VEGFA-binding molecule

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 February 19, 2022, the contents and elements of which are incorporated herein by reference for all purposes.

[0002] The present disclosure relates to the field of molecular biology, more particularly to the field of antigen binding molecule technology. The present invention also relates to methods of medical treatment and prophylaxis. [Background technology]

[0003] Anti-VEGF therapy is used to treat various conditions, especially in the fields of oncology and ophthalmology (1-3). It is desirable to develop humanized and stabilized antibody domains against VEGF due to their small size and modularity. For ophthalmic applications, small size and high stability are desirable as they can enable localized delivery of drugs (4, 5). Modularity, i.e., the ability of domain antibodies to fold autonomously and be fused to other domain antibodies or other proteins without compromising their integrity, is highly desirable for the development of multivalent and multispecific molecules. Indeed, fusing antibody domains in tandem to monoclonal antibodies or any other fusion protein simplifies the process of increasing valency and specificity (6-8). Summary of the Invention [Means for solving the problem]

[0004] In a first aspect, the present disclosure provides an antigen binding molecule that binds to VEGFA, optionally isolated, comprising the following CDRs: 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 The present invention provides an antigen-binding molecule comprising a single domain antibody sequence incorporating:

[0005] In some embodiments, the antigen binding molecule comprises or consists of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:49. In some embodiments, the antigen binding molecule has the following FR: 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:47 FR4 having the amino acid sequence of SEQ ID NO:44 The present invention includes a single domain antibody sequence incorporating the above.

[0006] In some embodiments, the antigen binding molecule has the following CDRs: 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 The present invention includes a single domain antibody sequence incorporating the above.

[0007] In some embodiments, the antigen binding molecule comprises or consists of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:53. In some embodiments, the antigen binding molecule comprises: (i) The following CDR: 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 single domain antibody sequence incorporating (ii) The following CDR: 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 single domain antibody sequence incorporating (iii) the following CDR: 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 sequences incorporating Includes.

[0008] In some embodiments, the antigen-binding molecule comprises or consists of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 16, 5 or 12. In some embodiments, the antigen binding molecule has the following CDRs: CDR1 having 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 The present invention includes a single domain antibody sequence incorporating the above.

[0009] In some embodiments, the antigen binding molecule comprises or consists of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:57. In some embodiments, the antigen binding molecule comprises: (i) The following CDR: 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 single domain antibody sequence incorporating (ii) The following CDR: 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 single domain antibody sequence incorporating (iii) the following CDR: 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 single domain antibody sequence incorporating (iv) the following CDR: 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 single domain antibody sequence incorporating (v) The following CDR: 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 sequences incorporating Includes.

[0010] In some embodiments, the antigen binding molecule comprises or consists of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 20, 24, 28, 32, or 36.

[0011] In some embodiments, the antigen binding molecule has 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 The present invention includes a single domain antibody sequence incorporating the above.

[0012] In some embodiments, the antigen binding molecule comprises or consists of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:1. In some embodiments, the antigen binding molecule has the following CDRs: 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 The present invention includes a single domain antibody sequence incorporating the above.

[0013] In some embodiments, the antigen binding molecule comprises or consists of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:9. In some embodiments, the antigen binding molecule has the following FR: 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 The present invention includes a single domain antibody sequence incorporating the above.

[0014] In some embodiments, the antigen binding molecule has the following FR: 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 The present invention includes a single domain antibody sequence incorporating the above.

[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 comprises an antigen binding domain specific for a target antigen other than VEGFA.

[0016] The present disclosure also provides a chimeric antigen receptor (CAR) comprising the antigen binding molecule described herein. The present disclosure also provides nucleic acids, optionally isolated, encoding the antigen-binding molecules or CARs described herein.

[0017] The disclosure also provides expression vectors comprising the nucleic acids described herein. The present disclosure also provides a cell comprising the antigen binding molecule, CAR, nucleic acid, or expression vector described herein.

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

[0019] The present disclosure also provides a composition comprising an antigen binding molecule, CAR, nucleic acid, expression vector, or cell described herein and a pharma- ceutically acceptable carrier, diluent, excipient, or adjuvant.

[0020] The present disclosure also provides an antigen binding molecule, a CAR, a nucleic acid, an expression vector, a cell or a composition described herein for use in a method of medical treatment or prevention. The present disclosure also provides an antigen binding molecule, CAR, nucleic acid, expression vector, cell or composition described herein for use in a method of treatment or prevention of a disease in which VEGFA / VEGFR-mediated signaling is pathologically involved.

[0021] The present disclosure also provides the use of an antigen binding molecule, CAR, nucleic acid, expression vector, cell or composition described herein in the manufacture of a medicament for treating or preventing a disease in which VEGFA / VEGFR-mediated signaling is pathologically implicated.

[0022] The present disclosure also provides a method for treating or preventing a disease in which VEGFA / VEGFR-mediated signaling is pathologically implicated, comprising administering to a subject a therapeutically or prophylactically effective amount of an antigen binding molecule, CAR, nucleic acid, expression vector, cell or composition described herein.

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

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

[0025] The present disclosure also provides the use of the antigen binding molecules described herein in methods for detecting, localizing or imaging VEGFA, or cells that contain or express VEGFA.

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

[0027] The present disclosure also provides the use of the antigen binding molecules described herein as diagnostic or prognostic agents in vitro or in vivo. The present disclosure also provides the use of the antigen binding molecules described herein in methods for detecting, localizing or imaging diseases / conditions characterized by expression of VEGFA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] We describe herein the generation of humanized, stabilized and autonomous VH domain antibodies that target human and mouse VEGFA with high affinity and can block VEGF-VEGFR interactions with high potency. These VEGF binding molecules can be used as building blocks to generate multivalent and multispecific molecules, as exemplified by bivalent anti-VEGFA molecules 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 the mRNA encoded by the human VEGFA gene results in 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 Claesson-Welsh and Welsh, J Intern Med. (2013) 273(2):114-27, both of which are incorporated by reference in their entireties.

[0031] Vascular endothelial growth factors (VEGFs) are a family of secreted polypeptides with a conserved receptor-binding cystine-knot structure. VEGFA monomers associate through interchain disulfide bonds to form homodimers. VEGFAs act through a family of cognate receptor kinases expressed by endothelial cells to stimulate angiogenesis.

[0032] VEGFA plays an important role not only in normal vascular development but also in diseases associated with abnormal vascular proliferation (e.g., cancer). VEGFA stimulates the proliferation of vascular endothelial cells derived from arteries, veins, and the lymphatic system, induces angiogenesis (i.e., the formation of thin-walled endothelium-lined structures) and induces a rapid increase in microvascular permeability in various in vivo models.

[0033] As used herein, "VEGFA" refers to VEGFA from any species, including isoforms, fragments, variants, or homologs from any species. In some embodiments, VEGFA is from a mammal (e.g., eutherian, placental, epithelial, monad, archontan, primate (rhesus, cynomolgus, non-human primate, or human)). In some embodiments, VEGFA is human VEGFA or mouse VEGFA.

[0034] As used herein, an isoform, fragment, variant, or homologue of a given reference protein can be characterized as having at least 70% sequence identity, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of the reference protein. A "fragment" generally refers to a portion of a reference protein. A "variant" generally refers to a protein having an amino acid sequence that contains one or more amino acid substitutions, insertions, deletions, or other modifications to the amino acid sequence of the reference protein, but retains a significant degree of sequence identity (e.g., at least 60%) to the amino acid sequence of the reference protein. An "isoform" generally refers to a variant of a reference protein that is expressed by the same species as the species of the reference protein. A "homolog" generally refers to a variant of a reference protein that is produced by a different species compared to the species of the reference protein. A homologue includes an ortholog.

[0035] VEGFA isoforms naturally include VEGF206 (UniProt P15692-1), VEGF189 (UniProt P15692-2), VEGF165 (UniProt P15692-4), and VEGF121 (UniProt P15692-9). VEGFA isoforms have also been identified as 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 P15692-16), and VEGFA isoform 20 (UniProt P15692-17). P15692-16), 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 to the amino acid sequence of an immature or mature VEGFA isoform from a given species, e.g., human.

[0037] In some embodiments, the VEGFA is human VEGFA. In some embodiments, the VEGFA is mouse VEGFA. The isoform, fragment, variant, or homologue may optionally be a functional isoform, fragment, variant, or homologue that has a functional property / activity of a reference VEGFA (e.g., human VEGF165), e.g., as determined by analysis of a suitable assay for functional property / activity. For example, an isoform, fragment, variant, or homologue of VEGFA may exhibit binding to a VEGF receptor (e.g., VEGFR1, VEGFR2, and / or VEGFR3).

[0038] In some embodiments, VEGFA comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 60, 61, 62 or 63.

[0039] In some embodiments, VEGFA or a fragment thereof comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 64, 65, 66 or 67.

[0040] VEGF exerts its biological effects through binding to the 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 for VEGF, a transmembrane sequence, and an intracellular tyrosine kinase portion. Binding of VEGF to the extracellular receptor domain results in receptor dimerization and phosphorylation of the intracellular tyrosine kinase portion. VEGFA has been shown to exert its biological effects primarily through VEGFR1 and VEGFR2.

[0041] As used herein, "VEGFR1", "VEGFR2" and "VEGFR3" refer to VEGFR1 / VEGFR2 / VEGFR3 from any species, including isoforms, fragments, variants or homologs from any species. In some embodiments, VEGFR1 / VEGFR2 / VEGFR3 is from a mammal (e.g., eutherian, placental, epitherian, monad, archaic, primate (rhesus, cynomolgus, non-human primate or human)). In some embodiments, VEGFR1 / VEGFR2 / VEGFR3 is human or mouse VEGFR1 / VEGFR2 / VEGFR3.

[0042] Isoforms, fragments, variants or homologs of VEGFR1 / VEGFR2 / VEGFR3 can optionally be characterized as having at least 70% amino acid sequence identity, 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 isoform of the relevant molecule from a given species, e.g., human.

[0043] As used herein, "VEGFA / VEGFR-mediated signaling" refers to signaling initiated by the binding of VEGFA to a VEGF receptor. "Signal transduction" refers to signal transduction 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 contents of which are incorporated herein by reference in their entirety. VEGFA / VEGFR-mediated signaling proceeds intracellularly through the PI3K / AKT, MAPK / ERK, and PLC-γ pathways, as well as through SCR and FAK, promoting cell survival, proliferation, and cytoskeletal reorganization, influencing changes in vascular permeability, vasodilation, and promoting angiogenesis.

[0045] antigen binding molecule The present disclosure provides an antigen binding molecule that can bind to VEGFA (i.e., binds to VEGFA). The present disclosure provides an antigen binding molecule that specifically binds to VEGFA. The antigen binding molecule described in the present disclosure can be provided in purified or isolated form, i.e., from other naturally occurring biological materials.

[0046] As used herein, "antigen-binding molecule" refers to a molecule that can bind to a target antigen. The term "antigen-binding molecule" includes monoclonal antibodies, polyclonal antibodies, monospecific antibodies 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), etc.) and aptamers.

[0047] More specifically, the antigen-binding molecule described in the present disclosure comprises an antigen-binding polypeptide portion, which may also be referred to as an "antigen-binding domain." In a preferred embodiment, the antigen-binding molecule described in the present disclosure comprises or consists of a single domain antibody that specifically binds to VEGFA.

[0048] Single domain antibodies (sdAbs), variously referred to in the art as "single variable domains on heavy chain antibodies", "VHHs", "nanobodies", and "heavy chain-only antibodies (HcAbs)", and sometimes referred to herein as "DotBody", 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 by reference in their entireties.

[0049] Single domain antibodies are formed from a single monomeric antibody variable domain. The first single domain antibodies were made from heavy chain antibodies found in camelids, although cartilaginous fish also have heavy chain antibodies.

[0050] A single domain antibody according to the present disclosure generally comprises three complementarity determining regions CDR: CDR1, CDR2 and CDR3. Together the three CDRs define the paratope of the molecule, which is the part that binds to the target antigen.

[0051] Single domain antibodies further comprise framework regions (FR) on either side of each CDR, providing a scaffold for the CDRs. From N-terminus to C-terminus, a single domain antibody comprises the following structure: N-terminus-[FR1]-[CDR1]-[FR2]-[CDR2]-[FR3]-[CDR3]-[FR4]-C-terminus.

[0052] There are several different definitions of antibody CDRs and FRs, such as those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), Chothia et al., J. Mol. Biol. 196:901-917 (1987), and in VBASE2, Retter et al., Nucl. Acids Res. (2005) 33 (supra 1): D671-D674.

[0053] In some embodiments, the antigen binding molecule comprises the CDR of the VEGFA-binding single domain antibody described herein or comprises the CDR derived from the VEGFA-binding single domain antibody described herein. In some embodiments, the antigen binding molecule comprises the FR of the VEGFA-binding single domain antibody described herein or comprises the FR derived from the VEGFA-binding single domain antibody described herein. In some embodiments, the antigen binding molecule comprises the CDR and FR of the VEGFA-binding single domain antibody described herein or comprises the CDR and FR derived from the VEGFA-binding single domain antibody described herein. That is, in some embodiments, the antigen binding molecule comprises the amino acid sequence of the VEGFA-binding single domain antibody described herein or comprises the amino acid sequence derived from the VEGFA-binding single domain antibody described herein. In some embodiments, the CDRs and FRs of the antigen binding molecules referred to herein are defined according to the IMGT information system (the International IMGT (ImMunoGeneTics) information system (described in LeFranc et al., Nucleic Acids Res. (2015) 43 (Database issue):D413-22), which uses the IMGT V-DOMAIN numbering convention described in Lefranc et al., Dev. Comp. Immunol. (2003) 27:55-77), the Kabat system (described in 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 (described in 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., 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 of positions 1 to 31; CDR1 is formed by the amino acid sequence of positions 32 to 36; FR2 is formed by the amino acid sequence of positions 37 to 50; CDR2 is formed by the amino acid sequence of positions 51 to 67; FR3 is formed by the amino acid sequence of positions 68 to 99; CDR3 is formed by the amino acid sequence of positions 100 to 119; FR4 is formed by the amino acid sequence of 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, e.g., 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 CDRs, FRs and / or the complete amino acid sequence of a VEGFA-binding 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 comprises the CDRs, FRs and / or complete amino acid sequence of a VEGFA-binding single domain antibody having an amino acid sequence set forth 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 comprises the CDRs (i.e., CDR1, 2, and 3) of a VEGFA-binding single domain antibody having an amino acid sequence set forth 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 comprises the FRs (i.e., FR1, 2, 3, and 4) of a VEGFA-binding single domain antibody having an amino acid sequence set forth 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 comprises the CDRs (i.e., CDR1, 2 and 3) and FRs (i.e., FR1, 2, 3 and 4) of a VEGFA-binding single domain antibody having an amino acid sequence set forth 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 according to one of the following (1) to (13): (1) (ConAII) A single domain antibody sequence comprising the following CDRs: 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 thereof in which one or two or three amino acids in one or more of CDR1, CDR2, or CDR3 are replaced with another amino acid. (2) (derived from Con16A2.1) A single domain antibody sequence comprising the following CDRs: 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 thereof in which one or two or three amino acids in one or more of CDR1, CDR2, or CDR3 are replaced with another amino acid. (3) (Con21 / 23) A single domain antibody sequence comprising the following CDRs: CDR1 having 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 thereof in which one or two or three amino acids in one or more of CDR1, CDR2, or CDR3 are replaced with another amino acid. (4) (13A6) A single domain antibody sequence comprising 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 thereof in which one or two or three amino acids in one or more of CDR1, CDR2, or CDR3 are replaced with another amino acid. (5) (16A2.1) A single domain antibody sequence comprising 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 or a variant thereof in which one or two or three amino acids in one or more of CDR1, CDR2, or CDR3 are replaced with another amino acid. (6) (16A6.1) A single domain antibody sequence comprising the following CDRs: 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 thereof in which one or two or three amino acids in one or more of CDR1, CDR2, or CDR3 are replaced with another amino acid. (7) (20A2.1) A single domain antibody sequence comprising 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 thereof in which one or two or three amino acids in one or more of CDR1, CDR2, or CDR3 are replaced with another amino acid. (8) (20A3.1) A single domain antibody sequence comprising 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 or a variant thereof in which one or two or three amino acids in one or more of CDR1, CDR2, or CDR3 are replaced with another amino acid. (9) (21A1.1) A single domain antibody sequence comprising 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 thereof in which one or two or three amino acids in one or more of CDR1, CDR2, or CDR3 are replaced with another amino acid. (10) (21A8.1) A single domain antibody sequence comprising 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 thereof in which one or two or three amino acids in one or more of CDR1, CDR2, or CDR3 are replaced with another amino acid. (11) (21D9.1) A single domain antibody sequence comprising 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 thereof in which one or two or three amino acids in one or more of CDR1, CDR2, or CDR3 are replaced with another amino acid. (12) (21E6.1) A single domain antibody sequence comprising 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 thereof in which one or two or three amino acids in one or more of CDR1, CDR2, or CDR3 are replaced with another amino acid. (13) (23D5.1) A single domain antibody sequence comprising 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 thereof in which one or two or three amino acids in one or more of CDR1, CDR2, or CDR3 are replaced with another amino acid.

[0059] In some embodiments, the antigen-binding molecule comprises or consists of a single domain antibody sequence according to one of the following (14) to (16): (14) (ConAII) Single domain antibody sequence containing the following FR: 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:47 FR4 having the amino acid sequence of SEQ ID NO:44 or a variant thereof in which one or two or three amino acids in one or more of FR1, FR2, FR3, or FR4 are replaced with another amino acid. (15) (13A6, 16A2.1, 20A2.1, 20A3.1, 21A1.1, 21A8.1, 21D9.1, 21E6.1) Single domain antibody sequences comprising the following FRs: 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 or a variant thereof in which one or two or three amino acids in one or more of FR1, FR2, FR3, or FR4 are replaced with another amino acid. (16) (16A6.1) A single domain antibody sequence comprising the following FR: 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 or a variant thereof in which one or two or three amino acids in one or more of FR1, FR2, FR3, or FR4 are replaced with another amino acid.

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

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

[0062] In some embodiments, the antigen-binding molecule comprises or consists of a single domain antibody sequence according to one of the following (30) to (42): (30) (ConAII) A single domain antibody sequence comprising an amino acid sequence having at least 70% sequence identity, 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 to the amino acid sequence of SEQ ID NO: 49. (31) (derived from Con16A2.1) A single domain antibody sequence comprising an amino acid sequence having at least 70% sequence identity, 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 to the amino acid sequence of SEQ ID NO: 53. (32) (Con21 / 23) A single domain antibody sequence comprising an amino acid sequence having at least 70% sequence identity, 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 to the amino acid sequence of SEQ ID NO:57. (33)(13A6) A single domain antibody sequence comprising an amino acid sequence having at least 70% sequence identity, 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 to the amino acid sequence of SEQ ID NO:1. (34) (16A2.1) A single domain antibody sequence comprising an amino acid sequence having at least 70% sequence identity, 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 to the amino acid sequence of SEQ ID NO:5. (35) (16A6.1) A single domain antibody sequence comprising an amino acid sequence having at least 70% sequence identity, 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 to the amino acid sequence of SEQ ID NO:9. (36) (20A2.1) A single domain antibody sequence comprising an amino acid sequence having at least 70% sequence identity, 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 to the amino acid sequence of SEQ ID NO: 12. (37) (20A3.1) A single domain antibody sequence comprising an amino acid sequence having at least 70% sequence identity, 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 to the amino acid sequence of SEQ ID NO: 16. (38) (21A1.1) A single domain antibody sequence comprising an amino acid sequence having at least 70% sequence identity, 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 to the amino acid sequence of SEQ ID NO: 20. (39) (21A8.1) A single domain antibody sequence comprising an amino acid sequence having at least 70% sequence identity, 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 to the amino acid sequence of SEQ ID NO: 24. (40) (21D9.1) A single domain antibody sequence comprising an amino acid sequence having at least 70% sequence identity, 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 to the amino acid sequence of SEQ ID NO: 28. (41) (21E6.1) A single domain antibody sequence comprising an amino acid sequence having at least 70% sequence identity, 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 to the amino acid sequence of SEQ ID NO: 32. (42) (23D5.1) A single domain antibody sequence comprising an amino acid sequence having at least 70% sequence identity, 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 to the amino acid sequence of SEQ ID NO: 36.

[0063] In embodiments described herein where one or more amino acids are replaced with another amino acid, the replacement may be a conservative replacement, for example according to the following table: In some embodiments, amino acids in the same block in the middle column are replaced. In some embodiments, amino acids in the same row in the rightmost column are replaced:

[0064] [Table 1]

[0065] In some embodiments, the substitutions may be functionally conservative, i.e., in some embodiments, the substitutions may not affect (or may not substantially affect) one or more functional properties (e.g., target binding) of an antigen-binding molecule that contains the substitution, compared to a comparable unsubstituted molecule.

[0066] In some embodiments, the antigen-binding molecule of the present disclosure comprises one or more regions (e.g., CH1, CH2 and / or CH3) of an immunoglobulin heavy chain constant sequence. In some embodiments, the immunoglobulin heavy chain constant sequence is or is derived from a heavy chain constant sequence of an IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM, such as a 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 a heavy chain constant sequence of a human IgG1 allotype (e.g., G1m1, G1m2, G1m3 or G1m17).

[0067] In some embodiments, the antigen binding molecule comprises an amino acid sequence having at least 70% sequence identity, 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 to the amino acid sequence of SEQ ID NO:69.

[0068] In some embodiments, the antigen-binding molecule comprises a CH1 region comprising an amino acid sequence having at least 70% sequence identity, 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, to the amino acid sequence of SEQ ID NO: 70. In some embodiments, the antigen-binding molecule comprises a hinge region comprising an amino acid sequence having at least 70% sequence identity, 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, to the amino acid sequence of SEQ ID NO: 71. In some embodiments, the antigen-binding molecule comprises a CH2 region comprising an amino acid sequence having at least 70% sequence identity, 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, to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the antigen-binding molecule comprises a CH3 region comprising an amino acid sequence having at least 70% sequence identity, 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, to the amino acid sequence of SEQ ID NO: 73.

[0069] In some embodiments, the antigen-binding molecule of the present disclosure comprises an Fc region.The Fc region is composed of a CH2 region and a CH3 region from one polypeptide and a CH2 region and a CH3 region from another polypeptide.The CH2 region and the CH3 region from the two polypeptides are combined to form an Fc region.

[0070] The Fc region provides interaction with Fc receptors and other molecules of the immune system, resulting in functional effects. IgG Fc-mediated effector functions are reviewed, for example, in Jefferis et al., Immunol Rev 1998 163:59-76 (incorporated herein in its entirety), and are 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 the interaction between the Fc region and the Fc receptors expressed by immune cells, recruitment of complement pathway components via binding of the Fc region to complement protein C1q, and the resulting activation of the complement cascade. Fc-mediated functions include Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), formation of the membrane attack complex (MAC), cell degranulation, production of cytokines and / or chemokines, and antigen processing and presentation.

[0071] In some embodiments, the antigen binding molecules described herein comprise an Fc region capable of enhancing / inducing one or more of ADCC, ADCP, CDC against cells that contain / express VEGFA (e.g., cells that express VEGFA and / or complexes that contain VEGFA on the cell surface), and / or enhancing the formation of a MAC on a cell or cell degranulation.

[0072] Modifications to antibody Fc regions that affect Fc-mediated functions are known in the art, such as, for example, those described in Wang et al., Protein Cell (2018) 9(1):63-73, which is incorporated by reference in its 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] Multispecific antigen-binding molecules are also contemplated. "Multispecific" 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 different antigen-binding domains.

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

[0075] It is understood that the antigen-binding molecules (e.g., multispecific antigen-binding molecules) described in this disclosure may include antigen-binding molecules capable of binding to a target for which the antigen-binding molecule is specific. For example, an antigen-binding molecule that binds to VEGFA and an antigen other than VEGFA may include (i) an antigen-binding molecule that binds to VEGFA, and (ii) an antigen-binding molecule that binds to an antigen other than VEGFA. In some embodiments, a component antigen-binding molecule of a larger antigen-binding molecule (e.g., a multispecific antigen-binding molecule) may be referred to, for example, as an "antigen-binding domain" or "antigen-binding region" of the larger antigen-binding molecule.

[0076] It will also be understood that an antigen-binding molecule (e.g., a multispecific antigen-binding molecule) according to the present disclosure may comprise an antigen-binding polypeptide or antigen-binding polypeptide complex capable of binding to a target for 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] A cancer cell antigen is an antigen that is expressed or overexpressed by a cancer cell. A cancer cell antigen can be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof. Expression of a cancer cell antigen can be associated with cancer. A cancer cell antigen can be aberrantly expressed by a cancer cell (e.g., a cancer cell antigen can be expressed with abnormal localization) or can be expressed with abnormal structure by a cancer cell. A cancer cell antigen can be capable of eliciting an immune response. In some embodiments, an antigen is expressed on the cell surface of a cancer cell (i.e., a cancer cell antigen is a cancer cell surface antigen). In some embodiments, the portion of an antigen that is bound by an antigen binding molecule described herein is presented on the outer surface of a cancer cell (i.e., is extracellular). A cancer cell antigen can be a cancer-associated antigen. In some embodiments, a cancer cell antigen is an antigen whose expression is associated with the onset, progression, or severity of a symptom of cancer. A cancer-associated antigen can be associated with the cause or pathology of cancer, or can be aberrantly expressed as a result of cancer. In some embodiments, a cancer cell antigen is an antigen whose expression (e.g., at the RNA and / or protein level) is upregulated by cancer cells, for example, when compared to the expression level by comparable non-cancerous cells (e.g., non-cancerous cells derived from the same tissue / cell type). In some embodiments, a cancer-associated antigen may be preferentially expressed by cancerous cells and not expressed by comparable non-cancerous cells (e.g., non-cancerous cells derived from the same tissue / cell type). In some embodiments, a cancer-associated antigen may be the product of a mutated oncogene or a mutated tumor suppressor gene. In some embodiments, a cancer-associated antigen may be the product of an overexpressed cellular protein, a cancer antigen produced by an oncogenic virus, an oncofetal antigen, or a cell surface glycolipid or glycoprotein.

[0079] The immune cell surface molecule may be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof expressed at 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 the present disclosure is on the outer surface of the immune cell (i.e., extracellular). The immune cell surface molecule may be expressed on the cell surface of any immune cell. In some embodiments, the immune cell may be a cell of hematopoietic origin, such as a neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, or monocyte. The lymphocyte may be, for example, a T cell, a B cell, a natural killer (NK) cell, a NKT cell, or an innate lymphoid cell (ILC), or a precursor thereof (e.g., a thymocyte or a pre-B cell). 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 an antigen other than VEGFA. Valency refers to the number of binding sites within an antigen-binding molecule for a given antigenic determinant.

[0081] In some embodiments, the antigen binding molecules include single domain antibodies (e.g., those described herein) capable of binding to VEGFA, and antigen binding regions (e.g., polypeptides (e.g., single domain antibodies), Fvs, Fabs or antibodies) capable of binding to antigens other than VEGFA.

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

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

[0084] In some embodiments, the antigen binding molecule comprises 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 comprises a target antigen binding domain and a CD3 polypeptide (typically CD3ε) binding domain through which the BiTE recruits T cells. Binding of the BiTE to its target antigen and to the CD3 polypeptide expressed by the T cell activates the T cell and ultimately directs T cell effector activity against cells expressing the target antigen. Other types of immune cell engagers are known in the art, including 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, hi some embodiments, the immune cell engager is a T cell engager.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0103] The antigen-binding molecules and polypeptides of the present disclosure may additionally comprise additional amino acids or sequences of amino acids. For example, the antigen-binding molecules and polypeptides may comprise an amino acid sequence(s) for facilitating expression, folding, transport, processing, purification, or detection of the antigen-binding molecules / polypeptides. For example, the antigen-binding molecules / polypeptides may comprise sequences encoding His (e.g., 6XHis), Myc, GST, MBP, FLAG, HA, E, or biotin tags, optionally at the N-terminus or C-terminus of the antigen-binding molecules / polypeptides. In some embodiments, the antigen-binding molecules / polypeptides comprise detectable moieties, such as fluorescent labels, luminescent labels, immunodetectable labels, radioactive labels, chemical labels, nucleic acid labels, or enzyme labels.

[0104] The antigen-binding molecules and polypeptides of the present disclosure may additionally contain a signal peptide (also known as a leader sequence or signal sequence). A signal peptide usually consists of a sequence of 5 to 30 hydrophobic amino acids and forms an alpha helix. Secreted proteins and proteins expressed on the cell surface often contain a signal peptide.

[0105] The signal peptide may be present at the N-terminus of the antigen-binding molecule / polypeptide. The signal peptide can provide efficient transport and secretion of the antigen-binding molecule / polypeptide. The signal peptide is typically removed by cleavage and is therefore not included in the mature antigen-binding molecule / polypeptide secreted from cells expressing the antigen-binding molecule.

[0106] Signal peptides are known for 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, for example, SignalP (Petersen et al., 2011 Nature Methods 8:785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24:2172-2176).

[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 may be covalently or non-covalently labeled with the detectable moiety.

[0108] Fluorescent labels include, for example, 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. 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 Examples of suitable labels include radioisotopes such as 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 112, 113, 114, 115, 116, 117, 120, 121, 122, 123, 124, 125, 126, 127, 130, 131, 132, 133, 134, 135, 14

[0109] In some embodiments, the antigen-binding molecule of the present disclosure is conjugated to a chemical moiety. The chemical moiety can be a moiety for providing a therapeutic effect. Antibody-drug conjugates are reviewed, for example, in Parslow et al., Biomedicines. September 2016;4(3):14. In some embodiments, the chemical moiety can be a drug moiety (e.g., a cytotoxic agent) such that the antigen-binding molecule is cytotoxic to cells that contain / express VEGFA (e.g., cells that express VEGFA and / or complexes that contain VEGFA on the cell surface). In some embodiments, the drug moiety can 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, duocarmycin, D6.5, and PBD.

[0110] Functional properties of antigen-binding molecules The antigen-binding molecules described herein can be characterized by reference 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); Inhibits the interaction between VEGFA and VEGFR (i.e., the receptor for VEGFA, e.g., VEGFR1); Inhibits VEGFA / VEGFR-mediated signaling; maintains binding to VEGFA after heat treatment; Decrease the number / percentage of cells expressing VEGFA; Increases cell death in cells expressing VEGFA.

[0111] It will be understood that a given antigen-binding molecule may exhibit more than one 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 a suitable assay. The assay can be, for example, an in vitro assay, which can be a cell-free assay or a cell-based assay. Alternatively, the assay can be, for example, an in vivo assay, i.e., carried out in an animal other than a human. The assay can use a species labeled with a detectable entity to facilitate detection.

[0112] Analysis of the results of such assays may include determining the concentration at which 50% of the maximal level of the relevant activity is achieved. The concentration of the antigen-binding molecule at which 50% of the maximal level of the relevant activity is achieved may be referred to as the "half maximal effective concentration" of the antigen-binding molecule with respect to the relevant activity, which may also be referred to as the "EC 50 For example, the EC of a given antigen-binding molecule for binding to VEGFA may be 50 may be the concentration at which 50% of the maximal level of binding to the relevant species is achieved.

[0113] Depending on the characteristics, EC 50 is the "half maximal inhibitory concentration" or "IC 50 ", which is the concentration of an antigen-binding molecule at which 50% of the maximal inhibition level of a given property is observed. For example, the IC of a given antigen-binding molecule for inhibition of the interaction between VEGFA and VEGFR (e.g., VEGFR1) is 50 may be the concentration at which 50% of the maximal inhibition 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 antigen and can be distinguished from non-specific binding to non-target antigens.An antigen-binding molecule / domain that specifically binds to target molecule preferably binds to target with higher affinity and / or longer duration than it binds 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 analysis. Such analysis allows binding to the given molecule to be measured and quantified. In some embodiments, binding can be a response detected in a given assay.

[0116] In some embodiments, the degree of binding of the antigen-binding molecule to the non-target molecule is less than about 10% of the binding of the antibody to the target molecule, as measured, for example, by ELISA, SPR, biolayer interferometry, or by RIA. Alternatively, the binding specificity is such that the antigen-binding molecule is at least 0.1 orders of magnitude (i.e., 0.1×10) greater than the KD of the antigen-binding molecule to the non-target molecule. n , where n is an integer representing an order of magnitude), this may be reflected in terms of binding affinity, which binds with a large dissociation constant (KD), which may optionally be 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 by biolayer interferometry, for example, as described in Example 10 of the present disclosure. In some embodiments, the antigen binding molecules described herein bind to VEGFA. In some embodiments, the antigen binding molecules described herein bind to a polypeptide complex that includes VEGFA.

[0118] In some embodiments, the antigen binding molecules described herein bind with submicromolar affinity, i.e., K D <1x10-6 In some embodiments, the antigen binding molecules described herein bind to VEGFA with an affinity in the nanomolar range, i.e., K D =9.9×10 -7 ~1×10 -9 In some embodiments, the antigen binding molecules described herein bind to VEGFA with subnanomolar affinity, i.e., K D <1x10 -9 In some embodiments, the antigen binding molecules described herein bind to VEGFA with an affinity in the picomolar range, i.e., K D =9.9×10 -10 ~1×10 -12 In some embodiments, the antigen binding molecules described herein bind to VEGFA with sub-picomolar affinity, i.e., K D <1x10 -12 Binds to VEGFA at M.

[0119] In some embodiments, the antigen binding molecules described herein have a K 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 In some embodiments, the antigen binding molecule binds to VEGFA (e.g., human VEGF165) at a K D = binds to VEGFA (e.g., human VEGF165) with an affinity of ≦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.

[0120] In some embodiments, the antigen-binding molecules described herein have a K determined for ranibizumab (i.e., the molecule formed by binding of the polypeptides of SEQ ID NOs: 74 and 75) in the same assay. D Similar to K D (e.g., as determined by BLI, e.g., as described in Example 10 of the present disclosure) binds to VEGFA (e.g., human VEGF165). In the same assay, the K determined for ranibizumab was D 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, 0.95 times or more and 1.1 times or less. D (e.g., as determined by BLI, e.g., as described in Example 10 of the present disclosure) binds to VEGFA (e.g., human VEGF165).

[0121] In some embodiments, the antigen binding molecules described herein have a K determined for ranibizumab in the same assay. D Lower than D In some embodiments, the antigen-binding molecule binds to VEGFA (e.g., human VEGF165) with a K (e.g., as determined by BLI, e.g., as described in Example 10 of the present disclosure). In some embodiments, the antigen-binding molecule binds to VEGFA (e.g., human VEGF165) with a K determined for ranibizumab in the same assay. D ≦0.99×, ≦0.95×, ≦0.9×, ≦0.85×, ≦0.8×, ≦0.75×, ≦0.7×, ≦0.65×, ≦0.6×, ≦0.55×, or ≦0.5×. D (e.g., as determined by BLI, e.g., as described in Example 10 of the present disclosure) binds to VEGFA (e.g., human VEGF165).

[0122] In some embodiments, the antigen-binding molecules described herein have a K determined for bevacizumab (i.e., the molecule formed by binding of the polypeptides of SEQ ID NOs: 76 and 77) in the same assay. D Similar to K D (e.g., as determined by BLI, e.g., as described in Example 10 of the present disclosure) binds to VEGFA (e.g., human VEGF165). In the same assay, the K determined for bevacizumab was D 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, 0.95 times or more and 1.1 times or less. D (e.g., as determined by BLI, e.g., as described in Example 10 of the present disclosure) binds to VEGFA (e.g., human VEGF165).

[0123] In some embodiments, the antigen binding molecules described herein have a K determined for bevacizumab in the same assay. D Lower than D In some embodiments, the antigen-binding molecule binds to VEGFA (e.g., human VEGF165) with a K determined for bevacizumab in the same assay (e.g., as determined by BLI, e.g., as described in Example 10 of the present disclosure). D ≦0.99×, ≦0.95×, ≦0.9×, ≦0.85×, ≦0.8×, ≦0.75×, ≦0.7×, ≦0.65×, ≦0.6×, ≦0.55×, or ≦0.5×. D (e.g., as determined by BLI, e.g., as described in Example 10 of the present disclosure) binds to VEGFA (e.g., human VEGF165).

[0124] The antigen binding molecule of the present disclosure can bind to a specific region of interest of VEGFA. The antigen binding molecule described in the present disclosure can bind to the linear epitope of VEGFA, which is composed of a continuous sequence of amino acids (i.e., primary amino acid sequence). In some embodiments, the antigen binding molecule can bind to the conformational epitope of VEGFA, which is composed of a discontinuous sequence of amino acids of amino acid sequence.

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

[0126] In some embodiments, the antigen binding molecules described herein bind to the same region of VEGFA, or overlapping regions of VEGFA, or to a region of VEGFA bound by an antigen binding molecule comprising the CDRs, FRs and / or the complete amino acid sequence of a VEGFA-binding 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, an antigen binding molecule described in the present disclosure binds to the region of VEGFA where VEGFA binds to VEGFR (eg, VEGFR1 and / or VEGFR2).

[0128] In some embodiments, the antigen binding molecule binds to VEGFA in the region where 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 where VEGFR (e.g., VEGFR1) is bound, thereby inhibiting the interaction between VEGFR (e.g., VEGFR1) and VEGFA. In some embodiments, the antigen binding molecule displaces VEGFR (e.g., VEGFR1) from a complex comprising 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 in the presence of an antibody / fragment or after incubation of one or both of the interaction partners with the antibody / fragment. 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 / reduction 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). A suitable analysis can be performed in vitro, for example, using recombinant interaction partners or using cells that express the interaction partners. The cells that express the interaction partners may be endogenously expressed or may be expressed from a nucleic acid introduced into the cells. For the purposes of such assays, one or both of the interaction partners and / or antigen-binding molecules can be labeled or used in conjunction with a detectable entity for the purposes of detecting and / or measuring the level of interaction.

[0130] In some embodiments, an antigen binding molecule described in the present disclosure inhibits the interaction between VEGFA and VEGFR1 (e.g., human VEGF121 and human VEGFR1) with an IC of 10 μM or less, preferably one of: ≦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 (eg, as determined by competitive ELISA, such as the competitive ELISA described in Example 11 of the present disclosure).

[0131] In some embodiments, the antigen binding molecules described herein are capable of inhibiting the interaction between VEGFA and VEGFR1 (e.g., human VEGF121 and human VEGFR1) in the same assay as determined for ranibizumab (i.e., the molecule formed by the binding of the polypeptides of SEQ ID NOs: 74 and 75). 50 and similar ICs 50 (e.g., as determined by competitive ELISA, e.g., the competitive ELISA described in Example 11 of the present 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 as measured by an IC50 value for inhibition of such interaction by ranibizumab. 50 0.5 times or more and less than 2 times the value, for example, 0.55 times or more and less than 1.9 times, 0.6 times or more and less than 1.8 times, 0.65 times or more and less than 1.7 times, 0.7 times or more and less than 1.6 times, 0.75 times or more and less than 1.5 times, 0.8 times or more and less than 1.4 times, 0.85 times or more and less than 1.3 times, 0.9 times or more and less than 1.2 times, 0.95 times or more and less than 1.1 times 50 (eg, as determined by 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, with an IC for inhibition of such an interaction. 50 Lower IC 50 (e.g., as determined by competitive ELISA, e.g., the competitive ELISA described in Example 11 of the present 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 as measured by an IC50 value for inhibition of such interaction by ranibizumab. 50An IC of less than 1x the value, e.g., ≤0.99x, ≤0.95x, ≤0.9x, ≤0.85x, ≤0.8x, ≤0.75x, ≤0.7x, ≤0.65x, ≤0.6x, ≤0.55x, or ≤0.5x 50 (eg, as determined by competitive ELISA, such as the competitive ELISA described in Example 11 of this disclosure).

[0133] In some embodiments, the antigen binding molecules described herein are capable of inhibiting 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 the polypeptides of SEQ ID NOs: 76 and 77), as determined by IC for inhibition of such interaction. 50 and similar ICs 50 (e.g., as determined by competitive ELISA, e.g., the competitive ELISA described in Example 11 of the present 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 as measured by an IC50 value for inhibition of such interaction by bevacizumab. 50 0.5 times or more and less than 2 times the value, for example, 0.55 times or more and less than 1.9 times, 0.6 times or more and less than 1.8 times, 0.65 times or more and less than 1.7 times, 0.7 times or more and less than 1.6 times, 0.75 times or more and less than 1.5 times, 0.8 times or more and less than 1.4 times, 0.85 times or more and less than 1.3 times, 0.9 times or more and less than 1.2 times, 0.95 times or more and less than 1.1 times 50 (eg, as determined by 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 determined for bevacizumab, with an IC for inhibition of such an interaction. 50 Lower IC 50(e.g., as determined by competitive ELISA, e.g., the competitive ELISA described in Example 11 of the present 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 as measured by an IC50 value for inhibition of such interaction by bevacizumab. 50 An IC of less than 1x the value, e.g., ≤0.99x, ≤0.95x, ≤0.9x, ≤0.85x, ≤0.8x, ≤0.75x, ≤0.7x, ≤0.65x, ≤0.6x, ≤0.55x, or ≤0.5x 50 (eg, as determined by competitive ELISA, such as the competitive ELISA described in Example 11 of this disclosure).

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

[0136] Suitable assays for examining 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 contacting VEGFR-expressing cells with the antigen binding molecules described in the present disclosure in the presence of VEGFA. Assays for examining VEGFA / VEGFR-mediated signaling may include analyzing signaling through PI3K / AKT, MAPK / ERK and / or PLC-γ pathways, and / or through SCR and / or FAK.

[0137] In some embodiments, the antigen binding molecules of the disclosure are capable of inhibiting VEGFA / VEGFR-mediated signaling (e.g., signaling mediated by binding of human VEGF121 to human VEGFR1) by 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, ≦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 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 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 ranibizumab (i.e., the molecule formed by the binding of the polypeptides of SEQ ID NOs: 74 and 75), with an IC for inhibition of such an interaction. 50 and similar ICs 50 In some embodiments, the antigen binding molecule inhibits VEGFA / VEGFR-mediated signaling (e.g., signaling mediated by binding of human VEGF121 to human VEGFR1) in the same assay as the IC for inhibition of such signaling by ranibizumab. 50 0.5 times or more and 2 times or less of the value, 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, 0.95 times or more and 1.1 times or less 50 and inhibits.

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

[0140] 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 (i.e., the molecule formed by the binding of the polypeptides of SEQ ID NOs: 76 and 77), with an IC for inhibition of such an interaction. 50 and similar ICs 50 In some embodiments, the antigen binding molecule inhibits VEGFA / VEGFR-mediated signaling (e.g., signaling mediated by binding of human VEGF121 to human VEGFR1) in the same assay as the IC for inhibition of such signaling by bevacizumab. 50 0.5 times or more and 2 times or less of the value, 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, 0.95 times or more and 1.1 times or less 50 and inhibits.

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

[0142] In some embodiments, the antigen binding molecules described herein bind VEGFA (e.g., human VEGFA) with similar affinity before and after heat treatment. Heat treatment may include incubation in an appropriate 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 performed as described in Example 12 of the present disclosure.

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

[0144] As used herein, a binding affinity that is "similar" to a reference binding affinity means a binding affinity that is within 50%, e.g., one of 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% of the reference binding affinity determined in the same assay.

[0145] K for binding to VEGFA (e.g., human VEGFA) D may be similar before and after heat treatment. As used herein, a "similar" K D The value can be one of 0.5 times or more and 2 times or less of the reference value, e.g., 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 in the present disclosure may enhance (i.e., upregulate and potentiate) cell killing of cells that contain / express VEGFA. In some embodiments, the antigen binding molecules described herein are capable of reducing the number / proportion of cells that contain / express VEGFA. In some embodiments, the antigen binding molecules described herein are capable of depleting / enhancing the depletion of such cells.

[0147] In some embodiments, an antigen binding molecule described herein reduces the number / proportion of cells that contain / express VEGFA in a given assay to less than 1-fold the number / proportion of such cells observed in the absence of the antigen binding molecule or in the presence of an equal amount of a suitable control antigen binding molecule, 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.

[0148] The antigen binding molecules described herein may comprise one or more moieties for enhancing the reduction of the number / proportion of cells that contain / express 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 molecule described herein comprises an Fc region capable of enhancing / inducing one or more of ADCC, ADCP, CDC against cells containing / expressing VEGFA, and / or enhancing MAC formation or cell degranulation on cells containing / expressing VEGFA.

[0150] In some embodiments, the antigen binding molecules described in the present disclosure are capable of enhancing / inducing ADCC against cells that contain / express VEGFA. In some embodiments, the antigen-binding molecule described in the present disclosure comprises a drug moiety. The antigen-binding molecule may be conjugated to a drug moiety. Antibody-drug conjugates are, for example, reviewed in Parslow et al., Biomedicines. September 2016;4(3):14 (incorporated herein by reference above). In some embodiments, the drug moiety is or comprises a cytotoxic agent, such that the antigen-binding molecule is cytotoxic to cells that contain / express VEGFA. In some embodiments, the drug moiety is or comprises a chemotherapeutic agent.

[0151] In some embodiments, the antigen binding molecule described herein comprises an immune cell engaging portion. In some embodiments, the antigen binding molecule comprises 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 in the present disclosure are capable of enhancing / inducing T cell-mediated cytolytic activity against cells containing / expressing VEGFA. In some embodiments, an antigen binding molecule described herein reduces the number / proportion of cells that contain / express VEGFA in a given assay to less than 1-fold the number / proportion of such cells observed in the absence of the antigen binding molecule or in the presence of an equal amount of a suitable control antigen binding molecule, 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.

[0153] In some embodiments, the antigen binding molecules described in this disclosure increase the level of killing of cells containing / expressing VEGFA in a given assay by more than 1-fold, e.g., ≧1.5-fold, ≧2-fold, ≧3-fold, ≧4-fold, ≧5-fold, ≧6-fold, ≧7-fold, ≧8-fold, ≧9-fold, ≧10-fold, ≧15-fold, ≧20-fold, ≧30-fold, ≧40-fold, or ≧50-fold, over the level of killing of such cells observed in the absence of the antigen binding molecule or in the presence of an equivalent amount of a suitable control antigen binding molecule.

[0154] Chimeric antigen receptors (CARs) The present disclosure also provides a chimeric antigen receptor (CAR) comprising an antigen-binding polypeptide of the present disclosure.

[0155] CAR is a recombinant receptor that provides both antigen binding and T cell activation functions. The structure and operation of CAR is reviewed, for example, in Dotti et al., Immunol Rev (2014) 257(1), which is incorporated herein by reference in its entirety. CAR comprises an antigen binding region linked to a cell membrane anchor region and a signal transduction region. An optional hinge region can provide a gap between the antigen binding region and the cell membrane anchor region and can function as a flexible linker.

[0156] The CAR of the present disclosure comprises an antigen-binding region that comprises or consists of an antigen-binding molecule of the present disclosure, or that comprises or consists of a single domain antibody sequence described in the present disclosure, i.e., the antigen-binding molecule / single domain antibody sequence described in the present disclosure is comprised in or constitutes the antigen-binding region of the CAR.

[0157] A cell membrane anchor region is provided between the antigen binding region and the signaling region of the CAR to anchor the CAR to the cell membrane of a cell expressing the CAR, where the antigen binding region is in the extracellular space and the signaling region is intracellular. In some embodiments, the CAR comprises a cell membrane anchor region that comprises, consists of, or is derived from an amino acid sequence of one of the transmembrane domain amino acid sequences of CD3-zeta, CD4, CD8, or CD28. As used herein, a region "derived from" a reference amino acid sequence comprises an amino acid sequence that has at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference sequence.

[0158] The signaling region of the 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 CARs (Haynes et al., 2001 J Immunol 166(1):182-187). The signaling region of the CAR may contain a costimulatory sequence derived from the signaling region of a costimulatory molecule to promote 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, the CAR is engineered to provide costimulation of different intracellular signaling pathways. For example, signaling associated with CD28 costimulation preferentially activates the phosphatidylinositol 3-kinase (PI3K) pathway, whereas 4-1BB-mediated signaling is via TNF receptor-associated factor (TRAF) adaptor proteins. Thus, the signaling region of a CAR may include costimulatory sequences derived from the signaling regions of multiple costimulatory molecules. In some embodiments, the CAR of the present disclosure includes one or more costimulatory sequences that include, consist of, or are derived from the amino acid sequence of one or more of the intracellular domains of CD28, OX40, 4-1BB, ICOS, and CD27. The optional hinge region can provide 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 CAR of the present disclosure includes a hinge region that includes, consists of, or is derived from the amino acid sequence of the hinge region of IgG1.

[0159] Also provided are cells comprising the CAR described in the present disclosure.The CAR described in the present disclosure can be used to generate CAR-expressing immune cells, such as CAR-T cells or CAR-NK cells.The engineering of CAR into immune cells can be performed during in vitro culture.

[0160] The antigen-binding region of the CAR of the present disclosure can be provided in any suitable format, e.g., as an scFv, scFab, etc. Nucleic acids and vectors The present disclosure provides nucleic acids encoding the antigen-binding molecules and CARs described herein. In some embodiments, the nucleic acids comprise or consist of DNA and / or RNA.

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

[0162] The nucleotide sequence of the nucleic acid described in this disclosure can be included in a vector, for example, an expression vector. As used herein, a "vector" is a nucleic acid molecule used as a vehicle to introduce exogenous nucleic acid into a cell. The vector can be a vector for expressing a nucleic acid in a cell. Such a vector can include a promoter sequence operably linked to the nucleotide sequence that codes for the sequence to be expressed. The vector can also include a stop codon and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon known in the art can be used to express a peptide or polypeptide from the vector described in this disclosure.

[0163] The term "operably linked" may include the situation 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 expression of the nucleic acid sequence is under the influence or control of the regulatory sequence (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to a selected nucleic acid sequence if the regulatory sequence can affect the transcription of the nucleic acid sequence. The resulting transcript can then be translated into the desired peptide(s) / polypeptide(s).

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

[0165] In some embodiments, the vector may be a eukaryotic vector, e.g., a vector that contains elements necessary for expression of a protein from the vector in a eukaryotic cell, hi some embodiments, the vector may be a mammalian expression vector, e.g., containing a cytomegalovirus (CMV) or SV40 promoter driving protein expression.

[0166] Cells containing / expressing antigen-binding molecules / CARs The present disclosure also provides cells that contain or express the antigen binding molecules and CARs described in this disclosure. Also provided are cells that contain or express the nucleic acids or vectors described in this disclosure.

[0167] The cell may be a eukaryotic cell, for example a mammalian cell. The mammal may be a primate (rhesus monkey, cynomolgus monkey, non-human primate or human) or a non-human mammal (e.g. rabbit, guinea pig, rat, mouse or other rodent (including any animal of the order Rodentia), cat, dog, pig, sheep, goat, cow (including, for example, cattle such as dairy cows, or any animal of the order Bos), horse (including any animal of the family Equidae), donkey, and non-human primate).

[0168] In some embodiments, the cell is or is derived from a cell type that is commonly used for the expression of polypeptides for use in human therapy.Exemplary cells are described, for example, in Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100:3451-3461 (hereby incorporated by reference in its entirety), and include, for example, CHO cells, HEK293 cells, PER.C6 cells, NS0 cells, and BHK cells.

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

[0170] The present disclosure also provides a method for producing a cell that expresses / contains an antigen binding molecule / CAR described in the present disclosure, comprising introducing a nucleic acid or vector described in the present disclosure into the cell. In some embodiments, the method further comprises culturing the cell under conditions suitable for expression of the nucleic acid / vector by the cell. In some embodiments, the method is performed in vitro.

[0171] The present disclosure also provides cells obtained or obtainable by the methods described in the present disclosure. Production of antigen-binding molecules and polypeptides The antigen-binding molecules and polypeptides described in this disclosure can be prepared by methods for producing polypeptides known to those of skill 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 methods described in, 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 production of polypeptides 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 Nat Methods. (2008); 5(2): 135-146, both of which are incorporated herein by reference in their entirety.Recombinant production methods of antigen-binding molecules are also described in Frenzel et al., Front Immunol. (2013); 4: 217, and Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100: 3451-3461, both of which are incorporated herein by reference in their entirety.

[0174] For recombinant production as described in this disclosure, any cell suitable for expression of a polypeptide may be used. The cell may be prokaryotic or eukaryotic. In some embodiments, the cell is a prokaryotic cell, such as an archaeal or bacterial cell. In some embodiments, the bacterium may be a gram-negative bacterium, such as a bacterium of the Enterobacteriaceae family, e.g., Escherichia coli. In some embodiments, the cell is a eukaryotic cell, such as a yeast cell, a plant cell, an insect cell, or a mammalian cell, e.g., a cell as described herein above.

[0175] In some cases, the cells may not be prokaryotic, as some prokaryotic cells are not capable of 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 by using appropriate tags. Specific plasmids that facilitate the secretion of proteins into the medium are also available.

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

[0177] Production may include the culture or fermentation of eukaryotic cells modified to express the polypeptide(s) of interest. The culture or fermentation may be carried out in a bioreactor provided with an appropriate supply of nutrients, air / oxygen and / or growth factors. Secreted proteins may be recovered by fractionating the medium / fermentation broth from the cells, extracting the protein content and separating the individual proteins to isolate the secreted polypeptide(s). Culture, fermentation and separation techniques 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, supra, incorporated herein by reference).

[0178] A bioreactor contains one or more vessels in which cells can be cultured. Cultivation in a bioreactor can be continuous, with a continuous inflow of reactants into the reactor and a continuous outflow of cultured cells from the reactor. Alternatively, the cultivation can be done in batches. Bioreactors monitor and control environmental conditions such as pH, oxygen, flow rates in and out of the vessel, and agitation within the vessel to provide optimal conditions for the cells being cultured.

[0179] After culturing the cells expressing the antigen-binding molecule, the antigen-binding molecule can be isolated or purified (e.g., from the cell culture supernatant). Any suitable method can be used to isolate / purify the polypeptide of interest produced by expression from the cultured cells.

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

[0181] It may then be desired to isolate the polypeptide of interest from the supernatant or culture medium, which may contain other protein and non-protein components. A common approach to separate protein components from the supernatant or culture medium is by precipitation. Proteins of different solubilities are precipitated at different concentrations of a precipitant, such as ammonium sulfate. For example, at low concentrations of the precipitant, water-soluble proteins are extracted. Thus, by adding different and increasing concentrations of the precipitant, proteins of different solubilities can be differentiated. Dialysis can then be used to remove the 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, which can be used as alternatives to precipitation or can be performed following precipitation.

[0183] If the polypeptide of interest has been isolated from 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 a pharma- ceutically acceptable carrier, diluent, excipient or adjuvant.

[0186] The compositions of the present disclosure may be formulated with one or more pharma- ceutically 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), antioxidants, or the like. (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] The term "pharmacologically acceptable" as used herein refers to compounds, ingredients, materials, compositions, dosage forms, etc., that are suitable for use in contact with the tissues of a subject (e.g., a human subject) within the scope of sound medical judgment, without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, antioxidant, lubricant, binder, stabilizer, solubilizer, surfactant, masking agent, colorant, flavoring, or sweetener of the compositions described in this disclosure must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, binders, stabilizers, solubilizers, surfactants, masking agents, colorants, flavorings or sweetening agents can be found in standard pharmaceutical texts, such as Remington's 'The Science and Practice of Pharmacy' (ed. A. Adejare), 23rd Edition (2020), Academic Press.

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

[0189] Suitable formulations may include antigen-binding molecules in a sterile medium or isotonic medium.Medicines 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 catheter) into selected areas of the human or animal body.

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

[0191] For example, a further aspect of the disclosure relates to a method of formulating or producing a medicament or pharmaceutical composition for use in treating a disease / condition (e.g., a disease / condition described herein), comprising formulating the pharmaceutical composition or medicament by mixing an antigen binding molecule, CAR, nucleic acid, expression vector or cell described herein with a pharma- ceutically acceptable carrier, adjuvant, excipient or diluent.

[0192] Therapeutic and prophylactic uses The antigen binding molecules, CARs, nucleic acids, expression vectors, cells and compositions described herein find use in therapeutic and prophylactic methods.

[0193] The present disclosure provides an antigen-binding molecule, a CAR, a nucleic acid, an expression vector, a cell, or a composition described herein for use in a method of medical treatment or prevention. Also provided is the use of an antigen-binding molecule, a CAR, a nucleic acid, an expression vector, a cell, or a composition described herein in the manufacture of a medicament for treating or preventing a disease or condition. Also provided is a method of treating or preventing a disease or condition, comprising administering to a subject a therapeutically or prophylactically effective amount of an antigen-binding molecule, a CAR, a nucleic acid, an expression vector, a cell, or a composition described herein.

[0194] Therapeutic or preventive intervention according to the present disclosure may be effective in reducing the onset or progression of a disease / condition, alleviating symptoms of a disease / condition, or reducing the pathology of a disease / condition. Intervention may be effective in preventing the progression of a disease / condition, for example, preventing the worsening of a disease / condition, or slowing the onset rate of a disease / condition. In some embodiments, the method may lead to an improvement of a disease / condition, for example, reducing symptoms of a disease / condition, or reducing other correlates of the severity / activity of a disease / condition. In some embodiments, the method may prevent the onset of a disease / condition at a later stage (e.g., a more severe stage, or a chronic stage).

[0195] As used herein, the terms "develop," "developing," and "development," for example, of a disorder, refer to both the onset of the disease and the progression, worsening, or deterioration of the disease state / its correlates.

[0196] It will be appreciated that the articles of the present disclosure can be used to treat / prevent any disease / condition that may result in a therapeutic or prophylactic benefit from a reduction in VEGFA levels, VEGFA / VEGFR-mediated signaling, a reduction in the number of cells containing / expressing VEGFA, and / or a reduction in the 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 implicated, for example, a disease / condition in which an increase in the level of VEGFA / VEGFR-mediated signaling and / or an increase in the number of cells containing / expressing VEGFA / VEGFR positively correlates 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 an increase in the level of VEGFA / VEGFR-mediated signaling and / or an increase in the number of cells containing / expressing VEGFA / VEGFR is a risk factor for the onset, development or progression of the disease / condition.

[0197] Treatment and prevention according to the aspects and embodiments disclosed herein primarily relates to diseases / conditions characterized by VEGFA / VEGFR mediated signaling. In some embodiments, the disease / condition treated / prevented by the present disclosure is a disease / condition characterized by, for example, an increased expression level of VEGFA compared to the expression level of VEGFA in the absence of the disease / condition. In some embodiments, the disease / condition treated / prevented by the present disclosure is a disease / condition characterized by, for example, an increased number / proportion / activity of cells expressing VEGFR compared to the number / proportion / activity of cells expressing VEGFR in the absence of the disease / condition.

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

[0199] VEGFA / VEGFR-mediated signaling has been implicated in the pathogenesis of several diseases: VEGFA promotes angiogenesis, blood-retinal barrier breakdown, inflammation, and vision loss in individuals with ocular diseases such as diabetic retinopathy and exudative age-related macular degeneration.

[0200] VEGF and VEGF receptors are also 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 and confirmed the importance of VEGFA in this process.

[0201] In some embodiments, the disease / condition treated by the present invention is a disease characterized by pathological (i.e., excessive) angiogenesis, cancer, VEGFA-expressing cancer (i.e., cancer comprising cells that express VEGFA; e.g., cancer comprising cells that have elevated levels of VEGFA compared to expression levels by comparable non-cancerous cells), VEGFR-expressing cancer (i.e., cancer comprising cells that express VEGFR; e.g., cancer comprising cells that have elevated levels of VEGFA compared to expression levels by comparable non-cancerous cells), or a combination of these. cancers containing cells with elevated levels of EGFR), 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, ocular tumors, corneal neovascularization, inflammatory diseases, autoimmune diseases, arthritis, rheumatoid arthritis, osteoarthritis, psoriasis, multiple sclerosis, sepsis, motor neuron disease, and amyotrophic lateral sclerosis.

[0202] It is understood that the specific diseases listed in the previous paragraph are interrelated. For example, diseases characterized by pathological angiogenesis include cancer and eye disease. As used herein, "pathological angiogenesis" refers to angiogenesis (i.e., the growth of new blood vessels from existing vascular plexus) that contributes to the development and / or progression of disease.

[0203] In some embodiments, the disease / condition treated / prevented by the present disclosure is cancer. Cancer can be any unwanted cell proliferation (or any disease manifested by unwanted cell proliferation), neoplasm or tumor. Cancer can be benign or malignant, primary or secondary (metastatic). Neoplasm or tumor is the abnormal growth or proliferation of cells and can be present in any tissue. The cancer may be of tissue / cells derived from, for example, the adrenal gland, 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, tonsils, trachea, uterus, vulva, 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 cancer / tumor can originate from other non-nervous tissues, and examples include melanoma, mesothelioma, lymphoma, myeloma, leukemia, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), hepatocellular carcinoma, epidermoid carcinoma, prostate cancer, breast cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, thymic carcinoma, NSCLC, blood cancer, and sarcoma.

[0205] The treatment / prophylaxis may 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 prolonging the survival of a subject.

[0206] In some embodiments, the cancer to be treated / prevented comprises cells expressing VEGFA. In some embodiments, the cancer to be treated / prevented comprises cells expressing VEGFR. In some embodiments, the cancer to be treated / prevented is a VEGFA-positive cancer. In some embodiments, the cancer to be treated / prevented is a 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 expression levels of the relevant factors that are higher than the expression levels by comparable non-cancerous cells / non-tumor tissues.

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

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

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

[0210] VEGFA / VEGFR targeted interventions are also being investigated to treat / prevent ocular diseases, as described, for example, in Cornel et al., Rom J Ophthalmol. (2015) 59(4):235-242, which is hereby incorporated by reference in its entirety. In some embodiments, the disease / condition treated / prevented by the present disclosure is 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 has also been implicated in the pathology 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 hereby incorporated by reference in their entireties. In some embodiments, the disease / condition treated / prevented by the present disclosure is 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 pathology of motor neuron diseases, such as amyotrophic lateral sclerosis, as described, for example, in Lambrechts et al., Nat Genet. (2003) 34(4):383-94. In some embodiments, the disease / condition treated / prevented by the present disclosure is a motor neuron disease or amyotrophic lateral sclerosis.

[0213] According to various aspects of the present disclosure, methods are provided that aim at or include (e.g., in the context of therapeutic / prophylactic interventions as 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] Also provided are agents according to the present disclosure for use in such methods, and the use of agents according to the present disclosure in the manufacture of compositions (e.g., medicaments) for use in such methods. In some embodiments, therapeutic / prophylactic interventions according to the present disclosure can be described as "associated with" one or more of the effects described in the preceding paragraph. Those skilled in the art can easily assess such characteristics using techniques routinely practiced in the art.

[0215] Administration of the articles of the present disclosure is preferably a "therapeutically effective" or "prophylactically effective" amount, which is sufficient to show a therapeutic or prophylactic benefit to the subject. The actual amount administered, the rate of administration, and the duration of administration will depend on the nature and severity of the disease / condition, and the particular article administered. Prescription of treatment, e.g., the determination of dosage, etc., is within the responsibility of general practitioners 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' (A. Adejare, ed.), 23rd Edition (2020), Academic Press.

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

[0217] Concurrent administration refers to administering the antigen-binding molecule, CAR, nucleic acid, expression vector, cell or composition of the present disclosure and other therapeutic agents together, for example, as a pharmaceutical composition containing both agents (i.e., in the case of a combined preparation), or immediately after each other, optionally via the same administration route, for example, into the same artery, vein, or other blood vessel. Sequential administration refers to administering one agent and then separately administering the other agent after a predetermined time interval. Although in some embodiments, it is not required that the two agents be administered by the same route. The time interval may be any time interval.

[0218] Multiple administrations of antigen-binding molecules, CARs, nucleic acids, expression vectors, cells or compositions can be provided. One or more or each administration can be accompanied by simultaneous or sequential administration of another therapeutic agent. The multiple administrations can be separated by a predetermined time interval, which can be selected to be one of 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. By way of example, administration can be performed once every 7, 14, 21, or 28 days (plus or minus 3, 2, or 1 day).

[0219] Detection Method The present disclosure also provides articles of the present disclosure for use in methods for detecting VEGFA or for detecting cells that contain / express VEGFA.

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

[0221] In such a case, a method is provided that includes contacting a sample containing or suspected of containing VEGFA and detecting the formation of a complex between the antigen-binding molecule and VEGFA. Also provided is a method that includes contacting a sample containing or suspected of containing a cell containing / expressing VEGFA and detecting the formation of a complex between the antigen-binding molecule and the cell containing / expressing VEGFA.

[0222] Suitable method formats are well known in the art and include sandwich assays, immunoassays such as ELISA. The method may include labeling the antigen-binding molecule, the target(s), or both with a detectable moiety, such as a fluorescent label, a phosphorescent label, a luminescent label, an immunodetectable label, a radioactive label, a chemical label, a nucleic acid label, or an enzyme 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 which involve detecting VEGFA, or cells which contain / express VEGFA, include methods for diagnosing / prognosing diseases / conditions in which VEGFA expression / activity is pathologically implicated.

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

[0225] Such methods may include, for example, detecting or quantifying one or more of VEGFA, cells that contain / express VEGFA in patient samples.When the method includes quantifying a related factor, the method may further include comparing the determined amount with a standard or reference value as part of diagnosis or prognosis.Other diagnostic / prognostic tests may be used in combination with the tests described herein to improve the accuracy of diagnosis or prognosis or to confirm the results obtained by using the tests described herein.

[0226] Detection in a sample can be used for purposes of diagnosing a disease / condition (e.g., cancer), a predisposition to a disease / condition, or to provide a prognosis (prediction of prognosis) of a disease / condition, such as a disease / condition 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 body fluid. Samples can include or be derived from: a volume of blood; a volume of serum from an individual's blood, which can include the liquid part of blood obtained after removing fibrin clots and blood cells; a tissue sample or biopsy; pleural effusion; cerebrospinal fluid (CSF); or cells isolated from said individual. In some embodiments, samples can be obtained or derived from one or more tissues affected by a disease / condition (e.g., one or more tissues in which symptoms of the disease appear, or one or more tissues involved in the pathogenesis of the disease / condition).

[0228] The present disclosure also provides a method for selecting / stratifying a subject for treatment with a VEGFA targeting drug. In some embodiments, a subject is selected for treatment / prevention according to the present disclosure or identified as a subject that would benefit from such treatment / prevention based on, for example, detection / quantification of VEGFA or cells containing / expressing VEGFA in a sample obtained from an individual.

[0229] subject The subject according to the present disclosure may be any animal. In some embodiments, the subject may be a mammal. In some embodiments, the subject may be a human. In some embodiments, the subject may be a non-human animal, e.g., a non-human mammal. The subject may be male or female.

[0230] The subject may be a patient. The patient may have a disease / condition as described herein. The subject may have been diagnosed with a disease / condition as described herein, may be suspected of having a disease / condition as described herein, or may be at risk of developing a disease / condition as described herein.

[0231] Subjects / patients may be selected for treatment / prevention according to the present disclosure based on marker characterization of the disease / condition described herein. In some embodiments according to the present disclosure, the subject is preferably a human subject. In some embodiments, the subject treated by the therapeutic or prophylactic methods of the present disclosure has a disease or is at risk of developing a disease as described herein.

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

[0233] In some embodiments, the kits may include materials for producing the antigen-binding molecules, nucleic acids, expression vectors, cells, or compositions described herein. The kit can be provided with instructions for administering the antigen binding molecule, nucleic acid, expression vector, cell or composition to a patient to treat a particular disease / condition.

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

[0235] Sequence identity As used herein, "sequence identity" refers to the percentage of nucleotides / amino acid residues in a subject sequence that are identical to the nucleotides / amino acid residues in a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage sequence identity between the sequences. Pairwise and multiple sequence alignment for the purpose of determining the percent sequence identity between two or more amino acid or nucleic acid sequences can be accomplished in a variety of ways known to those skilled in the art using publicly available computer software, such as, for example, ClustalOmega (Soding, J., Bioinformatics (2005) 21, 951-960), T-coffee (Notredame et al., J. Mol. Biol. (2000) 302, 205-217), Kalign (Lassmann and Sonnhammer, BMC Bioinformatics (2005) 6, 298), and MAFFT (Katoh and Standley, Molecular Biology and Evolution (2013) 30(4) 772-780) software. When using such software, default parameters, for example, gap penalties and extension penalties, are preferably used.

[0236] array

[0237] [Table 2-1]

[0238] [Table 2-2]

[0239] [Table 2-3]

[0240] [Table 2-4]

[0241] The present disclosure includes combinations of the described embodiments and preferred features unless such combinations are expressly not permitted or explicitly avoided. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0242] Aspects and embodiments of the present disclosure will now be described by way of example with reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this specification are incorporated herein by reference.

[0243] Throughout this specification, including the claims which follow, unless the context otherwise requires, the word "comprise", and variations such as "comprises" and "comprising", will be understood to mean the inclusion of a stated integer or step, or group of integers or steps, but not the exclusion of other integers or steps, or groups of integers or steps.

[0244] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. As used herein, ranges may be expressed as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, it will be understood that by using the antecedent "about," the particular value forms another embodiment.

[0245] When a nucleic acid sequence is disclosed herein, the reverse complement is also expressly contemplated. The methods described herein may preferably be performed in vitro. The term "in vitro" is intended to encompass procedures performed with cells in culture, whereas the term "in vivo" is intended to encompass procedures with or on intact multicellular organisms.

[0246] BRIEF DESCRIPTION OF THE DRAWINGS Embodiments and experiments illustrating the principles of the present disclosure will now be described with reference to the accompanying drawings. [Brief description of the drawings]

[0247] [Figure 1] 1 is a table summarizing the amino acid differences in the CDR1, CDR2 and CDR3 regions of library 1 and library 2, 4D5 (Trastuzumab) and the starting template. "Xaa" represents a randomized amino acid. [Figure 2A] 1A-1C are sensorgrams 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 listed in nM below the binding curves. [Figure 2B] 1A-1C are sensorgrams 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 listed in nM below the binding curves. [Figure 2C]1A-1C are sensorgrams 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 listed in nM below the binding curves. [Figure 2D] 1A-1C are sensorgrams 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 listed in nM below the binding curves. [Figure 2E] 1A-1C are sensorgrams 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 listed in nM below the binding curves. [Figure 2F] 1A-1C are sensorgrams 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 listed in nM below the binding curves. [Figure 2G]1A-1C are sensorgrams 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 listed in nM below the binding curves. [Figure 2H] 1A-1C are sensorgrams 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 listed in nM below the binding curves. [Figure 2I] 1A-1C are sensorgrams 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 listed in nM below the binding curves. [Figure 2J] 1A-1C are sensorgrams 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 listed in nM below the binding curves. [Figure 2K]1A-1C are sensorgrams 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 listed in nM below the binding curves. [Figure 3A] 1A-1J are sensorgrams showing 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 listed in nM below the binding curves. [Figure 3B] 1A-1J are sensorgrams showing 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 listed in nM below the binding curves. [Figure 3C] 1A-1J are sensorgrams showing 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 listed in nM below the binding curves. [Figure 3D]1A-1J are sensorgrams showing 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 listed in nM below the binding curves. [Figure 3E] 1A-1J are sensorgrams showing 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 listed in nM below the binding curves. [Figure 3F] 1A-1J are sensorgrams showing 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 listed in nM below the binding curves. [Figure 3G] 1A-1J are sensorgrams showing 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 listed in nM below the binding curves. [Figure 3H]1A-1J are sensorgrams showing 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 listed in nM below the binding curves. [Figure 3I] 1A-1J are sensorgrams showing 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 listed in nM below the binding curves. [Figure 3J] 1A-1J are sensorgrams showing 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 listed in nM below the binding curves. [Figure 4] 1 is a graph showing inhibition of the interaction between human VEGFA and VEGFR1 by 13A6, 16A2.1, 16A6.1 and ranibizumab in a competitive ELISA. [Figure 5A] Graph showing 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 a competitive ELISA. [Figure 5B]Graph showing 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 a competitive ELISA. [Figure 5C] Graph showing 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 a competitive ELISA. [Figure 5D] Graph showing 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 a competitive ELISA. [Figure 5E] Graph showing 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 a competitive ELISA. [Figure 5F] Graph showing 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 a competitive ELISA. [Figure 5G]Graph showing 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 a competitive ELISA. [Figure 5H] Graph showing 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 a competitive ELISA. [Figure 5I] Graph showing 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 a competitive ELISA. [Figure 6A] 1A-1C are sensorgrams showing 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] 1A-1C are sensorgrams showing 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]1A-1C are sensorgrams showing 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] 1A-1C are sensorgrams showing 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] 1A-1C are sensorgrams showing 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] 1A-1C are sensorgrams showing 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]1A-1C are sensorgrams showing 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] 1A-1C are sensorgrams showing 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: Generation of a naive synthetic DotBody phage display library Two DotBody phage display libraries were used to identify anti-VEGF DotBody. These libraries were 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 was based on the following VH domain template sequence (positions mutated to create the library are underlined): SEVQLVESGGGLVQPGGSLRLSSAISGF SISSTS IDWVRQAPGKGLEWVARI SPSSGSTS YADSVKGRFTISADTSKNTVYLQMNSLRAEDTAVYYT GRSSSAM DYRGQGTLVTVSS Library 2 was based on the following VH domain template sequence (positions mutated to create the library are underlined): SEVQLVESGGGLVQPGGSLRLSCAISGF SISSTS IDWVRQAPGKGLEWVARI SPSSGSTS YADSVKGRFTISADTSKNTVYLQMNSLRAEDTAVYYC GRSSSAM DYRGQGTLVTVSS CDR-1, CDR-2, and CDR-3 of the VH domain template were randomized by Kunkel mutagenesis according to the procedures by Bostrom J. et al. (14, 15) and Tonikian R. et al. (16) with 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 Library 1 contained approximately 1.37x10 clones with all CDRs mutated, whereas library 2 contained approximately 1.37x10 clones with all CDRs mutated. 10 Clones were included. The library was assessed 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 in Maxisorp Immuno tubes (Thermo Scientific) at 20 μg in 1 mL PBS for round 1 and 10 μg in 1 mL PBS for subsequent rounds overnight at 4°C. Tubes were washed twice with PBS and blocked with milk block buffer (MBB:PBST, i.e., 1% skim milk in PBS containing 0.05% Tween-20) for 1 h at room temperature (RT). Negative selection tubes were prepared in the same way as described for VEGF-121, except that PBS was used instead of VEGF-121 protein. 500 μL of library 1 and library 2 (approximately 2x10 13 pfu / mL) were precipitated with PEG / NaCl buffer (20% PEG8,000, 2.5 M NaCl), resuspended in MBB, transferred to negative selection tubes, and incubated at room temperature for 1 h. Phages were transferred to VEGF-121-coated immunotubes and incubated at room temperature for 2 h. 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). Eluted phages were used to culture phages in logarithmic growth phase (OD 600 Approximately 0.5) of 5 mL of TG1 bacterial cell culture (in 2YT medium) was infected for 30 min at 37 °C. From round 2 onwards, 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. The culture was incubated at OD 600 Incubate at 37°C with shaking until the pH is approximately 0.5, then add 1x10 10pfu / mL of M13K07 helper phage was used for infection for 30 min at 37°C. Infected TG1 cells were pelleted at 3,900g for 20 min at 4°C, resuspended in 500 μL of 2YT broth, and plated on 2x15 cm round 2YT agar plates supplemented with 100 μg / mL carbenicillin and 50 μg / mL kanamycin. After overnight incubation at 30°C, the bacterial lawn was resuspended in 25 mL of TBS. The generated phages were purified by precipitation with PEG / NaCl buffer. After two rounds of PEG / NaCl precipitation, the phages were resuspended in PBS+10% glycerol. The purified phages were used for the subsequent rounds of selection.

[0254] Four rounds of selection were performed against VEGF-121. From the second selection round onwards, the number of washes was increased as follows: Round 2: 4 rounds with MBB, 4 rounds with PBST, and 2 rounds with PBS.

[0255] Round 3: 6 times with MBB, 6 times with PBST, 2 times with PBS. Round 4: 7 times with MBB, 7 times with PBST, 2 times with PBS. From the second selection round onwards, the final concentration was 1 × 10 12 One mL of purified phage from the previous selection round was used in pfu / mL. The remaining panning procedures were the same.

[0256] Example 3: Identification of unique binders by monoclonal phage ELISA A monoclonal phage ELISA was used to identify unique binding DotBody selected from the naive library as well as the affinity maturation library. 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 for 2 hours in 1 mL of 2YT broth supplemented with 100 μg / mL carbenicillin, and then cultured at 1x10 10pfu / mL of M13K07 helper phage was used to infect the cells. Cultures were further supplemented with 50 μg / mL of kanamycin and incubated at 30° C. overnight.

[0257] Cells were pelleted by centrifugation at 1,100 g for 10 min at 4°C, and the supernatant was used for phage monoclonal ELISA. For phage monoclonal ELISA, VEGF-121 was immobilized at 1 μg / mL on Maxisorp 96-well plates (Thermo Scientific) overnight at 4°C, washed twice with PBS, and blocked with MBB for 1 h at room temperature. 25 μL of phage culture supernatant was mixed with 25 μL of MBB, added to the plate, and incubated for 2 h at room temperature. After washing the plate eight times with PBST, 50 μL of anti-M13 antibody HRP conjugate (GE Healthcare) was added at a 1:7,000 dilution in MBB and incubated for 1 h at room temperature. 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 min, the reaction was stopped by adding 50 μL of 2M H2SO4 and the signal was measured at absorbance at 450 nm. Monoclonal clones with high signal intensity (absorbance greater than 1) were sequenced by Sanger sequencing to identify the unique VH domains that bind to VEGF-121.

[0258] Example 4: Construction of an affinity maturation 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). An affinity-matured phage display library was generated by Kunkel mutagenesis according to Bostrom J. et al. (14) using the primers shown in Table 3. The library contained 1.1x10 mAbs, as estimated by serial dilutions upon library electroporation into TG1 cells, plating on 2YT agar supplemented with 100 μg / mL carbenicillin, and subsequent sequencing of plasmids from 30 colonies.8 It contained unique sequences.

[0259] [Table 5]

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

[0261] The remaining selection procedure was similar to the phage display selection from the naive synthetic DotBody phage display library, with certain modifications as summarized in Table 4.

[0262] [Table 6]

[0263] 13A6-based affinity maturation phage display selection gave rise to 16A2.1, 16C2.1 and 16A6.1. Example 6: Construction of a 16A2.1-based affinity maturation phage display library 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 maturation phage display library was generated by Kunkel mutagenesis according to Bostrom J. et al. (14) using the primers shown in Table 5. As CDR3 contained a potential N-glycosylation site (sequence "NST"), three primers were designed for this CDR, one of which left the parent sequence intact and the other two were modified as follows before randomized primer design was performed (changes are underlined): 16A2.1(AST):SEVQLVESGGGLVQPGGSLRLSCAISGFALAETDIDWVRQAPGKGLEWVARIFSSGGNTDYADSVKGRFTISADTSKNTVYLQMNSLRAEDTAVYYCGRSDSIAYNVILP A STYTARDYRGQGTLVTVSS 16A2.1(NSA):SEVQLVESGGGLVQPGGSLRLSCAISGFALAETDIDWVRQAPGKGLEWVARIFSSGGNTDYADSVKGRFTISADTSKNTVYLQMNSLRAEDTAVYYCGRSDSIAYNVILPNS A YTARDYRGQGTLVTVSS The resulting library contained 0.6x10 mutations, as estimated by serial dilution upon library electroporation into 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 unique sequences.

[0264] [Table 7]

[0265] Example 7: Construction of a 16C2.1-based affinity maturation 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). An affinity-matured phage display library was generated by Kunkel mutagenesis according to Bostrom J. et al. (14) using the primers shown in Table 6. The resulting library contained 1.1x10 mAbs, as estimated by serial dilutions upon library electroporation into 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. 8 It contained unique sequences.

[0266] [Table 8]

[0267] Example 8: Selection of 16A2.1- and 16C2.1-based affinity maturation phage display with heat challenge The 16A2.1-based and 16C2.1-based phage display libraries were panned separately following the same procedure.

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

[0269] The remaining selection procedure was similar to the selection of phage display from the naive synthetic DotBody phage display library, with certain modifications as summarized in Table 7.

[0270] [Table 9]

[0271] 16A2.1-based affinity maturation phage display selection with heat challenge gave rise to 20A2.1 and 20A3.1. 16C2.1-based affinity maturation phage display selection with heat challenge gave rise to 21A1.1, 21A8.1, 21D9.1 and 21E6.1.

[0272] Example 9: 16C2.1-based affinity maturation phage display selection without heat stress The 16C2.1-based phage display library was also panned without heat challenge as follows.

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

[0274] The remaining selection procedure was similar to the phage display selection from the naive synthetic DotBody phage display library, with certain modifications as summarized in Table 8.

[0275] [Table 10]

[0276] 16C2.1-based affinity maturation phage display selection without heat challenge gave rise to 23D5.1. Example 10: Protein production and characterization of binding kinetics by biolayer interferometry VEGFA-binding clones were cloned into a pET-based expression vector with an ATG codon 5' to the open reading frame and a sequence encoding a hexahistidine tag 3' to the open reading frame. They were recombinantly produced in E. coli and purified by immobilized metal affinity chromatography, followed by desalting in PBS. The bivalent molecule 16A2.1x2 was also produced in a similar manner.

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

[0279] [Table 11]

[0280] Example 11: Competitive ELISA To perform the competitive ELISA, VEGFR1 was immobilized at 2 μg / mL on Maxisorp 96-well plates (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 h at room temperature. Human VEGF-121 at a concentration of 0.5 nM was mixed with different concentrations of purified anti-VEGFA DotBody after a 1:3 serial dilution starting at 500 nM and ending at 0.008 nM, with 0 nM as the control. Ranibizumab was also mixed with human VEGF-121 using a 1:3 serial dilution starting at 30 nM and ending at 0.0005 nM, with 0 nM as the control. Samples were incubated for 2 h at room temperature and 50 μL was transferred to the VEGFR1-coated plates. After 2 h of incubation, the plates were washed three times with PBST. 50 μL of streptavidin-HRP conjugate (Thermo Scientific) was added at a 1:5,000 dilution in EBB and incubated for 1 h at room temperature. Plates were washed 5 times with PBST and developed with 50 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate (GeneTex). After 5-15 min, the reaction was stopped by adding 50 μL of 2M H2SO4 and the signal was measured by absorbance at 450 nm.

[0281] Data were plotted using Graphpad Prism 9 software and ICs were calculated using '[inhibitor] vs. response - variable slope (4 parameters)' nonlinear regression curves. 50 It was decided.

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

[0283] Based on the data in Figure 5, the IC determined for various molecules for inhibition of the interaction between human VEGF-121 and human VEGFR1. 50 The values ​​were as follows: 16A2.1=3.0nM 20A2.1=1.6nM 20A3.1=0.9nM 21A1.1=0.9nM 21A8.1=1.8nM 21D9.1=1.0nM 21E6.1=7.5nM 23D5.1=1.4nM Ranibizumab = 7.4 nM Example 12: Assessment of thermal stability by binding analysis to VEGFA after heat challenge VEGFA-conjugated 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 challenge). After heat challenge, samples were centrifuged at 15,000 g for 5 minutes and the supernatant was used for characterization of binding to human VEGF165 by BLI as described in Example 10 above.

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

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

[0286] To further improve the binding affinity for human and mouse VEGFA, 13A6 was subjected to affinity maturation by phage display. A new phage display library was generated in which each CDR position was mutated with approximately 50% of the residues present in the wild-type sequence of 13A6 and 50% of the other amino acids. Affinity maturation selection against human VEGFA was performed at increasing levels of stringency by decreasing phage concentration, antigen concentration and binding time with increasing wash times. This procedure identified several DotBody with improved affinities.

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

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

[0289] DotBody 16A2.1 and 16C2.1 were selected for further activity improvement. In addition, 16A2.1 contained a potential glycosylation site ("NST") in CDR3 that needed to be removed while maintaining binding. Two new phage display libraries were generated, in which each CDR position was mutated at approximately 50% of the residues present in the 16A2.1 and 16C2.1 sequences, respectively, and 50% of the other 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 except proline) (12,13). Selections based on binding and stability to human VEGFA were performed with decreasing antigen concentration, increasing wash times, and heat challenge at increasing temperatures to identify the most stable and high affinity anti-VEGF DotBody. Additional selections without heat challenge were performed on the 16C2.1-based library. The heat challenged library identified clones 20A2.1, 20A3.1, 21A1.1, 21A8.1, 21D9.1, and 21E9.1, while selections without heat challenge identified DotBody 23D5.1 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 DotBodys retained binding to human VEGFA with affinities ranging from 2.8 nM to 20 nM and to mouse VEGFA with affinities ranging from 8.3 nM to 46.1 nM. Their ability to block VEGF-VEGFR interactions was measured by competitive ELISA with IC50s ranging from 0.9 nM to 7.5 nM. When ranibizumab was used as a control, the measured IC50 was 7.4 nM. The parent DotBody 16A2.1 had an IC50=3 nM, and the newly identified DotBody 20A2.1 and 20A3.1 showed improved blocking activity (IC50=1.6 nM and IC50=0.9 nM, respectively). The following DotBody also showed high blocking activity: 21A1.1 (IC50=0.9 nM), 21D9.1 (IC50=1.0 nM) and 23D5.1 (IC50=1.4 nM).

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

[0292] In conclusion, through a series of adapted phage display library design and selection strategies, we obtained a broad range of anti-VEGF DotBodies with medium to low nanomolar affinity that bind to both human and mouse VEGFA. These DotBodies are capable of blocking VEGF-VEGFR interactions with IC50s in the low micromolar to subnanomolar range. Most of the identified DotBodies, including the five potent blockers 16A2.1, 20A2.1, 20A3.1, and 21D9.1, have high thermostability and retain binding to human VEGFA 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, comprising the following CDRs: (a) CDR1 having the amino acid sequence of SEQ ID NO:50 CDR2 having the amino acid sequence of SEQ ID NO:51 A CDR3 having the amino acid sequence of SEQ ID NO:52; or (b) CDR1 having the amino acid sequence of SEQ ID NO:54 CDR2 having the amino acid sequence of SEQ ID NO:55 A CDR3 having the amino acid sequence of SEQ ID NO:56; or (c) CDR1 having the amino acid sequence of SEQ ID NO:37 CDR2 having the amino acid sequence of SEQ ID NO:38 A CDR3 having the amino acid sequence of SEQ ID NO: 39; or (d) CDR1 having the amino acid sequence of SEQ ID NO:2 CDR2 having the amino acid sequence of SEQ ID NO:3 A CDR3 having the amino acid sequence of SEQ ID NO:4; or (e) 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 The antigen-binding molecule comprising a single domain antibody sequence incorporating:

2. (a) 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 or a single domain antibody sequence incorporating (b) 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 or a single domain antibody sequence incorporating (c) 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 sequences incorporating The antigen-binding molecule of claim 1 .

3. (a) 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 single domain antibody sequence incorporating (b) 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 single domain antibody sequence incorporating (c) 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 single domain antibody sequence incorporating (d) 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 Single domain antibody sequences incorporating The antigen-binding molecule of claim 1 .

4. The antigen-binding molecule of any one of claims 1 to 3, comprising or consisting of an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 53, 16, 5, 12, 57, 20, 24, 28, 32, 36, 1, or 9.

5. The following FR: 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 The antigen-binding molecule of claim 1 , comprising a single domain antibody sequence incorporating:

6. The following FR: 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 The antigen-binding molecule of claim 1, comprising a single domain antibody sequence incorporating:

7. The antigen-binding molecule of claim 1 , which inhibits the interaction between VEGFA and VEGFR.

8. The antigen-binding molecule of claim 1 , which is a multispecific antigen-binding molecule and further comprises an antigen-binding domain specific for a target antigen other than VEGFA.

9. A chimeric antigen receptor (CAR) comprising an antigen-binding molecule according to any one of claims 1 to 8.

10. A nucleic acid encoding an antigen-binding molecule according to any one of claims 1 to 8, or a CAR according to claim 9.

11. An expression vector comprising the nucleic acid of claim 10.

12. A cell comprising the antigen-binding molecule of any one of claims 1 to 8, the CAR of claim 9, the nucleic acid of claim 10, or the expression vector of claim 11.

13. A method for producing an antigen-binding molecule that binds to VEGFA, comprising culturing the cell of claim 12 under conditions suitable for expression of the antigen-binding molecule or CAR by the cell.

14. A composition comprising the antigen-binding molecule of any one of claims 1 to 8, the CAR of claim 9, the nucleic acid of claim 10, the expression vector of claim 11, or the cell of claim 12, and a pharma- ceutically acceptable carrier, diluent, excipient or adjuvant.

15. 15. 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 8, a CAR according to claim 9, a nucleic acid according to claim 10, an expression vector according to claim 11, a cell according to claim 12, or a composition according to claim 14.

16. 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 8, a CAR according to claim 9, a nucleic acid according to claim 10, an expression vector according to claim 11, a cell according to claim 12, or a composition according to claim 14.

17. 17. The pharmaceutical composition for use according to claim 16, wherein the disease is selected from diseases characterized by pathological angiogenesis, cancer, VEGFA-expressing cancer, VEGFR-expressing cancer, eye disease, retinopathy, diabetic retinopathy, macular degeneration, age-related macular degeneration, wet age-related macular degeneration, retinal vein occlusion, myopic choroidal neovascularization, retinopathy of prematurity, neovascular glaucoma, central serous retinopathy, ocular tumors, corneal neovascularization, inflammatory diseases, autoimmune diseases, arthritis, rheumatoid arthritis, osteoarthritis, psoriasis, multiple sclerosis, sepsis, motor neuron diseases and amyotrophic lateral sclerosis.

18. An in vitro complex comprising an antigen-binding molecule according to any one of claims 1 to 8 bound to VEGFA.

19. A method for detecting VEGFA in a sample, comprising contacting a sample containing or suspected of containing VEGFA in vitro with an antigen-binding molecule according to any one of claims 1 to 8, and detecting the formation of a complex between the antigen-binding molecule and VEGFA.

20. An agent comprising the antigen-binding molecule of any one of claims 1 to 8, for detecting, localizing or imaging VEGFA, or a cell containing or expressing VEGFA.

21. A method for selecting or stratifying a subject for treatment with a VEGFA-targeting drug, comprising contacting a sample from a subject in vitro with an antigen-binding molecule described in any one of claims 1 to 8, and detecting the formation of a complex between the antigen-binding molecule and VEGFA.

22. A drug comprising the antigen-binding molecule of any one of claims 1 to 8, for detecting, localizing or imaging a disease / condition characterized by expression of VEGFA.

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