule is tetravalent.

[0083] In some embodiments, the anti-Ang2 VHH comprises an amino acid sequence as set forth in SEQ ID NO: 19 or SEQ ID NO: 21, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with said amino acid sequence as set forth in SEQ ID NO: 19 or SEQ ID NO: 21, or consists of the amino acids as set forth in SEQ ID NO: 19 or SEQ ID NO: 21. In some embodiments, the VHH comprises an amino acid sequence having one or more (preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less) mutations, e.g. substitutions, deletions or additions, preferably substitutions, e.g. conservative substitutions, compared to the amino acid sequence as set forth in SEQ ID NO: 19 or SEQ ID NO: 21. In some preferred embodiments, the mutations are not present in the CDRs, e.g. HCDR1, HCDR2 or HCDR3.

[0084] In some embodiments, the VEGFR extracellular domain is an extracellular domain of VEGFR from human. In some embodiments, the VEGFR extracellular domain comprises a VEGFR1 second antibody-like domain (e.g., FLT1 domain 2) and a VEGFR2 third antibody-like domain (e.g., KDR domain 3). In some embodiments, the VEGFR extracellular domain comprises a human VEGFR1 second antibody-like domain and a human VEGFR2 third antibody-like domain. In some embodiments, the VEGFR extracellular domain comprises an amino acid sequence as shown 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 with the amino acid sequence as shown in SEQ ID NO:26, or consists of the amino acid sequence as shown 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, 1 or less) mutations, e.g., substitutions, deletions or additions, preferably substitutions, e.g., conservative substitutions, compared to the amino acid sequence set forth in SEQ ID NO: 26. Preferably, the VEGFR extracellular domain retains a binding affinity for VEGF similar to (e.g., has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) of the domain set forth in SEQ ID NO: 26.

[0085] In some embodiments, the Fc is an Fc derived from human IgG1, IgG2, IgG3, or IgG4, e.g., a wild-type Fc, or an Fc variant known in the art. In some embodiments, the Fc comprises the amino acid sequence set forth in SEQ ID NO:27, or 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, or consists of the amino acids set forth in SEQ ID NO:27.

[0086] In some embodiments, the VEGF R extracellular domain-Fc is a fusion protein of the VEGFR extracellular domain and Fc, such as Aflibercept or a derivative thereof.

[0087] In some embodiments, the VEGF R extracellular domain comprises an 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 with the amino acid sequence set forth in SEQ ID NO: 25, or consists of the amino acids set forth in SEQ ID NO: 25. In some embodiments, the VEGF R extracellular domain comprises an amino acid sequence having one or more (preferably not more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations, e.g. substitutions, deletions or additions, preferably substitutions, e.g. conservative substitutions, compared to the amino acid sequence set forth in SEQ ID NO: 25. Preferably, the VEGF R extracellular domain-Fc retains a binding affinity for VEGF similar to the domain set forth in SEQ ID NO: 25 (e.g. having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0088] In some embodiments, the linker comprises or consists of the amino acid sequence of SEQ ID NO: 23, eg, n=3.

[0089] In some embodiments, one chain of the anti-VEGF A×ANG2 bispecific binding molecule of the invention comprises an amino acid sequence of SEQ ID NO: 24 or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with said amino acid sequence shown in SEQ ID NO: 24 or consists of the amino acids shown in SEQ ID NO: 24. In some embodiments, said bispecific binding molecule comprises an amino acid sequence having one or more (preferably not more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1) mutations, e.g. substitutions, deletions or additions, preferably substitutions, e.g. conservative substitutions, compared to the amino acid sequence shown in SEQ ID NO: 24. In some preferred embodiments, said mutations are not present in the CDRs of the anti-Ang2 VHH. In some embodiments, the mutated VEGF R extracellular domain-Fc retains a binding affinity for VEGF similar to (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) the domain set forth in SEQ ID NO:25.

[0090] In one embodiment of the present invention, the antibody or binding molecule described herein comprises one or more amino acid mutations. In some embodiments, the amino acid mutation comprises an amino acid substitution, insertion, or deletion. Preferably, the amino acid mutation described herein is an amino acid substitution, preferably a conservative substitution.

[0091] In a preferred embodiment, the amino acid mutations described in the present invention occur in regions outside the CDRs (e.g., FRs). In some embodiments, the amino acid mutations described in the present invention occur in the antibody heavy chain constant region, such as the Fc region, and in a preferred embodiment, the amino acid mutations on the Fc region weaken or eliminate the ADCC and / or CDC activity of the antibody.

[0092] In some embodiments, the substitution is a conservative substitution. A conservative substitution refers to the replacement of one amino acid with another amino acid of the same kind, for example, the replacement of one acidic amino acid with another acidic amino acid, the replacement of one basic amino acid with another basic amino acid, or the replacement of one neutral amino acid with another neutral amino acid.

[0093] In some embodiments, Fc region variants can be generated by introducing one or more amino acid mutations into the Fc region of an antibody provided herein to alter one or more functional properties of the antibody, e.g., serum half-life, complement fixation, complement dependent cytotoxicity, Fc receptor binding and / or antibody dependent cellular cytotoxicity. The Fc region variants can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) that contains an amino acid mutation (e.g., a substitution) at one or more amino acid positions.

[0094] In some embodiments, it may be necessary to mutate the variable regions of an antibody to create disulfide bonds, such as generating an scFv that contains disulfide bond mutations.

[0095] In some embodiments, the antibody or binding molecule provided herein can be further modified to include other non-protein moieties that are known and readily available in the art. Suitable moieties for the 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, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerin), polyvinyl alcohol, and mixtures thereof.

[0096] IV. Properties of the VHH antibodies or bispecific binding molecules of the invention In some embodiments, the anti-Ang2 VHH antibodies of the invention can specifically bind to Ang2, eg, human Ang2, eg, with high affinity.

[0097] In some embodiments, the anti-VEGFA VHH antibodies of the invention can specifically bind to VEGF A, eg, human VEGF A, eg, with high affinity.

[0098] In some embodiments, a bispecific binding molecule of the invention can specifically bind to Ang2 and VEGFA, eg, human Ang2 and human VEGFA, eg, with high affinity.

[0099] In some embodiments, the anti-Ang2 or anti-VEGFA antibody or bispecific binding molecule of the invention has the following characteristics: (i) the anti-Ang2 antibody or bispecific binding molecule has an inhibitory effect on Ang2-induced Tie2 phosphorylation; (ii) the anti-Ang2 antibody or bispecific binding molecule has a blocking effect on the binding between Ang2 and Tie2; (iii) the anti-VEGFA antibody or bispecific binding molecule has a blocking effect on the VEGFA activation-associated receptor signaling pathway, e.g., as detected by a KDR reporter; (iv) the anti-VEGFA antibody or bispecific binding molecule has a blocking effect on the binding between VEGFA and VEGFR; (v) the anti-VEGFA antibody or bispecific binding molecule has an inhibitory effect on VEGFA-induced cell (e.g., primary cells, e.g., vascular endothelial cells, e.g., human umbilical vein endothelial cells, e.g., HUVEC) survival and proliferation; (vi) the bispecific binding molecule has an inhibitory effect on leakage of vascular endothelial cells (e.g., human umbilical vein endothelial cells, e.g., HUVEC); and (vii) The bispecific binding molecule has an inhibitory effect on angiogenesis in vivo or in vitro, e.g., inhibiting fundus or retina-choroidal neovascularization, e.g., inhibiting leakage caused by neovessels, e.g., preserving vascular integrity.

[0100] V. Nucleic Acids of the Invention and Host Cells Containing Same In one aspect, the invention provides a nucleic acid encoding any of the above VHH or bispecific binding molecules, or any of their chains. In one embodiment, a carrier is provided comprising said nucleic acid. In one embodiment, the carrier is an expression carrier, e.g., a eukaryotic expression carrier. Carriers include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs). In one embodiment, the vector is, e.g., pcDNA3.1. In one embodiment, a host cell is provided comprising said nucleic acid or said carrier. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells, e.g., CHO cells (e.g., CHO-S) or 293 cells (e.g., 293F or HEK293 cells), or other cells applied to the production of antibodies or fragments thereof. In another embodiment, the host cell is a prokaryotic cell, e.g., an E. coli cell, e.g., TG1.

[0101] For example, nucleic acids of the invention include nucleic acids encoding an amino acid sequence set forth in any one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:28, or nucleic acids encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in any one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:28.

[0102] In one embodiment, a host cell is provided that comprises said vector.Suitable host cells for cloning or expressing the vector encoding VHH or bispecific binding molecule include prokaryotic or eukaryotic cells as described herein.For example, VHH can be produced in bacteria.After expression, VHH is present in culture supernatant and can be further purified.

[0103] In one embodiment, the host cell is prokaryotic, for example a bacteria, such as an E. coli cell, for example TG1.

[0104] In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells, or other cells adapted for the production of antibodies or fragments thereof. Eukaryotic microorganisms, such as filamentous fungi or yeast, are suitable cloning or expression hosts for antibody-encoding vectors. For example, fungal and yeast strains in which the glycosylation pathway has already been "humanized" produce antibodies with partial or complete human glycosylation patterns. Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells may also be used as hosts. For example, mammalian cell lines modified for suspension growth can be used. Other examples of useful mammalian host cell lines are the SV40-transformed monkey kidney CV1 cell line (COS-7), human embryonic kidney cell lines (HEK293, 293F or 293T), etc. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells, CHO-S cells, ExpiCHO, etc., and myeloma cell lines such as Y0, NS0 and Sp2 / 0. Suitable mammalian host cell lines for the production of antibodies are known in the art.

[0105] VI. Production and purification of VHH or bispecific binding molecules of the invention In one embodiment, there is provided a method for producing a VHH or bispecific binding molecule of the invention, said method comprising culturing a host cell comprising a nucleic acid encoding said VHH or bispecific binding molecule (e.g. any one polypeptide chain and / or multiple polypeptide chains) or an expression vector for said nucleic acid under conditions suitable for expression of said VHH or bispecific binding molecule or chains thereof, as described above, and optionally recovering said VHH or bispecific binding molecule from said host cell (or host cell medium).

[0106] To recombinantly produce the molecules of the invention, the nucleic acid encoding the VHH or bispecific binding molecule of the invention (e.g., the molecules described above, e.g., any one polypeptide chain and / or multiple polypeptide chains) is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids are easy to isolate and sequence using conventional protocols.

[0107] The VHH or bispecific binding molecules produced as described herein can be purified by known conventional techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions for purifying a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to one of skill in the art. The purity of the antibody molecules of the invention can be determined by any one of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc.

[0108] VII.Measurement method Various assays known in the art can be used to identify, screen, or characterize the physical / chemical properties and / or biological activity of the VHHs or bispecific binding molecules provided herein. In one aspect, the VHHs or bispecific binding molecules of the invention are tested for their target (e.g., antigen) binding activity by known methods, such as, for example, biolayer interference technology, ELISA, etc. Binding to VEGF A and / or Ang2 can be measured by methods known in the art, and exemplary methods are disclosed herein. In some embodiments, it is measured by radioimmunoassay (RIA) or biolayer interferometry or MSD assay or surface plasmon resonance (SPR) or flow cytometry.

[0109] The present invention further provides assays for identifying VHHs or bispecific binding molecules with biological activity. Biological activity can be, for example, (i) the inhibitory effect of anti-Ang2 antibodies or bispecific binding molecules on Ang2-induced Tie2 phosphorylation; (ii) the blocking effect of the anti-Ang2 antibody or bispecific binding molecule on the binding between Ang2 and Tie2; (iii) the blocking effect of the anti-VEGFA antibody or bispecific binding molecule on the VEGFA activation-related receptor signaling pathway, e.g., as detected by a KDR reporter; (iv) the blocking effect of the anti-VEGFA antibody or bispecific binding molecule on the binding between VEGFA and VEGFR; (v) the inhibitory effect of the anti-VEGFA antibody or bispecific binding molecule on VEGFA-induced cell (e.g., primary cells, e.g., vascular endothelial cells, e.g., human umbilical vein endothelial cells, e.g., HUVEC) survival and proliferation; (vi) the inhibitory effect of the bispecific binding molecule on leakage of vascular endothelial cells (e.g., human umbilical vein endothelial cells, e.g., HUVEC); (vii) The bispecific binding molecule is selected from an inhibitory effect on angiogenesis in vivo or in vitro, e.g. inhibition of fundus or retina-choroidal neovascularization, e.g. inhibition of leakage caused by neovessels, e.g. preservation of vascular integrity.

[0110] The cells used in any of the above in vitro assays may be primary cells or cell lines, including cells that naturally express or overexpress an Ang2 receptor (e.g., Tie2) or a VEGFR (e.g., VEGFR2, i.e., KDR), such as 293 cells that overexpress Tie2 or KDR, e.g., HEK293 or Expi293, or vascular endothelial cells, e.g., human umbilical vein endothelial cells, e.g., HUVEC.

[0111] In some embodiments, the measurement can be performed using a marker such as biotin or hFc.

[0112] It should be noted that any of the above assays can be accomplished using a combination of the antibodies of the present invention and other active agents.

[0113] VIII. Immunoconjugates and Pharmaceutical Compositions In some embodiments, the present invention provides an immunoconjugate comprising any of the VHH or bispecific binding molecules described herein. Preferably, said immunoconjugate comprises one or more other therapeutic agents or markers.

[0114] In some embodiments, the invention provides a composition or a medicament or formulation comprising any of the VHH or bispecific binding molecules described herein, preferably the composition is a pharmaceutical composition. In one embodiment, the composition further comprises a pharmaceutical auxiliary material. In one embodiment, the composition, e.g. the pharmaceutical composition, comprises a combination of a VHH or bispecific binding molecule of the invention and one or more other therapeutic agents.

[0115] The present invention also includes compositions (including pharmaceutical compositions) or drugs or preparations comprising the VHH or bispecific binding molecules of the present invention. These compositions or drugs or preparations may also contain suitable pharmaceutical auxiliary materials, such as pharmaceutical vectors known in the art, pharmaceutical excipients including buffering agents.

[0116] As used herein, a "medicinal vector" includes any or all of physiologically compatible solvents, dispersion media, isotonic agents, absorption delaying agents, and the like.

[0117] For the use of pharmaceutical excipients and their applications, reference is also made to Handbook of Pharmaceutical Excipients, Eighth Edition, R. C. Rowe, P. J. Eskey and S. C. Wen, Pharmaceutical Press, London, Chicago.

[0118] The composition or drug or formulation of the present invention can be in various forms.These forms include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (for example, injections or eye drops), dispersions or suspensions, liposomes and suppositories.The preferred form is determined by the desired mode of administration and therapeutic use.For example, the composition or drug or formulation of the present invention can be eye drops.

[0119] A drug or formulation of the VHH or bispecific binding molecule described herein can be produced by mixing the VHH or bispecific binding molecule of the present invention having the desired purity with one or more optional pharmaceutical auxiliary materials, preferably in the form of a lyophilized formulation or an aqueous solution.

[0120] The compositions or medicaments or formulations of the present invention may further comprise one or more active ingredients, which are necessary for the particular indication being treated, and preferably have complementary activities that do not adversely affect each other, e.g., it may be desirable to provide other therapeutic agents as well.

[0121] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or in microcapsule form.

[0122] The present invention further provides a drug combination or drug combination product, comprising a VHH or bispecific binding molecule of the present invention and one or more other therapeutic agents. The present invention further provides a set of drug kits comprising said drug combination, e.g. said set of drug kits in the same package, - a first container containing a pharmaceutical composition comprising a VHH or a bispecific binding molecule of the invention, and a second container containing a pharmaceutical composition comprising another therapeutic agent.

[0123] IX. Use and Methods One aspect of the present invention provides a method for preventing or treating an eye disease in a subject, the method comprising administering to the subject an effective amount of an anti-VEGF VHH of the present invention, an anti-Ang2 VHH of the present invention, or a bispecific binding molecule of the present invention, or a composition, drug or formulation comprising them.

[0124] In some embodiments, the patient has elevated levels of VEGF, eg, VEGFA, and / or Ang2 (eg, elevated nucleic acid or protein levels).

[0125] In some embodiments, the ocular disease includes, but is not limited to, angiogenesis-associated ocular disease, such as, for example, corneal neovascularization-associated ocular disease.

[0126] In some embodiments, the treatment of the eye disease would benefit from suppressed nucleic acid or protein levels of VEGF, such as VEGFA, and / or Ang2.

[0127] In another aspect, the present invention provides the use of an anti-VEGF VHH of the invention, an anti-Ang2 VHH of the invention, or a bispecific binding molecule of the invention, or a composition comprising same, in the production or manufacture of a medicament, said medicament for a use as described herein, e.g. for the prevention or treatment of an associated disease or condition as described herein.

[0128] In some embodiments, the anti-VEGF VHH of the invention, the anti-Ang2 VHH of the invention, or the bispecific binding molecule of the invention, or a composition or drug or formulation comprising them, can delay the onset of a disease and / or symptoms associated with a disease.

[0129] In some embodiments, the anti-VEGF VHH of the invention, the anti-Ang2 VHH of the invention, or the bispecific binding molecule of the invention, or a composition or drug or formulation comprising them, may be further administered in combination with one or more other therapies, e.g., treatment regimens and / or other therapeutic agents, and used for the uses described herein, e.g., for the prevention or treatment of the associated diseases or conditions described herein.

[0130] The route of administration of the anti-VEGF VHH of the invention, the anti-Ang2 VHH of the invention, or the bispecific binding molecule of the invention, or a composition or drug or formulation comprising them, is according to known methods, e.g., topical administration, e.g. intraocular administration, ocular surface administration. In some embodiments, administration is by injection or instillation.

[0131] X. Methods and Compositions for Diagnosis and Detection In some embodiments, the anti-Ang2 VHH antibodies provided herein can also be used to detect the presence of Ang2 in a biological sample. In some embodiments, the anti-VEGFA VHH antibodies provided herein can be used to detect the presence of VEGFA in a biological sample. In some embodiments, the anti-bispecific binding molecules provided herein could be used to detect the presence of Ang2 and / or VEGFA in a biological sample.

[0132] The term "detection" as used herein includes quantitative or qualitative detection, and exemplary detection methods include immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), antibody molecule-conjugated magnetic beads, ELISA assays, PCR-techniques (e.g., RT-PCR). In some embodiments, the biological sample is a body fluid or an ocular tissue, such as vitreous humor.

[0133] In some embodiments, the method comprises contacting a biological sample with a VHH or bispecific binding molecule as described herein under conditions that permit binding to Ang2 or VEGFA, and detecting whether a complex is formed between the VHH or bispecific binding molecule and Ang2 or VEGFA. The formation of a complex indicates the presence of Ang2 or VEGFA. The method may be an in vitro or in vivo method. In one embodiment, the antibody of the invention is used to select subjects suitable for treatment with the VHH or bispecific binding molecule of the invention, e.g., where Ang2 or VEGFA is a biomarker for selecting said subjects.

[0134] In some embodiments, a labeled VHH or bispecific binding molecule is provided. Labels include, but are not limited to, directly detectable labels or moieties (e.g., fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels and radioactive labels) and indirectly detectable moieties, e.g., enzymes or ligands, e.g., by enzyme-catalyzed reactions or molecular interactions. In some embodiments, the label is a marker, e.g., biotin or hFc.

[0135] In some embodiments described herein, the sample is obtained prior to treatment with the VHH or bispecific binding molecule of the invention. In some embodiments, the sample is obtained prior to the use of other therapies. In some embodiments, the sample is obtained during or after treatment with other therapies.

[0136] In some embodiments, Ang2 and / or VEGF A are detected pre-treatment, for example, before treatment begins or pre-treatment after a treatment interval.

[0137] In some embodiments, a method of treating a disease of the present invention is provided, said method comprising determining Ang2 and / or VEGF A levels by testing a subject (e.g. a sample) (e.g. a subject sample) for the presence of Ang2 and / or VEGF A, comparing the Ang2 and / or VEGF A levels to a control value (e.g. a value in a normal individual), and if the Ang2 and / or VEGF A levels are greater than the control value, administering to the subject a therapeutically effective amount of a VHH or bispecific binding molecule according to the present invention, optionally in combination with one or more other therapies, thereby treating the disease.

[0138] These and other aspects and embodiments of the present invention are described in the drawings (followed by a brief description of the drawings) and the following detailed description of the invention, and are illustrated in the following examples. Any or all of the features described above and throughout this application can be combined in each embodiment of the present invention. The present invention is further described using the following examples, which are given by way of illustration and not by way of limitation, and it should be understood that various modifications can be made by those skilled in the art. EXAMPLES

[0139] Example 1. Preparation of a phage immunization library Construction of alpaca immune or synthetic libraries 1.1 Two healthy adult alpacas (Chengdu NBBIOLAB) were selected. 0.5mg of recombinant protein antigen VEGFA or Ang2 (Beijing Sino Biological Co., Ltd.) was mixed uniformly with Freund's adjuvant in a 1:1 ratio and subcutaneously injected into multiple sites on the back to immunize the alpacas four times, with an immunization interval of 2 weeks.

[0140] 1.2 Collect 50 ml of alpaca peripheral blood, separate lymphocytes, and obtain 2.5 x 10 viable cells. 71 mL of Trizol reagent was added per cell, and total RNA was extracted by chloroform / isopropyl alcohol precipitation. 10 μg of RNA was taken as a template and reverse transcription was performed using PrimeScript reverse transcription kit (Takara). The first PCR reaction was performed using cDNA as a template, and the first PCR product was obtained using forward primer Alp-VhL and reverse primer Alp-2b / 2cR. The second PCR reaction was performed using the first PCR product as a template, and the second PCR product was obtained using forward primer Alp-VhF and reverse primer Alp-JHR-SalI. The pC3-HF vector and the second PCR product were double-digested using SacI and SalI (Thermo), respectively, and the enzyme digestion product was added to T4 ligase (Thermo) and reacted, and TG1 competent cells were electrotransformed to construct a VHH antibody library, and the bacterial solution was frozen and stored at -80 °C. The revived bacterial liquid was inoculated into 100 ml of YT-AG medium (Shanghai Sangon Biotech), and M13KO7 helper phage was added to infect the cells. The cells were then resuspended in 2xYT-AK medium (Shanghai Sangon Biotech) and cultured overnight at 37°C and 200 rpm. The culture supernatant was collected, and recombinant phages were prepared using the PEG / NaCl precipitation method.

[0141] 1.3 The 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 an appropriate amount of biotin-labeled antigen were added to each tube, incubated at room temperature for 30 min, and then 1 × 10 12 cfu of recombinant phages were added and incubated at room temperature for 1 h. The resulting mixture was added to 1 ml of PBST and washed 10 times with 5 min each wash time. Finally, 0.5 ml of pH 2.5 glycine buffer was added to elute the antigen-bound recombinant phages, which were then infected with TG1 and cultured overnight to prepare recombinant phages for the next panning experiment to identify the TG1 bacterial clones with positive VHHs.

[0142] 1.4 Binding ELISA was used to detect binding activity and cloning sequencing. VEGF A was pretreated, and Ang2 antigen (Beijing Sino Biological Co., Ltd.) was diluted to 0.5 μg / ml in PBS buffer and coated on a 96-well ELISA plate and left in a refrigerator at 4 ° C overnight. The antigen-coated plate was washed three times with PBST, and 300 μl / well of blocking agent was added and blocked at room temperature for 1 hour. The plate was washed three times with PBST, and 80 μl of blocking agent + 20 μl of expression supernatant of TG1 bacteria of positive VHH identified in 1.3 above was added and shaken at room temperature for 1 hour. The plate was washed three times with PBST, and 100 μl / well of Anti-Flag / HRP secondary antibody (Sigma Co., Ltd.) diluted with blocking agent 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 and color development was allowed to continue for 5 to 15 minutes away from light. 100 μl / well of stop solution was further added. The OD450nm absorbance was measured by reading with a microplate reader, and bacterial clones with a reading greater than 0.5 were selected and sent to GeneWiz for sequencing, and TG1 bacterial clones containing each corresponding VHH sequence were selected, added with glycerol, and frozen at −80°C in a refrigerator.

[0143] Example 2. Prokaryotic antibody production and purification, and humanization The present invention utilizes molecular biology techniques to obtain antibody sequences within anti-VEGFA or Ang2 positive phages, and utilizes TG1 monoclonal expression and purification containing the above obtained positive VHH to obtain VHH antibody proteins.

[0144] TG1 bacteria containing the VHH expression plasmid identified in Example 1 were inoculated into 800 ml of LB-Amp medium and cultured at 37 ° C and 200 rpm until the OD600 value reached 0.5 to 0.6. 1 mM IPTG was added to the bacterial solution to induce expression, and the bacteria were cultured overnight at 28 ° C and 200 rpm. The culture supernatant was collected, and after centrifugation, 15 ml PB + 1 mg / ml polymyxin was added to resuspend the bacteria, centrifuged again, and filtered through a 0.22 μm filter membrane. The bacterial lysate was run through a 1 ml Ni Sepharose precolumn, washed twice with PBS, and the target protein was eluted by adding 0.5 M imidazole, and the protein concentration was measured using the ultraviolet method. The eluted target protein was measured for protein concentration using the ultraviolet method, divided into multiple tubes, and frozen at -40 ° C in a refrigerator. The obtained antibody solution is hereinafter referred to as the supernatant.

[0145] The amino acid sequences of CDR and VHH of two anti-VEGF A VHH antibodies (LA42F8 and LA46E11) and one anti-Ang2 VHH antibody (LA24C11) obtained in the present invention, as well as SEQ ID NOs, were referenced in the sequence listing.

[0146] Next, the immune library antibodies LA42F8, LA46E11, and LA24C11 obtained above were humanized according to the following steps: 1. Determine the CDR loop structure; 2. Find the closest homologous sequence for each V / J region of the heavy chain in a human germline sequence database; 3. Screening for the closest matching human germline heavy and light chains and the lowest amount of reversion mutations; 4. Assembling the CDR regions of the chimeric antibody onto human framework regions; 5. Using sequence and structural features to determine amino acid positions in the framework region that serve to maintain the CDRs; 6. Backmutating (reverting to the input amino acid type) at sequence positions determined to be important; 7. The amino acids at the risk sites were optimized.

[0147] The amino acid sequences of the CDRs and heavy chain variable regions of the humanized VHH antibodies LA42F8.5, LA46E11.8, and LA24C11.10 obtained from the three humanized antibodies of the present invention are shown in the attached sequence listing.

[0148] The above-mentioned LA42F8, LA46E11, LA24C11 and humanized antibodies LA42F8.5, LA46E11.8, LA24C11.10 were prepared for expression in eukaryotic cells as follows:

[0149] The humanized antibody sequences obtained above were cloned into pcDNA3.1 (Invitrogen) to obtain plasmids containing the antibody sequences.

[0150] Depending on the required transfection volume, passage Expi-293 cells (Invitrogen) to a cell density of 1.5 × 10 the day before transfection. 6 The cell density was adjusted to approximately 3 × 10 cells / mL on the day of transfection. 6The transfection concentration was 1000 cells / mL. 1 / 10 of the final volume of F17 medium (Gibco, A13835-01) was used as a transfection buffer, and the appropriate plasmid was added and mixed uniformly. An appropriate amount of polyethyleneimine (PEI) (Polysciences, 23966) was added to the plasmid (the ratio of plasmid to PEI in 293F cells was 1:3), mixed uniformly, and then incubated at room temperature for 10 minutes to obtain a DNA / PEI mixture. The cells were resuspended in the DNA / PEI mixture and cultured at 36.5°C and 8% CO2. After 24 hours, a feed medium (FEED) (Sigma) was added at 2% of the transfection volume, and the cells were cultured at 36.5°C, 120 rpm, and 8% CO2. On the 6th day of continuous culture or when the cell activity was below 60%, the cell supernatant was collected and purified. The gravity column used for purification was treated with 0.5M NaOH overnight, and the glass bottles were washed with distilled water and then dried at 180°C for 4 hours to obtain the purification column. Before purification, the collected cell supernatant was centrifuged at 4500 rpm for 30 minutes and the cells were discarded. The supernatant was then filtered through a 0.22 μl filter. A Protein A column (Hitrap Mabselect Sure 5*5ml, GE, 11-0034-95) was equilibrated with 10 ml of binding buffer (sodium phosphate 20 mM, NaCl 150 mM, pH 7.0). The filtered supernatant was added to the purification column and re-equilibrated with 15 ml of binding buffer. 5 ml of elution buffer (citric acid + sodium citrate 0.1 M, pH 3.5) was added to collect the eluate, and 80 μl of Tris-HCl was added per ml of eluate. The collected antibody was concentrated by ultrafiltration and exchanged into PBS (Gibco, 70011-044) to detect the concentration. Except for the antibodies of the present invention for detection in Table 3, Figure 2, Figure 4A, and Figure 5A, all antibodies used in the examples were purified antibodies for this expression unless specifically stated for the supernatant.

[0151] Similarly, the coding nucleic acids of negative control IgG, positive control BI-anti-VEGF, BI836880, and Faricimab (see sequence listing for sequences) were cloned into pcDNA3.1, transfected into Expi-293 cells, and then expressed and purified, and the method was the same as in Example 2.

[0152] Example 3 Measurement of binding kinetics between the chimeric antibody of the present invention and an antigen by biolayer interference technology The equilibrium dissociation constant (KD) of the antibody of the present invention binding to human Ang2 was measured using Biolayer Interferometry Technology (ForteBio). ForteBio affinity was measured according to a conventional method (Estep, P et al., High throughput solution based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2):p270-8).

[0153] Half an hour before the start of the experiment, an appropriate number of AMQ (Pall, 1506091) (for sample detection) or AHQ (Pall, 1502051) (for positive control detection) sensors were prepared according to the number of samples and immersed in SD buffer (PBS 1x, BSA 0.1%, polysorbate 20 0.05%).

[0154] 100 μl of SD buffer, VHH antibody prepared in Example 2 above, and antigen [including human Ang2 (Beijing Sino Biological), and human VEGF165 (R&D)] were taken and added to a 96-well black polystyrene half-volume microplate (Greiner, 675076) respectively. The plate was arranged according to the position of the sample, and the position of the sensor was determined. The instrument parameters were set as follows: the steps Baseline, Loading~1 nm, Baseline, Association, and Dissociation were performed, and the running time of each step was determined by the binding and dissociation speed of the sample, the rotation speed was 400 rpm, and the temperature was 30 °C. K was measured using the ForteBio analysis software.D The values ​​were analyzed.

[0155] In the experiments described in the above assay, the affinity of the antibodies is shown in Table 1. [Table 2] [Table 3]

[0156] Example 4 Anti-VEGF A VHH Antibody ELISA Blocking Experiment This example examined the blocking effect of the anti-VEGF A VHH of the present invention on the binding of hVEGFA to the receptor KDR. SA (Thermo Cat. No. 21125) was diluted to 1 μg / ml and plated in a microplate at 100 μl / well and placed at 4° C. overnight. After washing three times with PBST, 3% BSA was added for blocking for 1.5 hours. Washed 3 times with PBST, 50ng / ml biotin-labeled VEGF A165 (ACRO Cat. No. VE5-H8210) was added and incubated for 1.5 hours, and LA42F8 supernatant, LA46E11 supernatant, negative control IgG, and 50μl positive control BI-anti-VEGF prepared in Example 2 (initial concentration 150μg / ml, 3-fold serial dilution) were added to the plate after incubation with VEGFR-Fc (Beijing Sino Biological, Cat. No.: 10012-H02H, final concentration 0.2μg / ml) for 20 minutes. Washed 3 times with PBST, anti-human Fc HRP antibody (Bethyl Cat. No.: A80-104P) (1:10000) was added and incubated for 30 minutes. Washed 6 times with PBST, developed with TMB for 5 minutes, and read at OD450nm after stopping.

[0157] The blocking results of the three anti-VEGF VHH antibodies obtained according to the present invention are shown in Figure 2. Figure 2 shows that the candidate molecules LA42F8 and LA46E11 antibodies can completely block the binding of VEGF A to VEGFR2.

[0158] Example 5 Anti-Ang2 VHH Antibody ELISA Blocking Experiment In this example, the blocking 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 was examined.

[0159] Tie2-Fc (Beijing Sino Biological Catalog No.: 10700-H03H) was diluted to 2 μg / ml and plated on a microplate at 100 μl / well and left at 4°C overnight. Washed three times with PBST and blocked with 3% BSA for 1.5 hours. Washed three times with PBST, 50 μl each of purified LA24C11.10 and negative control IgG prepared in advance in Example 2 were incubated with hAng2-biotin (final concentration 0.2 μg / ml) for 20 minutes and then added to the plate. Washed three times with PBST, Avidin HRP (1:2000) was added and incubated for 35 minutes. Washed six times with PBST, developed with TMB for 5 minutes, and read at OD450nm after stopping.

[0160] The blocking results of the anti-Ang2 VHH antibodies obtained according to the present invention are shown in Table 3, and the candidate molecule LA24C11 had a blocking effect on the binding of Ang2 to its receptor Tie2. [Table 4]

[0161] Similar to Example 4 (using Ang2-Bio (R&D Cat. No.: BT623B / CF)), the effect of LA24C11.10 on blocking the binding of Ang2 and Tie2 was detected by ELISA blocking experiment, and the results are shown in Figure 3. It was found that LA24C11.10 has a blocking effect on the binding of Ang2 to its receptor Tie2.

[0162] Example 6 Phosphorylation experiment of anti-Ang2 VHH antibody This example shows the effect of the anti-Ang2 antibody of the present invention on hAng2-Fc-induced Tie2 phosphorylation. The inhibitory effect of VHH antibodies was demonstrated.

[0163] Phosphorylation experiments induced by hAng2 This study co-incubated antibodies and recombinant hAng2-Fc protein with Expi293 cells 293-Tie2 overexpressing Tie2, and detected the content of phosphorylated Tie2 in the system to reflect the inhibitory effects of different antibodies on Tie2 phosphorylation induced by hAng2-fc.

[0164] Expi-293 cells overexpressing human Tie2 293-Tie2 cells were generated by transfecting Expi-293 cells (Thermo) with the pCHO1.0 vector (Invitrogen) carrying the human Tie2 gene (Beijing Sino Biological Catalog No.: HG10700-M) cloned into the multiple cloning site MCS.

[0165] 293-Tie2 cells overexpressing human Tie2 were taken and cultured at 2 × 10 6 Dilute cells / ml and add 100 μl per well to a 96-well plate, centrifuge at 400 g for 5 minutes and remove the supernatant.

[0166] Expi293 medium (Thermo Cat. No. A1435102) was used to constitute the experimental medium, into which the test antibodies (LA24C11 (24C11) and LA24C11.0 (hz24C11.10) prepared in Example 2, as well as the negative control IgG, and the positive control Nesvacumab (prepared according to CN202010573625.2)) were added, with an initial concentration of 60 μg / ml, serially diluted 1:2, and the final concentration of hAng2-Fc (Beijing Sino Biological: 10691-H02H) was 2.5 μg / ml.

[0167] Cells were resuspended in 100 μl of experimental medium per well, incubated at 37°C for 15 min, centrifuged to remove medium, 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, placed on ice for 30 min, centrifuged at 2000g, and protein supernatants were collected and stored in a refrigerator at -80°C.

[0168] The pTie2 concentration was detected according to the instructions of the phosphorylated Tie2 ELISA kit (R&D Catalog No.: DYC2720E), and the capture antibody in the kit was coated on a microplate at a concentration of 4 μg / ml and left at 4 °C overnight. It was washed three times with PBST and blocked with 5% BSA for 1 h. 100 μl of the frozen and thawed protein supernatant obtained in the previous step and the control pTie2 (R&D Catalog No.: DYC2720E) were added and used to generate a standard curve and incubated at room temperature for 2 h (if the concentration of the sample pTie2 was too high and exceeded the detection range of the ELISA, the resulting mixture was diluted 2-3 times). It was washed three times with PBST, and 100 μl of anti-pTyr antibody conjugated with HRP (R&D Catalog No.: DYC2720E) was added and incubated at room temperature for 2 h. The plate was washed six times with PBST, and 100 μl of TMB (Solarbio Catalog Number: PR1200) was added to develop the color. After 15 minutes, 100 μl of stop buffer (Solarbio Catalog Number: C1058) was added to stop the reaction. OD450-OD620 was measured using a multi-function microplate reader SpectraMax i3. The experimental results are shown in Figure 4A (24C11 is the LA24C11 prokaryotic expression purified supernatant prepared in Example 2) and Figure 4B.

[0169] Thus, both anti-Ang2 VHH antibodies LA24C11 and LA24C11.10 of the present invention were able to effectively inhibit hAng2-Fc-induced phosphorylation of 293-Tie2 cells in vitro.

[0170] Example 7 Anti-VEGF A VHH antibody KDR reporter blocking experiment VEGF A binds to its associated receptor VEGFR2 (KDR), activates the VEGFR2 signaling pathway, and induces the survival, proliferation, and migration of vascular endothelial cells. In this study, we used the KDR reporter experimental system and NFAT-RE-luc2P / KDR HEK293 cells (Promega Cat CS181401) to detect the blocking effect of serially diluted antibodies on the VEGFA activation-associated receptor signaling pathway.

[0171] The experimental method was described in accordance with the manufacturer's (Promega) instructions.

[0172] Take NFAT-RE-luc2P / KDR HEK293 cells that were replaced with experimental medium (DMEM medium containing 10% FBS) 3 days ago, aspirate off the old medium, wash once with PBS, then use 1ml of Accutase solution (Sigma Cat. No.: A6964-500ML) to digest the cells until they become round and dewalled, stop the reaction with 5ml of dilution medium, aspirate the cells into a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, discard the medium, add 10ml of dilution medium (DMEM medium containing 10% FBS) to resuspend the cells, mix evenly and count, the cell viability should be more than 90%. Use dilution medium to reduce the cell density to 0.8×10 6 Cells / ml were adjusted and added at 50 μl / well to 96-well white cell culture plates according to the experimental layout.

[0173] A mixture of VEGF A at a concentration of 100ng / ml and gradient diluted test antibodies was prepared, left to stand for 30 minutes, and then added to a 96-well white cell culture plate containing cells at 50μl / well, and incubated at 37℃ in a 5% CO2 incubator for 6 hours. The test samples were negative control IgG, positive control BI-anti-VEGF, LA42F8, LA46E11, LA42F8.5, and LA46E11.8; Blank contained only the diluted medium, no VEGF A, no antibody; VEGF A 100ng / ml contained only 100ng / ml VEGFA.

[0174] The 96-well white cell culture plate incubated for 6 hours was removed from the carbon dioxide incubator and equilibrated to room temperature for 10-15 minutes. The Bio-Glo Luciferase Assay System, previously equilibrated to room temperature, was removed and added to the 96-well white cell culture plate at 100 μl / well according to the experimental layout, and incubated at room temperature for 5 minutes away from light. Fluorescence reading was performed using a multi-function microplate reader, the chemiluminescence mode was selected as the plate reading mode, the end point method was selected as the plate reading type, the wavelength was set to full wavelength, fluorescence was collected row by row, and the collection time for each row was 1000 ms.

[0175] In the experiment described in the above assay, the detection results are shown in FIG. 5, and the anti-VEGF VHH antibodies LA42F8, LA42F8.5, LA46E11, and LA46E11.8 were all able to block the activation of the KDR signaling pathway induced by VEGF A.

[0176] Example 8: Experimental study of inhibition of VEGF A-induced HUVEC survival and proliferation by anti-VEGF A VHH VEGF A can act on receptors related to VEGFR in vascular endothelial cells, promoting the survival, proliferation, and migration of vascular endothelial cells, thereby inducing angiogenesis. Based on the fact that VEGF induces the survival and proliferation of human umbilical vein endothelial cells (HUVEC), this experiment detected the inhibitory effect of antibodies on the survival and proliferation of primary cells induced by VEGF A.

[0177] In this example, CCK-8 was used to measure the survival and proliferation of HUVECs, and the specific method was that HUVEC cells (Allcells Cat. No.: H-001-CN) were treated one day in advance, plated in a 96-well culture plate at 2000 cells / well, and incubated in a 37°C, 5% carbon dioxide incubator for 24 hours.

[0178] After the cells attached to the wall, an experimental medium containing VEGF A at a final concentration of 10 ng / ml and gradient diluted antibodies (LA42F8 prepared in Example 2, initial concentration 80 μg / ml, diluted 1:3 at equal times, negative control IgG, positive control BI836880, a group containing only VEGF A at 10 ng / ml (VEGFA), and Blank without VEGF A and antibody addition) was prepared, replacing the endothelial cell medium in the 96-well plate, and incubated for 72 hours in a 37 ° C, 5% carbon dioxide incubator, and CCK-8 detection solution (Dojindo catalog number: CK04) was added at 10 μl / well, and incubated for 12 to 24 hours in a 37 ° C, 5% carbon dioxide incubator, and the absorbance OD450-OD620 was read using a multi-function microplate reader.

[0179] In the experiment described in the above measurement method, the detection results are shown in FIG. 6, which shows that the anti-VEGFA antibody LA42F8 can completely inhibit the survival and proliferation of HUVEC cells induced by VEGFA.

[0180] Example 9 Anti-VEGF A / Ang2 Bispecific Antibody HEK293-KDR Reporter Blocking Experiment 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 pcDNA3.1 vector and expressed in 293 cells as described in Example 2 and the bispecific binding molecules were purified.

[0181] In this example, the blocking effect of anti-VEGF A / Ang2 bispecific antibody on VEGF A was detected by HEK293-KDR reporter experiment. The experimental method was described in Example 7. The binding molecules or controls used were as follows: Blank: no VEGF A, no antibody, VEGF A: 100ng / ml VEGF A, Negative control IgG: Prepared as above Positive control Faricimab: Prepared as above, initial concentration 13.5 μg / ml, 1:3 gradient dilution, Positive control BI-836880: Prepared as above, initial concentration 13.5 μg / ml, 1:3 gradient dilution, IEX04-012: Prepared as above, initial concentration 13.5 μg / ml, 1:3 gradient dilution.

[0182] The detection results are shown in FIG. 7, and the bispecific binding molecule IEX04-012 inhibited the activation of the KDR signaling pathway induced by VEGF, with a better inhibitory ability compared with the control antibody BI836880.

[0183] Example 10 Anti-VEGF A / Ang2 bispecific binding molecule HUVEC proliferation inhibition experiment This study detected the inhibitory effect of anti-VEGF A / Ang2 bispecific binding molecule on VEGFA-induced survival and proliferation of HUVEC cells by HEK293-KDR reporter experiments.

[0184] The experimental method was the same as in Example 8, but the antibodies used were as follows: Panel A: IEX04-008, prepared as above, at an initial concentration of 20 ng / ml and diluted 1:3, negative control IgG, positive controls Faricimab and BI836880, blank (i.e., no antibody and VEGF A), and VEGF A group (i.e., only VEGFA at 20 ng / ml was added). Panel B: Negative control IgG, BI-anti-VEGF, IEX04-010, prepared as above, diluted 1:3 with an initial concentration of 80 μg / ml; Blank (i.e., no antibody and VEGF A); and VEGF A group (i.e., only 20 ng / ml VEGFA was added). Panel C: IEX04-012 prepared as above at an initial concentration of 20 nM and diluted 1:3, negative control IgG, positive controls Faricimab and BI836880, blank (i.e., no antibody and VEGF A), and VEGF A group (i.e., only 20 ng / ml VEGFA was added).

[0185] The detection results are shown in Figure 8. The bispecific binding molecules IEX04-008, IEX04-010, and IEX04-012 all inhibited the survival and proliferation of HUVEC cells induced by VEGF, and compared with the control antibodies BI836880 and Faricimab, IEX04-012 had a lower IC50 and better inhibitory ability.

[0186] Example 11 Anti-VEGF A / Ang2 Bispecific Antibody Ang2 Blocking Experiment Ang2 can bind to its natural receptor Tie2, and this study detected the blocking of the binding between Ang2 and Tie2 by anti-VEGF A / Ang2 bispecific binding molecules by ELISA and FACS.

[0187] (1) ELISA The ability of IEX04-008, IEX04-010 and IEX04-012, as well as the control antibodies BI-836880 and Faricimab to block the binding of human Ang2 to hTie2 was detected by ELISA.

[0188] hTie2 protein (Beijing Sino Biological) was resuspended in PBS to a concentration of 2 μg / mL and coated on a microplate overnight. Blocked with 5% BSA for 1 hour, biotin antigen Recombinant Biotinylated hAngiopoietin-2 protein (R&D) was diluted to 600 μg / mlM, 50 μl / well. Antibodies (IEX04-008, IEX04-010, IEX04-012, as well as positive control antibodies BI836880, Faricimab, and negative control IgG) prepared as above were diluted 1:2 from the highest concentration of 300 nM, for a total of 8 or 12 dilution gradients, 50 μl / well, and incubated in PBS on ice for 30 minutes, with the final concentration of biotin antigen being 300 ng / ml. The antigen-antibody mixture obtained above was incubated in a microplate for 90 minutes, washed three times with PBS, the supernatant was discarded, 100 μL of Avidin-HRP (Invitrogen) diluted 1:10000 was added to each well, incubated at room temperature for 30 minutes, and washed six times with PBS. Color was developed for 1 minute with 100 μL / well of TMB color development solution (solarbio), and stopped with 100 μL / well of stop solution (Solarbio).

[0189] The OD450 and OD620 of each well were read using a microplate reader.

[0190] The experimental results (see FIG. 9) showed that IEX04-008, IEX04-010, IEX04-012 and the control antibody BI836880 all had complete blocking effects, and the IC50s of the bispecific binding molecules of the invention were all significantly lower than that of the positive control antibody.

[0191] (2) Flow cytometry (FACS) The ability of IEX04-008, IEX04-010, IEX04-012, as well as the positive control antibodies BI-836880 and Faricimab and the negative control IgG to block binding of human Ang2-hFc to Tie2 on the cell surface was measured by FACS.

[0192] Antigen hAng2-Fc protein (Beijing Sino Biological, Catalog No.: 10691-H02H) was diluted to 4 μg / ml, 50 μl / well. Antibodies (IEX04-008, IEX04-010, IEX04-012, as well as positive control antibodies BI-836880 and Faricimab and negative control IgG) prepared as above were diluted 2-fold starting from the highest concentration of 800 nM, totaling 12 dilution gradients, 50 μl / well, and incubated in PBS on ice for 30 min, with the final concentration of antigen hAng2-Fc protein being 2 μg / ml, and the maximum final concentration of each antibody being 400 nM. 2 × 10 293-Tie2 cells prepared as above were incubated in PBS on ice for 30 min, with the final concentration of antigen hAng2-Fc protein being 2 μg / ml, and the maximum final concentration of each antibody being 400 nM. 5 The cells were adjusted to 100 μl / well. The cells were centrifuged at 300 g for 5 min, the supernatant was discarded, and the cells were resuspended in the antigen-antibody mixture. The cells were incubated on ice for 30 min, 100 μl / well of PBS was added, centrifuged at 300 g for 5 min, washed with PBS, and 100 μl of Goat anti-human IgG-PE (SouthernBiotech) diluted 1:200 was added per well, incubated in ice for 20 min, 100 μl of PBS was added per well, centrifuged at 300 g for 5 min, and washed with PBS. The cells were resuspended in 100 μl of PBS and the cell fluorescence signal value was detected with a flow cytometer (BD Biosciences). A concentration-dependent curve was fitted with GraphPad according to the MFI. The results are shown in Figure 10. As shown in the figure, the bispecific binding molecules IEX04-008, IEX04-010 and IEX04-012 were all able to effectively block the binding of human Ang2-hFc to Tie2 with IC50s lower than the positive control.

[0193] Example 12: Ang2 phosphorylation inhibition experiment of anti-VEGF A / Ang2 bispecific binding molecules In this example, hAng2-induced phosphorylation experiments were used to verify the inhibitory effect of the bispecific binding molecules of the present invention on hAng2-Fc-induced Tie2 phosphorylation.

[0194] In this study, the bispecific binding molecule and recombinant hAng2-Fc protein were co-incubated with Expi293 cells overexpressing Tie2, 293-Tie2, and the content of phosphorylated Tie2 in the system was detected to reflect the inhibitory effects of different antibodies on Tie2 phosphorylation induced by hAng2-Fc.

[0195] The overexpressing 293-Tie2 cells prepared as above were taken and diluted to 2×10 6 cells / ml, 100 μl per well was added to a 96-well plate, centrifuged at 400 g for 5 minutes, and the supernatant was removed.

[0196] Expi293 medium (Thermo Cat. No. A1435102) was used to prepare the experimental medium, into which the test antibodies (IEX04-012 prepared as above, positive control BI-836880 and Faricimab, negative control IgG) were added respectively to a maximum final concentration of 60 μg / ml, serially diluted 1:2 in equal parts, and the final concentration of hAng2-Fc (Beijing Sino Biological, Cat. No.: 10691-H02H) was 2.5 μg / ml.

[0197] Cells were resuspended in 100 μl of experimental medium per well, incubated at 37°C for 15 min, centrifuged to remove medium, 100 μl of NP-40 lysis solution containing 1% protease and phosphatase inhibitors was added, and placed on ice for 30 min. After centrifugation at 2000g, protein supernatants were collected and stored in a refrigerator at -80°C.

[0198] The concentration of pTie2 was detected according to the instructions of the phosphorylated Tie2 ELISA kit (R&D DYC2720E), and the capture antibody was coated on a microplate at a concentration of 4 μg / ml and left at 4 °C overnight. It was washed three times with PBST and blocked with 5% BSA for 1 hour. 100 μl of test sample and control pTie2 (R&D catalog number: DYC2720E) were added and used to generate a standard curve and incubated at room temperature for 2 hours (if the concentration of sample pTie2 was too high and exceeded the detection range of ELISA, the resulting mixture was diluted 2-3 times). It was washed three times with PBST, and 100 μl of anti-pTyr antibody conjugated with HRP (R&D catalog number: DYC2720E) was added and incubated at room temperature for 2 hours. It was washed six times with PBST, and 100 μl of TMB was added to develop the color, and 100 μl of stop buffer was added after 15 minutes to stop the reaction. The OD450-OD620 of each well was measured using a spectrophotometer.

[0199] The experimental results are shown in Figure 11. The antibody IEX04-012 of the present invention could effectively inhibit 293-Tie2 phosphorylation induced by hAng2-Fc in vitro, with IC50 superior to that of the positive control.

[0200] Example 13: Inhibition experiment of anti-VEGF A / Ang2 bispecific binding molecule against Ang2 vascular endothelial cell leakage This study identified the effect and function of anti-VEGF A / Ang2 bispecific binding molecule on vascular endothelial cell leakage by HUVEC-Tie2 leakage experiments.

[0201] HUVEC cells were transfected with lentivirus (Allcells Catalog Number: H-001-CN) to obtain HUVEC-Tie2 cells overexpressing Tie2.

[0202] Using a mini-well 96-well insert Petri dish, 300 μl of EGM-2 medium was plated on the bottom layer, digested with Accutase (Sigma) to obtain HUVEC-Tie2, and then cultured at 1 × 10 7The cells were resuspended at 100 cells / ml and plated in the upper layer of a Petri dish at 100 μl / well. The lower chamber medium (EGM-2 medium) was replaced every 24 hours, and after 24 hours the lower chamber medium was replaced with the experimental medium, the composition of which was as follows: Blank: EGM-2 medium (Lonza Catalog Number: CC-5035), VEGF Group A: EGM-2 medium + 20ng / ml VEGF (R&D Catalog Number: 293-VE), IgG group (VEGF A+IgG): EGM-2 medium+20ng / ml VEGF+10μg / ml IgG, Ang1 group (VEGF A + Ang1): EGM-2 medium + 20ng / ml VEGF (R&D Catalog No.: 293-VE) + 200ng / ml Ang1 (R&D Catalog No.: 923-AN), IEX04-012 group (VEGF A+IEX04-012): EGM-2 medium +20ng / ml VEGF+10μg / ml IEX04-012, BI-836880 group (VEGF A+BI-836880): EGM-2 medium+20ng / ml VEGF+10μg / ml BI-836880, Faricimab group (VEGF A+Faricimab): EGM-2 medium+20ng / ml VEGF+10μg / ml Faricimab. The above experimental medium was placed at 37°C and 5% CO2 for incubation.

[0203] After 24 hours, 1 μl of FITC-Dextran (Sigma Cat. No.: FD2000S-1G) (4 mg / ml) was added to each well of the upper chamber experimental medium, placed at 37 °C and 5% CO2, and after 30 minutes, the lower chamber medium was removed and diluted with PBS at 1:10, and then detected by a microplate reader, with the excitation light wavelength of 488 nm and the emission wavelength of 535 nm.

[0204] The experimental results, as shown in FIG. 12, demonstrated that the antibody IEX04-012 of the present invention can effectively reduce the permeability of vascular endothelial cells induced by VEGF.

[0205] Example 14. Efficacy test of laser-induced choroidal neovascularization This experiment used a rhesus monkey laser-induced choroidal neovascularization model to measure the anti-angiogenic effect of the bispecific binding molecule of the invention, IEX04-012.

[0206] Rhesus monkey: the species is Rhesus monkey, the grade is normal grade, the weight is 3.30-4.20kg at the time of purchase, and 3.35-4.35kg at the time of modeling, the origin is Sichuan Hengshu Bio-Technology Co., Ltd., the production permit number is SCXK (river) 2019-029, and the experimental animal quality certificate number is No. 0023356;

[0207] In this study, a laser was used to perform photocoagulation around the fovea of ​​the macula of the fundus of rhesus monkeys to induce choroidal neovascularization in the fundus, creating an animal model similar to human choroidal neovascularization. Fluorescein fundus angiography was performed before and 20 days after photocoagulation to determine the modeling status, and 20 rhesus monkeys (half male and half female) that were successfully modeled were selected and divided into five groups: model control group, IEX04-012 low dose group, IEX04-012 high dose group, Eylea group, and Faricimab group, with four monkeys in each group, half male and half female.

[0208] On the 21st day after photocoagulation, monkeys in each group were administered the doses shown in the table and were injected with IEX04-012, Eylea (Bayer) or Faricimab (all dissolved in 0.9% sodium chloride injection) into the vitreous of both eyes, and the model control group was administered the same volume of 0.9% sodium chloride injection. The animals in each group underwent color fundus photography, fluorescein fundus angiography (leakage spot statistics and leakage area measurement) (Robin J Goody, Wenzheng Hu, Afshin Shafiee et al. Optimization of laser-induced choroidal neovascularization in African green monkeys. Experimental Eye Research, Exp Eye Res. 2011 92(6):464-72), and optical coherence tomography (OCT, Wang Q, Lin X, Xiang W et al. Assessment of laser induction of Bruch's membrane disruption in monkey by spectral-domain optical coherence tomography. British Journal of Ophthalmology, 2015, 99(1):119-24) on the 7th, 14th, 21st, and 28th days after administration, respectively, to observe the inhibition of choroidal neovascularization by the test products. After euthanasia on the 29th day after administration, both eyes were taken for histological examination by immunohistochemistry (HE) staining. [Table 5]

[0209] As shown in Figures 13-15, the bispecific binding molecule of the present invention exhibited a significant antiangiogenic effect after 28 days of administration, and the statistics of 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 in the IEX04-012 treated animals was significantly lower than that in the control group and the positive control group. The results of OCT showed that the retinal thickness and the degree of retinal edema were significantly reduced in the IEX04-012 treated animals, and the effect was superior to that of the control (Figure 14). The results of fluorescein fundus angiography showed that the fundus leakage area in the IEX04-012 treated group was significantly reduced, and the effect was superior to that of the positive controls Eylea and Faricimab (Figure 15), indicating that the antibody of the present invention can significantly inhibit leakage caused by neovascularization. From the above, it was demonstrated that the antibody-combined anti-VEGF inhibitor of the present invention has a clear inhibitory effect on laser-induced ocular fundus neovascularization and at the same time has the function of protecting the integrity of blood vessels.

[0210] Twenty-nine days after administration, the rhesus monkeys were anesthetized with sodium pentobarbital according to body weight (approximately 30 mg / kg intravenously; the dose could be adjusted according to the health condition of the animals), euthanized by exsanguination from the abdominal aorta or femoral artery, gross observation was performed, and both eyes were removed.

[0211] Both eyes of some animals were fixed in modified Davidson's fixative, paraffin embedded, sectioned, and laser modeling areas were selected for histopathological examination, including routine HE staining and CD31 IHC staining.

[0212] In pathological sections, the antibodies of the present invention significantly reduced the area of ​​retinal lesions, alleviated retinal edema, and reduced tissue proliferation in the laser-damaged area compared to anti-VEGF treatment alone, with the results showing better retinal morphological improvement (see FIG. 16), inhibition of retinal-choroidal neovascularization, and improved vascular integrity (FIG. 17). [Table 6-1]

Table 6-2

Table 6-3

Table 6-4

Table 6-5

Claims

1. A VHH antibody against Ang2, comprising three CDRs of HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the sequence shown in SEQ ID NO: 16, HCDR2 comprises or consists of the sequence shown in SEQ ID NO: 17 or SEQ ID NO: 20, HCDR3 comprises or consists of the sequence shown in SEQ ID NO: 18, the VHH antibody.

2. Said VHH is (1) comprising the amino acid sequence shown in SEQ ID NO: 19 or SEQ ID NO: 21, or comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 19 or SEQ ID NO: 21, or consisting of the amino acids shown in SEQ ID NO: 19 or SEQ ID NO: 21, or (2) compared with the amino acid sequence shown in SEQ ID NO: 19 or SEQ ID NO: 21, comprising an amino acid sequence having one or more (preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less) mutations, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions, the VHH antibody according to claim 1.

3. A bispecific binding molecule that binds to VEGF A and Ang2, comprising a first target binding region that specifically binds to VEGF A and a second target binding region that specifically binds to Ang2, wherein the second target binding region is the VHH antibody according to claim 1, Optionally, the first target binding region is A VHH that specifically binds to VEGF A, An antigen-binding fragment of an antibody that specifically binds to VEGF A, such as an scFv, for example, said antibody is a fully human antibody or a humanized antibody, or A VEGF receptor (VEGFR) that specifically binds to VEGF A or its extracellular domain, or a fusion protein comprising its extracellular domain, for example, a fusion protein of its extracellular domain and Fc, the bispecific binding molecule selected from.

4. The bispecific binding molecule according to claim 3, which is a bispecific antibody.

5. The bispecific binding molecule according to claim 3, which is bivalent, trivalent or tetravalent.

6. The following structure: Variable light chain region VL of anti-VEGF antibody - linker - variable heavy chain region VH of anti-VEGF antibody - linker - anti-Ang2 VHH, or The bispecific binding molecule according to claim 4, having a heavy chain variable region VH of an anti-VEGF antibody - linker - a light chain variable region VL of an anti-VEGF antibody - linker - an anti-Ang2 VHH.

7. The light chain variable region VL of the anti-VEGF antibody includes LCDR1, LCDR2, and LCDR3, where LCDR1 includes or consists of the sequence shown in SEQ ID NO: 31, LCDR2 includes or consists of the sequence shown in SEQ ID NO: 32, LCDR3 includes or consists of the sequence shown in SEQ ID NO: 33, and / or The heavy chain variable region VH of the anti-VEGF antibody includes HCDR1, HCDR2, and HCDR3, where HCDR1 includes or consists of the sequence shown in SEQ ID NO: 35, HCDR2 includes or consists of the sequence shown in SEQ ID NO: 36, HCDR3 includes or consists of the sequence shown in SEQ ID NO: 37, the bispecific binding molecule according to claim 6.

8. The heavy chain variable region VH of the anti-VEGF antibody includes the amino acid sequence shown in SEQ ID NO: 34, or includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 34, or consists of the amino acids shown in SEQ ID NO: 34, or the heavy chain variable region VH has one or more (preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less) mutations compared to the amino acid sequence shown in SEQ ID NO: 34, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions, and includes an amino acid sequence. The light chain variable region VL of the anti-VEGF antibody includes the amino acid sequence shown in SEQ ID NO: 30, or includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 30, or consists of the amino acids shown in SEQ ID NO: 30, or the light chain variable region VL has one or more (preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less) mutations compared to the amino acid sequence shown in SEQ ID NO: 30, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions, and includes an amino acid sequence. or the anti-VEGF antibody is an scFv having the amino acid sequence shown in SEQ ID NO: 29, The bispecific binding molecule according to claim 6.

9. (1) The binding molecule comprises the amino acid sequence shown 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 with the amino acid sequence shown in SEQ ID NO: 28, or consists of the amino acids shown in SEQ ID NO: 28, or, (2) The bispecific binding molecule according to claim 6, which comprises an amino acid sequence having one or more (preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less) mutations, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions, compared to the amino acid sequence shown in SEQ ID NO:

28.

10. The following structure: Having a first anti-VEGF VHH-linker-a second anti-VEGF VHH-linker-anti-Ang2 VHH, wherein the first anti-VEGF VHH and the second anti-VEGF VHH are the same or different, the bispecific binding molecule according to claim 4.

11. The first anti-VEGF VHH or the second anti-VEGF VHH comprises HCDR1, HCDR2 and HCDR3, wherein, HCDR1 comprises or consists of the sequence shown in SEQ ID NO: 1, HCDR2 comprises or consists of the sequence shown in SEQ ID NO: 2, HCDR3 comprises or consists of the sequence shown in SEQ ID NO: 3, or, HCDR1 comprises or consists of the sequence shown in SEQ ID NO: 6, HCDR2 comprises or consists of the sequence shown in SEQ ID NO: 7 or SEQ ID NO: 10, HCDR3 comprises or consists of the sequence shown in SEQ ID NO: 8, the bispecific binding molecule according to claim 10.

12. The first anti-VEGF VHH or the second anti-VEGF VHH comprises an amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 9 or SEQ ID NO: 11, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 9 or SEQ ID NO: 11, or consists of the amino acids shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 9 or SEQ ID NO: 11, or, the VHH comprises an amino acid sequence having one or more (preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less) mutations, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions, as compared with the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 9 or SEQ ID NO: 11, the bispecific binding molecule according to claim 10.

13. (1) The binding molecule comprises an amino acid sequence shown 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 with the amino acid sequence shown in SEQ ID NO: 22, or consists of the amino acids shown 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, 1 or less) mutations, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions, as compared with the amino acid sequence shown in SEQ ID NO: 22, the bispecific binding molecule according to claim 10.

14. The following chains: One or two of VEGFR extracellular domain - Fc - linker - anti-Ang2 VHH, the bispecific binding molecule according to claim 3.

15. The VEGFR extracellular domain is the extracellular domain of human-derived VEGFR, preferably, the VEGFR extracellular domain comprises the second antibody-like domain of VEGFR1 and the third antibody-like domain of VEGFR2, more preferably, the VEGFR extracellular domain comprises the second antibody-like domain of human VEGFR1 and the third antibody-like domain of human VEGFR2, the bispecific binding molecule according to claim 14.

16. The VEGFR extracellular domain comprises the amino acid sequence shown 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 with the amino acid sequence shown in SEQ ID NO: 26, or consists of the amino acids shown in SEQ ID NO: 26, the bispecific binding molecule according to claim 14.

17. The Fc is an Fc derived from human IgG1, IgG2, IgG3, or IgG4. Preferably, the Fc comprises the amino acid sequence shown 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 with the amino acid sequence shown in SEQ ID NO: 27, or consists of the amino acids shown in SEQ ID NO: 27, the bispecific binding molecule according to claim 14.

18. The VEGFR extracellular domain-Fc is a fusion protein of the VEGFR extracellular domain and Fc, such as aflibercept or a derivative thereof, for example, comprises the amino acid sequence shown 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 with the amino acid sequence shown in SEQ ID NO: 25, or consists of the amino acids shown in SEQ ID NO: 25, the bispecific binding molecule according to claim 14.

19. (1) The binding molecule comprises the amino acid sequence shown 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 with the amino acid sequence shown in SEQ ID NO: 24, or consists of the amino acids shown 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, 1 or less) mutations, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions, compared to the amino acid sequence shown in SEQ ID NO: 24, the bispecific binding molecule according to claim 14.

20. The linker comprises or consists of the amino acid sequence of SEQ ID NO: 23, the bispecific binding molecule according to claim 3.

21. A VHH antibody against VEGF A, comprising three CDRs of HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the sequence shown in SEQ ID NO: 1, HCDR2 comprises or consists of the sequence shown in SEQ ID NO: 2, HCDR3 comprises or consists of the sequence shown in SEQ ID NO: 3, or HCDR1 comprises or consists of the sequence shown in SEQ ID NO: 6, HCDR2 comprises or consists of the sequence shown in SEQ ID NO: 7 or SEQ ID NO: 10, HCDR3 comprises or consists of the sequence shown in SEQ ID NO: 8, a VHH antibody.

22. (1) comprising the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 9 or SEQ ID NO: 11, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 9 or SEQ ID NO: 11, or consisting of the amino acids shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 9 or SEQ ID NO: 11, or (2) an amino acid sequence having one or more (preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less) mutations, such as substitutions, deletions or additions, preferably substitutions, such as conservative substitutions, compared with the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 9 or SEQ ID NO: 11, the VHH antibody according to claim 21.

23. A nucleic acid molecule encoding the VHH antibody according to any one of claims 1, 2, 21 and 22, or the bispecific binding molecule according to any one of claims 3 to 20 or a chain of said binding molecule.

24. An expression vector comprising the nucleic acid molecule according to claim 23, preferably, said expression vector is pcDNA3.1, an expression vector.

25. A host cell comprising the nucleic acid molecule according to claim 23, preferably, said host cell is prokaryotic or eukaryotic, such as a bacterium such as an E. coli cell, such as TG1, or a 293 cell, such as 293F or Expi-293 cell, a host cell. A host cell comprising the expression vector according to claim 24, preferably, the host cell is prokaryotic or eukaryotic, such as a bacterium such as an E. coli cell, such as TG1, or a 293 cell, such as 293F or Expi-293 cell.

27. A method for producing the VHH antibody according to any one of claims 1, 2, 21 and 22, or the bispecific binding molecule according to any one of claims 3 to 20, the method comprising culturing the host cell according to claim 25 under conditions suitable for the expression of the VHH antibody or bispecific binding molecule or its chain, and optionally recovering the VHH or bispecific binding molecule from the host cell (or host cell culture medium).

28. An immune complex comprising the VHH antibody according to any one of claims 1, 2, 21 and 22, or the bispecific binding molecule according to any one of claims 3 to 20.

29. A pharmaceutical composition or formulation comprising the VHH antibody according to any one of claims 1, 2, 21 and 22, or the bispecific binding molecule according to any one of claims 3 to 20, and optionally one or more other therapeutic agents, and optionally pharmaceutical adjuvants.

30. The pharmaceutical composition according to claim 29 for preventing or treating an eye disease in a subject.

31. The eye disease is selected from eye diseases associated with angiogenesis, such as eye diseases associated with corneal angiogenesis, or The subject has VEGF, such as VEGF A, and / or Ang2 (such as those with elevated levels, such as nucleic acid or protein levels), the pharmaceutical composition according to claim 30.

32. The pharmaceutical composition according to claim 29, wherein the pharmaceutical composition is administered to a subject in combination with one or more therapies, such as treatment modalities and / or other therapeutic agents.

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