HK40005903A - Method and means for detecting the level of total vegf-a - Google Patents

Description

Methods and means for detecting levels of total VEGF-A

Technical Field

The present invention relates to a method for measuring the level of VEGF-a in the presence of a VEGF-a antagonist, a kit comprising means for detecting VEGF-a in the presence of a VEGF-a antagonist, a composition of matter comprising a first and a second antibody suitable for detecting the level of VEGF-a in the presence of a VEGF-a antagonist, and a method of detecting a complex comprising human VEGF-a and a non-human or chimeric protein.

Background

Cancer is one of the most fatal threats to human health. Cancer affects nearly 130 million new patients annually in the united states alone and is the second cause of death after the endemic cardiovascular disease, accounting for 1 of approximately 4 deaths. Solid tumors are responsible for most of those deaths. Although medical treatment of certain cancers has been a significant advance, the overall 5-year survival rate for all cancers has improved only by about 10% over the past 20 years. Cancer (or malignant tumor) rapidly grows and metastasizes in an uncontrolled manner, making timely detection and treatment extremely difficult. Depending on the type of cancer, patients typically have several treatment options available to them, including chemotherapy, radiation, and antibody-based drugs. Diagnostic methods useful for predicting clinical outcomes from different treatment regimens would greatly benefit the clinical management of these patients.

It is now well established that angiogenesis is implicated in the pathogenesis of a variety of disorders. These include solid tumors, intraocular neovascular syndromes such as proliferative retinopathy or age-related macular degeneration (AMD), rheumatoid arthritis, 20 and psoriasis (Folkman et al, J.biol.chem.267: 10931-. In the case of solid tumors, neovascularization allows tumor cells to acquire a growth advantage and proliferative autonomy compared to normal cells. Thus, a correlation has been observed between the density of microvessels in tumor sections and patient survival in breast cancer as well as in several other tumors (Weidner et al, N.Engl. J.Med.324:1-6 (1991); Horak et al, Lancet340: 1120-52 1124 (1992); and Macchiarini et al, Lancet340:145-146 (1992)).

The critical role of Vascular Endothelial Growth Factor (VEGF) in the regulation of normal and abnormal angiogenesis has been established (Ferrara et al, endocr. Rev.18:4-25 (1997)). Even the finding that loss of one VEGF allele results in embryonic lethality points to the irreplaceable role this factor plays in the development and differentiation of the vascular system (Ferrara et al, supra). VEGF has also been shown to be a key mediator of neovascularization associated with tumors and intraocular disorders (Ferrara et al, supra). VEGF mRNA was overexpressed by most of the Human tumors examined (Berkman et al, J.Clin.Invest.91: 153-. Also, the concentration of VEGF in ocular fluids is highly correlated with the presence of active vascular proliferation in patients with diabetes and other ischemia-related retinopathies (Aiello et al, N.Engl. J.Med.331:1480-1487 (1994)). Moreover, studies have demonstrated the localization of VEGF in choroidal neovascular membranes in patients affected by Acute Macular Degeneration (AMD) (Lopez et al, invest. Ophtalmo. Vis. Sci.37:855-868 (1996)).

The ability to measure endogenous VEGF levels depends on the availability of sensitive and specific assays. Colorimetric, chemiluminescent, and fluorescent colorimetric-based enzyme-linked immunosorbent assays (ELISAs) for VEGF have been reported (Houck et al, supra (1992); Yeo et al, Clin.WO 2008/060777 PCT/US2007/080310Chem.38:71 (1992); Kondo et al, Biochim.Biophys.Acta1221:211 (1994); Baker et al, Obstet.Gvnecol.86:815 (1995); Hanatani et al, biosci.Biotechn.biochem.59: 1958 (1995); Leith and Michelson, Cell Prolif.28:415 (1995); Shien et al, J.Clin.Endocrinol.81: 3112 (1996); Takakaet al, Cancer et al, Cancer Res 56: 5: 187i, 1996, 187r et al, 1998: 18777: 19877, 1996) and 1996 et al, Cancer et al, 1998: 19858: 1996). For example, Houck et al, supra (1992), describe a colorimetric ELISA that appears to have ng/ml sensitivity, which may not be sufficient to detect endogenous VEGF levels. Yeo et al, supra (1992) describe a two-site time resolved immunofluorescent colorimetric assay, however, VEGF was not detected in normal serum (Yeo et al, Cancer Res.53:2912 (1993)). Using a modified version of this immunofluorescent colorimetric assay, Baker et al, supra (1995) report detectable levels of VEGF in plasma from pregnant women, with higher levels observed in women with preeclampsia. Similar data in pregnant women were reported by Anthony et al (Anthony et al, Ann. Clin. biochem.34:276(1997)) using radioimmunoassay. Hanatani et al, supra (1995) developed a chemiluminescent ELISA capable of measuring circulating VEGF and reported VEGF levels of 8-36pg/ml in serum from 30 normal individuals (male and female). Brekken et al, supra (1998) describe ELISA assays using antibodies with binding preferences for either VEGF alone or the VEGF: Flk-1 complex. An ELISA kit for VEGF detection is commercially available from R & DSystems (Minneapolis, MN). The R & D VEGF ELISA kit has been used in a sandwich assay, where a monoclonal antibody is used to capture the target VEGF antigen and a polyclonal antibody is used to detect VEGF. Webb et al, supra (1998). See also, e.g., Obermair et al, supra (1998). Keyt et al, J.biol.chem.271:7788-7795 (1996); keyt et al, J.biol.chem.271:5638 (1996); and Shifren et al, supra (1996) also developed a colorimetric ELISA based on a pair of double-stranded monoclonal antibodies. While this ELISA is able to detect elevated VEGF levels in cancer patients, it lacks the sensitivity required to measure endogenous levels of VEGF in normal individuals. Rodriguezet al, j.immunol.methods 219:45(1998) describes a two-site fluorescence colorimetry VEGF ELISA that produces a sensitivity of 10pg/ml VEGF in pure plasma or serum. However, this fluorescent colorimetric assay only detects the intact 165/165 and 165/110 species of VEGF (VEGF 165/165 has been reported to proteolytically cleave into three other forms: 165/110 heterodimer, 110/110 homodimer, and a 55 amino acid C-terminal fragment (Keyt et al, J.biol. chem.271: 7788-.

VEGF-A is a therapeutic antagonist that binds to VEGF-A and blocks its activity such as, for exampleThe target of (1). However, to date, no assay described or commercially available in the literature allows for the measurement of VEGF-A levels in the presence of such VEGF-A antagonists. Thus, there is an urgent need to develop assays and means to be used in these assays that allow the detection of VEGF-a levels in the presence of such antagonists, in particular in the presence of such therapeutic antagonists.

Summary of The Invention

In a first aspect, the invention relates to a method for measuring the level of VEGF-A in the presence of a VEGF-A antagonist, the method comprising incubating a sample with a first and a second antibody, wherein each of said first and said second antibody is capable of binding VEGF-A in the presence of the VEGF-A antagonist, and detecting the complex formed, thereby measuring the level of VEGF-A in the presence of the VEGF-A antagonist. In various embodiments, the binding of the first and the second antibody do not interfere with each other. In various embodiments, one of the antibodies is bound or capable of binding a solid phase and the other of the antibodies is detectably labeled. The detectably labeled complex formed comprises the first antibody, VEGF-a, and the second antibody.

In a second aspect, the invention relates to a kit for measuring the level of VEGF-A in the presence of a VEGF-A antagonist, the kit comprising a first and a second antibody, wherein both said first and said second antibody are capable of binding VEGF-A in the presence of the VEGF-A antagonist. In various embodiments, the binding of the first and the second antibody do not interfere with each other. In various embodiments, one of the antibodies is bound or capable of binding a solid phase and the other of the antibodies is detectably labeled.

In a third aspect, the invention relates to a composition of matter comprising a first and a second antibody, wherein both said first and said second antibody are capable of binding to VEGF-a or a variant thereof in the presence of a VEGF-a antagonist. In various embodiments, the binding of the first and the second antibody do not interfere with each other. In various embodiments, one of the antibodies is bound or capable of binding a solid phase and the other of the antibodies is detectably labeled.

In a fourth aspect, the invention relates to a method of detecting a complex comprising human VEGF-A and a non-human or chimeric protein, comprising the steps of (a) incubating a sample comprising said complex with a detectably labeled antibody that binds to or is capable of binding to the human VEGF-A and/or the non-human or chimeric protein, and (b) detecting the detectably labeled antibody or antigen-binding fragment thereof.

Brief Description of Drawings

FIG. 1A. assay design; assay design including VEGF-A receptor

FIG. 2 amino acid sequences of the light and heavy chains of antibodies M-13.2.5 and M-13.7.40; CDRs are highlighted in bold; FR is underlined

FIG. 3A schematic representation of Biacore sensorgram sequences with and without VEGF-A receptors R1 or R2

SEQ ID NO 1 human VEGF-A

Amino acid sequence of the light chain of SEQ ID NO 2M-13.2.5

Amino acid sequence of the heavy chain of SEQ ID NO 3M-13.2.5

Amino acid sequence of the light chain of SEQ ID NO 4: M-13.7.40

Amino acid sequence of the heavy chain of SEQ ID NO 5: M-13.7.40

Detailed Description

Definition of

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

Several documents are cited throughout the text of this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions for use, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. Some documents cited herein are characterized as "included by reference". In the event of a conflict between a definition or teaching of such incorporated reference and that set forth in this specification, the text of this specification takes precedence.

The elements of the present invention will be described below. These elements are listed in specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The wide variety of examples and preferred embodiments described should not be construed to limit the invention to only the explicitly described embodiments. Such description should be understood to support and encompass embodiments combining the explicitly described embodiments with any number of the disclosed and/or preferred elements. Moreover, any arrangement or combination of elements described in this application should be considered disclosed by the specification in this application unless the context indicates otherwise.

The word "comprising" will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise.

Concentrations, amounts, and other numerical data may be expressed or presented herein in a "range" format. It is to be understood that such range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. By way of illustration, a numerical range of "150 mg to 600 mg" should be interpreted to include not only the explicitly recited values of 150mg to 600mg, but also to include individual values and sub-ranges within the indicated range. Thus, included in this range of values are individual values such as 150,160,170,180,190, …,580,590,600mg and sub-ranges such as 150 to 200,150 to 250,250 to 300,350 to 600, and the like. This same principle applies to ranges reciting only one numerical value. Moreover, such interpretation should apply regardless of the breadth of the range or the features being described.

The term "about," when used in conjunction with a numerical value, is meant to encompass a range of values having a lower limit that is 5% less than the indicated value and an upper limit that is 5% greater than the indicated value.

The term "expression level" refers to the amount of gene product present in a body or sample at a certain point in time. The expression level can be measured/quantified/detected, for example, by means of the protein or mRNA expressed from the gene. The expression level can be quantified, for example, by normalizing the amount of the gene product of interest (e.g., mRNA or protein) present in a sample to the total amount of the gene product (total protein or mRNA) in the same category in the same sample or a reference sample (e.g., a sample taken from the same individual at the same time or a portion of the same sample of the same size (weight, volume), or by identifying the amount of the gene product of interest in each defined sample size (weight, volume, etc.). Expression levels can be measured or detected by any method known in the art, for example, methods for directly detecting and quantifying a gene product of interest (such as mass spectrometry) or methods for indirectly detecting and measuring a gene product of interest (typically operated via binding of a gene product of interest to one or more different molecules or detection means specific for a gene product of interest (e.g., primers, probes, antibodies, protein scaffolds)). It is also within the knowledge of the skilled person to determine the gene copy level of the gene product of interest, further comprising determining the deletion or presence of one or more fragments (e.g. via nucleic acid probes or primers, such as quantitative PCR, multiple ligation dependent probe amplification (MLPA) PCR).

In the context of the present invention, the term "peptide" refers to a short polymer of amino acids linked by peptide bonds. It has the same chemical (peptide) bond as the protein, but is generally shorter in length. The shortest peptide is a dipeptide, consisting of two amino acids linked by a peptide bond. Also, tripeptides, tetrapeptides, pentapeptides, etc. may be used. Typically, peptides have a length of up to 8,10,12,15,18 or 20 amino acids. The peptide has an amino terminus and a carboxyl terminus unless it is a cyclic peptide.

In the context of the present invention, the term "polypeptide" refers to a linear chain of amino acids bonded together by peptide bonds and typically comprises at least about 21 amino acids. The polypeptide may be one chain of a protein consisting of more than one chain, or it may be the protein itself if the protein consists of one chain.

In the context of the different aspects of the present invention, the term "protein" refers to a molecule comprising one or more polypeptides restoring secondary and tertiary structure and additionally to a protein consisting of several polypeptides, i.e. several subunits, forming a quaternary structure. Proteins are sometimes attached to non-peptide groups, which may be referred to as prosthetic groups or cofactors.

As used herein, the term "complex" refers to an ensemble comprising several individual components, portions or modules that are in close proximity to each other and perform a common or related function. The individual modules of the complexes may be of the same or different nature, i.e., they may be composed of the same, similar, or different chemical entities, such as, but not limited to, nucleotides, amino acids, nucleic acids, peptides, polypeptides, proteins, carbohydrates, or lipids. For example, a complex may comprise some protein of interest, or a mixture of one or more proteins and one or more nucleic acids, or a mixture of one or more proteins and one or more lipids and/or carbohydrates. It is understood that any other combination of the same, similar or different chemical entities is also contemplated. The individual modules of the composite may or may not be interconnected. Typically, the individual moieties of the complex are linked via covalent or non-covalent bonds. For example, a complex may comprise a protein and a receptor to which it binds, or a complex may comprise a protein and an antibody that binds an epitope of the protein.

The term "antigen" refers to any substance that raises the immune system to produce antibodies against it. The antigen may be derived from the in vivo ("self-antigen") or from the external environment ("non-self"). Antigen presenting cells present antigen as a peptide on a histocompatibility molecule. T cells of the adaptive immune system recognize antigens. Depending on the type of antigen and histocompatibility molecule, different types of T cells are activated.

An "epitope", also called "antigenic determinant", is a segment of a macromolecule, in particular a segment of an antigen, which is recognized by the immune system, in particular antibodies, B cells, or T cells. An epitope is typically part of an antigen and is capable of binding to an antibody or antigen-binding fragment thereof. In this context, the term "binding" preferably relates to specific binding. In the context of the present invention, the term "epitope" refers to a segment of a protein that is recognized by an antibody. Epitopes are usually composed of molecules, chemically active surface groupings such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that binding to the former, but not the latter, is lost in the presence of denaturing solvents.

As used herein, the term "variant" is to be understood as a polypeptide or polynucleotide that differs from the polypeptide or polynucleotide from which it was derived by one or more changes in its length or sequence. A polypeptide or polynucleotide from which a variant of the polypeptide or polynucleotide is derived is also referred to as a parent polypeptide or polynucleotide. The term "variant" encompasses a "fragment" or "derivative" of a parent molecule. Typically, a "fragment" is smaller in length or size than the parent molecule, while a "derivative" exhibits one or more differences in their sequence compared to the parent molecule. Also contemplated are modified molecules such as, but not limited to, post-translationally modified proteins (e.g., glycosylated, biotinylated, phosphorylated, ubiquinated, palmitoylated, or proteolytically cleaved proteins) and modified nucleic acids (such as methylated DNA). Mixtures of different molecules (such as, but not limited to, RNA-DNA hybrids) are also encompassed by the term "variant". Typically, variants are constructed artificially, preferably by means of genetic techniques, while the parent protein or polynucleotide is the wild-type protein or polynucleotide, or a consensus sequence thereof. However, naturally occurring variants are also to be understood as being encompassed by the term "variant" as used herein. Furthermore, variants which can be used in the present invention may also be derived from homologues, orthologues, or paralogues of the parent molecule or artificially constructed variants, provided that the variant exhibits at least one biological activity, i.e. is functionally active, of the parent molecule.

In particular, the terms "peptide variant", "polypeptide variant", "protein variant" are to be understood as a peptide, polypeptide, or protein that differs from the peptide, polypeptide, or protein from which it was derived by one or more changes in the amino acid sequence. The peptide, polypeptide, or protein from which the peptide, polypeptide, or protein variant is derived is also referred to as the parent peptide, polypeptide, or protein. Furthermore, variants that may be used in the present invention may also be derived from a homolog, ortholog, or paralog of a parent peptide, polypeptide, or protein or an artificially constructed variant, provided that the variant exhibits at least one biological activity of the parent peptide, polypeptide, or protein. The change in amino acid sequence may be an amino acid exchange, insertion, deletion, N-terminal truncation, or C-terminal truncation, or any combination of these changes, which may occur at one or several sites. A peptide, polypeptide, or protein variant may exhibit up to 50 (up to 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45, or 50) changes in amino acid sequence (i.e., exchanges, insertions, deletions, N-terminal truncations, and/or C-terminal truncations). Amino acid exchanges can be conservative, semi-conservative, and/or non-conservative. Semi-conservative and especially conservative amino acid substitutions are preferred, wherein an amino acid is substituted with a chemically related amino acid. Typical substitutions are between aliphatic amino acids, between amino acids having aliphatic hydroxyl side chains, between amino acids having acidic residues, between amide derivatives, between amino acids having basic residues, or between amino acids having aromatic residues. Typical semi-conservative and conservative substitutions are:

a, F, H, I, L, M, P, V, W or Y to C is semi-conserved if the new cysteine remains as a free thiol moreover, the skilled artisan will appreciate that the glycine at the sterically required position should not be replaced and that P should not be introduced in the part of the protein having the α helix or β sheet structure.

Alternatively, or in addition, as used herein, a "variant" may be characterized by some degree of sequence identity to the parent peptide, polypeptide, or protein from which it is derived. More precisely, a peptide, polypeptide, or protein variant exhibits at least 80% sequence identity with its parent peptide, polypeptide, or protein in the context of the present invention. In particular, the variant may exhibit at least 85%, 90%, 95%, 97%, or 99% sequence identity. The sequence identity of a peptide, polypeptide, or protein variant is over a contiguous stretch of 20,30,40,45,50,60,70,80,90,100 or more amino acids.

The terms "biomarker" or "indicator" are used interchangeably herein. In the context of the present invention, a biomarker may be defined as a substance within a biological system that serves as an indicator of the biological state of the system. In the art, the term "biomarker" is sometimes also applied to means for detecting said endogenous substance (e.g. antibodies, nucleic acid probes etc., imaging systems). In the context of the present invention, the term "biomarker" will only apply to a substance, not to a detection means. Thus, a biomarker may be any kind of molecule present in a living organism, such as a nucleic acid (DNA, mRNA, miRNA, rRNA, etc.), a protein (cell surface receptor, cytosolic protein, etc.), a metabolite or hormone (blood glucose, insulin, estrogen, etc.), a molecule characteristic of some modification of another molecule (e.g. a sugar module or phosphoryl residue on a protein, a methyl residue on genomic DNA) or a substance that has been internalized by the organism or a metabolite of such a substance.

The term "VEGF" refers to VEGF from human and non-human species such as mouse, rat or primate. Sometimes, VEGF from a particular species is indicated by terminology, such as hVEGF for human VEGF, mVEGF for murine VEGF, etc.

"VEGF biological activity" includes, but is not limited to, binding of VEGF to any VEGF receptor, and VEGF signaling activity, such as modulation of both normal and abnormal angiogenesis (angiogenisis) and vasculogenesis (vasculogenesis) (Ferrara and Davis-Smyth (1997) endothelial Rev.18: 4-25; Ferrara (1999) J.mol.Med.77: 527-543); promote embryonic vasculogenesis and angiogenesis (Carmeliet et al (1996) Nature380: 435-; and regulate periodic vascular proliferation in the female reproductive tract and for bone growth and cartilage formation (Ferrara et al (1998) Nature Med.4: 336-. In addition to being an angiogenic factor in angiogenesis and vasculogenesis, VEGF, as a pleiotropic growth factor, exhibits a variety of biological effects in other physiological processes, such as endothelial cell survival, vascular permeability and vasodilation, monocyte chemotaxis and calcium influx (Ferrara and Davis-Smyth (1997), supra and Cebe-Suarez et al (2006) cell. mol. Life Sci.63: 601-. Moreover, recent studies have reported mitogenic effects of VEGF on a few non-endothelial cell types, such as retinal pigment epithelial cells, pancreatic ductal cells, and Schwann cells (Guerrin et al (1995) J.cell Physiol.164: 385-.

In the context of the present invention, "VEGF-A" refers to vascular endothelial growth factor protein A, e.g., SEQ ID NO:1(Swiss Prot accession number P15692, Gene ID (NCBI): 7422). VEGF-a can form disulfide-linked homodimers and act as glycosylated mitogens that act specifically on endothelial cells and have a variety of effects including mediating increased vascular permeability, inducing angiogenesis, vasculogenesis and endothelial cell growth, promoting cell migration, and inhibiting apoptosis. The term "VEGF-A" encompasses proteins having the amino acid sequence of SEQ ID NO 1 as well as isoforms, fragments and variants thereof. Alternatively spliced transcripts encoding either freely secreted or cell associated isoforms have been characterized, e.g., splice isoforms of VEGF-A, e.g., VEGF₁₂₁,VEGF₁₄₅,VEGF₁₆₅,VEGF₁₈₉And VEGF₂₀₆Together with naturally occurring allelic and processed forms thereof. Fragments include, but are not limited to, cleavage of VEGF by plasmin₁₆₅The 110 amino acid human vascular endothelial cell growth factor produced was as described (Ferrara (2010) mol. biol. cell 21: 687; Leung et al (1989) Science 246: 1306; and Houck et al (1991) mol. Endocrin.5: 1806). The term "VEGF-A" thus relates to proteins having the amino acid sequence of SEQ ID NO:1 and to splicing isoforms of VEGF₁₂₁,VEGF₁₄₅,VEGF₁₆₅,VEGF₁₈₉And VEGF₂₀₆110 amino acid fragments thereof, and variants, splicing isoforms of the amino acid sequence of SEQ ID NO 1VEGF₁₂₁,VEGF₁₄₅,VEGF₁₆₅,VEGF₁₈₉And VEGF₂₀₆And variants of 110 amino acid fragments thereof.

The two best characterized VEGF receptors are "VEGFR 1" (also known as Flt-1) and "VEGFR 2" (the murine homologs also known as KDR and FLK-1). Each receptor has variations in specificity with respect to each VEGF family member, but VEGF-A binds both Flt-1 and KDR. The full-length Flt-1 receptor includes an extracellular domain with seven Ig domains, a transmembrane domain, and an intracellular domain with tyrosine kinase activity. The extracellular domain is involved in the binding of VEGF and the intracellular domain is involved in signal transduction. VEGF-A receptor molecules or fragments thereof that specifically bind VEGF-A may be used as VEGF-A inhibitors that bind and sequester VEGF-A protein, thereby preventing it from signaling. Also, the soluble form of the receptor exerts an inhibitory effect on the biological activity of the VEGF-a protein by binding to VEGF-a, thereby preventing it from binding to its native receptor present on the surface of the target cell.

The terms "disease" and "disorder" are used interchangeably herein to refer to an abnormal condition, particularly an abnormal medical condition, such as a disease or a lesion, in which a tissue, organ or individual is no longer able to effectively fulfill its function. Typically, but not necessarily, a disease is associated with a particular symptom or sign indicative of the presence of such a disease. The presence of such symptoms or signs may thus indicate that the tissue, organ or individual is suffering from a disease. Changes in these symptoms or signs may indicate the progression of such diseases. Progression of the disease is typically characterized by an increase or decrease in such symptoms or signs, which may indicate "worsening" or "worsening" of the disease. "deterioration" of a disease is characterized by a decreased ability of a tissue, organ or organism to effectively perform its function, while "improvement" of a disease is typically characterized by an increased ability of a tissue, organ or individual to effectively perform its function. Tissues, organs or individuals at "risk of developing disease" are in a healthy state but show the potential for disease manifestation. Typically, the risk of disease development is associated with early or weak signs or symptoms of such diseases. In such cases, the onset of the disease can still be prevented by treatment. Examples of diseases include, but are not limited to, traumatic diseases, inflammatory diseases, infectious diseases, skin conditions, endocrine diseases, intestinal diseases, neurological disorders, joint diseases, genetic disorders, autoimmune diseases, and various types of cancer.

The terms "cell proliferative disorder" and "proliferative disorder" refer to a disorder associated with some degree of abnormal cell proliferation.

As used herein, the term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive as referred to herein.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell cancer, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including, for example, gastrointestinal cancer), pancreatic cancer (including, for example, metastatic pancreatic cancer), glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (including locally advanced, recurrent or metastatic HER-2 negative breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic cancer, and various types of head and neck cancer, and B-cell lymphoma (including low-grade/follicular non-Hodgkin's lymphoma (NHL), Small Lymphocytic (SL) NHL, medium-grade/follicular NHL, medium-grade diffuse NHL, high-level immunocytocytic NHL, high-grade lymphoblastic NHL, high-grade small non-Hodgkin's lymphoma (NHL), and small-scale non-lymphocytic lymphomas A mitotic cell NHL; storage disease (bulk disease) NHL; mantle cell lymphoma; AIDS-related lymphomas; and waldenstrom's macroglobulinemia); chronic Lymphocytic Leukemia (CLL); acute Lymphoblastic Leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with scarring nevus, edema (such as that associated with brain tumors), and meigs' syndrome.

"symptoms" of a disease are suggestive of a disease perceptible to a tissue, organ, or organism having such a disease and include, but are not limited to, pain, weakness, sensitivity (tenderness), tension (strain), stiffness, and spasm in the tissue, organ, or individual. "signs" or "signals" of a disease include, but are not limited to, changes or alterations in a particular indicator, such as a biomarker or molecular marker, such as presence, absence, elevation or elevation, decrease or decline, or the occurrence, presence, or deterioration of a symptom. Symptoms of pain include, but are not limited to, unpleasant sensations that can be felt as persistent or changing burns, paroxysmal pain, itching, or stinging pain (burning, stabbing, itching).

As used herein, "patient" means any mammal, fish, reptile or bird that can benefit from the diagnosis or treatment disclosed herein. In particular, the "patient" is selected from the group consisting of laboratory animals (e.g. mice, rats, rabbits, or zebrafish), domesticated animals (including, for example, guinea pigs, rabbits, horses, donkeys, cattle, sheep, goats, pigs, chickens, camels, cats, dogs, turtles, terrapin, snakes, lizards, or goldfish), or primates (including chimpanzees, bonobos, gorillas, and humans). In particular, a "patient" is a human.

The terms "sample" or "sample of interest" are used interchangeably herein to refer to a portion or piece/sheet of a tissue, organ or individual, typically smaller than such tissue, organ or individual, intended to represent the entire tissue, organ or individual. Once analyzed, the sample provides information about the state of the tissue or the health or diseased state of the organ or individual. Examples of samples include, but are not limited to, fluid samples such as blood, serum, plasma, synovial fluid, urine, saliva, and lymph fluid, or solid samples such as tissue extracts. The analysis of the sample may be achieved based on visual inspection or chemistry. Visual analysis includes, but is not limited to, microscopic imaging or radiographic scanning of tissues, organs or individuals, which allows morphological assessment of the sample. Chemical analysis includes, but is not limited to, detecting the presence or absence of specific indicators or changes in their amounts or levels.

As used herein, the term "reference sample" refers to a sample that is analyzed in substantially the same manner as a sample of interest and whose information is compared to that of the sample of interest. The reference sample thus provides a standard allowing the evaluation of the information obtained from the sample of interest. The reference sample may be derived from a healthy or normal tissue, organ or individual, thereby providing a standard of health status of the tissue, organ or individual. A difference between the status of the normal reference sample and the status of the sample of interest may be indicative of the risk of disease development or the presence or further progression of such a disease or disorder. The reference sample may also be derived from the same tissue, organ, or individual as the sample of interest but collected at an earlier time point. A difference between the status of the reference sample taken earlier and the status of the sample of interest may indicate the progression of the disease, i.e. the disease becomes better or worse over time. In the case of a time lapse between the collection of the reference sample and the collection of the sample of interest, the reference sample is collected at an earlier or later point in time. Such time periods may represent months (1,2,3,4,5,6,7,8,9,10,11,12 months), weeks (e.g., 1,2,3,4,5,6,7,8 weeks), days (e.g., 1,2,3,4,5,10,15,20,25,30,35,40,45,50,60,70,80,90,100,150,200,250,300,350 days), or hours (1,2,3,4,5,6,7,8,9,10,11,12 hours).

The term "decreased" or "reduced" level of an indicator means that the level of such indicator in a sample is reduced as compared to a reference or reference sample. The term "elevated" or "elevated" level of an indicator means that the level of such indicator in a sample is high compared to a reference or reference sample.

The term "agonist" as used herein refers to a substance that causes an effect such as receptor signaling, gene expression, protein synthesis, and protein degradation in a tissue, organ, or individual. Typically, agonists act by binding to the active site or allosteric site of the receptor molecule, thereby triggering a specific response. Examples of agonists include, but are not limited to, nucleic acid molecules, such as mRNA or miRNA, or proteins, such as hormones, cytokines, growth factors, neurotransmitters, and transcription factors.

The term "antagonist" as used herein refers to a substance that blocks the action of an agonist. Typically, antagonists act by binding to the active site or allosteric site of the receptor molecule, or interacting with a unique binding site that is not normally involved in modulating the activity of the receptor. Typically, an antagonist competes with an agonist at a structurally defined binding site or alters the binding site of an agonist in such a way that, due to its binding, the agonist is unable to elicit the effect that it would normally elicit. Antagonist activity can be reversible or irreversible depending on the lifetime of the antagonist-receptor complex interaction. Examples of antagonists include, but are not limited to, nucleic acid molecules, such as siRNA or miRNA, or proteins, such as hormones, cytokines, growth factors or neurotransmitters, antibodies, or transcription factors.

The term "antagonist antibody" refers to an antibody that partially or completely reduces or completely prevents at least one functional activity of a molecule of interest (e.g., a peptide, polypeptide, or protein of interest). Typically, an antagonistic antibody binds to the active site of the receptor and thereby prevents binding of the agonist to the receptor, or binds to the receptor at a different site in a manner that sterically hinders the agonist from binding to the receptor.

The term "receptor" as used herein refers to a molecule, such as a protein or polynucleotide, to which one or more specific signaling molecules bind. Signaling molecules may function as agonists or antagonists, including but not limited to nucleic acid molecules such as siRNA or miRNA, or proteins such as hormones, cytokines, growth factors or neurotransmitters, antibodies or transcription factors. Receptors can be localized at the plasma membrane of a cell, in the cytosol and/or in the intracellular compartment.

The term "immunoglobulin (Ig)" as used herein refers to an immune-conferring glycoprotein of the immunoglobulin superfamily, "surface immunoglobulins" attach to the membrane of effector cells through their transmembrane regions and encompass molecules such as, but not limited to, B cell receptors, T cell receptors, Major Histocompatibility Complex (MHC) class I and II proteins, β -2 microglobulin (-2M), CD3, CD4 and CDs.

Typically, the term "antibody" as used herein refers to a secreted immunoglobulin that lacks a transmembrane region and is thus capable of being released into the blood stream and body cavities. Human antibodies are grouped into different isotypes based on the heavy chains they possess. There are five types of human Ig heavy chains that define the class of antibodies, i.e., IgA, IgD, IgE, IgG, and IgM antibodies, each performing a different role and directing the appropriate immune response against a different type of antigen. IgA is found in mucosal areas such as the intestine, respiratory and genitourinary tracts, as well as in saliva, tears, and milk and prevents pathogen colonization (Underdown & Schiff (1986) Annu. Rev. immunol.4: 389-417). IgD functions primarily as an antigen receptor on B cells that have not been exposed to an antigen and is involved in activating basophils and mast cells to produce antimicrobial factors (Geisberger et al (2006) Immunology 118: 429-437; Chen et al (2009) nat. immunol.10: 889-898). IgE is involved in an allergic reaction, which triggers the release of histamine from mast cells and basophils via its binding to allergens. IgE is also implicated in protection against parasitic worms (Pier et al (2004) Immunology, Infection, and Immunity, ASMPress). IgG provides the majority of antibody-based immunity to invasive pathogens and is the only isotype of antibody that can pass through the placenta to give passive immunity to the fetus (Pier et al (2004) Immunology, Infection, and Immunology, ASM Press). There are four different IgG subclasses in humans (IgG1,2,3, and 4), named in order of their abundance in serum, IgG1 is the most abundant (-66%), followed by IgG2 (-23%), IgG3 (-7%), and IgG (-4%). The biological profile of the different IgG classes is determined by the structure of the corresponding hinge region. IgM is expressed as a monomer on the surface of B cells and as a secreted pentamer with high affinity. IgM is involved in eliminating pathogens in the early stages of B cell-mediated (humoral) immunity before sufficient IgG production (Geisberger et al (2006) Immunology 118: 429-437). Not only are antibodies found as monomers, but they are also known to form dimers of two Ig units (e.g., IgA), tetramers of four Ig units (e.g., IgM for teleost fish), or pentamers of five Ig units (e.g., mammalian IgM).

Antibodies typically consist of four polypeptide chains, comprising two identical heavy chains and two identical light chains, which are linked via disulfide bonds and resemble a "Y" -shaped macromolecule, each chain comprising some immunoglobulin domains, some of which are constant domains and others of which are variable domains the immunoglobulin domains consist of a two-layer sandwich of 7 to 9 antiparallel chains arranged in two β sheets the heavy chains of an antibody typically comprise four Ig domains, three of which are constant (CH domains: CH1, CH2, CH3) and one of which is a variable domain (VH), the light chains typically comprise one constant Ig domain (CL) and one variable Ig domain (VL), e.g., a human IgG heavy chain consists of four Ig domains linked in the order VL1-CH1-CH2-CH3 from N to C-terminus, while a human IgG light chain consists of two immunoglobulin domains linked in the order VL-CL from N to C-terminus, or kappa (λ) or lambda (V-C) kappa-type kappa or V-C447 from N to C-terminal, e.g., a human IgG light chain comprises a constant amino acid.

The terms "hypervariable region (HVR or HV)" and "Complementarity Determining Region (CDR)" are used interchangeably herein and refer to regions of an antibody variable domain that are mutated in sequence and/or form structurally defined loops. Typically, an antibody comprises six HVRs or CDRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Among natural antibodies, H3 and L3 display most of the diversity of six HVRs, and H3 is specifically thought to play a unique role in conferring fine specificity to antibodies. See, e.g., Xu et al (2000) Immunity 13: 37-45; johnson and Wu, in Methods in Molecular Biology 248:1-25(Lo, ed., Human Press, Totowa, NJ, 2003). Indeed, naturally occurring camelid antibodies consisting of only the heavy chain are functional and stable in the absence of the light chain. See, for example, Hamers-Casterman et al, (1993) Nature 363:446 + 448 and Sheriff et al, (1996) Nature struct. biol.3:733 + 736. Some HVR depictions are used and encompassed herein. The HVRs, the Kabat Complementarity Determining Regions (CDRs), are based on sequence variability and are most commonly used (Kabat et al, Sequences of Proteins of Immunological Interest,5th Ed. public health service, National Institutes of health, Bethesda, Md. (1991)). Chothia instead refers to the location of the structural loops (Chothia and Lesk (1987) J.mol.biol.196: 901-. AbM HVR represents a compromise between Kabat CDR and Chothia structural loops and is obtained by Oxford Molecular AbM antibody simulation software use. The "contact" HVR is based on analysis of the crystal structure of the available complex. Residues from each of these HVRs are recorded below.

The HVRs may comprise "extended HVRs" as described below, 24-36 or 24-34(L1),46-56 or 50-56(L2), and 89-97 or 89-96(L3) in the VL, and 26-35(H1),50-65 or 49-65(H2), and 93-102,94-102, or 95-102(H3) in the VH. For each of these extended HVR definitions, the variable domain residues are numbered according to Kabat et al, supra.

Typically, four FRs separate three HVRs and form an β sheet structure, which act as a scaffold to hold the HV region in place in contact with the antigen.

The expression "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat", and variants thereof, refers to the numbering system of the heavy chain variable domain or the light chain variable domain as used in antibody compilation by Kabat et al, supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids, corresponding to a shortening or insertion of the FR or HVR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert after residue 52 of H2 (residue 52a according to Kabat) and residues inserted after heavy chain FR residue 82 (e.g., residues 82a,82b, and 82c, etc. according to Kabat). "EU index as in Kabat" refers to the residue numbering of the human IgG lEU antibody. Thus, in the context of IgG the CH domains are as follows, "CHI" refers to amino acid position 118-; "CH 2" refers to amino acid position 237-; and "CH 3" refers to amino acid position 341- "447 according to the EU index as in Kabat.

The term "binding affinity" generally refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including but not limited to surface plasmon resonance based assays (such as BIAcore assay as described in PCT application publication No. WO 2005/012359); enzyme-linked immunosorbent assay (ELISA); and competitive assays (e.g., of RIA). Low affinity antibodies generally bind antigen slowly and tend to dissociate readily, while high affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods for measuring binding affinity are known in the art, and all of them can be used for the purposes of the present invention. Specific exemplary and illustrative embodiments for measuring binding affinity are described below.

"Kd" or "Kd value" can be used at 25 ℃ in-10 Response Units (RU) using immobilized antigen CM5 chipsOrThe instrument (BIAcore, inc., Piscataway, NJ) was measured using surface plasmon resonance assay. Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) were activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen was diluted to 5 μ g/ml (. about.0.2. mu.M) with 10mM sodium acetate, pH 4.8, and then injected at a flow rate of 5 μ l/min to achieve approximately 10 Response Units (RU) of conjugated protein. After injection of the antigen, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions (0.78nM to 500nM) of Fab in PBS (PBST) containing 0.05% TWEEN 20. TM. surfactant were injected at 25 ℃ at a flow rate of about 25. mu.l/min. Using a simple one-to-one Langmuir (Langmuir) binding model (Evaluation Software version 3.2) calculate the association rate (k) by simultaneously fitting the association and dissociation sensorgrams_on) And dissociation rate (k)_off). As a ratio k_off/k_onThe equilibrium dissociation constant (Kd) was calculated. See, e.g., Chen et al (1999) J.mol.biol.293: 865-. If the binding rate according to the above surface plasmon resonance assay exceeds 10⁶M^-1s^-1The rate of binding can then be determined using fluorescence quenching techniques, i.e.according to a spectrometer such as an Aviv Instruments spectrophotometer or 8000 series SLM-AMINCO^TMMeasurement in a stirred cuvette in a spectrophotometer (ThermoSpectronic) measured the increase or decrease in fluorescence emission intensity (excitation 295 nM; emission 340nM,16nM bandpass) of 20nM anti-antigen antibody (Fab form) in PBS at pH 7.2 in the presence of increasing concentrations of antigen at 25 ℃.

"rate of incorporation", or "k_on"may also be used as described aboveOrSystematic (BIAcore, inc., Piscataway, NJ).

Typically, antibodies bind their target with sufficient binding affinity, e.g., with a K between 500nM and 1pM_dValues, i.e. 500nM,450nM,400nM,350nM,300nM,250nM,200nM,150nM,100nM,50nM,1nM,900pM,800pM,700pM,600pM,500pM,400pM,300pM,200pM,100pM,50pM,1 pM.

An "affinity matured" antibody is one that has one or more alterations in one or more of its HVRs, which result in an improvement in the affinity of the antibody for an antigen as compared to a parent antibody that does not possess those alterations. Affinity matured antibodies have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are generated by procedures known in the art. For example, Marks et al (1992) Bio/Technology10:779-783 describes affinity maturation by VH and VL domain shuffling. For example, Barbas et al (1994) Proc Nat. Acad. Sci. USA 91: 3809-; schier et al (1995) Gene 169: 147-155; yelton et al (1995) J.Immunol.155: 1994-2004; jackson et al (1995) J.Immunol.154(7): 3310-9; and Hawkins et al (1992) J.mol.biol.226:889-896 describe random mutagenesis of HVRs and/or framework residues.

The term antibody also encompasses "antigen-binding fragments" thereof. The term "antigen-binding fragment" of an antibody refers to a molecule that possesses the ability to bind antigen in a similar manner to an antibody, but is smaller in size than the intact antibody molecule. For example, three "antigen-binding fragments" of an antibody, i.e., two identical fragments, called "Fab fragments" (also called "Fab portions" or "Fab regions"), each having an antigen binding site, and the remaining "Fc fragment" (also called "Fc portion" or "Fc region"), the name reflecting its ability to crystallize readily, are obtained by papain digestion to produce three fragments. In IgG, IgA and IgD isotypes, the Fc region is composed of two identical protein fragments derived from the CH2 and CH3 domains of the two heavy chains of an antibody; in the IgM and IgE isotype, the Fc region contains three heavy chain constant domains (CH2-4) in each polypeptide chain. In addition, other antigen-binding fragments occur naturally or are constructed artificially. The term "Fab 'fragment" refers to a Fab fragment that additionally comprises the hinge region of an Ig molecule, whereas "F (ab')₂A fragment "is understood to comprise two Fab' fragments linked either chemically or via a disulfide bond. "Single domain antibodies (sdAbs)" (Desmyter et al (1996) nat. Structure biol.3: 803-. A bivalent single-chain variable fragment (di-scFv) can be engineered by linking two scFvs (scFvA-scFvB). This can be done by generating one peptide chain with two VH and two VL regions, resulting in a "tandem scFv" (VHA-VLA-VHB-VLB). Another possibility is to create an scFv with a linker that is too short for the two variable regions to fold together, forcing the scFv to dimerize. Typically, linkers having a length of 5 residues are used to generateThese dimers are formed. This type is called "diabodies". The still shorter linker (one or two amino acids) between the VH and VL domains results in the formation of a monospecific trimer, the so-called "triabody" or tribody. Bispecific diabodies are formed by expressing chains with the permutations VHA-VLB and VHB-VLA or VLA-VHB and VLB-VHA, respectively. Single chain diabodies (scDb) comprise VHA-VLB and VHB-VLA fragments (VHA-VLB-P-VHB-VLA) connected by a linker peptide (P) of 12-20 amino acids, preferably 14 amino acids. A "bispecific T cell engager (BITE)" is a fusion protein consisting of two scFv of different antibodies, one of which binds to T cells via the CD3 receptor and the other binds to tumor cells via a tumor-specific molecule (Kufer et al (2004) Trends Biotechnol.22: 238-244). Dual affinity retargeting molecules ("DART" molecules) are diabodies that are additionally stabilized via C-terminal disulfide bridges.

The terms "antibody" and "antigen-binding fragment thereof" also encompass variants of an antibody or variants of an antigen-binding fragment thereof. With respect to any protein variant as defined above, a variant of an antibody or a variant of an antigen-binding fragment thereof is also to be understood as an antibody or antigen-binding fragment which differs from the antibody or antigen-binding fragment from which it was derived by one or more changes in its length or sequence, as defined above in detail with respect to the protein variant. In addition, variants of an antibody or antigen-binding fragment thereof may exhibit varying degrees of sequence identity in different portions of the antibody or antigen-binding fragment. With respect to framework regions, a degree of variability is contemplated herein, i.e., individual FRs may comprise or consist of the specifically recited amino acid sequence or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 92.5%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical. It will be appreciated that for different FRs, different degrees of sequence identity may be tolerable, depending on the actual sequence and, for example, the length of the corresponding FR sequence, as well as its positioning within the corresponding variable chain domain. However, in variants of an antibody or antigen-binding fragment thereof, the CDRs may have the specifically recited sequences of the CDRs, or may be distinguished therefrom by at most one amino acid substitution. As such, one amino acid in each CDR may be replaced with a different amino acid. It will be appreciated that amino acid substitutions may be present in some but not all CDRs of a chain of an antibody.

The antibody or antigen-binding fragment thereof, or variant thereof, may be "detectably labeled". The term "detectably labeled" encompasses labels that are capable of direct or indirect detection. Suitable labels include, but are not limited to, molecules detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. Suitable labels include, but are not limited to, fluorescent dyes (e.g., GFT and its variants, FITC, TRITC, fluorescein and rhodamine, and the like), electron-dense reagents (e.g., gold), enzymes (e.g., as commonly used in ELISA), radionuclide-containing molecules (i.e., radioisotopes), chemiluminescent molecules, electrochemiluminescent molecules, biotin, digoxigenin/digoxigenin, or haptens and other entities that are or can become detectable. For example, the antibody may be biotinylated or ruthenated. Methods for labeling antibodies are well known and well described to those skilled in the art, for example, Haughland (2003) Molecular Probes Handbook of fluorescent Probes and Research Chemicals, Molecular Probes, Inc.; brinkley (1992) Bioconjugate chem.3: 2; garman (1997) Non-Radioactive labelling, A practical approach, Academic Press, London; means (1990) Bioconjugate chem.1: 2; glazer et al, Chemical Modification of proteins, laboratory Techniques in biochemistry and Molecular Biology (t.s.word and e.word, Eds.) American Elsevier publishing co., New York; lundblad, r.l. and nos. C.M (1984) Chemical Reagents for protein modification, vols.i and II, CRC Press, New York; pfleiderer, G. (1985) "chemical modification of Proteins", Modern Methods in Protein Chemistry, H.Tschescheschesche, Ed., Walter DeGruyter, Berlin and New York; and Wong (1991) Chemistry of protein conjugation and Cross-linking, CRC Press, Boca Raton, Fla.); Deleon-Rodriguez et al (2004) chem. Eur. J.10: 1149-; lewis et al (2001) bioconjugugatechem.12: 320-324; li et al (2002) Bioconjugate chem.13: 110-115; mier et al (2005) Bioconjugate chem.16: 240-.

Antibodies target potential biomarkers whose presence, absence, or level can be detected via various measurement methods, particularly immunoassay techniques. These include, but are not limited to, Western blotting (with or without immunoprecipitation), two-dimensional SDS-PAGE, immunoprecipitation, fluorescence-activated cell sorting (FACS), flow cytometry, and immunoassay protocols. The method allows for the determination of a wide variety of tissues and samples, including plasma or serum. A wide range of immunoassay techniques using such assay formats are available, see, e.g., U.S. Pat. nos. 4,016,043,4,424,279, and 4,018,653. These include both non-competitive types of single and two-site or "sandwich" assays, as well as traditional competitive binding assays. These assays also include direct binding of labeled antibodies to the target biomarkers.

"Sandwich assay" is one of the most useful and commonly used assays, covering a variety of variations of the sandwich assay technique. Briefly, in a typical assay, unlabelled antibodies are immobilized on a solid substrate and the sample to be tested is contacted with the bound molecules. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen complex, a second antibody labeled with a reporter molecule capable of producing a detectable signal is then added and incubated for a period of time sufficient for formation of another complex (antibody-antigen-labeled antibody). Any unreacted material may be washed away and the presence of the analyte determined by observing the signal generated by the reporter molecule. The results may be qualitative (by simply observing the visible signal) or may be quantified by comparison to a control sample containing known amounts of the biomarker.

A variation of the sandwich assay comprises a simultaneous assay, wherein both the sample to be analyzed and the labeled antibody are added to the immobilized antibody simultaneously. These techniques are well known to those skilled in the art and include any minor variations that would be apparent. In a typical sandwich assay, a first antibody directed against a first epitope on a biomarker is covalently or passively bound to a solid phase. The solid phase is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. The solid phase may be in the form of a tube, bead, plate of a microplate, or any other surface suitable for performing an immunoassay. The binding process is well known in the art and generally consists of cross-linking covalent binding or physisorption, washing the polymer-antibody complex, and preparing for the test sample. A small sample of the sample to be analyzed is then added to the solid phase complex and incubated under suitable conditions (e.g., between room temperature and 40 ℃, such as between 25 ℃ and 32 ℃) for a sufficient period of time (e.g., 2-40 minutes or overnight, if more convenient) to allow binding of any subunit present in the antibody. After the incubation period, the antibody subunits are washed and incubated with a second antibody specific for a different epitope on the biomarker. The secondary antibody is linked to a reporter molecule that indicates the binding of the secondary antibody to the analyte.

An alternative competitive method involves immobilizing the analyte on a solid phase and then exposing the immobilized target, together with the sample to be analyzed, to an antibody specific for the analyte, which may or may not be labeled with a reporter molecule. Depending on the amount of target molecule in the sample and the intensity of the reporter molecule signal, competition for the target molecule may be directly detectable via such labeled antibodies. Alternatively, a second labeled antibody specific for the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody ternary complex. The complex is detected by the signal emitted by the reporter molecule.

As used herein, the term "reporter molecule" or "directly detectable label" refers to a molecule that, by its chemical nature, provides an analytically identifiable signal, thereby allowing detection of antigen-bound antibodies. The most commonly used reporter molecules in this type of assay are enzymes, fluorophores or radionuclide containing molecules (i.e., radioisotopes) and chemiluminescent or electrochemiluminescent molecules.

In the case of an "Enzyme Immunoassay (EIA)", an enzyme is conjugated to a second antibody, typically by means of glutaraldehyde or periodate, however, as will be readily appreciated, there are a wide variety of different conjugation techniques that are readily available to the skilled artisan.

Furthermore, mRNA or DNA of the analyte of interest can be detected by a method selected from the group consisting of analysis using Northern, dot blot, or Polymerase Chain Reaction (PCR), array hybridization, RNase protection assay, or using a DNA microarray, which are commercially available, including DNA microarray snapshots. For example, real-time PCR (RT-PCR) assays such as quantitative PCR assays are well known in the art. Methods of detecting mRNA of an analyte of interest in a biological sample include, but are not limited to, generating cDNA from the sample by reverse transcription using at least one primer; amplifying the cDNA so generated; and detecting the presence of the amplified cDNA. In addition, such methods may include one or more steps that allow for the determination of the level of mRNA in a biological sample (e.g., by simultaneously examining the level of a comparative control mRNA sequence for a "housekeeping" gene, such as an actin family member). Additionally or alternatively, the sequence of the amplified cDNA may be determined. Northern blot analysis is a routine technique well known in the art and is described, for example, in Molecular Cloning, a Laboratory Manual, second edition,1989, Sambrook, Fritch, Maniatis, Cold Spring Harbor Press,10Skyline Drive, Plainview, NY 11803-. Typical protocols for assessing the status of genes and gene products can be found, for example, In Ausubel et al.eds.,1995, Current protocols In Molecular Biology, Units 2(Northern Blotting),4(southern Blotting),15 (immunology) and 18(PCR Analysis).

"particle" as used herein means a small, confined object to which a physical property such as volume, mass or average size can be attributed. The microparticles may thus be symmetrical, spherical, essentially spherical or spherically shaped, or irregular, asymmetrically shaped or shaped. The size of the particles encompassed by the present invention may vary. Typically, the microparticles have diameters in the nanometer and micrometer ranges. The microparticles may have a diameter of 50 nanometers to 50 micrometers, i.e., 50nm,100nm,200nm,300nm,400nm,500nm,600nm,700nm,800nm,900nm,1000nm (i.e., 1 μm),2 μm,3 μm,4 μm,5 μm,6 μm,7 μm,8 μm,9 μm,10 μm,15 μm,20 μm,25 μm,30 μm,35 μm,40 μm,45 μm, and 50 μm. In particular, the microparticles have a diameter of between 100nm and 10 μm, in particular from 200nm to 5 μm or from 750nm to 5 μm. The microparticles comprise or consist of any suitable material known to those skilled in the art, for example they comprise or consist of or consist essentially of an inorganic or organic material. Typically, they comprise or consist of or consist essentially of a metal or metal alloy, or an organic material, or comprise or consist of or consist essentially of a carbohydrate element. Examples of materials for the microparticles include, but are not limited to, agarose, polystyrene, latex, polyvinyl alcohol, silica and ferromagnetic metals, alloys or composites. The particles may also comprise or consist of magnetic or ferromagnetic metals, alloys or compositions. The material may have particular properties, such as being hydrophobic or hydrophilic, for example. Typically, the microparticles are dispersed in an aqueous solution and retain a small negative surface charge, keeping the microparticles apart and avoiding non-specific clustering. Magnetic or paramagnetic particles can be separated by magnetic force. A magnetic force is applied to pull the paramagnetic or magnetic particles out of the solution/suspension and to retain them while removing the liquid of the solution/suspension and, if desired, washing the particles, for example.

The term "buffer/buffering agent" or "buffer solution" refers to an aqueous solution comprising a mixture of a weak acid and its conjugate base (or vice versa). It is used to prevent the change in pH of a solution because its pH changes little when a small or medium amount of a strong acid or base is added to it. Buffer solutions are used as a means to maintain pH near constant values in a wide variety of chemical applications. Common buffer compounds used include, but are not limited to, TAPS, Bicine, Tris, Tricine, TAPSO, HEPES, TES, MOPS, PIPES, cacodylate, and MES.

A "kit" is any article of manufacture (e.g., a package or container) that contains at least one reagent, e.g., a drug for treating a condition, or a probe for specifically detecting a biomarker gene or protein of the invention. The article of manufacture is preferably distributed, marketed, or sold as a unit for practicing the methods of the present invention. Typically, the kit may further comprise carrier means which are compartmentalized to receive in close confinement one or more container means such as vials, tubes, and the like. In particular, each container means comprises one of the separate elements to be used in the method of the first aspect. The kit may further comprise one or more other containers comprising additional materials including, but not limited to, buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. A label may be present on the container to indicate that the composition is for a particular application, but may also indicate instructions for use in vivo or in vitro.

"package insert" is used to refer to instructions for use that are typically included in commercial packages of therapeutic products or drugs, which contain information about the indications, usage, dosage, administration, contraindications, other therapeutic products to be combined with the packaged product, and/or warnings, etc., regarding the use of such therapeutic products or drugs.

Detailed description of the preferred embodiments

In a first aspect, the invention relates to a method for measuring the level of VEGF-A in the presence of a VEGF-A antagonist. The method comprises incubating the sample with a first and a second antibody or fragments of a first and a second antibody in a first step. Both the first and the second antibodies are capable of binding to VEGF-A or a variant thereof in the presence of the VEGF-A antagonist. In particular, the first and the second antibodies bind to VEGF-A in the presence of the VEGF-A antagonist.

In various embodiments, the binding of the first antibody and the binding of the second antibody do not interfere with each other. In a particular embodiment, one of the first or the second antibody is capable of binding to a solid phase. In certain embodiments, the other of the first or second antibodies is detectably labeled. Once the sample is incubated with the first and the second antibodies, a detectably labeled complex is formed comprising the first antibody, VEGF-A or a variant thereof, and the second antibody.

In the second step of the method for measuring the level of VEGF-A in the presence of a VEGF-A antagonist, the complex formed in the first step is detected. This assay allows for the measurement of VEGF-A levels in the presence of a VEGF-A antagonist.

Thus, a first aspect relates to a method for measuring the level of VEGF-a in the presence of a VEGF-a antagonist, the method comprising incubating a sample with a first and a second antibody, wherein each of said first and said second antibody is capable of binding to VEGF-a in the presence of the VEGF-a antagonist and wherein the binding of said first and said second antibody do not interfere with each other, wherein one of said antibodies is bound or capable of binding to a solid phase and wherein the other of said antibodies is detectably labeled, thereby forming a detectably labeled complex comprising said first antibody, VEGF-a, and said second antibody, and detecting the complex formed, thereby measuring the level of VEGF-a in the presence of a VEGF-a antagonist.

In a particular embodiment, the VEGF-A is human VEGF-A or a variant thereof. In a particular embodiment, the VEGF-A comprises an amino acid sequence according to SEQ ID NO 1 or a variant thereof. In a particular embodiment, the VEGF-A consists of an amino acid sequence according to SEQ ID NO 1 or a variant thereof. In a particular embodiment, the variant of VEGF-A has the same functionality as VEGF-A, i.e., the variant is a functional variant. In a particular embodiment, the variant of VEGF-A exhibits at least 80% sequence identity with human VEGF-A, particularly with the amino acid sequence according to SEQ ID NO: 1. In particular embodiments, the variant of VEGF-A exhibits at least 85%, 90%, 95%, 98% or 99% sequence identity to human VEGF-A, particularly to the amino acid sequence according to SEQ ID NO: 1. In a particular embodiment, the variant of VEGF-A exhibits at least 85% or at least 95% sequence identity with human VEGF-A, particularly with the amino acid sequence according to SEQ ID NO. 1. In particular embodiments, the variant of VEGF-A exhibits 85%, 95%, or 98% sequence identity to human VEGF-A, particularly to the amino acid sequence according to SEQ ID NO: 1. In various embodiments of the invention, VEGF-A is present as a monomer or dimer, particularly a homodimer.

In particular embodiments, the VEGF-A is a human VEGF-A isoform or a variant thereof. In certain embodiments, the VEGF-A isoform is a human VEGF-A isoform VEGF₁₂₁,VEGF₁₄₅,VEGF₁₆₅,VEGF₁₈₉And/or VEGF₂₀₆Or a variant thereof. In a particular embodiment, the variant of the VEGF-A isoform has the same functionality as the corresponding VEGF-A isoform, i.e., the isoform variant is a functional isoform variant. In particular embodiments, the variant of the VEGF-A isoform exhibits at least 80% sequence identity to the amino acid sequence of the corresponding human VEGF-A isoform. In certain embodiments, the variant of the VEGF-A isoform is identical to the human VEGF-A isoformThe amino acid sequences of the isoforms exhibit at least 80% sequence identity. In particular embodiments, the variant of the VEGF-A isoform exhibits at least 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence of the corresponding human VEGF-A isoform. In particular embodiments, the variant of the VEGF-A isoform exhibits at least 85% or at least 95% sequence identity to the amino acid sequence of the corresponding human VEGF-A isoform. In particular embodiments, the variant of the VEGF-A isoform exhibits 85%, 95%, or 98% sequence identity to the amino acid sequence of the corresponding human VEGF-A isoform. Thus, in certain embodiments, the VEGF of VEGF-A₁₂₁Variants of the isoform and the human VEGF₁₂₁The amino acid sequences of the isoforms exhibit 85%, 95%, or 98% sequence identity; VEGF of the VEGF-A₁₄₅Variants of the isoform and the human VEGF₁₄₅The amino acid sequences of the isoforms exhibit 85%, 95%, or 98% sequence identity; VEGF of the VEGF-A₁₆₅Variants of the isoform and the human VEGF₁₆₅The amino acid sequences of the isoforms exhibit 85%, 95%, or 98% sequence identity; VEGF of the VEGF-A₁₈₉Variants of the isoform and the human VEGF₁₈₉The amino acid sequences of the isoforms exhibit 85%, 95%, or 98% sequence identity; and VEGF of the VEGF-A₂₀₆Variants of the isoform and the human VEGF₂₀₆The amino acid sequences of the isoforms exhibit 85%, 95%, or 98% sequence identity.

In particular embodiments, the VEGF-A is a human VEGF-A fragment or variant thereof. In a particular embodiment, the VEGF-A fragment is a 110 amino acid fragment of human VEGF-A or a variant thereof. In a particular embodiment, the variant of the VEGF-A fragment has the same functionality as the corresponding VEGF-A fragment, i.e., the fragment variant is a functional fragment variant. In particular embodiments, the variant of the VEGF-A fragment exhibits at least 80% sequence identity to the amino acid sequence of the corresponding human VEGF-A isoform. In particular embodiments, the variant of the VEGF-A fragment exhibits at least 80% sequence identity to the amino acid sequence of the human VEGF-A fragment. In particular embodiments, the variant of the VEGF-A fragment exhibits at least 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence of the corresponding human VEGF-A fragment. In particular embodiments, the variant of the VEGF-A fragment exhibits at least 85% or at least 95% sequence identity to the amino acid sequence of the corresponding human VEGF-A fragment. In particular embodiments, the variant of the VEGF-A fragment exhibits 85%, 95%, or 98% sequence identity to the amino acid sequence of the corresponding human VEGF-A fragment. Thus, in particular embodiments, the variant of the VEGF-A110 amino acid fragment exhibits 85%, 95%, or 98% sequence identity to the amino acid sequence of the human VEGF-A110 amino acid fragment.

In various embodiments, the VEGF-a antagonist prevents the interaction between VEGF-a and one or more VEGF receptors. In particular, the VEGF-a antagonist competes with VEGF-a at the binding site of the receptor or alters the binding site on VEGF-a for its receptor in such a way that it is no longer able to bind its receptor or trigger a functional effect normally caused by its binding. Thus, the VEGF-a antagonist can bind to an epitope of VEGF-a and thereby block binding of VEGF-a to its receptor, or the VEGF-a antagonist can bind to an epitope of the receptor and thereby block binding of VEGF-a to the receptor. In particular embodiments, the VEGF-A antagonist binds to an epitope on VEGF-A and thereby prevents its binding to a VEGF receptor. In particular embodiments, the VEGF-A receptor is VEGFA-R1 and/or VEGFA-R2.

In still further embodiments of the first aspect of the invention, the VEGF-a antagonist is selected from the group consisting of a polypeptide, a protein, a peptibody (peptibody), an immunoadhesin, a small molecule and an aptamer (aptamer).

In particular embodiments where the antagonist is a protein, the protein is an antibody. In a particular embodiment, the antibody is an anti-VEGF-A antibody. In particular, the anti-VEGF antibody is an antibody that binds VEGF-a with sufficient affinity and specificity. In various embodiments, the antibody has sufficient binding affinity for VEGF-A. In particular, the antibody or antigen-binding fragment thereof has a K between 100nM and 1pM_dValues, i.e., K at 100nM,50nM,1nM,900pM,800pM,700pM,600pM,500pM,400pM,300pM,200pM,100pM,50pM, or 1pM_dValues bound to hVEGF-A. In a particular embodimentThe antibody or antigen-binding fragment thereof has a K between 50nM and 50pM,1nM and 100pM, or between 700pM and 300pM_dValues bind to human VEGF-A (hVEGF-A).

In particular embodiments, the antagonistic VEGF-a antibody is monoclonal or polyclonal. In certain embodiments, the antagonistic VEGF-A antibody is recombinantly produced. In still other embodiments, the antagonistic VEGF-A antibody is a chimeric antibody, in particular a humanized anti-VEGF-A antibody. In certain embodiments, the antagonistic VEGF-a antibody comprises a mutated human IgG1 framework region. The antagonistic VEGF-a antibody further comprises antigen binding Complementarity Determining Regions (CDRs) from the murine anti-hVEGF monoclonal antibody a.4.6.1 that block the binding of human VEGF to its receptor. In a particular embodiment, 93% of the amino acid sequence of the antagonistic VEGF-A antibody, including the majority of the framework regions, is derived from human IgG1, and about 7% of the sequence is derived from the murine antibody A4.6.1In certain embodiments, the antagonistic VEGF-A antibody is glycosylated. In still other embodiments, the antagonistic VEGF-a antibody has a molecular weight of about 149,000 daltons. In particular embodiments, the antagonistic VEGF-A antibody is Bevacizumab (BV), also known as "rhuMAb VEGF" orIt is a recombinant humanized anti-VEGF monoclonal antibody produced according to Presta et al (1997) Cancer Res.57: 4593-4599.

In particular embodiments, the antagonistic VEGF-a antibody is an antibody fragment. The antibody fragment is selected from the group consisting of Fab fragment, Fab 'fragment, F (ab')₂Fragments, single domain antibodies (sdabs), nanobodies (nanobodies), single chain fv (scFv), bivalent single chain variable fragments (di-scFv), tandem scFv, diabodies (diabodies), bispecific diabodies, single chain diabodies (scDb), bispecific T cell engagers (BiTE), and DART molecules. In particular embodiments, the antagonistic antibody fragment is a Fab fragment or F (ab')₂Fragments, in particular humanized Fab fragments or humanized F (ab')₂Fragments。

In still other embodiments, the VEGF-A antagonist is selected from the group consisting of VEGF-trap, Mucagen, PTK787, SU11248, AG-013736, Bay 439006 (sorafenib), ZD-6474, CP632, CP-547632, AZD-2171, CDP-171, SU-14813, CHIR-258, AEE-788, SB786034, BAY579352, CDP-791, EG-3306, GW-786034, RWJ-417975/CT6758, and KRN-633.

In a particular embodiment of the first aspect, a first antibody directed against VEGF-a and a second antibody directed against VEGF-a are used, wherein the first and second antibodies bind VEGF-a at the same or different epitopes. In certain embodiments, the first antibody and the second antibody bind VEGF-a at different epitopes.

In a particular embodiment, the first and the second antibody do not interfere with each other. Thus, binding of one of these antibodies does not prevent or reduce binding of the corresponding other antibody. In various embodiments of the invention, the first and second antibodies bind two different epitopes on the same monomer and/or two different epitopes on each monomer of the dimer. Alternatively, the first and the second antibodies bind to the same or substantially the same epitope on different monomers of the dimer. In certain embodiments, the first antibody and the second antibody bind VEGF-a at different epitopes.

In a particular embodiment, the first and second antibodies bind to each other an epitope covered or bound by a VEGF receptor, in particular VEGF-a receptors VEGFA-R1 and/or VEGFA-R2, respectively. Thus, the first and second antibodies bind to the same epitope as each other respectively as the VEGF-A receptor, in particular VEGFA-R1 or VEGFA-R2. Alternatively, said first and said second antibodies bind to an epitope which is not directly bound by the VEGF-a receptor, such as for example VEGFA-R1 or VEGFA-R2, but which is covered by the receptor, respectively, to each other, such that binding of the first and/or second antibodies prevents binding of the VEGF-a receptor. Thus, in particular embodiments, the first antibody and/or the second antibody compete for binding of VEGFA-R1 and/or VEGFA-R2.

In a particular embodiment, the first and the second antibody bind to the same or different epitopes, respectively, as the VEGF-a antagonist, in particular bind to different epitopes, as the antagonist antibody. Where the first or the second antibody binds to the same epitope as the antagonist, particularly the antagonist antibody, it is contemplated that the first or the second antibody binds to the epitope with a lower Kd value than the antagonist. In a particular embodiment, the first or second antibody binds the epitope with a Kd value of 1.5nM or less, particularly 1nM or less, 0.75nM or less, particularly 0.5nM or less.

In embodiments of the first aspect, either the first antibody or the second antibody binds to an epitope bound by an antibody, the antibody comprises a sequence selected from the group consisting of SEQ ID NOs: 2, amino acids 46-51 of SEQ ID NO:2, amino acids 69-71 of SEQ ID NO:2, amino acid 108-116 of SEQ ID NO:3, amino acids 44-52 of SEQ ID NO:3, amino acids 70-76 of SEQ ID NO:3, amino acid 115-125 of SEQ ID NO:4, amino acids 47-52 of SEQ ID NO:4, amino acids 70-72 of SEQ ID NO:4, amino acid 109 and 117 of SEQ ID NO:5, amino acids 45-52 of SEQ ID NO:5 of the amino acids 70-77 of, and SEQ ID NO:5 amino acid 116 and 126.

In embodiments of the first aspect, the first antibody and/or the second antibody binds to an epitope bound by an antibody comprising a CDR selected from the group consisting of amino acids 46-51 of SEQ ID NO:2, amino acids 69-71 of SEQ ID NO:2, amino acids 108-116 of SEQ ID NO:2, amino acids 44-52 of SEQ ID NO:3, amino acids 70-76 of SEQ ID NO:3, and amino acids 115-125 of SEQ ID NO: 3. In a particular embodiment, the first antibody and/or the second antibody binds to an epitope bound by an antibody comprising a CDR consisting of amino acids 46-51 of SEQ ID NO:2, amino acids 69-71 of SEQ ID NO:2, amino acids 108-116 of SEQ ID NO:2, amino acids 44-52 of SEQ ID NO:3, amino acids 70-76 of SEQ ID NO:3, and amino acids 115-125 of SEQ ID NO: 3.

In embodiments of the first aspect, the first antibody and/or the second antibody binds to an epitope bound by an antibody comprising a CDR selected from the group consisting of amino acids 47-52 of SEQ ID NO:4, amino acids 70-72 of SEQ ID NO:4, amino acids 109-117 of SEQ ID NO:4, amino acids 45-52 of SEQ ID NO:5, amino acids 70-77 of SEQ ID NO:5, and amino acids 116-126 of SEQ ID NO: 5. In a particular embodiment, the first antibody and/or the second antibody binds to an epitope bound by an antibody comprising a CDR consisting of amino acids 47-52 of SEQ ID NO:4, amino acids 70-72 of SEQ ID NO:4, amino acids 109-117 of SEQ ID NO:4, amino acids 45-52 of SEQ ID NO:5, amino acids 70-77 of SEQ ID NO:5, and amino acids 116-126 of SEQ ID NO: 5.

In embodiments of the first aspect, the first and/or the second antibody binds to an epitope bound by an antibody comprising an FR selected from the group consisting of amino acids 20-45 of SEQ ID NO:2, amino acids 52-68 of SEQ ID NO:2, amino acids 72-107 of SEQ ID NO:2, amino acids 117-126 of SEQ ID NO:2, amino acids 19-43 of SEQ ID NO:3, amino acids 53-69 of SEQ ID NO:3, amino acids 77-114 of SEQ ID NO:3, and amino acids 126-136 of SEQ ID NO: 3. In a particular embodiment, the first antibody and/or the second antibody binds to an epitope bound by an antibody comprising an FR consisting of amino acids 20-45 of SEQ ID NO:2, amino acids 51-68 of SEQ ID NO:2, amino acids 72-107 of SEQ ID NO:2, amino acids 117-126 of SEQ ID NO:2, amino acids 19-43 of SEQ ID NO:3, amino acids 51-69 of SEQ ID NO:3, amino acids 77-114 of SEQ ID NO:3, and amino acids 126-136 of SEQ ID NO: 3.

In embodiments of the first aspect, the first antibody and/or the second antibody binds to an epitope bound by an antibody comprising an FR selected from the group consisting of amino acids 21-46 of SEQ ID NO:4, amino acids 53-69 of SEQ ID NO:4, amino acids 73-108 of SEQ ID NO:4, amino acids 118-127 of SEQ ID NO:4, amino acids 20-44 of SEQ ID NO:5, amino acids 53-69 of SEQ ID NO:5, amino acids 78-115 of SEQ ID NO:5, and amino acids 127-137 of SEQ ID NO: 5. In a particular embodiment, the first antibody and/or the second antibody binds to an epitope bound by an antibody comprising an FR consisting of amino acids 21-46 of SEQ ID NO:4, amino acids 53-69 of SEQ ID NO:4, amino acids 73-108 of SEQ ID NO:4, amino acids 118-127 of SEQ ID NO:4, amino acids 20-44 of SEQ ID NO:5, amino acids 53-69 of SEQ ID NO:5, amino acids 78-115 of SEQ ID NO:5, and amino acids 127-137 of SEQ ID NO: 5.

In a particular embodiment, the first antibody and/or the second antibody binds to an epitope bound by an antibody comprising a CDR consisting of amino acids 46-51 of SEQ ID NO:2, amino acids 69-71 of SEQ ID NO:2, amino acids 108-116 of SEQ ID NO:2, amino acids 44-52 of SEQ ID NO:3, amino acids 70-76 of SEQ ID NO:3, and amino acids 115-125 of SEQ ID NO: 3; and comprises the FRs consisting of amino acids 20-45 of SEQ ID NO.2, amino acids 52-68 of SEQ ID NO.2, amino acids 72-107 of SEQ ID NO.2, amino acids 117-126 of SEQ ID NO.2, amino acids 19-43 of SEQ ID NO. 3, amino acids 53-69 of SEQ ID NO. 3, amino acids 77-114 of SEQ ID NO. 3, and amino acids 126-136 of SEQ ID NO. 3.

In a particular embodiment, the first antibody and/or the second antibody binds to an epitope bound by an antibody comprising a CDR consisting of amino acids 47-52 of SEQ ID NO:4, amino acids 70-72 of SEQ ID NO:4, amino acids 109-117 of SEQ ID NO:4, amino acids 45-52 of SEQ ID NO:5, amino acids 70-77 of SEQ ID NO:5, and amino acids 116-126 of SEQ ID NO:5, and comprising an epitope consisting of amino acids 21-46 of SEQ ID NO:4, amino acids 53-69 of SEQ ID NO:4, amino acids 73-108 of SEQ ID NO:4, amino acids 118-127 of SEQ ID NO:4, amino acids 20-44 of SEQ ID NO:5, amino acids 53-69 of SEQ ID NO:5, amino acids 78-115 of SEQ ID NO. 5, and amino acids 127-137 of SEQ ID NO. 5.

In embodiments of the first aspect, the first antibody and/or the second antibody comprises an amino acid sequence selected from the group consisting of SEQ id nos 2,3,4 and 5.

In embodiments of the first aspect, the first and/or the second antibody comprises a light chain having an amino acid sequence selected from the group consisting of SEQ id nos 2 and 4.

In embodiments of the first aspect, the first and/or the second antibody comprises a heavy chain having an amino acid sequence selected from the group consisting of SEQ id nos 3 and 5.

In embodiments of the first aspect, the first or the second antibody comprises a light chain having the amino acid sequence of SEQ ID NO.2 and a heavy chain having the amino acid sequence of SEQ ID NO. 3.

In embodiments of the first aspect, the first or second antibody comprises a light chain having the amino acid sequence of SEQ ID NO.4 and a heavy chain having the amino acid sequence of SEQ ID NO. 5.

In embodiments of the first aspect, one of the first antibody or the second antibody is detectably labeled. The label may be a molecule detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. In particular embodiments, the first or the second antibody may be labeled with a fluorescent dye, an electron-dense reagent, an enzyme (e.g., as commonly used in an ELISA), biotin, digoxigenin/digoxigenin, or a hapten and other entity that is or is capable of being detectable. In particular embodiments, the first or second antibody is biotinylated or ruthenated. Methods for labeling antibodies are well known and well described to those skilled in the art, for example, Haughland (2003) Molecular Probes handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.; brinkley (1992) Bioconjugate chem.3: 2; garman (1997) Non-Radioactive labelling, active Approach, Academic Press, London; means (1990) Bioconjugate chem.1: 2; glazer et al, Chemical Modification of proteins, laboratory Techniques in biochemistry and Molecular Biology (T.S. word and E.word, Eds.) American Elsevierpublishing Co., New York; lundblad, r.l. and nos. C.M (1984) Chemical regensfor Protein Modification, vols.i and II, CRC Press, New York; pfleiderer, G. (1985) "Chemical Modification of Proteins", Modern Methods in protein chemistry, H.Tscheschesche, Ed., Walter DeGruyter, Berlin and New York; wong (1991) Chemistry of Protein Conjugation and Cross-linking, CRC Press, Boca Raton, Fla.); Deleon-Rodriguez et al, chem. Eur. J.10(2004) 1149-; lewis et al, Bioconjugate chem.12(2001) 320-324; li et al, Bioconjugate chem.13(2002) 110-115; and Mier et al, Bioconjugate chem.16(2005) 240-.

In particular embodiments, one of the first antibody or the second antibody is capable of binding to or to a solid phase.

In particular embodiments, the first antibody is capable of binding to or is bound to a solid phase, and the second antibody or antigen-binding fragment thereof is detectably labeled.

In still further embodiments of the first aspect, the first antibody is bound to or capable of binding to a solid phase and has a light chain comprising or consisting of a sequence according to SEQ ID No.2, and the second antibody is detectably labeled and has a light chain comprising or consisting of a sequence according to SEQ ID No. 4.

In still further embodiments of the first aspect, the first antibody is bound to or capable of binding to a solid phase and has a light chain comprising or consisting of a sequence according to SEQ ID No.4 and the second antibody is detectably labeled and has a light chain comprising or consisting of a sequence according to SEQ ID No. 2.

In still further embodiments of the first aspect, the first antibody is bound to or capable of binding to a solid phase and has a heavy chain comprising or consisting of a sequence according to SEQ ID No. 3, and the second antibody is detectably labeled and has a heavy chain comprising or consisting of a sequence according to SEQ ID No. 5.

In still further embodiments of the first aspect, the first antibody is bound to or capable of binding to a solid phase and has a heavy chain comprising or consisting of a sequence according to SEQ ID No. 5, and the second antibody is detectably labeled and has a heavy chain comprising or consisting of a sequence according to SEQ ID No. 3.

In still further embodiments of the first aspect, the first antibody is bound to or capable of binding to a solid phase and has a light chain comprising or consisting of a sequence according to SEQ ID No.2 and a heavy chain comprising or consisting of a sequence according to SEQ ID No. 3, and the second antibody is detectably labeled and has a light chain comprising or consisting of a sequence according to SEQ ID No.4 and a heavy chain comprising or consisting of a sequence according to SEQ ID No. 5.

In still further embodiments of the first aspect, the first antibody is bound to or capable of binding to a solid phase and has a light chain comprising or consisting of a sequence according to SEQ ID No.4 and a heavy chain comprising or consisting of a sequence according to SEQ ID No. 5, and the second antibody is detectably labeled and has a light chain comprising or consisting of a sequence according to SEQ ID No.2 and a heavy chain comprising or consisting of a sequence according to SEQ ID No. 3.

In still other embodiments, the solid phase is in a form selected from the group consisting of a bead, a tube, a plate of a microplate, and any other suitable surface (particularly suitable for performing immunoassays). In a particular embodiment, the bead is a microbead. Microbeads are microparticles having diameters in the nanometer and micrometer range. In various embodiments, the microparticles may have a diameter of 50 nanometers to 50 micrometers. In particular, the particles have a diameter of between 100nm and 10 μm, in particular from 200nm to 5 μm, or from 750nm to 5 μm. The microparticles comprise or consist of any suitable material known to those skilled in the art, for example they comprise or consist of or consist essentially of an inorganic or organic material. In particular, they comprise or consist of or consist essentially of a metal or metal alloy, or an organic material, or comprise or consist of or consist essentially of a carbohydrate element. In a particular embodiment, the material of the microparticles is selected from the group consisting of agarose, polystyrene, latex, polyvinyl alcohol, silica and ferromagnetic metals, alloys or composites. The particles may also comprise or consist of magnetic or ferromagnetic metals, alloys or compositions. The material may have specific properties such as, for example, being hydrophobic or hydrophilic. In particular embodiments, the microparticles are dispersed in an aqueous solution and retain a small negative surface charge, keeping the microparticles apart and avoiding non-specific clustering.

In a particular embodiment, the magnetic or paramagnetic microparticles are separated by magnetic force. A magnetic force is applied to pull the paramagnetic or magnetic particles out of the solution/suspension and to retain them while removing the liquid of the solution/suspension and, if desired, washing the particles, for example.

In a particular embodiment, the first or the second antibody is an IgG antibody.In a particular embodiment, the first or the second antibody is an IgG2 antibody. In a particular embodiment, the first or the second antibody is an IgG2b antibody, or an antigen-binding fragment thereof, particularly IgG2b-F (ab')₂And (3) fragment.

In embodiments of the first aspect, the sample is derived from or is a bodily fluid, in particular selected from the group consisting of whole blood, serum, plasma, urine, saliva and sputum. In particular embodiments, the sample is derived from or is a whole blood sample, serum, or plasma.

In various embodiments, the sample is derived from a healthy individual or patient. In a particular embodiment, the patient is suffering from a proliferative disorder, in particular cancer, in particular from a metastatic cancer. In particular embodiments, the patient is suffering from cancer, particularly metastatic cancer, and is treated with a VEGF-a antagonist. In particular embodiments, the patient is suffering from cancer, particularly metastatic cancer, and is treated with bevacizumab.

In particular embodiments, the cancer is selected from the group consisting of carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell cancer, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including, for example, gastrointestinal cancer), pancreatic cancer (including, for example, metastatic pancreatic cancer), glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (including locally advanced, recurrent or metastatic HER-2 negative breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic cancer, and various types of head and neck cancer, and B-cell lymphoma (including low-grade/follicular non-Hodgkin's lymphoma (NHL), Small Lymphocytic (SL) NHL, medium-grade/follicular NHL, medium-grade diffuse NHL, high-level immunocytocytic NHL, high-grade lymphoblastic NHL, high-grade small non-Hodgkin's lymphoma (NHL), and small-scale non-lymphocytic lymphomas A mitotic cell NHL; storage disease (bulk disease) NHL; mantle cell lymphoma; AIDS-related lymphomas; and waldenstrom's macroglobulinemia); chronic Lymphocytic Leukemia (CLL); acute Lymphoblastic Leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with scarring nevus, edema (such as that associated with brain tumors), and meigs' syndrome. In particular, the patient is suffering from a cancer selected from the group consisting of colon cancer, lung cancer, kidney cancer, ovarian cancer, and glioblastoma multiforme of the brain.

In particular embodiments, the patient is a mammal, reptile, bird or fish. In a particular embodiment, the patient is a mammal selected from the group consisting of a mouse, rat, rabbit, or zebrafish guinea pig, rabbit, horse, donkey, cow, sheep, goat, pig, chicken, camel, cat, dog, turtle, tortoise, snake, lizard, goldfish, and primate. In particular, the patient is a human.

In still further embodiments of the first aspect, the method for measuring the level of VEGF-a in the presence of a VEGF-a antagonist is an immunoassay, in particular a sandwich immunoassay in which antibody-antigen-antibody complexes (also referred to as sandwiches) are formed. The skilled person will appreciate that in a sandwich assay for the detection of VEGF-a, the first antibody may function as a capture antibody and the second antibody may function as a tracer antibody. Alternatively, the second antibody may function as a capture antibody and the first antibody may function as a tracer antibody.

In a particular embodiment, in which the level of VEGF-A in a sample is measured, the first and second antibodies are mixed with the sample to be analyzed.

In one embodiment, wherein the sandwich assay is performed without a washing step, such mixing/incubation is performed in one reaction vessel. The order of adding and mixing the three components (e.g., microparticles coated with the first antibody or antigen-binding fragment thereof, sample, second detectably labeled antibody or antigen-binding fragment thereof, respectively) is not critical. This mixture is incubated for a period of time sufficient for the first antibody (particularly the first antibody coated onto the microparticles) and the detectably labeled second antibody to bind to VEGF-A.

In another embodiment in which the sandwich assay is performed with a washing step, the addition and mixing of the first antibody (in particular the first antibody coated onto the microparticles), the sample and the detectably labeled second antibody or antigen-binding fragment thereof is performed sequentially in one reaction vessel. In the first step (analyte capture step), the microparticles coated with the first antibody are incubated with the sample to be analyzed for a period of time sufficient for the analyte, i.e., VEGF-A, to be bound. After the washing step, the detectably labeled secondary antibody is added and incubated for a period of time sufficient for the secondary antibody to bind to the analyte, i.e., VEGF-A. In various embodiments, the method of the first aspect is performed in a competitive assay format.

In various embodiments, the mixture is incubated for less than 60 minutes, i.e., less than 60,55,50,45,40,35,30,25,20,15,10, or 5 minutes. In particular embodiments, the mixture is incubated for 4 minutes to 1 hour (i.e., 4,5,6,7,8,9,10,15,20,25,30,35,40,45,50,55, or 60 minutes). In particular embodiments, the mixture is incubated for 5 minutes to 45 minutes, i.e., 5,6,7,8,9,10,15,20,25,30,35,40, or 45 minutes). In particular embodiments, the mixture is incubated for 5 minutes to 30 minutes, i.e., 5,6,7,8,9,10,15,20,25, or 30 minutes. In particular embodiments, the mixture is incubated for less than 9 or 18 minutes. In various embodiments, the mixture is incubated for 1-12 hours (i.e., 1,2,3,4,5,6,7,8,9,10,11, or 12 hours). In particular embodiments, the mixture is incubated for 1-4 hours or 8-12 hours.

In yet other embodiments, the mixture is incubated at a temperature of 3-40 ℃ (i.e., 3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39, or 40 ℃). In particular at 3 ℃ to 8 ℃ (i.e. 3,4,5,6,7 or 8), in particular at 4-5 ℃, or at 20 ℃ to 25 ℃ (i.e. at 20,21,22,23,24, or 25 ℃), in particular at 20-22 ℃, or at 35-37 ℃.

It is well known to the person skilled in the art that the incubation temperature and the incubation time depend on each other. Thus, in particular embodiments, the mixture is incubated at 20-25 ℃ for 10 minutes to 1 hour, i.e., the mixture is incubated at 20,21,22,23,24, or 25 ℃ for 10,15,20,25,30,35,40,45,50,55, or 60 minutes. In particular embodiments, the mixture is incubated at 22 ℃ for less than 10 minutes or less than 20 minutes. In each embodiment, the mixture is incubated at 3-8 ℃ for 1-12 hours. In particular, the mixture is incubated at 3-8 ℃, in particular at 4-5 ℃ for 1-4 hours or 8-12 hours.

Incubating the first and/or the second antibody for a period of time sufficient for the first antibody to coat onto the microparticles and the detectably labeled second antibody to bind VEGF-A in the sample.

In a particular embodiment, the first and/or the second antibody is comprised in a physiological solution, in particular in a physiological buffer and/or incubated therein. In particular embodiments, the buffer is selected from the group of TAPS, Bicine, Tris, Tricine, TAPSO, HEPES, TES, MOPS, PIPES, cacodylate, and MES. In a particular embodiment, the buffer is MES buffer. In a particular embodiment, the MES buffer comprises 50mM MES,150mM NaCl,2mM EDTA-Na₂(dihydrate), 0.1% N-Methylisothiazolon-HCl, 0.1% oxypyroon, 0.1% Polydocanol (thesit), 1.0% albumin RPLA 4 assay quality, 0.2% PAK<->R-IgG (DET), Millipore water, pH adjusted to 6.30 with 2N NaOH.

In a particular embodiment, the formed antibody-antigen-antibody complex, in particular the formed complex comprising the first antibody, VEGF-a and the second antibody, is detected via any method known in the art. In particular embodiments, the formed complex is detected via electrochemiluminescence, chemiluminescence, or fluorescence.

In a second aspect, the invention relates to a kit for measuring the level of VEGF-A in the presence of a VEGF-A antagonist. The kit comprises means for detecting VEGF-A in the presence of a VEGF-A antagonist.

In certain embodiments, the kit comprises a first and a second antibody. The first and second antibodies are capable of binding to VEGF-A in the presence of the VEGF-A antagonist. In particular, the binding of the first and the second antibody does not interfere with each other. Furthermore, one of the antibodies is bound or capable of binding a solid phase and wherein the other of the antibodies is detectably labeled. Thus, in a second aspect, the invention relates to a kit for measuring the level of VEGF-A in the presence of a VEGF-A antagonist, the kit comprising first and second antibodies, wherein said first and said second antibodies are capable of binding VEGF-A in the presence of the VEGF-A antagonist, and wherein the binding of said first and said second antibodies do not interfere with each other, and wherein one of said antibodies binds or is capable of binding a solid phase and wherein the other of said antibodies is detectably labeled.

In various embodiments, the kit further comprises carrier means partitioned to receive in close confinement one or more container means selected from the group consisting of vials and tubes. In a particular embodiment, the container means further comprises one of several separate elements to be used, in particular those selected from the group consisting of buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. A label may be present on the container to indicate that the composition is for a particular application, and may also indicate instructions for either in vivo or in vitro use.

In certain embodiments, the kit comprises at least one container, a label on said at least one container, and a composition contained within said at least one container, wherein the composition comprises at least one antibody that binds VEGF-a.

In particular, the kit comprises at least one container comprising the first and the second antibody, or the kit comprises at least two containers, wherein one container comprises the first antibody and a second container comprises the second antibody.

In particular, the label on the container indicates that the composition can be used to assess the presence of VEGF-a in a sample. In particular, the kit includes instructions for using the antibody to assess the presence of VEGF-a in a particular sample type. The kit may further comprise a set of instructions and materials for preparing a sample and applying the antibody to the sample.

In various embodiments, the kit further comprises a VEGF-a antagonist.

In particular embodiments, the kit comprises a container comprising the first antibody, the second antibody, and the VEGF-a antagonist, as described above in the context of the first aspect or as described below in the context of other aspects.

In certain embodiments, the kit comprises two containers, wherein a first container comprises the first antibody and the second antibody, and wherein a second container comprises the VEGF-a antagonist, as described above in the context of the first aspect or as described below in the context of another aspect.

In a particular embodiment, the kit comprises three containers, wherein a first container comprises the first antibody, a second container comprises the second antibody, and a third container comprises the VEGF-a antagonist, as described above in the context of the first aspect or as described below in the context of the third aspect.

In still other embodiments, the kit further comprises a component selected from the group consisting of one or more buffers (e.g., blocking buffer, wash buffer, substrate buffer, etc.), other reagents such as a substrate that is chemically altered by an enzyme label (e.g., chromogen), an epitope retrieval solution, a control sample (positive and/or negative control), a control slide, and the like. The kit may also include instructions for interpreting the results obtained using the kit.

In certain embodiments, the first antibody, the second antibody contained in the one or more containers, and/or the VEGF-a antagonist is present in a lyophilized form or in a solubilized form. In particular embodiments, the first antibody, the second antibody, and/or the VEGF-a antagonist contained in the one or more containers are contained in a solution, particularly a physiological solution. In a particular embodiment, the first antibody, the second antibody, and/or the VEGF-a antagonist contained in the one or more containers are contained in a physiological buffer, in particular in a buffer selected from TAPS, Bicine, Tris, Tricine, TAPSO, HEPES, TES, MOPS, PIPES, cacodylate, and MES. In certain embodiments, the first antibody, the second antibody, and/or the VEGF-a antagonist contained in the one or more containers are contained in a MES buffer. In a particular embodiment, the MES buffer comprises 50mM MES,150mM NaCl,2mM EDTA-Na₂(dihydrate), 0.1% N-Methylisothiazolon-HCl, 0.1% oxypyroon, 0.1% Polydocanol (thesit), 1.0% albumin RPLA 4 assay quality, 0.2% PAK<->R-IgG (DET), Millipore water, pH adjusted to 6.30 with 2N NaOH.

In various embodiments of the second aspect, the kit comprises an antibody as described above with respect to the first aspect and/or as described below with respect to the other aspects.

In various embodiments, the VEGF-a antagonist prevents the interaction between VEGF-a and one or more VEGF receptors. In particular, the VEGF-a antagonist competes with VEGF-a at the binding site of the receptor or alters the binding site on VEGF-a for its receptor in such a way that it is no longer able to bind its receptor or trigger a functional effect normally caused by its binding. Thus, the VEGF-a antagonist can bind to an epitope of VEGF-a and thereby block binding of VEGF-a to its receptor, or the VEGF-a antagonist can bind to an epitope of the receptor and thereby block binding of VEGF-a to the receptor. In particular embodiments, the VEGF-A antagonist binds to an epitope on VEGF-A and thereby prevents its binding to a VEGF receptor. In particular embodiments, the VEGF-A receptor is VEGFA-R1 and/or VEGFA-R2.

In still further embodiments of the second aspect of the invention, the VEGF-a antagonist is selected from the group consisting of a polypeptide, a peptibody (peptibody), an immunoadhesin, a small molecule and an aptamer (aptamer).

In particular embodiments where the antagonist is a polypeptide, the polypeptide is an antibody. In a particular embodiment, the antibody is an anti-VEGF-A antibody. In particular, the anti-VEGF antibody is an antibody that binds VEGF-a with sufficient affinity and specificity. In various embodiments, the antibody hasSufficient binding affinity for VEGF-A. In particular, the antibodies are raised against a K of between 100nM and 1pM_dValues, i.e., K at 100nM,50nM,1nM,900pM,800pM,700pM,600pM,500pM,400pM,300pM,200pM,100pM,50pM, or 1pM_dValues bound to hVEGF-A. In particular embodiments, the antibody has a K between 50nM and 50pM,1nM and 100pM, or 700pM and 300pM_dValues bind to human VEGF-A (hVEGF-A).

In particular embodiments, the antagonistic VEGF-a antibody is monoclonal or polyclonal. In certain embodiments, the antagonistic VEGF-A antibody is recombinantly produced. In still other embodiments, the antagonistic VEGF-A antibody is a chimeric antibody, in particular a humanized anti-VEGF-A antibody. In certain embodiments, the antagonistic VEGF-a antibody comprises a mutated human IgG1 framework region. The antagonistic VEGF-a antibody further comprises antigen binding Complementarity Determining Regions (CDRs) from the murine anti-hVEGF monoclonal antibody a.4.6.1 that block the binding of human VEGF to its receptor. In a particular embodiment, 93% of the amino acid sequence of the antagonistic VEGF-A antibody, including the majority of the framework regions, is derived from human IgG1, and about 7% of the sequence is derived from the murine antibody A4.6.1In certain embodiments, the antagonistic VEGF-A antibody is glycosylated. In still other embodiments, the antagonistic VEGF-a antibody has a molecular weight of about 149,000 daltons. In particular embodiments, the antagonistic VEGF-A antibody is Bevacizumab (BV), also known as "rhuMAb VEGF" orIt is a recombinant humanized anti-VEGF monoclonal antibody produced according to Presta et al (1997) Cancer Res.57: 4593-4599.

In particular embodiments, the antagonistic VEGF-a antibody is an antigen-binding antibody fragment. The antibody fragment is selected from the group consisting of Fab fragment, Fab 'fragment, F (ab')₂Fragments, single domain antibodies (sdabs), nanobodies (nanobodies), single chain fv (scFv), bivalent single chain variable fragments (di-scFv), tandem scfvs, diabodies (diabodies), bispecific diabodies, single chain diabodies(scDb), a bispecific T-cell engager (BiTE), and a DART molecule. In particular embodiments, the antagonistic antibody fragment is a Fab fragment or F (ab')₂Fragments, in particular humanized Fab fragments or humanized F (ab')₂And (3) fragment.

In still other embodiments, the VEGF-A antagonist is selected from the group consisting of VEGF-trap, Mucagen, PTK787, SU11248, AG-013736, Bay 439006 (sorafenib), ZD-6474, CP632, CP-547632, AZD-2171, CDP-171, SU-14813, CHIR-258, AEE-788, SB786034, BAY579352, CDP-791, EG-3306, GW-786034, RWJ-417975/CT6758, and KRN-633.

In a particular embodiment of the second aspect, the kit comprises a first antibody directed to VEGF-a and a second antibody directed to VEGF-a, wherein said first and said second antibodies bind VEGF-a at the same or different epitopes. In certain embodiments, the first antibody and the second antibody bind VEGF-a at different epitopes.

VEGF-A exists as a monomer or dimer, particularly a homodimer. In a particular embodiment, the first and the second antibody do not interfere with each other. Thus, binding of one of these antibodies does not prevent or reduce binding of the corresponding other antibody. In embodiments of the second aspect, the first and second antibodies bind two different epitopes on the same monomer and/or two different epitopes on each monomer of the dimer. Alternatively, the first and the second antibodies bind to the same or substantially the same epitope on different monomers of the homodimer. In certain embodiments, the first antibody and the second antibody bind VEGF-a at different epitopes.

In a particular embodiment, the first and the second antibody bind to the same or different epitopes, respectively, as the VEGF-a antagonist, in particular bind to different epitopes, as the antagonist antibody. Where the first or the second antibody binds to the same epitope as the antagonist, particularly the antagonist antibody, it is contemplated that the first or the second antibody binds to the epitope with a lower Kd value than the antagonist. In a particular embodiment, the first or second antibody binds the epitope with a Kd value of 1.5nM or less, particularly 1nM or less, 0.75nM or less, particularly 0.5nM or less.

In a particular embodiment, the first and second antibodies bind to each other an epitope covered or bound by a VEGF receptor, in particular VEGF-a receptors VEGFA-R1 and/or VEGFA-R2, respectively. Thus, the first and second antibodies bind to the same epitope as each other respectively as the VEGF-A receptor, in particular VEGFA-R1 or VEGFA-R2. Alternatively, said first and said second antibodies bind to an epitope which is not directly bound by the VEGF-a receptor, such as for example VEGFA-R1 or VEGFA-R2, but which is covered by the receptor, respectively, to each other, such that binding of the first and/or second antibodies prevents binding of the VEGF-a receptor. Thus, in particular embodiments, the first antibody and/or the second antibody compete for binding of VEGFA-R1 and/or VEGFA-R2.

In embodiments of the second aspect, either the first antibody or the second antibody binds to an epitope bound by an antibody, the antibody comprises a sequence selected from the group consisting of SEQ ID NOs: 2, amino acids 46-51 of SEQ ID NO:2, amino acids 69-71 of SEQ ID NO:2, amino acid 108-116 of SEQ ID NO:3, amino acids 44-52 of SEQ ID NO:3, amino acids 70-76 of SEQ ID NO:3, amino acid 115-125 of SEQ ID NO:4, amino acids 47-52 of SEQ ID NO:4, amino acids 70-72 of SEQ ID NO:4, amino acid 109 and 117 of SEQ ID NO:5, amino acids 45-52 of SEQ ID NO:5 of the amino acids 70-77 of, and SEQ ID NO:5 amino acid 116 and 126.

In embodiments of the second aspect, the first antibody and/or the second antibody binds to an epitope bound by an antibody comprising a CDR selected from the group consisting of amino acids 46-51 of SEQ ID NO:2, amino acids 69-71 of SEQ ID NO:2, amino acids 108-116 of SEQ ID NO:2, amino acids 44-52 of SEQ ID NO:3, amino acids 70-76 of SEQ ID NO:3, and amino acids 115-125 of SEQ ID NO: 3. In a particular embodiment, the first antibody and/or the second antibody binds to an epitope bound by an antibody comprising a CDR consisting of amino acids 46-51 of SEQ ID NO:2, amino acids 69-71 of SEQ ID NO:2, amino acids 108-116 of SEQ ID NO:2, amino acids 44-52 of SEQ ID NO:3, amino acids 70-76 of SEQ ID NO:3, and amino acids 115-125 of SEQ ID NO: 3.

In embodiments of the second aspect, the first antibody and/or the second antibody binds to an epitope bound by an antibody comprising a CDR selected from the group consisting of amino acids 47-52 of SEQ ID NO:4, amino acids 70-72 of SEQ ID NO:4, amino acids 109-117 of SEQ ID NO:4, amino acids 45-52 of SEQ ID NO:5, amino acids 70-77 of SEQ ID NO:5, and amino acids 116-126 of SEQ ID NO: 5. In a particular embodiment, the first antibody and/or the second antibody binds to an epitope bound by an antibody comprising a CDR consisting of amino acids 47-52 of SEQ ID NO:4, amino acids 70-72 of SEQ ID NO:4, amino acids 109-117 of SEQ ID NO:4, amino acids 45-52 of SEQ ID NO:5, amino acids 70-77 of SEQ ID NO:5, and amino acids 116-126 of SEQ ID NO: 5.

In embodiments of the second aspect, the first and/or the second antibody binds to an epitope bound by an antibody comprising an FR selected from the group consisting of amino acids 20-45 of SEQ ID NO:2, amino acids 52-68 of SEQ ID NO:2, amino acids 72-107 of SEQ ID NO:2, amino acids 117-126 of SEQ ID NO:2, amino acids 19-43 of SEQ ID NO:3, amino acids 53-69 of SEQ ID NO:3, amino acids 77-114 of SEQ ID NO:3, and amino acids 126-136 of SEQ ID NO: 3. In a particular embodiment, the first antibody and/or the second antibody binds to an epitope bound by an antibody comprising an FR consisting of amino acids 20-45 of SEQ ID NO:2, amino acids 51-68 of SEQ ID NO:2, amino acids 72-107 of SEQ ID NO:2, amino acids 117-126 of SEQ ID NO:2, amino acids 19-43 of SEQ ID NO:3, amino acids 51-69 of SEQ ID NO:3, amino acids 77-114 of SEQ ID NO:3, and amino acids 126-136 of SEQ ID NO: 3.

In embodiments of the second aspect, the first antibody and/or the second antibody binds to an epitope bound by an antibody comprising an FR selected from the group consisting of amino acids 21-46 of SEQ ID NO:4, amino acids 53-69 of SEQ ID NO:4, amino acids 73-108 of SEQ ID NO:4, amino acids 118-127 of SEQ ID NO:4, amino acids 20-44 of SEQ ID NO:5, amino acids 53-69 of SEQ ID NO:5, amino acids 78-115 of SEQ ID NO:5, and amino acids 127-137 of SEQ ID NO: 5. In a particular embodiment, the first antibody and/or the second antibody binds to an epitope bound by an antibody comprising an FR consisting of amino acids 21-46 of SEQ ID NO:4, amino acids 53-69 of SEQ ID NO:4, amino acids 73-108 of SEQ ID NO:4, amino acids 118-127 of SEQ ID NO:4, amino acids 20-44 of SEQ ID NO:5, amino acids 53-69 of SEQ ID NO:5, amino acids 78-115 of SEQ ID NO:5, and amino acids 127-137 of SEQ ID NO: 5.

In a particular embodiment, the first antibody and/or the second antibody binds to an epitope bound by an antibody comprising a CDR consisting of amino acids 46-51 of SEQ ID NO:2, amino acids 69-71 of SEQ ID NO:2, amino acids 108-116 of SEQ ID NO:2, amino acids 44-52 of SEQ ID NO:3, amino acids 70-76 of SEQ ID NO:3, and amino acids 115-125 of SEQ ID NO: 3; and comprises the FRs consisting of amino acids 20-45 of SEQ ID NO.2, amino acids 52-68 of SEQ ID NO.2, amino acids 72-107 of SEQ ID NO.2, amino acids 117-126 of SEQ ID NO.2, amino acids 19-43 of SEQ ID NO. 3, amino acids 53-69 of SEQ ID NO. 3, amino acids 77-114 of SEQ ID NO. 3, and amino acids 126-136 of SEQ ID NO. 3.

In a particular embodiment, the first antibody and/or the second antibody binds to an epitope bound by an antibody comprising a CDR consisting of amino acids 47-52 of SEQ ID NO:4, amino acids 70-72 of SEQ ID NO:4, amino acids 109-117 of SEQ ID NO:4, amino acids 45-52 of SEQ ID NO:5, amino acids 70-77 of SEQ ID NO:5, and amino acids 116-126 of SEQ ID NO:5, and comprising an epitope consisting of amino acids 21-46 of SEQ ID NO:4, amino acids 53-69 of SEQ ID NO:4, amino acids 73-108 of SEQ ID NO:4, amino acids 118-127 of SEQ ID NO:4, amino acids 20-44 of SEQ ID NO:5, amino acids 53-69 of SEQ ID NO:5, amino acids 78-115 of SEQ ID NO. 5, and amino acids 127-137 of SEQ ID NO. 5.

In embodiments of the second aspect, the first antibody and/or the second antibody comprises an amino acid sequence selected from the group consisting of SEQ id nos 2,3,4 and 5.

In embodiments of the second aspect, the first and/or the second antibody comprises a light chain having an amino acid sequence selected from the group consisting of SEQ id nos 2 and 4.

In embodiments of the second aspect, the first and/or the second antibody comprises a heavy chain having an amino acid sequence selected from the group consisting of SEQ id nos 3 and 5.

In embodiments of the second aspect, the first or the second antibody comprises a light chain having the amino acid sequence of SEQ ID NO.2 and a heavy chain having the amino acid sequence of SEQ ID NO. 3.

In embodiments of the second aspect, the first or second antibody comprises a light chain having the amino acid sequence of SEQ ID NO.4 and a heavy chain having the amino acid sequence of SEQ ID NO. 5.

In embodiments of the second aspect, one of the first antibody or the second antibody is detectably labeled. The label may be a molecule detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. In particular embodiments, the first or the second antibody may be labeled with a fluorescent dye, an electron-dense reagent, an enzyme (e.g., as commonly used in an ELISA), biotin, digoxigenin/digoxigenin, or a hapten and other entity that is or is capable of being detectable. In particular embodiments, the first or second antibody is biotinylated or ruthenated. Methods for labeling antibodies are well known and well described to those skilled in the art, for example, Haughland (2003) Molecular Probes handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.; brinkley (1992) Bioconjugate chem.3: 2; garman (1997) Non-Radioactive labelling, active Approach, Academic Press, London; means (1990) Bioconjugate chem.1: 2; glazer et al, Chemical Modification of proteins, laboratory Techniques in biochemistry and Molecular Biology (T.S. word and E.word, Eds.) American Elsevierpublishing Co., New York; lundblad, r.l. and nos. C.M (1984) Chemical regensfor Protein Modification, vols.i and II, CRC Press, New York; pfleiderer, G. (1985) "Chemical Modification of Proteins", Modern Methods in protein chemistry, H.Tscheschesche, Ed., Walter DeGruyter, Berlin and New York; wong (1991) Chemistry of Protein Conjugation and Cross-linking, CRC Press, Boca Raton, Fla.); Deleon-Rodriguez et al, chem. Eur. J.10(2004) 1149-; lewis et al, Bioconjugate chem.12(2001) 320-324; li et al, Bioconjugate chem.13(2002) 110-115; and Mier et al, Bioconjugate chem.16(2005) 240-.

In particular embodiments, one of the first antibody or the second antibody is capable of binding to or to a solid phase.

In particular embodiments, the first antibody is capable of binding to or is bound to a solid phase, and the second antibody or antigen-binding fragment thereof is detectably labeled.

In still further embodiments of the second aspect, the first antibody is bound to or capable of binding to a solid phase and has a light chain comprising or consisting of a sequence according to SEQ ID No.2, and the second antibody is detectably labeled and has a light chain comprising or consisting of a sequence according to SEQ ID No. 4.

In still further embodiments of the second aspect, the first antibody is bound to or capable of binding to a solid phase and has a light chain comprising or consisting of a sequence according to SEQ ID No.4 and the second antibody is detectably labeled and has a light chain comprising or consisting of a sequence according to SEQ ID No. 2.

In still further embodiments of the second aspect, the first antibody is bound to or capable of binding to a solid phase and has a heavy chain comprising or consisting of a sequence according to SEQ ID No. 3, and the second antibody is detectably labeled and has a heavy chain comprising or consisting of a sequence according to SEQ ID No. 5.

In still further embodiments of the second aspect, the first antibody is bound to or capable of binding to a solid phase and has a heavy chain comprising or consisting of a sequence according to SEQ ID No. 5, and the second antibody is detectably labeled and has a heavy chain comprising or consisting of a sequence according to SEQ ID No. 3.

In still further embodiments of the second aspect, the first antibody is bound to or capable of binding to a solid phase and has a light chain comprising or consisting of a sequence according to SEQ ID No.2 and a heavy chain comprising or consisting of a sequence according to SEQ ID No. 3, and the second antibody is detectably labeled and has a light chain comprising or consisting of a sequence according to SEQ ID No.4 and a heavy chain comprising or consisting of a sequence according to SEQ ID No. 5.

In still further embodiments of the second aspect, the first antibody is bound to or capable of binding to a solid phase and has a light chain comprising or consisting of a sequence according to SEQ ID No.4 and a heavy chain comprising or consisting of a sequence according to SEQ ID No. 5, and the second antibody is detectably labeled and has a light chain comprising or consisting of a sequence according to SEQ ID No.2 and a heavy chain comprising or consisting of a sequence according to SEQ ID No. 3.

In still other embodiments, the solid phase is in a form selected from the group consisting of a bead, a tube, a plate of a microplate, and any other suitable surface (particularly suitable for performing immunoassays). In a particular embodiment, the bead is a microbead. Microbeads are microparticles having diameters in the nanometer and micrometer range. In various embodiments, the microparticles may have a diameter of 50 nanometers to 50 micrometers. In particular, the particles have a diameter of between 100nm and 10 μm, in particular from 200nm to 5 μm, or from 750nm to 5 μm. The microparticles comprise or consist of any suitable material known to those skilled in the art, for example they comprise or consist of or consist essentially of an inorganic or organic material. In particular, they comprise or consist of or consist essentially of a metal or metal alloy, or an organic material, or comprise or consist of or consist essentially of a carbohydrate element. In a particular embodiment, the material of the microparticles is selected from the group consisting of agarose, polystyrene, latex, polyvinyl alcohol, silica and ferromagnetic metals, alloys or composites. The particles may also comprise or consist of magnetic or ferromagnetic metals, alloys or compositions. The material may have specific properties such as, for example, being hydrophobic or hydrophilic. In particular embodiments, the microparticles are dispersed in an aqueous solution and retain a small negative surface charge, keeping the microparticles apart and avoiding non-specific clustering.

In a particular embodiment, the magnetic or paramagnetic microparticles are separated by magnetic force. A magnetic force is applied to pull the paramagnetic or magnetic particles out of the solution/suspension and to retain them while removing the liquid of the solution/suspension and, if desired, washing the particles, for example.

In a particular embodiment, the first or the second antibody is an IgG antibody. In a particular embodiment, the first or the second antibody or antigen-binding fragment thereof is an IgG2 antibody. In a particular embodiment, the first or the second antibody is an IgG2b antibody, or an antigen-binding fragment thereof, particularly IgG2b-F (ab')₂And (3) fragment.

In a particular embodiment, the first and/or the second antibody is comprised in a physiological solution, in particular in a physiological buffer. In particular embodiments, the buffer is selected from the group of TAPS, Bicine, Tris, Tricine, TAPSO, HEPES, TES, MOPS, PIPES, cacodylate, and MES. In a particular embodiment, the buffer is MES buffer. In a particular embodiment, the MES buffer comprises 50mM MES,150mM NaCl,2mM EDTA-Na₂(dihydrate), 0.1% N-Methylisothiazolon-HCl, 0.1% oxypyroon, 0.1% Polydocanol (thesit), 1.0% albumin RPLA 4 assay quality, 0.2% PAK<->R-IgG (DET), Millipore water, pH adjusted to 6.30 with 2N NaOH.

In certain embodiments, the kit of the second aspect as disclosed above is for use in a method of measuring the level of VEGF-a in the presence of a VEGF-a antagonist as disclosed above in relation to the first aspect. Thus, in a particular embodiment, the kit of the second aspect as disclosed above is for use in a method for measuring the level of VEGF-a in the presence of a VEGF-a antagonist, the method comprising incubating a sample with said first and said second antibodies, wherein both said first and said second antibodies are capable of binding to VEGF-a in the presence of the VEGF-a antagonist and wherein the binding of said first and said second antibodies do not interfere with each other, wherein one of said antibodies is bound or capable of binding to a solid phase and wherein the other of said antibodies is detectably labeled, thereby forming a detectably labeled complex comprising said first antibody, VEGF-a, and said second antibody, and detecting the complex formed, thereby measuring the level of VEGF-a in the presence of a VEGF-a antagonist.

In a particular embodiment, the VEGF-A is human VEGF-A or a variant thereof. In a particular embodiment, the VEGF-A comprises an amino acid sequence according to SEQ ID NO 1 or a variant thereof. In a particular embodiment, the VEGF-A consists of an amino acid sequence according to SEQ ID NO 1 or a variant thereof. In a particular embodiment, the variant of VEGF-A has the same functionality as VEGF-A, i.e., the variant is a functional variant. In a particular embodiment, the variant of VEGF-A exhibits at least 80% sequence identity with human VEGF-A, particularly with the amino acid sequence according to SEQ ID NO: 1. In particular embodiments, the variant of VEGF-A exhibits at least 85%, 90%, 95%, 98% or 99% sequence identity to human VEGF-A, particularly to the amino acid sequence according to SEQ ID NO: 1. In a particular embodiment, the variant of VEGF-A exhibits at least 85% or at least 95% sequence identity with human VEGF-A, particularly with the amino acid sequence according to SEQ ID NO. 1. In particular embodiments, the variant of VEGF-A exhibits 85%, 95%, or 98% sequence identity to human VEGF-A, particularly to the amino acid sequence according to SEQ ID NO: 1. In various embodiments of the invention, VEGF-A is present as a monomer or dimer, particularly a homodimer.

In particular embodiments, the VEGF-A is a human VEGF-A isoform or a variant thereof. In certain embodiments, the VEGF-A isoform is a human VEGF-A isoform VEGF₁₂₁,VEGF₁₄₅,VEGF₁₆₅,VEGF₁₈₉And/or VEGF₂₀₆Or a variant thereof. In a particular embodiment, the variant of the VEGF-A isoform has the same functionality as the corresponding VEGF-A isoform, i.e., the isoform variant is a functional isoform variant. In particular embodiments, the variant of the VEGF-A isoform exhibits at least 80% sequence identity to the amino acid sequence of the corresponding human VEGF-A isoform. In particular embodiments, the variant of the VEGF-A isoform exhibits at least 80% sequence identity to the amino acid sequence of the human VEGF-A isoform. In particular embodiments, the variant of the VEGF-A isoform exhibits at least 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence of the corresponding human VEGF-A isoform.In particular embodiments, the variant of the VEGF-A isoform exhibits at least 85% or at least 95% sequence identity to the amino acid sequence of the corresponding human VEGF-A isoform. In particular embodiments, the variant of the VEGF-A isoform exhibits 85%, 95%, or 98% sequence identity to the amino acid sequence of the corresponding human VEGF-A isoform. Thus, in certain embodiments, the VEGF of VEGF-A₁₂₁Variants of the isoform and the human VEGF₁₂₁The amino acid sequences of the isoforms exhibit 85%, 95%, or 98% sequence identity; VEGF of the VEGF-A₁₄₅Variants of the isoform and the human VEGF₁₄₅The amino acid sequences of the isoforms exhibit 85%, 95%, or 98% sequence identity; VEGF of the VEGF-A₁₆₅Variants of the isoform and the human VEGF₁₆₅The amino acid sequences of the isoforms exhibit 85%, 95%, or 98% sequence identity; VEGF of the VEGF-A₁₈₉Variants of the isoform and the human VEGF₁₈₉The amino acid sequences of the isoforms exhibit 85%, 95%, or 98% sequence identity; and VEGF of the VEGF-A₂₀₆Variants of the isoform and the human VEGF₂₀₆The amino acid sequences of the isoforms exhibit 85%, 95%, or 98% sequence identity.

In particular embodiments, the VEGF-A is a human VEGF-A fragment or variant thereof. In a particular embodiment, the VEGF-A fragment is a 110 amino acid fragment of human VEGF-A or a variant thereof. In a particular embodiment, the variant of the VEGF-A fragment has the same functionality as the corresponding VEGF-A fragment, i.e., the fragment variant is a functional fragment variant. In particular embodiments, the variant of the VEGF-A fragment exhibits at least 80% sequence identity to the amino acid sequence of the corresponding human VEGF-A isoform. In particular embodiments, the variant of the VEGF-A fragment exhibits at least 80% sequence identity to the amino acid sequence of the human VEGF-A fragment. In particular embodiments, the variant of the VEGF-A fragment exhibits at least 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence of the corresponding human VEGF-A fragment. In particular embodiments, the variant of the VEGF-A fragment exhibits at least 85% or at least 95% sequence identity to the amino acid sequence of the corresponding human VEGF-A fragment. In particular embodiments, the variant of the VEGF-A fragment exhibits 85%, 95%, or 98% sequence identity to the amino acid sequence of the corresponding human VEGF-A fragment. Thus, in particular embodiments, the variant of the VEGF-A110 amino acid fragment exhibits 85%, 95%, or 98% sequence identity to the amino acid sequence of the human VEGF-A110 amino acid fragment.

In various embodiments, the VEGF-a antagonist prevents the interaction between VEGF-a and one or more VEGF receptors. In particular, the VEGF-a antagonist competes with VEGF-a at the binding site of the receptor or alters the binding site on VEGF-a for its receptor in such a way that it is no longer able to bind its receptor or trigger a functional effect normally caused by its binding. Thus, the VEGF-a antagonist can bind to an epitope of VEGF-a and thereby block binding of VEGF-a to its receptor, or the VEGF-a antagonist can bind to an epitope of the receptor and thereby block binding of VEGF-a to the receptor. In particular embodiments, the VEGF-A antagonist binds to an epitope on VEGF-A and thereby prevents its binding to a VEGF receptor. In particular embodiments, the VEGF-A receptor is VEGFA-R1 and/or VEGFA-R2.

In still further embodiments of the second aspect of the invention, the VEGF-a antagonist is selected from the group consisting of a polypeptide, a peptibody (peptibody), an immunoadhesin, a small molecule and an aptamer (aptamer).

In particular embodiments where the antagonist is a polypeptide, the polypeptide is an antibody. In a particular embodiment, the antibody is an anti-VEGF-A antibody. In particular, the anti-VEGF antibody is an antibody that binds VEGF-a with sufficient affinity and specificity. In various embodiments, the antibody has sufficient binding affinity for VEGF-A. In particular, the antibody or antigen-binding fragment thereof has a K between 100nM and 1pM_dValues, i.e., K at 100nM,50nM,1nM,900pM,800pM,700pM,600pM,500pM,400pM,300pM,200pM,100pM,50pM, or 1pM_dValues bound to hVEGF-A. In particular embodiments, the antibody or antigen-binding fragment thereof binds human VEGF-A (hVEGF-A) with a Kd value between 50nM and 50pM,1nM and 100pM, or 700pM and 300 pM.

In certain embodiments, the antagonistic VEGF-A antibody isMonoclonal or polyclonal. In certain embodiments, the antagonistic VEGF-A antibody is recombinantly produced. In still other embodiments, the antagonistic VEGF-A antibody is a chimeric antibody, in particular a humanized anti-VEGF-A antibody. In certain embodiments, the antagonistic VEGF-a antibody comprises a mutated human IgG1 framework region. The antagonistic VEGF-a antibody further comprises antigen binding Complementarity Determining Regions (CDRs) from the murine anti-hVEGF monoclonal antibody a.4.6.1 that block the binding of human VEGF to its receptor. In a particular embodiment, 93% of the amino acid sequence of the antagonistic VEGF-A antibody, including the majority of the framework regions, is derived from human IgG1, and about 7% of the sequence is derived from the murine antibody A4.6.1In certain embodiments, the antagonistic VEGF-A antibody is glycosylated. In still other embodiments, the antagonistic VEGF-a antibody has a molecular weight of about 149,000 daltons. In particular embodiments, the antagonistic VEGF-A antibody is Bevacizumab (BV), also known as "rhuMAb VEGF" orIt is a recombinant humanized anti-VEGF monoclonal antibody produced according to Presta et al (1997) Cancer Res.57: 4593-4599.

In particular embodiments, the antagonistic VEGF-a antibody is an antibody fragment. The antibody fragment is selected from the group consisting of Fab fragment, Fab 'fragment, F (ab')₂Fragments, single domain antibodies (sdabs), nanobodies (nanobodies), single chain fv (scFv), bivalent single chain variable fragments (di-scFv), tandem scFv, diabodies (diabodies), bispecific diabodies, single chain diabodies (scDb), bispecific T cell engagers (BiTE), and DART molecules. In particular embodiments, the antagonistic antibody fragment is a Fab fragment or F (ab')₂Fragments, in particular humanized Fab fragments or humanized F (ab')₂And (3) fragment.

In still other embodiments, the VEGF-A antagonist is selected from the group consisting of VEGF-trap, Mucagen, PTK787, SU11248, AG-013736, Bay 439006 (sorafenib), ZD-6474, CP632, CP-547632, AZD-2171, CDP-171, SU-14813, CHIR-258, AEE-788, SB786034, BAY579352, CDP-791, EG-3306, GW-786034, RWJ-417975/CT6758, and KRN-633.

In embodiments of the second aspect, the sample is derived from or is a bodily fluid, in particular selected from the group consisting of whole blood, serum, plasma, urine, saliva and sputum. In particular embodiments, the sample is derived from or is a whole blood sample, serum, or plasma.

In various embodiments, the sample is derived from a healthy individual or patient. In a particular embodiment, the patient is suffering from a proliferative disorder, in particular cancer, in particular from a metastatic cancer. In particular embodiments, the patient is suffering from cancer, particularly metastatic cancer, and is treated with a VEGF-a antagonist. In particular embodiments, the patient is suffering from cancer, particularly metastatic cancer, and is treated with bevacizumab.

In particular embodiments, the cancer is selected from the group consisting of carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell cancer, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including, for example, gastrointestinal cancer), pancreatic cancer (including, for example, metastatic pancreatic cancer), glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (including locally advanced, recurrent or metastatic HER-2 negative breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic cancer, and various types of head and neck cancer, and B-cell lymphoma (including low-grade/follicular non-Hodgkin's lymphoma (NHL), Small Lymphocytic (SL) NHL, medium-grade/follicular NHL, medium-grade diffuse NHL, high-level immunocytocytic NHL, high-grade lymphoblastic NHL, high-grade small non-Hodgkin's lymphoma (NHL), and small-scale non-lymphocytic lymphomas A mitotic cell NHL; storage disease (bulk disease) NHL; mantle cell lymphoma; AIDS-related lymphomas; and waldenstrom's macroglobulinemia); chronic Lymphocytic Leukemia (CLL); acute Lymphoblastic Leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with scarring nevus, edema (such as that associated with brain tumors), and meigs' syndrome. In particular, the patient is suffering from a cancer selected from the group consisting of colon cancer, lung cancer, kidney cancer, ovarian cancer, and glioblastoma multiforme of the brain.

In particular embodiments, the patient is a mammal, reptile, bird or fish. In a particular embodiment, the patient is a mammal selected from the group consisting of a mouse, rat, rabbit, or zebrafish guinea pig, rabbit, horse, donkey, cow, sheep, goat, pig, chicken, camel, cat, dog, turtle, tortoise, snake, lizard, goldfish, and primate. In particular, the patient is a human.

In further embodiments, the method for measuring the level of VEGF-a in the presence of a VEGF-a antagonist using the kit of the second aspect is an immunoassay, in particular a sandwich immunoassay in which antibody-antigen-antibody complexes (also referred to as sandwiches) are formed. The skilled person will appreciate that in a sandwich assay for the detection of VEGF-a, the first antibody may function as a capture antibody and the second antibody may function as a tracer antibody. Alternatively, the second antibody may function as a capture antibody and the first antibody may function as a tracer antibody.

In a particular embodiment, in which the level of VEGF-A in a sample is measured, the first and second antibodies are mixed with the sample to be analyzed.

In one embodiment, wherein the sandwich assay is performed without a washing step, such mixing/incubation is performed in one reaction vessel. The order of adding and mixing the three components (e.g., microparticles coated with the first antibody or antigen-binding fragment thereof, sample, second detectably labeled antibody or antigen-binding fragment thereof, respectively) is not critical. This mixture is incubated for a period of time sufficient for the first antibody (particularly the first antibody coated onto the microparticles) and the detectably labeled second antibody to bind to VEGF-A.

In another embodiment in which the sandwich assay is performed with a washing step, the addition and mixing of the first antibody (in particular the first antibody coated onto the microparticles), the sample and the detectably labeled second antibody or antigen-binding fragment thereof is performed sequentially in one reaction vessel. In the first step (analyte capture step), the microparticles coated with the first antibody are incubated with the sample to be analyzed for a period of time sufficient for the analyte, i.e., VEGF-A, to be bound. After the washing step, the detectably labeled secondary antibody is added and incubated for a period of time sufficient for the secondary antibody to bind to the analyte, i.e., VEGF-A. In various embodiments, the method of the first aspect is performed in a competitive assay format.

In various embodiments, the mixture is incubated for less than 60 minutes, i.e., less than 60,55,50,45,40,35,30,25,20,15,10, or 5 minutes. In particular embodiments, the mixture is incubated for 4 minutes to 1 hour (i.e., 4,5,6,7,8,9,10,15,20,25,30,35,40,45,50,55, or 60 minutes). In particular embodiments, the mixture is incubated for 5 minutes to 45 minutes, i.e., 5,6,7,8,9,10,15,20,25,30,35,40, or 45 minutes). In particular embodiments, the mixture is incubated for 5 minutes to 30 minutes, i.e., 5,6,7,8,9,10,15,20,25, or 30 minutes. In particular embodiments, the mixture is incubated for 9 or 18 minutes. In various embodiments, the mixture is incubated for 1-12 hours (i.e., 1,2,3,4,5,6,7,8,9,10,11, or 12 hours). In particular embodiments, the mixture is incubated for 1-4 hours or 8-12 hours.

In yet other embodiments, the mixture is incubated at a temperature of 3-40 ℃ (i.e., 3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39, or 40 ℃). In particular at 3 ℃ to 8 ℃ (i.e. 3,4,5,6,7 or 8), in particular at 4-5 ℃, or at 20 ℃ to 25 ℃ (i.e. at 20,21,22,23,24, or 25 ℃), in particular at 20-22 ℃, or at 35-37 ℃.

It is well known to the person skilled in the art that the incubation temperature and the incubation time depend on each other. Thus, in particular embodiments, the mixture is incubated at 20-25 ℃ for 10 minutes to 1 hour, i.e., the mixture is incubated at 20,21,22,23,24, or 25 ℃ for 10,15,20,25,30,35,40,45,50,55, or 60 minutes. In particular embodiments, the mixture is incubated at 22 ℃ for less than 10 minutes or less than 20 minutes. In each embodiment, the mixture is incubated at 3-8 ℃ for 1-12 hours. In particular, the mixture is incubated at 3-8 ℃, in particular at 4-5 ℃ for 1-4 hours or 8-12 hours.

Incubating the first and/or the second antibody for a period of time sufficient for the first antibody to coat onto the microparticles and the detectably labeled second antibody to bind VEGF-A in the sample.

In a particular embodiment, the first and/or the second antibody is comprised in a physiological solution, in particular in a physiological buffer and/or incubated therein. In particular embodiments, the buffer is selected from the group of TAPS, Bicine, Tris, Tricine, TAPSO, HEPES, TES, MOPS, PIPES, cacodylate, and MES. In a particular embodiment, the buffer is MES buffer. In a particular embodiment, the MES buffer comprises 50mM MES,150mM NaCl,2mM EDTA-Na₂(dihydrate), 0.1% N-Methylisothiazolon-HCl, 0.1% oxypyroon, 0.1% Polydocanol (thesit), 1.0% albumin RPLA 4 assay quality, 0.2% PAK<->R-IgG (DET), Millipore water, pH adjusted to 6.30 with 2N NaOH.

In a particular embodiment, the formed antibody-antigen-antibody complex, in particular the formed complex comprising the first antibody, VEGF-a and the second antibody, is detected via any method known in the art. In particular embodiments, the formed complex is detected via electrochemiluminescence, chemiluminescence, or fluorescence.

In a third aspect, the invention relates to a composition of matter comprising a first and a second antibody. In particular, the first and the second antibodies are capable of binding VEGF-A in the presence of a VEGF-A antagonist. In particular, the binding of the first and the second antibody does not interfere with each other. In particular, one of the antibodies is bound or capable of binding a solid phase and wherein the other of the antibodies is detectably labeled. Thus, in a third aspect, the invention relates to a composition of matter comprising a first antibody and a second antibody, wherein said first and said second antibody are capable of binding to VEGF-a in the presence of a VEGF-a antagonist, wherein the binding of said first and said second antibody do not interfere with each other, and wherein one of said antibodies is bound or capable of binding to a solid phase and wherein the other of said antibodies is detectably labeled.

In a particular embodiment, the first and the second antibody do not interfere with each other. Thus, binding of one of these antibodies does not prevent or reduce binding of the corresponding other antibody. In a particular embodiment of the third aspect, the composition of matter comprises a first antibody directed to VEGF-a and a second antibody directed to VEGF-a, wherein said first antibody and said second antibody bind VEGF-a at the same or different epitopes. In various embodiments of the invention, the first and second antibodies bind two different epitopes on the same monomer and/or two different epitopes on each monomer to the dimer. Alternatively, the first and the second antibodies bind to the same or substantially the same epitope on different monomers of the dimer. In certain embodiments, the first antibody and the second antibody bind VEGF-a at different epitopes.

In a particular embodiment, the first and the second antibody bind to the same or different epitopes, respectively, as the VEGF-a antagonist, in particular bind to different epitopes, as the antagonist antibody. Where the first or the second antibody binds to the same epitope as the antagonist, particularly the antagonist antibody, it is contemplated that the first or the second antibody binds to the epitope with a lower Kd value than the antagonist. In a particular embodiment, the first or second antibody binds the epitope with a Kd value of 1.5nM or less, particularly 1nM or less, 0.75nM or less, particularly 0.5nM or less.

In a particular embodiment, the first and second antibodies bind to each other an epitope covered or bound by a VEGF receptor, in particular VEGF-a receptors VEGFA-R1 and/or VEGFA-R2, respectively. Thus, the first and second antibodies bind to the same epitope as each other respectively as the VEGF-A receptor, in particular VEGFA-R1 or VEGFA-R2. Alternatively, said first and said second antibodies bind to an epitope which is not directly bound by the VEGF-a receptor, such as for example VEGFA-R1 or VEGFA-R2, but which is covered by the receptor, respectively, to each other, such that binding of the first and/or second antibodies prevents binding of the VEGF-a receptor. Thus, in particular embodiments, the first antibody and/or the second antibody compete for binding of VEGFA-R1 and/or VEGFA-R2.

VEGF-A exists as a monomer or dimer, particularly a homodimer. Thus, in embodiments of the third aspect, the first antibody and the second antibody bind to two different epitopes on the same monomer and/or two different epitopes on each monomer of the dimer. Alternatively, the first and the second antibodies bind to the same or substantially the same epitope on different monomers of the dimer.

In various embodiments of the third aspect, either the first antibody or the second antibody binds to an epitope bound by an antibody, the antibody comprises a sequence selected from the group consisting of SEQ ID NOs: 2, seq id NO:2, amino acids 69-71 of SEQ ID NO:2, amino acid 108-116 of SEQ ID NO:3, amino acids 44-52 of SEQ ID NO:3, amino acids 70-76 of SEQ ID NO:3, amino acid 115-125 of SEQ ID NO:4, amino acids 47-52 of SEQ ID NO:4, amino acids 70-72 of SEQ ID NO:4, amino acid 109 and 117 of SEQ ID NO:5, amino acids 45-52 of SEQ ID NO:5 of the amino acids 70-77 of, and SEQ ID NO:5 amino acid 116 and 126.

In various embodiments of the third aspect, the first antibody and/or the second antibody binds to an epitope bound by an antibody comprising a CDR selected from the group consisting of amino acids 46-51 of SEQ ID NO:2, amino acids 69-71 of SEQ ID NO:2, amino acids 108-116 of SEQ ID NO:2, amino acids 44-52 of SEQ ID NO:3, amino acids 70-76 of SEQ ID NO:3, and amino acids 115-125 of SEQ ID NO: 3. In a particular embodiment, the first antibody and/or the second antibody binds to an epitope bound by an antibody comprising a CDR consisting of amino acids 46-51 of SEQ ID NO:2, amino acids 69-71 of SEQ ID NO:2, amino acids 108-116 of SEQ ID NO:2, amino acids 44-52 of SEQ ID NO:3, amino acids 70-76 of SEQ ID NO:3, and amino acids 115-125 of SEQ ID NO: 3.

In various embodiments of the third aspect, the first antibody and/or the second antibody binds to an epitope bound by an antibody comprising a CDR selected from the group consisting of amino acids 47-52 of SEQ ID NO:4, amino acids 70-72 of SEQ ID NO:4, amino acids 109-117 of SEQ ID NO:4, amino acids 45-52 of SEQ ID NO:5, amino acids 70-77 of SEQ ID NO:5, and amino acids 116-126 of SEQ ID NO: 5. In a particular embodiment, the first antibody and/or the second antibody binds to an epitope bound by an antibody comprising a CDR consisting of amino acids 47-52 of SEQ ID NO:4, amino acids 70-72 of SEQ ID NO:4, amino acids 109-117 of SEQ ID NO:4, amino acids 45-52 of SEQ ID NO:5, amino acids 70-77 of SEQ ID NO:5, and amino acids 116-126 of SEQ ID NO: 5.

In embodiments of the third aspect, the first and/or the second antibody binds to an epitope bound by an antibody comprising an FR selected from the group consisting of amino acids 20-45 of SEQ ID NO:2, amino acids 52-68 of SEQ ID NO:2, amino acids 72-107 of SEQ ID NO:2, amino acids 117-126 of SEQ ID NO:2, amino acids 19-43 of SEQ ID NO:3, amino acids 53-69 of SEQ ID NO:3, amino acids 77-114 of SEQ ID NO:3, and amino acids 126-136 of SEQ ID NO: 3. In a particular embodiment, the first antibody and/or the second antibody binds to an epitope bound by an antibody comprising an FR consisting of amino acids 20-45 of SEQ ID NO:2, amino acids 51-68 of SEQ ID NO:2, amino acids 72-107 of SEQ ID NO:2, amino acids 117-126 of SEQ ID NO:2, amino acids 19-43 of SEQ ID NO:3, amino acids 51-69 of SEQ ID NO:3, amino acids 77-114 of SEQ ID NO:3, and amino acids 126-136 of SEQ ID NO: 3.

In embodiments of the third aspect, the first antibody and/or the second antibody binds to an epitope bound by an antibody comprising an FR selected from the group consisting of amino acids 21-46 of SEQ ID NO:4, amino acids 53-69 of SEQ ID NO:4, amino acids 73-108 of SEQ ID NO:4, amino acids 118-127 of SEQ ID NO:4, amino acids 20-44 of SEQ ID NO:5, amino acids 53-69 of SEQ ID NO:5, amino acids 78-115 of SEQ ID NO:5, and amino acids 127-137 of SEQ ID NO: 5. In a particular embodiment, the first antibody and/or the second antibody binds to an epitope bound by an antibody comprising an FR consisting of amino acids 21-46 of SEQ ID NO:4, amino acids 53-69 of SEQ ID NO:4, amino acids 73-108 of SEQ ID NO:4, amino acids 118-127 of SEQ ID NO:4, amino acids 20-44 of SEQ ID NO:5, amino acids 53-69 of SEQ ID NO:5, amino acids 78-115 of SEQ ID NO:5, and amino acids 127-137 of SEQ ID NO: 5.

In a particular embodiment, the first antibody and/or the second antibody binds to an epitope bound by an antibody comprising a CDR consisting of amino acids 46-51 of SEQ ID NO:2, amino acids 69-71 of SEQ ID NO:2, amino acids 108-116 of SEQ ID NO:2, amino acids 44-52 of SEQ ID NO:3, amino acids 70-76 of SEQ ID NO:3, and amino acids 115-125 of SEQ ID NO: 3; and comprises the FRs consisting of amino acids 20-45 of SEQ ID NO.2, amino acids 52-68 of SEQ ID NO.2, amino acids 72-107 of SEQ ID NO.2, amino acids 117-126 of SEQ ID NO.2, amino acids 19-43 of SEQ ID NO. 3, amino acids 53-69 of SEQ ID NO. 3, amino acids 77-114 of SEQ ID NO. 3, and amino acids 126-136 of SEQ ID NO. 3.

In a particular embodiment, the first antibody and/or the second antibody binds to an epitope bound by an antibody comprising a CDR consisting of amino acids 47-52 of SEQ ID NO:4, amino acids 70-72 of SEQ ID NO:4, amino acids 109-117 of SEQ ID NO:4, amino acids 45-52 of SEQ ID NO:5, amino acids 70-77 of SEQ ID NO:5, and amino acids 116-126 of SEQ ID NO:5, and comprising an epitope consisting of amino acids 21-46 of SEQ ID NO:4, amino acids 53-69 of SEQ ID NO:4, amino acids 73-108 of SEQ ID NO:4, amino acids 118-127 of SEQ ID NO:4, amino acids 20-44 of SEQ ID NO:5, amino acids 53-69 of SEQ ID NO:5, amino acids 78-115 of SEQ ID NO. 5, and amino acids 127-137 of SEQ ID NO. 5.

In embodiments of the third aspect, the first antibody and/or the second antibody comprises an amino acid sequence selected from the group consisting of SEQ id nos 2,3,4 and 5.

In various embodiments of the third aspect, the first and/or the second antibody comprises a light chain having an amino acid sequence selected from the group consisting of SEQ id nos 2 and 4.

In various embodiments of the third aspect, the first and/or the second antibody comprises a heavy chain having an amino acid sequence selected from the group consisting of SEQ id nos 3 and 5.

In embodiments of the third aspect, the first or the second antibody comprises a light chain having the amino acid sequence of SEQ ID NO.2 and a heavy chain having the amino acid sequence of SEQ ID NO. 3.

In embodiments of the third aspect, the first or second antibody comprises a light chain having the amino acid sequence of SEQ ID NO.4 and a heavy chain having the amino acid sequence of SEQ ID NO. 5.

In various embodiments of the third aspect, one of the first antibody or the second antibody is detectably labeled. The label may be a molecule detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. In particular embodiments, the first or the second antibody may be labeled with a fluorescent dye, an electron-dense reagent, an enzyme (e.g., as commonly used in an ELISA), biotin, digoxigenin/digoxigenin, or a hapten and other entity that is or is capable of being detectable. In particular embodiments, the first or second antibody is biotinylated or ruthenated. Methods for labeling antibodies are well known and well described to those skilled in the art, for example, Haughland (2003) Molecular Probes handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.; brinkley (1992) Bioconjugate chem.3: 2; garman (1997) Non-Radioactive labelling, active Approach, Academic Press, London; means (1990) Bioconjugate chem.1: 2; glazer et al, Chemical Modification of proteins, laboratory Techniques in biochemistry and Molecular Biology (T.S. word and E.word, Eds.) American Elsevierpublishing Co., New York; lundblad, r.l. and nos. C.M (1984) Chemical regensfor Protein Modification, vols.i and II, CRC Press, New York; pfleiderer, G. (1985) "Chemical Modification of Proteins", Modern Methods in protein chemistry, H.Tscheschesche, Ed., Walter DeGruyter, Berlin and New York; wong (1991) Chemistry of Protein Conjugation and Cross-linking, CRC Press, Boca Raton, Fla.); Deleon-Rodriguez et al, chem. Eur. J.10(2004) 1149-; lewis et al, Bioconjugate chem.12(2001) 320-324; li et al, Bioconjugate chem.13(2002) 110-115; and Mier et al, Bioconjugate chem.16(2005) 240-.

In particular embodiments, one of the first antibody or the second antibody is capable of binding to or to a solid phase.

In particular embodiments, the first antibody is capable of binding to or is bound to a solid phase, and the second antibody is detectably labeled.

In still further embodiments of the third aspect, the first antibody is bound to or capable of binding to a solid phase and has a light chain comprising or consisting of a sequence according to SEQ ID No.2, and the second antibody is detectably labeled and has a light chain comprising or consisting of a sequence according to SEQ ID No. 4.

In still further embodiments of the third aspect, the first antibody is bound to or capable of binding to a solid phase and has a light chain comprising or consisting of a sequence according to SEQ ID No.4 and the second antibody is detectably labeled and has a light chain comprising or consisting of a sequence according to SEQ ID No. 2.

In still further embodiments of the third aspect, the first antibody is bound to or capable of binding to a solid phase and has a heavy chain comprising or consisting of a sequence according to SEQ ID No. 3, and the second antibody is detectably labeled and has a heavy chain comprising or consisting of a sequence according to SEQ ID No. 5.

In still further embodiments of the third aspect, the first antibody is bound to or capable of binding to a solid phase and has a heavy chain comprising or consisting of a sequence according to SEQ ID No. 5, and the second antibody is detectably labeled and has a heavy chain comprising or consisting of a sequence according to SEQ ID No. 3.

In still further embodiments of the third aspect, the first antibody is bound to or capable of binding to a solid phase and has a light chain comprising or consisting of a sequence according to SEQ ID No.2 and a heavy chain comprising or consisting of a sequence according to SEQ ID No. 3, and the second antibody is detectably labeled and has a light chain comprising or consisting of a sequence according to SEQ ID No.4 and a heavy chain comprising or consisting of a sequence according to SEQ ID No. 5.

In still further embodiments of the third aspect, the first antibody is bound to or capable of binding to a solid phase and has a light chain comprising or consisting of a sequence according to SEQ ID No.4 and a heavy chain comprising or consisting of a sequence according to SEQ ID No. 5, and the second antibody is detectably labeled and has a light chain comprising or consisting of a sequence according to SEQ ID No.2 and a heavy chain comprising or consisting of a sequence according to SEQ ID No. 3.

In still other embodiments, the solid phase is in a form selected from the group consisting of a bead, a tube, a plate of a microplate, and any other suitable surface (particularly suitable for performing immunoassays). In a particular embodiment, the bead is a microbead. Microbeads are microparticles having diameters in the nanometer and micrometer range. In various embodiments, the microparticles may have a diameter of 50 nanometers to 50 micrometers. In particular, the particles have a diameter of between 100nm and 10 μm, in particular from 200nm to 5 μm, or from 750nm to 5 μm. The microparticles comprise or consist of any suitable material known to those skilled in the art, for example they comprise or consist of or consist essentially of an inorganic or organic material. In particular, they comprise or consist of or consist essentially of a metal or metal alloy, or an organic material, or comprise or consist of or consist essentially of a carbohydrate element. In a particular embodiment, the material of the microparticles is selected from the group consisting of agarose, polystyrene, latex, polyvinyl alcohol, silica and ferromagnetic metals, alloys or composites. The particles may also comprise or consist of magnetic or ferromagnetic metals, alloys or compositions. The material may have specific properties such as, for example, being hydrophobic or hydrophilic. In particular embodiments, the microparticles are dispersed in an aqueous solution and retain a small negative surface charge, keeping the microparticles apart and avoiding non-specific clustering.

In a particular embodiment, the magnetic or paramagnetic microparticles are separated by magnetic force. A magnetic force is applied to pull the paramagnetic or magnetic particles out of the solution/suspension and to retain them while removing the liquid of the solution/suspension and, if desired, washing the particles, for example.

In a particular embodiment, the first or the second antibody is an IgG antibody. In a particular embodiment, the first or the second antibody is an IgG2 antibody. In a particular embodiment, the first or the second antibody is an IgG2b antibody, or an antigen-binding fragment thereof, particularly IgG2b-F (ab')₂And (3) fragment.

In certain embodiments, the composition of matter further comprises a VEGF-a antagonist.

In various embodiments, the VEGF-a antagonist prevents the interaction between VEGF-a and one or more VEGF receptors. In particular, the VEGF-a antagonist competes with VEGF-a at the binding site of the receptor or alters the binding site on VEGF-a for its receptor in such a way that it is no longer able to bind its receptor or trigger a functional effect normally caused by its binding. Thus, the VEGF-a antagonist can bind to an epitope of VEGF-a and thereby block binding of VEGF-a to its receptor, or the VEGF-a antagonist can bind to an epitope of the receptor and thereby block binding of VEGF-a to the receptor. In particular embodiments, the VEGF-A antagonist binds to an epitope on VEGF-A and thereby prevents its binding to a VEGF receptor. In particular embodiments, the VEGF-A receptor is VEGFA-R1 and/or VEGFA-R2.

In still further embodiments of the third aspect of the invention, the VEGF-a antagonist is selected from the group consisting of a polypeptide, a peptibody (peptibody), an immunoadhesin, a small molecule and an aptamer (aptamer).

In particular embodiments where the antagonist is a polypeptide, the polypeptide is an antibody. In a particular embodiment, the antibody is an anti-VEGF-A antibody. In particular, the anti-VEGF antibody is an antibody or antigen-binding fragment thereof that binds VEGF-a with sufficient affinity and specificity. In various embodiments, the antibody or antigen-binding fragment thereof has sufficient binding affinity for VEGF-A. In particular, the antibody or antigen-binding fragment thereof has a K between 100nM and 1pM_dValues, i.e., K at 100nM,50nM,1nM,900pM,800pM,700pM,600pM,500pM,400pM,300pM,200pM,100pM,50pM, or 1pM_dValue binding hVEGF-A. In particular embodiments, the antibody or antigen-binding fragment thereof has a K between 50nM and 50pM,1nM and 100pM, or 700pM and 300pM_dValues bind to human VEGF-A (hVEGF-A).

In particular embodiments, the antagonistic VEGF-a antibody is monoclonal or polyclonal. In certain embodiments, the antagonistic VEGF-a antibody or antigen-binding fragment thereof is recombinantly produced. In still other embodiments, the antagonistic VEGF-A antibody is a chimeric antibody, in particular a humanized anti-VEGF-A antibody. In certain embodiments, the antagonistic VEGF-a antibody comprises a mutated human IgG1 framework region. The antagonistic VEGF-a antibody further comprises antigen binding Complementarity Determining Regions (CDRs) from the murine anti-hVEGF monoclonal antibody a.4.6.1 that block the binding of human VEGF to its receptor. In a particular embodiment, 93% of the amino acid sequence of the antagonistic VEGF-A antibody, including the majority of the framework regions, is derived from human IgG1, and about 7% of the sequence is derived from the murine antibody A4.6.1In certain embodiments, the antagonistic VEGF-A antibody is glycosylated. In still other embodiments, the antagonistic VEGF-a antibody has a molecular weight of about 149,000 daltons. In particular embodiments, the antagonistic VEGF-A antibody is Bevacizumab (BV), also known as "rhuMAbVEGF" orIt is a recombinant humanized anti-VEGF monoclonal antibody produced according to Presta et al (1997) Cancer Res.57: 4593-4599.

In particular embodiments, the antagonistic VEGF-a antibody is an antibody fragment. The antibody fragment is selected from the group consisting of Fab fragment, Fab 'fragment, F (ab')₂Fragments, single domain antibodies (sdabs), nanobodies (nanobodies), single chain fv (scFv), bivalent single chain variable fragments (di-scFv), tandem scFv, diabodies (diabodies), bispecific diabodies, single chain diabodies (scDb), bispecific T cell engagers (BiTE), and DART molecules. In particular embodiments, the antagonistic antibody fragment is a Fab fragment or F (ab')₂Fragments, in particularHumanized Fab fragments or humanized F (ab')₂And (3) fragment.

In still other embodiments, the VEGF-A antagonist is selected from the group consisting of VEGF-trap, Mucagen, PTK787, SU11248, AG-013736, Bay 439006 (sorafenib), ZD-6474, CP632, CP-547632, AZD-2171, CDP-171, SU-14813, CHIR-258, AEE-788, SB786034, BAY579352, CDP-791, EG-3306, GW-786034, RWJ-417975/CT6758, and KRN-633.

In a particular embodiment, the first and/or the second antibody and/or the VEGF-a antagonist are contained in a physiological solution, in particular in a physiological buffer. In particular embodiments, the buffer is selected from the group of TAPS, Bicine, Tris, Tricine, TAPSO, HEPES, TES, MOPS, PIPES, cacodylate, and MES. In a particular embodiment, the buffer is MES buffer. In a particular embodiment, the MES buffer comprises 50mM MES,150mM NaCl,2mM EDTA-Na₂(dihydrate), 0.1% N-Methylisothiazolon-HCl, 0.1% oxypyroon, 0.1% Polydocanol (thesit), 1.0% albumin RPLA 4 assay quality, 0.2% PAK<->R-IgG (DET), Millipore water, pH adjusted to 6.30 with 2N NaOH.

In certain embodiments, the composition of matter as disclosed above is for use in a method of measuring the level of VEGF-a in the presence of a VEGF-a antagonist as disclosed above in relation to the first aspect. Thus, in a particular embodiment, a composition of matter as disclosed above is for use in a method for measuring the level of VEGF-a in the presence of a VEGF-a antagonist, the method comprising incubating a sample with said first and said second antibodies, wherein both said first and said second antibodies are capable of binding to VEGF-a in the presence of the VEGF-a antagonist and wherein the binding of said first and said second antibodies do not interfere with each other, wherein one of said antibodies binds or is capable of binding to a solid phase and wherein the other of said antibodies is detectably labeled, thereby forming a detectably labeled complex comprising said first antibody, VEGF-a, and said second antibody, and detecting the formed complex, thereby measuring the level of VEGF-a in the presence of a VEGF-a antagonist.

In a particular embodiment, the VEGF-A is human VEGF-A or a variant thereof. In a particular embodiment, the VEGF-A comprises an amino acid sequence according to SEQ ID NO 1 or a variant thereof. In a particular embodiment, the VEGF-A consists of an amino acid sequence according to SEQ ID NO 1 or a variant thereof. In a particular embodiment, the variant of VEGF-A has the same functionality as VEGF-A, i.e., the variant is a functional variant. In a particular embodiment, the variant of VEGF-A exhibits at least 80% sequence identity with human VEGF-A, particularly with the amino acid sequence according to SEQ ID NO: 1. In particular embodiments, the variant of VEGF-A exhibits at least 85%, 90%, 95%, 98% or 99% sequence identity to human VEGF-A, particularly to the amino acid sequence according to SEQ ID NO: 1. In a particular embodiment, the variant of VEGF-A exhibits at least 85% or at least 95% sequence identity with human VEGF-A, particularly with the amino acid sequence according to SEQ ID NO. 1. In particular embodiments, the variant of VEGF-A exhibits 85%, 95%, or 98% sequence identity to human VEGF-A, particularly to the amino acid sequence according to SEQ ID NO: 1. In various embodiments of the invention, VEGF-A is present as a monomer or dimer, particularly a homodimer.

In particular embodiments, the VEGF-A is a human VEGF-A isoform or a variant thereof. In certain embodiments, the VEGF-A isoform is a human VEGF-A isoform VEGF₁₂₁,VEGF₁₄₅,VEGF₁₆₅,VEGF₁₈₉And/or VEGF₂₀₆Or a variant thereof. In a particular embodiment, the variant of the VEGF-A isoform has the same functionality as the corresponding VEGF-A isoform, i.e., the isoform variant is a functional isoform variant. In particular embodiments, the variant of the VEGF-A isoform exhibits at least 80% sequence identity to the amino acid sequence of the corresponding human VEGF-A isoform. In particular embodiments, the variant of the VEGF-A isoform exhibits at least 80% sequence identity to the amino acid sequence of the human VEGF-A isoform. In particular embodiments, the variant of the VEGF-A isoform exhibits at least 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence of the corresponding human VEGF-A isoform. In particular embodiments, the variant of the VEGF-A isoform exhibits at least 85% or at least 95% sequence identity to the amino acid sequence of the corresponding human VEGF-A isoform. In a particular embodimentIn embodiments, the variant of the VEGF-A isoform exhibits 85%, 95%, or 98% sequence identity to the amino acid sequence of the corresponding human VEGF-A isoform. Thus, in certain embodiments, the VEGF of VEGF-A₁₂₁Variants of the isoform and the human VEGF₁₂₁The amino acid sequences of the isoforms exhibit 85%, 95%, or 98% sequence identity; VEGF of the VEGF-A₁₄₅Variants of the isoform and the human VEGF₁₄₅The amino acid sequences of the isoforms exhibit 85%, 95%, or 98% sequence identity; VEGF of the VEGF-A₁₆₅Variants of the isoform and the human VEGF₁₆₅The amino acid sequences of the isoforms exhibit 85%, 95%, or 98% sequence identity; VEGF of the VEGF-A₁₈₉Variants of the isoform and the human VEGF₁₈₉The amino acid sequences of the isoforms exhibit 85%, 95%, or 98% sequence identity; and VEGF of the VEGF-A₂₀₆Variants of the isoform and the human VEGF₂₀₆The amino acid sequences of the isoforms exhibit 85%, 95%, or 98% sequence identity.

In particular embodiments, the VEGF-A is a human VEGF-A fragment or variant thereof. In a particular embodiment, the VEGF-A fragment is a 110 amino acid fragment of human VEGF-A or a variant thereof. In a particular embodiment, the variant of the VEGF-A fragment has the same functionality as the corresponding VEGF-A fragment, i.e., the fragment variant is a functional fragment variant. In particular embodiments, the variant of the VEGF-A fragment exhibits at least 80% sequence identity to the amino acid sequence of the corresponding human VEGF-A isoform. In particular embodiments, the variant of the VEGF-A fragment exhibits at least 80% sequence identity to the amino acid sequence of the human VEGF-A fragment. In particular embodiments, the variant of the VEGF-A fragment exhibits at least 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence of the corresponding human VEGF-A fragment. In particular embodiments, the variant of the VEGF-A fragment exhibits at least 85% or at least 95% sequence identity to the amino acid sequence of the corresponding human VEGF-A fragment. In particular embodiments, the variant of the VEGF-A fragment exhibits 85%, 95%, or 98% sequence identity to the amino acid sequence of the corresponding human VEGF-A fragment. Thus, in particular embodiments, the variant of the VEGF-A110 amino acid fragment exhibits 85%, 95%, or 98% sequence identity to the amino acid sequence of the human VEGF-A110 amino acid fragment.

In various embodiments, the VEGF-a antagonist prevents the interaction between VEGF-a and one or more VEGF receptors. In particular, the VEGF-a antagonist competes with VEGF-a at the binding site of the receptor or alters the binding site on VEGF-a for its receptor in such a way that it is no longer able to bind its receptor or trigger a functional effect normally caused by its binding. Thus, the VEGF-a antagonist can bind to an epitope of VEGF-a and thereby block binding of VEGF-a to its receptor, or the VEGF-a antagonist can bind to an epitope of the receptor and thereby block binding of VEGF-a to the receptor. In particular embodiments, the VEGF-A antagonist binds to an epitope on VEGF-A and thereby prevents its binding to a VEGF receptor. In particular embodiments, the VEGF-A receptor is VEGFA-R1 and/or VEGFA-R2.

In still further embodiments of the third aspect of the invention, the VEGF-a antagonist is selected from the group consisting of a polypeptide, a peptibody (peptibody), an immunoadhesin, a small molecule and an aptamer (aptamer).

In particular embodiments where the antagonist is a polypeptide, the polypeptide is an antibody. In a particular embodiment, the antibody is an anti-VEGF-A antibody. In particular, the anti-VEGF antibody is an antibody that binds VEGF-a with sufficient affinity and specificity. In various embodiments, the antibody has sufficient binding affinity for VEGF-A. In particular, the antibody or antigen-binding fragment thereof has a K between 100nM and 1pM_dValues, i.e., binding to hVEGF-A with Kd values of 100nM,50nM,1nM,900pM,800pM,700pM,600pM,500pM,400pM,300pM,200pM,100pM,50pM, or 1 pM. In particular embodiments, the antibody or antigen-binding fragment thereof has a K between 50nM and 50pM,1nM and 100pM, or 700pM and 300pM_dValues bind to human VEGF-A (hVEGF-A).

In particular embodiments, the antagonistic VEGF-a antibody is monoclonal or polyclonal. In certain embodiments, the antagonistic VEGF-A antibody is recombinantly produced. In still other embodiments, the antagonistic VEGF-A antibody is a chimeric anti-VEGF-A antibodyA body, in particular a humanized anti-VEGF-A antibody. In certain embodiments, the antagonistic VEGF-a antibody comprises a mutated human IgG1 framework region. The antagonistic VEGF-a antibody further comprises antigen binding Complementarity Determining Regions (CDRs) from the murine anti-hVEGF monoclonal antibody a.4.6.1 that block the binding of human VEGF to its receptor. In a particular embodiment, 93% of the amino acid sequence of the antagonistic VEGF-A antibody, including the majority of the framework regions, is derived from human IgG1, and about 7% of the sequence is derived from the murine antibody A4.6.1In certain embodiments, the antagonistic VEGF-A antibody is glycosylated. In still other embodiments, the antagonistic VEGF-a antibody has a molecular weight of about 149,000 daltons. In particular embodiments, the antagonistic VEGF-A antibody is Bevacizumab (BV), also known as "rhuMAb VEGF" orIt is a recombinant humanized anti-VEGF monoclonal antibody produced according to Presta et al (1997) Cancer Res.57: 4593-4599.

In particular embodiments, the antagonistic VEGF-a antibody is an antibody fragment. The antibody fragment is selected from the group consisting of Fab fragment, Fab 'fragment, F (ab')₂Fragments, single domain antibodies (sdabs), nanobodies (nanobodies), single chain fv (scFv), bivalent single chain variable fragments (di-scFv), tandem scFv, diabodies (diabodies), bispecific diabodies, single chain diabodies (scDb), bispecific T cell engagers (BiTE), and DART molecules. In particular embodiments, the antagonistic antibody fragment is a Fab fragment or F (ab')₂Fragments, in particular humanized Fab fragments or humanized F (ab')₂And (3) fragment.

In still other embodiments, the VEGF-A antagonist is selected from the group consisting of VEGF-trap, Mucagen, PTK787, SU11248, AG-013736, Bay 439006 (sorafenib), ZD-6474, CP632, CP-547632, AZD-2171, CDP-171, SU-14813, CHIR-258, AEE-788, SB786034, BAY579352, CDP-791, EG-3306, GW-786034, RWJ-417975/CT6758, and KRN-633.

In embodiments of the third aspect, the sample is derived from or is a bodily fluid, in particular selected from the group consisting of whole blood, serum, plasma, urine, saliva and sputum. In particular embodiments, the sample is derived from or is a whole blood sample, serum, or plasma.

In various embodiments, the sample is derived from a healthy individual or patient. In a particular embodiment, the patient is suffering from a proliferative disorder, in particular cancer, in particular from a metastatic cancer. In particular embodiments, the patient is suffering from cancer, particularly metastatic cancer, and is treated with a VEGF-a antagonist. In particular embodiments, the patient is suffering from cancer, particularly metastatic cancer, and is treated with bevacizumab.

In particular embodiments, the cancer is selected from the group consisting of carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell cancer, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (including, for example, gastrointestinal cancer), pancreatic cancer (including, for example, metastatic pancreatic cancer), glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (including locally advanced, recurrent or metastatic HER-2 negative breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic cancer, and various types of head and neck cancer, and B-cell lymphoma (including low-grade/follicular non-Hodgkin's lymphoma (NHL), Small Lymphocytic (SL) NHL, medium-grade/follicular NHL, medium-grade diffuse NHL, high-level immunocytocytic NHL, high-grade lymphoblastic NHL, high-grade small non-Hodgkin's lymphoma (NHL), and small-scale non-lymphocytic lymphomas A mitotic cell NHL; storage disease (bulk disease) NHL; mantle cell lymphoma; AIDS-related lymphomas; and waldenstrom's macroglobulinemia); chronic Lymphocytic Leukemia (CLL); acute Lymphoblastic Leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with scarring nevus, edema (such as that associated with brain tumors), and meigs' syndrome. In particular, the patient is suffering from a cancer selected from the group consisting of colon cancer, lung cancer, kidney cancer, ovarian cancer, and glioblastoma multiforme of the brain.

In particular embodiments, the patient is a mammal, reptile, bird or fish. In a particular embodiment, the patient is a mammal selected from the group consisting of a mouse, rat, rabbit, or zebrafish guinea pig, rabbit, horse, donkey, cow, sheep, goat, pig, chicken, camel, cat, dog, turtle, tortoise, snake, lizard, goldfish, and primate. In particular, the patient is a human.

In still further embodiments, the method for measuring the level of VEGF-a in the presence of a VEGF-a antagonist using a composition of matter as disclosed above is an immunoassay, in particular a sandwich immunoassay in which antibody-antigen-antibody complexes (also referred to as sandwiches) are formed. The skilled person will appreciate that in a sandwich assay for the detection of VEGF-a, the first antibody may function as a capture antibody and the second antibody may function as a tracer antibody. Alternatively, the second antibody may function as a capture antibody and the first antibody may function as a tracer antibody.

In a particular embodiment, in which the level of VEGF-A in a sample is measured, the first and second antibodies are mixed with the sample to be analyzed.

In one embodiment, wherein the sandwich assay is performed without a washing step, such mixing/incubation is performed in one reaction vessel. The order of adding and mixing the three components (e.g., microparticles coated with the first antibody or antigen-binding fragment thereof, sample, second detectably labeled antibody or antigen-binding fragment thereof, respectively) is not critical. This mixture is incubated for a period of time sufficient for the first antibody (particularly the first antibody coated onto the microparticles) and the detectably labeled second antibody to bind to VEGF-A.

In another embodiment in which the sandwich assay is performed with a washing step, the addition and mixing of the first antibody (in particular the first antibody coated onto the microparticles), the sample and the detectably labeled second antibody or antigen-binding fragment thereof is performed sequentially in one reaction vessel. In the first step (analyte capture step), the microparticles coated with the first antibody are incubated with the sample to be analyzed for a period of time sufficient for the analyte, i.e., VEGF-A, to be bound. After the washing step, the detectably labeled secondary antibody is added and incubated for a period of time sufficient for the secondary antibody to bind to the analyte, i.e., VEGF-A. In various embodiments, the method of the first aspect is performed in a competitive assay format.

In various embodiments, the mixture is incubated for less than 60 minutes, i.e., less than 60,55,50,45,40,35,30,25,20,15,10, or 5 minutes. In particular embodiments, the mixture is incubated for 4 minutes to 1 hour (i.e., 4,5,6,7,8,9,10,15,20,25,30,35,40,45,50,55, or 60 minutes). In particular embodiments, the mixture is incubated for 5 minutes to 45 minutes, i.e., 5,6,7,8,9,10,15,20,25,30,35,40, or 45 minutes). In particular embodiments, the mixture is incubated for 5 minutes to 30 minutes, i.e., 5,6,7,8,9,10,15,20,25, or 30 minutes. In particular embodiments, the mixture is incubated for 9 or 18 minutes. In various embodiments, the mixture is incubated for 1-12 hours (i.e., 1,2,3,4,5,6,7,8,9,10,11, or 12 hours). In particular embodiments, the mixture is incubated for 1-4 hours or 8-12 hours.

In yet other embodiments, the mixture is incubated at a temperature of 3-40 ℃ (i.e., 3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39, or 40 ℃). In particular at 3 ℃ to 8 ℃ (i.e. 3,4,5,6,7 or 8), in particular at 4-5 ℃, or at 20 ℃ to 25 ℃ (i.e. at 20,21,22,23,24, or 25 ℃), in particular at 20-22 ℃, or at 35-37 ℃.

It is well known to the person skilled in the art that the incubation temperature and the incubation time depend on each other. Thus, in particular embodiments, the mixture is incubated at 20-25 ℃ for 10 minutes to 1 hour, i.e., the mixture is incubated at 20,21,22,23,24, or 25 ℃ for 10,15,20,25,30,35,40,45,50,55, or 60 minutes. In particular embodiments, the mixture is incubated at 22 ℃ for less than 10 minutes or less than 20 minutes. In each embodiment, the mixture is incubated at 3-8 ℃ for 1-12 hours. In particular, the mixture is incubated at 3-8 ℃, in particular at 4-5 ℃ for 1-4 hours or 8-12 hours.

Incubating the first and/or the second antibody for a period of time sufficient for the first antibody to coat onto the microparticles and the detectably labeled second antibody to bind VEGF-A in the sample.

In a particular embodiment, the first and/or the second antibody is comprised in a physiological solution, in particular in a physiological buffer and/or incubated therein. In particular embodiments, the buffer is selected from the group of TAPS, Bicine, Tris, Tricine, TAPSO, HEPES, TES, MOPS, PIPES, cacodylate, and MES. In a particular embodiment, the buffer is MES buffer. In a particular embodiment, the MES buffer comprises 50mM MES,150mM NaCl,2mM EDTA-Na₂(dihydrate), 0.1% N-Methylisothiazolon-HCl, 0.1% oxypyroon, 0.1% Polydocanol (thesit), 1.0% albumin RPLA 4 assay quality, 0.2% PAK<->R-IgG (DET), Millipore water, pH adjusted to 6.30 with 2N NaOH.

In a particular embodiment, the formed antibody-antigen-antibody complex, in particular the formed complex comprising the first antibody, VEGF-a and the second antibody, is detected via any method known in the art. In particular embodiments, the formed complex is detected via electrochemiluminescence, chemiluminescence, or fluorescence.

In a fourth aspect, the present invention relates to a method for detecting a protein complex comprising human VEGF-a as a first protein and a non-human or chimeric protein as a second protein, comprising the steps of:

(a) incubating a sample comprising said complex with a detectably labeled antibody binding to or capable of binding to VEGF-A and/or a second non-human or chimeric protein of said complex, and

(b) detecting the detectably labeled antibody bound to the complex.

In a particular embodiment, the VEGF-A is human VEGF-A or a variant thereof. In a particular embodiment, the VEGF-A comprises an amino acid sequence according to SEQ ID NO 1 or a variant thereof. In a particular embodiment, the VEGF-A consists of an amino acid sequence according to SEQ ID NO 1 or a variant thereof. In a particular embodiment, the variant of VEGF-A has the same functionality as VEGF-A, i.e., the variant is a functional variant. In a particular embodiment, the variant of VEGF-A exhibits at least 80% sequence identity with human VEGF-A, particularly with the amino acid sequence according to SEQ ID NO: 1. In particular embodiments, the variant of VEGF-A exhibits at least 85%, 90%, 95%, 98% or 99% sequence identity to human VEGF-A, particularly to the amino acid sequence according to SEQ ID NO: 1. In a particular embodiment, the variant of VEGF-A exhibits at least 85% or at least 95% sequence identity with human VEGF-A, particularly with the amino acid sequence according to SEQ ID NO. 1. In particular embodiments, the variant of VEGF-A exhibits 85%, 95%, or 98% sequence identity to human VEGF-A, particularly to the amino acid sequence according to SEQ ID NO: 1.

In a particular embodiment of the fourth aspect, the non-human or chimeric protein is an antibody that binds VEGF-A.

Thus, in particular embodiments, the complex to be detected comprises human VEGF-A or a variant thereof and a non-human or chimeric antibody that binds VEGF-A.

In certain embodiments, the detectably labeled antibody or antigen-binding fragment thereof binds VEGF-A.

In various embodiments wherein the detectably labeled antibody binds VEGF-A, the non-human or chimeric antibody and the detectably labeled antibody bind VEGF-A at the same or different epitopes. In certain embodiments, the non-human or chimeric antibody and the detectably labeled antibody bind VEGF-A at different epitopes.

In certain embodiments, the non-human or chimeric antibody and the detectably labeled antibody do not interfere with each other. Thus, binding of one of these antibodies does not prevent or reduce binding of the corresponding other antibody. In various embodiments of the invention, the non-human or chimeric antibody and the detectably labeled antibody bind two different epitopes on the same monomer and/or two different epitopes on each monomer of the dimer. Alternatively, the non-human or chimeric antibody and the detectably labeled antibody bind the same or substantially the same epitope on different monomers of the dimer. In certain embodiments, the non-human or chimeric antibody and the detectably labeled antibody bind VEGF-A at different epitopes.

In particular embodiments, the non-human or chimeric antibody and the detectably labeled antibody bind to the same or different epitopes, respectively, as the VEGF-a antagonist, particularly bind to different epitopes, as the antagonist antibody. Where the non-human or chimeric antibody and the detectably labeled antibody bind the same epitope as the antagonist, particularly the antagonist antibody, it is contemplated that the non-human or chimeric antibody and the detectably labeled antibody bind the epitope with a lower Kd value than the antagonist. In particular embodiments, the non-human or chimeric antibody and the detectably labeled antibody bind to the epitope with a Kd value of 1.5nM or less, particularly 1nM or less, 0.75nM or less, particularly 0.5nM or less.

In a particular embodiment, the non-human or chimeric antibody and the detectably labeled antibody bind to each other epitopes covered or bound by VEGF receptors, in particular VEGF-a receptors VEGFA-R1 and/or VEGFA-R2, respectively. Thus, the non-human or chimeric antibody and the detectably labeled antibody bind to the same epitope as the VEGF-A receptor, in particular VEGFA-R1 or VEGFA-R2. Alternatively, the non-human or chimeric antibody and the detectably labeled antibody bind to an epitope that is not directly bound by the VEGF-a receptor, such as, for example, VEGFA-R1 or VEGFA-R2, but is covered by the receptor, such that binding of the first and/or second antibody prevents binding of the VEGF-a receptor. Thus, in certain embodiments, the non-human or chimeric antibody and the detectably labeled antibody compete for binding of VEGFA-R1 and/or VEGFA-R2.

VEGF-A exists as a monomer or dimer, particularly a homodimer. Thus, in various embodiments of the fourth aspect, the non-human or chimeric antibody and the detectably labeled antibody bind two different epitopes on the same monomer and/or two different epitopes on each monomer of the dimer. Alternatively, the first and the second antibodies bind to the same or substantially the same epitope on different monomers of the dimer.

In various embodiments of the fourth aspect, either the non-human or chimeric antibody and the detectably labeled antibody binds to an epitope that is bound by an antibody, the antibody comprises a sequence selected from the group consisting of SEQ ID NOs: 2, amino acids 46-51 of SEQ ID NO:2, amino acids 69-71 of SEQ ID NO:2, amino acid 108-116 of SEQ ID NO:3, amino acids 44-52 of SEQ ID NO:3, amino acids 70-76 of SEQ ID NO:3, amino acid 115-125 of SEQ ID NO:4, amino acids 47-52 of SEQ ID NO:4, amino acids 70-72 of SEQ ID NO:4, amino acid 109 and 117 of SEQ ID NO:5, amino acids 45-52 of SEQ ID NO:5 of the amino acids 70-77 of, and SEQ ID NO:5 amino acid 116 and 126.

In various embodiments of the fourth aspect, the non-human or chimeric antibody and the detectably labeled antibody bind an epitope which is bound by an antibody comprising a CDR selected from the group consisting of amino acids 46-51 of SEQ ID NO:2, amino acids 69-71 of SEQ ID NO:2, amino acids 108-116 of SEQ ID NO:2, amino acids 44-52 of SEQ ID NO:3, amino acids 70-76 of SEQ ID NO:3, and amino acids 115-125 of SEQ ID NO: 3. In a particular embodiment, the first antibody and/or the second antibody binds to an epitope bound by an antibody comprising a CDR consisting of amino acids 46-51 of SEQ ID NO:2, amino acids 69-71 of SEQ ID NO:2, amino acids 108-116 of SEQ ID NO:2, amino acids 44-52 of SEQ ID NO:3, amino acids 70-76 of SEQ ID NO:3, and amino acids 115-125 of SEQ ID NO: 3.

In various embodiments of the fourth aspect, the non-human or chimeric antibody and the detectably labeled antibody bind an epitope which is bound by an antibody comprising a CDR selected from the group consisting of amino acids 47-52 of SEQ ID NO:4, amino acids 70-72 of SEQ ID NO:4, amino acids 109-117 of SEQ ID NO:4, amino acids 45-52 of SEQ ID NO:5, amino acids 70-77 of SEQ ID NO:5, and amino acids 116-126 of SEQ ID NO: 5. In a particular embodiment, the first antibody and/or the second antibody binds to an epitope bound by an antibody comprising a CDR consisting of amino acids 47-52 of SEQ ID NO:4, amino acids 70-72 of SEQ ID NO:4, amino acids 109-117 of SEQ ID NO:4, amino acids 45-52 of SEQ ID NO:5, amino acids 70-77 of SEQ ID NO:5, and amino acids 116-126 of SEQ ID NO: 5.

In various embodiments of the fourth aspect, the non-human or chimeric antibody and the detectably labeled antibody bind an epitope which is bound by an antibody comprising an FR selected from the group consisting of amino acids 20-45 of SEQ ID NO:2, amino acids 52-68 of SEQ ID NO:2, amino acids 72-107 of SEQ ID NO:2, amino acids 117-126 of SEQ ID NO:2, amino acids 19-43 of SEQ ID NO:3, amino acids 53-69 of SEQ ID NO:3, amino acids 77-114 of SEQ ID NO:3, and amino acids 126-136 of SEQ ID NO: 3. In a particular embodiment, the first antibody and/or the second antibody binds to an epitope bound by an antibody comprising an FR consisting of amino acids 20-45 of SEQ ID NO:2, amino acids 51-68 of SEQ ID NO:2, amino acids 72-107 of SEQ ID NO:2, amino acids 117-126 of SEQ ID NO:2, amino acids 19-43 of SEQ ID NO:3, amino acids 51-69 of SEQ ID NO:3, amino acids 77-114 of SEQ ID NO:3, and amino acids 126-136 of SEQ ID NO: 3.

In various embodiments of the fourth aspect, the non-human or chimeric antibody and the detectably labeled antibody bind an epitope which is bound by an antibody comprising an FR selected from the group consisting of amino acids 21-46 of SEQ ID NO:4, amino acids 53-69 of SEQ ID NO:4, amino acids 73-108 of SEQ ID NO:4, amino acids 118-127 of SEQ ID NO:4, amino acids 20-44 of SEQ ID NO:5, amino acids 53-69 of SEQ ID NO:5, amino acids 78-115 of SEQ ID NO:5, and amino acids 127-137 of SEQ ID NO: 5. In a particular embodiment, the first antibody and/or the second antibody binds to an epitope bound by an antibody comprising an FR consisting of amino acids 21-46 of SEQ ID NO:4, amino acids 53-69 of SEQ ID NO:4, amino acids 73-108 of SEQ ID NO:4, amino acids 118-127 of SEQ ID NO:4, amino acids 20-44 of SEQ ID NO:5, amino acids 53-69 of SEQ ID NO:5, amino acids 78-115 of SEQ ID NO:5, and amino acids 127-137 of SEQ ID NO: 5.

In a particular embodiment, the non-human or chimeric antibody and the detectably labeled antibody bind an epitope that is bound by an antibody comprising a CDR consisting of amino acids 46-51 of SEQ ID NO:2, amino acids 69-71 of SEQ ID NO:2, amino acids 108-116 of SEQ ID NO:2, amino acids 44-52 of SEQ ID NO:3, amino acids 70-76 of SEQ ID NO:3, and amino acids 115-125 of SEQ ID NO: 3; and comprises the FRs consisting of amino acids 20-45 of SEQ ID NO.2, amino acids 52-68 of SEQ ID NO.2, amino acids 72-107 of SEQ ID NO.2, amino acids 117-126 of SEQ ID NO.2, amino acids 19-43 of SEQ ID NO. 3, amino acids 53-69 of SEQ ID NO. 3, amino acids 77-114 of SEQ ID NO. 3, and amino acids 126-136 of SEQ ID NO. 3.

In a particular embodiment, the non-human or chimeric antibody and the detectably labeled antibody bind an epitope which is bound by an antibody which comprises a CDR consisting of amino acids 47-52 of SEQ ID NO:4, amino acids 70-72 of SEQ ID NO:4, amino acid 109-117 of SEQ ID NO:4, amino acids 45-52 of SEQ ID NO:5, amino acids 70-77 of SEQ ID NO:5, and amino acid 116-126 of SEQ ID NO:5, and which comprises amino acids 21-46 of SEQ ID NO:4, amino acids 53-69 of SEQ ID NO:4, amino acids 73-108 of SEQ ID NO:4, amino acids 118-127 of SEQ ID NO:4, amino acids 20-44 of SEQ ID NO:5, amino acids 53-69 of SEQ ID NO:5, amino acids 78-115 of SEQ ID NO:5, and FR consisting of amino acids 127-137 of SEQ ID NO: 5.

In various embodiments of the fourth aspect, the non-human or chimeric antibody and the detectably labeled antibody comprise amino acid sequences selected from the group consisting of SEQ ID NOs: 2,3,4 and 5.

In various embodiments of the fourth aspect, the non-human or chimeric antibody and the detectably labeled antibody comprise a light chain having the amino acid sequence of SEQ ID NO.2 and a heavy chain having the amino acid sequence of SEQ ID NO. 3.

In various embodiments of the fourth aspect, the non-human or chimeric antibody and the detectably labeled antibody comprise a light chain having the amino acid sequence of SEQ ID NO.4 and a heavy chain having the amino acid sequence of SEQ ID NO. 5.

In various embodiments of the fourth aspect, the non-human or chimeric antibody and/or the detectably labeled antibody is labeled with a molecule detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. In particular embodiments, the non-human or chimeric antibody and/or the detectably labeled antibody is labeled with a fluorescent dye, an electron-dense reagent, an enzyme (e.g., as commonly used in an ELISA), biotin, digoxigenin/digoxigenin, or a hapten and other entity that is or is capable of being detectable. In particular embodiments, the non-human or chimeric antibody and/or the detectably labeled antibody is biotinylated or ruthenated. Methods for labeling antibodies are well known and well described to those skilled in the art, for example, Haughland (2003) Molecular Probes Handbook of Fluorescent Probes and research Chemicals, Molecular Probes, Inc.; brinkley (1992) Bioconjugate chem.3: 2; garman (1997) Non-Radioactive labelling, A Practical Approach, Academic Press, London; means (1990) Bioconjugate chem.1: 2; glazer et al, Chemical Modificationof proteins laboratory Techniques in Biochemistry and Molecular Biology (T.S. word and E.word, Eds.) American Elsevier Publishing Co., New York; lundblad, r.l. and nos. C.M (1984) Chemical Reagents for Protein Modification, vols.i. andII, CRC Press, New York; pfleiderer, G. (1985) "Chemical Modification of Proteins", Modern Methods in Protein Chemistry, H.Tscheschesche, Ed., Walter DeGruyter, Berlin and New York; wong (1991) Chemistry of Protein Conjugation and Cross-linking, CRCPress, Boca Raton, Fla.); Deleon-Rodriguez et al, chem. Eur. J.10(2004) 1149-; lewis et al, Bioconjugate chem.12(2001) 320-324; li et al, Bioconjugate chem.13(2002) 110-115; and Mier et al, Bioconjugate chem.16(2005) 240-.

In particular embodiments, the non-human or chimeric antibody and the detectably labeled antibody are capable of binding to or to a solid phase.

In particular embodiments, the non-human or chimeric antibody is capable of binding to or is bound to a solid phase.

In still further embodiments of the fourth aspect, the non-human or chimeric antibody is bound to or capable of binding to a solid phase and has a light chain comprising or consisting of a sequence according to SEQ ID NO.2, and the detectable antibody has a light chain comprising or consisting of a sequence according to SEQ ID NO. 4.

In still further embodiments of the fourth aspect, the non-human or chimeric antibody is bound to or capable of binding to a solid phase and has a light chain comprising or consisting of a sequence according to SEQ ID NO.4, and the detectably labeled antibody has a light chain comprising or consisting of a sequence according to SEQ ID NO. 2.

In still further embodiments of the fourth aspect, the non-human or chimeric antibody is bound to or capable of binding to a solid phase and has a heavy chain comprising or consisting of a sequence according to SEQ ID NO. 3, and the detectably labeled antibody has a heavy chain comprising or consisting of a sequence according to SEQ ID NO. 5.

In still further embodiments of the fourth aspect, the non-human or chimeric antibody is bound to or capable of binding to a solid phase and has a heavy chain comprising or consisting of a sequence according to SEQ ID NO. 5, and the detectably labeled antibody has a heavy chain comprising or consisting of a sequence according to SEQ ID NO. 3.

In still further embodiments of the fourth aspect, the non-human or chimeric antibody is bound to or capable of binding to a solid phase and has a light chain comprising or consisting of a sequence according to SEQ ID NO.2 and a heavy chain comprising or consisting of a sequence according to SEQ ID NO. 3, and the detectably labeled antibody has a light chain comprising or consisting of a sequence according to SEQ ID NO.4 and a heavy chain comprising or consisting of a sequence according to SEQ ID NO. 5.

In still further embodiments of the fourth aspect, the non-human or chimeric antibody is bound to or capable of binding to a solid phase and has a light chain comprising or consisting of a sequence according to SEQ ID NO.4 and a heavy chain comprising or consisting of a sequence according to SEQ ID NO. 5, and the detectably labeled antibody has a light chain comprising or consisting of a sequence according to SEQ ID NO.2 and a heavy chain comprising or consisting of a sequence according to SEQ ID NO. 3.

In still other embodiments, the solid phase is in a form selected from the group consisting of a bead, a tube, a plate of a microplate, and any other suitable surface (particularly suitable for performing immunoassays). In a particular embodiment, the bead is a microbead. Microbeads are microparticles having diameters in the nanometer and micrometer range. In various embodiments, the microparticles may have a diameter of 50 nanometers to 50 micrometers. In particular, the particles have a diameter of between 100nm and 10 μm, in particular from 200nm to 5 μm, or from 750nm to 5 μm. The microparticles comprise or consist of any suitable material known to those skilled in the art, for example they comprise or consist of or consist essentially of an inorganic or organic material. In particular, they comprise or consist of or consist essentially of a metal or metal alloy, or an organic material, or comprise or consist of or consist essentially of a carbohydrate element. In a particular embodiment, the material of the microparticles is selected from the group consisting of agarose, polystyrene, latex, polyvinyl alcohol, silica and ferromagnetic metals, alloys or composites. The particles may also comprise or consist of magnetic or ferromagnetic metals, alloys or compositions. The material may have specific properties such as, for example, being hydrophobic or hydrophilic. In particular embodiments, the microparticles are dispersed in an aqueous solution and retain a small negative surface charge, keeping the microparticles apart and avoiding non-specific clustering.

In a particular embodiment, the magnetic or paramagnetic microparticles are separated by magnetic force. A magnetic force is applied to pull the paramagnetic or magnetic particles out of the solution/suspension and to retain them while removing the liquid of the solution/suspension and, if desired, washing the particles, for example.

In particular embodiments, the non-human or chimeric antibody and/or the detectably labeled antibody is an IgG antibody. In certain embodiments, the non-human or chimeric antibody and/or the detectably labeled antibody is an IgG2 antibody. In particular embodiments, the non-human or chimeric antibody and/or the detectably labeled antibody is an IgG2b antibody, or an antigen-binding fragment thereof, particularly IgG2b-F (ab')₂And (3) fragment.

In a particular embodiment of the fourth aspect, the sample comprising said complex incubated in step (a) is further incubated with a VEGF-a antagonist. In particular embodiments, the incubation of the sample comprising said complex with the VEGF-a antagonist is performed prior to, simultaneously with, or subsequent to the incubation with the detectably labeled antibody or antigen-binding fragment thereof.

In various embodiments, the VEGF-a antagonist prevents the interaction between VEGF-a and one or more VEGF receptors. In particular, the VEGF-a antagonist competes with VEGF-a at the binding site of the receptor or alters the binding site on VEGF-a for its receptor in such a way that it is no longer able to bind its receptor or trigger a functional effect normally caused by its binding. Thus, the VEGF-a antagonist can bind to an epitope of VEGF-a and thereby block binding of VEGF-a to its receptor, or the VEGF-a antagonist can bind to an epitope of the receptor and thereby block binding of VEGF-a to the receptor. In particular embodiments, the VEGF-A antagonist binds to an epitope on VEGF-A and thereby prevents its binding to a VEGF receptor. In particular embodiments, the VEGF-A receptor is VEGFA-R1 and/or VEGFA-R2.

In still further embodiments of the fourth aspect, the VEGF-a antagonist is selected from the group consisting of a polypeptide, a peptibody (peptibody), an immunoadhesin, a small molecule, and an aptamer (aptamer).

In particular embodiments where the antagonist is a polypeptide, the polypeptide is an antibody. In a particular embodiment, the antibody is an anti-VEGF-A antibody. In particular, the anti-VEGF antibody is an antibody or antigen-binding fragment thereof that binds VEGF-a with sufficient affinity and specificity. In various embodiments, the antibody or antigen-binding fragment thereof has sufficient binding affinity for VEGF-A. In particular, the antibody or antigen-binding fragment thereof binds hVEGF-A with a Kd value between 100nM and 1pM, i.e., with a Kd value of 100nM,50nM,1nM,900pM,800pM,700pM,600pM,500pM,400pM,300pM,200pM,100pM,50pM, or 1 pM. In particular embodiments, the antibody or antigen-binding fragment thereof has a K between 50nM and 50pM,1nM and 100pM, or 700pM and 300pM_dValues bind to human VEGF-A (hVEGF-A).

In particular embodiments, the antagonistic VEGF-a antibody is monoclonal or polyclonal. In certain embodiments, the antagonistic VEGF-A antibody is recombinantly produced. In yet other embodiments, the antagonismThe VEGF-A antibodies are chimeric, in particular humanized anti-VEGF-A antibodies. In certain embodiments, the antagonistic VEGF-a antibody comprises a mutated human IgG1 framework region. The antagonistic VEGF-a antibody further comprises antigen binding Complementarity Determining Regions (CDRs) from the murine anti-hVEGF monoclonal antibody a.4.6.1 that block the binding of human VEGF to its receptor. In a particular embodiment, 93% of the amino acid sequence of the antagonistic VEGF-A antibody, including the majority of the framework regions, is derived from human IgG1, and about 7% of the sequence is derived from the murine antibody A4.6.1In certain embodiments, the antagonistic VEGF-A antibody is glycosylated. In still other embodiments, the antagonistic VEGF-a antibody has a molecular weight of about 149,000 daltons. In particular embodiments, the antagonistic VEGF-A antibody is Bevacizumab (BV), also known as "rhuMAb VEGF" orIt is a recombinant humanized anti-VEGF monoclonal antibody produced according to Presta et al (1997) Cancer Res.57: 4593-4599.

In particular embodiments, the antagonistic VEGF-a antibody is an antibody fragment. The antibody fragment is selected from the group consisting of Fab fragment, Fab 'fragment, F (ab')₂Fragments, single domain antibodies (sdabs), nanobodies (nanobodies), single chain fv (scFv), bivalent single chain variable fragments (di-scFv), tandem scFv, diabodies (diabodies), bispecific diabodies, single chain diabodies (scDb), bispecific T cell engagers (BiTE), and DART molecules. In particular embodiments, the antagonistic antibody fragment is a Fab fragment or F (ab')₂Fragments, in particular humanized Fab fragments or humanized F (ab')₂And (3) fragment.

In still other embodiments, the VEGF-A antagonist is selected from the group consisting of VEGF-trap, Mucagen, PTK787, SU11248, AG-013736, Bay 439006 (sorafenib), ZD-6474, CP632, CP-547632, AZD-2171, CDP-171, SU-14813, CHIR-258, AEE-788, SB786034, BAY579352, CDP-791, EG-3306, GW-786034, RWJ-417975/CT6758, and KRN-633.

In a particular embodiment, the complex, the detectably labeled antibody and/or the VEGF-a antagonist are contained in a physiological solution, in particular in a physiological buffer. In particular embodiments, the buffer is selected from the group of TAPS, Bicine, Tris, Tricine, TAPSO, HEPES, TES, MOPS, PIPES, cacodylate, and MES. In a particular embodiment, the buffer is MES buffer. In a particular embodiment, the MES buffer comprises 50mM MES,150mM NaCl,2mM EDTA-Na₂(dihydrate), 0.1% N-Methylisothiazolon-HCl, 0.1% oxypyroon, 0.1% Polydocanol (thesit), 1.0% albumin RPLA 4 assay quality, 0.2% PAK<->R-IgG (DET), Millipore water, pH adjusted to 6.30 with 2N NaOH.

In a particular embodiment of step (a), the detectably labeled antibody is mixed with the sample comprising said complex. The mixture is incubated for a period of time sufficient for the detectably labeled antibody to bind to the complex.

In various embodiments, the mixture is incubated for less than 60 minutes, i.e., less than 60,55,50,45,40,35,30,25,20,15,10, or 5 minutes. In particular embodiments, the mixture is incubated for 4 minutes to 1 hour (i.e., 4,5,6,7,8,9,10,15,20,25,30,35,40,45,50,55, or 60 minutes). In particular embodiments, the mixture is incubated for 5 minutes to 45 minutes, i.e., 5,6,7,8,9,10,15,20,25,30,35,40, or 45 minutes). In particular embodiments, the mixture is incubated for 5 minutes to 30 minutes, i.e., 5,6,7,8,9,10,15,20,25, or 30 minutes. In particular embodiments, the mixture is incubated for less than 20 minutes or less than 10 minutes. In particular embodiments, the mixture is incubated for 18 or 9 minutes.

In various embodiments, the mixture is incubated for 1-12 hours (i.e., 1,2,3,4,5,6,7,8,9,10,11, or 12 hours). In particular embodiments, the mixture is incubated for 1-4 hours or 8-12 hours.

In yet other embodiments, the mixture is incubated at a temperature of 3-40 ℃ (i.e., 3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39, or 40 ℃). In particular at 3 ℃ to 8 ℃ (i.e. 3,4,5,6,7 or 8), in particular at 4-5 ℃, or at 20 ℃ to 25 ℃ (i.e. at 20,21,22,23,24, or 25 ℃), in particular at 20-22 ℃, or at 35-37 ℃.

It is well known to the person skilled in the art that the incubation temperature and the incubation time depend on each other. Thus, in particular embodiments, the mixture is incubated at 20-25 ℃ for 10 minutes to 1 hour, i.e., the mixture is incubated at 20,21,22,23,24, or 25 ℃ for 10,15,20,25,30,35,40,45,50,55, or 60 minutes. In particular embodiments, the mixture is incubated at 22 ℃ for less than 10 minutes or less than 20 minutes. In yet other embodiments, the mixture is incubated at 3-8 ℃ for 8-12 hours, i.e., the mixture is incubated at 3,4,5,6,7, or 8 ℃ for 8,9,10,11, or 12 hours. In a particular embodiment, the mixture is incubated at 4 ℃ for 12 hours. In particular, the mixture is incubated at 3-8 ℃, in particular at 4-5 ℃ for 1-4 hours or 8-12 hours.

In a particular embodiment, the complex and/or the detectably labeled antibody are contained in a physiological solution, in particular in a physiological buffer and/or incubated therein. In particular embodiments, the buffer is selected from the group of TAPS, Bicine, Tris, Tricine, TAPSO, HEPES, TES, MOPS, PIPES, cacodylate, and MES. In a particular embodiment, the buffer is MES buffer. In a particular embodiment, the MES buffer comprises 50mM MES,150mM NaCl,2mM EDTA-Na₂(dihydrate), 0.1% N-Methylisothiazolon-HCl, 0.1% oxypyroon, 0.1% Polydocanol (thesit), 1.0% albumin RPLA 4 assay quality, 0.2% PAK<->R-IgG (DET), Millipore water, pH adjusted to 6.30 with 2N NaOH.

In a particular embodiment, the formed antibody-antigen-antibody complex, in particular the formed complex comprising the complex of human VEGF-a and the non-human or chimeric antibody, and the detectably labeled antibody or antigen-binding fragment thereof, is detected in step (b) via any method known in the art. In particular embodiments, the formed complex is detected via electrochemiluminescence, chemiluminescence, or fluorescence.

In particular, the present invention relates to the following items:

1. a method for measuring the level of VEGF-a in the presence of a VEGF-a antagonist, the method comprising:

incubating the sample with the first and second antibodies,

wherein said first and said second antibodies are capable of binding VEGF-A or a variant thereof in the presence of the VEGF-A antagonist, and wherein the binding of said first and said second antibodies do not interfere with each other,

wherein one of said antibodies is detectably labeled, thereby forming a detectably labeled complex comprising the first antibody, VEGF-A or a variant thereof, and the second antibody, and

detecting the formed complex, thereby measuring the level of VEGF-A in the presence of a VEGF-A antagonist.

2. The method of item 1, wherein the VEGF-a antagonist is a VEGF-a binding polypeptide, particularly a VEGF-a binding antibody.

3. The method of item 1 or 2, wherein the VEGF-a antagonist is the antibody bevacizumab.

4. The method of any one of items 1 to 3, wherein either the first and/or the second antibody binds to an epitope covered or bound by a VEGF receptor.

5. The method of any one of items 1 to 4, wherein either the first or the second antibody comprises a CDR consisting of amino acids 46-51 of SEQ ID NO:2, amino acids 69-71 of SEQ ID NO:2, amino acids 108-116 of SEQ ID NO:2, amino acids 44-52 of SEQ ID NO:3, amino acids 70-76 of SEQ ID NO:3, amino acids 115-125 of SEQ ID NO:3, or

Comprises CDRs consisting of amino acids 47-52 of SEQ ID NO.4, amino acids 70-72 of SEQ ID NO.4, amino acids 109-117 of SEQ ID NO.4, amino acids 45-52 of SEQ ID NO. 5, amino acids 70-77 of SEQ ID NO. 5, and amino acids 116-126 of SEQ ID NO. 5.

6. The method of any one of items 1 to 5, wherein either the first antibody and/or the second antibody binds to an epitope bound by an antibody comprising a CDR consisting of amino acids 46-51 of SEQ ID NO:2, amino acids 69-71 of SEQ ID NO:2, amino acids 108-116 of SEQ ID NO:2, amino acids 44-52 of SEQ ID NO:3, amino acids 70-76 of SEQ ID NO:3, amino acids 115-125 of SEQ ID NO:3, and comprising amino acids 20-45 of SEQ ID NO:2, amino acids 52-68 of SEQ ID NO:2, amino acids 72-107 of SEQ ID NO:2, amino acids 117-126 of SEQ ID NO:2, amino acids 19-43 of SEQ ID NO:3, amino acids 53-69 of SEQ ID NO:3, amino acids 77 to 114 of SEQ ID NO. 3 and amino acid 126 and 136 of SEQ ID NO. 3, or

Comprises a CDR consisting of amino acids 47 to 52 of SEQ ID NO.4, amino acids 70 to 72 of SEQ ID NO.4, amino acids 109 and 117 of SEQ ID NO.4, amino acids 45 to 52 of SEQ ID NO. 5, amino acids 70 to 77 of SEQ ID NO. 5, and amino acids 116 and 126 of SEQ ID NO. 5, and comprises amino acids 21 to 46 of SEQ ID NO.4, amino acids 53 to 69 of SEQ ID NO.4, amino acids 73 to 108 of SEQ ID NO.4, amino acids 118 and 127 of SEQ ID NO.4, amino acids 20 to 44 of SEQ ID NO. 5, amino acids 53 to 69 of SEQ ID NO. 5, amino acids of SEQ ID NO. 5

78-115, and amino acid 127-137 of SEQ ID NO: 5.

7. The method of any one of items 1 to 6, wherein either the first or the second antibody comprises a light chain having the amino acid sequence of SEQ ID NO.2 and a heavy chain having the amino acid sequence of SEQ ID NO. 3.

8. The method of any one of items 1 to 6, wherein either the first or the second antibody comprises a light chain having the amino acid sequence of SEQ ID NO.4 and a heavy chain having the amino acid sequence of SEQ ID NO. 5.

9. The method according to any one of items 1 to 8, wherein the first or the second antibody is bound or capable of binding to a solid phase.

10. The method according to any one of items 1 to 9, wherein the first antibody binds or is capable of binding to a solid phase and comprises a light chain having the amino acid sequence of SEQ ID NO.2 and a heavy chain having the amino acid sequence of SEQ ID NO. 3, and wherein the second antibody is detectably labeled and comprises a light chain having the amino acid sequence of SEQ ID NO.4 and a heavy chain having the amino acid sequence of SEQ ID NO. 5.

11. The method according to any one of items 1 to 10, wherein one of the first antibody or the second antibody is biotinylated.

12. The method according to any one of items 1 to 10, wherein one of the first antibody or the second antibody is ruthenated.

13. The method according to any one of items 1 to 12, wherein the sample is derived from a patient treated with a VEGF-a antagonist.

14. The method according to any one of items 1 to 13, wherein the sample is a bodily fluid, in particular whole blood, serum or plasma.

15. The method of any one of items 1 to 14, wherein the method for measuring the level of VEGF-a or a variant thereof in the presence of a VEGF-a antagonist is a sandwich immunoassay.

16. The method of any one of items 1 to 15, wherein the method for measuring the level of VEGF-a in the presence of a VEGF-a antagonist comprises detecting the formed complex via electrochemiluminescence.

17. A kit for measuring the level of VEGF-A in the presence of a VEGF-A antagonist, the kit comprising a first and a second antibody,

wherein both said first and said second antibodies are capable of binding VEGF-A in the presence of the VEGF-A antagonist, and wherein the binding of said first and said second antibodies do not interfere with each other,

wherein one of the antibodies is detectably labeled.

18. A kit according to item 17, wherein the kit comprises an antibody as defined in any one of items 4 to 12.

19. A composition of matter comprising a first and a second antibody,

wherein said first and said second antibodies are both capable of binding VEGF-A or a variant thereof in the presence of a VEGF-A antagonist,

wherein the binding of said first and said second antibodies do not interfere with each other, and

wherein one of the antibodies is detectably labeled.

20. The composition of matter of item 19, wherein either the first and/or the second antibody binds to an epitope covered or bound by a VEGF receptor.

21. The composition of matter of any one of items 19 or 20, wherein either the first or the second antibody comprises a CDR consisting of amino acids 46-51 of SEQ ID NO:2, amino acids 69-71 of SEQ ID NO:2, amino acids 108-116 of SEQ ID NO:2, amino acids 44-52 of SEQ ID NO:3, amino acids 70-76 of SEQ ID NO:3, amino acids 115-125 of SEQ ID NO:3, or

Comprises CDRs consisting of amino acids 47-52 of SEQ ID NO.4, amino acids 70-72 of SEQ ID NO.4, amino acids 109-117 of SEQ ID NO.4, amino acids 45-52 of SEQ ID NO. 5, amino acids 70-77 of SEQ ID NO. 5, and amino acids 116-126 of SEQ ID NO. 5.

22. The composition of matter of any one of items 19 to 21, wherein either the first antibody and/or the second antibody binds to an epitope bound by an antibody comprising a CDR consisting of amino acids 46-51 of SEQ ID NO:2, amino acids 69-71 of SEQ ID NO:2, amino acids 108-116 of SEQ ID NO:2, amino acids 44-52 of SEQ ID NO:3, amino acids 70-76 of SEQ ID NO:3, amino acids 115-125 of SEQ ID NO:3, and comprising an epitope consisting of amino acids 20-45 of SEQ ID NO:2, amino acids 52-68 of SEQ ID NO:2, amino acids 72-107 of SEQ ID NO:2, amino acids 117-126 of SEQ ID NO:2, amino acids 19-43 of SEQ ID NO:3, amino acids 53-69 of SEQ ID NO:3, amino acids 77 to 114 of SEQ ID NO. 3 and amino acid 126 and 136 of SEQ ID NO. 3, or

Comprises the CDR consisting of amino acids 47 to 52 of SEQ ID NO.4, amino acids 70 to 72 of SEQ ID NO.4, amino acids 109 to 117 of SEQ ID NO.4, amino acids 45 to 52 of SEQ ID NO. 5, amino acids 70 to 77 of SEQ ID NO. 5 and amino acids 116 to 126 of SEQ ID NO. 5 and comprises the FR consisting of amino acids 21 to 46 of SEQ ID NO.4, amino acids 53 to 69 of SEQ ID NO.4, amino acids 73 to 108 of SEQ ID NO.4, amino acids 118 to 127 of SEQ ID NO.4, amino acids 20 to 44 of SEQ ID NO. 5, amino acids 53 to 69 of SEQ ID NO. 5, amino acids 78 to 115 of SEQ ID NO. 5 and amino acids 127 to 137 of SEQ ID NO. 5.

23. The composition of matter of any one of claims 19 to 22, wherein either the first or the second antibody comprises a light chain having the amino acid sequence of SEQ ID No.2 and a heavy chain having the amino acid sequence of SEQ ID No. 3.

24. The composition of matter of any one of claims 19 to 23, wherein either the first or the second antibody comprises a light chain having the amino acid sequence of SEQ ID No.4 and a heavy chain having the amino acid sequence of SEQ ID No. 5.

25. The composition of matter according to any one of items 19 to 24, wherein the first or the second antibody is bound or capable of binding to a solid phase.

26. The composition of matter of any one of claims 19 to 25, wherein the first antibody or fragment thereof binds or is capable of binding to a solid phase and comprises a light chain having the amino acid sequence of SEQ ID No.2 and a heavy chain having the amino acid sequence of SEQ ID No. 3, and wherein the second antibody is detectably labeled and comprises a light chain having the amino acid sequence of SEQ ID No.4 and a heavy chain having the amino acid sequence of SEQ ID No. 5.

27. The composition of matter according to any one of clauses 19 to 26, wherein one of the first antibody or the second antibody is biotinylated.

28. The composition of matter according to any one of clauses 19 to 27, wherein one of the first antibody or the second antibody is ruthenated.

29. The composition of matter according to any one of claims 19 to 28, wherein the composition further comprises a VEGF-a antagonist.

30. The composition of matter according to item 29, wherein the VEGF-a antagonist is the antibody bevacizumab.

31. A method of detecting a complex comprising human VEGF-a and a non-human or chimeric protein, comprising the steps of:

(a) incubating a sample comprising said complex with a detectably labeled antibody that binds or is capable of binding to human VEGF-A or a variant thereof and/or the non-human or chimeric protein, and

(b) detecting the antigen binding protein.

32. The method of item 31, wherein the complex comprises human VEGF-a and a non-human or chimeric antibody or antigen-binding fragment thereof that binds VEGF-a.

33. The method of item 31 or 32, wherein both the non-human or chimeric protein and the detectably labeled antibody are capable of binding VEGF-A in the presence of a VEGF-A antagonist.

34. The method of any one of items 31 to 33, wherein either the non-human or chimeric protein or the detectably labeled antibody binds to an epitope bound by an antibody comprising a CDR consisting of amino acids 46-51 of SEQ ID NO:2, amino acids 69-71 of SEQ ID NO:2, amino acids 108-116 of SEQ ID NO:2, amino acids 44-52 of SEQ ID NO:3, amino acids 70-76 of SEQ ID NO:3, amino acids 115-125 of SEQ ID NO:3, or

Comprises CDRs consisting of amino acids 47-52 of SEQ ID NO.4, amino acids 70-72 of SEQ ID NO.4, amino acids 109-117 of SEQ ID NO.4, amino acids 45-52 of SEQ ID NO. 5, amino acids 70-77 of SEQ ID NO. 5, and amino acids 116-126 of SEQ ID NO. 5.

35. Method according to any of items 31 to 34, wherein either the non-human or chimeric protein or the detectably labeled antibody comprises a CDR consisting of amino acids 46-51 of SEQ ID NO:2, amino acids 69-71 of SEQ ID NO:2, amino acids 108-116 of SEQ ID NO:2, amino acids 44-52 of SEQ ID NO:3, amino acids 70-76 of SEQ ID NO:3, amino acids 115-125 of SEQ ID NO:3 and comprises amino acids 20-45 of SEQ ID NO:2, amino acids 52-68 of SEQ ID NO:2, amino acids 72-107 of SEQ ID NO:2, amino acids 117-126 of SEQ ID NO:2, amino acids 19-43 of SEQ ID NO:3, amino acids 53-69 of SEQ ID NO:3, amino acids 77-114 of SEQ ID NO:3, and amino acids 126 and 136 of SEQ ID NO:3, or

Comprises the CDR consisting of amino acids 47 to 52 of SEQ ID NO.4, amino acids 70 to 72 of SEQ ID NO.4, amino acids 109 to 117 of SEQ ID NO.4, amino acids 45 to 52 of SEQ ID NO. 5, amino acids 70 to 77 of SEQ ID NO. 5 and amino acids 116 to 126 of SEQ ID NO. 5 and comprises the FR consisting of amino acids 21 to 46 of SEQ ID NO.4, amino acids 53 to 69 of SEQ ID NO.4, amino acids 73 to 108 of SEQ ID NO.4, amino acids 118 to 127 of SEQ ID NO.4, amino acids 20 to 44 of SEQ ID NO. 5, amino acids 53 to 69 of SEQ ID NO. 5, amino acids 78 to 115 of SEQ ID NO. 5 and amino acids 127 to 137 of SEQ ID NO. 5.

36. The method of any one of claims 31 to 35, wherein either the non-human or chimeric protein or the detectably labeled antibody comprises a light chain having the amino acid sequence of SEQ ID No.2 and a heavy chain having the amino acid sequence of SEQ ID No. 3.

37. The method of any one of claims 31 to 36, wherein either the non-human or chimeric protein or the detectably labeled antibody comprises a light chain having the amino acid sequence of SEQ ID No.4 and a heavy chain having the amino acid sequence of SEQ ID No. 5.

38. The method of any one of claims 31 to 37, wherein either the non-human or chimeric protein or the detectably labeled antibody is bound or capable of binding to a solid phase.

39. The method of any one of items 31 to 38, wherein the non-human or chimeric protein binds or is capable of binding to a solid phase and comprises a light chain having the amino acid sequence of SEQ ID NO 2 and a heavy chain having the amino acid sequence of SEQ ID NO 3, and wherein the second antibody is detectably labeled and comprises a light chain having the amino acid sequence of SEQ ID NO 4 and a heavy chain having the amino acid sequence of SEQ ID NO 5.

40. The method of any one of items 31 to 39, wherein the non-human or chimeric protein or the detectably labeled antibody is biotinylated or ruthenated.

41. The method of any one of items 31 to 40, wherein the sample comprising said complex is further incubated with a VEGF-A antagonist prior to, simultaneously with, or after step (a).

42. The method of item 41, wherein the VEGF-A antagonist is the antibody bevacizumab.

Examples

The following examples are provided to illustrate, but not to limit, the presently claimed invention.

Example 1 Biacore epitope binning

Ternary epitope binning experiments were performed using a Biacore T200 instrument (GE Healthcare) to assess epitope accessibility of 3 monoclonal antibody or antibody fragment conjugates on dimeric VEGF-a 121 (see fig. 1). The antibodies described herein that have affinity for VEGF-A are rH-4.6.1-IgG (Avastin),13.2.5-F (ab') 2-Bi, and 13.7.40-Ru. The SC1 sensor was placed into the Biacore system according to the manufacturer's instructions and normalized with HBSN buffer (10mM HEPES pH 7.4,150mM NaCl). The system buffer was changed to PBS buffer pH 7.4 containing 5% DMSO and 0.05% Tween 20. The sample buffer was system buffer supplemented with 1mg/ml CMD (carboxy methyl dextran, Sigma # 86524). The system was run at 25 ℃. The antibody capture system was immobilized on the positioned SC1 sensor. 1600RU monoclonal mouse anti-human FC gamma-pan capture antibody (MAHFcg-pan, Roche) was immobilized on all sensor flow chambers by conventional EDC/NHS chemistry as described by the supplier. The capture system was regenerated by injecting 10mM NaOH at 20. mu.l/min for 15 seconds followed by injection of 10mM glycine buffer pH 2.5 twice at 20. mu.l/min for 1 minute each.

75nM of anti-rH-4.6.1-IgG was injected at 30. mu.l/min for 1 min at 5. mu.l/min on flow cell 1 and flow cell 2. The capture system was saturated on both flow chambers by injecting for 3 minutes at 30 μ l/min using a mixture of 2 μ M human normal IgG (Roche) and 1 μ M M-33(Roche) in sample buffer. At this stage of preparation, the assay set up on the flow cell 1 serves as an antigen-free reference. 150nM VEGF-A (VEGF-A121, Peprotech) was injected into the flow cell at 30. mu.l/min for 2 minutes to be displayed by rH-4.6.1-IgG on the sensor. To saturate the binding stability of the second antibody on the potential accessible dimeric VEGF 121 displayed, 75nM rH-4.6.1-IgG was again injected into the flow cell 1 and 2 at 20. mu.l/min for 3 min binding time. 75nM biotinylated antibody fragment 13.2.5-F (ab') 2-Bi was injected into the flow chamber at 20. mu.l/min for 3 minutes, followed by sequential injections of 75nM 13.7.40-Ru at 30. mu.l/min for 3 minutes. The black line in the image (FIG. 2) shows the sequential injection of the first, 13.2.5-F (ab') 2-Bi conjugate and the second, 13.7.40-Ru conjugate into flow chamber 2 after preparation of the sensor surface as described above. The black dotted line shows the signal level of two sequential injections of 13.2.5-F (ab') 2-Bi and 13.7.40-Ru in the absence of VEGF-A121 on flow cell 1 as a reference. On flow cell 2, 13.7.40-Ru showed an elevated binding signal, which was higher than the expected signal saturation level for the previous 13.2.5-F (ab') 2-Bi injection. This indicates the accessibility of the VEGF-a 121 epitope to both conjugates. On flow cell 1, omitting the VEGF-A antigen, there was no detectable interaction. Control experiments with either no VEGF-A present (FIG. 2B), or with VEGFA-R1 (FIG. 2C) or VEGFA-R2 (FIG. 2D) demonstrated that binding of the test antibody is VEGF-A specific and that the antibody binds to VEGF-A receptor binding or covers the epitope of VEGF-A that is covered by binding of VEGF-A receptor.

Example 2 Elecsys Competition assay

Measurements were performed in a sandwich assay format.Signal detection in the e601 analyzer is based on electrochemiluminescence. In this sandwich assay, a biotin conjugate (i.e., a capture antibody) is immobilized on the surface of streptavidin-coated magnetic beads. The detection antibody carries a complexed ruthenium cation as a signal moiety. In the presence of an analyte, a chromophoric ruthenium complex is bridged to a solid phaseThe e601 analyzer emits light at 620nm after excitation at platinum electrodes contained in the measurement chamber. The signal output is in arbitrary light units.

An experimental VEGF-A antigen assay was performed as follows. Incorporation of at least 50-fold molar excess of VEGF-A antagonist into antigen-positive samples and incubation for 30 minutes to allow equilibrium binding to VEGF-A. In that50 μ l of the sample preincubated with VEGF-A antagonist was measured on an e601 analyzer in physiological buffer at pH 7.0 containing 100mM potassium phosphate and 225mM KCl with 50 μ l of 1 μ g/ml capture antibody-biotin conjugate and 50 μ l of 1 μ g/ml detection antibody-ruthenium label conjugate. After a 9 minute incubation time, 50. mu.l streptavidin-coated paramagnetic microparticles were added and incubated for a further 9 minutes. Thereafter, the VEGF-A antigen was detected (via the generation of an electrochemiluminescent signal in these experiments). VEGF-A was detected in the presence of Avastin using the Elecsys competition assay.

Sequence listing

<110> Haofmii Roche GmbH (F. Hoffmann-La Roche AG)

<120> methods and means for detecting the level of total VEGF-A

<130>P33749-WO

<150>EP16179780.8

<151>2016-07-15

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Claims (15)

1. A method for measuring the level of VEGF-a in the presence of a VEGF-a antagonist, the method comprising:

incubating the sample with the first and second antibodies,

wherein said first and said second antibodies are capable of binding VEGF-A in the presence of the VEGF-A antagonist, and wherein the binding of said first and said second antibodies do not interfere with each other, wherein one of said antibodies is detectably labeled, thereby forming a detectably labeled complex comprising said first antibody, VEGF-A, and said second antibody, and

detecting the formed complex, thereby measuring the level of VEGF-A in the presence of a VEGF-A antagonist.

2. The method of claim 1, wherein the VEGF-a antagonist is a VEGF-a binding polypeptide, particularly a VEGF-a binding antibody.

3. The method of claim 1 or 2, wherein the VEGF-a antagonist is the antibody bevacizumab (bevacizumab).

4. The method of any one of claims 1 to 3, wherein either the first and/or the second antibody binds to an epitope covered or bound by a VEGF receptor.

5. The method according to any one of claims 1 to 4, wherein either the first or the second antibody comprises a CDR consisting of amino acids 46-51 of SEQ ID NO 2, amino acids 69-71 of SEQ ID NO 2, amino acids 108-116 of SEQ ID NO 2, amino acids 44-52 of SEQ ID NO 3, amino acids 70-76 of SEQ ID NO 3, amino acids 115-125 of SEQ ID NO 3, or

Comprises CDRs consisting of amino acids 47-52 of SEQ ID NO.4, amino acids 70-72 of SEQ ID NO.4, amino acids 109-117 of SEQ ID NO.4, amino acids 45-52 of SEQ ID NO. 5, amino acids 70-77 of SEQ ID NO. 5, and amino acids 116-126 of SEQ ID NO. 5.

6. The method of any one of claims 1 to 5, wherein either the first or the second antibody comprises a light chain having the amino acid sequence of SEQ ID NO 2 and a heavy chain having the amino acid sequence of SEQ ID NO 3.

7. The method of any one of claims 1 to 6, wherein either the first or the second antibody comprises a light chain having the amino acid sequence of SEQ ID NO.4 and a heavy chain having the amino acid sequence of SEQ ID NO. 5.

8. The method according to any one of claims 1 to 7, wherein the first or the second antibody is bound or capable of binding to a solid phase.

9. The method according to any one of claims 1 to 8, wherein either the first antibody or the second antibody is biotinylated and wherein the other antibody is ruthenated.

10. The method according to any one of claims 1 to 9, wherein the sample is derived from a patient treated with a VEGF-a antagonist.

11. The method according to any one of claims 1 to 10, wherein the sample is a bodily fluid, in particular whole blood, serum or plasma.

12. A kit for measuring the level of VEGF-A, or a variant thereof, in the presence of a VEGF-A antagonist, the kit comprising first and second antibodies,

wherein said first and said second antibodies are both capable of binding to VEGF-A in the presence of the VEGF-A antagonist and wherein the binding of said first and said second antibodies do not interfere with each other, and

wherein one of the antibodies is detectably labeled.

13. A kit according to claim 12, wherein the kit comprises an antibody as defined in any one of claims 4 to 9.

14. A composition of matter comprising a first and a second antibody,

wherein said first and said second antibodies are both capable of binding VEGF-A in the presence of a VEGF-A antagonist,

wherein the binding of said first and said second antibodies do not interfere with each other, and

wherein one of the antibodies is detectably labeled.

15. A method of detecting a complex comprising human VEGF-a and a non-human or chimeric protein, comprising the steps of:

(a) incubating a sample comprising said complex with a detectably labeled antibody or antigen-binding fragment thereof that binds or is capable of binding to human VEGF-A and/or the non-human or chimeric protein, and

(b) detecting the antigen binding protein.