VEGFA binding molecules

Abstract

VEGFA-binding molecules are disclosed. Also disclosed are nucleic acids and expression vectors encoding the VEGFA-binding molecules, compositions comprising the VEGFA-binding molecules, and methods of using the VEGFA-binding molecules.

Description

[Technical Field]

[0001] This application claims priority to SG10202101681W, filed on 19 February 2022, the contents and elements thereof being incorporated herein by reference for all purposes.

[0002] This disclosure relates to the field of molecular biology, and more specifically, to the field of antigen-binding molecular technology. The present invention also relates to methods for medical treatment and prevention. [Background technology]

[0003] Anti-VEGF therapy is used to treat a variety of conditions, particularly in the fields of oncology and ophthalmology (1-3). The development of humanized and stabilized antibody domains against VEGF is desirable due to their small size and modularity. For ophthalmic applications, small size and high stability are desirable because they enable local drug delivery (4, 5). Modularity, i.e., the ability of domain antibodies to autonomously fold and fuse with other domain antibodies or other proteins without compromising their integrity, is highly desirable for the development of multivalent and multispecific molecules. Indeed, tandem fusion of antibody domains with monoclonal antibodies or other arbitrary fusion proteins simplifies the process of increasing titer and specificity (6-8). [Overview of the project] [Means for solving the problem]

[0004] In the first aspect, the present disclosure relates to an optionally isolated antigen-binding molecule that binds to VEGFA, the following CDR: CDR1 having the amino acid sequence of SEQ ID NO: 45 CDR2 having the amino acid sequence of SEQ ID NO: 46 CDR3 having the amino acid sequence of SEQ ID NO: 48 This invention provides an antigen-binding molecule containing a single-domain antibody sequence incorporating [a specific element].

[0005] In some embodiments, the antigen-binding molecule includes, or consists of, an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 49. In some embodiments, the antigen-binding molecule is as follows: FR1 has the amino acid sequence of SEQ ID NO: 40 FR2 having the amino acid sequence of SEQ ID NO: 41 FR3 has the amino acid sequence of SEQ ID NO: 47 FR4 having the amino acid sequence of SEQ ID NO: 44 It contains a single-domain antibody sequence incorporating [the specified element].

[0006] In some embodiments, the antigen-binding molecule is the following CDR: CDR1 having the amino acid sequence of SEQ ID NO: 50 CDR2 having the amino acid sequence of SEQ ID NO: 51 CDR3 having the amino acid sequence of SEQ ID NO: 52 It contains a single-domain antibody sequence incorporating [the specified element].

[0007] In some embodiments, the antigen-binding molecule comprises or consists of an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 53. In some embodiments, the antigen-binding molecule is (i) The following CDRs: CDR1 having the amino acid sequence of SEQ ID NO: 17 CDR2 having the amino acid sequence of SEQ ID NO: 18 CDR3 having the amino acid sequence of SEQ ID NO: 19 A single-domain antibody sequence incorporating; or (ii) The following CDRs: CDR1 having the amino acid sequence of SEQ ID NO: 6 CDR2 having the amino acid sequence of SEQ ID NO: 7 CDR3 having the amino acid sequence of SEQ ID NO: 8 A single-domain antibody sequence incorporating; or (iii) The following CDRs: CDR1 having the amino acid sequence of SEQ ID NO: 13 CDR2 having the amino acid sequence of SEQ ID NO: 14 CDR3 having the amino acid sequence of SEQ ID NO: 15 Single-domain antibody sequence incorporating Includes.

[0008] In some embodiments, the antigen-binding molecule comprises or consists of an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 16, 5, or 12. In some embodiments, the antigen-binding molecule is the following CDR: CDR1 with the amino acid sequence of SEQ ID NO: 54 CDR2 having the amino acid sequence of SEQ ID NO: 55 CDR3 having the amino acid sequence of SEQ ID NO: 56 It contains a single-domain antibody sequence incorporating [the specified element].

[0009] In some embodiments, the antigen-binding molecule includes, or consists of, an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 57. In some embodiments, the antigen-binding molecule is (i) The following CDRs: CDR1 having the amino acid sequence of SEQ ID NO: 21 CDR2 having the amino acid sequence of SEQ ID NO: 22 CDR3 having the amino acid sequence of SEQ ID NO: 23 A single-domain antibody sequence incorporating; or (ii) The following CDRs: CDR1 having the amino acid sequence of SEQ ID NO: 25 CDR2 having the amino acid sequence of SEQ ID NO: 26 CDR3 having the amino acid sequence of SEQ ID NO: 27 A single-domain antibody sequence incorporating; or (iii) The following CDRs: CDR1 having the amino acid sequence of SEQ ID NO: 29 CDR2 having the amino acid sequence of SEQ ID NO: 30 CDR3 having the amino acid sequence of SEQ ID NO: 31 A single-domain antibody sequence incorporating; or (iv) The following CDRs: CDR1 having the amino acid sequence of SEQ ID NO: 33 CDR2 having the amino acid sequence of SEQ ID NO: 34 CDR3 having the amino acid sequence of SEQ ID NO: 35 A single-domain antibody sequence incorporating; or (v) The following CDRs: CDR1 having the amino acid sequence of SEQ ID NO: 37 CDR2 having the amino acid sequence of SEQ ID NO: 38 CDR3 having the amino acid sequence of SEQ ID NO: 39 Single-domain antibody sequence incorporating Includes.

[0010] In some embodiments, the antigen-binding molecule comprises or consists of an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NOs. 20, 24, 28, 32, or 36.

[0011] In some embodiments, the antigen-binding molecule is the following CDR: CDR1 having the amino acid sequence of SEQ ID NO: 2 CDR2 having the amino acid sequence of SEQ ID NO: 3 CDR3 having the amino acid sequence of SEQ ID NO: 4 It contains a single-domain antibody sequence incorporating [the specified element].

[0012] In some embodiments, the antigen-binding molecule includes, or consists of, an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 1. In some embodiments, the antigen-binding molecule is the following CDR: CDR1 having the amino acid sequence of SEQ ID NO: 2 CDR2 having the amino acid sequence of SEQ ID NO: 10 CDR3 having the amino acid sequence of SEQ ID NO: 11 It contains a single-domain antibody sequence incorporating [the specified element].

[0013] In some embodiments, the antigen-binding molecule includes, or consists of, an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 9. In some embodiments, the antigen-binding molecule is as follows: FR1 has the amino acid sequence of SEQ ID NO: 40 FR2 having the amino acid sequence of SEQ ID NO: 41 FR3 has the amino acid sequence of SEQ ID NO: 42 FR4 having the amino acid sequence of SEQ ID NO: 44 It contains a single-domain antibody sequence incorporating [the specified element].

[0014] In some embodiments, the antigen-binding molecule is as follows: FR1 has the amino acid sequence of SEQ ID NO: 40 FR2 having the amino acid sequence of SEQ ID NO: 41 FR3 has the amino acid sequence of SEQ ID NO: 43 FR4 having the amino acid sequence of SEQ ID NO: 44 It contains a single-domain antibody sequence incorporating [the specified element].

[0015] In some embodiments, the antigen-binding molecule inhibits the interaction between VEGFA and VEGFR. In some embodiments, the antigen-binding molecule is a multispecific antigen-binding molecule that further includes antigen-binding domains specific to target antigens other than VEGFA.

[0016] This disclosure also provides chimeric antigen receptors (CARs) comprising the antigen-binding molecules described herein. This disclosure also provides optionally isolated nucleic acids encoding antigen-binding molecules or CARs as described herein.

[0017] This disclosure also provides expression vectors comprising the nucleic acids described herein. This disclosure also provides cells comprising antigen-binding molecules, CARs, nucleic acids, or expression vectors as described herein.

[0018] This disclosure also provides a method for producing an antigen-binding molecule that binds to VEGFA, the method comprising the step of culturing the cells described herein under conditions suitable for the expression of the antigen-binding molecule by the cells.

[0019] This disclosure also provides compositions comprising antigen-binding molecules, CARs, nucleic acids, expression vectors, or cells described herein, and pharmaceutically acceptable carriers, diluents, excipients, or adjuvants.

[0020] This disclosure also provides antigen-binding molecules, CARs, nucleic acids, expression vectors, cells, or compositions described herein for use in methods of medical treatment or prevention. This disclosure also provides antigen-binding molecules, CARs, nucleic acids, expression vectors, cells, or compositions described herein for use in methods of treating or preventing diseases in which VEGFA / VEGFR-mediated signaling is pathologically involved.

[0021] This disclosure also provides the use of the antigen-binding molecules, CARs, nucleic acids, expression vectors, cells, or compositions described herein in the manufacture of pharmaceuticals for treating or preventing diseases in which VEGFA / VEGFR-mediated signaling is pathologically involved.

[0022] This disclosure also provides a method for treating or preventing diseases in which VEGFA / VEGFR-mediated signaling is pathologically involved, the method comprising the step of administering a therapeutic or prophylactic effective amount of an antigen-binding molecule, CAR, nucleic acid, expression vector, cell, or composition described herein to the target.

[0023] In some embodiments, the disease is selected from diseases characterized by pathological angiogenesis, cancer, VEGFA-expressing cancer, VEGFR-expressing cancer, eye diseases, retinopathy, diabetic retinopathy, macular degeneration, age-related macular degeneration, exudative age-related macular degeneration, retinal vein occlusion, myopic choroidal neovascularization, retinopathy of prematurity, neovascular glaucoma, central serous retinopathy, eye tumors, corneal neovascularization, inflammatory diseases, autoimmune diseases, arthritis, rheumatoid arthritis, osteoarthritis, psoriasis, multiple sclerosis, sepsis, motor neuron diseases, and amyotrophic lateral sclerosis.

[0024] This disclosure also provides in vitro complexes comprising, optionally isolated, VEGFA-bound antigen-binding molecules described herein. The disclosure also provides a method for detecting VEGFA in a sample, comprising the steps of contacting a sample containing or suspected to contain VEGFA with an antigen-binding molecule described herein, and detecting the formation of a complex between the antigen-binding molecule and VEGFA.

[0025] This disclosure also provides the use of the antigen-binding molecules described herein in methods for detecting, localizing, or imaging VEGFAs, or cells containing or expressing VEGFAs.

[0026] The disclosure also provides a method for selecting or stratifying subjects for treatment with a VEGFA-targeted drug, comprising the steps of contacting a sample from the subject in vitro with an antigen-binding molecule described herein, and detecting the formation of a complex between the antigen-binding molecule and VEGFA.

[0027] This disclosure also provides the use of the antigen-binding molecules described herein as diagnostic or prognostic agents in vitro or in vivo. This disclosure also provides the use of the antigen-binding molecules described herein in methods for detecting, localizing, or imaging diseases / conditions characterized by VEGFA expression. [Modes for carrying out the invention]

[0028] The inventors hereby describe the production of humanized, stabilized, and autonomous VH domain antibodies that target human and mouse VEGFA with high affinity and can strongly block VEGF-VEGFR interactions. These VEGF-binding molecules can be used as building blocks to produce multivalent and multispecific molecules, as exemplified by a bivalent anti-VEGFA molecule constructed as a tandem of two VH domain antibodies.

[0029] VEGFA Vascular endothelial growth factor A (VEGFA) is a protein identified by UniProt P15692. Alternative splicing of mRNA encoded by the human VEGFA gene yields four major VEGFA isoforms: VEGF206 (SEQ ID NO: 60), VEGF189 (SEQ ID NO: 61), VEGF165 (SEQ ID NO: 62), and VEGF121 (SEQ ID NO: 63). After processing to remove the N-terminal 26-amino acid signal peptide (SEQ ID NO: 68), VEGF206, VEGF189, VEGF165, and VEGF121 each contain the amino acid sequences shown in SEQ ID NOs. 64-67. VEGF165 appears to be the major VEGFA isoform.

[0030] VEGFA is a growth factor, and is described, for example, in Holme and Zachary Genome Biol. (2005) 6(2):209, and in Claesson-Welsh and Welsh, J Intern Med. (2013) 273(2):114-1127, both of which are incorporated herein by reference in their entirety.

[0031] Vascular endothelial growth factor (VEGF) is a family of secreted polypeptides that possess a conserved receptor-binding cystine knot structure. VEGFA monomers bind via interchain disulfide bonds to form homodimers. VEGFA acts through a family of homologous receptor kinases expressed by endothelial cells to stimulate angiogenesis.

[0032] VEGFAs play a crucial role not only in normal vascular development but also in diseases involving abnormal vascular proliferation (e.g., cancer). VEGFAs stimulate the proliferation of vascular endothelial cells derived from arteries, veins, and lymphatic systems, inducing angiogenesis (i.e., the formation of thin-walled endothelial structures) and a rapid increase in microvascular permeability in various in vivo models.

[0033] In this specification, "VEGFA" means VEGFA derived from any species, and includes isoforms, fragments, variants, or homologs derived from any species. In some embodiments, VEGFA is VEGFA derived from mammals (e.g., eutherians, placentalians, epithelialians, anthropotherians, archontans, primates (rhesus macaques, crab-eating macaques, non-human primates, or humans)). In some embodiments, VEGFA is human VEGFA or mouse VEGFA.

[0034] As used herein, an isoform, fragment, variant, or homolog of a given reference protein can be characterized as having at least 70% sequence identity with respect to the amino acid sequence of the reference protein, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity. “Fragment” generally refers to a portion of a reference protein. “Variant” generally refers to a protein having an amino acid sequence that includes one or more amino acid substitutions, insertions, deletions, or other modifications to the amino acid sequence of a reference protein, but retains a considerable degree (e.g., at least 60%) of sequence identity with respect to the amino acid sequence of the reference protein. “Isoform” generally refers to a variant of a reference protein expressed by the same species as the reference protein. “Homolog” generally refers to a variant of a reference protein produced by a different species compared to the species of the reference protein. Homologs include orthologues.

[0035] VEGFA isoforms naturally include VEGF206 (UniProt P15692-1), VEGF189 (UniProt P15692-2), VEGF165 (UniProt P15692-4), and VEGF121 (UniProt P15692-9). VEGFA isoforms also include VEGF183 (UniProt P15692-3), VEGF148 (UniProt P15692-5), VEGF145 (UniProt P15692-6), VEGF165B (UniProt P15692-8), VEGF111 (UniProt P15692-10), L-VEGF165 (UniProt P15692-11), L-VEGF121 (UniProt P15692-12), L-VEGF189 (UniProt P15692-13), L-VEGF206 (UniProt P15692-14), VEGFA isoform 15 (UniProt P15692-15), VEGFA isoform 16 (UniProt Includes VEGFA isoform 17 (UniProt P15692-17) and VEGFA isoform 18 (UniProt P15692-18).

[0036] VEGFA isoforms, fragments, variants, or homologs can optionally be characterized as having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the amino acid sequence of an immature or mature VEGFA isoform of a given species, such as human.

[0037] In some embodiments, VEGFA is human VEGFA. In some embodiments, VEGFA is mouse VEGFA. Isoforms, fragments, variants, or homologs may optionally be functional isoforms, fragments, variants, or homologs that possess the functional properties / activities of a reference VEGFA (e.g., human VEGF165), as determined, for example, by analysis by an appropriate assay for functional properties / activity. For example, isoforms, fragments, variants, or homologs of VEGFA may exhibit binding to VEGF receptors (e.g., VEGFR1, VEGFR2, and / or VEGFR3).

[0038] In some embodiments, the VEGFA comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NOs. 60, 61, 62, or 63, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity.

[0039] In some embodiments, the VEGFA or its fragment comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NOs. 64, 65, 66, or 67, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity.

[0040] VEGF exerts its biological effects by binding to VEGF receptors (VEGFRs) VEGFR1 (AAH39007.1 GI:24660372), VEGFR2 (P35968.2 GI:9087218), and VEGFR3 (AAA85215.1 GI:1150991). Each receptor has an extracellular binding domain, a transmembrane sequence, and an intracellular tyrosine kinase moiety for VEGF. Binding of VEGF to the extracellular receptor domain causes the receptor to dimerize and the intracellular tyrosine kinase moiety to phosphorylate. VEGF has been shown to exert its biological effects primarily through VEGFR1 and VEGFR2.

[0041] In this specification, “VEGFR1,” “VEGFR2,” and “VEGFR3” refer to VEGFR1 / VEGFR2 / VEGFR3 from any species, and include isoforms, fragments, variants, or homologs from any species. In some embodiments, VEGFR1 / VEGFR2 / VEGFR3 are derived from mammals (e.g., eutherians, placental mammals, epithelial mammals, anthropothermic mammals, primates (rhesus monkeys, crab-eating macaques, non-human primates, or humans)). In some embodiments, VEGFR1 / VEGFR2 / VEGFR3 are human or mouse VEGFR1 / VEGFR2 / VEGFR3.

[0042] VEGFR1 / VEGFR2 / VEGFR3 isoforms, fragments, variants, or homologs can optionally be characterized as having at least 70% amino acid sequence identity, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity, with the amino acid sequence of an immature or mature isoform of a relevant molecule of a given species, e.g., human.

[0043] In this specification, “VEGFA / VEGFR-mediated signaling” refers to signaling initiated by the binding of VEGFA to the VEGF receptor. “Signaling” refers to signaling and other cellular processes that govern cellular activity.

[0044] VEGFA / VEGFR-mediated signaling is described, for example, in Geindreau et al., Int J Mol Sci. (2021) 22(9):4871, the entire content of which is incorporated herein by reference. VEGFA / VEGFR-mediated signaling proceeds within cells via the PI3K / AKT, MAPK / ERK, and PLC-γ pathways, as well as via SCR and FAK, promoting cell survival, proliferation, and cytoskeletal reorganization, influencing changes in vascular permeability and vasodilation, and promoting angiogenesis.

[0045] antigen binding molecule This disclosure provides antigen-binding molecules that can bind to (i.e., bind to) VEGFA. This disclosure provides antigen-binding molecules that specifically bind to VEGFA. The antigen-binding molecules described in this disclosure can be provided in purified or isolated forms, i.e., from other naturally occurring biological materials.

[0046] As used herein, “antigen-binding molecule” refers to a molecule capable of binding to a target antigen. The term “antigen-binding molecule” encompasses monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., Fv, scFv, Fab, scFab, F(ab')2, Fab2, diabody, triabody, scFv-Fc, minibody, single-domain antibody (VHH)), and aptamers.

[0047] More specifically, the antigen-binding molecule described herein comprises an antigen-binding polypeptide moiety, which may also be called an "antigen-binding domain." In preferred embodiments, the antigen-binding molecule described herein comprises or consists of a single-domain antibody that specifically binds to VEGFA.

[0048] Single-domain antibodies (sdAbs) are variously referred to in this art as "single variable domain on a heavy-chain antibody," "VHH," "nanobody," and "heavy-chain-only antibody (HcAb)," and are sometimes referred to herein as "DotBody." These are described, for example, in Henry and MacKenzie, Front Immunol. (2018) 9:41, and Bever et al., Anal Bioanal Chem. (2016) 408(22):5985-6002, both of which are incorporated herein by reference in their entirety.

[0049] Single-domain antibodies are formed from a single monomeric antibody variable domain. The first single-domain antibodies were created from heavy-chain antibodies found in camels, but cartilaginous fish also possess heavy-chain antibodies.

[0050] The single-domain antibodies described in this disclosure generally include three complementarity-determining regions (CDRs): CDR1, CDR2, and CDR3. Together, the three CDRs define a paratope of the molecule that is the portion that binds to the target antigen.

[0051] A single-domain antibody further includes framework regions (FRs) on both sides of each CDR, providing a scaffold for the CDR. From the N-terminus to the C-terminus, a single-domain antibody has the following structure: N-terminus-[FR1]-[CDR1]-[FR2]-[CDR2]-[FR3]-[CDR3]-[FR4]-C-terminus.

[0052] There are several different definitions of CDR and FR for antibodies, such as those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), and Chothia et al., J. Mol. Biol. 196: pp. 901-917 (1987). Similarly, there are several different definitions for VBASE2, such as those described in Retter et al., Nucl. Acids Res. (2005) 33 (see above 1): pp. D671-D674.

[0053] In some embodiments, the antigen-binding molecule includes the CDR of the VEGFA-binding single-domain antibody described herein, or includes the CDR derived from the VEGFA-binding single-domain antibody described herein. In some embodiments, the antigen-binding molecule includes the FR of the VEGFA-binding single-domain antibody described herein, or includes the FR derived from the VEGFA-binding single-domain antibody described herein. In some embodiments, the antigen-binding molecule includes the CDR and FR of the VEGFA-binding single-domain antibody described herein, or includes the CDR and FR derived from the VEGFA-binding single-domain antibody described herein. That is, in some embodiments, the antigen-binding molecule includes the amino acid sequence of the VEGFA-binding single-domain antibody described herein, or includes the amino acid sequence derived from the VEGFA-binding single-domain antibody described herein. In some embodiments, the CDR and FR of antigen-binding molecules referred to herein are defined according to the IMGT information system (International IMGT (ImMunoGeneTics) information system (LeFranc et al., Nucleic Acids Res. (2015) 43 (Database issue): D413-22), which uses the IMGT V-DOMAIN numbering rules described in Lefranc et al., Dev. Comp. Immunol. (2003) 27:55-77), the Kabat system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)), or the Chothia system (Chothia et al., J. Mol. Biol. 196:901-917 (1987)).

[0054] In some embodiments, the CDR and FR of the antigen-binding molecule (e.g., a single-domain antibody) referred to herein are defined according to the Kabat system. In some embodiments, in the amino acid sequences of SEQ ID NOs: 1, 5, 9, 12, 16, 20, 24, 28, 32, 36, 49, 53 and 57, FR1 is formed by the amino acid sequence from positions 1 to 31; CDR1 is formed by the amino acid sequence from positions 32 to 36; FR2 is formed by the amino acid sequence from positions 37 to 50; CDR2 is formed by the amino acid sequence from positions 51 to 67; FR3 is formed by the amino acid sequence from positions 68 to 99; CDR3 is formed by the amino acid sequence from positions 100 to 119; and FR4 is formed by the amino acid sequence from positions 120 to 130.

[0055] As used herein, an amino acid sequence / domain "derived" from a reference amino acid sequence / domain includes an amino acid sequence having at least 60% sequence identity with the reference sequence, for example, one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0056] In some embodiments, the antigen-binding molecule comprises the CDR, FR, and / or complete amino acid sequence of a VEGFA-conjugated single-domain antibody selected from 13A6, 16A2.1, 16A6.1, 20A2.1, 20A3.1, 21A1.1, 21A8.1, 21D9.1, 21E6.1, and 23D5.1.

[0057] In some embodiments, the antigen-binding molecule includes the CDR, FR, and / or complete amino acid sequence of a VEGFA-binding single-domain antibody having the amino acid sequence described in one of SEQ ID NOs: 1, 5, 9, 12, 16, 20, 24, 28, 32, 36, 49, 53, or 57. In some embodiments, the antigen-binding molecule includes the CDR (i.e., CDR1, 2, and 3) of a VEGFA-binding single-domain antibody having the amino acid sequence described in one of SEQ ID NOs: 1, 5, 9, 12, 16, 20, 24, 28, 32, 36, 49, 53, or 57. In some embodiments, the antigen-binding molecule includes the FR (i.e., FR1, 2, 3, and 4) of a VEGFA-binding single-domain antibody having the amino acid sequence described in one of SEQ ID NOs: 1, 5, 9, 12, 16, 20, 24, 28, 32, 36, 49, 53, or 57. In some embodiments, the antigen-binding molecule includes CDRs (i.e., CDR1, 2, and 3) and FRs (i.e., FR1, 2, 3, and 4) of a VEGFA-binding single-domain antibody having the amino acid sequence described in one of SEQ ID NOs: 1, 5, 9, 12, 16, 20, 24, 28, 32, 36, 49, 53, or 57.

[0058] In some embodiments, the antigen-binding molecule comprises or consists of a single-domain antibody sequence described in one of the following (1) to (13): (1) Single-domain antibody sequences containing the following CDRs: (ConAII) CDR1 having the amino acid sequence of SEQ ID NO: 45 CDR2 having the amino acid sequence of SEQ ID NO: 46 CDR3 having the amino acid sequence of SEQ ID NO: 48 Or a variant in which one or more of CDR1, CDR2, or CDR3 amino acids are substituted with other amino acids. (2) Single-domain antibody sequences containing the following CDRs (derived from Con16A2.1): CDR1 having the amino acid sequence of SEQ ID NO: 50 CDR2 having the amino acid sequence of SEQ ID NO: 51 CDR3 having the amino acid sequence of SEQ ID NO: 52 Or a variant in which one or more of CDR1, CDR2, or CDR3 amino acids are substituted with other amino acids. (3) Single-domain antibody sequences containing the following CDRs: (Con21 / 23) CDR1 with the amino acid sequence of SEQ ID NO: 54 CDR2 having the amino acid sequence of SEQ ID NO: 55 CDR3 having the amino acid sequence of SEQ ID NO: 56 Or a variant in which one or more of CDR1, CDR2, or CDR3 amino acids are substituted with other amino acids. (4) Single-domain antibody sequences containing the following CDRs: CDR1 having the amino acid sequence of SEQ ID NO: 2 CDR2 having the amino acid sequence of SEQ ID NO: 3 CDR3 having the amino acid sequence of SEQ ID NO: 4 Or a variant in which one or more of CDR1, CDR2, or CDR3 amino acids are substituted with other amino acids. (5) Single-domain antibody sequences containing the following CDRs: (16A2.1) CDR1 having the amino acid sequence of SEQ ID NO: 6 CDR2 having the amino acid sequence of SEQ ID NO: 7 CDR3 having the amino acid sequence of SEQ ID NO: 8 Or a variant in which one or more of CDR1, CDR2, or CDR3 amino acids are substituted with other amino acids. (6) Single-domain antibody sequences containing the following CDRs: (16A6.1) CDR1 having the amino acid sequence of SEQ ID NO: 2 CDR2 having the amino acid sequence of SEQ ID NO: 10 CDR3 having the amino acid sequence of SEQ ID NO: 11 Or a variant in which one or more of CDR1, CDR2, or CDR3 amino acids are substituted with other amino acids. (7) Single-domain antibody sequences containing the following CDRs: CDR1 having the amino acid sequence of SEQ ID NO: 13 CDR2 having the amino acid sequence of SEQ ID NO: 14 CDR3 having the amino acid sequence of SEQ ID NO: 15 Or a variant in which one or more of CDR1, CDR2, or CDR3 amino acids are substituted with other amino acids. (8) Single-domain antibody sequences containing the following CDRs: (20A3.1) CDR1 having the amino acid sequence of SEQ ID NO: 17 CDR2 having the amino acid sequence of SEQ ID NO: 18 CDR3 having the amino acid sequence of SEQ ID NO: 19 Or a variant in which one or more of CDR1, CDR2, or CDR3 amino acids are substituted with other amino acids. (9)(21A1.1) Single-domain antibody sequences containing the following CDRs: CDR1 having the amino acid sequence of SEQ ID NO: 21 CDR2 having the amino acid sequence of SEQ ID NO: 22 CDR3 having the amino acid sequence of SEQ ID NO: 23 Or a variant in which one or more of CDR1, CDR2, or CDR3 amino acids are substituted with other amino acids. (10)(21A8.1) Single-domain antibody sequences containing the following CDRs: CDR1 having the amino acid sequence of SEQ ID NO: 25 CDR2 having the amino acid sequence of SEQ ID NO: 26 CDR3 having the amino acid sequence of SEQ ID NO: 27 Or a variant in which one or more of CDR1, CDR2, or CDR3 amino acids are substituted with other amino acids. (11)(21D9.1) Single-domain antibody sequences containing the following CDRs: CDR1 having the amino acid sequence of SEQ ID NO: 29 CDR2 having the amino acid sequence of SEQ ID NO: 30 CDR3 having the amino acid sequence of SEQ ID NO: 31 Or a variant in which one or more of CDR1, CDR2, or CDR3 amino acids are substituted with other amino acids. (12)(21E6.1) Single-domain antibody sequences containing the following CDRs: CDR1 having the amino acid sequence of SEQ ID NO: 33 CDR2 having the amino acid sequence of SEQ ID NO: 34 CDR3 having the amino acid sequence of SEQ ID NO: 35 Or a variant in which one or more of CDR1, CDR2, or CDR3 amino acids are substituted with other amino acids. (13)(23D5.1) Single-domain antibody sequences containing the following CDRs: CDR1 having the amino acid sequence of SEQ ID NO: 37 CDR2 having the amino acid sequence of SEQ ID NO: 38 CDR3 having the amino acid sequence of SEQ ID NO: 39 Or a variant in which one or more of CDR1, CDR2, or CDR3 amino acids are substituted with other amino acids.

[0059] In some embodiments, the antigen-binding molecule comprises or consists of a single-domain antibody sequence described in one of (14) to (16) below: (14)(ConAII) Single-domain antibody sequences containing FR below: FR1 has the amino acid sequence of SEQ ID NO: 40 FR2 having the amino acid sequence of SEQ ID NO: 41 FR3 has the amino acid sequence of SEQ ID NO: 47 FR4 having the amino acid sequence of SEQ ID NO: 44 Or a variant in which one or more of FR1, FR2, FR3, or FR4 amino acids are substituted with other amino acids. (15) Single-domain antibody sequences containing the following FRs: (13A6, 16A2.1, 20A2.1, 20A3.1, 21A1.1, 21A8.1, 21D9.1, 21E6.1) FR1 has the amino acid sequence of SEQ ID NO: 40 FR2 having the amino acid sequence of SEQ ID NO: 41 FR3 has the amino acid sequence of SEQ ID NO: 42 FR4 having the amino acid sequence of SEQ ID NO: 44 Or a variant in which one or more of FR1, FR2, FR3, or FR4 amino acids are substituted with other amino acids. (16)(16A6.1) Single-domain antibody sequences containing FR below: FR1 has the amino acid sequence of SEQ ID NO: 40 FR2 having the amino acid sequence of SEQ ID NO: 41 FR3 has the amino acid sequence of SEQ ID NO: 43 FR4 having the amino acid sequence of SEQ ID NO: 44 Or a variant in which one or more of FR1, FR2, FR3, or FR4 amino acids are substituted with other amino acids.

[0060] In some embodiments, the antigen-binding molecule comprises or consists of a single-domain antibody sequence comprising the CDR described in one of (1) to (13) above and the FR described in one of (14) to (16) above.

[0061] In some embodiments, the antigen-binding molecule comprises or consists of a single-domain antibody sequence described in one of (17) to (29) below: A single-domain antibody sequence comprising the CDR described in (17)(ConAII)(1) and the FR described in (14). (18) A single-domain antibody sequence containing the CDR described in (2) and the FR described in (15) (derived from Con16A2.1). A single-domain antibody sequence comprising the CDR described in (19)(Con21 / 23)(3) and the FR described in (15). A single-domain antibody sequence comprising the CDR described in (20)(13A6)(4) and the FR described in (15). A single-domain antibody sequence comprising the CDR described in (21)(16A2.1)(5) and the FR described in (15). A single-domain antibody sequence comprising the CDR described in (22)(16A6.1)(6) and the FR described in (16). A single-domain antibody sequence comprising the CDR described in (23)(20A2.1)(7) and the FR described in (15). A single-domain antibody sequence comprising the CDR described in (24)(20A3.1)(8) and the FR described in (15). A single-domain antibody sequence comprising the CDR described in (25)(21A1.1)(9) and the FR described in (15). A single-domain antibody sequence comprising the CDR described in (26)(21A8.1)(10) and the FR described in (15). A single-domain antibody sequence containing the CDR described in (27)(21D9.1)(11) and the FR described in (15). A single-domain antibody sequence comprising the CDR described in (28)(21E6.1)(12) and the FR described in (15). A single-domain antibody sequence containing the CDR described in (29)(23D5.1)(13) and the FR described in (15).

[0062] In some embodiments, the antigen-binding molecule comprises or consists of a single-domain antibody sequence described in one of (30) to (42) below: (30) A single-domain antibody sequence comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of (ConAII) SEQ ID NO: 49, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. (31) A single-domain antibody sequence comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of Sequence ID No. 53 (derived from Con16A2.1), more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. (32) A single-domain antibody sequence comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of (Con21 / 23) Sequence ID No. 57, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. (33)(13A6) A single-domain antibody sequence comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of Sequence ID No. 1, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. (34)(16A2.1) A single-domain antibody sequence comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of Sequence ID No. 5, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. (35)(16A6.1) A single-domain antibody sequence comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of Sequence ID No. 9, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. (36)(20A2.1) A single-domain antibody sequence comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of Sequence ID No. 12, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. (37)(20A3.1) A single-domain antibody sequence comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of Sequence ID No. 16, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. (38)(21A1.1) A single-domain antibody sequence comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of Sequence ID No. 20, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. (39)(21A8.1) A single-domain antibody sequence comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of Sequence ID No. 24, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. (40)(21D9.1) A single-domain antibody sequence comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of Sequence ID No. 28, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. (41)(21E6.1) A single-domain antibody sequence comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of Sequence ID No. 32, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. (42)(23D5.1) A single-domain antibody sequence comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of Sequence ID No. 36, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0063] In embodiments of this disclosure in which one or more amino acids are substituted with other amino acids, the substitutions may be conservative substitutions, for example, as shown in the table below. In some embodiments, amino acids in the same block in the middle column are substituted. In some embodiments, amino acids in the same row in the rightmost column are substituted:

[0064] [Table 1]

[0065] In some embodiments, substitutions may be functionally conserved. That is, in some embodiments, substitutions may not affect (or substantially affect) one or more functional properties (e.g., target binding) of the antigen-binding molecule containing the substitution compared to an equivalent unsubstituted molecule.

[0066] In some embodiments, the antigen-binding molecule of the Disclosure comprises one or more regions of an immunoglobulin heavy chain constant sequence (e.g., CH1, CH2, and / or CH3). In some embodiments, the immunoglobulin heavy chain constant sequence is or is derived from the heavy chain constant sequence of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM, for example, human IgG (e.g., hIgG1, hIgG2, hIgG3, hIgG4), hIgA (e.g., hIgA1, hIgA2), hIgD, hIgE, or hIgM. In some embodiments, the immunoglobulin heavy chain constant sequence is or is derived from the heavy chain constant sequence of a human IgG1 allotype (e.g., G1m1, G1m2, G1m3, or G1m17).

[0067] In some embodiments, the antigen-binding molecule includes an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 69, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0068] In some embodiments, the antigen-binding molecule includes a CH1 region containing an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 70, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the antigen-binding molecule includes a hinge region containing an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 71, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the antigen-binding molecule includes a CH2 region containing an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 72, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the antigen-binding molecule includes a CH3 region containing an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 73, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0069] In some embodiments, the antigen-binding molecule of this disclosure includes an Fc region. The Fc region consists of CH2 and CH3 regions derived from one polypeptide and CH2 and CH3 regions derived from another polypeptide. The CH2 and CH3 regions from the two polypeptides combine to form the Fc region.

[0070] The Fc region provides interactions with Fc receptors and other molecules of the immune system, resulting in functional effects. The IgG Fc-mediated effector function is outlined, for example, in Jefferis et al., Immunol Rev 1998 163:59-76 (the entire work is incorporated herein by reference), and is mediated through Fc-mediated recruitment and activation of immune cells (e.g., macrophages, dendritic cells, neutrophils, basophils, eosinophils, platelets, mast cells, NK cells, and T cells) through interactions between the Fc region and Fc receptors expressed by immune cells, recruitment of complement pathway components via binding of the Fc region to complement protein C1q, and consequently activation of the complement cascade. Fc-mediated functions include Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cell-mediated cytotoxicity (CDC), membrane attack complex (MAC) formation, cell degranulation, cytokine and / or chemokine production, and antigen processing and presentation.

[0071] In some embodiments, the antigen-binding molecules described herein include an Fc region that can enhance / induce one or more of ADCC, ADCP, and CDC to cells containing / expressing VEGFA (e.g., cells expressing VEGFA and / or complexes containing VEGFA on the cell surface), and / or enhance MAC formation or cell degranulation on cells.

[0072] Modifications to the antibody Fc region that affect Fc-mediated function are known in the art, such as those described in Wang et al., Protein Cell (2018) 9(1):63-73, which are incorporated herein by reference in their entirety. Exemplary Fc region modifications known to affect antibody effector function are summarized in Table 1 of Wang et al., Protein Cell (2018) 9(1):63-73.

[0073] Multiple-specific antigen-binding molecules are also being considered. "Multi-specific" means that the antigen-binding molecule exhibits specific binding to multiple targets. In some embodiments, the antigen-binding molecule is a bispecific antigen-binding molecule. In some embodiments, the antigen-binding molecule comprises at least two distinct antigen-binding domains.

[0074] In some embodiments, the antigen-binding molecule binds to both VEGFA and another target (e.g., an antigen other than VEGFA), and is therefore at least bispecific. The term "bispecific" means that the antigen-binding molecule can specifically bind to at least two different antigenic determinants.

[0075] It will be understood that the antigen-binding molecules described herein (e.g., multispecific antigen-binding molecules) may include antigen-binding molecules capable of binding to targets for which the antigen-binding molecule is specific. For example, antigen-binding molecules that bind to VEGFA and antigens other than VEGFA may include (i) an antigen-binding molecule that binds to VEGFA, and (ii) an antigen-binding molecule that binds to antigens other than VEGFA. In some embodiments, the antigen-binding molecules that are components of a larger antigen-binding molecule (e.g., a multispecific antigen-binding molecule) may be referred to, for example, as the “antigen-binding domain” or “antigen-binding region” of the larger antigen-binding molecule.

[0076] Furthermore, it will be understood that the antigen-binding molecules described in this disclosure (e.g., multispecific antigen-binding molecules) may include antigen-binding polypeptides or antigen-binding polypeptide complexes that are capable of binding to targets to which the antigen-binding molecule is specific.

[0077] In some embodiments, the antigen other than VEGFA in the multispecific antigen-binding molecule is an immune cell surface molecule. In some embodiments, the antigen is a cancer cell antigen. In some embodiments, the antigen is a receptor molecule, such as a cell surface receptor. In some embodiments, the antigen is a cell signaling molecule, such as a cytokine, chemokine, interferon, interleukin, or lymphokine. In some embodiments, the antigen is a growth factor or hormone.

[0078] Cancer cell antigens are antigens expressed or overexpressed by cancer cells. Cancer cell antigens can be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof. The expression of cancer cell antigens may be associated with cancer. Cancer cell antigens may be abnormally expressed by cancer cells (e.g., cancer cell antigens may be expressed with abnormal localization) or abnormally structured by cancer cells. Cancer cell antigens may be able to induce an immune response. In some embodiments, the antigen is expressed on the cell surface of the cancer cell (i.e., the cancer cell antigen is a cancer cell surface antigen). In some embodiments, the portion of the antigen bound by the antigen-binding molecule described herein is presented on the outer surface of the cancer cell (i.e., extracellular). Cancer cell antigens may be cancer-associated antigens. In some embodiments, a cancer cell antigen is an antigen whose expression is associated with the onset, progression, or severity of cancer symptoms. Cancer-associated antigens may be associated with the cause or pathology of cancer, or may be abnormally expressed as a result of cancer. In some embodiments, cancer cell antigens are antigens whose expression is upregulated (e.g., at the RNA and / or protein level) by cancer cells compared to the expression levels of equivalent non-cancerous cells (e.g., non-cancerous cells from the same tissue / cell type). In some embodiments, cancer-associated antigens may be preferentially expressed by cancer cells and not expressed by equivalent non-cancerous cells (e.g., non-cancerous cells from the same tissue / cell type). In some embodiments, cancer-associated antigens may be products of mutated oncogenes or mutated tumor suppressor genes. In some embodiments, cancer-associated antigens may be products of overexpressed cellular proteins, cancer antigens produced by oncogenic viruses, carcinoembryonic antigens, or glycolipids or glycoproteins on the cell surface.

[0079] Immune cell surface molecules can be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof expressed on or on the cell surface of an immune cell. In some embodiments, the portion of the immune cell surface molecule bound by the antigen-binding molecule of this disclosure is located on the outer surface of the immune cell (i.e., extracellular). Immune cell surface molecules can be expressed on the cell surface of any immune cell. In some embodiments, the immune cell may be a hematopoietic cell, e.g., neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, or monocyte. Lymphocytes may be, for example, T cells, B cells, natural killer (NK) cells, NKT cells, or innate lymphocytes (ILCs), or their precursors (e.g., thymocytes or pre-B cells). In some embodiments, the antigen is a CD3 polypeptide (e.g., CD3ε, CD3δ, CD3γ, or CD3ζ).

[0080] In some embodiments, the multispecific antigen-binding molecules described herein exhibit at least monovalent binding to VEGFA and at least monovalent binding to antigens other than VEGFA. Binding titer refers to the number of binding sites within the antigen-binding molecule for a given antigenic determinant.

[0081] In some embodiments, the antigen-binding molecule includes a single-domain antibody capable of binding to VEGFA (e.g., those described herein) and an antigen-binding region capable of binding to antigens other than VEGFA (e.g., a polypeptide (e.g., a single-domain antibody), Fv, Fab, or antibody).

[0082] In some embodiments, the antigen-binding molecule includes an immune cell-engaging portion. In some embodiments, the antigen-binding molecule is an immune cell-engager. Immune cell-engagers are outlined, for example, in Goebeler and Bargou, Nat. Rev. Clin. Oncol. (2020) 17: pp. 418–434, and Ellerman, Methods (2019) 154: pp. 102–117, both of which are incorporated herein by reference in their entirety.

[0083] Immune cell engager molecules contain an antigen-binding domain for a target antigen of interest and an antigen-binding domain for recruiting / engaging the target immune cells. Immune cell engagers recruit / engage immune cells through antigen-binding domains specific to immune cell surface molecules.

[0084] In some embodiments, the antigen-binding molecule includes a CD3 polypeptide-binding moiety (e.g., an antigen-binding domain capable of binding to a CD3 polypeptide). The most well-studied immune cell engager is the bispecific T cell engager (BiTE), which includes a target antigen-binding domain and a CD3 polypeptide (typically CD3ε)-binding domain, through which the BiTE recruits T cells. When the BiTE binds to its target antigen and the CD3 polypeptide expressed by the T cell, the T cell is activated, ultimately directing T cell effector activity against cells expressing the target antigen. Other types of immune cell engagers are well-known in the art and include natural killer cell engagers such as bispecific killer engagers (BiKE), which recruit and activate NK cells.

[0085] In some embodiments, the immune cells engaged by the immune cell engager are T cells or NK cells. In some embodiments, the immune cell engager is a T cell engager.

[0086] Specific exemplary embodiments of antigen-binding molecules In some embodiments, the antigen-binding molecule of the present disclosure comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NO: 49, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity.

[0087] In some embodiments, the antigen-binding molecule of the present disclosure comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NO: 53, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity.

[0088] In some embodiments, the antigen-binding molecule of the present disclosure comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NO: 57, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity.

[0089] In some embodiments, the antigen-binding molecule of the present disclosure comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NO: 1, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity.

[0090] In some embodiments, the antigen-binding molecule of the present disclosure comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NO: 5, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity.

[0091] In some embodiments, the antigen-binding molecule of the present disclosure comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NO: 9, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity.

[0092] In some embodiments, the antigen-binding molecule of the present disclosure comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NO: 12, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity.

[0093] In some embodiments, the antigen-binding molecule of the present disclosure comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NO: 16, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity.

[0094] In some embodiments, the antigen-binding molecule of the present disclosure comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NO: 20, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity.

[0095] In some embodiments, the antigen-binding molecule of the present disclosure comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NO: 24, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity.

[0096] In some embodiments, the antigen-binding molecule of the present disclosure comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NO: 28, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity.

[0097] In some embodiments, the antigen-binding molecule of the present disclosure comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NO: 32, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity.

[0098] In some embodiments, the antigen-binding molecule of the present disclosure comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NO: 36, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity.

[0099] Linker and additional arrays The antigen-binding molecule may include additional amino acid / amino acid sequences in addition to the amino acid sequence required for binding to the target antigen. In some embodiments, such additional amino acid / amino acid sequences are provided at the N-terminus of the single-domain antibody sequence described in this disclosure. In some embodiments, such additional amino acid / amino acid sequences are provided at the C-terminus of the single-domain antibody sequence described in this disclosure. In some embodiments, such additional amino acid / amino acid sequences are provided at both the N-terminus and C-terminus of the single-domain antibody sequence described in this disclosure.

[0100] In some embodiments, the antigen-binding molecule includes one or more linker sequences between amino acid subsequences. For example, the linker sequences may be provided at one or both ends of the antigen-binding domain of the antigen-binding molecule described herein.

[0101] Linker sequences are known to those skilled in the art, for example, as described in Chen et al., Adv Drug Deliv Rev (2013) 65(10): pp. 1357-1369, which is incorporated herein by reference in its entirety. In some embodiments, the linker sequence may be a flexible linker sequence. Flexible linker sequences allow for the relative movement of the amino acid sequence linked by the linker sequence. Flexible linkers are known to those skilled in the art, and some are identified in Chen et al., Adv Drug Deliv Rev (2013) 65(10): pp. 1357-1369. Flexible linker sequences often contain a high proportion of glycine and / or serine residues.

[0102] In some embodiments, the linker sequence includes at least one glycine residue and / or at least one serine residue. In some embodiments, the linker sequence consists of a glycine residue and a serine residue. In some embodiments, the linker sequence includes one or more copies (e.g., one of 1, 2, 3, 4, 5, 6, or 7) of a sequence motif consisting of a glycine residue and a serine residue, e.g., G4S, in tandem. In some embodiments, the linker sequence has a length of 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or 1-30 amino acids. In some embodiments, the linker sequence includes or consists of an amino acid sequence having at least 70% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 58, preferably one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity.

[0103] The antigen-binding molecules and polypeptides of this disclosure may additionally include further amino acids or sequences of amino acids. For example, the antigen-binding molecules and polypeptides may include amino acid sequences(s) to facilitate the expression, folding, transport, processing, purification, or detection of the antigen-binding molecule / polypeptide. For example, the antigen-binding molecule / polypeptide may optionally include a sequence encoding His (e.g., 6XHis), Myc, GST, MBP, FLAG, HA, E, or a biotin tag at the N-terminus or C-terminus of the antigen-binding molecule / polypeptide. In some embodiments, the antigen-binding molecule / polypeptide includes a detectable portion, such as a fluorescent label, luminescent label, immunodetectable label, radioactive label, chemical label, nucleic acid label, or enzymatic label.

[0104] The antigen-binding molecules and polypeptides of this disclosure may additionally include signal peptides (also known as leader sequences or signal sequences). Signal peptides typically consist of a sequence of 5 to 30 hydrophobic amino acids forming a single alpha-helix. Secretory proteins and proteins expressed on the cell surface often contain signal peptides.

[0105] Signal peptides can be present at the N-terminus of antigen-binding molecules / polypeptides. Signal peptides can facilitate the efficient transport and secretion of antigen-binding molecules / polypeptides. Signal peptides are typically removed by cleavage and are therefore not present in mature antigen-binding molecules / polypeptides secreted from cells expressing the antigen-binding molecule.

[0106] Signal peptides are known in many proteins and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, InterPro, and / or can be identified / predicted using amino acid sequence analysis tools such as SignalP (Petersen et al., Nature Methods 8:785-786, 2011) or Signal-BLAST (Frank and Sippl, Bioinformatics 24:2172-2176, 2008).

[0107] Labels and conjugates In some embodiments, the antigen-binding molecules of the present disclosure additionally comprise a detectable moiety. In some embodiments, the antigen-binding molecule comprises a detectable moiety such as a fluorescent label, a phosphorescent label, a luminescent label, an immunodetectable label (e.g., an epitope tag), a radioactive label, a chemical label, a nucleic acid label, or an enzyme label. The antigen-binding molecule can be labeled covalently or non-covalently with the detectable moiety.

[0108] Examples of fluorescent labels include fluorescein, rhodamine, allophycocyanin, eosin and NDB, green fluorescent protein (GFP), rare earth chelates such as europium (Eu), terbium (Tb) and samarium (Sm), tetramethylrhodamine, Texas red, 4-methylumbelliferone, 7-amino-4-methylcoumarin, Cy3, and Cy5. Examples of radioactive labels include iodine 123 , iodine 125 , iodine 126 , iodine 131 , iodine 133 , bromine 77 , technetium 111 , indium 111 , indium 113m , gallium 67 , gallium 68 , ruthenium 95 , ruthenium 97 , ruthenium103 ,ruthenium 105 ,mercury 207 ,mercury 203 ,rhenium 99m ,rhenium 101 ,rhenium 105 ,scandium 47 ,tellurium 121m ,tellurium 122m ,tellurium 125m ,thulium 165 ,thulium 167 ,thulium 168 ,copper 67 fluorine 18 ,yttrium 90 ,palladium 100 bismuth 217 Antimony 211 Radioactive isotopes such as [list of radioactive isotopes] are included. Luminescent labels include radioluminescent labels, chemiluminescent labels (e.g., acridinium esters, luminols, isoluminols), and bioluminescent labels. Immunodetectable labels include haptens, peptides / polypeptides, antibodies, receptors, and ligands, such as biotin, avidin, streptavidin, or digoxigenin. Nucleic acid labels include aptamers. Enzyme labels include, for example, peroxidase, alkaline phosphatase, glucose oxidase, beta-galactosidase, and luciferase.

[0109] In some embodiments, the antigen-binding molecule of this disclosure is conjugated to a chemical moiety. The chemical moiety may be a moiety for providing a therapeutic effect. Antibody drug conjugates are outlined, for example, in Parslow et al., Biomedicines. September 2016; 4(3): pp. 14. In some embodiments, the chemical moiety may be a drug moiety (e.g., a cytotoxic agent) such that the antigen-binding molecule exhibits cytotoxicity against cells containing / expressing VEGFA (e.g., cells expressing VEGFA and / or complexes containing VEGFA on the cell surface). In some embodiments, the drug moiety may be a chemotherapeutic agent. In some embodiments, the drug moiety is selected from calicheamicin, DM1, DM4, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), SN-38, doxorubicin, duocalmycin, D6.5, and PBD.

[0110] Functional properties of antigen-binding molecules The antigen-binding molecules described herein can be characterized by referring to certain functional properties. In some embodiments, the antigen-binding molecules described herein may possess one or more of the following properties: Binds to VEGFA (e.g., human VEGFA and / or mouse VEGFA); Inhibit the interaction between VEGFA and VEGFR (i.e., the receptor for VEGFA, e.g., VEGFR1); Inhibits signal transduction mediated by VEGFA / VEGFR; Maintains bonding to VEGFA even after heat treatment; Reduce the number / percentage of cells expressing VEGFA; It increases cell death in cells that express VEGFA.

[0111] It will be understood that a given antigen-binding molecule may exhibit several of the properties described in the previous paragraph. A given antigen-binding molecule can be evaluated for the properties described in the previous paragraph using an appropriate assay. The assay may be, for example, an in vitro assay, which may be a cell-free assay or a cell-based assay. Alternatively, the assay may be, for example, an in vivo assay, i.e., performed in an animal other than a human. In the assay, species labeled with a detectable entity may be used to facilitate detection.

[0112] Analysis of the results of such assays may involve determining the concentration at which 50% of the maximum level of the relevant activity is achieved. The concentration of the antigen-binding molecule at which 50% of the maximum level of the relevant activity is achieved may be called the “half-maximal effective concentration” of the antigen-binding molecule with respect to the relevant activity, and this is also known as the “EC 50 It can also be called "[...]. For example, the EC of a given antigen-binding molecule regarding binding to VEGFA. 50 This may be the concentration at which 50% of the maximum level of binding to the relevant species is achieved.

[0113] Depending on the characteristics, EC 50 This refers to the "half-maximum inhibitory concentration" or "IC2". 50 This is sometimes also called the IC50, which is the concentration of an antigen-binding molecule at which 50% of the maximum inhibitory level of a given property is observed. For example, the IC50 of a given antigen-binding molecule for inhibiting the interaction between VEGFA and VEGFR (e.g., VEGFR1). 50 This may be the concentration at which 50% of the maximum inhibitory level is achieved.

[0114] The antigen-binding molecules and antigen-binding domains described herein preferably exhibit specific binding to VEGFA. As used herein, “specific binding” refers to binding that is selective to an antigen and can be distinguished from nonspecific binding to a non-target antigen. Antigen-binding molecules / domains that specifically bind to a target molecule preferably bind to the target with higher affinity and / or longer duration than binding to other non-target molecules.

[0115] The ability of a given polypeptide to specifically bind to a given molecule can be determined by methods known in the art, such as ELISA, surface plasmon resonance (SPR; Hearty et al., Methods Mol Biol (2012) 907:411-442), biolayer interferometry (e.g., Lad et al., (2015) J Biomol Screen 20(4):498-507), flow cytometry, or radiolabeled antigen binding assay (RIA) enzyme-linked immunosorbent assay. Such analyses can measure and quantify binding to a given molecule. In some embodiments, binding may be a response detected in a given assay.

[0116] In some embodiments, the degree of binding of the antigen-binding molecule to a non-target molecule is less than 10% of the binding of the antibody to the target molecule, as measured, for example, by ELISA, SPR, biolayer interferometry, or RIA. Alternatively, the binding specificity of the antigen-binding molecule is at least 0.1 orders of magnitude greater than the KD of the antigen-binding molecule to the non-target molecule (i.e., 0.1 × 10⁻¹⁰). n (wherein the formula, n is an integer representing the digits) This can be reflected in terms of bond affinity, which is bonded with a large dissociation constant (KD). This may be, at any choice, at least one of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, or 2.0.

[0117] Binding to VEGFA can be determined, for example, by biolayer interferometry, as described in Example 10 of this disclosure. In some embodiments, the antigen-binding molecule described herein binds to VEGFA. In some embodiments, the antigen-binding molecule described herein binds to a polypeptide complex containing VEGFA.

[0118] In some embodiments, the antigen-binding molecules described herein have an affinity of less than a micromolar, i.e., K D <1x10-6 It binds to VEGFA at M. In some embodiments, the antigen-binding molecules described herein have an affinity in the nanomolar range, i.e., K D = 9.9 × 10 -7 ~1 × 10 -9 It binds to VEGFA at M. In some embodiments, the antigen-binding molecules described herein have an affinity of less than nanomolar, i.e., K D <1x10 -9 It binds to VEGFA at M. In some embodiments, the antigen-binding molecules described herein are bound at picomolar affinity, i.e., K D = 9.9 × 10 -10 ~1 × 10 -12 It binds to VEGFA at M. In some embodiments, the antigen-binding molecules described herein have an affinity of less than picomoles, i.e., K D <1x10 -12 M is used to combine with VEGFA.

[0119] In some embodiments, the antigen-binding molecule described herein has a K content of 5 μM or less, preferably ≤5 μM, ≤2 μM, ≤1 μM, ≤500 nM, ≤100 nM, ≤75 nM, ≤50 nM, ≤40 nM, ≤30 nM, ≤20 nM, ≤15 nM, ≤12.5 nM, ≤10 nM, ≤9 nM, ≤8 nM, ≤7 nM, ≤6 nM, ≤5 nM, ≤4 nM, ≤3 nM, ≤2 nM, ≤1 nM, ≤500 pM, ≤400 pM, ≤300 pM, ≤200 pM, ≤100 pM, ≤75 pM, ≤50 pM, ≤45 pM, ≤40 pM, ≤35 pM, ≤30 pM, ≤25 pM, ≤20 pM, ≤15 pM, or ≤10 pM. D It binds to VEGFA (e.g., human VEGF165). In some embodiments, the antigen-binding molecule is K D = Binds to VEGFA (e.g., human VEGF165) with affinity of ≤1nM, ≤500pM, ≤400pM, ≤300pM, ≤200pM, ≤100pM, ≤75pM, ≤50pM, ≤45pM, ≤40pM, ≤35pM, ≤30pM, ≤25pM, ≤20pM, ≤15pM, or ≤10pM.

[0120] In some embodiments, the antigen-binding molecule described herein is determined in the same assay as the K2 molecule formed by the binding of ranibizumab (i.e., the molecule formed by the binding of polypeptides SEQ ID NOs. 74 and 75). D Similar to K D It binds to VEGFA (e.g., human VEGF165) (determined by BLI, for example, as described in Example 10 of this disclosure). In the same assay, the K determined for ranibizumab D K is one of the following ranges: 0.5 times or more and 2 times or less, for example, 0.55 times or more and 1.9 times or less, 0.6 times or more and 1.8 times or less, 0.65 times or more and 1.7 times or less, 0.7 times or more and 1.6 times or less, 0.75 times or more and 1.5 times or less, 0.8 times or more and 1.4 times or less, 0.85 times or more and 1.3 times or less, 0.9 times or more and 1.2 times or less, and 0.95 times or more and 1.1 times or less. D It binds to VEGFA (e.g., human VEGF165) (for example, as determined by BLI, for example, as described in Example 10 of this disclosure).

[0121] In some embodiments, the antigen-binding molecule described in this disclosure is determined for ranibizumab in the same assay as K D Lower K D It binds to VEGFA (e.g., human VEGF165) (determined, for example, by BLI, as described in Example 10 of this disclosure). In some embodiments, the antigen-binding molecule is determined for ranibizumab in the same assay. D K is less than 1 times, for example, one of ≤0.99, ≤0.95, ≤0.9, ≤0.85, ≤0.8, ≤0.75, ≤0.7, ≤0.65, ≤0.6, ≤0.55, or ≤0.5 times. D It binds to VEGFA (e.g., human VEGF165) (for example, as determined by BLI, for example, as described in Example 10 of this disclosure).

[0122] In some embodiments, the antigen-binding molecule described herein is determined in the same assay as the K2 molecule formed by the binding of bevacizumab (i.e., the polypeptide of SEQ ID NOs. 76 and 77). D Similar to K D It binds to VEGFA (e.g., human VEGF165) (determined, for example, by BLI, as described in Example 10 of this disclosure). In the same assay, the K determined for bevacizumab D K is one of the following ranges: 0.5 times or more and 2 times or less, for example, 0.55 times or more and 1.9 times or less, 0.6 times or more and 1.8 times or less, 0.65 times or more and 1.7 times or less, 0.7 times or more and 1.6 times or less, 0.75 times or more and 1.5 times or less, 0.8 times or more and 1.4 times or less, 0.85 times or more and 1.3 times or less, 0.9 times or more and 1.2 times or less, and 0.95 times or more and 1.1 times or less. D It binds to VEGFA (e.g., human VEGF165) (for example, as determined by BLI, for example, as described in Example 10 of this disclosure).

[0123] In some embodiments, the antigen-binding molecule described in this disclosure is determined for bevacizumab in the same assay. D Lower K D It binds to VEGFA (e.g., human VEGF165) (determined, for example, by BLI, as described in Example 10 of this disclosure). In some embodiments, the antigen-binding molecule is determined for bevacizumab in the same assay. D K is less than 1 times, for example, one of ≤0.99, ≤0.95, ≤0.9, ≤0.85, ≤0.8, ≤0.75, ≤0.7, ≤0.65, ≤0.6, ≤0.55, or ≤0.5 times. D It binds to VEGFA (e.g., human VEGF165) (for example, as determined by BLI, for example, as described in Example 10 of this disclosure).

[0124] The antigen-binding molecules of this disclosure may bind to a specific region of interest of VEGFA. The antigen-binding molecules described herein may bind to linear epitopes of VEGFA consisting of a continuous sequence of amino acids (i.e., a primary amino acid sequence). In some embodiments, the antigen-binding molecules may bind to structural epitopes of VEGFA consisting of a discontinuous sequence of amino acids in the amino acid sequence.

[0125] The region of a given target molecule to which an antigen-binding molecule binds can be determined by those skilled in the art using a variety of methods well known in the art, including X-ray cocrystallization of antibody-antigen complexes, peptide scanning, mutagenesis mapping, hydrogen-deuterium exchange analysis by mass spectrometry, phage display, competitive ELISA, and "protection" methods based on proteolysis. Such methods are described, for example, by Gershoni et al., BioDrugs, 2007, 21(3):145-156, which are incorporated herein by reference in their entirety.

[0126] In some embodiments, the antigen-binding molecule described herein binds to the same region of VEGFA, or an overlapping region of VEGFA, or to a region of VEGFA to which the antigen-binding molecule is bound, comprising the CDR, FR, and / or complete amino acid sequence of a VEGFA-conjugating single-domain antibody selected from 13A6, 16A2.1, 16A6.1, 20A2.1, 20A3.1, 21A1.1, 21A8.1, 21D9.1, 21E6.1, and 23D5.1.

[0127] In some embodiments, the antigen-binding molecule described herein binds to a region of VEGFA that binds to VEGFRs (e.g., VEGFR1 and / or VEGFR2).

[0128] In some embodiments, the antigen-binding molecule binds to the region of VEGFA to which a VEGFR (e.g., VEGFR1) is bound. In some embodiments, the antigen-binding molecule inhibits the interaction between VEGFR (e.g., VEGFR1) and VEGFA. In some embodiments, the antigen-binding molecule is a competitive inhibitor of the binding of VEGFR (e.g., VEGFR1) to VEGFA. In some embodiments, the antigen-binding molecule blocks VEGFA from binding to VEGFR (e.g., VEGFR1). In some embodiments, the antigen-binding molecule occupies the region of VEGFA to which VEGFR (e.g., VEGFR1) is bound, thereby inhibiting the interaction between VEGFR (e.g., VEGFR1) and VEGFA. In some embodiments, the antigen-binding molecule replaces VEGFR (e.g., VEGFR1) from a complex containing VEGFA and VEGFR (e.g., VEGFR1).

[0129] The ability of an antigen-binding molecule to inhibit the interaction between two factors can be determined, for example, by analyzing the interaction after incubation with an antibody / fragment in the presence of an antibody / fragment, or with one or both of the interaction partners. An example of a suitable assay for determining whether a given antigen-binding molecule inhibits the interaction between two interaction partners is a competitive ELISA assay. An antigen-binding molecule that inhibits a given interaction (e.g., between VEGFA and VEGFR) is identified by observing a decrease / decrease in the level of interaction between the interaction partners in the presence of the antigen-binding molecule, or after incubation of one or both of the interaction partners with the antigen-binding molecule, compared to the level of interaction in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule). Appropriate analyses can be performed in vitro, for example, using recombinant interaction partners or using cells that express the interaction partners. Cells that express the interaction partners may express them endogenously or from nucleic acids introduced into the cells. For the purposes of such assays, one or both of the interaction partner and / or antigen-binding molecules can be labeled or used in combination with a detectable entity for the purpose of detecting and / or measuring the level of interaction.

[0130] In some embodiments, the antigen-binding molecule described herein facilitates the interaction between VEGFA and VEGFR1 (e.g., human VEGF121 and human VEGFR1) with an IC of 10 μM or less, preferably one of the following ICs: ≤5 μM, ≤2 μM, ≤1 μM, ≤500 nM, ≤100 nM, ≤75 nM, ≤50 nM, ≤40 nM, ≤30 nM, ≤20 nM, ≤15 nM, ≤12.5 nM, ≤10 nM, ≤9 nM, ≤8 nM, ≤7 nM, ≤6 nM, ≤5 nM, ≤4 nM, or ≤3 nM. 50 Inhibition (for example, determined by a competitive ELISA, e.g., the competitive ELISA described in Example 11 of this disclosure).

[0131] In some embodiments, the antigen-binding molecules described herein inhibit the interaction between VEGFA and VEGFR1 (e.g., human VEGF121 and human VEGFR1) in the same assay as ranibizumab (i.e., the molecule formed by the binding of polypeptides of SEQ ID NOs. 74 and 75), according to ICs for inhibition of such interaction. 50 Similar IC 50 (For example, inhibited by competitive ELISA, e.g., the competitive ELISA described in Example 11 of this disclosure). In some embodiments, the antigen-binding molecule inhibits the interaction between VEGFA and VEGFR1 (e.g., human VEGF121 and human VEGFR1) in the same assay, and an IC for inhibition of such interaction by ranibizumab is obtained. 50 One IC that is 0.5 times or more but less than 2 times the value, for example, 0.55 times or more but 1.9 times or less, 0.6 times or more but 1.8 times or less, 0.65 times or more but 1.7 times or less, 0.7 times or more but 1.6 times or less, 0.75 times or more but 1.5 times or less, 0.8 times or more but 1.4 times or less, 0.85 times or more but 1.3 times or less, 0.9 times or more but 1.2 times or less, or 0.95 times or more but 1.1 times or less. 50 Inhibition (for example, determined by a competitive ELISA, such as the competitive ELISA described in Example 11 of this disclosure).

[0132] In some embodiments, the antigen-binding molecules described herein inhibit the interaction between VEGFA and VEGFR1 (e.g., human VEGF121 and human VEGFR1) in the same assay as determined for ranibizumab, and an IC for inhibition of such interaction was obtained. 50 Lower IC 50 (For example, inhibited by competitive ELISA, e.g., the competitive ELISA described in Example 11 of this disclosure). In some embodiments, the antigen-binding molecule inhibits the interaction between VEGFA and VEGFR1 (e.g., human VEGF121 and human VEGFR1) in the same assay, and an IC for inhibition of such interaction by ranibizumab is obtained. 50An IC less than 1 times the value, for example, one of the following ICs: ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, or ≤0.5 times. 50 Inhibition (for example, determined by a competitive ELISA, such as the competitive ELISA described in Example 11 of this disclosure).

[0133] In some embodiments, the antigen-binding molecules described herein inhibit the interaction between VEGFA and VEGFR1 (e.g., human VEGF121 and human VEGFR1) in the same assay as bevacizumab (i.e., the molecule formed by the binding of polypeptides of SEQ ID NOs. 76 and 77), according to ICs for inhibition of such interaction. 50 Similar IC 50 (For example, inhibited by competitive ELISA, e.g., the competitive ELISA described in Example 11 of this disclosure). In some embodiments, the antigen-binding molecule inhibits the interaction between VEGFA and VEGFR1 (e.g., human VEGF121 and human VEGFR1) in the same assay, and an IC for inhibition of such interaction by bevacizumab is obtained. 50 One IC that is 0.5 times or more but less than 2 times the value, for example, 0.55 times or more but 1.9 times or less, 0.6 times or more but 1.8 times or less, 0.65 times or more but 1.7 times or less, 0.7 times or more but 1.6 times or less, 0.75 times or more but 1.5 times or less, 0.8 times or more but 1.4 times or less, 0.85 times or more but 1.3 times or less, 0.9 times or more but 1.2 times or less, or 0.95 times or more but 1.1 times or less. 50 Inhibition (for example, determined by a competitive ELISA, such as the competitive ELISA described in Example 11 of this disclosure).

[0134] In some embodiments, the antigen-binding molecules described herein inhibit the interaction between VEGFA and VEGFR1 (e.g., human VEGF121 and human VEGFR1) in the same assay as bevacizumab, according to IC for inhibition of such interaction. 50 Lower IC 50(For example, inhibited by competitive ELISA, e.g., the competitive ELISA described in Example 11 of this disclosure). In some embodiments, the antigen-binding molecule inhibits the interaction between VEGFA and VEGFR1 (e.g., human VEGF121 and human VEGFR1) in the same assay, and an IC for inhibition of such interaction by bevacizumab is obtained. 50 An IC less than 1 times the value, for example, one of the following ICs: ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, or ≤0.5 times. 50 Inhibition (for example, determined by a competitive ELISA, such as the competitive ELISA described in Example 11 of this disclosure).

[0135] In some embodiments, antigen-binding molecules inhibit VEGFA / VEGFR-mediated signaling (i.e., signaling mediated by the binding of VEGFA to VEGFR). VEGFA / VEGFR-mediated signaling can be analyzed, for example, using VEGFR-expressing cells in assays that detect and / or quantify VEGFA / VEGFR-mediated signaling.

[0136] Appropriate assays for investigating VEGFA / VEGFR-mediated signaling include assays for detecting phosphorylation / activation / expression of factors that are phosphorylated / activated / expressed as a result of VEGFA / VEGFR-mediated signaling. Such assays may include the step of contacting VEGFR-expressing cells with the antigen-binding molecules described herein in the presence of VEGFA. Assays for investigating VEGFA / VEGFR-mediated signaling may include analyzing signaling via the PI3K / AKT, MAPK / ERK and / or PLC-γ pathways, as well as via SCR and / or FAK.

[0137] In some embodiments, the antigen-binding molecules of the present disclosure inhibit VEGFA / VEGFR-mediated signaling (e.g., signaling mediated by the binding of human VEGF121 to human VEGFR1) to less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold of the level of such signaling in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule).

[0138] In some embodiments, the antigen-binding molecules described in the present disclosure inhibit VEGFA / VEGFR-mediated signaling (e.g., signaling mediated by the binding of human VEGF121 to human VEGFR1) with an IC 50 similar to that 50 determined for ranibizumab (i.e., the molecule formed by the binding of the polypeptides of SEQ ID NOs: 74 and 75) for inhibition of such interaction in the same assay. In some embodiments, the antigen-binding molecule inhibits VEGFA / VEGFR-mediated signaling (e.g., signaling mediated by the binding of human VEGF121 to human VEGFR1) with an IC 50 value that is 0.5-fold or more and 2-fold or less, e.g., 0.55-fold or more and 1.9-fold or less, 0.6-fold or more and 1.8-fold or less, 0.65-fold or more and 1.7-fold or less, 0.7-fold or more and 1.6-fold or less, 0.75-fold or more and 1.5-fold or less, 0.8-fold or more and 1.4-fold or less, 0.85-fold or more and 1.3-fold or less, 0.9-fold or more and 1.2-fold or less, 0.95-fold or more and 1.1-fold or less of the IC 50 for inhibition of such signaling by ranibizumab in the same assay.

[0139] In some embodiments, the antigen-binding molecules described in the present disclosure inhibit VEGFA / VEGFR-mediated signaling (e.g., signaling mediated by the binding of human VEGF121 to human VEGFR1) with an IC 50 lower than that determined for ranibizumab for inhibition of such interaction in the same assay. In some embodiments, the antigen-binding molecule inhibits VEGFA / VEGFR-mediated signaling (e.g., signaling mediated by the binding of human VEGF121 to human VEGFR1) with an IC 50 that is less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, or ≤0.5-fold of the IC 50 value determined for inhibition of such signaling by ranibizumab in the same assay. 50 In some embodiments, the antigen-binding molecules described in the present disclosure inhibit VEGFA / VEGFR-mediated signaling (e.g., signaling mediated by the binding of human VEGF121 to human VEGFR1) with an IC

[0140] [[ID=II]] similar to the IC 50 determined for bevacizumab (i.e., the molecule formed by the binding of the polypeptides of SEQ ID NOs: 76 and 77) for inhibition of such interaction in the same assay. In some embodiments, the antigen-binding molecule inhibits VEGFA / VEGFR-mediated signaling (e.g., signaling mediated by the binding of human VEGF121 to human VEGFR1) with an IC 50 that is 0.5-fold or more and 2-fold or less, e.g., 0.55-fold or more and 1.9-fold or less, 0.6-fold or more and 1.8-fold or less, 0.65-fold or more and 1.7-fold or less, 0.7-fold or more and 1.6-fold or less, 0.75-fold or more and 1.5-fold or less, 0.8-fold or more and 1.4-fold or less, 0.85-fold or more and 1.3-fold or less, 0.9-fold or more and 1.2-fold or less, 0.95-fold or more and 1.1-fold or less of the IC 50 value determined for inhibition of such signaling by bevacizumab in the same assay. 50 In some embodiments, the antigen-binding molecules described in the present disclosure inhibit VEGFA / VEGFR-mediated signaling (e.g., signaling mediated by the binding of human VEGF121 to human VEGFR1) with an IC

[0141] In some embodiments, the antigen-binding molecules described herein inhibit VEGFA / VEGFR-mediated signaling (e.g., signaling mediated by the binding of human VEGF121 to human VEGFR1) in the same assay as determined for bevacizumab in IC for inhibition of such interactions. 50 Lower IC 50 Inhibits the signal. In some embodiments, the antigen-binding molecule inhibits VEGFA / VEGFR-mediated signaling (e.g., signaling mediated by the binding of human VEGF121 to human VEGFR1) in the same assay, and in the IC for inhibition of such signaling by bevacizumab. 50 An IC less than 1 times the value, for example, one of the following ICs: ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, or ≤0.5 times. 50 It inhibits it.

[0142] In some embodiments, the antigen-binding molecules described herein bind to VEGFA (e.g., human VEGFA) with similar affinity before and after heat treatment. Heat treatment may include incubation in a suitable buffer (e.g., a buffer containing 0.1% BSA and 0.01% Tween-20 in PBS) at room temperature, 60°C, 70°C, or 80°C for 1 hour. Heat treatment may be carried out as described in Example 12 of this disclosure.

[0143] In some embodiments, the antigen-binding molecule exhibits similar affinity to VEGFA before heat treatment and after heat treatment at room temperature for 1 hour. In some embodiments, the antigen-binding molecule exhibits similar affinity to VEGFA before heat treatment and after heat treatment at 60°C for 1 hour. In some embodiments, the antigen-binding molecule exhibits similar affinity to VEGFA before heat treatment and after heat treatment at 70°C for 1 hour. In some embodiments, the antigen-binding molecule exhibits similar affinity to VEGFA before heat treatment and after heat treatment at 80°C for 1 hour.

[0144] In this specification, "reference binding affinity" and "similar" binding affinity mean one binding affinity within 50% of the reference binding affinity determined in the same assay, for example, 40%, 45%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0145] K for binding to VEGFA (e.g., human VEGFA) D This may be similar before and after heat treatment. In this specification, the reference value and "similar" K D The value can be one of the following: 0.5 times or more and 2 times or less of the reference value, for example, 0.7 times or more and 1.5 times or less, 0.75 times or more and 1.25 times or less, 0.8 times or more and 1.2 times or less, 0.85 times or more and 1.15 times or less, 0.9 times or more and 1.1 times or less, 0.91 times or more and 1.09 times or less, 0.92 times or more and 1.08 times or less, 0.93 times or more and 1.07 times or less, 0.94 times or more and 1.06 times or less, 0.95 times or more and 1.05 times or less, 0.96 times or more and 1.04 times or less, 0.97 times or more and 1.03 times or less, 0.98 times or more and 1.02 times or less, or 0.99 times or more and 1.01 times or less.

[0146] In some embodiments, the antigen-binding molecules described herein may enhance (i.e., upregulate and enhance) cell death in cells containing / expressing VEGFA. In some embodiments, the antigen-binding molecules described herein can reduce the number / percentage of cells containing / expressing VEGFA. In some embodiments, the antigen-binding molecules described herein can deplete / enhance such cells.

[0147] In some embodiments, the antigen-binding molecules described herein reduce the number / percentage of cells containing / expressing VEGFA to less than 1x of the number / percentage of such cells observed in a given assay in the absence of the antigen-binding molecule or in the presence of an equal amount of a suitable control antigen-binding molecule, for example, ≤0.99x, ≤0.95x, ≤0.9x, ≤0.85x, ≤0.8x, ≤0.75x, ≤0.7x, ≤0.65x, ≤0.6x, ≤0.55x, ≤0.5x, ≤0.45x, ≤0.4x, ≤0.35x, ≤0.3x, ≤0.25x, ≤0.2x, ≤0.15x, ≤0.1x, ≤0.05x, or ≤0.01x.

[0148] The antigen-binding molecules described herein may comprise one or more moieties for enhancing the reduction of the number / percentage of cells containing / expressing VEGFA. For example, the antigen-binding molecules described herein may comprise, for example, an Fc region and / or a drug moiety.

[0149] In some embodiments, the antigen-binding molecules described herein include an Fc region that can enhance / induce one or more of ADCC, ADCP, and CDC in cells containing / expressing VEGFA, and / or enhance MAC formation or cell degranulation on cells containing / expressing VEGFA.

[0150] In some embodiments, the antigen-binding molecules described herein can enhance / induce ADCC in cells containing / expressing VEGFA. In some embodiments, the antigen-binding molecule described herein includes a drug moiety. The antigen-binding molecule may be conjugated to the drug moiety. Antibody-drug conjugates are outlined, for example, in Parslow et al., Biomedicines. September 2016; 4(3): 14 (incorporated herein by the above reference). In some embodiments, the drug moiety is or includes a cytotoxic agent such that the antigen-binding molecule exhibits cytotoxicity against cells containing / expressing VEGFA. In some embodiments, the drug moiety is or includes a chemotherapeutic agent.

[0151] In some embodiments, the antigen-binding molecule described herein includes an immune cell-engaging portion. In some embodiments, the antigen-binding molecule includes a CD3 polypeptide-binding portion (e.g., an antigen-binding domain capable of binding to a CD3 polypeptide).

[0152] In some embodiments, the antigen-binding molecules described herein can enhance / induce T cell-mediated cytolytic activity against cells containing / expressing VEGFA. In some embodiments, the antigen-binding molecules described herein reduce the number / percentage of cells containing / expressing VEGFA to less than 1x of the number / percentage of such cells observed in a given assay in the absence of the antigen-binding molecule or in the presence of an equal amount of a suitable control antigen-binding molecule, for example, ≤0.99x, ≤0.95x, ≤0.9x, ≤0.85x, ≤0.8x, ≤0.75x, ≤0.7x, ≤0.65x, ≤0.6x, ≤0.55x, ≤0.5x, ≤0.45x, ≤0.4x, ≤0.35x, ≤0.3x, ≤0.25x, ≤0.2x, ≤0.15x, ≤0.1x, ≤0.05x, or ≤0.01x.

[0153] In some embodiments, the antigen-binding molecules described herein increase the level of cell death of cells containing / expressing VEGFA by more than 1x the level of such cell death observed in a given assay in the absence of the antigen-binding molecule or in the presence of an equal amount of a suitable control antigen-binding molecule, for example, by more than ≥1.5x, ≥2x, ≥3x, ≥4x, ≥5x, ≥6x, ≥7x, ≥8x, ≥9x, ≥10x, ≥15x, ≥20x, ≥30x, ≥40x, or ≥50x.

[0154] Chimeric antigen receptor (CAR) This disclosure also provides chimeric antigen receptors (CARs) comprising the antigen-binding polypeptides of this disclosure.

[0155] CARs are recombinant receptors that provide both antigen-binding and T-cell activation functions. The structure and operation of CARs are outlined, for example, in Dotti et al., Immunol Rev (2014) 257(1), which is incorporated herein by reference in its entirety. CARs include an antigen-binding region linked to a cell membrane anchoring region and a signaling region. Any hinge region can provide a separation between the antigen-binding region and the cell membrane anchoring region and can function as a flexible linker.

[0156] The CARs of this disclosure include, or consist of, the antigen-binding molecules of this disclosure, or include, or consist of, an antigen-binding region of a single-domain antibody sequence described in this disclosure. That is, the antigen-binding molecules / single-domain antibody sequences described in this disclosure are included in or constitute the antigen-binding region of the CAR.

[0157] A cell membrane anchoring region is provided between the antigen-binding region and the signaling region of the CAR, and is provided for immobilizing the CAR to the cell membrane of a cell expressing the CAR, with the antigen-binding region located in extracellular space and the signaling region located intracellular. In some embodiments, the CAR includes a cell membrane anchoring region that includes, consists of, or derives from, one transmembrane amino acid sequence of CD3-ζ, CD4, CD8, or CD28. As used herein, a region “derived” from a reference amino acid sequence includes an amino acid sequence having at least 60% sequence identity with the reference sequence, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0158] The signaling region of CAR can activate T cells. The CAR signaling region may contain the amino acid sequence of the intracellular domain of CD3-ζ, which provides an immunoreceptor tyrosine-based activation motif (ITAM) for phosphorylation and activation of CAR-expressing T cells. Signaling regions containing sequences of other ITAM-containing proteins, such as FcγRI, have also been used in CAR (Haynes et al., 2001 J Immunol 166(1):182-187). The CAR signaling region may also contain a costimulatory sequence derived from the signaling region of a costimulatory molecule to facilitate activation of CAR-expressing T cells upon binding to a target protein. Suitable costimulatory molecules include CD28, OX40, 4-1BB, ICOS, and CD27. In some cases, CARs are engineered to provide costimulation of different intracellular signaling pathways. For example, CD28-mediated signaling preferentially activates the phosphatidylinositol 3-kinase (PI3K) pathway, while 4-1BB-mediated signaling is via the TNF receptor-associated factor (TRAF) adapter protein. Therefore, the signaling region of a CAR may include costimulatory sequences derived from the signaling regions of multiple costimulatory molecules. In some embodiments, the CARs of this disclosure include, consist of, or are derived from, amino acid sequences of one or more intracellular domains of CD28, 0X40, 4-1BB, ICOS, and CD27, or include one or more costimulatory sequences consisting of such sequences. Any hinge region can provide a separation between the antigen-binding domain and the transmembrane domain and can function as a flexible linker. The hinge region may be derived from IgG1. In some embodiments, the CARs of this disclosure include, consist of, or are derived from, amino acid sequences of the hinge region of IgG1, or include a hinge region consisting of such sequences.

[0159] Cells containing the CARs described in this disclosure are also provided. The CARs described in this disclosure can be used to generate CAR-expressing immune cells, such as CAR-T cells or CAR-NK cells. Manipulation of the CARs into immune cells can be carried out during in vitro culture.

[0160] The antigen-binding region of the CAR of this disclosure may be provided in any suitable form, such as scFv, scFab, etc. nucleic acids and vectors This disclosure provides nucleic acids encoding antigen-binding molecules and CARs as described herein. In some embodiments, the nucleic acids include or consist of DNA and / or RNA.

[0161] This disclosure also provides a vector comprising the nucleic acid described in the preceding paragraph. The nucleic acids and vectors described herein may be provided in purified or isolated forms, i.e., from other nucleic acids or natural biological materials.

[0162] The nucleotide sequences of nucleic acids described herein may be contained in a vector, for example, an expression vector. As used herein, “vector” is a nucleic acid molecule used as a vehicle for introducing exogenous nucleic acids into cells. A vector may be a vector for expressing nucleic acids in cells. Such a vector may include a promoter sequence operably ligated to a nucleotide sequence encoding the sequence to be expressed. A vector may also include a stop codon and an expression enhancer. Peptides or polypeptides can be expressed from the vectors described herein using any suitable vector, promoter, enhancer, and stop codon known in the art.

[0163] The term "operably linked" may include situations where a selected nucleic acid sequence and a regulatory nucleic acid sequence (e.g., a promoter and / or enhancer) are covalently linked in such a way that the expression of the nucleic acid sequence is under the influence or control of the regulatory sequence (thereby forming an expression cassette). Therefore, if the regulatory sequence may affect the transcription of the nucleic acid sequence, the regulatory sequence is operably linked to the selected nucleic acid sequence. The resulting transcript can then be translated into the desired peptide / polypeptide(s).

[0164] Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g., gamma retrovirus vectors (e.g., mouse leukemia virus (MLV)-derived vectors), lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors, and herpesvirus vectors)), transposon-based vectors, and artificial chromosomes (e.g., yeast artificial chromosomes).

[0165] In some embodiments, the vector may be a eukaryotic vector, for example, a vector containing elements necessary for protein expression from the vector in eukaryotic cells. In some embodiments, the vector may be a mammalian expression vector, for example, containing a cytomegalovirus (CMV) or SV40 promoter that drives protein expression.

[0166] Cells that contain / express antigen-binding molecules / CARs This disclosure also provides cells that contain or express the antigen-binding molecules and CARs described herein. Furthermore, cells that contain or express the nucleic acids or vectors described herein are also provided.

[0167] The cells may be eukaryotic cells, for example, mammalian cells. Mammals may be primates (rhesus macaques, crab-eating macaques, non-human primates or humans) or non-human mammals (for example, rabbits, guinea pigs, rats, mice or other rodents (including any animals of the order Rodentia), cats, dogs, pigs, sheep, goats, cattle (for example, cattle such as dairy cows or any animals of the order Bos), horses (including any animals of the family Equidae), donkeys, and non-human primates).

[0168] In some embodiments, the cells are or are derived from cell types commonly used for polypeptide expression for therapeutic use in humans. Exemplary cells are described, for example, in Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100:3451–3461 (the whole of which is thus incorporated herein by reference), and include, for example, CHO cells, HEK293 cells, PER.C6 cells, NS0 cells, and BHK cells.

[0169] The Disclosure also provides a method for producing cells comprising the nucleic acid or vector described herein, the method comprising the step of introducing the nucleic acid or vector described herein into cells. In some embodiments, the step of introducing the isolated nucleic acid or vector described herein into cells comprises transformation, transfection, electroporation, or transduction (e.g., retroviral transduction).

[0170] The Disclosure also provides a method for producing cells expressing / containing an antigen-binding molecule / CAR described herein, the method comprising the step of introducing a nucleic acid or vector described herein into cells. In some embodiments, the method further comprises the step of culturing cells under conditions suitable for the expression of nucleic acid / vectors by cells. In some embodiments, the method is carried out in vitro.

[0171] This disclosure also provides cells that can be obtained or obtained by the methods described herein. Production of antigen-binding molecules and polypeptides The antigen-binding molecules and polypeptides described herein can be prepared by polypeptide production methods known to those skilled in the art.

[0172] Polypeptides can be prepared by chemical synthesis, for example, liquid-phase synthesis or solid-phase synthesis. For example, peptides / polypeptides can be synthesized using the method described, for example, Chandrudu et al., Molecules (2013), 18:4373-4388, which is incorporated herein by reference in its entirety.

[0173] Alternatively, antigen-binding molecules and polypeptides can be produced by recombinant expression. Molecular biology techniques suitable for recombinant polypeptide production are well known in the art and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th edition), Cold Spring Harbor Press, 2012, and in Nat Methods. (2008); 5(2): pp. 135-146, both of which are incorporated herein by reference in their entirety. Methods for recombinant production of antigen-binding molecules are also described in Frenzel et al., Front Immunol. (2013); 4: pp. 217, and in Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100: pp. 3451-3461, both of which are incorporated herein by reference in their entirety.

[0174] Any cells suitable for polypeptide expression may be used for the recombinant production described herein. The cells may be prokaryotes or eukaryotes. In some embodiments, the cells are prokaryotic cells, such as archaeal or bacterial cells. In some embodiments, the bacteria may be bacteria of the Enterobacteriaceae family, such as Gram-negative bacteria such as Escherichia coli. In some embodiments, the cells are eukaryotic cells, such as yeast cells, plant cells, insect cells, or mammalian cells, such as the cells described above herein.

[0175] In some cases, cells may not be prokaryotic because some prokaryotic cells are unable to perform the same folding or post-translational modifications as eukaryotic cells. Furthermore, very high expression levels are possible in eukaryotes, and proteins can be easily purified from eukaryotes using appropriate tags. Specific plasmids that promote the secretion of proteins into culture media are also available.

[0176] In some embodiments, polypeptides can be prepared by cell-free protein synthesis (CFPS) using, for example, the system described in Zemella et al. Chembiochem (2015) 16(17):2420-2431, which is thus incorporated herein by reference in its entirety.

[0177] Production may involve culturing or fermenting eukaryotic cells modified to express the target polypeptide(s). Culturing or fermentation can be carried out in a bioreactor provided with adequate supply of nutrients, air / oxygen, and / or growth factors. Secreted proteins can be recovered by fractionating the culture medium / fermentation broth from the cells, extracting the protein contents, and separating individual proteins to isolate the secreted polypeptide(s). Techniques for culturing, fermentation, and isolation are well known to those skilled in the art and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th edition, incorporated by reference herein).

[0178] The bioreactor includes one or more containers in which cells can be cultured. Culturing in the bioreactor can be carried out continuously by continuous inflow of reactants into the reactor and continuous outflow of cultured cells from the reactor. Alternatively, the culture can be carried out in batches. The bioreactor monitors and controls environmental conditions such as pH, oxygen, the flow rates of inflow and outflow to the container, and agitation within the container so as to provide optimal conditions for the cells to be cultured.

[0179] After culturing cells expressing an antigen-binding molecule, the antigen-binding molecule can be isolated or purified (for example, from the cell culture supernatant). Any suitable method for isolating / purifying the polypeptide of interest produced by expression from the cultured cells can be used.

[0180] To isolate the polypeptide, it may be necessary to separate the cells from the nutrient medium. If the polypeptide is secreted from the cells, the cells can be separated from the medium containing the secreted polypeptide of interest by centrifugation. If the polypeptide of interest accumulates intracellularly, protein isolation may include centrifugation to separate the cells from the cell culture medium, treatment of the cell pellet with lysis buffer, and cell disruption by, for example, sonication, rapid freeze-thaw, or osmotic lysis.

[0181] [[ID=I5]]Subsequently, it may be desirable to isolate the polypeptide of interest from the supernatant or culture medium which may contain other protein components and non-protein components. A common approach to separating protein components from the supernatant or culture medium is by precipitation. Proteins with different solubilities precipitate at different concentrations of a precipitating agent such as ammonium sulfate. For example, when the concentration of the precipitating agent is low, water-soluble proteins are extracted. Thus, proteins with different solubilities can be distinguished by adding different increasing concentrations of the precipitating agent. Subsequently, dialysis can be used to remove ammonium sulfate from the separated proteins.

[0182] Other methods for distinguishing different proteins are known in the art, such as ion exchange chromatography and size chromatography. These can be used as an alternative to precipitation or can be carried out following precipitation.

[0183] When the polypeptide of interest is isolated from a culture, it may be desirable or necessary to concentrate the polypeptide. Many methods for concentrating proteins are known in the art, such as ultrafiltration or lyophilization.

[0184] Composition The present disclosure also provides compositions comprising the antigen-binding molecules, CARs, nucleic acids, expression vectors and cells described herein.

[0185] The antigen-binding molecules, CARs, nucleic acids, expression vectors and cells described herein can be formulated as pharmaceutical compositions or medicaments for clinical use and may include pharmaceutically acceptable carriers, diluents, excipients or adjuvants.

[0186] The compositions disclosed herein include one or more pharmaceutically acceptable carriers (e.g., liposomes, micelles, microspheres, nanoparticles), diluents / excipients (e.g., starch, cellulose, cellulose derivatives, polyols, dextrose, maltodextrin, magnesium stearate), adjuvants, fillers, buffers, preservatives (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methylparaben, propylparaben), and antioxidants. It may contain (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium), lubricants (e.g., magnesium stearate, talc, silica, stearic acid, vegetable stearin), binders (e.g., sucrose, lactose, starch, cellulose, gelatin, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), xylitol, sorbitol, mannitol), stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, or colorants (e.g., titanium dioxide).

[0187] As used herein, the term “pharmaceutically acceptable” means, within the bounds of sound medical judgment, a compound, component, material, composition, dosage form, etc., that is appropriate for use in contact with the tissue of the subject (e.g., a human subject) and that provides a reasonable benefit / risk ratio without excessive toxicity, irritation, allergic reaction, or other problems or complications. Each of the carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, binders, stabilizers, solubilizers, surfactants, masking agents, colorants, fragrances, or sweeteners of the compositions described herein must also be “acceptable” in the sense that they are compatible with the other components of the formulation. Appropriate carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, binders, stabilizers, solubilizers, surfactants, masking agents, colorants, fragrances, or sweeteners can be found in standard pharmaceutical texts, such as Remington's 'The Science and Practice of Pharmacy' (edited by A. Adejare), 23rd edition (2020), Academic Press.

[0188] The composition can be formulated via local administration, parenteral administration, systemic administration, intracavitary administration, intravenous administration, intra-arterial administration, intramuscular administration, intrathecal administration, intraocular administration, intraconjunctival administration, intratumoral administration, subcutaneous administration, intradermal administration, intrathecal administration, oral administration, or transdermal administration. In some embodiments, the pharmaceutical composition / pharmaceutical can be formulated for administration by injection or infusion, or for oral ingestion.

[0189] A suitable formulation may contain antigen-binding molecules in a sterile or isotonic medium. Pharmaceuticals and pharmaceutical compositions can be formulated in fluid form, including gels. Fluid formulations can be formulated for administration by injection or infusion (e.g., via a catheter) into a selected area of ​​the human or animal body.

[0190] In some embodiments, the composition is formulated, for example, for injection or infusion into a target blood vessel or tissue / organ. The disclosure also provides a method for producing a pharmaceutically useful composition, which may comprise one or more steps selected from: producing an antigen-binding molecule, CAR, nucleic acid, expression vector, or cell as described herein; isolating an antigen-binding molecule, CAR, nucleic acid, expression vector, or cell as described herein; and / or mixing an antigen-binding molecule, CAR, nucleic acid, expression vector, or cell as described herein with a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

[0191] For example, further aspects of this disclosure relate to a method for formulating or producing a pharmaceutical or pharmaceutical composition for use in the treatment of a disease / condition (e.g., a disease / condition as described herein), the method comprising the step of formulating the pharmaceutical composition or pharmaceutical by mixing an antigen-binding molecule, CAR, nucleic acid, expression vector, or cell as described herein with a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

[0192] Therapeutic and preventive uses The antigen-binding molecules, CARs, nucleic acids, expression vectors, cells, and compositions described herein have been found to be used in therapeutic and prophylactic methods.

[0193] This disclosure provides antigen-binding molecules, CARs, nucleic acids, expression vectors, cells, or compositions described herein for use in methods of medical treatment or prevention. It also provides the use of antigen-binding molecules, CARs, nucleic acids, expression vectors, cells, or compositions described herein in the manufacture of pharmaceuticals for the treatment or prevention of diseases or conditions. Furthermore, it provides methods for treating or preventing diseases or conditions, comprising the step of administering a therapeutic or prophylactic effective amount of an antigen-binding molecule, CAR, nucleic acid, expression vector, cell, or composition described herein to a target.

[0194] The therapeutic or preventive interventions described herein may be effective in reducing the onset or progression of a disease / condition, alleviating the symptoms of a disease / condition, or reducing the pathology of a disease / condition. Interventions may be effective in preventing the progression of a disease / condition, e.g., preventing the worsening of a disease / condition, or slowing the rate of disease / condition onset. In some embodiments, this method may lead to improvement of a disease / condition, e.g., reduction of symptoms of a disease / condition, or reduction of other correlations of disease / condition severity / activity. In some embodiments, this method may prevent the onset of a disease / condition in later stages (e.g., more severe or chronic stages).

[0195] As used herein, the terms “onset,” “having onset,” and “onset” of a disorder refer to both the onset of the disease and the progression, exacerbation, or worsening of the disease state / correlation.

[0196] It will be understood that the articles of this disclosure may be used to treat / prevent any disease / condition in which therapeutic or preventive benefits may be obtained from reduced VEGFA levels, reduced VEGFA / VEGFR-mediated signaling, reduced number of cells containing / expressing VEGFA, and / or reduced activity of cells expressing VEGFR. The disease / condition may be, for example, a disease / condition in which VEGFA, VEGFA / VEGFR-mediated signaling, and / or cells containing / expressing VEGFA / VEGFR are pathologically involved, for example, a disease / condition in which elevated VEGFA / VEGFR-mediated signaling levels and / or increased number of cells containing / expressing VEGFA / VEGFR are positively correlated with the onset, development, or progression of the disease / condition, and / or the severity of one or more symptoms of the disease / condition, or a disease / condition in which elevated VEGFA / VEGFR-mediated signaling levels and / or increased number of cells containing / expressing VEGFA / VEGFR are risk factors for the onset, development, or progression of the disease / condition.

[0197] The treatments and preventions described herein in terms of the aspects and embodiments are primarily related to diseases / conditions characterized by VEGFA / VEGFR-mediated signaling. In some embodiments, the disease / condition treated / prevented by this disclosure is, for example, a disease / condition characterized by an increased level of VEGFA expression compared to the level of VEGFA expression in the absence of the disease / condition. In some embodiments, the disease / condition treated / prevented by this disclosure is, for example, a disease / condition characterized by an increased number / percentage / activity of VEGFR-expressing cells compared to the number / percentage / activity of VEGFR-expressing cells in the absence of the disease / condition.

[0198] The role of VEGFA / VEGFR-mediated signaling and disease is outlined, for example, in Karaman Development (2018) 145(14):dev151019, Ferrara and Adamis, Nat Rev Drug Discov. (2016) 15(6):pp. 385–403, and Claesson-Welsh and Welsh, J Intern Med. (2013) 273(2):pp. 114–27, all of which are thus incorporated herein by reference in their entirety.

[0199] VEGFA / VEGFR-mediated signaling is involved in the pathogenesis of several diseases. VEGFA promotes angiogenesis, blood-retinal barrier disruption, inflammation, and vision loss in individuals with ophthalmic diseases such as diabetic retinopathy and exudative age-related macular degeneration.

[0200] VEGF and VEGF receptors are expressed in non-endothelial cells, including some tumor cells. VEGFA secreted by tumor cells stimulates the proliferation and survival of endothelial cells, leading to the formation of new blood vessels and promoting tumor growth. The development and use of neutralizing antibodies against VEGFA provided the first direct evidence that tumor growth is dependent on angiogenesis, confirming the importance of VEGFA in this process.

[0201] In some embodiments, the diseases / conditions treated by the present invention are diseases, cancers, VEGFA-expressing cancers (i.e., cancers containing cells that express VEGFA; for example, cancers containing cells with elevated levels of VEGFA expression compared to equivalent non-cancerous cells), and VEGFR-expressing cancers (i.e., cancers containing cells that express VEGFR; for example, cancers containing cells with elevated levels of VEGFA expression compared to equivalent non-cancerous cells). The following conditions are selected from cancers (including cells with elevated EGFR expression levels), eye diseases, retinopathy, diabetic retinopathy, macular degeneration, age-related macular degeneration, exudative (i.e., neovascular) age-related macular degeneration, retinal vein occlusion, myopic choroidal neovascularization, retinopathy of prematurity, neovascular glaucoma, central serous retinopathy, eye tumors, corneal neovascularization, inflammatory diseases, autoimmune diseases, arthritis, rheumatoid arthritis, osteoarthritis, psoriasis, multiple sclerosis, sepsis, motor neuron diseases, and amyotrophic lateral sclerosis.

[0202] It will be understood that the specific diseases mentioned in the previous paragraph are interrelated. For example, diseases characterized by pathological angiogenesis include cancer and eye diseases. As used herein, “pathological angiogenesis” refers to the growth of new blood vessels from an existing vascular plexus that contributes to the onset and / or progression of a disease.

[0203] In some embodiments, the disease / condition treated / prevented by the present disclosure is cancer. Cancer can be any unwanted cell growth (or any disease manifested by unwanted cell growth), neoplasm or tumor. Cancer can be benign or malignant and can be primary or secondary (metastatic). A neoplasm or tumor is an abnormal growth or proliferation of cells and can be present in any tissue. Cancer can be, for example, of adrenal, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (with or without the brain), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g., renal epithelium), gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph node, lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testis, thymus, thyroid, tongue, tonsil, trachea, uterus, vulva, tissue / cells derived from white blood cells.

[0204] The tumor to be treated can be a nervous system tumor or a non-nervous system tumor. Nervous system tumors can occur in either the central nervous system or the peripheral nervous system and include, for example, glioma, medulloblastoma, meningioma, neurofibroma, ependymoma, schwannoma, neurofibrosarcoma, astrocytoma, and oligodendroglioma. Non-nervous system cancers / tumors can be derived from other non-nervous tissues and examples include melanoma, mesothelioma, lymphoma, myeloma, leukemia, non-Hodgkin lymphoma (NHL), Hodgkin lymphoma, chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), hepatoma, epidermoid carcinoma, prostate cancer, breast cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, thymic cancer, NSCLC, blood cancer, and sarcoma.

[0205] The treatment / prevention can be aimed at one or more of delaying / preventing the onset / progression of cancer symptoms, reducing the severity of cancer symptoms, reducing cancer cell survival / growth / invasion / metastasis, reducing the number of cancer cells, and / or extending the survival of the subject.

[0206] In some embodiments, the cancer to be treated / prevented includes cells expressing VEGFA. In some embodiments, the cancer to be treated / prevented includes cells expressing VEGFR. In some embodiments, the cancer to be treated / prevented is VEGFA-positive cancer. In some embodiments, the cancer to be treated / prevented is VEGFR-positive cancer. In some embodiments, the cancer overexpresses VEGFA. In some embodiments, the cancer overexpresses VEGFR. Overexpression of VEGFA and / or VEGFR can be determined by detecting higher levels of expression of the relevant factors than those expressed by equivalent non-cancerous cells / non-tumor tissues.

[0207] VEGFA and / or VEGFR expression can be determined by any suitable means. Expression may be gene expression or protein expression. Gene expression can be determined, for example, by quantitative real-time PCR (qRT-PCR), for example, by detection of mRNA encoding VEGFA and / or VEGFR. Protein expression can be determined, for example, by antibody-based methods, for example, by detecting VEGFA and / or VEGFR by Western blotting, immunohistochemistry, immunocytochemistry, flow cytometry, or ELISA.

[0208] In some embodiments, patients may be selected for the treatment described herein based, for example, on the detection of cancer expressing VEGFA and / or VEGFR, or cancer overexpressing VEGFA and / or VEGFR, in a sample taken from the subject.

[0209] VEGFA / VEGFR antagonists are being studied as drugs to treat / prevent a wide variety of cancers, as described, for example, Kieran et al., Cold Spring Harb Perspect Med. December 2012;2(12):a006593 (the entire article is incorporated herein by reference; see, for example, Table 2). In some embodiments, the cancers treated / prevented by this disclosure are selected from solid tumors, hematological malignancies, myeloid hematological malignancies, acute myeloid leukemia, multiple myeloma, breast cancer, kidney cancer, renal cell carcinoma, lung cancer, non-small cell lung cancer, thyroid cancer, medullary thyroid cancer, brain / spinal cord cancer, glioblastoma, glioma, high-grade glioma, head and neck cancer, skin cancer, melanoma, squamous cell carcinoma, liver cancer, hepatocellular carcinoma, pancreatic cancer, stomach cancer, intestinal cancer, colon cancer, rectal cancer, colorectal cancer, bile duct cancer, cholangiocarcinoma, bone cancer, sarcoma, ovarian cancer, cervical cancer, peritoneal cancer, prostate cancer, urothelial carcinoma, and neuroendocrine cancer. In some embodiments, the cancers treated / prevented are primary cancers. In some embodiments, the cancers treated / prevented are secondary cancers (i.e., metastatic cancers).

[0210] VEGFA / VEGFR targeted interventions have also been studied to treat / prevent ocular diseases, as described, for example, in Cornel et al., Rom J Ophthalmol. (2015) 59(4):235-242, which is thus incorporated herein by reference in its entirety. In some embodiments, the diseases / conditions treated / prevented by this disclosure are selected from ocular diseases, retinopathy, diabetic retinopathy, macular degeneration, age-related macular degeneration, exudative (i.e., neovascular) age-related macular degeneration, retinal vein occlusion, myopic choroidal neovascularization, retinopathy of prematurity, neovascular glaucoma, central serous retinopathy, ocular tumors, and corneal neovascularization.

[0211] VEGFA / VEGFR-mediated signaling is also involved in the pathogenesis of inflammatory and autoimmune conditions, as described, for example, in Le and Kwon, Int J Mol Sci. (2021) 22(10):5387, Marina et al., Clujul Med. (2015) 88(3):247-252, Ferrara, Endocr Rev. (2004) 25(4):581-611, and Azimi et al., Neurol Sci. (2020) 41(6):1459-1465, all of which are thus incorporated herein by reference in their entirety. In some embodiments, the diseases / conditions treated / prevented by this disclosure are selected from inflammatory diseases, autoimmune diseases, arthritis, rheumatoid arthritis, osteoarthritis, psoriasis, multiple sclerosis, and sepsis.

[0212] VEGFA / VEGFR-mediated signaling is also involved in the pathogenesis of motor neuron diseases, such as amyotrophic lateral sclerosis (ALS), as described, for example, in Lambrechts et al., Nat Genet. (2003) 34(4):383-394. In some embodiments, the disease / condition to be treated / prevented by this disclosure is motor neuron disease or amyotrophic lateral sclerosis.

[0213] Various aspects of this disclosure provide methods aimed at, or including (for example, in the context of therapeutic / preventive interventions described herein), inhibiting the interaction between VEGFA and VEGFR (i.e., a receptor for VEGFA, e.g., VEGFR1) and / or inhibiting VEGFA / VEGFR-mediated signaling.

[0214] Furthermore, the use of the agents according to the Disclosure in the manufacture of agents according to the Disclosure for use in such a manner, and in the manufacture of compositions (e.g., pharmaceuticals) for use in such a manner is also provided. In some embodiments, the therapeutic / preventive intervention according to the Disclosure can be described as "related" to one or more of the effects described in the preceding paragraph. Those skilled in the art can readily assess such properties using techniques routinely practiced in the art.

[0215] The administration of the articles of this disclosure is preferably a “therapeutic effective” dose or a “preventive effective” dose, which is sufficient to demonstrate a therapeutic or preventive benefit to the subject. The actual dose, rate of administration, and duration of administration will depend on the nature and severity of the disease / condition, as well as the specific article being administered. The determination of the prescription of treatment, e.g., the dose, is the responsibility of the general practitioner and other physicians, and typically takes into account the disease / disorder to be treated, the condition of the individual subject, the site of delivery, the method of administration, and other factors known to the practitioner. Examples of the above techniques and protocols can be found in Remington's 'The Science and Practice of Pharmacy' (edited by A. Adejare), 23rd edition (2020), Academic Press.

[0216] The administration may be alone or, depending on the condition being treated, may be simultaneous or sequentially administered in combination with other therapies. The antigen-binding molecules or compositions and therapeutic agents described herein may be administered simultaneously or sequentially.

[0217] Co-administration means administering the antigen-binding molecules, CARs, nucleic acids, expression vectors, cells, or compositions of this disclosure together with other therapeutic agents, for example, as a pharmaceutical composition containing both agents (i.e., in the case of a combination formulation), or immediately after each other, via the same route of administration, for example, into the same artery, vein, or other blood vessel, at the discretion of the two. Sequential administration means administering one agent, followed by the other agent separately after a predetermined time interval. It is not necessary for the two agents to be administered via the same route, as is the case in some embodiments. The time interval may be any time interval.

[0218] Multiple doses of antigen-binding molecules, CARs, nucleic acids, expression vectors, cells, or compositions can be provided. Single or multiple doses, or each dose, may be accompanied by the simultaneous or sequential administration of another therapeutic agent. Multiple doses can be separated by predetermined time intervals, which can be selected to be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days, or 1, 2, 3, 4, 5, or 6 months. For example, doses may be given once every 7, 14, 21, or 28 days (plus or minus 3, 2, or 1 day).

[0219] Detection method The Disclosure also provides articles of the Disclosure for use in methods for detecting VEGFAs or for methods for detecting cells containing / expressing VEGFAs.

[0220] The antigen-binding molecules described herein can be used in a method that includes the step of detecting the binding of the antigen-binding molecule to VEGFA. Such a method may include the detection of a bound complex between the antigen-binding molecule and VEGFA.

[0221] In such cases, a method is provided comprising the steps of contacting a sample containing or suspected to contain VEGFA and detecting the formation of a complex between an antigen-binding molecule and VEGFA. A method is also provided comprising the steps of contacting a sample containing or suspected to contain cells containing / expressing VEGFA and detecting the formation of a complex between an antigen-binding molecule and cells containing / expressing VEGFA.

[0222] Appropriate methods are well known in the art and include immunoassays such as sandwich assays and ELISA. These methods may involve labeling an antigen-binding molecule, target(s), or both with a detectable portion, such as a fluorescent label, phosphorescent label, luminescent label, immunodetection label, radioactive label, chemical label, nucleic acid label, or enzymatic label as described herein. Detection techniques are well known to those skilled in the art and can be selected to correspond to the labeling agent.

[0223] Methods that include the step of detecting cells containing / expressing VEGFA, or VEGFA, include methods for diagnosing / prognosing diseases / conditions in which VEGFA expression / activity is pathologically involved.

[0224] This type of method can be performed in vitro on patient samples or after processing the patient samples. Once the sample is collected, the patient does not need to be present for the in vitro procedure to be performed, and therefore this method may not be performed on human or animal bodies. In some embodiments, this method is performed in vivo.

[0225] Such methods may include, for example, a step of detecting or quantifying one or more of VEGFAs or VEGFA-containing / expressing cells in a patient sample. If the method includes quantifying relevant factors, the method may further include a step of comparing the determined amount to a standard or reference value as part of a diagnosis or prognosis. Other diagnostic / prognostic tests can be used in combination with the tests described herein to improve the accuracy of the diagnosis or prognosis, or to confirm the results obtained by using the tests described herein.

[0226] Detection in a sample can be used for the purpose of diagnosing a disease / condition (e.g., cancer), predisposition to a disease / condition, or to provide a prognosis (prognosis prediction) for a disease / condition, such as those described herein. The diagnosis or prognosis may be related to an existing (previously diagnosed) disease / condition.

[0227] Samples can be taken from any tissue or bodily fluid. Samples may include, or be derived from: a volume of blood; a volume of serum derived from the blood of an individual, which may include the liquid portion of the blood obtained after removal of fibrin clots and blood cells; a tissue sample or biopsy; pleural fluid; cerebrospinal fluid (CSF); or cells isolated from the individual. In some embodiments, samples may be obtained from or derived from one or more tissues affected by the disease / condition (e.g., one or more tissues on which symptoms of the disease appear, or one or more tissues involved in the pathogenesis of the disease / condition).

[0228] This disclosure also provides a method for selecting / stratifying subjects for treatment with VEGFA-targeted drugs. In some embodiments, subjects are selected for treatment / prevention according to this disclosure, or identified as subjects who would benefit from such treatment / prevention, based on the detection / quantification of VEGFA or cells containing / expressing VEGFA, for example, in a sample obtained from an individual.

[0229] subject The subjects described in this disclosure may be any animal. In some embodiments, the subjects may be mammals. In some embodiments, the subjects may be humans. In some embodiments, the subjects may be non-human animals, such as non-human mammals. The subjects may be male or female.

[0230] The subjects may be patients. Patients may have the diseases / conditions described herein. The subjects may have been diagnosed with, be suspected of having, or be at risk of developing the diseases / conditions described herein.

[0231] Subjects / patients may be selected for treatment / prevention according to this disclosure based on the characterization of disease / condition markers described herein. In some embodiments of this disclosure, the subject is preferably a human subject. In some embodiments, the subject treated by the therapeutic or preventive method of this disclosure is a subject that has or is at risk of developing one of the diseases described herein.

[0232] kit In some embodiments of this disclosure, a kit of parts is provided. In some embodiments, the kit may have at least one container having a predetermined amount of an antigen-binding molecule, nucleic acid, expression vector, cell, or composition as described herein.

[0233] In some embodiments, the kit may include materials for producing antigen-binding molecules, nucleic acids, expression vectors, cells, or compositions described herein. The kit may provide antigen-binding molecules, nucleic acids, expression vectors, cells, or compositions, along with instructions for administering them to a patient, in order to treat a specific disease / condition.

[0234] The kits described herein may include, for example, instructions for use in the form of a manual or leaflet. The instructions for use may include protocols for carrying out any one or more of the methods described herein.

[0235] Sequence identity As used herein, “sequence identity” means the percentage of nucleotide / amino acid residues in a target sequence that are identical to nucleotide / amino acid residues in a reference sequence after the sequences have been aligned and gaps introduced where necessary to achieve the maximum sequence identity percentage between the sequences. Pairwise and multiple sequence alignments for the purpose of determining the sequence identity percentage between two or more amino acid sequences or nucleic acid sequences can be achieved in various ways known to those skilled in the art using publicly available computer software such as ClustalOmega (Soding, J., Bioinformatics (2005) 21, pp. 951-960), T-coffee (Notredame et al., J. Mol. Biol. (2000) pp. 302, pp. 205-217), Kalign (Lassmann and Sonnhammer, BMC Bioinformatics (2005) 6, p. 298), and MAFFT (Katoh and Standley, Molecular Biology and Evolution (2013) 30(4) pp. 772-780). When using such software, default parameters are preferably used for, for example, gap penalties and extension penalties.

[0236] array

[0237] [Table 2-1]

[0238] [Table 2-2]

[0239] [Table 2-3]

[0240] [Table 2-4]

[0241] This disclosure includes combinations of the described embodiments and preferred features, unless such combinations are clearly unacceptable or expressly avoided. The section headings used in this specification are for organizational purposes only and should not be construed as limiting the subjects described herein.

[0242] Aspects and embodiments of this disclosure are described herein by reference to the accompanying drawings. Further aspects and embodiments will be obvious to those skilled in the art. All documents referenced herein are incorporated herein by reference.

[0243] Throughout this Spec., including the subsequent claims, unless otherwise required by context, the word “comprise,” and variations such as “comprises” and “comprising,” mean to include the integer or step, or group of integers or steps, described herein, but not to exclude other integers or steps, or groups of integers or steps.

[0244] Note that, as used herein and in the appended claims, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include plural demonstratives. Herein, ranges may be expressed as from a certain “approximate” value to and / or another particular “approximate” value. Where such ranges are expressed, alternative embodiments include from a certain value to and / or other particular values. Similarly, where values ​​are expressed as approximations, the use of the antecedent “approximate” will be understood to mean that a particular value forms an alternative embodiment.

[0245] Where a nucleic acid sequence is disclosed herein, its reverse complement is also clearly intended. The methods described herein can preferably be carried out in vitro. The term “in vitro” is intended to encompass procedures carried out using cultured cells, while the term “in vivo” is intended to encompass procedures using or on intact multicellular organisms.

[0246] Embodiments and experiments illustrating the principles of this disclosure will be described here with reference to the attached drawings. [Brief explanation of the drawing]

[0247] [Figure 1] This table summarizes the amino acid differences in the CDR1, CDR2, and CDR3 regions of Library 1, Library 2, 4D5 (trastuzumab), and the initiation template. "Xaa" represents the randomized amino acid. [Figure 2A] This is a sensorgram showing the binding of (2A)13A6, (2B)16A2.1, (2C)16A6.1 and (2D)16A2.1x2, (2E)20A2.1, (2F)20A3.1, (2G)21A1.1, (2H)21A8.1, (2I)21D9.1, (2J)21E6.1 and (2K)23D5.1 to human VEGFA, as measured by biolayer interferometry (BLI). The concentrations tested for each DotBody are indicated in nM below the binding curve. [Figure 2B] This is a sensorgram showing the binding of (2A)13A6, (2B)16A2.1, (2C)16A6.1 and (2D)16A2.1x2, (2E)20A2.1, (2F)20A3.1, (2G)21A1.1, (2H)21A8.1, (2I)21D9.1, (2J)21E6.1 and (2K)23D5.1 to human VEGFA, as measured by biolayer interferometry (BLI). The concentrations tested for each DotBody are indicated in nM below the binding curve. [Figure 2C]This is a sensorgram showing the binding of (2A)13A6, (2B)16A2.1, (2C)16A6.1 and (2D)16A2.1x2, (2E)20A2.1, (2F)20A3.1, (2G)21A1.1, (2H)21A8.1, (2I)21D9.1, (2J)21E6.1 and (2K)23D5.1 to human VEGFA, as measured by biolayer interferometry (BLI). The concentrations tested for each DotBody are indicated in nM below the binding curve. [Figure 2D] This is a sensorgram showing the binding of (2A)13A6, (2B)16A2.1, (2C)16A6.1 and (2D)16A2.1x2, (2E)20A2.1, (2F)20A3.1, (2G)21A1.1, (2H)21A8.1, (2I)21D9.1, (2J)21E6.1 and (2K)23D5.1 to human VEGFA, as measured by biolayer interferometry (BLI). The concentrations tested for each DotBody are indicated in nM below the binding curve. [Figure 2E] This is a sensorgram showing the binding of (2A)13A6, (2B)16A2.1, (2C)16A6.1 and (2D)16A2.1x2, (2E)20A2.1, (2F)20A3.1, (2G)21A1.1, (2H)21A8.1, (2I)21D9.1, (2J)21E6.1 and (2K)23D5.1 to human VEGFA, as measured by biolayer interferometry (BLI). The concentrations tested for each DotBody are indicated in nM below the binding curve. [Figure 2F] This is a sensorgram showing the binding of (2A)13A6, (2B)16A2.1, (2C)16A6.1 and (2D)16A2.1x2, (2E)20A2.1, (2F)20A3.1, (2G)21A1.1, (2H)21A8.1, (2I)21D9.1, (2J)21E6.1 and (2K)23D5.1 to human VEGFA, as measured by biolayer interferometry (BLI). The concentrations tested for each DotBody are indicated in nM below the binding curve. [Figure 2G]This is a sensorgram showing the binding of (2A)13A6, (2B)16A2.1, (2C)16A6.1 and (2D)16A2.1x2, (2E)20A2.1, (2F)20A3.1, (2G)21A1.1, (2H)21A8.1, (2I)21D9.1, (2J)21E6.1 and (2K)23D5.1 to human VEGFA, as measured by biolayer interferometry (BLI). The concentrations tested for each DotBody are indicated in nM below the binding curve. [Figure 2H] This is a sensorgram showing the binding of (2A)13A6, (2B)16A2.1, (2C)16A6.1 and (2D)16A2.1x2, (2E)20A2.1, (2F)20A3.1, (2G)21A1.1, (2H)21A8.1, (2I)21D9.1, (2J)21E6.1 and (2K)23D5.1 to human VEGFA, as measured by biolayer interferometry (BLI). The concentrations tested for each DotBody are indicated in nM below the binding curve. [Figure 2I] This is a sensorgram showing the binding of (2A)13A6, (2B)16A2.1, (2C)16A6.1 and (2D)16A2.1x2, (2E)20A2.1, (2F)20A3.1, (2G)21A1.1, (2H)21A8.1, (2I)21D9.1, (2J)21E6.1 and (2K)23D5.1 to human VEGFA, as measured by biolayer interferometry (BLI). The concentrations tested for each DotBody are indicated in nM below the binding curve. [Figure 2J] This is a sensorgram showing the binding of (2A)13A6, (2B)16A2.1, (2C)16A6.1 and (2D)16A2.1x2, (2E)20A2.1, (2F)20A3.1, (2G)21A1.1, (2H)21A8.1, (2I)21D9.1, (2J)21E6.1 and (2K)23D5.1 to human VEGFA, as measured by biolayer interferometry (BLI). The concentrations tested for each DotBody are indicated in nM below the binding curve. [Figure 2K]This is a sensorgram showing the binding of (2A)13A6, (2B)16A2.1, (2C)16A6.1 and (2D)16A2.1x2, (2E)20A2.1, (2F)20A3.1, (2G)21A1.1, (2H)21A8.1, (2I)21D9.1, (2J)21E6.1 and (2K)23D5.1 to human VEGFA, as measured by biolayer interferometry (BLI). The concentrations tested for each DotBody are indicated in nM below the binding curve. [Figure 3A] These are sensorgrams showing the binding of (3A)13A6, (3B)16A2.1, (3C)16A6.1, (3D)20A2.1, (3E)20A3.1, (3F)21A1.1, (3G)21A8.1, (3H)21D9.1, (3I)21E6.1, and (3J)23D5.1 to mouse VEGFA, as measured by biolayer interferometry (BLI). The concentrations tested for each DotBody are indicated in nM below the binding curve. [Figure 3B] These are sensorgrams showing the binding of (3A)13A6, (3B)16A2.1, (3C)16A6.1, (3D)20A2.1, (3E)20A3.1, (3F)21A1.1, (3G)21A8.1, (3H)21D9.1, (3I)21E6.1, and (3J)23D5.1 to mouse VEGFA, as measured by biolayer interferometry (BLI). The concentrations tested for each DotBody are indicated in nM below the binding curve. [Figure 3C] These are sensorgrams showing the binding of (3A)13A6, (3B)16A2.1, (3C)16A6.1, (3D)20A2.1, (3E)20A3.1, (3F)21A1.1, (3G)21A8.1, (3H)21D9.1, (3I)21E6.1, and (3J)23D5.1 to mouse VEGFA, as measured by biolayer interferometry (BLI). The concentrations tested for each DotBody are indicated in nM below the binding curve. [Figure 3D]These are sensorgrams showing the binding of (3A)13A6, (3B)16A2.1, (3C)16A6.1, (3D)20A2.1, (3E)20A3.1, (3F)21A1.1, (3G)21A8.1, (3H)21D9.1, (3I)21E6.1, and (3J)23D5.1 to mouse VEGFA, as measured by biolayer interferometry (BLI). The concentrations tested for each DotBody are indicated in nM below the binding curve. [Figure 3E] These are sensorgrams showing the binding of (3A)13A6, (3B)16A2.1, (3C)16A6.1, (3D)20A2.1, (3E)20A3.1, (3F)21A1.1, (3G)21A8.1, (3H)21D9.1, (3I)21E6.1, and (3J)23D5.1 to mouse VEGFA, as measured by biolayer interferometry (BLI). The concentrations tested for each DotBody are indicated in nM below the binding curve. [Figure 3F] These are sensorgrams showing the binding of (3A)13A6, (3B)16A2.1, (3C)16A6.1, (3D)20A2.1, (3E)20A3.1, (3F)21A1.1, (3G)21A8.1, (3H)21D9.1, (3I)21E6.1, and (3J)23D5.1 to mouse VEGFA, as measured by biolayer interferometry (BLI). The concentrations tested for each DotBody are indicated in nM below the binding curve. [Figure 3G] These are sensorgrams showing the binding of (3A)13A6, (3B)16A2.1, (3C)16A6.1, (3D)20A2.1, (3E)20A3.1, (3F)21A1.1, (3G)21A8.1, (3H)21D9.1, (3I)21E6.1, and (3J)23D5.1 to mouse VEGFA, as measured by biolayer interferometry (BLI). The concentrations tested for each DotBody are indicated in nM below the binding curve. [Figure 3H]These are sensorgrams showing the binding of (3A)13A6, (3B)16A2.1, (3C)16A6.1, (3D)20A2.1, (3E)20A3.1, (3F)21A1.1, (3G)21A8.1, (3H)21D9.1, (3I)21E6.1, and (3J)23D5.1 to mouse VEGFA, as measured by biolayer interferometry (BLI). The concentrations tested for each DotBody are indicated in nM below the binding curve. [Figure 3I] These are sensorgrams showing the binding of (3A)13A6, (3B)16A2.1, (3C)16A6.1, (3D)20A2.1, (3E)20A3.1, (3F)21A1.1, (3G)21A8.1, (3H)21D9.1, (3I)21E6.1, and (3J)23D5.1 to mouse VEGFA, as measured by biolayer interferometry (BLI). The concentrations tested for each DotBody are indicated in nM below the binding curve. [Figure 3J] These are sensorgrams showing the binding of (3A)13A6, (3B)16A2.1, (3C)16A6.1, (3D)20A2.1, (3E)20A3.1, (3F)21A1.1, (3G)21A8.1, (3H)21D9.1, (3I)21E6.1, and (3J)23D5.1 to mouse VEGFA, as measured by biolayer interferometry (BLI). The concentrations tested for each DotBody are indicated in nM below the binding curve. [Figure 4] This graph shows the inhibition of the interaction between human VEGFA and VEGFR1 by 13A6, 16A2.1, 16A6.1, and ranibizumab in competitive ELISA. [Figure 5A] This graph shows the inhibition of the interaction between human VEGFA and VEGFR1 by (5A)16A2.1, (5B)20A2.1, (5C)20A3.1, (5D)21A1.1, (5E)21A8.1, (5F)21D9.1, (5G)21E6.1, (5H)23D5.1 and (5I) ranibizumab in competitive ELISA. [Figure 5B]This graph shows the inhibition of the interaction between human VEGFA and VEGFR1 by (5A)16A2.1, (5B)20A2.1, (5C)20A3.1, (5D)21A1.1, (5E)21A8.1, (5F)21D9.1, (5G)21E6.1, (5H)23D5.1 and (5I) ranibizumab in competitive ELISA. [Figure 5C] This graph shows the inhibition of the interaction between human VEGFA and VEGFR1 by (5A)16A2.1, (5B)20A2.1, (5C)20A3.1, (5D)21A1.1, (5E)21A8.1, (5F)21D9.1, (5G)21E6.1, (5H)23D5.1 and (5I) ranibizumab in competitive ELISA. [Figure 5D] This graph shows the inhibition of the interaction between human VEGFA and VEGFR1 by (5A)16A2.1, (5B)20A2.1, (5C)20A3.1, (5D)21A1.1, (5E)21A8.1, (5F)21D9.1, (5G)21E6.1, (5H)23D5.1 and (5I) ranibizumab in competitive ELISA. [Figure 5E] This graph shows the inhibition of the interaction between human VEGFA and VEGFR1 by (5A)16A2.1, (5B)20A2.1, (5C)20A3.1, (5D)21A1.1, (5E)21A8.1, (5F)21D9.1, (5G)21E6.1, (5H)23D5.1 and (5I) ranibizumab in competitive ELISA. [Figure 5F] This graph shows the inhibition of the interaction between human VEGFA and VEGFR1 by (5A)16A2.1, (5B)20A2.1, (5C)20A3.1, (5D)21A1.1, (5E)21A8.1, (5F)21D9.1, (5G)21E6.1, (5H)23D5.1 and (5I) ranibizumab in competitive ELISA. [Figure 5G]This graph shows the inhibition of the interaction between human VEGFA and VEGFR1 by (5A)16A2.1, (5B)20A2.1, (5C)20A3.1, (5D)21A1.1, (5E)21A8.1, (5F)21D9.1, (5G)21E6.1, (5H)23D5.1 and (5I) ranibizumab in competitive ELISA. [Figure 5H] This graph shows the inhibition of the interaction between human VEGFA and VEGFR1 by (5A)16A2.1, (5B)20A2.1, (5C)20A3.1, (5D)21A1.1, (5E)21A8.1, (5F)21D9.1, (5G)21E6.1, (5H)23D5.1 and (5I) ranibizumab in competitive ELISA. [Figure 5I] This graph shows the inhibition of the interaction between human VEGFA and VEGFR1 by (5A)16A2.1, (5B)20A2.1, (5C)20A3.1, (5D)21A1.1, (5E)21A8.1, (5F)21D9.1, (5G)21E6.1, (5H)23D5.1 and (5I) ranibizumab in competitive ELISA. [Figure 6A] These are sensorgrams showing the binding of anti-VEGFA DotBody(6A)16A2.1, (6B)20A2.1, (6C)20A3.1, (6D)21A1.1, (6E)21A8.1, (6F)21D9.1, (6G)21E9.1, and (6H)23D5.1 to human VEGFA at a single concentration of 250 nM after incubation for 1 hour at room temperature, 60°C, 70°C, or 80°C. Measurements were performed by BLI as described in Example 10. [Figure 6B] These are sensorgrams showing the binding of anti-VEGFA DotBody(6A)16A2.1, (6B)20A2.1, (6C)20A3.1, (6D)21A1.1, (6E)21A8.1, (6F)21D9.1, (6G)21E9.1, and (6H)23D5.1 to human VEGFA at a single concentration of 250 nM after incubation for 1 hour at room temperature, 60°C, 70°C, or 80°C. Measurements were performed by BLI as described in Example 10. [Figure 6C]These are sensorgrams showing the binding of anti-VEGFA DotBody(6A)16A2.1, (6B)20A2.1, (6C)20A3.1, (6D)21A1.1, (6E)21A8.1, (6F)21D9.1, (6G)21E9.1, and (6H)23D5.1 to human VEGFA at a single concentration of 250 nM after incubation for 1 hour at room temperature, 60°C, 70°C, or 80°C. Measurements were performed by BLI as described in Example 10. [Figure 6D] These are sensorgrams showing the binding of anti-VEGFA DotBody(6A)16A2.1, (6B)20A2.1, (6C)20A3.1, (6D)21A1.1, (6E)21A8.1, (6F)21D9.1, (6G)21E9.1, and (6H)23D5.1 to human VEGFA at a single concentration of 250 nM after incubation for 1 hour at room temperature, 60°C, 70°C, or 80°C. Measurements were performed by BLI as described in Example 10. [Figure 6E] These are sensorgrams showing the binding of anti-VEGFA DotBody(6A)16A2.1, (6B)20A2.1, (6C)20A3.1, (6D)21A1.1, (6E)21A8.1, (6F)21D9.1, (6G)21E9.1, and (6H)23D5.1 to human VEGFA at a single concentration of 250 nM after incubation for 1 hour at room temperature, 60°C, 70°C, or 80°C. Measurements were performed by BLI as described in Example 10. [Figure 6F] These are sensorgrams showing the binding of anti-VEGFA DotBody(6A)16A2.1, (6B)20A2.1, (6C)20A3.1, (6D)21A1.1, (6E)21A8.1, (6F)21D9.1, (6G)21E9.1, and (6H)23D5.1 to human VEGFA at a single concentration of 250 nM after incubation for 1 hour at room temperature, 60°C, 70°C, or 80°C. Measurements were performed by BLI as described in Example 10. [Figure 6G]These are sensorgrams showing the binding of anti-VEGFA DotBody(6A)16A2.1, (6B)20A2.1, (6C)20A3.1, (6D)21A1.1, (6E)21A8.1, (6F)21D9.1, (6G)21E9.1, and (6H)23D5.1 to human VEGFA at a single concentration of 250 nM after incubation for 1 hour at room temperature, 60°C, 70°C, or 80°C. Measurements were performed by BLI as described in Example 10. [Figure 6H] These are sensorgrams showing the binding of anti-VEGFA DotBody(6A)16A2.1, (6B)20A2.1, (6C)20A3.1, (6D)21A1.1, (6E)21A8.1, (6F)21D9.1, (6G)21E9.1, and (6H)23D5.1 to human VEGFA at a single concentration of 250 nM after incubation for 1 hour at room temperature, 60°C, 70°C, or 80°C. Measurements were performed by BLI as described in Example 10. [Examples]

[0248] Example 1: Preparation of a naively synthesized DotBody phage display library Two DotBody phage display libraries were used to identify anti-VEGF DotBody. These libraries are based on a humanized, stabilized autonomous VH domain template derived from the trastuzumab VH domain (e.g., the “DotBody scaffold patent” described in WO2016 / 072938A1).

[0249] Library 1 is based on the following VH domain template sequence (mutation locations for library creation are underlined): SEVQLVESGGGLVQPGGSLRLSSAISGF SISSTS IDWVRQAPGKGLEWVARI SPSSGSTS YADSVKGRFTISADTSKNTVYLQMNSLRAEDTAVYYT GRSSSAM DYRGQGTLVTVSS Library 2 is based on the following VH domain template sequence (mutation locations for library creation are underlined): SEVQLVESGGGLVQPGGSLRLSCAISGF SISSTS IDWVRQAPGKGLEWVARI SPSSGSTS YADSVKGRFTISADTSKNTVYLQMNSLRAEDTAVYYC GRSSSAM DYRGQGTLVTVSS The VH domain templates CDR-1, CDR-2, and CDR-3 were randomized by Kunkel mutagenesis according to the procedure by Bostrom J. et al. (14, 15) and Tonikian R. et al. (16), using the design shown in Figure 1. The primers used for Library 1 are shown in Table 1, and the primers used for Library 2 are shown in Table 2.

[0250] Library 1 contains approximately 2.87x10 10 It contains clones, while Library 2 contains approximately 1.37x10 with all CDRs mutated. 10 Clones were included. The library was evaluated by serial dilution and colony counting after library transformation.

[0251] [Table 3]

[0252] [Table 4]

[0253] Example 2: Phage display selection from a naive synthetic DotBody phage display library Human VEGF-121 (Acro Biosystems) was immobilized overnight at 4°C in Maxisorp Immuno tubes (Thermo Scientific) at 20 μg in 1 mL of PBS for Round 1, and at 10 μg in 1 mL of PBS for subsequent rounds. The tubes were washed twice with PBS and blocked at room temperature (RT) for 1 hour with milk block buffer (MBB:PBST, i.e., 1% skim milk in PBS containing 0.05% Tween-20). Negative selection tubes were prepared in the same manner as described for VEGF-121, but using PBS instead of VEGF-121 protein. 500 μL of Library 1 and Library 2 (approximately 2 x 10⁶) were used. 13 The phages (pfu / mL) were precipitated in PEG / NaCl buffer (20% PEG8,000, 2.5M NaCl), resuspended in MBB, and transferred to negative selection tubes, where incubated at room temperature for 1 hour. The phages were transferred to VEGF-121 coated immunotubes and incubated at room temperature for 2 hours. The tubes were then washed three times with MBB, three times with PBST, and twice with PBS to remove unbound phages. Bound phages were eluted with 1 mg / mL trypsin in trypsin buffer (TBS + 2 mM CaCl2). The eluted phages were used to induce the logarithmic growth phase (OD). 600 Approximately 0.5) 5 mL of TG1 bacterial cell culture (in 2YT medium) was infected at 37°C for 30 minutes. From round 2 onward, 1.2 mL of infected TG1 cells were stored at -80°C with 20% glycerol for use in monoclonal screening (glycerol stock for monoclonal screening). The remaining infected TG1 cells were transferred to 50 mL of 2YT medium. The culture was then OD 600 After incubation at 37°C with shaking until the temperature reaches approximately 0.5, 1x10 10M13K07 helper phage at pfu / mL was used to infect cells at 37°C for 30 minutes. Infected TG1 cells were pelleted at 3,900 g for 20 minutes at 4°C, resuspended in 500 μL of 2YT broth, and plated onto 2 x 15 cm round 2YT agar plates supplemented with 100 μg / mL of carbenicillin and 50 μg / mL of kanamycin. After incubation overnight at 30°C, the bacterial community was resuspended in 25 mL of TBS. The generated phages were purified by precipitation with PEG / NaCl buffer. After repeating PEG / NaCl precipitation twice, the phages were resuspended in PBS + 10% glycerol. The purified phages were used in subsequent selection rounds.

[0254] Four rounds of selection were performed on VEGF-121. From the second selection round onward, the number of washes was increased as follows: Round 2: 4 times at MBB, 4 times at PBST, 2 times at PBS.

[0255] Round 3: 6 times at MBB, 6 times at PBST, 2 times at PBS. Round 4: 7 wins in MBB, 7 wins in PBST, 2 wins in PBS. From the second selection round onward, the final concentration is 1 × 10⁻¹⁰. 12 We used 1 mL of phage purified from the previous selection round at pfu / mL. The remaining panning procedure was the same.

[0256] Example 3: Identification of specific binders by monoclonal phage ELISA Monoclonal phage ELISA was used to identify specific binding DotBody cells selected from naive and affinity-mature libraries. Glycerol stocks for monoclonal screening were plated onto 2YT agar plates supplemented with 100 μg / mL carbenicillin and incubated overnight at 37°C. Individual colonies were grown in 1 mL of 2YT broth supplemented with 100 μg / mL carbenicillin for 2 hours, followed by 1 x 10⁶ cells. 10The cultures were infected with M13K07 helper phage at pfu / mL. 50 μg / mL of kanamycin was added to the cultures, and they were incubated overnight at 30°C.

[0257] Cells were pelleted by centrifugation at 1,100 g at 4°C for 10 minutes, and the supernatant was used for phage monoclonal ELISA. For phage monoclonal ELISA, VEGF-121 was immobilized at a concentration of 1 μg / mL in a Maxisorp 96-well plate (Thermo Scientific) overnight at 4°C, washed twice with PBS, and blocked with MBB at room temperature for 1 hour. 25 μL of phage culture supernatant was mixed with 25 μL of MBB and added to the plate, and incubated at room temperature for 2 hours. After washing the plate eight times with PBST, 50 μL of anti-M13 antibody HRP conjugate (GE Healthcare) was added in MBB at a dilution of 1:7,000 and incubated at room temperature for 1 hour. The plate was washed eight times with PBST and developed with 50 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate (GeneTex). After 5-15 minutes, the reaction was stopped by adding 50 μL of 2 M H2SO4, and the signal was measured by absorbance at 450 nm. Monoclonal clones with high signal intensity (absorbance greater than 1) were sequenced by Sanger sequencing, and the specific VH domain that binds to VEGF-121 was identified.

[0258] Example 4: Construction of an affinity-matured phage display library Anti-VEGF DotBody 13A6 was selected for affinity maturation because its binding affinity was less than 50n and it also blocked the interaction between VEGFA and VEGF receptor 1 (VEGFR1). The affinity-matured phage display library was prepared by Kunkel mutagenesis using the primers shown in Table 3, according to Bostrom J. et al. (14). The library was estimated to have a size of 1.1 x 10⁶ by serial dilution during library electroporation of TG1 cells, plating on 2YT agar supplemented with 100 μg / mL carbenicillin, and subsequent plasmid sequencing from 30 colonies.8 It contained a unique sequence.

[0259] [Table 5]

[0260] Example 5: Selection of 13A6-based affinity matured phage display Nutraavidin was immobilized overnight at 4°C in Maxisorp Immuno tubes (Thermo Scientific) at 10 μg in 1 mL of PBS. The tubes were washed twice with PBS and blocked in MBB at room temperature (RT) for 1 hour. Biotinylated VEGF-121 (Acro Biosystems) was added at different concentrations depending on the panning round (see Table 4). The proteins were incubated at room temperature for 1 hour, and unbound proteins were removed by washing twice with PBS. Negative selection tubes were prepared as described for biotinylated VEGF-121, but with PBS added instead of biotinylated VEGF-121.

[0261] The remaining selection procedure is similar to phage display selection from the naively synthesized DotBody phage display library, but with certain modifications, as summarized in Table 4.

[0262] [Table 6]

[0263] Affinity-based matured phage display selection based on 13A6 resulted in the generation of 16A2.1, 16C2.1, and 16A6.1. Example 6: Construction of an affinity-matured phage display library based on 16A2.1 Anti-VEGF DotBody 16A2.1 was selected for affinity maturation because its binding affinity was less than 5 nM and it also blocked the interaction between VEGFA and VEGF receptor 1 (VEGFR1). The affinity-matured phage display library was constructed by Kunkel mutagenesis using the primers shown in Table 5, following Bostrom J. et al. (14). Since CDR3 contained a potential N-glycosylation site (sequence "NST"), three primers were designed for this CDR. One of these was retained as the parent sequence, while the other two were modified as follows before randomized primer design was performed (modifications are underlined): 16A2.1(AST):SEVQLVESGGGLVQPGGSLRLSCAISGFALAETDIDWVRQAPGKGLEWVARIFSSGGNTDYADSVKGRFTISADTSKNTVYLQMNSLRAEDTAVYYCGRSDSIAYNVILP A STYTARDYRGQGTLVTVSS 16A2.1(NSA):SEVQLVESGGGLVQPGGSLRLSCAISGFALAETDIDWVRQAPGKGLEWVARIFSSGGNTDYADSVKGRFTISADTSKNTVYLQMNSLRAEDTAVYYCGRSDSIAYNVILPNS A YTARDYRGQGTLVTVSS The obtained library was estimated to have a mutation rate of 0.6 x 10⁶ by serial dilution during library electroporation of TG1 cells, plating onto 2YT agar supplemented with 100 μg / mL carbenicillin, and subsequent sequencing of plasmids from several colonies to determine the mutation rate. 8 It contained a unique sequence.

[0264] [Table 7]

[0265] Example 7: Construction of a 16C2.1-based affinity matured phage display library Anti-VEGF DotBody 16C2.1 was selected for affinity maturation because its binding affinity was less than 5 nM and it also blocked the interaction between VEGFA and VEGF receptor 1 (VEGFR1). The affinity-matured phage display library was prepared by Kunkel mutagenesis using the primers shown in Table 6, according to Bostrom J. et al. (14). The obtained library was estimated by serial dilution during library electroporation of TG1 cells, plating on 2YT agar supplemented with 100 μg / mL carbenicillin, and subsequent sequencing of plasmids from several colonies to determine the mutation rate, which was 1.1 x 10⁶. 8 It contained a unique sequence.

[0266] [Table 8]

[0267] Example 8: Selection of 16A2.1-based and 16C2.1-based affinity matured phage displays with thermal loading. 16A2.1-based and 16C2.1-based phage display libraries were panned separately using the same procedure.

[0268] Nutraavidin was immobilized overnight at 4°C in Maxisorp Immuno tubes (Thermo Scientific) at 10 μg in 1 mL of PBS. The tubes were washed twice with PBS and blocked in MBB at room temperature (RT) for 1 hour. Biotinylated VEGF-121 (Acro Biosystems) was added at different concentrations depending on the panning round (see Table 7). The proteins were incubated at room temperature for 1 hour, and unbound proteins were removed by washing twice with PBS. Negative selection tubes were prepared as described for biotinylated VEGF-121, but with PBS added instead of biotinylated VEGF-121.

[0269] The remaining selection procedure is similar to selecting a phage display from a naively synthesized DotBody phage display library, but with certain modifications, as summarized in Table 7.

[0270] [Table 9]

[0271] Affinity-mature phage display selection based on 16A2.1 with thermal loading resulted in the generation of 20A2.1 and 20A3.1. Affinity-mature phage display selection based on 16C2.1 with thermal loading resulted in 21A1.1, 21A8.1, 21D9.1, and 21E6.1.

[0272] Example 9: Selection of a 16C2.1-based affinity matured phage display without thermal load. The 16C2.1-based phage display library was also panned without thermal load as follows:

[0273] Nutraavidin was immobilized overnight at 4°C in Maxisorp Immuno tubes (Thermo Scientific) at 10 μg in 1 mL of PBS. The tubes were washed twice with PBS and blocked in MBB at room temperature (RT) for 1 hour. Biotinylated VEGF-165 (Acro Biosystems) was added at different concentrations depending on the panning round (see Table 8). The proteins were incubated at room temperature for 1 hour, and unbound proteins were removed by washing twice with PBS. Negative selection tubes were prepared as described for biotinylated VEGF-165, but with PBS added instead of biotinylated VEGF-165.

[0274] The remaining selection procedure is similar to phage display selection from the naively synthesized DotBody phage display library, but with certain modifications, as summarized in Table 8.

[0275] [Table 10]

[0276] 23D5.1 was generated by 16C2.1-based affinity-mature phage display selection without thermal load. Example 10: Characterization of protein synthesis and binding kinetics by biolayer interferometry VEGFA-binding clones were cloned into pET-based expression vectors containing a sequence encoding an ATG codon at 5' and a hexahistidine tag at 3' of the open reading frame. These were recombinantly generated in E. coli, purified by immobilized metal affinity chromatography, and subsequently desalted in PBS. The divalent molecule 16A2.1x2 was generated in a similar manner.

[0277] Binding characterization was performed using BLI (Satorius) at room temperature with a flow rate of 1000 rpm. Biotinylated human VEGF-165 (Acro Biosystem) was immobilized on streptavidin-coated tips at a concentration of 3 μg / mL for 60 seconds in BLI buffer (0.1% BSA and 0.01% Tween-20 in PBS). After a baseline of 30 seconds, anti-VEGFA DotBody was bound in BLI buffer for 60 seconds at eight different concentrations, including a blank reference, followed by dissociation in BLI buffer for 400 seconds. Binding kinetics were calculated using BLI analysis software with a global fit according to a 1:1 binding model, subtracting the background buffer signal using a 0 nM reference concentration. The divalent molecule 16A2.1x2 was also characterized as described here, but with a 600-second dissociation period. The same procedure was used to characterize the binding of all clones to mouse VEGFA, but the immobilized target was replaced with biotinylated mouse VEGF-164 (Acro Biosystem).

[0278] The sensorgrams are shown in Figures 2 and 3, and the combined data is shown in the table below:

[0279] [Table 11]

[0280] Example 11: Competitive ELISA To perform competitive ELISA, VEGFR1 was immobilized at a concentration of 2 μg / mL in a Maxisorp 96-well plate (Thermo Scientific) overnight at 4°C, washed three times with PBS, and blocked with ELISA Block Buffer (EBB: 0.2% BSA in PBST) for 1 hour at room temperature. Human VEGF-121 at a concentration of 0.5 nM was mixed with purified anti-VEGFA DotBody at different concentrations after 1:3 serial dilutions starting at 500 nM and ending at 0.008 nM, with a control of 0 nM. Ranibizumab was also mixed with human VEGF-121 using 1:3 serial dilutions starting at 30 nM and ending at 0.0005 nM, with a control of 0 nM. The samples were incubated at room temperature for 2 hours, and 50 μL was transferred to a plate coated with VEGFR1. After 2 hours of incubation, the plate was washed three times with PBST. 50 μL of streptavidin-HRP conjugate (Thermo Scientific) was added to EBB at a 1:5,000 dilution and incubated at room temperature for 1 hour. The plate was washed five times with PBST and developed with 50 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate (GeneTex). After 5–15 minutes, the reaction was stopped by adding 50 μL of 2 M H2SO4, and the signal was measured by absorbance at 450 nm.

[0281] The data was plotted using Graphpad Prism 9 software, and an IC was calculated using a nonlinear regression curve "[Inhibitor] vs. Response - Variable Gradient (4 Parameters)". 50 We made that decision.

[0282] The results are shown in Figures 4 and 5. Based on the data in Figure 4, the inhibition of the interaction between human VEGF-121 and human VEGFR1 was determined for various molecules. 50 The values ​​were as follows: 13A6 = 5.9 μM 16A2.1 = 12.2 nM 16A6.1 = 41.4 nM Ranibizumab = 6.8 nM.

[0283] Based on the data in Figure 5, the inhibition of the interaction between human VEGF-121 and human VEGFR1 was determined for various molecules. 50 The values ​​were as follows: 16A2.1 = 3.0 nM 20A2.1 = 1.6 nM 20A3.1 = 0.9 nM 21A1.1 = 0.9 nM 21A8.1 = 1.8 nM 21D9.1 = 1.0 nM 21E6.1 = 7.5nM 23D5.1 = 1.4nM Ranibizumab = 7.4 nM Example 12: Evaluation of thermal stability by binding analysis to VEGFA after thermal loading VEGFA-bound DotBody was incubated in BLI buffer at a concentration of 250 nM for 1 hour at room temperature, 60°C, 70°C, or 80°C (heat loading). After heat loading, the sample was centrifuged at 15,000 g for 5 minutes, and the supernatant was used to characterize the binding to human VEGF165 by BLI as described in Example 10 above.

[0284] The results are shown in Figure 6. Example 13: Conclusion The inventors have generated a stabilized VH domain antibody that specifically binds to human VEGFA and cross-reacts with mouse VEGFA.

[0285] Of the antigen-binding molecules generated, clone 13A6 has an affinity of 38.9 nM for human VEGFA and 248 nM for mouse VEGFA. When measured by competitive ELISA, clone 13A6 inhibits the interaction between VEGFA and VEGF receptor 1 (VEGFR1) and IC 50 The concentration was 5.9 μM.

[0286] To further improve binding affinity to human and mouse VEGFA, 13A6 was subjected to affinity maturation by phage display. A new phage display library was constructed in which approximately 50% of the residues present in the wild-type sequence of 13A6 and 50% of the other amino acids were mutated at the CDR positions. Affinity maturation selection for human VEGFA was performed by increasing the stringency level by decreasing phage concentration, antigen concentration, and binding time while increasing the number of washes. This procedure identified several DotBody molecules with improved affinity.

[0287] Of these, clones 16A2.1 and 16A6.1 had affinities of 2.5 nM and 14.7 nM, respectively, to human VEGFA. Their affinities to mouse VEGFA were 16.6 nM and 18.4 nM, respectively. Their ability to block the VEGFA-VEGFR1 interaction was significantly improved, and their IC50s were estimated to be 12.2 nM for 16A2.1 and 41.4 nM for 16A6.1.

[0288] To demonstrate the modularity of the DotBody generated for VEGFA, clone 16A2.1 was generated as a divalent molecule containing two copies of the 16A2.1 sequence linked by a flexible "(GGGGS)x6-GGGG" linker. This molecule could be recombinantly generated in E. coli, and its affinity for VEGFA was improved to 301 pM. Since VEGFA exists as a homodimer (11), this improvement in binding affinity suggests that the divalent 16A2.1x2 molecule may be simultaneously bound to both VEGF monomers.

[0289] DotBody 16A2.1 and 16C2.1 were selected for further activity improvement. Furthermore, 16A2.1 contained a potential glycosylation site ("NST") in CDR3, which needed to be removed while maintaining binding. Two new phage display libraries were constructed, and each CDR site was mutated in approximately 50% of the residues present in the 16A2.1 and 16C2.1 sequences, and 50% of the remaining amino acids. For 16A2.1, the "NST" sequence in CDR3 was replaced with either "AST" or "NSA" during affinity maturation library design, with the aim of removing either Asn(N) or Ser(S) / Thr(T) in the typical Nx(T / S)N-glycosylation motif (where "x" is any amino acid other than proline) (12,13). Selection based on binding and stability to human VEGFA was performed under thermal load by increasing temperature while decreasing antigen concentration and increasing the number of washes to identify the most stable and high-affinity anti-VEGF DotBody. Additional selection without thermal load was performed on a 16C2.1-based library. In the thermally loaded library, clones 20A2.1, 20A3.1, 21A1.1, 21A8.1, 21D9.1, and 21E9.1 were identified, while in the selection without thermal load, DotBody 23D5.1 was identified as the most enriched sequence. Importantly, DotBody 20A2.1 and 20A3.1 did not contain the potential N-glycosylation sites present in the parent clone 16A2.1.

[0290] All identified DotBody molecules retained binding to human VEGFA with affinity ranging from 2.8 nM to 20 nM and to mouse VEGFA with affinity ranging from 8.3 nM to 46.1 nM. Their ability to block VEGF-VEGFR interactions was measured by competitive ELISA, with IC50 values ​​ranging from 0.9 nM to 7.5 nM. When ranibizumab was used as a control, the measured IC50 was 7.4 nM. The parental DotBody 16A2.1 had an IC50 of 3 nM, while the newly identified DotBody molecules 20A2.1 and 20A3.1 showed improved blocking activity (IC50 of 1.6 nM and 0.9 nM, respectively). The following DotBody samples also showed high blocking activity: 21A1.1 (IC50=0.9nM), 21D9.1 (IC50=1.0nM), and 23D5.1 (IC50=1.4nM).

[0291] All DotBody molecules identified by thermal selection retained their binding to VEGFA after incubation at temperatures ranging from room temperature to 80°C. 23D5.1 was identified by selection without thermal selection, but lost most of its binding activity above 60°C.

[0292] In conclusion, through a series of adapted phage display library designs and selection strategies, we obtained a broad range of anti-VEGFDotBodies with medium to low nanomolar affinity that bind to both human and mouse VEGFA. These DotBodies can block VEGF-VEGFR interactions with IC50s in the low micromolar to sub-nanomolar range. The majority of the identified DotBodies, including five potent blockers 16A2.1, 20A2.1, 20A3.1, and 21D9.1, exhibit high thermal stability and retain binding to human VEGFA even after incubation at temperatures ranging from room temperature to 80°C. These DotBodies are modular and can be used to generate multivalent and / or multispecific molecules by linking multiple DotBodies in tandem (e.g., by introducing linker sequences between DotBodies).

[0293] References 1. Fogli, S., Del Re, M., Rofi, E., Posarelli, C., Figus, M., and Danesi, R. (2018) Clinical pharmacology of intravitreal anti-VEGF drugs. Eye (Lond) 32, 1010-1020 2. Osaadon, P., Fagan, X. J., Lifshitz, T., and Levy, J. (2014) A review of anti-VEGF agents for proliferative diabetic retinopathy. Eye (Lond) 28, 510-520 3. Meadows, K. L., and Hurwitz, H. I. (2012) Anti-VEGF therapies in the clinic. Cold Spring Harb Perspect Med 2 4. Williams, K. A., Brereton, H. M., Farrall, A., Standfield, S. D., Taylor, S. D., Kirk, L. A., and Coster, D. J. (2005) Topically applied antibody fragments penetrate into the back of the rabbit eye. Eye (Lond) 19, 910-913 5. Thiel, M. A., Coster, D. J., Standfield, S. D., Brereton, H. M., Mavrangelos, C., Zola, H., Taylor, S., Yusim, A., and Williams, K. A. (2002) Penetration of engineered antibody fragments into the eye. Clin Exp Immunol 128, 67-74 6. Kontermann, R. E. (2012) Dual targeting strategies with bispecific antibodies. mAbs 4, 182-197 7. Brinkmann, U., and Kontermann, R. E. (2017) The making of bispecific antibodies. mAbs 9, 182-212 8. Deshaies, R. J. (2020) Multispecific drugs herald a new era of biopharmaceutical innovation. Nature 580, 329-338 9. Cornvik, T., Dahlroth, S. L., Magnusdottir, A., Herman, M. D., Knaust, R., Ekberg, M., and Nordlund, P. (2005) Colony filtration blot: a new screening method for soluble protein expression in Escherichia coli. Nat Methods 2, 507-509 10. Asial, I., Cheng, Y. X., Engman, H., Dollhopf, M., Wu, B., Nordlund, P., and Cornvik, T. (2013) Engineering protein thermostability using a generic activity-independent biophysical screen inside the cell. Nature communications 4, 2901 11. Muller, Y. A., Li, B., Christinger, H. W., Wells, J. A., Cunningham, B. C., and de Vos, A. M. (1997) Vascular endothelial growth factor: crystal structure and functional mapping of the kinase domain receptor binding site. Proc Natl Acad Sci U S A 94, 7192-7197 12. Bause, E. (1983) Structural requirements of N-glycosylation of proteins. Studies with proline peptides as conformational probes. Biochem J 209, 331-336 13. Bause, E., and Hettkamp, H. (1979) Primary structural requirements for N-glycosylation of peptides in rat liver. FEBS Lett 108, 341-344 14. Bostrom, J., Lee, C. V., Haber, L., and Fuh, G. (2009) Improving antibody binding affinity and specificity for therapeutic development. Methods Mol Biol 525, 353-376, xiii 15. Bostrom, J., and Fuh, G. (2009) Design and construction of synthetic phage-displayed Fab libraries. Methods Mol Biol 562, 17-35 16. Tonikian, R., Zhang, Y., Boone, C., and Sidhu, S. S. (2007) Identifying specificity profiles for peptide recognition modules from phage-displayed peptide libraries. Nature protocols 2, 1368-1386

Claims

1. An antigen-binding molecule that binds to VEGFA, and the following CDR: (a) CDR1 having the amino acid sequence of SEQ ID NO: 17 CDR2 having the amino acid sequence of SEQ ID NO: 18 CDR3 having the amino acid sequence of SEQ ID NO: 19; or (b) CDR1 having the amino acid sequence of SEQ ID NO: 6 CDR2 having the amino acid sequence of SEQ ID NO: 7 CDR3 having the amino acid sequence of SEQ ID NO: 8; or (c) CDR1 having the amino acid sequence of SEQ ID NO: 13 CDR2 having the amino acid sequence of SEQ ID NO: 14 CDR3 having the amino acid sequence of SEQ ID NO: 15; or (d) CDR1 having the amino acid sequence of SEQ ID NO: 21 CDR2 having the amino acid sequence of SEQ ID NO: 22 CDR3 having the amino acid sequence of SEQ ID NO: 23; or (e) CDR1 having the amino acid sequence of SEQ ID NO: 25 CDR2 having the amino acid sequence of SEQ ID NO: 26 CDR3 having the amino acid sequence of SEQ ID NO: 27; or (f) CDR1 having the amino acid sequence of SEQ ID NO: 29 CDR2 having the amino acid sequence of SEQ ID NO: 30 CDR3 having the amino acid sequence of SEQ ID NO: 31; or (g) CDR1 having the amino acid sequence of SEQ ID NO: 33 CDR2 having the amino acid sequence of SEQ ID NO: 34 CDR3 having the amino acid sequence of SEQ ID NO: 35; or (h) CDR1 having the amino acid sequence of SEQ ID NO: 2 CDR2 having the amino acid sequence of SEQ ID NO: 3 CDR3 having the amino acid sequence of SEQ ID NO: 4 Includes a single-domain antibody sequence incorporating The single-domain antibody sequence is as follows: FR1 having the amino acid sequence of SEQ ID NO: 40 FR2 having the amino acid sequence of SEQ ID NO: 41 FR3 having the amino acid sequence of SEQ ID NO: 42 FR4 having the amino acid sequence of SEQ ID NO: 44 To incorporate The aforementioned antigen-binding molecule.

2. An antigen-binding molecule that binds to VEGFA, the following CDR: CDR1 having the amino acid sequence of SEQ ID NO: 2 CDR2 having the amino acid sequence of SEQ ID NO: 10 CDR3 having the amino acid sequence of SEQ ID NO: 11 Includes a single-domain antibody sequence incorporating The single-domain antibody sequence is as follows: FR1 having the amino acid sequence of SEQ ID NO: 40 FR2 having the amino acid sequence of SEQ ID NO: 41 FR3 having the amino acid sequence of SEQ ID NO: 43 FR4 having the amino acid sequence of SEQ ID NO: 44 To incorporate The aforementioned antigen-binding molecule.

3. An antigen-binding molecule according to claim 1 or claim 2, which inhibits the interaction between VEGFA and VEGFR.

4. The antigen-binding molecule according to any one of claims 1 to 3, which is a multispecific antigen-binding molecule and further comprises an antigen-binding domain specific to a target antigen other than VEGFA.

5. A chimeric antigen receptor (CAR) comprising the antigen-binding molecule described in any one of claims 1 to 4.

6. An antigen-binding molecule according to any one of claims 1 to 4, or a nucleic acid encoding a CAR according to claim 5.

7. An expression vector comprising the nucleic acid described in claim 6.

8. A cell comprising an antigen-binding molecule according to any one of claims 1 to 4, a CAR according to claim 5, a nucleic acid according to claim 6, or an expression vector according to claim 7.

9. A method for producing an antigen-binding molecule that binds to VEGFA, comprising the step of culturing the cells described in claim 8 under conditions suitable for the expression of the antigen-binding molecule or CAR by the cells.

10. A composition comprising an antigen-binding molecule according to any one of claims 1 to 4, a CAR according to claim 5, a nucleic acid according to claim 6, an expression vector according to claim 7, or a cell according to claim 8, and a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

11. A pharmaceutical composition for use in a method of medical treatment or prevention, comprising an antigen-binding molecule according to any one of claims 1 to 4, a CAR according to claim 5, a nucleic acid according to claim 6, an expression vector according to claim 7, a cell according to claim 8, or the composition according to claim 10.

12. A pharmaceutical composition for use in a method for treating or preventing a disease in which VEGFA / VEGFR-mediated signaling is pathologically involved, comprising an antigen-binding molecule according to any one of claims 1 to 4, a CAR according to claim 5, a nucleic acid according to claim 6, an expression vector according to claim 7, a cell according to claim 8, or the composition according to claim 10.

13. A pharmaceutical composition for use according to claim 12, wherein the disease is selected from a disease characterized by pathological angiogenesis, cancer, VEGFA-expressing cancer, VEGFR-expressing cancer, eye disease, retinopathy, diabetic retinopathy, macular degeneration, age-related macular degeneration, exudative age-related macular degeneration, retinal vein occlusion, myopic choroidal neovascularization, retinopathy of prematurity, neovascular glaucoma, central serous retinopathy, eye tumor, corneal neovascularization, inflammatory disease, autoimmune disease, arthritis, rheumatoid arthritis, osteoarthritis, psoriasis, multiple sclerosis, sepsis, motor neuron disease, and amyotrophic lateral sclerosis.

14. An in vitro complex comprising an antigen-binding molecule according to any one of claims 1 to 4, bound to VEGFA.

15. A method for detecting VEGFA in a sample, comprising the steps of: contacting a sample containing or suspected to contain VEGFA in vitro with an antigen-binding molecule described in any one of claims 1 to 4; and detecting the formation of a complex of the antigen-binding molecule and VEGFA.

16. A drug comprising an antigen-binding molecule according to any one of claims 1 to 4, for detecting, localizing, or imaging VEGFA, or cells containing or expressing VEGFA.

17. A method for selecting or stratifying subjects for treatment with a VEGFA-targeted drug, comprising the steps of: contacting a sample from the subjects in vitro with an antigen-binding molecule described in any one of claims 1 to 4; and detecting the formation of a complex of the antigen-binding molecule and VEGFA.

18. A drug comprising an antigen-binding molecule according to any one of claims 1 to 4, for detecting, localizing, or imaging a disease / condition characterized by the expression of VEGFA.

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