VEGFA binding molecules

Abstract

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

Description

[Technical Field]

[0001] This application claims priority to SG10202101681W, filed 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, and 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 a variety of conditions, particularly in the fields of oncology and ophthalmology (1-3). Developing humanized and stabilized antibody domains against VEGF is desirable due to their small size and modularity. For ophthalmic applications, small size and high stability are desirable because they can enable localized drug delivery (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, tandem fusion of antibody domains 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: 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.

[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:16. In some embodiments, the antigen binding molecule has the following FR: FR1 having the amino acid sequence of SEQ ID NO: 9 FR2 having the amino acid sequence of SEQ ID NO: 10 FR3 having the amino acid sequence of SEQ ID NO: 11 FR4 having the amino acid sequence of SEQ ID NO: 12 The present invention includes a single domain antibody sequence incorporating:

[0006] 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:

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

[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:5. In some embodiments, the antigen binding molecule inhibits the interaction between VEGFA and VEGFR.

[0009] 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. The present disclosure also provides a chimeric antigen receptor (CAR) comprising the antigen-binding molecule described herein.

[0010] The present disclosure also provides nucleic acids, optionally isolated, encoding the antigen-binding molecules or CARs described herein. The present disclosure also provides expression vectors comprising the nucleic acids described herein.

[0011] The present disclosure also provides a cell comprising the antigen-binding molecule, CAR, nucleic acid, or expression vector described herein. 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.

[0012] The present disclosure also provides compositions comprising the antigen-binding molecule, CAR, nucleic acid, expression vector, or cell described herein and a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

[0013] 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 for treating or preventing a disease in which VEGFA / VEGFR-mediated signaling is pathologically involved.

[0014] The present disclosure also provides the use of the 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 involved.

[0015] The present disclosure also provides a method for treating or preventing a disease in which VEGFA / VEGFR-mediated signaling is pathologically involved, the method 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.

[0016] In some embodiments, the disease is selected from diseases characterized by pathological angiogenesis, cancer, VEGFA-expressing cancer, VEGFR-expressing cancer, ocular 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.

[0017] 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 the steps of 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.

[0018] The present disclosure also provides the use of the antigen-binding molecules described herein in methods for detecting, localizing, or imaging VEGFA, or cells containing or expressing VEGFA.

[0019] The present disclosure also provides a method for selecting or stratifying a subject for treatment with a VEGFA-targeted drug, the method comprising the steps of contacting a sample from the subject in vitro with an antigen-binding molecule described herein and detecting the formation of a complex between the antigen-binding molecule and VEGFA.

[0020] 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 INVENTION

[0021] We describe here 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 a bivalent anti-VEGFA molecule constructed as a tandem of two VH domain antibodies.

[0022] 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: 17), VEGF189 (SEQ ID NO: 18), VEGF165 (SEQ ID NO: 19), and VEGF121 (SEQ ID NO: 20). After processing to remove the N-terminal 26-amino acid signal peptide (SEQ ID NO: 25), VEGF206, VEGF189, VEGF165, and VEGF121 contain the amino acid sequences set forth in SEQ ID NOs: 21-24, respectively. VEGF165 appears to be the major VEGFA isoform.

[0023] 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 herein by reference in their entireties.

[0024] Vascular endothelial growth factors (VEGFs) are a family of secreted polypeptides with a conserved receptor-binding cystine knot structure. VEGFA monomers associate via interchain disulfide bonds to form homodimers. VEGFAs act through a family of cognate receptor kinases expressed by endothelial cells to stimulate angiogenesis.

[0025] VEGFA not only plays an important role 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, and induces angiogenesis (i.e., the formation of thin-walled endothelial-lined structures) and a rapid increase in microvascular permeability in various in vivo models.

[0026] 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., eutherians, placental mammals, epithelia, monotherians, archontans, primates (rhesus monkeys, cynomolgus monkeys, non-human primates, or humans)). In some embodiments, VEGFA is human VEGFA or mouse VEGFA.

[0027] As used herein, an isoform, fragment, variant, or homolog of a given reference protein can be characterized as having at least 70% sequence identity, 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 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 relative to the amino acid sequence of the reference protein, but that 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 reference protein. A "homolog" generally refers to a variant of a reference protein that is produced by a different species than the species of the reference protein. A homolog includes an ortholog.

[0028] Isoforms of VEGFA naturally include VEGF206 (UniProt P15692-1), VEGF189 (UniProt P15692-2), VEGF165 (UniProt P15692-4), and VEGF121 (UniProt P15692-9). VEGFA isoforms also include VEGF183 (UniProt P15692-3), VEGF148 (UniProt P15692-5), VEGF145 (UniProt P15692-6), VEGF165B (UniProt P15692-8), VEGF111 (UniProt P15692-10), L-VEGF165 (UniProt P15692-11), L-VEGF121 (UniProt P15692-12), L-VEGF189 (UniProt P15692-13), L-VEGF206 (UniProt P15692-14), VEGFA isoform 15 (UniProt P15692-15), and VEGFA isoform 16 (UniProt P15692-16). P15692-16), VEGFA isoform 17 (UniProt P15692-17), and VEGFA isoform 18 (UniProt P15692-18).

[0029] VEGFA isoforms, fragments, variants or homologs can optionally be characterized as having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence of an immature or mature VEGFA isoform from a given species, e.g., human.

[0030] 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 with 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).

[0031] 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: 17, 18, 19 or 20.

[0032] 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: 21, 22, 23 or 24.

[0033] 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 moiety. Binding of VEGF to the extracellular receptor domain results in receptor dimerization and phosphorylation of the intracellular tyrosine kinase moiety. VEGF has been shown to exert its biological effects primarily through VEGFR1 and VEGFR2.

[0034] 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., a eutherian, a placental, an epithelial, a monotherian, a hominid, a primate (rhesus monkey, cynomolgus monkey, non-human primate, or human)). In some embodiments, VEGFR1 / VEGFR2 / VEGFR3 is human or mouse VEGFR1 / VEGFR2 / VEGFR3.

[0035] 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 a related molecule from a given species, e.g., human.

[0036] 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.

[0037] 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 via the PI3K / AKT, MAPK / ERK, and PLC-γ pathways, as well as via SCR and FAK, promoting cell survival, proliferation, and cytoskeletal reorganization, affecting changes in vascular permeability and vasodilation, and promoting angiogenesis.

[0038] antigen binding molecule The present disclosure provides antigen-binding molecules that can bind to VEGFA (i.e., bind to VEGFA). The present disclosure provides antigen-binding molecules that specifically bind to VEGFA. The antigen-binding molecules described in the present disclosure can be provided in purified or isolated form, i.e., from other naturally occurring biological materials.

[0039] 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), as well as antibody fragments (e.g., Fv, scFv, Fab, scFab, F(ab')2, Fab2, diabody, triabody, scFv-Fc, minibody, single domain antibody (VHH), etc.) and aptamers.

[0040] More specifically, the antigen-binding molecules described herein comprise an antigen-binding polypeptide portion, which may also be referred to as an "antigen-binding domain." In a preferred embodiment, the antigen-binding molecules described herein comprise or consist of a single-domain antibody that specifically binds to VEGFA.

[0041] 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 herein by reference in their entireties.

[0042] 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.

[0043] Single domain antibodies according to the present disclosure generally comprise three complementarity determining regions (CDRs): CDR1, CDR2 and CDR3. Together, the three CDRs define the paratope of the molecule, which is the portion that binds to the target antigen.

[0044] Single domain antibodies further comprise framework regions (FRs) on either side of each CDR, providing a scaffold for the CDRs. From N- to C-terminus, a single domain antibody comprises the following structure: N-terminus-[FR1]-[CDR1]-[FR2]-[CDR2]-[FR3]-[CDR3]-[FR4]-C-terminus.

[0045] 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 VBASE2, Retter et al., Nucl. Acids Res. (2005) 33 (supra 1): D671-D674.

[0046] In some embodiments, the antigen-binding molecule comprises the CDRs of the VEGFA-binding single domain antibodies described herein or comprises CDRs derived from the VEGFA-binding single domain antibodies described herein. In some embodiments, the antigen-binding molecule comprises the FRs of the VEGFA-binding single domain antibodies described herein or comprises FRs derived from the VEGFA-binding single domain antibodies described herein. In some embodiments, the antigen-binding molecule comprises the CDRs and FRs of the VEGFA-binding single domain antibodies described herein or comprises CDRs and FRs derived from the VEGFA-binding single domain antibodies described herein. That is, in some embodiments, the antigen-binding molecule comprises the amino acid sequence of the VEGFA-binding single domain antibodies described herein or comprises an amino acid sequence derived from the VEGFA-binding single domain antibodies 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 (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)).

[0047] In some embodiments, the CDRs and FRs of the antigen-binding molecules (e.g., single-domain antibodies) referred to herein are defined according to the Kabat system. In some embodiments, in the amino acid sequences of SEQ ID NOs: 1, 5, and 16, 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; and FR4 is formed by the amino acid sequence of positions 120 to 130.

[0048] As used herein, an amino acid sequence / domain that is "derived from" a reference amino acid sequence / domain includes an amino acid sequence that has at least 60% sequence identity with the reference sequence, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0049] 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 16C2.1 and 21A5.1.

[0050] 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 having the amino acid sequence set forth in one of SEQ ID NOs: 1, 5 or 16. In some embodiments, the antigen binding molecule comprises the CDRs (i.e., CDR1, 2 and 3) of a VEGFA-binding single domain antibody having the amino acid sequence set forth in one of SEQ ID NOs: 1, 5 or 16. 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 the amino acid sequence set forth in one of SEQ ID NOs: 1, 5 or 16. 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 the amino acid sequence set forth in one of SEQ ID NOs: 1, 5 or 16.

[0051] In some embodiments, the antigen-binding molecule comprises or consists of a single domain antibody sequence according to one of the following (1) to (3): (1) (Con) 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, two or three amino acids in one or more of CDR1, CDR2 or CDR3 are replaced with another amino acid. (2) (16C2.1) 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: 3 CDR3 having the amino acid sequence of SEQ ID NO: 4 or a variant thereof in which one, two or three amino acids in one or more of CDR1, CDR2 or CDR3 are replaced with another amino acid. (3) (21A5.1) 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, two or three amino acids in one or more of CDR1, CDR2 or CDR3 are replaced with another amino acid.

[0052] In some embodiments, the antigen-binding molecule comprises or consists of a single domain antibody sequence as set forth in (4) below: (4) A single domain antibody sequence comprising the following FR: FR1 having the amino acid sequence of SEQ ID NO: 9 FR2 having the amino acid sequence of SEQ ID NO: 10 FR3 having the amino acid sequence of SEQ ID NO: 11 FR4 having the amino acid sequence of SEQ ID NO: 12 or a variant thereof in which one, two, or three amino acids in one or more of FR1, FR2, FR3, or FR4 are substituted with another amino acid.

[0053] In some embodiments, the antigen-binding molecule comprises or consists of a single domain antibody sequence comprising a CDR described in one of (1) to (3) above and a FR described in (4) above.

[0054] In some embodiments, the antigen-binding molecule comprises or consists of a single domain antibody sequence according to one of the following (5) to (7): (5) (Con) A single domain antibody sequence comprising the CDRs described in (1) and the FRs described in (4). (6) (2) A single domain antibody sequence comprising the CDRs described in (2) and the FRs described in (4). (7) (3) A single domain antibody sequence comprising the CDR described in (3) and the FR described in (4).

[0055] In some embodiments, the antigen-binding molecule comprises or consists of a single domain antibody sequence according to one of the following (8) to (10): (8) (Con) 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. (9) (16C2.1) 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:1. (10) (21A5.1) 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.

[0056] In embodiments described herein in which one or more amino acids are substituted with another amino acid, the substitution may be a conservative substitution, for example, according to the following table: In some embodiments, amino acids in the same block in the middle column are substituted. In some embodiments, amino acids in the same row in the right-most column are substituted:

[0057] [Table 1]

[0058] In some embodiments, substitutions may be functionally conservative, i.e., they may not affect (or may not substantially affect) one or more functional properties (e.g., target binding) of an antigen-binding molecule containing the substitution, compared to a comparable unsubstituted molecule.

[0059] 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 the heavy chain constant sequence of an IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM, such as human IgG (e.g., hIgG1, hIgG2, hIgG3, hIgG4), hIgA (e.g., hIgA1, hIgA2), hIgD, hIgE, or hIgM. In some embodiments, the immunoglobulin heavy chain constant sequence is or is derived from the heavy chain constant sequence of a human IgG1 allotype (e.g., G1m1, G1m2, G1m3, or G1m17).

[0060] 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: 26.

[0061] 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: 27. 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: 28. 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: 29. 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: 30.

[0062] 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 together form the Fc region.

[0063] The Fc region provides interaction with Fc receptors and other molecules of the immune system, resulting in functional effects. IgG Fc-mediated effector function is reviewed, for example, in Jefferis et al., Immunol Rev 1998, 163:59-76 (incorporated herein by reference in its entirety), and is mediated by the 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 Fc receptors expressed by immune cells, the recruitment of complement pathway components via binding of the Fc region to the 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), membrane attack complex (MAC) formation, cell degranulation, cytokine and / or chemokine production, and antigen processing and presentation.

[0064] 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 containing / expressing VEGFA (e.g., cells expressing VEGFA and / or complexes containing VEGFA on the cell surface), and / or enhancing MAC formation on cells or cell degranulation.

[0065] Modifications to antibody Fc regions that affect Fc-mediated function are known in the art, such as those described in Wang et al., Protein Cell (2018) 9(1):63-73, which is incorporated herein 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.

[0066] Multispecific antigen-binding molecules are also contemplated. "Multispecific" means that an antigen-binding molecule exhibits specific binding to multiple targets. In some embodiments, an antigen-binding molecule is a bispecific antigen-binding molecule. In some embodiments, an antigen-binding molecule comprises at least two different antigen-binding domains.

[0067] In some embodiments, the antigen-binding molecule is at least bispecific, as it binds to VEGFA and another target (e.g., an antigen other than VEGFA). The term "bispecific" means that the antigen-binding molecule can specifically bind to at least two different antigenic determinants.

[0068] It will be understood that antigen-binding molecules (e.g., multispecific antigen-binding molecules) described herein may include antigen-binding molecules capable of binding to a target for which the antigen-binding molecule is specific. For example, antigen-binding molecules that bind to VEGFA and antigens other than VEGFA may include (i) antigen-binding molecules that bind to VEGFA and (ii) antigen-binding molecules that bind to antigens other than VEGFA. In some embodiments, component antigen-binding molecules of a larger antigen-binding molecule (e.g., a multispecific antigen-binding molecule) may be referred to, for example, as the "antigen-binding domain" or "antigen-binding region" of the larger antigen-binding molecule.

[0069] 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.

[0070] 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.

[0071] A cancer cell antigen is an antigen expressed or overexpressed by cancer cells. 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 cancer cells (e.g., the cancer cell antigen can be expressed with abnormal localization) or with abnormal structure. A cancer cell antigen can potentially elicit an immune response. In some embodiments, the antigen is expressed on the cell surface of a cancer cell (i.e., the cancer cell antigen is a cancer cell surface antigen). In some embodiments, the portion of the antigen bound by the antigen binding molecule described herein is displayed on the outer surface of the 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 cancer symptom. 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 expression levels 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.

[0072] The immune cell surface molecule can be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof expressed on or at 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 can be expressed on the cell surface of any immune cell. In some embodiments, the immune cell can be a cell of hematopoietic origin, such as a neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, or monocyte. The lymphocyte can 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).

[0073] In some embodiments, the multispecific antigen-binding molecules described herein exhibit at least monovalent binding to VEGFA and at least monovalent binding to antigens other than VEGFA. Valency refers to the number of binding sites within an antigen-binding molecule for a given antigenic determinant.

[0074] In some embodiments, the antigen binding molecule comprises a single domain antibody (e.g., as described herein) capable of binding to VEGFA, and an antigen binding region (e.g., a polypeptide (e.g., a single domain antibody), Fv, Fab, or antibody) capable of binding to an antigen other than VEGFA.

[0075] In some embodiments, the antigen-binding molecule comprises an immune cell-engaging moiety. 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 entireties.

[0076] 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.

[0077] 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 engagers are bispecific T cell engagers (BiTEs), which comprise a target antigen-binding domain and a CD3 polypeptide (typically CD3ε)-binding domain through which the BiTE recruits T cells. Binding of a BiTE to its target antigen and the CD3 polypeptide expressed by the T cell activates the T cell, ultimately directing T cell effector activity against cells expressing the target antigen. Other types of immune cell engagers are known in the art and include natural killer cell engagers, such as bispecific killer engagers (BiKEs), which recruit and activate NK cells.

[0078] 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.

[0079] Certain 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 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 16.

[0080] 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 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:1.

[0081] In some embodiments, the antigen-binding molecule of the present disclosure comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:5.

[0082] Linkers and additional sequences An antigen-binding molecule may contain additional amino acids / amino acid sequences in addition to the amino acid sequence required for binding to a target antigen. In some embodiments, such additional amino acids / amino acid sequences are provided at the N-terminus of the single domain antibody sequences described herein. In some embodiments, such additional amino acids / amino acid sequences are provided at the C-terminus of the single domain antibody sequences described herein. In some embodiments, such additional amino acids / amino acid sequences are provided at the N-terminus and C-terminus of the single domain antibody sequences described herein.

[0083] In some embodiments, the antigen-binding molecule comprises one or more linker sequences between the amino acid subsequences. For example, linker sequences may be provided at one or both ends of the antigen-binding domain of the antigen-binding molecule described herein.

[0084] 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 may be a flexible linker sequence. The flexible linker sequence allows the relative movement of the amino acid sequences 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.

[0085] 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 one or more (e.g., tandem) 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 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 25, or 1 to 30 amino acids.

[0086] The antigen-binding molecules and polypeptides of the present disclosure may additionally comprise additional amino acids or amino acid sequences. 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 molecule / polypeptide. For example, the antigen-binding molecule / polypeptide may optionally comprise a sequence encoding His (e.g., 6XHis), Myc, GST, MBP, FLAG, HA, E, or biotin tag at the N-terminus or C-terminus of the antigen-binding molecule / polypeptide. In some embodiments, the antigen-binding molecule / polypeptide comprises a detectable moiety, such as a fluorescent label, a luminescent label, an immunodetectable label, a radioactive label, a chemical label, a nucleic acid label, or an enzyme label.

[0087] 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 typically consists of a sequence of 5 to 30 hydrophobic amino acids and forms a single alpha helix. Secreted proteins and proteins expressed on the cell surface often contain a signal peptide.

[0088] A signal peptide may be present at the N-terminus of an 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.

[0089] 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, etc., and / or can be identified / predicted using amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8:785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24:2172-2176).

[0090] 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 enzymatic label. The antigen-binding molecule can be covalently or non-covalently labeled with the detectable moiety.

[0091] 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 fluoroisotopes, ...

[0092] 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) that causes the antigen-binding molecule to exhibit cytotoxicity against cells containing / expressing VEGFA (e.g., cells expressing VEGFA and / or complexes containing VEGFA on the cell surface). In some embodiments, the drug moiety 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.

[0093] Functional properties of antigen-binding molecules The antigen-binding molecules described herein can be characterized by 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., receptors 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 VEGFA-expressing cells.

[0094] It will be understood that a given antigen-binding molecule may exhibit more than one of the properties described in the preceding paragraph. A given antigen-binding molecule can be evaluated for the properties described in the preceding paragraph using an appropriate 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., performed in an animal other than a human. The assay can use a species labeled with a detectable entity to facilitate detection.

[0095] 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.

[0096] 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 level of inhibition of a given property is observed. For example, the IC of a given antigen-binding molecule for inhibiting the interaction between VEGFA and VEGFR (e.g., VEGFR1) is 50 may be the concentration at which 50% of the maximum inhibition level is achieved.

[0097] 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 the antigen and can be distinguished from non-specific binding to non-target antigens.An antigen-binding molecule / domain that specifically binds to target molecules preferably binds to target with higher affinity and / or longer duration than binding to other non-target molecules.

[0098] 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 can measure and quantitate binding to a given molecule. In some embodiments, binding can be a response detected in a given assay.

[0099] 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 RIA. Alternatively, the binding specificity is such that the KD of the antigen-binding molecule for the non-target molecule is at least 0.1 orders of magnitude (i.e., 0.1 x 10 n , where n is an integer representing an order of magnitude), which may be reflected in terms of binding affinity to bind 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.

[0100] Binding to VEGFA can be determined by biolayer interferometry, for example, as described in Example 8 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 comprising VEGFA.

[0101] In some embodiments, the antigen binding molecules described herein bind with submicromolar affinity, i.e., K D <1×10-6 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 <1×10 -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 <1×10 -12 Binds to VEGFA at M.

[0102] 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.

[0103] In some embodiments, the antigen-binding molecules described herein have a K that is greater than or equal to the 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 8 of the present disclosure) binds to VEGFA (e.g., human VEGF165). In the same assay, the K determined for ranibizumab 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 8 of the present disclosure) binds to VEGFA (e.g., human VEGF165).

[0104] In some embodiments, the antigen binding molecules described herein have a K that is higher than that 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) at a K (e.g., as determined by BLI, e.g., as described in Example 8 of the present disclosure). In some embodiments, the antigen-binding molecule binds to VEGFA (e.g., human VEGF165) at a K (e.g., as determined for ranibizumab in the same assay). D K is less than 1 time, for example, ≦0.99 times, ≦0.95 times, ≦0.9 times, ≦0.85 times, ≦0.8 times, ≦0.75 times, ≦0.7 times, ≦0.65 times, ≦0.6 times, ≦0.55 times, or ≦0.5 times. D (e.g., as determined by BLI, e.g., as described in Example 8 of the present disclosure) binds to VEGFA (e.g., human VEGF165).

[0105] In some embodiments, the antigen-binding molecules described herein have a K that is greater than or equal to the 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 8 of the present disclosure) binds to VEGFA (e.g., human VEGF165). In the same assay, the K determined for bevacizumab 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 8 of the present disclosure) binds to VEGFA (e.g., human VEGF165).

[0106] In some embodiments, the antigen binding molecules described herein have a K 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 8 of the present disclosure) that is higher than the K determined for bevacizumab in the same assay. D K is less than 1 time, for example, ≦0.99 times, ≦0.95 times, ≦0.9 times, ≦0.85 times, ≦0.8 times, ≦0.75 times, ≦0.7 times, ≦0.65 times, ≦0.6 times, ≦0.55 times, or ≦0.5 times. D (e.g., as determined by BLI, e.g., as described in Example 8 of the present disclosure) binds to VEGFA (e.g., human VEGF165).

[0107] The antigen binding molecules of the present disclosure can bind to specific regions of interest in VEGFA. The antigen binding molecules described in the present disclosure can bind to linear epitopes of VEGFA consisting of a continuous sequence of amino acids (i.e., a primary amino acid sequence). In some embodiments, the antigen binding molecules can bind to conformational epitopes of VEGFA consisting of a discontinuous sequence of amino acids in the amino acid sequence.

[0108] The region of a given target molecule to which an antigen-binding molecule binds can be determined by those skilled in the art using a variety of methods well known in the art, including X-ray cocrystallography of antibody-antigen complexes, peptide scanning, mutagenesis mapping, mass spectrometric hydrogen-deuterium exchange analysis, 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.

[0109] In some embodiments, the antigen binding molecules described herein bind to the same region of VEGFA, or overlapping regions of VEGFA, or regions of VEGFA that are bound by antigen binding molecules comprising the CDRs, FRs and / or the complete amino acid sequence of a VEGFA-binding single domain antibody selected from 16C2.1 and 21A5.1.

[0110] In some embodiments, an antigen binding molecule described herein binds to the region of VEGFA where VEGFA binds to VEGFR (eg, VEGFR1 and / or VEGFR2).

[0111] In some embodiments, the antigen binding molecule binds to VEGFA in the region where VEGFR (e.g., VEGFR1) binds. 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) binds, 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).

[0112] 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 interaction level in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule). Suitable analyses can be performed in vitro, for example, using recombinant interaction partners or cells expressing the interaction partners. Cells expressing interaction partners may express them endogenously or may be expressed from nucleic acids 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.

[0113] In some embodiments, the antigen binding molecule described herein inhibits the interaction between VEGFA and VEGFR1 (e.g., human VEG121 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 9 of this disclosure).

[0114] In some embodiments, the antigen binding molecules described herein inhibit the interaction between VEGFA and VEGFR1 (e.g., human VEGFR1 and human VEGFR1) by an IC50 value greater than or equal to 100% for inhibition of such interaction as determined for ranibizumab (i.e., the molecule formed by the binding of the polypeptides of SEQ ID NOs: 74 and 75) in the same assay. 50 and similar ICs 50 In some embodiments, the antigen binding molecule inhibits the interaction between VEGFA and VEGFR1 (e.g., human VEGFR1 and human VEGFR1) in the same assay (e.g., as determined by competitive ELISA, e.g., the competitive ELISA described in Example 9 of the present disclosure). In some embodiments, the antigen binding molecule inhibits the interaction between VEGFA and VEGFR1 (e.g., human VEGFR1 and human VEGFR1) in the same assay (e.g., as determined by competitive ELISA, e ... determined by competitive ELISA described in Example 9 of the present disclosure) in the same assay (e.g., as determined by competitive ELISA, e.g., as determined by competitive ELISA described in Example 9 of the present disclosure). 50 0.5 times or more but less than 2 times the value, for example, 0.55 times or more but less than 1.9 times, 0.6 times or more but less than 1.8 times, 0.65 times or more but less than 1.7 times, 0.7 times or more but less than 1.6 times, 0.75 times or more but less than 1.5 times, 0.8 times or more but less than 1.4 times, 0.85 times or more but less than 1.3 times, 0.9 times or more but less than 1.2 times, 0.95 times or more but less than 1.1 times 50 (eg, as determined by competitive ELISA, such as the competitive ELISA described in Example 9 of this disclosure).

[0115] In some embodiments, the antigen binding molecules described herein inhibit the interaction between VEGFA and VEGFR1 (e.g., human VEG121 and human VEGFR1) in an assay that is higher than the IC50 for inhibition of such an interaction as determined for ranibizumab in the same assay. 50 Lower IC 50 In some embodiments, the antigen binding molecule inhibits the interaction between VEGFA and VEGFR1 (e.g., human VEGFR1 and human VEGFR1) in the same assay (e.g., as determined by competitive ELISA, e.g., the competitive ELISA described in Example 9 of the present disclosure). In some embodiments, the antigen binding molecule inhibits the interaction between VEGFA and VEGFR1 (e.g., human VEGFR1 and human VEGFR1) in the same assay (e.g., as determined by competitive ELISA, e ... determined by competitive ELISA described in Example 9 of the present disclosure) in the same assay (e.g., as determined by competitive ELISA, e.g., as determined by competitive ELISA described in Example 9 of the present disclosure). 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 9 of this disclosure).

[0116] In some embodiments, the antigen binding molecules described herein inhibit the interaction between VEGFA and VEGFR1 (e.g., human VEG121 and human VEGFR1) by an IC50 value greater than or equal to 100 for inhibition of such interaction as determined for bevacizumab (i.e., the molecule formed by the binding of the polypeptides of SEQ ID NOs: 76 and 77) in the same assay. 50 and similar ICs 50 In some embodiments, the antigen binding molecule inhibits the interaction between VEGFA and VEGFR1 (e.g., human VEGFR1 and human VEGFR1) in the same assay as measured by IC50 for inhibition of such interaction by bevacizumab (e.g., as determined by competitive ELISA, e.g., the competitive ELISA described in Example 9 of the present disclosure). 50 0.5 times or more but less than 2 times the value, for example, 0.55 times or more but less than 1.9 times, 0.6 times or more but less than 1.8 times, 0.65 times or more but less than 1.7 times, 0.7 times or more but less than 1.6 times, 0.75 times or more but less than 1.5 times, 0.8 times or more but less than 1.4 times, 0.85 times or more but less than 1.3 times, 0.9 times or more but less than 1.2 times, 0.95 times or more but less than 1.1 times 50 (eg, as determined by competitive ELISA, such as the competitive ELISA described in Example 9 of this disclosure).

[0117] In some embodiments, the antigen binding molecules described herein inhibit the interaction between VEGFA and VEGFR1 (e.g., human VEG121 and human VEGFR1) in an assay that is higher than or equal to the IC50 for inhibition of such an interaction as determined for bevacizumab in the same assay. 50 Lower IC 50In some embodiments, the antigen binding molecule inhibits the interaction between VEGFA and VEGFR1 (e.g., human VEGFR1 and human VEGFR1) in the same assay as measured by IC50 for inhibition of such interaction by bevacizumab (e.g., as determined by competitive ELISA, e.g., the competitive ELISA described in Example 9 of the present disclosure). 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 9 of this disclosure).

[0118] In some embodiments, the antigen binding molecule inhibits VEGFA / VEGFR-mediated signaling (i.e., signaling mediated by the binding of VEGFA to VEGFR). VEGFA / VEGFR-mediated signaling can be analyzed, for example, using VEGFR-expressing cells in assays that detect and / or quantify VEGFA / VEGFR-mediated signaling.

[0119] Suitable assays for examining VEGFA / VEGFR-mediated signal transduction include assays for detecting the phosphorylation / activation / expression of factors phosphorylated / activated / expressed as a result of VEGFA / VEGFR-mediated signal transduction. Such assays may include contacting VEGFR-expressing cells with an antigen binding molecule described herein in the presence of VEGFA. Assays for examining VEGFA / VEGFR-mediated signal transduction may include analyzing signal transduction via the PI3K / AKT, MAPK / ERK, and / or PLC-γ pathways, and / or via SCR and / or FAK.

[0120] In some embodiments, an antigen binding molecule of the present disclosure can inhibit VEGFA / VEGFR-mediated signaling (e.g., signaling mediated by binding of human VEGFl21 to human VEGFRl) 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).

[0121] 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 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 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 IC50 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.

[0122] 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, with an IC for inhibition of such interaction determined for ranibizumab. 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 IC50 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.

[0123] 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 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 interaction determined for bevacizumab. 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 IC50 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.

[0124] 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, with an IC for inhibition of such interaction determined for bevacizumab. 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 IC50 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.

[0125] In some embodiments, the antigen-binding molecules described herein bind to VEGFA (e.g., human VEGFA) with similar affinity before and after heat treatment. Heat treatment may include incubation in 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 10 of the present disclosure.

[0126] In some embodiments, the antigen-binding molecule exhibits 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 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 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 similar affinity for VEGFA before heat treatment and after heat treatment at 80°C for 1 hour.

[0127] As used herein, a binding affinity that is "similar" to a reference binding affinity means a binding affinity that is within 50%, e.g., 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.

[0128] 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.

[0129] In some embodiments, the antigen binding molecules described in the present disclosure may enhance (ie, upregulate and potentiate) cell killing of cells containing / expressing VEGFA. In some embodiments, the antigen binding molecules described herein are capable of reducing the number / proportion of cells containing / expressing VEGFA, hi some embodiments, the antigen binding molecules described herein are capable of depleting / enhancing the depletion of such cells.

[0130] In some embodiments, an antigen binding molecule described herein reduces the number / proportion of cells containing / expressing 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, for example, by ≦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.

[0131] The antigen-binding molecules described herein may comprise one or more moieties for enhancing the reduction of the number / proportion of cells containing / expressing VEGFA. For example, the antigen-binding molecules described herein may comprise, for example, an Fc region and / or a drug moiety.

[0132] 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 containing / expressing VEGFA, and / or enhancing MAC formation or cell degranulation on cells containing / expressing VEGFA.

[0133] In some embodiments, the antigen binding molecules described herein are capable of enhancing / inducing ADCC against cells containing / expressing VEGFA. In some embodiments, the antigen-binding molecule described herein comprises a drug moiety. The antigen-binding molecule may be conjugated to a drug moiety. Antibody-drug conjugates are generally described, for example, 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 exhibits cytotoxicity against cells containing / expressing VEGFA. In some embodiments, the drug moiety is or comprises a chemotherapeutic agent.

[0134] 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).

[0135] In some embodiments, the antigen binding molecules described herein 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 containing / expressing 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, for example, by ≦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.

[0136] In some embodiments, an antigen binding molecule described in the present disclosure increases 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, the level of killing 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.

[0137] Chimeric antigen receptor (CAR) The present disclosure also provides a chimeric antigen receptor (CAR) comprising an antigen-binding polypeptide of the present disclosure.

[0138] CAR is a recombinant receptor that provides both antigen binding function and T cell activation function.The structure and operation of CAR are, for example, outlined 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.

[0139] 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 included in or constitutes the antigen-binding region of the CAR.

[0140] The 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, with the antigen-binding region located in the extracellular space and the signaling region located intracellularly. In some embodiments, the CAR comprises a cell membrane anchor region that comprises, consists of, or is derived from the transmembrane region amino acid sequence of one of CD3-zeta, CD4, CD8, or CD28. As used herein, a region "derived from" a reference amino acid sequence includes 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.

[0141] The signaling region of a CAR can activate T cells. The CAR signaling region may contain the amino acid sequence of the intracellular domain of CD3-zeta, which provides an immunoreceptor tyrosine-based activation motif (ITAM) for phosphorylation and activation of CAR-expressing T cells. Signaling regions containing sequences from 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 a CAR may also 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, CARs are engineered to provide costimulation of different intracellular signaling pathways. For example, signaling associated with CD28 costimulation preferentially activates the phosphatidylinositol 3-kinase (PI3K) pathway, while 4-1BB-mediated signaling is via the TNF receptor-associated factor (TRAF) adaptor protein. 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 comprise, consist of, or are derived from the amino acid sequence of one or more intracellular domains of CD28, 0X40, 4-1BB, ICOS, and CD27.

[0142] The optional hinge region can provide a gap between the antigen-binding domain and the transmembrane domain, and can function as a flexible linker.The hinge region can be derived from IgG1.In some embodiments, the CAR of the present disclosure comprises a hinge region that comprises or consists of the amino acid sequence of the hinge region of IgG1 or is derived from it.

[0143] Also provided are cells comprising the CARs described in the present disclosure.The CARs described in the present disclosure can be used to generate CAR-expressing immune cells, such as CAR-T cells or CAR-NK cells.The manipulation of CARs into immune cells can be carried out during in vitro culture.

[0144] The antigen-binding region of the CAR of the present disclosure can be provided in any suitable format, e.g., 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.

[0145] The present 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 sources.

[0146] The nucleotide sequence of the nucleic acid described in this disclosure can be contained in a vector, for example, an expression vector. As used herein, a "vector" is a nucleic acid molecule used as a vehicle for introducing exogenous nucleic acid into a cell. The vector can be a vector for expressing a nucleic acid in a cell. Such a vector may contain a promoter sequence operably linked to the nucleotide sequence encoding the sequence to be expressed. The vector may also contain 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.

[0147] The term "operably linked" can include situations in which 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 it can affect the transcription of the nucleic acid sequence. The resulting transcript can then be translated into the desired peptide(s) / polypeptide(s).

[0148] Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g., gammaretroviral 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).

[0149] In some embodiments, the vector may be a eukaryotic vector, e.g., a vector containing 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.

[0150] 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.

[0151] 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 Rodents), cat, dog, pig, sheep, goat, cattle (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).

[0152] In some embodiments, the cell is or is derived from a cell type commonly used for expressing 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.

[0153] 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, introducing the isolated nucleic acid(s) or vector(s) described herein into a cell comprises transformation, transfection, electroporation, or transduction (e.g., retroviral transduction).

[0154] The present disclosure also provides a method for producing a cell that expresses / contains an antigen-binding molecule / CAR described in the present disclosure, the method 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.

[0155] 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 skilled in the art.

[0156] 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.

[0157] Alternatively, antigen-binding molecules and polypeptides can be produced by recombinant expression.Molecular biology techniques suitable for the 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 for 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.

[0158] For recombinant production as described herein, any cell suitable for expressing 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 an Enterobacteriaceae bacterium, 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 described herein above.

[0159] In some cases, the cells may not be prokaryotic, as some prokaryotic cells are unable to fold or post-translationally modify proteins in the same way as eukaryotic cells. Furthermore, very high expression levels are possible in eukaryotic organisms, and proteins can be easily purified from eukaryotic organisms by using appropriate tags. Specific plasmids that facilitate secretion of proteins into the culture medium are also available.

[0160] 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.

[0161] Production may involve 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 ed., supra, incorporated herein by reference).

[0162] 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, cultivation can be performed in batches. Bioreactors monitor and control environmental conditions, such as pH, oxygen, inflow and outflow flow rates to and from the vessel, and agitation within the vessel, to provide optimal conditions for the cultured cells.

[0163] 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 cultured cells.

[0164] 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 inside cells, protein isolation can include centrifugation to separate cells from cell culture medium, treating cell pellet with lysis buffer, and disrupting cells by, for example, sonication, rapid freeze-thawing, or osmotic lysis.

[0165] It may then be desirable 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 separating protein components from the supernatant or culture medium is by precipitation. Proteins of different solubilities are precipitated with different concentrations of a precipitant, such as ammonium sulfate. For example, low concentrations of the precipitant extract water-soluble proteins. Therefore, by adding different increasing concentrations of the precipitant, proteins of different solubilities can be differentiated. Subsequently, ammonium sulfate can be removed from the separated proteins using dialysis.

[0166] Other methods for distinguishing between different proteins are known in the art, such as ion exchange chromatography and size chromatography, which can be used as an alternative to precipitation or can be performed following precipitation.

[0167] If the polypeptide of interest is isolated from a culture, it may be desirable or necessary to concentrate the polypeptide. Many methods for concentrating proteins are known in the art, such as ultrafiltration or lyophilization.

[0168] composition The present disclosure also provides compositions comprising the antigen-binding molecules, CARs, nucleic acids, expression vectors, and cells described herein.

[0169] The antigen-binding molecules, CARs, nucleic acids, expression vectors, and cells described herein can be formulated as pharmaceutical compositions or medicaments for clinical use and may include pharmaceutically acceptable carriers, diluents, excipients, or adjuvants.

[0170] The compositions of the present disclosure may be formulated in a pharmaceutical composition containing one or more pharmaceutically acceptable carriers (e.g., liposomes, micelles, microspheres, nanoparticles), diluents / excipients (e.g., starch, cellulose, cellulose derivatives, polyols, dextrose, maltodextrin, magnesium stearate), adjuvants, fillers, buffering agents, 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).

[0171] The term "pharmaceutically acceptable," as used herein, refers to compounds, ingredients, materials, compositions, dosage forms, etc., that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of a subject (e.g., a human subject) 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, flavorant, or sweetener of the compositions described herein 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, flavors, or sweeteners can be found in standard pharmaceutical texts, such as Remington's 'The Science and Practice of Pharmacy' (A. Adejare, ed.), 23rd Edition (2020), Academic Press.

[0172] 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.

[0173] Suitable formulations may contain antigen-binding molecules in a sterile or isotonic medium. Medicaments and pharmaceutical compositions can be formulated in fluid form, including gels. Fluid formulations can be formulated for administration by injection or infusion (e.g., via a catheter) into a selected region of the human or animal body.

[0174] In some embodiments, the composition is formulated for injection or infusion, for example, into a blood vessel or tissue / organ of interest. The present disclosure also provides methods for producing a pharmaceutically useful composition, which may include one or more steps selected from: producing the antigen-binding molecule, CAR, nucleic acid, expression vector, or cell described herein; isolating the antigen-binding molecule, CAR, nucleic acid, expression vector, or cell described herein; and / or mixing the antigen-binding molecule, CAR, nucleic acid, expression vector, or cell described herein with a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

[0175] For example, a further aspect of the present 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 the step of formulating the pharmaceutical composition or medicament by mixing an antigen-binding molecule, CAR, nucleic acid, expression vector, or cell described herein with a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

[0176] 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.

[0177] The present disclosure provides antigen-binding molecules, CARs, nucleic acids, expression vectors, cells, or compositions described herein for use in methods of medical treatment or prevention. Also provided is the use of antigen-binding molecules, CARs, nucleic acids, expression vectors, cells, or compositions 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, CAR, nucleic acid, expression vector, cell, or composition described herein.

[0178] Therapeutic or prophylactic intervention according to the present disclosure may be effective in reducing the onset or progression of a disease / condition, alleviating the symptoms of a disease / condition, or reducing the pathology of a disease / condition. The intervention may be effective in preventing the progression of a disease / condition, e.g., preventing the worsening of a disease / condition, or slowing the rate of onset of a disease / condition. In some embodiments, the method may lead to an improvement in a disease / condition, e.g., a reduction in the symptoms of a disease / condition, or a reduction in 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).

[0179] As used herein, the terms "develop," "developing," and "development," e.g., of a disorder, refer to both the onset of the disease and the progression, worsening, or deterioration of the disease state / its correlates.

[0180] 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 reduced 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, such as a disease / condition in which increased levels of VEGFA / VEGFR-mediated signaling and / or increased numbers of cells containing / expressing VEGFA / VEGFR are positively correlated with the onset, development, or progression of the disease / condition and / or the severity of one or more symptoms of the disease / condition, or a disease / condition in which increased levels of VEGFA / VEGFR-mediated signaling and / or increased numbers of cells containing / expressing VEGFA / VEGFR are risk factors for the onset, development, or progression of the disease / condition.

[0181] 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.

[0182] 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.

[0183] VEGFA / VEGFR-mediated signaling is involved 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.

[0184] 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 depends on angiogenesis and confirmed the importance of VEGFA in this process.

[0185] 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 the expression level 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 VEGFR compared to the expression level 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.

[0186] It will be understood that the specific diseases listed in the preceding paragraph are interrelated. For example, diseases characterized by pathological angiogenesis include cancer and eye diseases. As used herein, "pathological angiogenesis" refers to angiogenesis (ie, the growth of new blood vessels from pre-existing vascular plexuses) that contributes to the development and / or progression of disease.

[0187] 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, and can be primary or secondary (metastatic). A neoplasm or tumor is an abnormal growth or proliferation of cells and can be present in any tissue. The cancer can 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.

[0188] The tumors to be treated may be nervous system tumors or non-nervous system tumors. Nervous system tumors can occur in either the central or peripheral nervous system, such as glioma, medulloblastoma, meningioma, neurofibroma, ependymoma, schwannoma, neurofibrosarcoma, astrocytoma, and oligodendroglioma. Non-nervous system cancers / tumors may originate from other non-nervous tissues, including 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 cancer, NSCLC, blood cancer, and sarcoma.

[0189] Treatment / prevention 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.

[0190] In some embodiments, the cancer to be treated / prevented comprises cells that express VEGFA. In some embodiments, the cancer to be treated / prevented comprises cells that express 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.

[0191] 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 by, for example, detecting the mRNA encoding VEGFA and / or VEGFR, for example, by quantitative real-time PCR (qRT-PCR).Protein expression can be determined by detecting VEGFA and / or VEGFR, for example, by antibody-based method, for example, by Western blot, immunohistochemistry, immunocytochemistry, flow cytometry or ELISA.

[0192] 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 overexpresses VEGFA and / or VEGFR in a sample taken from the subject.

[0193] VEGFA / VEGFR antagonists are being investigated as agents for treating / preventing 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 cord 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).

[0194] 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.

[0195] 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.

[0196] 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 motor neuron disease or amyotrophic lateral sclerosis.

[0197] According to various aspects of the present disclosure, methods are provided that aim at or involve (e.g., in the context of therapeutic / prophylactic interventions described herein) inhibiting the interaction between VEGFA and VEGFR (i.e., a receptor for VEGFA, e.g., VEGFR1) and / or inhibiting VEGFA / VEGFR-mediated signaling.

[0198] 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 properties using techniques routinely practiced in the art.

[0199] Administration of the articles of the present disclosure is preferably a "therapeutically effective" or "prophylactically effective" amount, which is sufficient to provide a therapeutic or prophylactic benefit to the subject. The actual amount, rate, and duration of administration will depend on the nature and severity of the disease / condition and the specific article being administered. Prescribing treatment, e.g., determining dosage, 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.

[0200] 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 agents can be administered simultaneously or sequentially.

[0201] Simultaneous 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, followed by separately administering the other agent after a predetermined time interval. Although this is the case in some embodiments, it is not required that the two agents be administered by the same route. The time interval can be any time interval.

[0202] 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. Multiple administrations can be separated by a predetermined time interval, which can be selected to be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days, or 1, 2, 3, 4, 5, or 6 months. For example, administration can be performed once every 7, 14, 21, or 28 days (plus or minus 3, 2, or 1 day).

[0203] Detection Method The present disclosure also provides articles of the present disclosure for use in methods for detecting VEGFA or for detecting cells containing / expressing VEGFA.

[0204] The antigen-binding molecules described herein can be used in methods comprising detecting the binding of the antigen-binding molecule to VEGFA. Such methods can include detecting a binding complex between the antigen-binding molecule and VEGFA.

[0205] In such cases, methods are provided that include 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 are methods that include contacting a sample containing or suspected of containing cells containing / expressing VEGFA and detecting the formation of a complex between the antigen-binding molecule and cells containing / expressing VEGFA.

[0206] Suitable method formats are well known in the art and include sandwich assays, immunoassays such as ELISA, etc. 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.

[0207] Methods that involve detecting VEGFA, or cells that contain / express VEGFA, include methods for diagnosing / prognosing diseases / conditions in which VEGFA expression / activity is pathologically implicated.

[0208] Such methods 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.

[0209] Such methods may include, for example, detecting or quantifying one or more of VEGFA, cells containing / expressing VEGFA in a patient sample. If 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 a 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.

[0210] 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 outcome) 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.

[0211] The sample can be taken from any tissue or body fluid.The sample can include or be derived from: a volume of blood; a volume of serum from an individual's blood, which can include the liquid portion 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, the sample can be obtained or derived from one or more tissues affected by a disease / condition (for example, one or more tissues in which the symptoms of the disease appear, or one or more tissues involved in the pathogenesis of the disease / condition).

[0212] The present disclosure also provides methods for selecting / stratifying subjects for treatment with VEGFA-targeting drugs. In some embodiments, a subject is selected for or identified as a subject who would benefit from treatment / prevention according to the present disclosure based on, for example, detection / quantification of VEGFA or cells containing / expressing VEGFA in a sample obtained from the individual.

[0213] subject The subject described in this disclosure can be any animal. In some embodiments, the subject can be a mammal. In some embodiments, the subject can be a human. In some embodiments, the subject can be a non-human animal, for example, a non-human mammal. The subject can be male or female.

[0214] The subject may be a patient. The patient may have a disease / condition described herein. The subject may have been diagnosed with a disease / condition described herein, may be suspected of having a disease / condition described herein, or may be at risk of developing a disease / condition described herein.

[0215] Subjects / patients may be selected for treatment / prevention according to the present disclosure based on the characterization of the markers for 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 is a subject who has or is at risk of developing a disease described herein.

[0216] 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 containing a predetermined amount of an antigen-binding molecule, nucleic acid, expression vector, cell, or composition described herein.

[0217] 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.

[0218] 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.

[0219] 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 percent sequence identity between the sequences. Pairwise and multiple sequence alignment for determining the percent sequence identity between two or more amino acid sequences or nucleic acid sequences can be achieved by various methods known to those skilled in the art, using publicly available computer software such as 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 are preferably used, for example, for gap penalties and extension penalties.

[0220] array

[0221] [Table 2-1]

[0222] [Table 2-2]

[0223] The present disclosure includes combinations of the described embodiments and preferred features unless such combinations are expressly disallowed 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.

[0224] 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.

[0225] 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.

[0226] 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. Ranges may be expressed herein 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, by use of the antecedent "about," it will be understood that the particular value forms another embodiment.

[0227] When a nucleic acid sequence is disclosed herein, the reverse complement thereof 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, while the term "in vivo" is intended to encompass procedures with or on intact multicellular organisms.

[0228] Embodiments and experiments illustrating the principles of the present disclosure will now be described with reference to the accompanying drawings. [Brief explanation of the drawings]

[0229] [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] Figure 2A is a sensorgram showing the binding of (2A)16C2.1 to human VEGFA as measured by biolayer interferometry (BLI). The concentrations tested for each DotBody are listed in nM below the binding curve. Figure 2B is a sensorgram showing the binding of (2B)21A5.1 to human VEGFA as measured by biolayer interferometry (BLI). The concentrations tested for each DotBody are listed in nM below the binding curve. [Figure 2B] Same as above. [Figure 3A] Figure 3A is a sensorgram showing the binding of (3A)16C2.1 to mouse VEGFA as measured by biolayer interferometry (BLI). The concentrations tested for each DotBody are listed in nM below the binding curve. Figure 3B is a sensorgram showing the binding of (3B)21A5.1 to mouse VEGFA as measured by biolayer interferometry (BLI). The concentrations tested for each DotBody are listed in nM below the binding curve. [Figure 3B] Same as above. [Figure 4] 1 is a graph showing inhibition of the interaction between human VEGFA and VEGFR1 by 16A2.1 and ranibizumab in a competitive ELISA. [Figure 5A] 5A-5C are graphs showing inhibition of the interaction between human VEGFA and VEGFR1 by (5A) 16C2.1, (5B) 21A5.1, and (5C) ranibizumab in a competitive ELISA. [Figure 5B]5A-5C are graphs showing inhibition of the interaction between human VEGFA and VEGFR1 by (5A) 16C2.1, (5B) 21A5.1, and (5C) ranibizumab in a competitive ELISA. [Figure 5C] 5A-5C are graphs showing inhibition of the interaction between human VEGFA and VEGFR1 by (5A) 16C2.1, (5B) 21A5.1, and (5C) ranibizumab in a competitive ELISA. [Figure 6A] Sensorgrams showing binding of anti-VEGFA DotBody (6A)16AC.1 and (6B)21A5.1 to human VEGFA at a single concentration of 250 nM after 1 hour incubation at room temperature, 60°C, 70°C, or 80°C. Measurements were performed by BLI as described in Example 8. [Figure 6B] Sensorgrams showing binding of anti-VEGFA DotBody (6A)16AC.1 and (6B)21A5.1 to human VEGFA at a single concentration of 250 nM after 1 hour incubation at room temperature, 60°C, 70°C, or 80°C. Measurements were performed by BLI as described in Example 8. [Example]

[0230] 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).

[0231] Library 1 was based on the following VH domain template sequence (positions mutated to create the library are underlined): SEVQLVESGGGLVQPGGSLRLSSAISGF SISSTS IDWVRQAPGKGLEWVARI SPSSGSTSYADSVKGRFTISADTSKNTVYLQMNSLRAEDTAVYYT 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 described by Bostrom J. et al. (14, 15) and Tonikian R. et al. (16) using the design shown in Figure 1. The primers used for library 1 are listed in Table 1, and the primers used for library 2 are listed in Table 2.

[0232] Library 1 contains approximately 2.87 × 10 10 Library 1 contained approximately 1.37 × 10 clones in which all CDRs were mutated, while Library 2 contained approximately 1.37 × 10 clones in which all CDRs were mutated. 10 The clones were included. The library was evaluated by serial dilution and colony counting after library transformation.

[0233] [Table 3]

[0234] [Table 4]

[0235] Example 2: Phage display selection from a naive synthetic DotBody phage display library Human VEGF-121 (Acro Biosystems) was immobilized in Maxisorp Immunotubes (Thermo Scientific) at 20 μg in 1 mL of PBS for round 1 and 10 μg in 1 mL of 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 hour at room temperature (RT). Negative selection tubes were prepared in the same manner as described for VEGF-121, except that PBS was used instead of VEGF-121 protein. 500 μL of Library 1 and Library 2 (approximately 2 × 10 13 The phage (pfu / mL) were precipitated with PEG / NaCl buffer (20% PEG 8,000, 2.5 M NaCl), resuspended in MBB, transferred to a negative selection tube, and incubated at room temperature for 1 hour. The phage were transferred to a VEGF-121-coated immunotube and incubated at room temperature for 2 hours. The tube was then washed three times with MBB, three times with PBST, and twice with PBS to remove unbound phage. Bound phage were eluted with 1 mg / mL trypsin in trypsin buffer (TBS + 2 mM CaCl). The eluted phage were used to culture phage in the logarithmic growth phase (OD 600 Approximately 0.5% of 5 mL of TG1 bacterial cell culture (in 2YT medium) was infected for 30 minutes 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 1 x 10 10The cells were infected with pfu / mL of M13K07 helper phage for 30 minutes at 37°C. Infected TG1 cells were pelleted at 3,900 g for 20 minutes at 4°C, resuspended in 500 μL of 2YT broth, and plated onto 2 × 15 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 phage was purified by precipitation with PEG / NaCl buffer. After two rounds of PEG / NaCl precipitation, the phage were resuspended in PBS + 10% glycerol. The purified phage were used for subsequent rounds of selection.

[0236] 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.

[0237] Round 3: 6 rounds with MBB, 6 rounds with PBST, and 2 rounds with PBS. · Round 4: 7 rounds in MBB, 7 rounds in PBST, 2 rounds in PBS. From the second selection round onwards, a final concentration of 1 x 10 12 One mL of purified phage from the previous selection round was used at pfu / mL. The rest of the panning procedure was the same.

[0238] Example 3: Identification of unique binders by monoclonal phage ELISA Monoclonal phage ELISA was used to identify unique binding DotBody clones selected from the naive and affinity-matured libraries. Monoclonal screening glycerol stocks 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, after which 1 × 10 10pfu / mL of M13K07 helper phage was used to infect the cells. The cultures were further supplemented with 50 μg / mL of kanamycin and incubated at 30° C. overnight.

[0239] Cells were pelleted by centrifugation at 1,100 g for 10 minutes 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 a Maxisorp 96-well plate (Thermo Scientific) overnight at 4°C, washed twice with PBS, and blocked with MBB for 1 hour at room temperature. 25 μL of phage culture supernatant was mixed with 25 μL of MBB, added to the plate, and incubated for 2 hours 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 hour 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 2 M H2SO4, and the signal was measured at 450 nm absorbance. 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.

[0240] 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 1.1 x 10 nucleotides, 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 30 colonies.8 It contained unique sequences.

[0241] [Table 5]

[0242] Example 5: Selection of 13A6-based affinity maturation phage display Neutravidin was immobilized in Maxisorp Immunotubes (Thermo Scientific) at 10 μg in 1 mL of PBS overnight at 4°C. Tubes were washed twice with PBS and blocked in MBB for 1 hour 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 hour at room temperature, and unbound protein was removed by washing twice with PBS. Negative selection tubes were prepared as described for biotinylated VEGF-121, except that PBS was added instead of biotinylated VEGF-121.

[0243] 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.

[0244] [Table 6]

[0245] 13A6-based affinity maturation phage display selection gave rise to 16C2.1. Example 6: 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 5. The resulting library contained 1.1 x 10 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:

[0246] [Table 7]

[0247] Example 7: Selection of 16C2.1-based affinity maturation phage display with heat challenge The 16C2.1-based phage display library was panned as follows.

[0248] Neutravidin was immobilized in Maxisorp Immunotubes (Thermo Scientific) at 10 μg in 1 mL of PBS overnight at 4°C. Tubes were washed twice with PBS and blocked in MBB at room temperature (RT) for 1 hour. Biotinylated VEGF-121 (Acro Biosystems) was added at different concentrations depending on the panning round (see Table 6). Proteins were incubated for 1 hour at room temperature, and unbound protein was removed by washing twice with PBS. Negative selection tubes were prepared as described for biotinylated VEGF-121, except that PBS was added instead of biotinylated VEGF-121.

[0249] 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 6.

[0250] [Table 8]

[0251] 16C2.1-based affinity maturation phage display selection with heat challenge gave rise to 21A5.1. Example 8: Protein production and characterization of binding kinetics by biolayer interferometry VEGFA-binding clones were cloned into pET-based expression vectors containing 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, purified by immobilized metal affinity chromatography, and subsequently desalted in PBS. The divalent molecule 16A2.1x2 was also produced in a similar manner.

[0252] Binding characterization was performed using a BLI (Satorius) at a flow rate of 1000 rpm at room temperature. Biotinylated human VEGF-165 (Acro Biosystems) was immobilized on a streptavidin-coated chip at 3 μg / mL in BLI buffer (0.1% BSA and 0.01% Tween-20 in PBS) for 60 seconds. After a 30-second baseline, anti-VEGFA DotBody was bound for 60 seconds in BLI buffer at eight different concentrations, including a blank reference, followed by dissociation for 400 seconds in BLI buffer. The background buffer signal was subtracted using a 0 nM 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 performed for 600 seconds. 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 Biosystems).

[0253] The sensorgrams are shown in Figures 2 and 3 and the binding data are shown in the table below:

[0254] [Table 9]

[0255] Example 9: Competitive ELISA To perform the competitive ELISA, VEGFR1 was immobilized at 2 μg / mL on a Maxisorp 96-well plate (Thermo Scientific) overnight at 4°C, washed three times with PBS, and blocked with ELISA Block buffer (EBB: 0.2% BSA in PBST) for 1 hour at room temperature. Human VEGF-121 at a concentration of 0.5 nM was mixed with purified anti-VEGFA DotBody at different concentrations after a 1:3 serial dilution starting at 500 nM and ending at 0.008 nM, with a 0 nM 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 a 0 nM control. Samples were incubated for 2 hours at room temperature, and 50 μL was transferred to the VEGFR1-coated plate. After the 2-hour incubation, the plate was washed three times with PBST. 50 μL of streptavidin-HRP conjugate (Thermo Scientific) was added at a 1:5,000 dilution to EBB and incubated for 1 hour at room temperature. The plate was washed five times with PBST and developed with 50 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate (GeneTex). After 5–15 minutes, the reaction was stopped by adding 50 μL of 2M H2SO4, and the signal was measured at 450 nm absorbance.

[0256] Data were plotted using Graphpad Prism 9 software and IC values ​​were calculated using a "[inhibitor] vs. response - variable slope (4 parameters)" nonlinear regression curve. 50 It was decided that:

[0257] 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: 16C2.1=2.3nM Ranibizumab = 6.8 nM.

[0258] 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: 16C2.1=1.8nM 21A5.1=12.8nM Ranibizumab = 7.4 nM Example 10: Evaluation of thermal stability by VEGFA binding analysis 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 15,000g for 5 minutes, and the supernatant was used to characterize binding to human VEGF165 by BLI, as described in Example 8 above.

[0259] The results are shown in Figure 6. Example 11: Conclusion The present inventors have generated stabilized VH domain antibodies that specifically bind to human VEGFA and cross-react with mouse VEGFA.

[0260] 16C2.1 has an affinity for human VEGFA of 3.0 nM and for mouse VEGFA of 14.6 nM. 16C2.1 blocks the VEGFA-VEGFR1 interaction with an estimated IC50 of 2.3 nM, which is 2-3 times better than the FDA-approved anti-VEGFA antibody ranibizumab, which had an IC50 of 6.8 nM in the same assay.

[0261] 16C2.1 was selected for further activity improvement. A new phage display library was generated in which each CDR position was mutated at approximately 50% of the residues present in 16C2.1 and 50% of the other amino acids. Selection based on binding to human VEGFA and stability was performed using decreasing antigen concentration, increasing wash cycles, and heat stress at increasing temperatures to identify the most stable and high-affinity anti-VEGF DotBody. This heat stress library identified clone 21A5.1.

[0262] 21A5.1 retained binding to human VEGFA with an affinity of 3.37 nM and to mouse VEGFA with an affinity of 15.3 nM. The ability of 21A5.1 to block the VEGF-VEGFR interaction was measured by competitive ELISA, which confirmed that it blocked the VEGFA-VEGFR1 interaction with an estimated IC50 of 12.8 nM. When ranibizumab was used as a control, the measured IC50 was 7.4 nM.

[0263] Both 16C2.1 and 21A5.1 retained the ability to bind VEGFA after incubation at temperatures ranging from room temperature to 80°C. In conclusion, through a series of adapted phage display library design and selection strategies, we obtained anti-VEGF DotBody 16C2.1 and 21A5.1, which bind to VEGFA with mid- to low-nanomolar affinity and to both human and mouse VEGFA. These DotBody 16C2.1 and 21A5.1 also show blocking of the VEGF-VEGFR interaction with IC50s in the low nanomolar range. 16C2.1 and 21A5.1 are highly thermostable, retaining binding to human VEGFA after incubation at temperatures ranging from room temperature to 80°C.

[0264] 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: 2, CDR2 having the amino acid sequence of SEQ ID NO:3, and A CDR3 having the amino acid sequence of SEQ ID NO: 4; or (b) CDR1 having the amino acid sequence of SEQ ID NO: 6, CDR2 having the amino acid sequence of SEQ ID NO: 7, and CDR3 having the amino acid sequence of SEQ ID NO:8 An antigen-binding molecule comprising a single domain antibody sequence incorporating:

2. The antigen-binding molecule of claim 1, comprising or consisting of an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

5.

3. The following FR: FR1 having the amino acid sequence of SEQ ID NO:9; FR2 having the amino acid sequence of SEQ ID NO: 10; FR3 having the amino acid sequence of SEQ ID NO: 11; and FR4 having the amino acid sequence of SEQ ID NO: 12 The antigen-binding molecule of claim 1 or claim 2, comprising a single domain antibody sequence incorporating:

4. The antigen-binding molecule of any one of claims 1 to 3, which inhibits the interaction between VEGFA and VEGFR.

5. 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.

6. A nucleic acid encoding the antigen-binding molecule described in any one of claims 1 to 5.

7. An expression vector comprising the nucleic acid of claim 6.

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

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

10. 10. An antigen-binding molecule according to any one of claims 1 to 5, a nucleic acid according to claim 6, an expression vector according to claim 7, a cell according to claim 8, or a composition according to claim 9, for use in medical treatment or prevention.

11. 10. An antigen-binding molecule according to any one of claims 1 to 5, a nucleic acid according to claim 6, an expression vector according to claim 7, a cell according to claim 8, or a composition according to claim 9, for use in the treatment or prevention of a disease in which VEGFA / VEGFR-mediated signaling is pathologically involved.

12. 12. The antigen-binding molecule, nucleic acid, expression vector, cell, or composition for use according to claim 11, wherein the disease is selected from diseases characterized by pathological angiogenesis, cancer, VEGFA-expressing cancer, VEGFR-expressing cancer, ocular 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.

13. 13. The antigen-binding molecule, nucleic acid, expression vector, cell, or composition for use according to claim 12, wherein the cancer is selected from solid tumors, hematological malignancies, myeloid hematological malignancies, acute myeloid leukemia, multiple myeloma, breast cancer, kidney cancer, renal cell carcinoma, lung cancer, non-small cell lung cancer, thyroid cancer, medullary thyroid cancer, brain / spinal cord cancer, glioblastoma, glioma, high-grade glioma, head and neck cancer, skin cancer, melanoma, squamous cell carcinoma, liver cancer, hepatocellular carcinoma, pancreatic cancer, gastric cancer, intestinal cancer, colon cancer, rectal cancer, colorectal cancer, bile duct cancer, cholangiocarcinoma, bone cancer, sarcoma, ovarian cancer, cervical cancer, peritoneal cancer, prostate cancer, urothelial cancer, or neuroendocrine cancer.

14. 6. The antigen-binding molecule of claim 1 , for use in a method for detecting, localizing, or imaging VEGFA, or cells / diseases / conditions containing / characterized by, or expressing VEGFA, or a method for selecting or stratifying subjects for treatment with a VEGFA-targeted drug, the method comprising detecting the formation of a complex between the antigen-binding molecule and VEGFA; or for use as an in vitro or in vivo diagnostic or prognostic agent.

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