TWI429655B - Antibodies to egfl7,composition comprising,polynucleotide encoding,and methods of making and using the same - Google Patents

Description

Anti-EGFL7 antibody, composition comprising the same, polynucleotide encoding the same, and method of making and using same

The present invention is directed to compositions and methods for modulating vascular development. In particular, the invention relates to antibodies that bind to an epidermal growth factor-like domain 7 (EGFL7) polypeptide. The invention further relates to the diagnosis and treatment of conditions and diseases associated with angiogenesis.

The development of vascular connections is a fundamental requirement for many physiological and pathological processes. Actively growing tissues such as embryos and tumors require an appropriate blood supply. They meet this need by creating pro-angiogenic factors that promote the formation of new blood vessels via a process called angiogenesis. Angiogenesis is a complex but ordered biological event involving all or more of the following steps: a) endothelial cells (EC) are proliferated by existing EC or differentiated by progenitor cells; b) EC migrates and coalesces to form a bundle structure; c) then the vascular bundle undergoes tubulogenesis to form a vessel with a central lumen; d) the existing bundle or vessel protrudes into a new branch to form a secondary vessel; e) the primitive vascular bundle undergoes further remodeling and reshaping; The recruitment of outer endothelial cells into the endothelial tube provides maintenance and regulation to the blood vessels; these cells include pericytes of small capillaries, smooth muscle cells of larger blood vessels, and cardiomyocytes in the heart. Hanahan, Science 277: 48-50 (1997); Hogan and Kolodziej, Nat. Rev. Genet. 3: 513-23 (2002); Lubarsky and Krasnow, Cell 112: 19-28 (2003).

It has been well established that the pathogenesis of a variety of conditions involves angiogenesis. These conditions include solid tumors and metastases; atherosclerosis; post-lens fibrous tissue hyperplasia; hemangioma; chronic inflammation; intraocular neovascular diseases such as proliferative retinopathy (eg diabetic retinopathy), age-related macular Degeneration (AMD), neovascular glaucoma; immune rejection of transplanted corneal tissue and other tissues; rheumatoid arthritis; and psoriasis. Folkman et al, J. Biol. Chem. 267: 10931-34 (1992); Klagsbrun et al, Annu. Rev. Physiol . 53: 217-39 (1991) and Garner A., Pathobiology of Ocular Disease, "Vascular Diseases". A Dynamic Approach, Garner A., Klintworth GK, 2nd ed. (Marcel Dekker, NY, 1994), pp. 1625-1710.

In the case of tumor growth, angiogenesis appears to be critical for the conversion of hyperplasia into neoplasia and nutrition for tumor growth and metastasis. Folkman et al, Nature 339: 58 (1989). Neovascularization allows tumor cells to gain growth and proliferation autonomy compared to normal cells. Tumors usually start with a single abnormal cell that can only proliferate to a few cubic millimeters due to the distance from the available capillary bed, and the tumor can remain "sleeping" for a longer period of time without further growth. spread. Some tumor cells then switch to an angiogenic phenotype to activate endothelial cells, which proliferate and mature into new capillaries. These newly formed blood vessels not only allow the primary tumor to continue to grow, but also allow the metastatic tumor cells to spread and re-emerge. Accordingly, a correlation between microvessel density in tumor sections and patient survival has been observed in breast cancer as well as in several other tumors. Weidner et al, N. Engl. J. Med. 324: 1-6 (1991); Horak et al, Lancet 340: 1120-24 (1992); Macchiarini et al, Lancet 340: 145-46 (1992). The exact mechanism controlling angiogenesis switching is not fully understood, but it is believed that the neovascularization of the tumor mass is caused by a net difference between a large number of angiogenic stimulators and inhibitors (Folkman, Nat. Med. 1(1): 27-31 ( 1995)).

The process of vascular development is strictly regulated. Up to now, it has been shown that a large number of molecules (mainly secreted by peripheral cells) regulate the differentiation, proliferation, migration and coalescence of EC into a bundle structure. For example, vascular endothelial growth factor (VEGF) has been identified as a key factor involved in stimulating angiogenesis and inducing vascular permeability. Ferrara et al., Endocr. Rev. 18: 4-25 (1997). The discovery that even single VEGF allele deletions also lead to embryonic death indicates an irreplaceable role for this factor in vascular system development and differentiation. In addition, VEGF has been shown to be a key mediator of neovascularization associated with tumors and intraocular disorders. Ferrara et al., Endocr. Rev., supra. Most of the human tumors examined showed excessive expression of VEGF mRNA. Berkman et al, J. Clin. Invest. 91: 153-59 (1993); Brown et al, Human Pathol. 26: 86-91 (1995); Brown et al, Cancer Res. 53: 4727-35 (1993) Mattern et al, Brit . J. Cancer 73: 931-34 (1996); Dvorak et al, Am. J. Pathol. 146: 1029-39 (1995).

In addition, the VEGF concentration in the eye fluid is highly correlated with the presence of vascular active hyperplasia in patients with diabetes and other ischemic-related retinopathy. Aiello et al., N. Engl. J. Med. 331:1480-87 (1994). In addition, studies have demonstrated that VEGF is localized in the choroidal neovascular membrane of patients with AMD. Lopez et al, Invest. Ophthalmol . Vis . Sci. 37: 855-68 (1996).

Anti-VEGF neutralizing antibodies inhibit the growth of a variety of human tumor cell lines in nude mice (Kim et al, Nature 362: 841-44 (1993); Warren et al, J. Clin. Invest. 95: 1789-97 (1995). ); Borgstr M et al, Cancer Res. 56:4032-39 (1996); Melnyk et al, Cancer Res. 56:921-24 (1996)), and also inhibits intraocular angiogenesis in a model of ischemic retinopathy (Adamis) Et al., Arch. Ophthalmol. 114: 66-71 (1996)). Thus, anti-VEGF monoclonal antibodies or other inhibitors of VEGF action are desirable candidates for the treatment of tumors and various intraocular neovascular disorders. Such antibodies are described in, for example, EP 817,648, published Jan. 14, 1998, and WO 98/45331 and WO 98/45332, issued October 15, 1998. An anti-VEGF antibody, bevacizumab, has been approved by the FDA in combination with a chemotherapeutic regimen for the treatment of metastatic colorectal cancer (CRC). Bevacizumab, which is used to treat various cancer indications, is also being investigated in a number of ongoing clinical trials.

Extracellular matrix (ECM) is known to play an important role in the process of angiogenesis. Madri, Transpl. Immunol. 5: 179-83 (1997). The EC is surrounded by a temporary ECM during its migration and adheres to the newly synthesized vascular basement membrane after formation of the lumen. In addition to providing stents during capillary morphogenesis, ECM has also been shown to have complex local control of EC function. For example, ECM is capable of regulating the availability of soluble angiogenic mediators for EC and defining the nature and type of interaction with integrins and cell adhesion molecules. It has also been shown that the survival of EC is regulated by the synergy between growth factor receptors and integrins, which in turn is controlled by the composition of local ECM. Stupack and Cheresh, Oncogene 22:9022-29 (2003).

Despite many advances in the field of angiogenesis, certain steps in the process of angiogenesis remain to be clearly understood. In particular, how to regulate tube formation - how the vascular bundle progresses into a tube and what factors regulate this change is poorly understood. In view of the role of angiogenesis in many diseases and conditions, there is a need for a method of reducing or inhibiting one or more of the biological effects that cause such processes. There is also a need for a method of assaying the presence of pathogenic polypeptides in normal and disease conditions, and particularly in cancer. There is also a need for compositions and methods that enhance the efficacy of existing anti-angiogenic therapies.

The invention is based, in part, on identifying an anti-EGFL7 antibody having properties indicative of its particular benefit to treatment.

In one aspect, the invention provides an antibody produced by fusion tumor anti-EGFL7 mumab 4F11.1.8, anti-EGFL7 mumab 10G9.1.6, and anti-EGFL7 mumab 18F7.1.8.

In one aspect, the invention provides an anti-EGFL7 antibody comprising one or more complementarity determining regions (CDRs) selected from the group consisting of: (a) 4F11 CDR-L1 sequence KASQSVDYDGDSYMS (SEQ ID NO: 5) (b) 4F11 CDR-L2 sequence GASNLES (SEQ ID NO: 6); (c) 4F11 CDR-L3 sequence QQNNEDPYT (SEQ ID NO: 7); (d) 4F11 CDR-H1 sequence TYGMS (SEQ ID NO: 8); (e) 4F11 CDR-H2 sequence WINTHSGVPTYADDFKG (SEQ ID NO: 9); and (f) 4F11 CDR-H3 sequence LGSSA (SEQ ID NO: 10). In some embodiments, the light chain of the antibody comprises at least one, at least two, or all three CDR sequences selected from the group consisting of: KASQSVDYDGDSYMS (SEQ ID NO: 5), GASNLES (SEQ ID NO: 6), and QQNNEDPYT ( SEQ ID NO: 7). In some embodiments, the heavy chain of the antibody comprises at least one, at least two or all three CDR sequences selected from the group consisting of TYGMS (SEQ ID NO: 8), WINTHSGVPTYADDFKG (SEQ ID NO: 9), and LGSSA ( SEQ ID NO: 10). In some embodiments, the light chain of the antibody comprises at least one, at least two, or all three CDR sequences selected from the group consisting of: KASQSVDYDGDSYMS (SEQ ID NO: 5), GASNLES (SEQ ID NO: 6), and QQNNEDPYT ( SEQ ID NO: 7); and the heavy chain of the antibody comprises at least one, at least two or all three CDR sequences selected from the group consisting of TYGMS (SEQ ID NO: 8), WINTHSGVPTYADDFKG (SEQ ID NO: 9) and LGSSA (SEQ ID NO: 10). In some embodiments, the light chain of the antibody comprises the sequence: DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMSWYQQKPGQPPKLLIYGASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQNNEDPYTFGGGTKVEIKR (SEQ ID NO: 1). In some embodiments, the heavy chain of the antibody comprises the sequence: QIQLVQSGPELKKPGETVKISCKASGHTFTTYGMSWVKQAPGKGLKWMGWINTHSGVPTYADDFKGRFAFSLETSASTAHLQINNLKNEDTATYFCARLGSSAVDYWGQGTTVTVSS (SEQ ID NO: 2).

In one aspect, the invention provides an anti-EGFL7 antibody comprising one or more complementarity determining regions (CDRs) selected from the group consisting of: (a) 10G9 CDR-L1 sequence RSSQSLVHTNGITYLH (SEQ ID NO: 11 (b) 10G9 CDR-L2 sequence KVSNRFS (SEQ ID NO: 12); (c) 10G9 CDR-L3 sequence SQSTHVPLT (SEQ ID NO: 13); (d) 10G9 CDR-H1 sequence DYYMNSDYYMN (SEQ ID NO: 14); (e) 10G9 CDR-H2 sequence DINPKNGGTTYNQKFKG (SEQ ID NO: 15); and (f) 10G9 CDR-H3 sequence (SEQ ID NO: 16). In some embodiments, the light chain of the antibody comprises at least one, at least two, or all three CDR sequences selected from the group consisting of: RSSQSLVHTNGITYLH (SEQ ID NO: 11), KVSNRFS (SEQ ID NO: 12), and SQSTHVPLT ( SEQ ID NO: 13). In some embodiments, the heavy chain of the antibody comprises at least one, at least two, or all three CDR sequences selected from the group consisting of: DYYMNSDYYMN (SEQ ID NO: 14), DINPKNGGTTYNQKFKG (SEQ ID NO: 15), and ALGVFDY ( SEQ ID NO: 16). In some embodiments, the light chain of the antibody comprises at least one, at least two, or all three CDR sequences selected from the group consisting of: RSSQSLVHTNGITYLH (SEQ ID NO: 11), KVSNRFS (SEQ ID NO: 12), and SQSTHVPLT ( SEQ ID NO: 13); and the heavy chain of the antibody comprises at least one, at least two or all three CDR sequences selected from the group consisting of DYYMNSDYYMN (SEQ ID NO: 14), DINPKNGGTTYNQKFKG (SEQ ID NO: 15) and ALGVFDY (SEQ ID NO: 16). In some embodiments, the light chain of the antibody comprises the sequence: DIVMTQTPLSLPVSLGDQASISCRSSQSLVHTNGITYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPLTFGAGTKVEIKR (SEQ ID NO: 3). In some embodiments, the heavy chain of the antibody comprises the sequence: EVQLQQSGPELVKPGASVKISCKASGYTFSDYYMNSDYYMNWVKQSHGKSLEWIGDINPKNGGTTYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCAREADYDPIYYAMDYWGQGTTLTVSA (SEQ ID NO: 4).

In some embodiments, the invention provides an anti-EGFL7 antibody that specifically binds to a polypeptide comprising one of the following amino acid sequences: CCP, TIY, and ACS. In some embodiments, the invention provides an isolated antibody that binds to the same epitope on human EGFL7 as other antibodies of the invention. In some embodiments, the invention provides isolated antibodies that compete with other antibodies of the invention for binding to EGFL7.

In some embodiments, the antibodies of the invention are monoclonal antibodies. In some embodiments, an antibody of the invention is a chimeric antibody, a humanized antibody, an affinity matured antibody, a human antibody, or a bispecific antibody. In some embodiments, the antibody is an antibody fragment.

In some embodiments, the invention provides a pharmaceutical composition comprising an anti-EGFL7 antibody of the invention. In some embodiments, the pharmaceutical composition additionally comprises an anti-angiogenic agent. In some embodiments, the anti-angiogenic agent is bevacizumab or ranibizumab.

In some embodiments, the invention provides a polynucleotide encoding an antibody of the invention. In some embodiments, the invention provides vectors comprising such polynucleotides. In some embodiments, the vector is a performance vector. In some embodiments, the invention provides host cells comprising such vectors, including prokaryotic and eukaryotic cells, including mammalian cells. In some embodiments, the invention provides a method of making an anti-EGFL7 antibody comprising (a) rendering a performance vector in a suitable host cell; and (b) recovering the antibody.

In some embodiments, the invention provides a method of reducing or inhibiting angiogenesis in a subject having a pathological condition associated with angiogenesis, comprising administering to the subject an effective amount of an anti-EGFL7 antibody of the invention Or a pharmaceutical composition comprising an anti-EGFL7 antibody of the invention. In some embodiments, the pathological condition is a neoplasm, such as a carcinoma. In some embodiments, the pathological condition is associated with the eye, such as an intraocular neovascular disorder. In some embodiments, an anti-angiogenic agent is administered to a subject in addition to the anti-EGFL7 antibody of the invention. In some embodiments, the anti-angiogenic agent is an antagonist of vascular endothelial growth factor (VEGF), such as an anti-VEGF antibody (including bevacizumab and Raney strain). In some embodiments, the anti-angiogenic agent is administered before or after administration of the anti-EGFL7 antibody. In some embodiments, the anti-angiogenic agent is administered concurrently with an anti-EGFL7 antibody.

In some embodiments, the invention provides a method of enhancing the efficacy of an anti-angiogenic agent in a subject having a pathological condition associated with angiogenesis, comprising administering to the subject an anti-EGFL7 antibody of the invention or A pharmaceutical composition comprising an anti-EGFL7 antibody of the invention. In some embodiments, the pathological condition is a neoplasm, such as a carcinoma. In some embodiments, the pathological condition is associated with the eye, such as an intraocular neovascular disorder. In some embodiments, an anti-angiogenic agent is administered to a subject in addition to the anti-EGFL7 antibody of the invention. In some embodiments, the anti-angiogenic agent is an antagonist of vascular endothelial growth factor (VEGF), such as an anti-VEGF antibody (including bevacizumab and Raney strain). In some embodiments, the anti-angiogenic agent is administered before or after administration of the anti-EGFL7 antibody. In some embodiments, the anti-angiogenic agent is administered concurrently with an anti-EGFL7 antibody. In some embodiments, other treatments, such as corticosteroids or photodynamic therapy, are also administered.

The invention provides anti-EGFL7 antibodies useful for, for example, treating or preventing a disease state associated with the performance and/or activity of EGFL7, such as enhanced and undesirable performance and/or activity of performance and/or activity. In some embodiments, the antibodies of the invention are useful for treating tumors, cancer, and/or cell proliferative disorders.

In another aspect, the anti-EGFL7 antibodies of the invention are useful as reagents for detecting and/or isolating EGFL7, such as EGFL7 in various tissues and cell types.

The invention further provides methods of making an anti-EGFL7 antibody, a polynucleotide encoding an anti-EGFL7 antibody, and a cell comprising a polynucleotide encoding an anti-EGFL7 antibody.

common technology

Those skilled in the art are generally well aware of and typically use conventional techniques to use the techniques and procedures described or referenced herein, such as Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd Edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYCURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FMAusubel et al., (2003)); METHODS IN ENZYMOLOGY series (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hames and GRTaylor Editor ( 1995)), Harlow and Lane ed. (1988) ANTIBODIES, A LABORATORY MANUAL and ANIMAL CELL CULTURE (RI Freshneyb ed. (1987)).

definition

An "isolated" antibody is one that has been identified and separated from components of its natural environment and/or recovered from components of the natural environment. The contaminating component of its natural environment is a substance that will interfere with the diagnostic or therapeutic use of the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody will: (1) be purified to greater than 95% by weight of the antibody as determined by the Lowry method and sometimes greater than 99% by weight; (2) purified to be sufficient to be rotated by use The cup sequencer obtains at least 15 residues of the N-terminal or internal amino acid sequence; or (3) by Coomassie blue or silver staining under reduced or non-reducing conditions by SDS The -PAGE method was purified to homogeneity. Since at least one component of the antibody's natural environment will not be present, the isolated antibody includes antibodies that are in situ in recombinant cells. However, isolated antibodies are typically prepared by at least one purification step.

An "isolated" nucleic acid molecule is a nucleic acid molecule that has been identified and separated from at least one contaminant nucleic acid molecule that is normally associated with it in the natural source of the antibody nucleic acid. The form or configuration of the isolated nucleic acid molecule is different from the form or configuration it sees in nature. The isolated nucleic acid molecule is thus distinguished from the nucleic acid molecule present in the native cell. However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in a cell that typically expresses an antibody, wherein, for example, the nucleic acid molecule is in a different chromosomal location than the nucleic acid molecule of the native cell.

The term "variable domain residues according to Kabat number" or "amino acid sites according to Kabat number" or its variant system refers to Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health , Bethesda, MD. (1991) Numbering system for antibody-encoded heavy chain variable domains or light chain variable domains. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the FR or CDR shortening or insertion of the variable domains. For example, the heavy chain variable domain can include a single amino acid insertion after H2 residue 52 (residue 52a according to Kabat) and an insertion residue following residue 82 of heavy chain FR (eg, residues according to Kabat) 82a, 82b, 82c, etc.). The Kabat numbering of residues in a given antibody can be determined by aligning the homologous region of the antibody sequence to a "standard" Kabat numbering sequence.

The phrase "substantially similar" or "substantially identical" as used herein means that two numerical values (generally one numerical value is associated with an antibody of the invention and the other is related to a reference/comparative antibody) is sufficiently high. Similarity such that one skilled in the art can recognize that there is little or no biologically and/or statistically significant difference between the two values within the range of biological characteristics measured by the equivalent (e.g., Kd value). . The difference between the two values as a function of the reference/compare antibody value is generally less than about 50%, about 40%, about 30%, about 20%, or about 10%.

"Binding affinity" generally refers to the total intensity of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). As used herein, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between a binding pair (eg, an antibody and an antigen) member, unless otherwise indicated. The affinity of the molecule X for its conjugate Y is generally represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including the methods described herein. Low affinity antibodies generally bind slowly to the antigen and tend to dissociate easily, while high affinity antibodies generally bind faster to the antigen and tend to remain bound for a longer period of time. A variety of methods for measuring binding affinity are known in the art, and any of these methods can be used to achieve the objectives of the present invention. Specific exemplary embodiments are described below.

In one embodiment, the "Kd" or "Kd value" of the invention is measured by radiolabeled antigen binding assay (RIA) with a Fab version of the antibody of interest and its antigen as described below: Measuring the binding affinity of Fab to antigen by equilibrating the Fab with a minimal concentration of ( ^125I ) labeled antigen in the presence of a continuous titration of the unlabeled antigen, followed by capture of the antigen bound to the culture plate coated with the anti-Fab antibody. (Chen et al, J. Mol. Biol. 293:865-81 (1999)). To establish assay conditions, a microtiter plate (Dynex) was coated overnight with 5 μg/ml capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and then at room temperature (about 23 ° C) 2% (w/v) bovine serum albumin in PBS was blocked for 2 to 5 hours. Mix 100 μM or 26 pM [ ¹²⁵ I] antigen with serially diluted Fab in a non-sorbent culture plate (Nunc #269620) (eg consistent with the evaluation of anti-VEGF antibody Fab-12, Presta et al. , Cancer Res. 57:4593-99 (1997)). The Fab of interest is then incubated overnight; however, incubation can last for a longer period of time (eg 65 hours) to ensure equilibrium. Thereafter, the mixture is transferred to a capture culture dish at room temperature for incubation (eg, for 1 hour). The solution was then removed and the plate was washed 8 times with 0.1% Tween-20 in PBS. When the plate had dried, 150 μl of scintillant (MicroScint-20; Packard) was added to each well, and the plate was counted on a Topcount gamma counter (Packard) for 10 minutes. Each Fab concentration providing a maximum binding of less than or equal to 20% is selected for competitive binding assays. According to another embodiment, by using surface plasmon resonance assays using BIAcore ^TM -2000 or a ^{BIAcore TM -3000 (BIAcore, Inc.,} Piscataway, NJ) at 25 deg.] C to about 10 the reaction unit (RU) of immobilized antigen The CM5 wafer measures the Kd or Kd value. Briefly, N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (N-hydroxyethyl succinimide) were used according to the supplier's instructions. NHS) Activated carboxymethylated dextran biosensor wafer (CM5, BIAcore Inc). The antigen was diluted to 5 μg/ml (about 0.2 μM) with 10 mM sodium acetate (pH 4.8), followed by injection at a flow rate of 5 μl per minute to achieve about 10 reaction units (RU) of coupled protein. After the antigen was injected, 1 M ethanolamine was injected to block the unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) in 0.05% Tween 20 in PBS (PBST) were injected at a flow rate of about 25 μl/min at 25 °C. The association rate ( _kon ) and dissociation rate ( _koff ) were calculated using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software version 3.2) by simultaneously fitting the association and dissociation induction spectra. The equilibrium dissociation constant (Kd) is calculated from the ratio of k _off / k _on . See, for example, Chen, Y. et al, J. Mol. Biol. 293:865-881 (1999). If the association rate obtained by the above surface plasma resonance test exceeds 10 ⁶ M ^-1 S ^-1 , it can be in a spectrometer such as a spectrophotometer equipped with a flow stop device (Aviv Instruments) or an 8000 with a stirring colorimetric tube. Fluorescence emission of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) measured at 25 ° C in the presence of increasing concentrations of antigen in a series of SLM-Aminco spectrophotometers (ThermoSpectronic) Fluorescence quenching techniques for increasing or decreasing intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) were used to determine the association rate.

According to the present invention may also be used BIAcore ^TM -2000 or a ^{BIAcore TM -3000 (BIAcore, Inc.,} Piscataway, NJ) to the same surface plasmon resonance technique described above using approximately 10 reaction unit (RU) at 25 deg.] C of The immobilized antigen CM5 wafer was measured for "association rate" or "k _on ". Briefly, N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (N-hydroxyethyl succinimide) were used according to the supplier's instructions. NHS) Activated carboxymethylated dextran biosensor wafer (CM5, BIAcore Inc). The antigen was diluted to 5 μg/ml (about 0.2 μM) with 10 mM sodium acetate (pH 4.8), followed by injection at a flow rate of 5 μl per minute to achieve about 10 reaction units (RU) of coupled protein. After the antigen was injected, 1 M ethanolamine was injected to block the unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) in 0.05% Tween 20 in PBS (PBST) were injected at a flow rate of about 25 μl/min at 25 °C. The association rate ( _kon ) and dissociation rate ( _koff ) were calculated using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software version 3.2) by simultaneously fitting the association and dissociation induction spectra. The equilibrium dissociation constant (Kd) is calculated from the ratio of k _off / k _on . See, for example, Chen, Y. et al, J. Mol. Biol. 293:865-81 (1999). However, if the association rate obtained by the above surface plasma resonance test exceeds 10 ⁶ M ^-1 S ^-1 , it is generally in a spectrometer such as a spectrophotometer equipped with a flow stop device (Aviv Instruments) or with a stirring colorimetric tube The 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) was measured at 25 ° C in the presence of an increasing concentration of the 8000 Series SLM-Aminco Spectrophotometer (ThermoSpectronic). Fluorescence quenching techniques for increasing or decreasing the intensity of light emission (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) were used to determine the association rate.

The term "vector," as used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it is linked. One type of vector is a "plastid", which refers to a circular double stranded DNA loop that can engage additional DNA segments. Another type of vector is a phage vector. Another type of vector is a viral vector in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which such vectors are introduced (e.g., bacterial vectors having a bacterial origin of replication and free mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell upon introduction into the host cell and thereby replicated along with the host genome. In addition, certain vectors are capable of directing the performance of genes to which they are operably linked. These vectors are referred to herein as "recombinant expression vectors" (or simply "recombinant vectors"). In general, expression vectors useful in recombinant DNA techniques are typically in the form of plastids. In this specification, "plastid" and "carrier" are used interchangeably because of the most commonly used carrier form of the mass system.

"Polynucleotide" or "nucleic acid" as used interchangeably herein, refers to a polymer of nucleotides of any length, and includes DNA and RNA. The nucleotide may be a deoxyribonucleotide, a ribonucleotide, a modified nucleotide or base, and/or an analog thereof, or may be incorporated into the polymer by DNA or RNA polymerase or a synthetic reaction. Any substrate. Polynucleotides can comprise modified nucleotides, such as methylated nucleotides and analogs thereof. If modifications to the nucleotide structure are present, the modifications can be made before or after assembly into the polymer. The nucleosidase sequence can be interrupted by a non-nucleotide component. The polynucleotide may be further modified after synthesis, such as by conjugation with a label. Other types of modifications include, for example, "capping"; one or more naturally occurring nucleotides are substituted with analogs; internucleotide modifications, such as having uncharged linkages (eg, methyl phosphonate, phosphoric acid) Polyesters such as esters, phospholysines, urethanes, and the like, and charged bonds (eg, phosphorothioates, phosphorodithioates, etc.), such as proteins (eg, nucleases, toxins, antibodies) a polynucleotide of a lateral portion of a signal peptide, a poly-L-lysine, or the like, a polynucleotide having an intercalating agent (for example, acridine, psoralen, etc.), and a chelating agent ( a polynucleotide such as a metal, a radioactive metal, boron, an oxidizing metal, or the like, a polynucleotide containing an alkylating agent, a modified-bonded polynucleotide (for example, a α-helical heteromeric nucleic acid, etc.) Unmodified form of the polynucleotide. In addition, any of the hydroxyl groups normally present in the sugar may, for example, be substituted with a phosphonate group, a phosphate group, protected with a standard protecting group or activated to make an additional bond with an additional nucleotide; or The solid or semi-solid support is conjugated. The 5' and 3' terminal OH groups may be phosphorylated or partially substituted with an amine or an organic capping group having from 1 to 20 carbon atoms. Other hydroxyl groups can also be derivatized as standard protecting groups. Polynucleotides may also contain similar forms of ribose or deoxyribose commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- Or 2'-azido-ribose; carbocyclic sugar analogue; alpha-raceomeric sugar; epimerized sugar, such as arabinose, xylose or lyxose; palladium; furanose; Heptulose; acyclic analog; and a basic nucleoside analog such as methyl riboside. One or more phosphodiester linkages may be replaced by an alternative linking group. Such alternative linking groups include, but are not limited to, phosphate esters via P(O)S ("thiol esters"), P(S)S ("dithiol esters"), (O)NR ₂ (" amides compounds "), P (O) R , P (O) oR ', CO or CH ₂ (" Swap embodiment methylal "), wherein each R or R' independently contain an ether is H or optionally A substituted or unsubstituted alkyl (1-20 C), aryl, alkenyl, cycloalkyl, cycloalkenyl or aralkyl group of the (-O-) linkage. All linkages in the polynucleotide need not be the same. The above description applies to all polynucleotides mentioned herein, including RNA and DNA.

As used herein, "oligonucleotide" generally refers to a short, generally single-stranded, generally synthetic polynucleotide that is generally, but not necessarily, less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is equally and fully applicable to oligonucleotides.

The term "EGFL7" (interchangeably referred to as "Epidermal Growth Factor-like 7") as used herein, unless otherwise specifically indicated or indicated otherwise in the context, refers to any of those described, for example, in WO 2005/117968. Natural or variant (natural or synthetic) EGFL7 polypeptides, the disclosure of which is hereby incorporated by reference in its entirety for all purposes. The term "native sequence" specifically encompasses naturally occurring truncated or secreted forms (eg, extracellular domain sequences), naturally occurring variant forms (eg, alternative spliced forms), and naturally occurring allelic variants. The term "wild-type EGFL7" generally refers to a polypeptide comprising an amino acid sequence of a naturally occurring EGFL7 protein. The term "wild type EGFL7 sequence" generally refers to the amino acid sequence found in naturally occurring EGFL7.

The terms "antibody" and "immunoglobulin" are used interchangeably and include monoclonal antibodies (eg full-length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (eg bispecific antibodies) As long as it exhibits the desired biological activity, and may also include certain antibody fragments (as described in more detail herein). The antibody can be a human antibody, a humanized antibody, and/or an affinity matured antibody.

The term "variable" refers to the fact that the sequences of certain portions of the variable domains between antibodies are generally different and that such portions are used for the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable domain. It focuses on three segments of the light and heavy chain variable domains called complementarity determining regions (CDRs) or hypervariable regions. The highly conserved part of the variable domain is called the framework (FR). The variable domains of the native heavy and light chains each comprise four FR regions joined by three CDRs, primarily in a beta-sheet configuration, which form a loop junction and in some cases form part of a beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR region and, together with the CDRs of the other chains, contribute to the formation of the antigen binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition). , National Institute of Health, Bethesda, MD (1991)). The binding of an antibody to an antigen does not directly involve a constant domain, but it exhibits various effector functions, such as the involvement of antibodies in antibody-dependent cellular cytotoxicity.

Papaya enzyme digestion of the antibody produces two identical antigen-binding fragments each having a single antigen-binding site called a "Fab" fragment, and the remaining "Fc" fragment, the name of which reflects its ability to crystallize readily. Pepsin treatment results in an F(ab')2 fragment that has two antigen combining sites and is still capable of cross-linking the antigen.

"Fv" is the smallest antibody fragment containing the entire antigen recognition site and binding site. In the double-stranded Fv species, this region consists of a dimer of one heavy chain variable domain that is tightly, non-covalently associated with one light chain variable domain. In the single-chain Fv species, one heavy chain variable domain and one light chain variable domain can be covalently linked via an elastin linker such that the light and heavy chains can be similar to the structure of the two-chain Fv species. Poly" structural association. The three CDRs of each variable domain define the antigen binding site on the surface of the VH-VL dimer by interaction in this configuration. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or only one of the three antigen-specific CDRs, half Fv) has the ability to recognize and bind antigen, but its affinity is lower than the intact binding site.

The Fab fragment also contains a light chain constant domain and a heavy chain first constant domain (CH1). The Fab' fragment differs from the Fab fragment by the addition of a small number of residues (including one or more of the cysteine from the antibody hinge region) at the carboxy terminus of the heavy chain CH1 domain. Fab'-SH is herein referred to as the Fab' of the free thiol group of the cysteine residue of the constant domain. The original F(ab')2 antibody fragment was made into a Fab' fragment pair with hinged cysteine. Other chemical couplings of antibody fragments are also known.

The "light chain" of antibodies (immunoglobulins) of any vertebrate species can be divided into two distinct types based on the amino acid sequence of the constant domain (called And one of λ).

Depending on the amino acid sequence of the heavy chain constant domain, immunoglobulins can be classified into different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into subclasses (isotypes): for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structure and three-dimensional configuration of different classes of immunoglobulins are well known.

An "antibody fragment" comprises only a portion of an intact antibody, wherein the portion preferably retains at least one, preferably most or all of the functions normally associated with the portion when present in the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments, bifunctional antibodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. In one embodiment, the antibody fragment comprises the antigen binding site of the intact antibody and thus retains the ability to bind antigen. In another embodiment, an antibody fragment (eg, a fragment comprising an Fc region) retains at least one biological function normally associated with the Fc region when present in an intact antibody, such as FcRn binding, antibody half-life regulation, ADCC function, and complement binding. In one embodiment, the antibody fragment is a monovalent antibody having an in vivo half-life substantially similar to an intact antibody. For example, such antibody fragments can comprise an antigen binding arm linked to an Fc sequence that confers in vivo stability to the fragment.

The term "hypervariable region", "HVR" or "HV" as used herein refers to a region of a sequence that is hypervariable and/or forms an antibody variable domain of a structurally defined loop. In general, antibodies contain six hypervariable regions, three in VH (H1, H2, H3) and three in VL (L1, L2, L3). A variety of depictions of hypervariable regions are used herein and are covered. Kabat complementarity determining regions (CDRs) are based on sequence variability and are most commonly used (Kabat et al, Sequences of Proteins of Immunological Interest, Fifth Edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia is involved in the location of the structural loop (Chothia and Lesk J. Mol. Biol. 196:901-17 (1987)). The AbM hypervariable region represents a compromise between the Kabat CDR and the Chothia structural loop and is used by the Oxford Molecular's AbM antibody modeling software. The "contact" hypervariable region is based on an analysis of the available complex crystal structures.

The hypervariable region may include the following "prolonged hypervariable regions": 24-36 (L1), 46-56 (L2), and 89-97 (L3) in VL; and 26-35 (H1), 49- in VH. 65 or 50 to 65 (H2) and 93-102 (H3). Each of these definitions can number variable domain residues according to Kabat et al. (supra).

"Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as defined herein.

A "humanized" form of a non-human (eg, murine) antibody is a chimeric antibody containing minimal sequence obtained from a non-human immunoglobulin. To a large extent, humanized antibodies are human immunoglobulins (recipient antibodies) in which the residues of the hypervariable region of the recipient are passed through a non-human species (donor antibody) having the desired specificity, affinity and ability (such as small Residue replacement in the hypervariable region of murine, rat, rabbit or non-human primate. In some cases, the framework region (FR) residues of human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further improve the efficacy of the antibody. In general, a humanized antibody will comprise substantially all of at least one and usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to a hypervariable loop of a non-human immunoglobulin, and all or substantially all of the FR The FR of the human immunoglobulin sequence. The humanized antibody will also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin. For further details, see Jones et al, Nature 321:522-25 (1986); Riechmann et al, Nature 332:323-29 (1988) and Presta, Curr. Op. Struct. Biol. 2:593-96 (1992) ). See also the following review papers and references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-15 (1998); Harris, Biochem. Soc . Transactions 23:1035-38 (1995) Hurle and Gross, Curr. Op . Biotech . 5: 428-33 (1994).

A "chimeric" antibody (immunoglobulin) has a portion of a heavy chain and/or a light chain that is identical or homologous to a corresponding sequence in a particular species or antibody obtained from a particular antibody class or subclass, with the remainder of the chain The antibodies obtained from another species or belonging to another antibody class or subclass and the corresponding sequences in fragments of such antibodies are identical or homologous as long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567 and Morrison) Et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Humanized antibodies as used herein are a subset of chimeric antibodies.

A "single-chain Fv" or "scFv" antibody fragment comprises the VH and VL domains of an antibody, wherein such domains are present in a single polypeptide chain. In general, the scFv polypeptide additionally comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For comments on svFv, see Pl Ckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

An "antigen" is a predetermined antigen to which an antibody can selectively bind. The antigen of interest may be a polypeptide, a sugar, a nucleic acid, a lipid, a hapten or other naturally occurring or synthetic compound. In general, the antigen of interest is a polypeptide.

An "antigenic determinant" is an antigenic portion to which an antibody selectively binds. For polypeptide antigens, the epitope is typically a peptide moiety having from about 4 to 10 amino acids.

The term "bifunctional antibody" refers to a small antibody fragment having two antigen binding sites comprising a heavy chain variable domain linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). (VH). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of the other chain and create two antigen-binding sites. Bifunctional antibodies are described in more detail in, for example, EP 404,097, WO 93/11161 and Hollinger et al, Proc. Natl. Acad. Sci. USA , 90:6444-48 (1993).

A "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and/or using any of the techniques for making human antibodies disclosed herein. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues.

An "affinity mature" antibody is one that has one or more changes in one or more CDRs, and such changes result in improved affinity of the antibody for the antigen as compared to a parent antibody that does not have such a change. Preferred affinity matured antibodies will have narm or even picomolar affinity for the antigen of interest. Affinity matured antibodies can be made by procedures known in the art. Marks et al. Bio/Technology 10:779-83 (1992) describe affinity maturation by VH and VL domain shuffling. Random mutations in CDR and/or framework residues are described in Barbas et al, Proc Nat . Acad. Sci. USA 91: 3809-13 (1994); Schier et al. Gene 169: 147-55 (1995); Yelton et al, J. Immunol. 155: 1994-2004 (1995); Jackson et al, J. Immunol. 154(7): 3310-19 (1995) and Hawkins et al, J. Mol. Biol. 226: 889 -96 (1992).

Antibody "effector function" refers to the biological activity caused by the Fc region of an antibody (the native sequence Fc region or the amino acid sequence variant Fc region), and which varies with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (eg, B cell receptors), and B cell activation.

"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cytotoxic form in which it is present on certain cytotoxic cells (eg, natural killer (NK) cells, neutrophils, and macrophages). The secreted Ig bound by the Fc receptor (FcR) enables these cytotoxic effector cells to specifically bind to the target cell carrying the antigen and subsequently kill the target cell with the cytotoxin. The antibody is "equipped with" cytotoxic cells and is required for complete killing. The primary cells (NK cells) that mediate ADCC display only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). In order to assess the ADCC activity of the molecule of interest, an in vitro ADCC assay can be performed, such as described in U.S. Patent No. 5,500,362 or U.S. Patent No. 5,821,337, issued toU.S. Pat. Effector cells used in such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Additionally or alternatively, the molecule of interest may be assessed in vivo, for example, in an animal model such as the animal model disclosed in Clynes et al, Proc. Natl. Acad. Sci. USA 95:652-56 (1998). ADCC activity.

A "human effector cell" is a white blood cell that exhibits one or more FcRs and performs effector functions. Preferably, the cells exhibit at least FcyRIII and perform an ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, with PBMC and NK cells being preferred. Effector cells can be isolated from natural sources such as blood.

"Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. Preferably, the FcR is a native sequence human FcR. Furthermore, preferred FcRs are FcRs (gamma receptors) that bind to IgG antibodies and include receptors for the FcyRI, FcyRII and FcyRIII subclasses, including allelic variants of such receptors and additional spliced forms. Fc[gamma]RII receptors include FcyRIIA ("Activated Receptor") and FcyRIIB ("Inhibition Receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domain. The cellular domain of the activated receptor FcyRIIA contains an immunoreceptor tyrosine-based activating unit (ITAM). The immunoreceptor tyrosine-based inhibition motif (ITIM) is contained in the cytoplasmic domain of the inhibitory receptor FcγRIIB (see review M. Da Ron, Annu. Rev. Immunol. 15:203-34 (1997)). FcR is discussed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (l991); Capel et al, Immunomethods 4:25-34 (1994) and de Haas et al, J. Lab. Clin. Med. : 330-41 (1995). Other FcRs are encompassed by the term "FcR" herein, including FcRs that are identified in the future. The term also encompasses the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG into the fetus (Guyer et al, J. Immunol. 117:587 (1976) and Kim et al, J. Immunol. 24:249 (1994)) And regulate the dynamic stability of immunoglobulin.

WO 00/42072 (Presta) describes improved or reduced antibody variants that bind to FcR. The contents of this patent publication are expressly incorporated herein by reference. See also Shields et al . J. Biol. Chem. 9(2): 6591-6604 (2001).

Methods for measuring binding to FcRn are known (see, for example, Ghetie and Ward, Immunol. Today 18:592-8 (1997)). In vivo binding to human FcRn and high affinity binding of human FcRn can be assayed, for example, in a transgenic mouse or a transfected human cell line expressing a human FcRn or a primate having administered an Fc variant polypeptide. The serum half-life of the polypeptide.

"Complement dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass) that binds to its cognate antigen. To assess complement activation, a CDC assay can be performed, for example as described in Gazzano-Santoro et al, J. Immunol. Methods 202: 163 (1996).

Polypeptide variants having a modified Fc region amino acid sequence and enhanced or reduced C1q binding ability are described in U.S. Patent No. 6,194,551 B1 and WO 99/51642. The contents of their patent publications are specifically incorporated herein by reference. See also Idusogie et al, J. Immunol. 164: 4178-84 (2000).

A "blocking" antibody or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. Preferably, the blocking antibody or antagonist antibody substantially or completely inhibits the biological activity of the antigen.

"Disease" or "disease" is any condition that would benefit from treatment with a substance/molecule or method of the invention. This includes chronic and acute conditions or diseases, including those pathological conditions that predispose a mammal to the condition. Non-limiting examples of conditions to be treated herein include malignant and benign tumors, carcinomas, blastomas, and sarcomas.

The terms "cell proliferative disorder" and "proliferative disorder" refer to a disorder associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

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

The terms "cancer" and "cancerous" refer to or describe a physiological condition in a mammal that is typically characterized by unregulated cell growth/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 carcinoma, small cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma, peritoneal cancer, hepatocytes. Cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney Cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, brain cancer, endometrial cancer, testicular cancer, cholangiocarcinoma, gallbladder cancer, stomach cancer, melanoma and various types of head and neck cancer. An dysfunctional angiogenesis can cause a number of conditions that can be treated by the compositions and methods of the invention. These conditions include non-neoplastic and neoplastic conditions. The neoplastic condition includes, but is not limited to, the conditions described above. Non-neoplastic conditions include (but are not limited to) inappropriate or abnormal hypertrophy, arthritis, rheumatoid arthritis (RA), psoriasis, psoriasis plaque, sarcoma-like disease, atherosclerosis, atherosclerotic plaque, diabetes And other proliferative retinopathy (including retinopathy of prematurity), post-lens fibrous tissue hyperplasia, neovascular glaucoma, age-related macular degeneration, diabetic macular edema, corneal neovascularization, corneal graft neovascularization, corneal transplantation Rejection, retinal/choroidal neovascularization, angina neovascularization (iris redness), intraocular neovascular disease, vascular restenosis, arteriovenous malformation (AVM), meningioma, hemangioma, angiofibroma, thyroid Proliferation (including Grave's disease), corneal and other tissue transplantation, chronic inflammation, pneumonia, acute lung injury/ARDS, sepsis, primary pulmonary hypertension, malignant pulmonary effusion, cerebral edema (eg Associated with acute stroke/closed head injury/trauma), synovitis, vasospasm formation in RA, ossifying myositis, hypertrophic bone Formation, osteoarthritis (OA), refractory ascites, polycystic ovary disease, endometriosis, 3rd spacing of fluid diseases (pancreatitis, septal syndrome, burns, bowel disease), uterus Fibroids, premature labor, chronic inflammation such as IBD (Crohn's disease and ulcerative colitis), renal allograft rejection, inflammatory bowel disease, renal disease, improper or abnormal tissue growth (non-cancer) , hemophilic joints, hypertrophic scars, inhibition of hair growth, Osler-Weber syndrome, purulent granuloma, posterior fibrous tissue hyperplasia, scleroderma, trachoma, vascular adhesion, synovitis, Dermatitis, pre-eclampsia, ascites, pericardial effusion (such as pericardial effusion associated with pericarditis) and pleural effusion.

The term "consumptive" condition (eg, wasting syndrome, cachexia, sarcopenia) refers to a condition caused by improper and/or unhealthy weight loss or somatic cell mass loss. In the elderly and in AIDS and cancer patients, wasting disease can lead to inappropriate weight loss, including weight loss between fat and fat-free intervals. The wasting disease can be caused by inappropriate food intake and/or metabolic changes associated with the disease and/or aging process. Cancer patients and AIDS patients, as well as patients with extensive infections, chronic infections, immune diseases, hyperthyroidism, Crohn's disease, mental illness, chronic heart failure or other serious trauma, are usually suffering from wasting diseases, and consumptive diseases sometimes Also known as cachexia, metabolic disorders and sometimes referred to as eating disorders. In addition, cachexia is characterized by hypermetabolism and catabolism. Although cachexia and wasting disorders are often used interchangeably to refer to wasting conditions, there is at least one body study that distinguishes cachexia from wasting syndromes based on de-lipid body weight and especially somatic mass loss (Mayer, J. Nutr. 129 (1S supplement): 256S-59S (1999)). Muscle reduction (another condition that affects aging individuals) is usually characterized by a reduction in muscle mass. The terminally wasting disease as described above can be developed in individuals who are suffering from cachexia or sarcopenia.

"Treatment," as used herein, refers to a clinical intervention that attempts to alter the natural course of the individual or cell being treated, and may be performed for prophylactic purposes or in the course of clinical pathology. The desired therapeutic effect includes preventing the occurrence or recurrence of the disease, alleviating the symptoms, reducing any direct or indirect pathological outcome of the disease, reducing the rate of progression of the disease, improving or ameliorating the condition of the disease, and improving the condition or improving the prognosis. In some embodiments, the use of an antibody of the invention delays the progression of a disease or condition.

An "individual", "subject" or "patient" is a vertebrate, such as a mammal, especially a human. Mammals include, but are not limited to, humans, domestic animals, and farm animals, as well as zoo animals, competitive animals, or pets, such as dogs, horses, cats, cows, and the like.

"Effective amount" means the amount of dosage and time necessary to effectively achieve the desired therapeutic or prophylactic result.

The "therapeutically effective amount" of a substance/molecule, agonist or antagonist of the present invention may vary depending on factors such as the disease state, age, sex and weight of the individual, and the substance/molecule, agonist or antagonist in the individual. The ability to induce a desired response. A therapeutically effective amount is also one in which the therapeutic benefit of the substance/molecule, agonist or antagonist exceeds any of its toxic or detrimental effects.

"Prophylactically effective amount" means the amount of dosage and time necessary to effectively achieve the desired prophylactic result. Since the prophylactic dose is administered to the subject prior to or prior to the disease, the prophylactically effective amount will generally (but not necessarily) be less than the therapeutically effective amount.

A "cytotoxic agent" as used herein refers to a substance that inhibits or impairs cellular function and/or causes cell destruction. The term is intended to include radioisotopes (eg, radioisotopes of At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ^{186 ,} Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ^{32 ,} and Lu); chemotherapeutic agents such as methotrexate (methotrexate), adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, mefa Melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents; enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; and toxins Small molecule toxins or enzymatically active toxins (including fragments and/or variants thereof) of bacterial, fungal, plant or animal origin; and various anti-tumor or anti-cancer agents disclosed below. Other cytotoxic agents are described below. Tumor killing agents cause destruction of tumor cells.

A "chemotherapeutic agent" is a compound that can be used to treat cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN Cyclosphosphamide; alkyl sulfonate such as busulfan, improsulfan, and piposulfan; aziridine, such as benzodopa, Carboquone, meturedopa and uredopa; ethyl imino and methyl melamine, including altretamine, triethylenemelamine, and sirolimus Trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenin (especially bullatacin and bran) Bartanacinone; δ-9-tetrahydrocannabinol (dronabinol), MARINOl ; β-lapachone; lapachol; colchicine; betulinic acid; camptothecin (including synthetic analogue topotecan (topotecan) ) (HYCAMTIN ), CPT-11 (irinotecan CAMPTOSAR) ), acetyl camptothecin, scopolectin and 9-aminocamptothecin; bryostatin; calallystatin; CC-1065 (including its adolescence) (adozelesin), carzelesin and bizelesin synthetic analogues; podophyllotoxin; podophyllinic acid; teniposide; nocturnal ring Peptide (cryptophycin) (especially Candida cyclic peptide 1 and Nostoccal cyclic peptide 8); dolastatin; doocarmycin (including synthetic analogues, KW-2189 and CB1-TM1); Eleutherobin; pancraticatin; sarcodictyin; spongistatin; nitrogen mustard, such as chlorambucil, chlornaphazine, Cholophosphamide, estramustine, ifosfamide, mechlorethamine, nitric oxide mustard hydrochloride, melphalan, neombibichin, Phenosterine, prednimustine, trofosfamide, urinary pyrimidine Mustard mustard; nitrosurea, such as carmustine, chlorozotocin, fotemustine, lomustine, nimos Nimustine and ranimnustine; antibiotics, such as enediyne antibiotics (eg calicheamicin), especially calicheamicin γ1I and calicheamicin ΩI1 (eg See Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)), dynemicin (including dynemicin A), esperamicin and Neocarzinostatin chromophore and related chromophore dioxin antibiotic chromophores, aclacinomysin, actinomycin, authramycin , azaserine, bleomycin, cactinomycin, caracalcin, carminomycin, carzinophilin, color mold Chromomycinis, actinomycin D, daunorubicin, detobicin n), 6-diazo-5-sideoxy-L-positive acid, ADRIAMYCIN Doxorubicin (including morpholinyl-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolyl-doxorubicin, and deoxydoxantine), epirubicin ( Epirubicin), esorubicin, idarubicin, marcellomycin, mitomycin (such as mitomycin C), mycophenolic acid , nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, arro Rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, levubicin ( Zorubicin); antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denoptinin, methotrexate, pteropterin, trimethoate (trimetrexate); purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; Analogs such as ancitabine, azacitidine, 6-azide uridine, carmofur, cytarabine, dihydrouridine, deoxyfluoride Doxyl fluridine, enocitabine, floxuridine; androgens, such as calutosterone, dromostanolone propionate, epitisolol, Mepitiostane, testolactone; anti-adrenal agents, such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folic acid; vinegar Aceglatone; aldophosphamide glycoside; aminolevulinic acid; 5-acetylene uracil; amsacrine; betribucin ); bisantrene; edatraxate; defofamine; demecolcine; diziquone; elfornithine; Elliptinium acetate); epothilone; relying on gru (e Toglucid); hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocin; Mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; piranthine (pirarubicin); losoxantrone; 2-ethyl hydrazine; procarbazine; PSK Polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid Triaziquone; 2,2',2"-trichlorotriethylamine; trichothecene (especially T-2 toxin, verracurin A, lacquer) Roridin A and anguidine (ureuidine); urathan; vindesine (ELDISINE) FILDESIN ); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; guacittosine ; arabinoside ("Ara-C");thiotepa; taxoids, such as TAXOL Taxol (paclitaxel) (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE TM paclitaxel free of polyoxyethylene cetyl alcohol ether of ethylene albumin engineering inert nanoparticles formulation (American Pharmaceutical Partners, Schaumberg, Illinois ) , and TAXOTERE Doxetaxel (Rh ne-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine (GEMZAR) 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine ); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN) ); oxaliplatin; formazan tetrahydrofolate (vincovovin); vinorelbine (NAVELBINE) ;;novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine (XELODA) a pharmaceutically acceptable salt, acid or derivative of any of the above; and combinations of two or more of the foregoing, such as CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisolone) abbreviations prednisolone (prednisolone) of the combination therapy) and abbreviations FOLFOX (Orsay force platinum (ELOXATIN ^TM) with 5-FU and acyl a treatment regimen of a combination of tetrahydro folate).

Anti-hormonal agents are also included in this definition for regulating, reducing, blocking or inhibiting the action of hormones that promote cancer growth and are typically in the form of systemic or holistic treatments. It can be the hormone itself. Examples include anti-estrogen and selective estrogen receptor modulators (SERMs) including, for example, tamoxifen (including NOLVADEX) Tamoxifen), EVISTA Raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, leroxifene hydrochloride, LY117018, onastroxone Onapristone) and FARESTON Toremifene; antiprogestin; estrogen receptor downregulator (ERD); an agent for inhibiting or preventing ovaries, such as luteinizing hormone releasing hormone (LHRH) agonist, such as LUPRON And ELIGARD Leuprolide acetate, goserelin acetate, buserelin acetate, and tripterelin; other antiandrogens, such as flutamide , nilutamide and bicalutamide; and aromatase inhibitors that inhibit the modulation of aromatase produced by estrogen in the adrenal gland, such as 4(5)-imidazole, amine ubmet, MEGASE Megestrol acetate, AROMASIN Exemestane, formestanie, fadrozole, RIVISOR Vorozole, FEMARA Letrozole and ARIMIDEX Anmeta ingot (anastrozole). In addition, this chemotherapeutic definition also includes bisphosphonates such as clodronate (eg BONEFOS) Or OSTAC ), DIDROCAL Etidronate, NE-58095, ZOMETA Zoledronic acid/zoledronate, FOSAMAX Alendronate, AREDIA Pamidronate, SKELID Tiluronate or ACTONEL Risedronate; and troxacitabine (1,3-dioxolan cytosine analog); antisense oligonucleotides, especially for inhibition of abnormal cell proliferation Antisense oligonucleotides expressed by genes in the signal pathway, such as PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE Vaccine and gene therapy vaccines (eg ALLOVECTIN) Vaccine, LEUVECTIN Vaccine and VAXID Vaccine); LURTOTECAN Topoisomerase 1 inhibitor; ABARELIX rmRH; lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosine kinase small molecule inhibitor, also known as GW572016); and pharmaceuticals of any of the above An acceptable salt, acid or derivative.

As used herein, "growth inhibitor" refers to a compound or composition that inhibits the growth of cells, such as cells expressing EGFL7, in vitro or in vivo. Thus, the growth inhibitor can be an agent that significantly reduces the percentage of cells in the S phase, such as cells expressing EGFL7. Examples of growth inhibitors include agents that block cell cycle progression (other than S phase), such as agents that induce G1 arrest and M arrest. Classical M-term blockers include vinblastine (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors (such as doxorubicin, epirubicin, daunorubicin, Etoposide and bleomycin). These agents that arrest G1 are also associated with S-phase arrest, such as DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, nitrogen mustard, cisplatin, methotrexate, 5 - Fluorouracil and ara-C. Additional information can be found in Murakami et al., The Molecular Basis of Cancer, edited by Mendelsohn and Israel, Chapter 1, entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs" (WB Saunders: Philadelphia, 1995), especially on page 13. . Taxanes (paclitaxel and docetaxel) are anticancer drugs derived from yew trees. Originated from the European yew, Docetaxel (TAXOTERE) , Rhone-Poulenc Rorer) is paclitaxel (TAXOL) , semi-synthetic analog of Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which leads to cell mitosis inhibition.

"Doxorubicin" is an anthracycline antibiotic. The complete chemical name of doxorubicin is (8S-cis)-10-[(3-amino-2,3,6-trideoxy-α-L-lais-hexanpyranosyl)oxy]- 7,8,9,10-Tetrahydro-6,8,11-trihydroxy-8-(hydroxyethenyl)-1-methoxy-5,12-tetrapentanedione.

The term "polypeptide comprising an Fc region" refers to a polypeptide comprising an Fc region, such as an antibody or immunoadhesin (as defined herein). The C-terminal amide acid of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during purification of the polypeptide or by recombinant engineering of the nucleic acid encoding the polypeptide. Thus, a composition comprising a polypeptide having an Fc region according to the invention may comprise a mixture having K447, a polypeptide from which all K447 has been removed, or a polypeptide having and without a K447 residue.

Composition of the present invention and method of producing the same

The invention encompasses compositions (including pharmaceutical compositions) comprising an anti-EGFL7 antibody and a polynucleotide comprising a sequence encoding an anti-EGFL7 antibody. As used herein, a composition comprises one or more antibodies that bind to EGFL7 and/or one or more polynucleotides comprising a sequence encoding one or more antibodies that bind to EGFL7. Such compositions may additionally comprise suitable carriers well known in the art, such as pharmaceutically acceptable excipients (including buffers).

Embodiments of isolated antibodies and polynucleotides are also contemplated by the invention. Embodiments of substantially pure antibodies and polynucleotides are also contemplated by the present invention.

The anti-EGFL7 antibody of the present invention is preferably a monoclonal antibody. Also contemplated within the scope of the invention are Fab, Fab', Fab'-SH and F(ab')2 fragments of the anti-EGFL7 antibodies provided herein. Such antibody fragments can be produced by conventional means such as enzymatic digestion or can be produced by recombinant techniques. Such antibody fragments can be chimeric or humanized antibody fragments. These fragments can be used to achieve the diagnostic and therapeutic purposes described below.

Individual antibodies can be obtained from a population of substantially homogeneous antibodies (i.e., a population comprising individual antibodies, except for mutations that may naturally occur in minor amounts). Thus, the modifier "single plant" means a feature that is not an antibody in the form of a mixture of discrete antibodies.

The anti-EGFL7 monoclonal antibody of the present invention can be produced by the fusion tumor method first described by Kohler et al., Nature 256:495 (1975), or can be produced by recombinant DNA method (U.S. Patent No. 4,816,567).

In the fusion tumor method, a mouse or other appropriate host animal, such as a hamster, is immunized to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Anti-EGFL7 antibodies are typically produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of EGFL7 and adjuvants. EGFL7 can be prepared using methods well known in the art, some of which are further described herein. For example, the recombinant manufacturing of EGFL7 will be described below. In one embodiment, the animal is immunized with an EGFL7 derivative comprising an extracellular domain (ECD) of EGFL7 fused to the Fc portion of the immunoglobulin heavy chain. In one embodiment, the animal is immunized with an EGFL7-IgG1 fusion protein. Animals are typically produced against immunogenic conjugates or derivatives of EGFL7 and monophosphorus lipid A (MPL) / trehalose bisphenolic mycolate (TDM) (Ribi Immunochem. Research, Inc., Hamilton, MT). Immunize and inject the solution into the cortex at multiple sites. The animals were boosted two weeks later. Animals were bled for 7 to 14 days and serum anti-EGFL7 titers were assayed. Strengthen the immunization of animals until the price reaches a stable stage.

Alternatively, lymphocytes can be immunized in vitro. Lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form fusion tumor cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The thus prepared fusion tumor is inoculated and grown in a suitable medium preferably containing one or more substances which inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the medium used for fusion tumors will usually include hypoxanthine, aminopterin and thymidine. (HAT medium), these substances prevent the growth of HGPRT-deficient cells.

Preferred myeloma cells are effective fusions which allow selected antibody-producing cells to stably produce high levels of antibodies and to be sensitive to myeloma cells such as HAT medium. Among them, preferred myeloma cell lines are murine myeloma strains such as MOPC-21 and MPC-11 mouse tumors purchased from Salk Institute Cell Distribution Center, San Diego, California USA and purchased from American Type Culture Collection, Rockville. , Cell line obtained from SP-2 or X63-Ag8-653 cells of Maryland USA. Human myeloma and mouse-human hybrid myeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Pages 51-63 (Marcel Dekker, Inc., New York, 1987)).

The production of monoclonal antibodies against EGFL7 in the medium in which the fusion tumor cells were grown was assayed. The binding specificity of monoclonal antibodies produced by fusion tumor cells is preferably determined by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

For example, the binding affinity of a monoclonal antibody can be determined by Scatchard analysis by Munson et al., Anal. Biochem. 107: 220 (1980).

Once a fusion cell producing an antibody having the desired specificity, affinity and/or activity is identified, the pure line can be subcultured by a limiting dilution procedure and by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, 59-103) Page (Academic Press, 1986)) makes it grow. Media suitable for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the fusion tumor cells can be grown in vivo in the form of ascites tumors in animals.

The monoclonal antibodies secreted by the sub-pure lines are appropriately separated from the culture medium, ascites or serum by a conventional immunoglobulin purification program such as protein A-agarose, hydroxyapatite chromatography, gel electrophoresis, dialysis. Or affinity chromatography.

The anti-EGFL7 antibodies of the invention can be prepared by screening combinatorial libraries for synthetic antibody-free lines having the desired activity. In general, synthetic antibody-derived lines are selected by screening phage libraries containing phage displaying various fragments of the antibody variable region (Fv) of the fusion phage sheath protein. These phage libraries are screened by affinity chromatography against the desired antigen. A pure line that expresses an Fv fragment capable of binding to the desired antigen is adsorbed to the antigen and thus is separated from the unbound pure line in the library. The bound pure line is then eluted from the antigen and further enriched by additional antigen adsorption/dissolution cycles. The phage-pure line of interest can be selected by designing an appropriate antigen screening program, followed by the use of Fv sequences from the phage-pure line of interest and Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication 91-3242, Bethesda MD (1991), appropriate constant region (Fc) sequences as described in Volumes 1-3 construct a full length anti-EGFL7 antibody line to obtain certain anti-EGFL7 antibodies of the invention.

The antigen binding domain of the antibody is formed by two variable (V) regions of about 110 amino acids, the two variable regions being derived from the light chain (VL) and the heavy chain (VH), respectively. Hypervariable loop or complementarity determining region (CDR). The variable domain can be made as a single-chain Fv (scFv) fragment in which VH and VL are covalently linked via a short elastin peptide, as described in Winter et al, Ann. Rev. Immunol . 12:433-55 (1994). Or functionally presented on phage in the form of Fab fragments in which each is fused to a constant domain and non-covalently interacted. As used herein, the scFv-encoding phage-pure line and the Fab-encoding phage are collectively referred to as "Fv phage pure line" or "Fv pure line".

Be by the polymerase chain reaction (PCR) separately cloned VH and VL gene repertoire, and the recombinant randomly in phage libraries, which can then be as described in Winter et al., Ann.Rev.Immunol 12:. 433-55 (1994 ) The antigen-binding pure line is searched for from it. Libraries from immunized sources provide high affinity antibodies against the immunogen without the need to construct fusion tumors. Alternatively, the natural lineage can be selected to provide a single source of human antibodies against a wide range of non-autologous and autoantigens without any immunization, such as Griffiths et al, EMBO J. 12: 725-34 (1993) Said. Finally, natural libraries such as Hoogenboom and Winter can also be synthesized by selecting unrearranged V gene segments from stem cells and encoding high variable CDR3 regions using a PCR primer containing random sequences and achieving in vitro rearrangement. J. Mol. Biol. 227:381-88 (1992).

Antibody fragments are presented by fusion with a small amount of the sheath protein pIII using filamentous phage. Antibody fragments can be presented as single-chain Fv fragments in which the VH and VL domains are joined by the elastic polypeptide spacer on the same polypeptide chain, for example, as in Marks et al, J. Mol. Biol. 222: 581-597 (1991). Said; or presented as a Fab fragment, wherein one strand is fused to pIII and the other strand is secreted into the periplasm of the bacterial host cell, in which the Fab-sphingin structure is assembled, which is translocated via certain wild-type sheath proteins. Presented on the surface of the phage, for example as described by Hoogenboom et al., Nucl. Acids Res. 19:4133-37 (1991).

In general, nucleic acids encoding antibody gene fragments are obtained freely from immune cells harvested by humans or animals. If a library that favors the EGFL7 pure line is desired, the subject is immunized with EGFL7 to generate an antibody response, and spleen cells and/or circulating B cells or other peripheral blood lymphocytes (PBL) are recovered for library construction. In a preferred embodiment, the anti-human EGFL7 antibody response is produced in a transgenic mouse having a functional human immunoglobulin gene array (and lacking a functional endogenous antibody production system) such that the EGFL7 Immunization results in the production of B cells against human antibodies to EGFL7, thus obtaining a library of human antibody gene fragments that are prone to anti-human EGFL7 pure lines. The production of a transgenic mouse producing a human antibody will be described below.

B cells expressing EGFL7-specific membrane-bound antibodies can be isolated by using appropriate screening procedures (eg, by isolating cells with EGFL7 affinity chromatography or by adsorbing cells to fluorescent dye-labeled EGFL7 followed by flow-activated cell sorting) FACS)) to obtain additional enrichment of the anti-EGFL7 reactive cell population.

Alternatively, spleen cells and/or B cells or other PBLs using unimmunized donors provide a better display of possible antibody lineages, and also allow the construction of antibody libraries using any animal (human or non-human) species that are not antigenic in EGFL7 . For libraries incorporating in vivo antibody gene construction, stem cells are harvested from the subject to provide nucleic acids encoding the unrearranged antibody gene segments. The immune cells of interest can be obtained from a variety of animal species, such as humans, mice, rats, rabbits, wolves, canines, felines, pigs, cattle, horses, and bird species.

Nucleic acids encoding antibody variable gene segments (including VH and VL segments) are recovered from the cells of interest and amplified. In the case of rearranged VH and VL gene libraries, the polymerase chain can be performed by isolating genomic DNA or mRNA from lymphocytes and then using primers that match the 5' and 3' ends of the rearranged VH and VL genes. Reaction (PCR) (as described by Orlandi et al, Proc. Natl. Acad. Sci. USA 86:3833-37 (1989)), thereby producing a variety of V gene lineages for expression to obtain the desired DNA. The V gene can be amplified from cDNA and genomic DNA, wherein the post-primer is encoded at the 5' end of the mature V domain and the forward primer is within the J segment, as described by Orlandi et al. (1989) and Ward et al. Nature 341: 544-46 (1989). However, for amplification by cDNA, the post-introduction can also be in the exon of the leader sequence as described in Jones et al., Biotechnol. 9:88-89 (1991), and the forward primers are, for example, Sastry et al. In the constant region described in Proc. Natl. Acad. Sci. USA 86: 5728-32 (1989). To maximize complementarity, degeneracy can be incorporated into the primers as described by Orlandi et al. (1989) or Sastry et al. (1989). Library diversity is preferably maximized by using PCR primers targeted to amplify each V gene family of all available VH and VL sequences present in the nucleic acid nucleic acid sample, for example, as Marks et al., J The method described in .Mol. Biol. 222: 581-97 (1991) or as described in the method of Orum et al., Nucl. Acids Res. 21: 4491-4498 (1993). For the selection of amplified DNA into a expression vector, a rare restriction site can be introduced into the PCR primer as a label for one end as described in Orlandi et al. (1989), or as Clackson et al., Nature 352:624- Further introduction by PCR amplification with a labeled primer is described in 628 (1991).

A synthetic rearranged V gene lineage can be obtained from a V gene segment in vitro. Most human VH gene segments have been cloned and sequenced (reported in Tomlinson et al, J. Mol. Biol. 227:776-98 (1992)) and localized (reported in Matsuda et al, Nature Genet) . 3: 88-94 (1993)); these may be used to generate more of the VH gene was cloned segments (including all the major conformations) having a plurality of H3 sequence and length of PCR primers encoding the ring H1 and H2 loop of Lineage, as described in Hoogenboom and Winter, J. Mol. Biol. 227:381-88 (1992). All VH lineages in which sequence diversity is concentrated in a single long length H3 loop can also be made, as described in Barbas et al, Proc. Natl. Acad. Sci. USA 89: 4457-61 (1992). Has been on human V The Vλ segment is housed and sequenced (reported in Williams and Winter, Eur. J. Immunol. 23: 1456-61 (1993)) and can be used to make synthetic light chain lines. A synthetic V gene lineage based on a range of VH and VL folds and L3 and H3 lengths will encode antibodies with substantial structural diversity. After amplification of the V gene-encoding DNA, the germline V gene segment can be rearranged in vitro according to the method of Hoogenboom and Winter, J. Mol. Biol. 227:381-88 (1992).

The antibody fragment lineage can be constructed in several ways by combining the VH and VL gene lineages. Each lineage can be produced in a different vector, and the vectors are in vitro (for example, as described in Hogrefe et al, Gene 128: 119-26 (1993)) or by in vivo infection (eg, as Recombination of the loxP system described in Waterhouse et al., Nucl. Acids Res. 21: 2265-66 (1993). The in vivo recombination method utilizes the double-stranded nature of the Fab fragment to overcome the limitations imposed on the size of the library imposed by the E. coli transformation power. The native VH and VL lineages were individually isolated, one was selected into the phagemid and the other was selected into the phage vector. The two libraries are then combined by phage infection of bacteria containing phagemids such that each cell contains different combinations and the library size is limited only by the number of cells present (about 10 ¹² pure lines). Both vectors contain an in vivo recombination signal such that the VH and VL genes are recombined on a single replicon and co-packaged in phage virions. These large libraries provide a large number of antibodies with good affinity (Kd ^-1 is about 10 ^-8 M).

Alternatively, the lineage can be subsequently cloned into the same vector, for example as described in Barbas et al, Proc. Natl. Acad. Sci. USA 88:7978-82 (1991); or the lineage can be assembled by PCR Together and then colonized, for example as described in Clackson et al, Nature 352:624-28 (1991). The VH and VL DNA can also be ligated together with the DNA encoding the elastin spacer using PCR assembly to form a single-chain FV (scFv) lineage. In another technique, the "intracellular PCR assembly" is used to combine the VH and VL genes in lymphocytes by PCR and then to select the lineage of the linked genes, such as Embleton et al., Nucl. Acids Res. 20:3831- 37 (1992).

Of an antibody (natural or synthetic) of naturally produced libraries can be of moderate affinity (Kd ^-1 of about ¹⁰⁶ to 10 ⁷ M ^-1), but may also be performed by a secondary library constructed from both in vitro and simulation selected from affinity Mature, as in Winter et al. (1994), as described above. For example, by the method of Hawkins et al, J. Mol. Biol. 226: 889-96 (1992) or by Gram et al, Proc. Natl. Acad. Sci USA 89: 3576-80 (1992) Mutations were randomly introduced in vitro using a pro-error polymerase (reported in Leung et al., Technique 1: 11-15 (1989)). In addition, affinity maturation can be performed by, for example, using a primer having a random sequence spanning the CDRs of interest, using PCR to randomly mutate one or more CDRs in a selected individual Fv line and screening for a higher affinity line. WO 96/07754 (published Mar. 14, 1996) describes a method of inducing mutations in the immunoglobulin light chain complementarity determining region to create a light chain gene library. Another effective method is to recombine the VH or VL domain selected by the phage to a naturally occurring V domain variant lineage obtained from an unimmunized donor and to screen for higher affinity in a number of rounds of reconfiguration, As described in Marks et al., Biotechnol. 10:779-83 (1992). This technique allows the production of antibodies and antibody fragments with affinities in the range of ^10-9 M.

The nucleic acid sequence encoding EGFL7 can be designed using the amino acid sequence in the desired region of EGFL7. Alternatively, a cDNA sequence (or a fragment thereof) can be used. Additional EGFL7 sequences are additionally disclosed, for example, in NM_022963 and Xie et al, Cytokine 11:729-35 (1999). DNA encoding EGFL7 can be prepared by a variety of methods known in the art. Such methods include, but are not limited to, chemical synthesis by any of the methods described in Engels et al., Agnew. Chem. Int. Ed. Engl. 28: 716-34 (1989), such as triesters, phosphites. , Aminophosphate and H-phosphonate methods. In one embodiment, the EGFL7 encoding DNA is designed using a codon that is preferred for the expression of the host cell. Alternatively, DNA encoding EGFL7 can be isolated from a genomic or cDNA library.

After constructing a DNA molecule encoding EGFL7, the DNA molecule is operably linked to a expression control sequence in a expression vector, such as a plastid, wherein the control sequence is recognized by the host cell transformed with the vector. In general, plastid vectors contain replication and control sequences obtained from species compatible with the host cell. Vectors typically have a replication site and a sequence encoding a protein capable of providing phenotypic selection in transformed cells. Suitable vectors for expression in prokaryotic and eukaryotic host cells are known in the art, and certain vectors will be further described herein. Cells obtained from eukaryotic organisms (such as yeast) or from multicellular organisms (such as mammals) can be used.

The DNA encoding EGFL7 is operably linked to the secretory leader sequence as appropriate to cause the host cell to secrete the expression product into the culture medium. Examples of secretory leader sequences include stII, ecotin, lamB, herpes GD, lpp, alkaline phosphatase, invertase, and alpha factor. The 36 amino acid leader sequence of Protein A (Abrahmsen et al., EMBO J. 4: 3901 (1985)) is also suitable for use herein.

Transfecting and preferably transforming a host cell with the expression or selection vector of the invention described above and culturing it to be adapted to induce a promoter, select a transformant or amplify a gene encoding the desired sequence In the conventional nutrient medium.

Transfection refers to the absorption of a display vector by a host cell that does or does not exhibit any coding sequence. A variety of transfection methods are known to those skilled in the art, such as CaPO ₄ precipitation and electroporation. Transfection is generally recognized to be successful when any indication of the operation of the vector occurs within the host cell. Transfection methods are well known in the art and some methods are further described herein.

Transformation means the introduction of DNA into an organism such that the DNA can be replicated as an extrachromosomal element or by a chromosomal component. Depending on the host cell used, transformation is carried out using standard techniques appropriate for such cells. Transformation methods are well known in the art, and certain methods are further described herein.

Prokaryotic host cells for the production of EGFL7 can be cultured as generally described in Sambrook et al. (supra).

Mammalian host cells for the production of EGFL7 can be cultured in a variety of media well known in the art, and certain media are described herein.

Host cells referred to in the present disclosure encompass cells in in vitro culture as well as cells in host animals.

Purification of EGFL7 can be accomplished using technically recognized methods.

The purified EGFL7 can be attached to a suitable substrate for affinity chromatography to isolate the phage from a pure line, such as agarose beads, acrylamide beads, glass beads, cellulose, various acrylic copolymers, methacrylic acid hydroxyl groups. Ester gels, polyacrylic acid and polymethacrylic copolymers, nylon, neutral and ionic carriers and the like. Attachment of the EGFL7 protein to the substrate can be accomplished by the method described in Meth. Enzymol., Vol. 44 (1976). A common technique for attaching a protein ligand to a polysaccharide matrix, such as agarose, dextran or cellulose, involves activating the carrier with a cyanogen halide and subsequently subjecting the peptide ligand to a primary aliphatic or aromatic amine to the activated matrix. Coupling.

Alternatively, EGFL7 can be used to coat the wells of the adsorption plate, either on the host cells immobilized on the adsorption plate or for cell sorting, or conjugated with biotin to coat the streptavidin. Microbead capture, or any other method known in the art for screening phage display libraries.

The phage library sample is contacted with immobilized EGFL7 under conditions suitable for binding at least a portion of the phage particles to the adsorbent. In general, conditions including pH, ionic strength, temperature, and the like are selected to simulate physiological conditions. The phage bound to the solid phase are washed and then eluted with acid (for example, as described in Barbas et al, Proc. Natl. Acad. Sci USA 88: 7978-82 (1991)) or dissolved with an alkali (eg, such as Marks) Et al., J. Mol. Biol. 222: 581-97 (1991)) or competitively dissociated by EGFL antigen (for example, in competition with the antigen of Clackson et al, Nature 352:624-28 (1991) Similar procedures). Enrichment of 20-1,000 fold phage in a single round of selection. In addition, the enriched phage can be grown in bacterial culture and subjected to another round of selection.

The efficiency of selection depends on a number of factors, including the dissociation kinetics during washing and the ability of multiple antibody fragments on a single phage to simultaneously bind to the antigen. Antibodies with rapid dissociation kinetics (and weak binding affinities) can be retained by using short washes, multivalent phage presentation, and high coating density of the antigen in the solid phase. High density not only stabilizes the phage via multivalent interactions, but also facilitates recombination of the dissociated phage. It can be presented by using long-term washing and monovalent phage display (as described in Bass et al., Proteins 8: 309-14 (1990) and WO 92/09690) and low antigen coating density (eg, Marks et al., Biotechnol. 10) . :779-83 (1992)) Promotes the selection of antibodies with slow dissociation kinetics (and good binding affinity).

Selection can be made between phage antibodies that have different affinities for EGFL7, even with slightly different affinities. However, random mutations in selected antibodies (e.g., as performed in certain affinity maturation techniques as described above) may result in a number of mutants, most of which bind to the antigen and a few have a higher affinity. Using restricted EGFL7, one can compete for the elimination of very few high affinity phage. To retain all mutants with higher affinity, phage can be incubated with excess EGFL7 bound to biotin, but EGFL7 bound to biotin has a concentration of molar concentration below the constant target molar affinity of EGFL7. The high affinity binding phage can then be captured by the streptavidin coated paramagnetic microbeads. This "equilibrium capture" enables the selection of antibodies with sensitivity based on the binding affinity of the antibody, which allows for the isolation of mutant lines with only twice the high affinity from a large number of excess phage with lower affinity. The conditions used in the wash and solid phase combined phage can also be manipulated to distinguish based on dissociation kinetics. The anti-EGFL7 pure line can also be selected according to the activity.

The monoclonal antibody or phage derived from the fusion tumor of the present invention can be readily isolated and present in Fv pure line, and can be used in a conventional procedure (for example, by using a specific amplification designed to be specifically amplified by a fusion tumor or phage DNA template). The oligonucleotide primers of the heavy and light chain coding regions are sequenced. After isolation, the DNA can be placed in an expression vector, and then the expression vector is transfected into a host cell (such as E. coli cells, 猿COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells) that does not otherwise produce immunoglobulins. The synthesis of the desired monoclonal antibody is obtained in a recombinant host cell. Review papers on the recombinant expression of antibody-encoding DNA in bacteria include Skerra et al., Curr. Opinion in Immunol. 5:256 (1993) and Pl Ckthun, Immunol. Rev. 130: 151 (1992).

A DNA encoding an Fv pure line of the invention can be combined with a DNA sequence known to encode a constant region of a heavy chain and/or a light chain (e.g., a suitable DNA sequence can be obtained from Kabat et al. (supra)) to form a full-length or partial length Pure chain and / or light chain. It will be appreciated that any isotype constant region can be used to achieve this, including IgG, IgM, IgA, IgD, and IgE constant regions, and such constant regions can be obtained from any human or animal species. As used herein, a definition of a "chimeric" antibody and a "hybrid" antibody is obtained from variable domain DNA of an animal (such as a human) species and then fused to constant region DNA of another animal species to form a "hybrid" Fv pure line of coding sequence (full length heavy chain and/or light chain). In a preferred embodiment, the Fv pure lineage obtained from human variable DNA is fused to human constant region DNA to form a fully human coding sequence, i.e., a full length or partial length heavy and/or light chain.

For example, a homologous murine sequence obtained by substituting a pure line of a fusion tumor with a coding sequence of a human heavy and light chain constant domain can also be used (for example, as Morrison et al., Proc. Natl. Acad. Sci. USA 81). : Method of 6851-55 (1984)) to modify the DNA encoding the anti-EGFL7 antibody obtained from the self-fused tumor of the present invention. DNA encoding an antibody or fragment obtained from a fusion tumor or Fv pure line can be further modified by covalently linking all or part of the coding sequence of the non-immunoglobulin polypeptide to the immunoglobulin coding sequence. In this way, a "chimeric" or "hybrid" antibody having the binding specificity of the antibody obtained from the Fv pure line or the fusion line of the present invention is prepared.

Antibody fragment

The invention encompasses antibody fragments. In some cases, the use of antibody fragments rather than the use of whole antibodies has advantages. Fragments of smaller size allow for rapid clearance and can result in improvements in access to solid tumors.

A variety of techniques for making antibody fragments have been developed. Traditionally, such fragments have been obtained by proteolytic digestion of intact antibodies (see, for example, Morimoto et al, J. Biochem. Biophys . Meth . 24: 107-17 (1992) and Brennan et al, Science 229: 81 (1985)). However, such fragments can now be produced directly by recombinant host cells. Fab, Fv and ScFv antibody fragments can all be expressed in E. coli and secreted by E. coli, whereby a large number of such fragments can be easily produced. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al, Bio/Technology 10: 163-67 (1992)). According to another method, the F(ab')2 fragment can be isolated directly from recombinant host cell culture. Fab and F(ab')2 fragments comprising a salvage receptor binding epitope residue having an increased in vivo half-life are described in U.S. Patent No. 5,869,046. Other techniques for generating antibody fragments should be apparent to those skilled in the art. In other embodiments, the antibody selected is a single chain Fv fragment (scFv). See WO 93/16185, U.S. Patent Nos. 5,571,894 and 5,587,458. Fv and sFv are the only species that lack a constant region but have a complete combinatorial site; therefore, they are suitable for achieving reduced non-specific binding during in vivo use. The sFv fusion protein can be constructed to obtain fusion of the effector protein at the sFv amine or carboxy terminus. See Antibody Engineering, edited by Borrebaeck, WH Freeman and Company (1992). The antibody fragment can also be a "linear antibody", for example, as described in U.S. Patent No. 5,641,870. Such linear antibody fragments can be monospecific or bispecific.

Humanized antibody

The invention encompasses humanized antibodies. A variety of methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced therein from a non-human source. Such non-human amino acid residues are often referred to as "input" residues, which are typically taken from the "input" variable domain. Basically according to the method of Winter and colleagues (Jones et al, Nature 321:522-25; Riechmann et al, Nature 332:323-27 (1988); Verhoeyen et al, Science 239: 1534-36 (1988)) Humanization is performed by substituting the corresponding sequence of the human antibody with a hypervariable region sequence. Thus, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein sequences substantially smaller than the entire human variable domain have been substituted from corresponding sequences of non-human species. In fact, humanized antibodies are typically human antibodies that are certain hypervariable region residues and possibly some FR residues that are substituted with residues from analogous sites in rodent antibodies.

The selection of human variable domains (light and heavy) for the production of humanized antibodies is extremely important to reduce antigenicity. The variable domain sequences of rodent antibodies were screened against a complete library of known human variable domain sequences according to the so-called "best fit" method. The human sequence closest to the rodent sequence is then accepted as a human framework for humanized antibodies (Sims et al, J. Immunol. 151: 2296 (1993); Chothia et al, J. Mol. Biol. 196: 901 (1987). )). Another method uses a specific framework obtained from a consensus sequence of all human antibodies with a particular light chain or heavy chain subpopulation. The same framework can be used for several different humanized antibodies (Carter et al, Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); Presta et al, J. Immunol., 151: 2623 (1993)).

In addition, antibodies are highly important to maintain high affinity for antigens and other beneficial biological properties. To achieve this goal, humanized antibodies are prepared according to one method by analyzing the parental sequence and various conceptual humanized products using a three-dimensional model of the parental sequence and the humanized sequence. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. There are computer programs that describe and present the approximate three-dimensional configuration of the selected candidate immunoglobulin sequences. Examination of such presentations allows analysis of the possible role of residues in the function of candidate immunoglobulin sequences, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind to its antigen. In this manner, FR residues can be selected and combined from the recipient and the input sequence to achieve desired antibody characteristics (such as increased affinity for the target antigen). In general, the effects on antigen binding directly and most substantially involve hypervariable region residues.

Human antibody

The human anti-EGFL7 antibody of the present invention can be constructed by combining an Fv pure line variable domain sequence selected from a human-derived phage display library with a known human constant domain sequence as described above. Alternatively, the human monoclonal anti-EGFL7 antibody of the present invention can be produced by the fusion tumor method. Human myeloma and mouse-human hybrid myeloma cell lines for producing human monoclonal antibodies have been described by, for example, Kozbor J. Immunol ., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987) and Boerner et al, J. Immunol. , 147: 86 (1991).

It is now possible to produce a transgenic animal (e.g., a mouse) that is capable of producing a whole human antibody lineage without immunization of the endogenous immunoglobulin. For example, homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice has been described to result in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene array into such germline mutant mice will result in the production of human antibodies following antigen challenge. See, for example, Jakobovits et al, Proc. Natl. Acad. Sci USA , 90:2551 (1993); Jakobovits et al, Nature , 362: 255 (1993); Bruggermann et al, Year in Immunol. , 7:33 (1993) .

Human antibodies can also be obtained by gene shuffling from non-human (e.g., rodent) antibodies, wherein the human antibodies have similar affinities and specificities as the original non-human antibodies. According to this method (also referred to as "antigenic stencil"), the human V domain gene lineage is substituted for the heavy or light chain variable region of a non-human antibody fragment obtained by the phage display technology as described above, Thereby a non-human chain/human chain scFv or Fab chimera population is produced. Selection with an antigen can result in the isolation of a non-human chain/human chain chimeric scFv or Fab, wherein the human chain restores the antigen binding site that is disrupted when the primary phage exhibits a corresponding non-human chain in the pure line, ie, the epitope is determined (Imprint) The choice of human chain collocation. Human antibodies are obtained when this process is repeated to replace the remaining non-human chains (see PCT WO 93/06213, published Apr. 1, 1993). Unlike traditional transplantation of humanized non-human antibodies by CDRs, this technology provides intact human antibodies that do not have FR or CDR residues of non-human origin.

Bispecific antibody

A bispecific antibody is a monoclonal antibody having binding specificity for at least two different antigens, preferably a human or humanized antibody. In the present case, one of the binding specificities is for EGFL7 and the other for any other antigen. An exemplary bispecific antibody can bind to two different epitopes of the EGFL7 protein. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing EGFL7. Such antibodies have an EGFL7 binding arm and an arm that binds to a cytotoxic agent such as saporin, anti-interferon-[alpha], vinblastine, ricin-A chain, methotrexate or radioisotope hapten. Bispecific antibodies (eg, F(ab')2 bispecific antibodies) can be prepared in the form of full length antibodies or antibody fragments.

Methods for making bispecific antibodies are known in the art. Traditionally, recombinant production of bispecific anti-systems has been based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537 (1983)). Due to the random distribution of immunoglobulin heavy and light chains, these fusion tumors (quaternary hybridomas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, usually by affinity chromatography steps, is quite cumbersome and the product yield is low. A similar procedure is disclosed in WO 93/08829, published May 13, 1993, and in Traunecker et al, EMBO J., 10:3655 (1991).

The antibody variable domain having the desired binding specificity (antibody-antigen combining site) is fused to an immunoglobulin constant domain sequence according to a different and better method. The fusion is preferably fused to an immunoglobulin heavy chain constant domain comprising at least a portion of the hinge region, the CH2 and CH3 regions. Preferably, the first heavy chain constant region (CH1) containing the site necessary for light chain binding is present in at least one of the fusions. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain is inserted into a separate expression vector and co-transfected into a suitable host organism. In the examples where the unequal ratios of the three polypeptide chains used in the construction provide the best yield, this provides great flexibility in the adjustment of the ratio of the three polypeptide fragments to each other. However, when at least two polypeptide chains are expressed in equal ratios to produce high yields or when such ratios are not of particular importance, the coding sequences for two or all three polypeptide chains can be inserted into an expression vector.

In some embodiments of the method, the bispecific anti-system consists of a hybrid immunoglobulin heavy chain having a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair in the other arm (providing Two binding specificities constitute. It has been found that since the immunoglobulin light chain is only present in half of the bispecific molecule to provide a convenient means of separation, the asymmetric structure promotes the separation of the desired bispecific compound from the immunoglobulin chain combination desired by the non-human. This method is disclosed in WO 94/04690. For additional details regarding the production of bispecific antibodies, see, for example, Suresh et al, Meth. Enzymol, 121:210 (1986).

According to another approach, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. A preferred interface comprises at least a portion of the CH3 domain of the antibody constant domain. In this way, one or more of the small amino acid side chains of the first antibody molecule interface are replaced with a larger side chain (eg, tyrosine or tryptophan). Compensating for the same or similar size to the larger side chain at the interface of the second antibody molecule by replacing the larger amino acid side chain with a smaller amino acid side chain (eg, alanine or threonine) Hole". This provides a mechanism for increasing the yield of heterodimers over other non-human end products, such as homodimers.

Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to an antibiotic, and the other antibody can be coupled to biotin. For example, such antibodies have been proposed to target immune system cells that are not desired cells (U.S. Patent No. 4,676,980) and for the treatment of HIV infection (WO 91/00360 and WO 92/00373). The heteroconjugated antibody can be made using any suitable crosslinking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Patent No. 4,676,980 and the numerous cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkages. Brennan et al, Science , 229: 81 (1985) describe a procedure for proteolytic cleavage of intact antibodies to produce F(ab')2 fragments. These fragments are reduced in the presence of the dithiol conjugate sodium arsenite to stabilize the adjacent dithiol and prevent intermolecular disulfide bond formation. The resulting Fab' fragment is then converted to a thionitrobenzoate (TNB) derivative. One of the Fab'-TNB derivatives is then reconverted to Fab'-thiol by reduction with mercaptoethylamine and mixed with another molar amount of another Fab'-TNB derivative to form a bispecific antibody. The bispecific antibody produced can be used as an agent for selectively immobilizing an enzyme.

Recent developments have facilitated the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al, J. Exp. Med. 175: 217-25 (1992) describe the production of a fully humanized bispecific antibody F(ab')2 molecule. E. coli secretes each Fab' fragment separately and undergoes in vitro directed chemical coupling to form a bispecific antibody. The bispecific antibody thus formed is capable of binding to cells overexpressing the HER2 receptor and normal human T cells, and also triggers the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

A variety of techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, leucine zippers have been used to generate bispecific antibodies. Kostelny et al. , J. Immunol. 148(5): 1547-53 (1992). The leucine zipper peptide from the Fos and Jun proteins was ligated to the Fab' portion of two different antibodies by gene fusion. The antibody homodimer is reduced in the hinge region to form a monomer, and then reoxidized to form an antibody heterodimer. This method can also be used to generate antibody homodimers. The "bifunctional antibody" technique described by Hollinger et al, Proc. Natl. Acad. Sci. USA 90:6444-48 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy chain variable domain (VH) joined to the light chain variable domain (VL) via a linker that is too short to allow pairing between the two domains on the same chain. Thus, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen binding sites. Another strategy for making bispecific antibody fragments by using single-chain Fv (sFv) dimers has also been reported. See Gruber et al. , J. Immunol. 152: 5368 (1994).

The invention also encompasses antibodies that are greater than bivalent. For example, a trispecific antibody can be prepared. Tutt et al . J. Immunol. 147: 60 (1991).

Multivalent antibody

Cells that exhibit antigen-bound antigens as compared to bivalent antibodies can internalize (and/or catabolize) multivalent antibodies more rapidly. The antibody of the present invention may be a multivalent antibody (except for an antibody of the IgM class) (for example, a tetravalent antibody) having three or more antigen-binding sites, and the multivalent antibody can be easily encoded by a recombinant expression to encode an antibody polypeptide chain. Made from nucleic acids. A multivalent antibody can comprise a dimerization domain and three or more antigen binding sites. Preferred dimerization domains comprise (or consist of) an Fc region or a hinge region. In this case, the antibody will comprise an Fc region and three or more antigen binding sites at the amino terminus of the Fe region. Preferred multivalent antibodies herein comprise from (or consist of) from three to about eight, but preferably four antigen binding sites. A multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain comprises two or more variable domains. For example, the polypeptide chain may comprise VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, and Fc is a polypeptide chain of the Fc region, X1 and X2 represent an amino acid or a polypeptide, and n is 0 or 1. For example, the polypeptide chain can comprise a VH-CH1-elastic linker-VH-CH1-Fc region chain or a VH-CH1-VH-CH1-Fc region chain. The multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable domain polypeptides. A multivalent antibody herein can, for example, comprise from about two to about eight light chain variable domain polypeptides. A light chain variable domain polypeptide encompassed herein comprises a light chain variable domain and, as the case may be, additionally comprises a CL domain.

Antibody variant

In some embodiments, the invention encompasses amino acid sequence modifications of the antibodies described herein. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of antibodies can be prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to achieve the final construct as long as the final construct has the desired characteristics. Amino acid changes can be introduced into the target antibody amino acid sequence at the time of manufacture of the sequence.

A method that can be used to identify certain residues or regions of an antibody that is a preferred location of mutation is referred to as a "alanine scanning mutation" as described by Cunningham and Wells, Sc Ience , 244: 1081-85 (1989). In this regard, identify residues or groups of target residues (eg, charged residues such as arg, asp, his, lys, and glu) and neutralize them with a neutral or negatively charged amino acid (preferably alanine or polyalanine) Replacement to affect the interaction of the amino acid with the antigen. The amino acid positions that exhibit functional sensitivity to the substitution are then improved by introducing additional or other variants at the substitution sites. Therefore, although the site of the change in the amino acid sequence has been scheduled to be introduced, the nature of the mutation itself does not need to be predetermined. For example, to analyze the potency of a mutation at a locus, an ala scan or random mutagenesis is performed at the target codon or region and the displayed immunoglobulin is screened for the desired activity.

Insertion of an amino acid sequence into an amino-terminal and/or carboxy-terminal fusion comprising a length ranging from one residue to a polypeptide having one hundred or more residues and insertion of a single or multiple amino acid residues within the sequence . Examples of terminal insertions include antibodies having N-terminal methionine residues or antibodies fused to cytotoxic polypeptides. Other insertion variants of the antibody molecule include fusions of the N-terminus or C-terminus of the antibody with an enzyme (eg, for ADEPT) or a polypeptide that increases the serum half-life of the antibody.

Another type of antibody amino acid variant alters the original glycosylation pattern of the antibody. Such changes include deletion of one or more sugar moieties present in the antibody and/or addition of a glycosylation site that is not present in one or more antibodies.

The glycosylation of a polypeptide is typically an N- or O-linked. N linkage refers to the attachment of a sugar moiety to the side chain of an aspartic acid residue. Tripeptide sequence aspartic acid-X-serine and aspartic acid-X-threonine (where X is any amino acid other than proline) is a sugar moiety and aspartic acid Identification sequence for side chain enzymatic attachment. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxylamino acid, most commonly seric acid or threonine, but 5-hydroxyguanamine can also be used. Acid or 5-hydroxy lysine.

The addition of a glycosylation site (for N-linked glycosylation sites) is conveniently achieved by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences. The change can also be made by adding one or more serine or threonine residues to or replacing the sequence of the original antibody (for O-linked glycosylation sites).

Where the antibody comprises an Fc region, the sugar attached thereto can be varied. For example, an antibody having a mature sugar structure that lacks fucose attached to the Fc region of an antibody has been described in US Patent Application No. US 2003/0157108 (Presta, L.). See also US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). An antibody having a bipartite N-acetylglucosamine (GlcNAc) in a sugar attached to an Fc region of an antibody is disclosed in WO 2003/011878, Jean-Mairet et al., and U.S. Patent No. 6,602,684. Mentioned in a et al. Antibodies having at least one galactose residue in oligosaccharides attached to the Fc region of an antibody are reported in WO 97/30087, Patel et al. For antibodies having altered sugars attached to the Fc region of an antibody, see also WO 98/58964 (Raju, S.) and WO 99/22764 (Raju, S.). For antigen-binding molecules with modified glycosylation, see also US 2005/0123546 (Uma a et al).

Preferred glycosylation variants herein comprise an Fc region in which the sugar structure attached to the Fc region lacks fucose. These variants have improved ADCC function. Optionally, the Fc region additionally comprises one or more amino acid substitutions that further improve ADCC, such as substitution of the 298 region 298, 333 and/or 334 (EU numbering of residues). Examples of publications relating to "defucosylation" or "fucose-deficient" antibodies include: US 2003/0157108, WO 2000/61739, WO 2001/29246, US 2003/0115614, US 2002/0164328, US 2004/ 00936221, US 2004/0132140, US 2004/0110704, US 2004/0110282, US 2004/0109865, WO 2003/085119, WO 2003/084570, WO 2005/035586, WO 2005/035778, WO 2005/053742, Okazaki et al. J. Mol. Biol. 336:1239-49 (2004); Yamane-Ohnuki et al, Biotech. Bioeng. 87:614 (2004). Examples of cell lines which produce de-alcoholized antibodies include Lec13 CHO cells which are defective in protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249: 533-45 (1986); U.S. Patent Application No. US 2003 /0157108 A1, Presta, L and WO 2004/056312 A1, Adams et al, especially Example 11) and gene knockout cell lines, such as α-1,6-fucosyltransferase gene (FUT8) gene knockout CHO Cells (Yamane-Ohnuki et al . Biotech. Bioeng. 87:614 (2004)).

Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue substituted with a different residue in the antibody molecule. The most interesting substitution sites include hypervariable regions, but also cover FR changes. The conservative substitutions are shown under the heading "Preferred substitutions" in Table 1. If such substitutions result in a change in biological activity, then a more substantial change, further described as described below with reference to the amino acid class, and the screening of the product can be introduced in Table 1.

Substantial modifications to the biological properties of an antibody can be achieved by selective substitutions that differ significantly in their role in maintaining the following: (a) the structure of the polypeptide backbone in the substitution region, such as a folded or helical configuration; b) the charge or hydrophobicity at the molecular target site; or (c) the side chain volume. Naturally occurring residues can be grouped based on common side chain properties: (1) hydrophobic: orthanoic acid, met, ala, val, leu, ile; (2) neutral hydrophilicity: Cys, Ser, Thr, Asn , Gln; (3) acidity: asp, glu; (4) basic: his, lys, arg; (5) residues that affect chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe .

Non-conservative substitutions will require members of one of these categories to be exchanged with another.

One type of substitution variant involves the substitution of one or more hypervariable region residues of a parent antibody (eg, a humanized antibody or a human antibody). In general, the resulting variants selected for further study should have improved biological properties relative to the parent antibody from which they are produced. One suitable way to generate such substitution variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (eg, 6-7 sites) are mutated to produce all possible amino acid substitutions at each point. When fused to the gene III product of M13 packaged in each filamentous phage particle, the resulting antibody is presented from the filamentous phage particle. Phage-presented variants are then screened for the biological activity (e.g., binding affinity) of the antibodies as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutations can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Additionally or alternatively, it may be useful to analyze the crystal structure of the antigen-antibody complex to identify the point of contact between the antibody and the antigen. These contact residues and adjacent residues are candidates that are substituted according to the techniques detailed herein. After generating such variants, the variant combinations are subjected to screening as described herein, and antibodies with superior properties can be selected for further investigation in one or more relevant assays.

Nucleic acid molecules encoding antibody amino acid sequence variants can be prepared by a variety of methods known in the art. Such methods include, but are not limited to, isolation from a natural source (in the case of a naturally occurring amino acid sequence variant) or mediated by a variant or non-variant type of oligonucleotide of a previously prepared antibody (or site-directed) mutations, PCR mutations and cassette mutagenesis were prepared.

It may be desirable to introduce one or more amino acid modifications into the Fc region of an immunoglobulin polypeptide of the invention, thereby producing an Fc region variant. An Fc region variant may comprise a human Fc region sequence comprising an amino acid modification (eg, a substitution) at one or more amino acid positions (including hinge cysteine positions) (eg, human IgGl, IgG2, IgG3, or IgG4 Fc) Area).

In accordance with the teachings of this specification and the teachings of the art, it is contemplated that in some embodiments the antibodies used in the methods of the invention may comprise one or more, for example, changes in the Fc region as compared to wild-type counterpart antibodies. However, such antibodies will retain substantially the same therapeutic utility characteristics as their wild-type counterparts. For example, it is believed that certain changes that would result in a change (ie, improvement or reduction) in C1q binding and/or complement dependent cytotoxicity (CDC) can be made in the Fc region, for example as described in WO 99/51642 Said. For further examples of Fc region variants, see also Duncah and Winter Nature 322: 738-40 (1988); U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO 94/29,351. WO 00/42072 (Presta) and WO 2004/056312 (Lowman) describe modified or reduced antibody variants that bind to FcR. The contents of these patent publications are expressly incorporated herein by reference. See also Shields et al . J. Biol. Chem. 9(2): 6591-6604 (2001). An antibody having an increased half-life and improved binding to a neonatal Fc receptor (FcRn) is described in US 2005/0014934 A1 (Hinton et al.), wherein FcRn is responsible for the transfer of maternal IgG into the fetus (Guyer et al., J. Immunol). 117: 587 (1976) and Kim et al., J.Immunol 24:. 249 (1994 )). Such antibodies comprise an Fc region having one or more substitutions that modify the binding of the Fc region to FcRn. Polypeptide variants having a modified Fc region amino acid sequence and increased or decreased C1q binding ability are described in U.S. Patent No. 6,194,551 B1, WO 99/51642. The contents of their patent publications are expressly incorporated herein by reference. See also Idusogie et al . J. Immunol. 164: 4178-84 (2000).

Antibody derivative

The antibodies of the invention may be further modified to contain additional non-protein portions known in the art and readily available. The moiety suitable for derivatizing the antibody is preferably a water soluble polymer. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone , poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer) and Glycan or poly(N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, polyoxypropylene/ethylene oxide copolymer, polyoxyethylene polyol (such as glycerin), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde has advantages in manufacturing due to its stability in water. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be modified, whether the antibody derivative will be used in a given condition, etc. .

Screen for antibodies with the desired properties

The antibodies of the invention bind to EGFL7 and, in some embodiments, can modulate one or more aspects associated with EGFL7, including, but not limited to, disrupting any biologically relevant EGFL7 biological pathway, and/or treatment and/or Or preventing a tumor, a cell proliferative disorder or cancer, and/or treating or preventing a disorder associated with EGFL7 expression and/or activity, such as increased EGFL7 expression and/or activity. For example, antibodies of the invention can be screened for antibodies that block HUVEC cell adhesion to EGFL7 and HUVECs on EGFL7 protein coated plates as described herein.

The purified antibody can additionally be characterized by a series of assays including, but not limited to, N-terminal sequencing, amino acid analysis, non-denaturing size exclusion, high pressure liquid chromatography (HPLC), mass spectrometry, ion exchange. Chromatography and digestion of papaya enzymes.

In certain embodiments of the invention, the biological activity of the antibodies made herein is analyzed. In some embodiments, the antigen binding activity of an antibody of the invention is tested. Antigen binding assays known in the art and useful herein include, but are not limited to, any direct or competitive binding assays, such as Western blotting techniques, radioimmunoassays, ELISA (enzyme-linked immunosorbent) Verification), "sandwich" immunoassay, immunoprecipitation assay, fluorescent immunoassay, and protein A immunoassay. An illustrative antigen binding assay is provided in the Examples section below.

In some embodiments, the invention provides an anti-EGFL7 antibody that competes with EGFL7 for binding to an antibody comprising a light chain variable domain and a heavy chain variable domain, the light chain variable domain comprising SEQ ID NO: 1 And the sequence of SEQ ID NO: 3 and the heavy chain variable domain comprises a sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4. Such competing antibodies can be obtained by screening anti-EGFL7 fusion tumor supernatants for binding to a labeled antibody that comprises a light chain variable domain and a heavy chain variable domain that competes with the immobilized EGFL7, the light chain variable domain A sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 3 and the heavy chain variable domain comprises a sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4. Such competing antibodies include antibodies that recognize the EGFL7 epitope that is identical to or overlaps with the EGFL7 epitope recognized by the antibody. The fusion cell supernatant containing the competitor antibody will reduce the detectable binding in the target competitive binding mixture as compared to the amount of bound, labeled antibody detected in the control binding mixture containing the irrelevant (or no) antibody. The amount of bound, labeled antibody. Any of the competitive binding assays described herein are applicable to the foregoing procedures.

The anti-EGFL7 antibody of the present invention having the properties described herein can be obtained by any suitable method by screening the anti-EGFL7 fusion tumor pure line for the desired property. For example, an anti-EGFL7 monoclonal antibody that competes with or does not compete with EGFL7 for binding to an antibody comprising a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain comprises a SEQ ID The sequence of NO: 1 and SEQ ID NO: 3 and the heavy chain variable domain comprises a sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4, and the candidate antibody can be tested in a binding competition assay. Competitive assays are well known in the art.

Other functional assays for determining the ability of anti-EGFL7 antibodies to inhibit are known in the art, and certain assays will be exemplified herein.

In some embodiments, the invention encompasses altered antibodies that have some, but not all, of the effector functions, which renders the half-life of the antibody in vivo extremely important while certain effector functions (such as complement and ADCC) are not required Or desirable candidates in a variety of applications that are harmful. In certain embodiments, the Fc activity of the produced immunoglobulin is measured to ensure that only desired characteristics are maintained. In vitro and/or in vivo cytotoxicity assays can be performed to confirm reduction/depletion of CDC and/or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcyR binding (and thus may lack ADCC activity) but still retains FcRn binding ability. Primary cells (NK cells) that mediate ADCC display only FcRIII, while monocytes express FcRI, FcRII, and FcRIII. The FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). An example of an in vitro assay for assessing the ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 or U.S. Patent No. 5,821,337. Effector cells that can be used for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Additionally or alternatively, the molecule of interest can be assessed in vivo, for example, in an animal model such as the animal model disclosed in Clynes et al, Proc. Natl. Acad. Sci. USA 95:652-656 (1998). ADCC activity. A C1q binding assay can also be performed to confirm that the antibody is unable to bind to C1q and thus lacks CDC activity. For assessing complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al, J. Immunol. Meth. 202: 163 (1996). FcRn binding and in vivo clearance/half life assays can also be performed using methods known in the art.

In some embodiments, the invention provides a modified antibody having enhanced effector function and/or increased half-life.

Vector, host cell and recombinant method

For recombinant production of an antibody of the invention, the nucleic acid encoding the antibody is isolated and inserted into a replicable vector for further selection (amplification of DNA) or expression. The DNA encoding the antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibody). A variety of carriers are available. The choice of vector will depend on the host cell used. In general, preferred host cells are of prokaryotic or eukaryotic (generally mammalian) origin. It will be appreciated that any isotype constant region can be used to achieve this, including IgG, IgM, IgA, IgD, and IgE constant regions, and such constant regions can be obtained from any human or animal species.

a. Using prokaryotic host cells to produce antibodies

i. Vector Construction A polynucleotide sequence encoding a polypeptide component of an antibody of the invention can be obtained using standard recombinant techniques. The desired polynucleotide sequence can be isolated from the antibody-producing cells (such as fusion tumor cells) and sequenced. Alternatively, the polynucleotide can be synthesized using a nucleotide synthesizer or PCR technique. After obtaining the sequence encoding the polypeptide, it is inserted into a recombinant vector capable of replicating in a prokaryotic host and expressing the heterologous polynucleotide. A wide variety of vectors that are available and are known in the art can be used in the present invention. The choice of appropriate vector will depend primarily on the size of the nucleic acid inserted into the vector and the particular host cell transformed by the vector. Each vector will contain a plurality of components depending on its function (a heterologous polynucleotide amplification or expression or both) and its compatibility with the particular host cell in which it is retained. Vector components generally include, but are not limited to, an origin of replication, a selectable marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, a heterologous nucleic acid insert, and a transcription termination sequence.

In general, plastid vectors containing replicons and control sequences obtained from species compatible with the host cell are used with such hosts. Vectors typically have a replication site and a marker sequence capable of providing phenotypic selection in transformed cells. For example, pBR322, a plastid obtained from E. coli species, is typically used to transform E. coli. pBR322 contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance and thus provides a convenient means of identifying transformed cells. pBR322, its derivatives or other microbial plastids or phage may also contain or be modified to contain a promoter which can be used by a microbial organism to express an endogenous protein. An example of a pBR 322 derivative for the expression of a specific antibody is described in detail in U.S. Patent No. 5,648,237 to Carter et al.

In addition, phage vectors containing replicons and control sequences compatible with the host microorganism can be used as transformation vectors with such hosts. For example, bacteriophage such as λGEM ^TM -11 for the manufacture of can be used to transform susceptible host cells (such as E. coli LE392) of the recombinant vector.

The expression vector of the present invention may comprise two or more promoter-cistronic pairs encoding each polypeptide component. The promoter is an untranslated regulatory sequence located upstream (5') of the cistron that regulates its expression. Prokaryotic promoters are generally divided into two categories, namely, inducible and constitutive. An inducible promoter is a promoter that initiates a cistron to be transcribed in increasing amounts under the control of its response to changes in culture conditions, such as the presence or absence of nutrients or temperature changes.

A large number of promoters that can be distinguished by a variety of potential host cells are well known. The promoter can be operably linked to the coding light or heavy chain by removing the selected promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the present invention. Cistron DNA. A natural promoter sequence can be used to direct amplification and/or expression of a target gene with a variety of heterologous promoters. In some embodiments, a heterologous promoter is typically used to allow for faster transcription and higher yields of the expressed target gene as compared to the native target polypeptide promoter.

Promoters suitable for use in prokaryotic hosts include the phoA promoter, beta-galactanase and lactose promoter systems, tryptophan ( trp ) promoter systems, and hybrid promoters (such as the tac or trc promoter). However, other promoters that are functional in bacteria, such as other known bacterial or bacteriophage promoters, are also suitable. Nucleotide sequences thereof have been published, whereby those skilled in the art will be able to operably associate a cistron encoding a target light and heavy chain with a linker or adaptor that provides any desired restriction site. (Siebenlist et al, Cell 20: 269 (1980)).

In one aspect of the invention, each cistron in the recombinant vector comprises a secretion signal sequence component that directs translocation of the expressed polypeptide across the membrane. In general, the signal sequence can be a component of the vector, or it can be part of the DNA of the polypeptide of interest inserted into the vector. The signal sequence selected for the purposes of the present invention shall be a signal sequence which is recognized and processed by the host cell (i.e., cleaved by a signal peptidase). For prokaryotic host cells that do not recognize and handle the native signal sequence of a heterologous polypeptide, the signal sequence is replaced, for example, by a prokaryotic signal sequence selected from the group consisting of alkaline phosphatase, penicillinase, Ipp or heat. Stable enterotoxin II (STII) leader sequence, LamB, PhoE, PelB, OmpA and MBP. In one embodiment of the invention, the signal sequence used in the two cistrons of the expression system is the STII signal sequence or a variant thereof.

In another aspect, the production of the immunoglobulin of the invention can occur in the cytoplasm of the host cell, and thus there is no need for a secretion signal sequence to be present in each cistron. In this regard, the light and heavy chains of immunoglobulins are expressed, folded, and assembled in the cytoplasm to form functional immunoglobulins. Certain host strains (e.g., E. coli trxB ^- strain) provide cytoplasmic conditions that facilitate disulfide bond formation, thereby allowing proper folding and assembly of the expressed protein subunits. Proba and Pl Ckthun, Gene 159: 203 (1995).

Prokaryotic host cells suitable for expression of the antibodies of the invention include archaea bacteria and Eubacteria, such as Gram-negative or Gram-positive organisms. Examples of suitable bacteria include Escherichia (Escherichia) (e.g., E. coli), Bacillus (Bacilli) (such as Bacillus subtilis (B. subtilis)), intestinal bacteria (Enterobacteria), Pseudomonas (of Pseudomonas) species (e.g. P. aeruginosa ), Salmonella typhimurium , Serratia marcescens , Klebsiella , Proteus , Shigella , Rhizobium (rhizobia), Vitreoscilla (Vitreoscilla) or Paracoccus (Paracoccus). In some embodiments, Gram-negative cells are used. In some embodiments, E. coli cells are used as a host of the invention. Examples of E. coli strains include the W3110 strain (Bachmann, Cellular and Molecular Biology, Vol. 2 (Washington, DC: American Society for Microbiology, 1987), pages 1190-1219; ATCC Accession No. 27,325) and its derivatives include a 33D3 strain having the genotype W3110ΔfhuA(ΔtonA)ptr3 lac Iq lacL8ΔompTΔ(nmpc-fepE)degP41 kanR (U.S. Patent No. 5,639,635). Other strains and their derivatives, such as E. coli 294 (ATCC) 31,446), E. coli B, E. coli λ 1776 (ATCC 31,537) and E. coli RV308 (ATCC 31,608) also applies. These examples are for purposes of illustration and not limitation. Methods for constructing any of the above-described derivatives of bacteria having the specified genotype are known in the art and are described, for example, in Bass et al, Proteins , 8:309-14 (1990). It is generally necessary to consider the reproducibility of the replicon in bacterial cells to select the appropriate bacteria. For example, when using the well-known plasmid (such as pBR322, pBR325, pACYC177, or pKN410) providing replication midnight, E. coli, Serratia (SERRATIA) or Salmonella (Salmonella) adapted to be used as the host species. In general, the host cell should secrete a minimal amount of proteolytic enzyme, and additional protease inhibitors may be suitably incorporated into the cell culture.

Ii. Antibody Production Host cells are transformed with the above expression vectors and cultured in a conventional nutrient medium modified to be suitable for inducing a promoter, selecting a transformant, or amplifying a gene encoding a desired sequence.

Transformation means the introduction of DNA into a prokaryotic host such that the DNA can be replicated as an extrachromosomal element or by a chromosomal component. Depending on the host cell used, transformation is carried out using standard techniques appropriate for such cells. Calcium treatment with calcium chloride is generally used for bacterial cells containing parenchymal cell wall barriers. Another method of transformation uses polyethylene glycol/DMSO. Another technique used is electroporation.

Prokaryotic cells used to produce the polypeptides of the invention are grown in a medium known in the art and suitable for culturing selected host cells. Examples of suitable media include Luria Broth (LB) supplemented with essential nutrient supplements. In some embodiments, the medium also contains a selection agent selected based on the construction of the expression vector to selectively permit growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the culture medium to grow cells expressing the ampicillin resistance gene.

Any necessary supplements other than carbon, nitrogen and inorganic phosphate sources, introduced alone or in a mixture with another supplement or medium, such as a complex nitrogen source, may also be included. Optionally, the medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, dithioerythritol, and dithiothreitol. alcohol.

Prokaryotic host cells are cultured at appropriate temperatures. For the growth of E. coli, for example, a temperature in the range of from about 20 ° C to about 39 ° C is generally employed, usually from about 25 ° C to about 37 ° C, for example about 30 ° C. The pH of the medium can be any pH in the range of from about 5 to about 9 depending on the host organism. For E. coli, the pH is typically from about 6.8 to about 7.4, and is typically about 7.0.

If an inducible promoter is used in the expression vector of the present invention, protein expression is induced under conditions suitable for activation of the promoter. In one aspect of the invention, the phoA promoter is used to control transcription of the polypeptide. Thus, the transformed host cells are cultured in phosphate-limited medium for induction. The phosphate-restricted medium is generally CRAP medium (see, for example, Simmons et al. , J. Immunol. Meth . 263: 133-47 (2002)). As is known in the art, a variety of other inducers can be used depending on the carrier construct used.

In one embodiment, the expressed polypeptide of the invention is secreted into the host cell periplasm and recovered from the periplasm. Protein recovery typically involves splitting the microorganisms generally by means such as osmotic shock, ultrasonic treatment or dissolution. After dividing the cells, the cell debris or whole cells can be removed by centrifugation or filtration. The protein can be further purified, for example, by affinity resin chromatography. Alternatively, the protein can be transferred to a medium and isolated therein. The cells can be removed from the culture and the culture supernatant filtered and concentrated to further purify the resulting protein. The expressed polypeptide can be further isolated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assays.

In one aspect of the invention, the manufacture of a plurality of antibodies is carried out by a fermentation process. Recombinant proteins can be produced using a variety of large-scale feed batch fermentation procedures. Large-scale fermentation has a capacity of at least 1,000 liters, preferably about 1,000 to 100,000 liters. These fermenters use a blender impeller to distribute oxygen and nutrients, especially glucose (a common carbon/energy source). Small-scale fermentation generally refers to fermentation in a fermentor that is no greater than about 100 liters and can range from about 1 liter to about 100 liters.

In Po fermentation process was generally grown under appropriate conditions to the starting cells for the induction of protein expression after the desired density (e.g. OD ₅₅₀ of about 180-220), cell lines at this stage in the early stationary phase. As is known in the art and as described above, a variety of inducers can be used depending on the vector construct used. Cells can be grown for a short period of time prior to induction. Although longer or shorter induction times can be used, the cells are typically induced for about 12 to 50 hours.

In order to improve the yield and quality of the polypeptide of the present invention, various fermentation conditions can be varied. For example, to improve proper assembly and folding of a secreted antibody polypeptide, host prokaryotic cells can be co-transformed with an additional vector that overexpresses a companion protein, such as a Dsb protein (DsbA, DsbB, DsbC, DsbD, and/or DsbG). Or FkpA (peptidyl amidoxime cis-trans isomerase with concomitant activity). The accompanying protein has been shown to promote proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al., J Bio Chem 274: 19601-05 (1999); Georgiou et al., U.S. Patent No. 6,083,715; Georgiou et al., U.S. Patent No. 6,027,888; Bothmann and Pl Ckthun J.Biol.Chem . 275:17100-05 (2000); Ramm and Pl Ckthun J. Biol. Chem. 275: 17106-13 (2000); Arie et al, Mol. Microbiol. 39: 199-210 (2001).

To minimize proteolysis of heterologous proteins (especially for proteolytically sensitive proteins), certain proteolytic enzyme-deficient host strains can be used in the present invention. For example, host cell strains can be modified to effect gene mutations encoding genes of known bacterial proteases such as Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V, Protease VI, and combinations thereof. Certain E. coli protease deficient strains can be used and are described, for example, in Joly et al., (1998), supra; Georgiou et al., U.S. Patent No. 5,264,365; Georgiou et al., U.S. Patent No. 5,508,192; Hara et al. , Microbial Drug Resistance , 2: 63-72 (1996).

In one embodiment, a proteolytic enzyme-deficient E. coli strain exhibiting one or more plastid transformations with accompanying proteins is used as a host cell in the expression system of the present invention.

Iii. Antibody Purification Standard protein purification methods known in the art can be used. The following procedure is an exemplary appropriate purification procedure: immunoaffinity column or ion exchange column fractionation, ethanol precipitation, reverse phase HPLC, vermiculite or cation exchange resin (such as DEAE) chromatography, chromatofocusing, SDS-PAGE, Ammonium sulfate precipitation and gel filtration using, for example, Sephadex G-75.

In one aspect, protein A immobilized on a solid phase is used for immunoaffinity purification of the full length antibody product of the invention. Protein A is a 41 kD cell wall protein from Staphylococcus aureas that binds to the Fc region of the antibody with high affinity. Lindmark et al. , J. Immunol. Meth . 62: 1-13 (1983). The solid phase of the immobilized protein A is preferably a column comprising a glass or vermiculite surface, more preferably a controlled microporous glass column or a citrate column. In some applications, the tubing is coated with a reagent such as glycerin in an effort to prevent non-specific adhesion of contaminants.

As a first step of purification, the preparation obtained from the cell culture as described above can be applied to the solid phase of immobilized protein A to specifically bind the antibody of interest to protein A. The solid phase is then washed to remove contaminants that are non-specifically bound to the solid phase. Finally, the antibody of interest is recovered from the solid phase by dissolution.

b. Production of antibodies using eukaryotic host cells

Vector components generally include, but are not limited to, one or more of the following components: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

(i) Signal sequence component The vector used in a eukaryotic host cell may also contain a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide of interest. The heterologous signal sequence selected is preferably a signal sequence that can be recognized and processed by the host cell (i.e., cleaved by a signal peptidase). In mammalian cell expression, mammalian signal sequences can be used as well as viral secretion leader sequences, such as the herpes simplex gD signal.

The DNA of the previous flooding region is ligated to the DNA encoding the antibody in the reading frame.

(ii) Origin of replication Generally, mammalian expression vectors do not require replication of the starting component. For example, the SV40 origin can usually be used only because it contains an early promoter.

(iii) Selection of the genome The performance and selection vector may contain a selection gene, also known as a selectable marker. A typical selection gene encodes a protein that has the following functions: (a) confers resistance to antibiotics or other toxins (eg, ampicillin, neomycin, methotrexate or tetracycline); (b) supplements auxotrophy (at When relevant; or (c) provide critical nutrients that are not available from the complex medium.

An example of a selection process is the use of drugs to arrest the growth of host cells. Those cells that have been successfully transformed by a heterologous gene can produce a protein that exhibits drug resistance and thus survive in this alternative. Examples of this dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.

Another example of a selectable marker suitable for use in mammalian cells is a selectable marker that enables the identification of cells capable of absorbing antibody nucleic acids, such as DHFR, thymidine kinase, metallothionein-I and II (preferably primate) An animal metallothionein gene), adenosine deaminase, ornithine decarboxylase, and the like.

For example, cells transformed with the DHFR selection gene are first identified by culturing all transformants in a medium containing methotrexate (Mtx) (a competitive antagonist of DHFR). When wild-type DHFR is used, one of the appropriate host cells is a Chinese hamster ovary (CHO) cell line (eg, ATCC) that lacks DHFR activity. CRL-9096).

Alternatively, the encoded antibody, wild type, can be selected by performing cell growth in a medium containing a selection agent for a selectable marker, such as an aminoglycoside antibiotic, such as kanamycin, neomycin or G418. A host cell (or in particular a wild-type host containing endogenous DHFR) transformed or co-transformed with the DNA sequence of the DHFR protein and another selectable marker such as aminoglycoside 3'-phosphotransferase (APH). See U.S. Patent No. 4,965,199.

(iv) Promoter components The expression and selection vectors typically contain a promoter that is recognized by the host organism and operably linked to the antibody polypeptide nucleic acid. Promoter sequences for use in eukaryotes are known. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream of the transcription start site. Another sequence that can be found 70 to 80 bases upstream of the transcription initiation point of a plurality of genes is the CNCAAT region (SEQ ID NO: 19), wherein N can be any nucleotide. At the 3' end of most eukaryotic genes is the AATAAA sequence (SEQ ID NO: 20), which can be a signal that adds a poly A tail to the 3' end of the coding sequence. All such sequences are suitable for insertion into a eukaryotic expression vector.

The antibody polypeptide from a vector in a mammalian host cell is transcribed (for example) from a virus (such as polyomavirus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus). , retrovirus, hepatitis B virus and prion 40 (SV40) genomes, promoters derived from heterologous mammalian promoters (eg, actin promoter or immunoglobulin promoter), derived from heat shock promoters Control, the constraints of which are compatible with the host cell system for such promoters.

The early and late promoters of the SV40 virus are conveniently obtained in the form of SV40 restriction fragments that also contain the SV40 viral origin of replication. The immediate early promoter of human cytomegalovirus is conveniently obtained in the form of a HindIII E restriction fragment. A system for expressing DNA in a mammalian host using bovine papilloma virus as a vector is disclosed in U.S. Patent No. 4,419,446. Modifications to this system are described in U.S. Patent No. 4,601,978. Alternatively, a long terminal repeat of Rous Sarcoma Virus can be used as a promoter.

(v) Enhancer element component Transcription of higher eukaryotes DNA encoding the antibody polypeptide of this invention can generally by inserting an enhancer sequence into the vector to increase. A variety of enhancer sequences from mammalian genes (blood globulin, elastase, albumin, alpha-fetoprotein, and insulin) are known. However, we will usually use enhancers from eukaryotic cells. Examples include the SV40 enhancer on the back of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the posterior side of the replication origin, and the adenovirus enhancer. See also Yaniv, Nature 297: 17-18 (1982) for reinforcing elements for activation of eukaryotic promoters. The enhancer can be spliced into the vector at the 5' or 3' position of the antibody polypeptide coding sequence, but is preferably located 5' to the promoter.

(vi) Transcription termination components Expression vectors for use in eukaryotic host cells typically also contain sequences necessary for termination of transcription and stabilization of mRNA. Such sequences are typically available from 5' and sometimes 3' non-translated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylation fragments in the non-translated portion of the mRNA encoding the antibody. One suitable transcription termination component is the bovine growth hormone polyadenylation region. See WO 94/11026 and the expression vectors disclosed therein.

(vii) Selection and Transformation of Host Cells Host cells suitable for use in the selection or expression of DNA in the vectors herein include higher eukaryotic cells as described herein, including vertebrate host cells. It has become a routine procedure to propagate vertebrate cells in culture (tissue culture). An example of a suitable mammalian host cell strain is a SV40 transformed monkey kidney CV1 cell line (COS-7, ATCC). CRL 1651), human embryonic kidney cell line (293 cells or 293 cells that have been subcultured in suspension culture, Graham et al, J. Gen Virol. 36:59 (1977)), baby hamster kidney cells ( BHK, ATCC CCL 10), Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al, Proc. Natl. Acad. Sci. USA 77: 4216 (1980)), mouse sertoli cell (TM4, Mather, Biol. Reprod. 23: 243-251 (1980)), monkey kidney cells (CV1 ATCC) CCL 70), African Green Monkey Kidney Cells (VERO-76, ATCC CRL-1587), human cervical cancer cells (HELA, ATCC) CCL 2), canine kidney cells (MDCK, ATCC) CCL 34), buffalo rat liver cell (BRL 3A, ATCC) CRL 1442), human lung cells (W138, ATCC) CCL 75), human hepatocytes (Hep G2, HB 8065), mouse mammary tumors (MMT 060562, ATCC) CCL51), TRI cells (Mather et al, Annals NYAcad. Sci. 383: 44-68 (1982)), MRC 5 cells, FS4 cells, and human liver tumor cell lines (Hep G2).

The host cell is transformed with the above-described expression or selection vector for producing the antibody, and cultured in a conventional nutrient medium modified to be suitable for inducing a promoter, selecting a transformant, or amplifying a gene encoding a desired sequence.

(viii) Culturing Host Cells Host cells for producing the antibodies of the present invention can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimum Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM), Sigma Both are suitable for culturing host cells. Furthermore, any of the media described in the following literature can be used as a medium for host cells: Ham et al, Meth. Enzymol. 58: 44 (1979); Barnes et al, Anal. Biochem. 102: 255 (1980); No. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469, WO 90/03430, WO 87/00195, or U.S. Patent No. 30,985. Any such medium may optionally be supplemented with hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN ^TM drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range exists), and glucose or an equivalent energy source. Any other necessary supplements that will be of a suitable concentration known to those skilled in the art may also be included. Culture conditions, such as temperature, pH, and the like, are conditions previously selected for use by the host cell for expression and will be apparent to those of ordinary skill.

(ix) Antibody purification When recombinant techniques are employed, antibodies can be produced intracellularly or directly secreted into the culture medium. If the antibody is produced intracellularly, as a first step, the host cell or the fragmented particles of the fragment are removed, for example by centrifugation or ultrafiltration. Where the antibody is secreted into the culture medium, the supernatant from such performance systems is typically first concentrated using a commercially available protein concentration filter (eg, Amicon or Millipore Pellicon ultrafiltration unit). Protease inhibitors (such as PMSF) can be included in any of the foregoing steps to inhibit proteolysis and can include antibiotics to prevent the growth of foreign contaminants.

The antibody composition prepared from the cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, wherein affinity chromatography is a preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and the isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human gamma 1, gamma 2 or gamma 4 heavy chains (Lindmark et al, J. Immunol. Meth . 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and human gamma 3 (Guss et al, EMBO J. 5: 1567-75 (1986)). The matrix to which the affinity ligand is attached is most commonly agarose, but other matrices may also be used. Mechanically stable matrices such as controlled microporous glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times compared to the flow rates and processing times achievable with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABX ^TM resin used (JTBaker, Phillipsburg, NJ) for purification. Depending on the antibody to be recovered may be of, other proteins may also be used for purification of techniques, such as ion-exchange column fractionation, ethanol precipitation, reverse phase HPLC, chromatography, chert, heparin SEPHAROSE ^TM chromatography on an anion or cation exchange resin (such as polyaspartic acid column) chromatography, chromatofocusing, SDS-PAGE and ammonium sulfate precipitation.

After any preliminary purification step, the mixture comprising the antibody of interest and the contaminant can be subjected to a dissolution buffer having a pH between about 2.5 and 4.5, preferably at a low salt concentration (e.g., about 0-0.25 M salt). Low pH hydrophobic interaction chromatography.

Immune conjugate

The invention also provides immunoconjugates (also referred to interchangeably as "antibody-drug conjugates" or ADCs) comprising cytotoxic agents, such as chemotherapeutic agents, drugs, growth inhibitors, toxins (eg, bacteria, fungi, Any anti-EGFL7 antibody described herein conjugated with a plant or animal derived enzymatic toxin or fragment thereof or a radioisotope (ie, a radioactive conjugate).

Local delivery of cytotoxic agents or cytostatic agents (i.e., drugs used to kill or inhibit tumor cells in cancer therapy) using antibody-drug conjugates (Syrigos and Epenetos, Anticancer Research 19: 605-14 (1999); Niculescu-Duvaz and Springer, Adv. Drg Del. Rev. 26:151-72 (1997); U.S. Patent No. 4,975,278), which allows the delivery of a drug moiety to a tumor and in which intracellular accumulation is achieved, wherein such non-conjugated Systemic administration of a drug produces unacceptable levels of toxicity to normal cells and tumor cells that are intended to be eliminated (Baldwin et al, Lancet , pp. (March 15, 1986): 603-05 (1986); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", Monoclonal Antibodies '84: Biological And Clinical A pplications, A. Pinchera et al. (eds.), pp. 475-506 (1985)). This also seeks maximum efficacy and minimal toxicity. Multiple antibodies and monoclonal antibodies have been reported to be suitable for such strategies (Rowland et al, Cancer Immunol. Immunother . 21: 183-87 (1986)). The drugs used in these methods include daunorubicin, doxorubicin, methotrexate and vindesine (Rowland et al., (1986) supra). Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin; phytotoxins such as ricin; small molecule toxins such as geldanamycin (Mandler et al., J. Nat . Cancer) Inst. 92(19): 1573-81 (2000); Mandler et al, Bioorganic & Med. Chem . Letters 10: 1025-28 (2000); Mandler et al, Bioconjugate Chem. 13:786-91 (2002)) , maytansinoids (EP 1391213; Liu et al, Proc. Natl. Acad. Sci. USA 93: 8618-8623 (1996)) and calicheamicin (Lode et al, Cancer Res. 58: 2928 (1998) Hinman et al, Cancer Res. 53:3336-3342 (1993)). Toxins can achieve their cytotoxicity and cell growth inhibition by a mechanism including tubulin binding, DNA binding or topoisomerase inhibition. Certain cytotoxic drugs tend to be inactive or less active when conjugated to a large antibody or protein receptor ligand.

ZEVALIN ^TM (ibritumomab (ibritumomab tiuxetan), Biogen / Idec ) An antibody-based - radioisotope conjugates, which is based by a thiourea linker - chelating agents for the binding seen in the surface of normal and malignant B lymphocytes Murine IgG1 on CD20 antigen Monoclonal antibodies are composed of ¹¹¹ In or ⁹⁰ Y radioisotopes (Wiseman et al, Eur. Jour. Nucl. Med. 27(7): 766-77 (2000); Wiseman et al, Blood 99(12): 4336-42 (2002); Witzig et al, J. Clin. Oncol. 20(10): 2453-63 (2002); Witzig et al, J. Clin. Oncol. 20(15): 3262-69 (2002)). Although ZEVALIN is active against B-cell non-Hodgkin's Lymphoma (NHL), administration causes severe and long-term blood cell reduction in most patients. MYLOTARG ^TM (gemtuzumab (gemtuzumab ozogamicin), Wyeth Pharmaceuticals) system, a conjugate is a hu CD33 antibody drug antibody nematocysts constitutes connected to neomycin, was approved in 2000 for the treatment of acute myeloid leukemia injection ( Drugs of the Future 25 (7): 686 (2000); U.S. Patent Nos. 4,970,198, 5,079,233, 5,558,089, 5,560,040, 5,693,762, 5,739,116, 5,767,285, 5,773,001. Cantuzumab mertansine (Immunogen, Inc.) is an antibody drug conjugate consisting of a huC242 antibody linked to the maytansinoid moiety DM1 via a disulfide linker SPP, which has been used to treat CanAg Phase II trial of cancers such as colon, pancreas, stomach, and other cancers. MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.) is an antibody drug conjugate consisting of an anti-prostate specific membrane antigen (PSMA) monoclonal antibody linked to the maytansinoid moiety DM1. The potential treatment of prostate tumors is under investigation. Auristatin peptide, auristatin E (AE) and monomethyl auristatin (MMAE) (a synthetic analog of dolastatin) and chimeric monoclonal antibody cBR96 (Lewis Y for cancer) Specificity) and cAC10 (specific for CD30 for hematological malignancies) are conjugated (Doronina et al, Nature Biotech. 21(7): 778-784 (2003)) and therapeutic studies are underway.

Chemotherapeutic agents that can be used to generate immunoconjugates are described herein (eg, above). The enzyme-active toxins and fragments thereof can be used, including the diphtheria toxin A chain, the non-binding active fragment of diphtheria toxin, the exotoxin A chain (from Pseudomonas aeruginosa), the ricin A chain, the abrin toxin A chain, and the lotus root toxic protein. (modeccin) A chain, aspergillus broom hormone (α-sarcin), tung oil (Aleurites fordii) protein, carnation (dianthin) proteins, Phytolacca foreign (Phytolacca americana) proteins (PAPI, PAPII, and PAP-S), bitter melon (Momordica Charantia ) inhibitors, curcin , crotin, sapaonaria officinalis inhibitors, gelonin, mitogen (mitogellin), constrictocin (restrictocin), phenol Phenomycin, enomycin and trichothecene. See, for example, WO 93/21232, published October 28, 1993. A variety of radionuclides can be used to make radioconjugated antibodies. Examples include ²¹² Bi, ¹³¹ I, ¹³¹ In, ⁹⁰ Y, and ¹⁸⁶ Re. The conjugate of the antibody and the cytotoxic agent can be prepared using a variety of bifunctional protein coupling agents such as 3-(2-pyridyldithio)propionic acid N-succinimide (SPDP). , iminothiolane cyclopentane (IT), a bifunctional derivative of sulfhydryl ester (such as dimethyl dimethyl imidate hydrochloride), an active ester (such as disuccinimide suberate) Ester), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis-(p-azidobenzylidene) hexamethylenediamine), bis-diazonium derivatives (such as double-(pairs) Diazonium salt benzhydryl)-ethylenediamine), diisocyanate (such as toluene 2,6-diisocyanate) and bis-active fluorine compound (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al, Science , 238: 1098 (1987). Carbon 14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO 94/11026.

Also encompassed herein are antibodies and one or more small molecule toxins (such as echinomycin, maytansinoids, dolastatin, auristatin, trichothecene, and CC1065, and derivatives of toxins of such toxins) Conjugate.

i. Mordenol and Maytansin Compounds In some embodiments, an immunoconjugate comprises an antibody (full length or fragment) of the invention in combination with one or more maytansinoid molecules.

Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansin was first isolated from the East African shrub Maytenus serrata (U.S. Patent No. 3,896,111). Subsequently, certain microorganisms were also found to produce maytansinoids such as maytansinol and C-3 maytansinol (U.S. Patent No. 4,151,042). Synthetic maytansinol and its derivatives and analogs are disclosed in, for example, U.S. Patent Nos. 4,137,230, 4,248,870, 4,256,746, 4,260,608, 4,265,814, 4,294,757, 4,307,016, 4,308,268. Nos. 4,308,269, 4,309,428, 4,313,946, 4,315,929, 4,317,821, 4,322,348, 4,331,598, 4,361,650, 4,364,866, 4,424,219, 4,450,254, 4,362,663, and 4,371,533 No.

The maytansin moiety is the most interesting drug moiety in the antibody drug conjugate because it is: (i) relatively easy to prepare by fermentation or chemical modification, derivatization of the fermentation product; Ii) readily apt to be derivatized by a functional group conjugated to the antibody via a non-disulfide linker; (iii) stable in plasma; and (iv) effective against a variety of tumor cell lines.

An immunoconjugate comprising a maytansinoid, a method for its manufacture, and a therapeutic use thereof are disclosed, for example, in U.S. Patent Nos. 5,208,020, 5,416,064, and European Patent No. EP 0 425 235 B1, the disclosures of The manner of citation is expressly incorporated herein. Liu et al, Proc. Natl. Acad. Sci. USA 93:8618-23 (1996) describes an immunoconjugate comprising a maytansinoid compound (designated DM1) linked to a monoclonal antibody C242 against human colorectal cancer. . This conjugate has been found to be highly cytotoxic to cultured colon cancer cells and exhibits anti-tumor activity in in vivo tumor growth assays. Chari et al, Cancer Research 52: 127-31 (1992) describe immunoconjugates in which a maytansinoid is conjugated to a murine antibody A7 that binds to an antigen on a human colon cancer cell line via a disulfide linker. Or conjugated with another murine monoclonal antibody TA.1 that binds to the HER-2/neu oncogene. The cytotoxicity of the TA.1-maytansinoid conjugate was tested in vitro on human breast cancer cell line SK-BR-3, which showed 3 x 10 ⁵ HER-2 surface antigen per cell. The drug conjugate achieves a degree of cytotoxicity similar to that of the free maytansinoid, which can be increased by increasing the number of molecules of each antibody molecule maytansinoid. The A7-maytansinoid conjugate exhibited low systemic cytotoxicity in mice.

The antibody-maytansinoid conjugate can be prepared by chemically linking the antibody to the maytansinoid molecule without significantly reducing the biological activity of the antibody or maytansinoid molecule. See, for example, U.S. Patent No. 5,208,020, the disclosure of which is expressly incorporated herein by reference. On average, conjugated 3-4 maytansinoid molecules per antibody molecule have been shown to enhance the cytotoxicity of target cells without adversely affecting antibody function or solubility, but it is expected that even one molecule of toxin/antibody can be Enhances cytotoxicity over the use of naked antibodies. Maytansinoids are well known in the art and can be synthesized by known techniques or isolated from natural sources. Suitable maytansinoids are disclosed, for example, in U.S. Patent No. 5,208,020 and other patents and non-patent publications mentioned above. Preferred maytansinoids are maytansinol and maytansinoids (such as various maytansinol) which have been modified in the aromatic ring of the maytansinol molecule or elsewhere.

A variety of linkers for the manufacture of antibody-maytansinoid conjugates are known in the art, including, for example, the linkers disclosed in U.S. Patent No. 5,208,020 or EP Patent No. 0 425 235 B1; Chari et al, Cancer Research 52: 127-131 (1992) and US 2005/0169933 A1, the disclosures of each of which are expressly incorporated herein by reference. An antibody-maytansinoid conjugate comprising a linker component SMCC can be prepared as disclosed in US 2005/0169933 A1. The linker includes a disulfide group, a thioether group, an acid labile group, a photolabile group, a peptidase labile group, or an esterase labile group, as described in the above identified patents. It is preferred to disclose disulfide groups and thioether groups. Additional linkers are described and exemplified herein.

The conjugate of the antibody and the maytansinoid compound can be prepared using a variety of bifunctional protein coupling agents such as 3-(2-pyridyldithio)propionic acid N-succinimide ester. (SPDP), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide (SMCC), iminothiolane pentane (IT), sulphate Bifunctional derivatives (such as dimethyl dimethyl imidate hydrochloride), active esters (such as disuccinimide suberate), aldehydes (such as glutaraldehyde), bis- azide compounds (such as bis-(p-azidobenzylidene) hexamethylenediamine), bis-diazonium salt derivatives (such as bis-(p-diazonium benzylidene)-ethylenediamine), diisocyanates (such as Toluene 2,6-diisocyanate) and a double active fluorine compound (such as 1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agents include 3-(2-pyridyldithio)propionic acid N-succinimide (SPDP) which provides a disulfide bond (Carlsson et al, Biochem. J. 173:723-37 ( 1978)) and 4-(2-pyridylthio)pentanoic acid N-succinimide (SPP).

The linker is attached to various positions of the maytansinoid molecule depending on the type of visual connection. For example, an ester bond can be formed by reaction with a hydroxyl group using conventional coupling techniques. The reaction can occur at a C-3 position having a hydroxyl group, a C-14 position modified by a hydroxymethyl group, a C-15 position modified by a hydroxyl group, and a C-20 position having a hydroxyl group. In a preferred embodiment, the bond is formed at the C-3 position of the maytansinol or maytansinol analog.

Ii. Auristatin and Dolastatin In some embodiments, the immunoconjugate comprises an antibody of the invention conjugated to a peptide analog of a dolastatin or a rabbit toxin and a derivative of auristatin (U.S. Patent No. 5,635,483) , No. 5,780,588). Dolphin toxin and auristatin have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al, Antimicrob. Agents Chemother. 45(12): 3580-3584 (2001)) and have anticancer (US) 5,663,149) and antifungal activity (Pettit et al, Antimicrob. Agents Chemother. 42:2961-2965 (1998)). The dolastatin or auristatin drug moiety can be attached to the antibody via the N (amino) terminus or C (carboxyl) terminus of the peptide drug moiety (WO 02/88172).

An exemplary auristatin embodiment includes an N-terminally linked monomethyl auristatin drug moiety DE and DF, which is disclosed in "Monomethylvaline Compounds Capable of Conjugation to Ligands", US 2005/0238649, the disclosure of which is incorporated by reference in its entirety. The manner is expressly incorporated herein.

In general, a peptidyl drug moiety can be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can, for example, be based on liquid phase synthesis methods well known in the art of peptide chemistry (see E. Schr Der and KL Bke, "The Peptides", Vol. 1, pp. 76-136, 1965, Academic Press). The auristatin/conne toxin drug moiety can be prepared according to the following literature: US 5,635,483, US 5,780,588, Pettit et al, JA m. Chem. Soc. 111:5463-65 (1989); Pettit et al, Anti-Cancer Drug Design 13: 243-77 (1998); Pettit et al, Synthesis 719-25 (1996) and Pettit et al, J. Chem. Soc. Perkin Trans. 1 5: 859-863 (1996). See also Doronina, Nature Biotechnol. 21(7): 778-84 (2003); "Monomethylvaline Compounds Capable of Conjugation to Ligands", US 2005/0238649, which is incorporated herein by reference in its entirety herein in And a method of preparing a monomethylproline acid compound such as MMAE and MMAF conjugated to a linker).

Iii. Citricin In other embodiments, the immunoconjugate comprises an antibody of the invention conjugated to one or more echinomycin molecules. Antibiotics from the echinomycin family are capable of producing double-stranded DNA breaks at sub-picmole concentrations. For the preparation of the echinomycin family conjugates, see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001 and 5,877,296 (both issued American Cyanamid Company). Structural analogs of echinomycin that may be used include, but are not limited to, γ1I, α2I, α3I, N-ethylindolyl-γ1I, PSAG, and θI1 (Hinman et al, Cancer Research 53: 3336-42 (1993); Lode et al, Cancer Research 58: 2925-28 (1998) and the above-referenced U.S. Patent to American Cyanamid). Another anti-tumor drug that can be conjugated to an antibody is QFA, which is an antifolate. Both echinomycin and QFA have intracellular sites of action and are not susceptible to crossing the plasma membrane. Thus, uptake of such agents via antibody-mediated internalization significantly enhances their cytotoxic effects.

Iv. Other cytotoxic agents Other anti-tumor agents that can be conjugated to the antibodies of the invention include BCNU, streptozoticin, vincristine, and 5-fluorouracil, as described in U.S. Patent Nos. 5,053,394, 5,770,710. A family of agents collectively referred to as the LL-E33288 complex and isparticin (U.S. Patent No. 5,877,296).

The enzyme-active toxins and fragments thereof can be used, including the diphtheria toxin A chain, the non-binding active fragment of diphtheria toxin, the exotoxin A chain (from Pseudomonas aeruginosa), the ricin A chain, the abrin toxin A chain, and the lotus root toxic protein. A chain, bacteriocin, tung oil protein, carnation protein, foreign commercial protein (PAPI, PAPII and PAP-S), bitter melon inhibitor, jatropha, croton toxin, saponin inhibitor, paradise fruit protein, mitosis , limited to bacteriocin, phenolic acid, INOmycin and mycotoxins. See, for example, WO 93/21232, published October 28, 1993.

The invention further encompasses immunoconjugates formed between an antibody and a compound having nuclear degrading activity, such as a ribonuclease or endonuclease, such as a deoxyribonuclease; a DNase.

To selectively destroy a tumor, the antibody can comprise highly radioactive atoms. Radioactive conjugated antibodies can be produced using a variety of radioisotopes. Examples include radioisotopes of At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and Lu. When conjugates are used for detection, they may contain radioactive atoms for scintigraphy studies, such as tc ^99m or I ¹²³ ; or for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri) Spin labels such as iodine-123 and iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, cesium, manganese or iron.

Radiolabels or other labels can be incorporated into the conjugate in a known manner. For example, the peptide can be biosynthesized or can be synthesized by chemical amino acid synthesis using a suitable amino acid precursor (eg, involving the replacement of hydrogen with a fluorine-19 amino acid). A label such as tc ^99m or I ¹²³ , Re ¹⁸⁶ , Re ¹⁸⁸ and In ¹¹¹ can be attached via a cysteine residue in the peptide. The 钇-90 can be attached via an amine acid residue. Iodine-123 can be incorporated using the IODOGEN method (Fraker et al, Biochem. Biophys. Res. Commun. 80: 49-57 (1978)). Other methods are described in detail in "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989).

The conjugate of the antibody and the cytotoxic agent can be prepared using a variety of bifunctional protein coupling agents such as 3-(2-pyridyldithio)propionic acid N-succinimide (SPDP). ), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid amber sulfite (SMCC), imidothioindole cyclopentane (IT), bismuth imidate Functional derivatives (such as dimethyl dimethyl imidate hydrochloride), active esters (such as dinonyl succinimide), aldehydes (such as glutaraldehyde), bis- azide compounds (such as Bis-(p-azidobenzylidene) hexamethylenediamine), bis-diazonium salt derivatives (such as bis-(p-diazonium benzylidene)-ethylenediamine), diisocyanates (such as toluene 2) , 6-diisocyanate) and a double active fluorine compound (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al, Science , 238: 1098 (1987). Carbon 14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO 94/11026. A linker can be a "cleavable linker" that promotes the release of a cytotoxic drug in a cell. For example, an acid labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker or a disulfide-containing linker can be used (Chari et al, Cancer Research 52: 127- 31 (1992); U.S. Patent No. 5,208,020).

The compounds of the invention specifically encompass, but are not limited to, ADCs prepared using the following crosslinker reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfonate Acid-EMCS, sulfonate-GMBS, sulfonate-KMUS, sulfonate-MBS, sulfonate-SIAB, sulfonate-SMCC and sulfonate-SMPB and SVSB ((4-vinylguanidine) Ammonium benzoate imidate), such crosslinker reagents are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., USA). See 2003-2004 Applications Handbook and Catalog, pp. 467-498.

v. Preparation of Antibody Drug Conjugates In the antibody drug conjugate (ADC) of the present invention, the antibody (Ab) is conjugated to one or more drug moiety (D) via a linker (L), for example, each antibody About 1 to about 20 drug parts. The ADC of Formula I can be prepared by several routes using organic chemical reactions, conditions and reagents known to those skilled in the art, including: (1) nucleophilic groups of antibodies and divalent linker reagents Reaction to form Ab-L via a covalent bond, followed by reaction with drug moiety D; and (2) reacting a nucleophilic group of the drug moiety with a divalent linker reagent to form D-L via a covalent bond, followed by antibody The nucleophilic group reacts. Additional methods for preparing ADCs are described herein.

Ab(LD)p I

A linker can be composed of one or more linker components. Exemplary linker components include 6-maleimido hexamethylene ("MC"), maleimide propyl ketone ("MP"), lysine- citrulline ("val- Cit"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), 4-(2-pyridylthio)pentanoic acid N-succinimide ("SPP"), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid N-succinimide ("SMCC") and (4-iodo-ethenyl)amine N-Amber succinimide ("SIAB"). Additional linker components are known in the art and some are described herein. See also "Monomethylvaline Compounds Capable of Conjugation to Ligands", US 2005/0238649, the contents of which are incorporated herein by reference in its entirety.

In some embodiments, the linker can comprise an amino acid residue. Exemplary amino acid linker components include dipeptides, tripeptides, tetrapeptides or pentapeptides. Exemplary dipeptides include valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe). Exemplary tripeptides include glycine-proline-glycine (gly-val-cit) and glycine-glycine-glycine. Amino acid residues comprising an amino acid linker component include naturally occurring amino acids as well as minor amino acids and non-naturally occurring amino acid analogs such as citrulline. The amino acid linker component can be designed and optimized for its selectivity for enzymatic cleavage of specific enzymes such as tumor-associated proteases, cathepsins B, C and D or plasmin.

Nucleophilic groups on the antibody include, but are not limited to, (i) an N-terminal amine group; (ii) a side chain amine group, such as an amine acid; (iii) a side chain thiol group, such as cysteine And (iv) a saccharide hydroxyl or amine group in which the antibody is glycosylated. The amine group, the thiol group and the hydroxyl group are nucleophilic groups and are capable of reacting with the electrophilic group on the linker moiety and the linker reagent to form a covalent bond, the electrophiles comprising: (i) an active ester such as NHS Esters, HOBt esters, haloformates and acid halides; (ii) alkyl and benzyl halides such as haloacetamide; (iii) aldehydes, ketones, carboxyl groups and maleimine groups. Certain antibodies have a reducible interchain disulfide bond, a cysteine bridge. The antibody can be reacted with a linker reagent by treatment with a reducing agent such as DTT (dithiothreitol). Thus, theoretically each cysteine bridge will form two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into the antibody by conversion of the amine to the thiol by reaction of the amine acid with 2-iminothioindole cyclopentane (Traut reagent). By introducing one, two, three, four or more cysteine residues (for example, preparing a mutant antibody comprising one or more non-natural cysteine amino acid residues) The reactive thiol group is introduced into the antibody (or a fragment thereof).

The antibody drug conjugates of the invention can also be prepared by modifying the antibody to introduce an electrophilic moiety reactive with a nucleophilic substituent on the linker reagent or drug. The sugar of the glycosylated antibody can be oxidized, for example, with a periodate oxidizing reagent to form an aldehyde or ketone group reactive with the linker reagent or the amine group of the drug moiety. The resulting imine Schiff base group can form a stable bond or can be reduced by, for example, a borohydride reagent to form a stable amine bond. In one embodiment, the reaction of the sugar moiety of the glycosylated antibody with galactose oxidase or sodium metaperiodate produces a carbonyl (aldehyde and ketone group) in the protein that reacts with a suitable group on the drug ( Hermanson, Bioconjugate Techniques). In another embodiment, a protein containing an N-terminal serine acid or a threonine residue can be reacted with sodium metaperiodate to produce an aldehyde that replaces the first amino acid (Geoghegan and Stroh, Bioconjugate Chem. 3: 138-46, (1992) US 5,362,852). This aldehyde can be reacted with a drug moiety or a linker nucleophile.

Likewise, nucleophilic groups on the drug moiety include, but are not limited to, amines, thiols, hydroxyl groups, hydrazine, hydrazine, hydrazine, thiosemicarbazone, bismuth carboxylate and aryl hydrazine groups, A covalent bond can be formed by reaction with an electrophilic group on the linker moiety and the linker reagent, the electrophiles comprising: (i) an active ester such as an NHS ester, a HOBt ester, a haloformate, and an acid halide (ii) an alkyl group and a benzyl halide, such as a halogenated acetamide; (iii) an aldehyde, a ketone, a carboxyl group, and a maleimine group.

Alternatively, a fusion protein comprising an antibody and a cytotoxic agent can be made, for example, by recombinant techniques or peptide synthesis. The length of the DNA may comprise separate regions encoding two portions of the conjugate, which regions may be adjacent to each other or separated by a region encoding a linker peptide that does not destroy the desired properties of the conjugate.

In another embodiment, the antibody can be conjugated to a "receptor", such as streptavidin, for pretargeting the tumor, wherein the antibody-receptor conjugate is administered to the patient, followed by a scavenger The unbound conjugate is removed from the circulation, and then a "ligand" (eg, avidin) conjugated to a cytotoxic agent (eg, a radioactive nucleotide) is administered.

Medical formulation

Prepared as an aqueous solution, frozen by mixing an antibody of the desired purity with a physiologically acceptable optional carrier, excipient or stabilizer (Remington: The Science and Practice of Pharmacy 20th Edition (2000)) A therapeutic formulation comprising an antibody of the invention in dry or other dry formulation form for storage. Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosages and concentrations employed, and include buffers such as phosphates, citrates, histidines, and other organic acids; antioxidants, Including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexahydrochloroammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol or Benzyl alcohol, alkyl paraben (such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate), catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol); low a molecular weight (less than about 10 residues) polypeptide; a protein such as serum albumin, gelatin or immunoglobulin; a hydrophilic polymer such as polyvinylpyrrolidone; an amino acid such as glycine, glutamic acid, Aspartic acid, histidine, arginine or lysine; monosaccharides, disaccharides and other sugars, including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol , trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (eg Zn) - protein complexes); and / or non-ionic surfactant, such as TWEEN ^TM, PLURONICS ^TM or polyethylene glycol (PEG).

The formulations herein may also contain more than one active compound for the particular indication being treated, preferably an active compound having complementary activities that do not adversely affect each other. These molecules are suitable for being present in the combination in an amount effective for the intended purpose.

The active ingredient may also be entrapped in a gelled drug delivery system (eg, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions (eg, by coacervation techniques or interfacial polymerization). The microcapsules (for example, hydroxymethylcellulose or gelatin-microcapsules and poly(methyl methacrylate) microcapsules, respectively). Such techniques are disclosed in Remington: The Science and Practice of Pharmacy 20th Edition (2000).

Formulations to be administered in vivo must be sterile. This is easily accomplished by filtration through a sterile filter.

Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices containing solid hydrophobic polymers of the immunoglobulins of the invention, which matrices are in the form of shaped articles, such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactide (U.S. Patent No. 3,773,919), L-Bran Copolymer of aminic acid with γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymer (such as LUPRON DEPOT ^TM (from lactic acid-glycolic acid copolymer and acetonitrile ⁾ Injectable microspheres composed of ruthenium) and poly-D-(-)-3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable the release of molecules for more than 100 days, while certain hydrogels release proteins for shorter periods of time. When the encapsulated immunoglobulin is maintained in the body for a prolonged period of time, it can be denatured or agglomerated by exposure to moisture at 37 ° C, resulting in loss of biological activity and possible alteration of immunogenicity. A reasonable stability strategy can be designed depending on the mechanism involved. For example, if the agglutination mechanism is found to form an intermolecular S-S bond via a thio-disulfide interchange, the thiol residue can be modified, lyophilized from an acidic solution, moisture content controlled, and appropriate additives used. And forming a specific polymer matrix composition to achieve stability.

use

The antibodies of the invention are useful, for example, in in vitro, ex vivo and in vivo therapeutic methods.

In some embodiments, the invention provides a method of reducing or inhibiting angiogenesis in a subject having a pathological condition associated with angiogenesis comprising administering to the subject an effective amount of an anti-EGFL7 antibody of the invention. Such conditions include, for example, neoplasms (including cancers) and certain eye diseases.

Cancers that can be ameliorated by the treatment of the invention include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specifically, examples of such cancers include breast cancer, colon cancer, rectal cancer, colorectal cancer, kidney cancer, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), squamous Cell carcinoma (eg, epithelial squamous cell carcinoma), cervical cancer, ovarian cancer, prostate cancer, liver cancer, bladder cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer), pancreatic cancer, head and neck cancer, glia Cell tumor, retinoblastoma, astrocytoma, vesicular cell tumor, ovarian male cell tumor, liver tumor, hematological malignancies (including non-Hodgkin's lymphoma (NHL), multiple myeloma, and acute blood Malignant disease), endometrial cancer or uterine cancer, endometriosis, fibrosarcoma, choriocarcinoma, salivary gland cancer, vulvar cancer, thyroid cancer, esophageal cancer, liver cancer, anal cancer, penile cancer, nasopharyngeal carcinoma, Laryngeal cancer, Kaposi's sarcoma, melanoma, skin cancer, schwannomas, oligodendroglioma, neuroblastoma, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcoma, urethra cancer, thyroid Cancer, prestige Wilm's tumor and abnormal vascular proliferation associated with maternal disease, edema (such as edema associated with brain tumors) and Meigs' syndrome. Preferably, the cancer is selected from the group consisting of breast cancer, colorectal cancer, non-small cell lung cancer, non-Hodgkin's lymphoma (NHL), renal cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian tumor, mesothelioma. And a group of multiple myeloma. Cancer is better for colorectal cancer. Cancer conditions that can be ameliorated by the treatment of the invention include metastatic cancer. The invention is particularly suitable for treating angioed tumors.

Eye diseases that can be improved by the treatment of the present invention include intraocular neovascular diseases including, but not limited to, age-related macular degeneration, diabetic macular edema, proliferative diabetic retinopathy, central retinal vein occlusion with macular cystic edema, retina Branch vein occlusion complicated by cystoid macular edema, iris reddenation, pathological myopia/CNV, Von Hippel Lindau Syndrome, pterygium, POHS (tissue bacteremia)/CNV, choroidal vessels Retinal disease, retinopathy of prematurity (ROP), radiation-induced retinopathy, intraocular tumors (eg melanoma, retinoblastoma, metastases and orbital cavernous hemangioma), polypoid choroidal lesions, idiopathic near-concave capillaries Dilation, Eales' Disease, orbital cavernous hemangioma, orbital lymphangioma, orbital capillary hemangioma, corneal graft angiogenesis, corneal graft neovascularization, Coats Disease And wound healing issues associated with glaucoma surgery.

Furthermore, at least some of the antibodies of the invention may bind antigens from other species. Thus, the antibodies of the invention can be used, for example, in cell cultures containing antigens, in human subjects, or in other mammalian subjects having antigens that cross-react with antibodies of the invention (e.g., chimpanzees, baboons, velvets) Monkeys, macaques and rhesus monkeys, pigs or mice) bind to specific antigenic activity in vivo. In some embodiments, an antibody can be used to inhibit antigenic activity by contacting the antibody of the invention with an antigen to inhibit antigenic activity. The antigen is preferably a human protein molecule.

In some embodiments, the antibody of the present invention can be used in a method of binding an antigen in a subject suffering from a condition associated with an increase in antigenic expression and/or activity, the method comprising administering to the subject an antibody of the present invention Binds to the antigen in the subject. The antigen is preferably a human protein molecule and the subject is a human subject. Alternatively, the subject can be a mammal that exhibits an antigen that binds to an antibody of the invention. In addition, the subject may be a mammal into which an antigen has been introduced (for example, by administering an antigen or by expressing an antigen-transforming gene). The antibodies of the invention can be administered to human subjects for therapeutic purposes. Furthermore, an antibody of the invention can be administered to a non-human mammal (e.g., a primate, pig or mouse) that exhibits an antigen that cross-reacts with the immunoglobulin for veterinary purposes or as an animal model of human disease. . In the latter case, such animal models can be used to assess the therapeutic efficacy of the antibodies of the invention (e.g., test dosing amount and time course).

The antibodies of the invention are useful for treating, inhibiting, delaying the progression of a disease, disorder or condition associated with the performance and/or activity of one or more antigenic molecules, preventing/delaying their recurrence, ameliorating or preventing such diseases, disorders or conditions.

In certain embodiments, an immunoconjugate comprising an antibody conjugated to one or more cytotoxic agents is administered to a patient. In some embodiments, the immunoconjugate and/or antigen bound thereto are internalized by the cell, thereby causing an increase in therapeutic efficacy of the immunoconjugate to kill the target cell to which it binds. In one embodiment, the cytotoxic agent targets or interferes with nucleic acids in the target cell. In one embodiment, the cytotoxic agent targets or interferes with microtubule polymerization. Examples of such cytotoxic agents include any of the chemotherapeutic agents described herein (such as maytansinoids, auristatin, dolastatin or echinomycin), radioisotopes or ribonucleases or DNA endonucleases .

The antibodies of the invention may be used alone or in combination with other compositions for treatment. For example, an antibody of the invention can be administered in combination with a chemotherapeutic agent (including a mixture of chemotherapeutic agents), other cytotoxic agents, anti-antigen agents, cytokines, and/or growth inhibitors. Such combination therapies described above include combination administration (in which two or more agents are included in the same or separate formulations) and administration alone, and in the latter case administration of the antibody of the present invention may be administered in the administration Perform before and/or after the therapy.

The antibodies (and adjuvant therapies) of the invention can be administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, if desired, administered intralesionally for topical treatment. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In addition, antibodies are also suitable for administration by pulse infusion, particularly in decreasing doses. Partially, depending on the short-term or long-term nature of administration, administration can be by any suitable route (e.g., by injection, such as intravenous or subcutaneous injection).

The antibody compositions of the invention can be formulated, administered and administered in a manner consistent with good medical practice. Factors to be considered in this regard include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition, the location of the delivery of the agent, the method of administration, the schedule of administration, and other factors known to the physician. The antibody is not required, but may optionally be formulated with one or more agents currently used to prevent or treat the condition. The effective amount of such other agents will depend on the amount of the antibody of the invention present in the formulation, the type of disorder or treatment, and other factors discussed above. These agents are generally employed in the same dosages and routes of administration as used hereinabove, or in amounts of from about 1% to about 99% of the dosages used hereinabove.

For the prevention or treatment of a disease, the appropriate dose of the antibody of the invention (when used alone or in combination with other agents) will depend on the type of disease being treated, the type of antibody, the severity and duration of the disease, for prevention or treatment purposes. The antibody, prior therapy, clinical history of the patient, and response to the antibody, as well as the judgment of the attending physician. The antibody is suitable for administration to a patient once or via a series of treatments. Depending on the type and severity of the disease, antibodies from about 1 μg/kg to 15 mg/kg (eg 0.1 mg/kg to 10 mg/kg) can be administered, for example, by one or more separate administrations or by continuous The infusion is administered to the patient's initial candidate dose. A typical daily dose may range from about 1 μg/kg to 100 mg/kg or higher, depending on the factors mentioned above. For repeated administration over several days or longer, depending on the condition, treatment will continue until the desired disease symptoms are inhibited. An exemplary dosage of one of the antibodies will range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, or 10 mg/kg (or any combination thereof) can be administered to a patient. Such doses can be administered intermittently, for example weekly or every three weeks (e.g., subjecting the patient to about two doses to about twenty doses, such as about six doses of antibody). The higher initial loading dose can be followed by one or more lower doses. An exemplary dosing regimen comprises administering an antibody at an initial loading dose of about 4 mg/kg followed by administration of a weekly maintenance dose of about 2 mg/kg of the antibody. However, other dosage regimens are also available. The progress of this therapy is easily monitored by conventional techniques and assays.

The anti-EGFL7 antibodies of the invention can be used in assays (such as diagnostic or prognostic assays) for detecting the expression of EGFL7 in a particular cell or tissue, wherein the antibody is labeled and/or immobilized on an insoluble substrate as described below.

The invention provides a method of detecting EGFL7, the method comprising detecting an EGFL7-anti-EGFL7 antibody complex in a sample. The term "detecting" as used herein includes qualitative and/or quantitative detection (measurement content) with or without reference to a control.

The invention provides methods of diagnosing a condition associated with EGFL7 expression and/or activity, the methods comprising detecting an EGFL7-anti-EGFL7 antibody complex in a biological sample from a patient having or suspected of having the condition. In some embodiments, EGFL7 behaves as an increased performance or an abnormal (undesirable) performance.

The invention provides any of the anti-EGFL7 antibodies described herein, wherein the anti-EGFL7 antibody comprises a detectable label.

The invention provides a complex of any of the anti-EGFL7 antibodies described herein and EGFL7. In some embodiments, the complex is an in vivo or in vitro complex. In some embodiments, the complex comprises cancer cells. In some embodiments, the anti-EGFL7 antibody is detectably labeled.

EGFL7 can be detected using an anti-EGFL7 antibody in any of a variety of well-known detection assays. For example, a sample can be obtained from a desired source, and the sample can be mixed with an anti-EGFL7 antibody to form an antibody/EGFL7 complex with any EGFL7 present in the mixture, and any antibody/EGFL7 complex present in the mixture can be detected. To determine EGFL7 in a biological sample. Biological samples can be prepared for assay by methods known in the art to be suitable for a particular sample. The method of mixing the sample with the antibody and the method of detecting the antibody/EGFL7 complex are selected depending on the type of assay used. These tests include immunohistochemistry, competition and mezzanine assays, and space suppression assays.

Analytical methods for EGFL7 use one or more of the following reagents: labeled EGFL7 analog, immobilized EGFL7 analog, labeled anti-EGFL7 antibody, immobilized anti-EGFL7 antibody, and a spatial conjugate. Labeled reagents are also known as "tracers."

The label used is any detectable functional group that does not interfere with the binding of EGFL7 to the anti-EGFL7 antibody. A variety of labels are known for use in immunoassays, examples of which are directly detectable, such as fluorescent dyes, chemiluminescent agents, and radioactive labels; and moieties that must be detected by reaction or derivatization, such as enzymes.

The label used is any detectable functional group that does not interfere with the binding of EGFL7 to the anti-EGFL7 antibody. A variety of labels are known for use in immunoassays, examples of which are directly detectable, such as fluorescent dyes, chemiluminescent agents, and radioactive labels; and moieties that must be detected by reaction or derivatization, such as enzymes. Examples of such labels include the radioisotopes ³² P, ¹⁴ C, ¹²⁵ I, ³ H and ¹³¹ I; fluorophores such as rare earth chelating agents or luciferins and their derivatives, rhodamine and its derivatives , tanshinyl, ketone; luciferase, such as firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456); luciferin; 2,3-dihydropyridazinedione; spicy Root peroxidase (HRP); alkaline phosphatase; β-galactosidase; glucoamylase; lysozyme; sugar oxidase, such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase a heterocyclic oxidase, such as uricase and xanthine oxidase, coupled to an enzyme (such as HRP, lactoperoxidase or microperoxidase) using a hydrogen peroxide dye precursor; biotin/antibiotic Biotin; spin label; phage label; stable free radicals and their analogs.

These labels can be covalently bound to a protein or polypeptide using conventional methods. For example, a coupling agent can be used to link the antibody to the above-described fluorescent, chemiluminescent, and enzymatic labels, such as dialdehyde, carbodiimide, dimaleimide, bismuth imidate. , double nitrided benzidine and its analogues. See, for example, U.S. Patent Nos. 3,940,475 (fluorescence assay) and 3,645,090 (enzyme method); Hunter et al., Nature 144:945 (1962); David et al., Biochemistry 13: 1014-21 (1974); Pain et al. Human, J. Immunol. Meth . 40: 219-30 (1981) and Nygren. J. Histochem. and Cytochem. 30: 407-12 (1982). Preferred labels herein are enzymes such as horseradish peroxidase and alkaline phosphatase. Conjugation of this marker (including enzymes) to antibodies is a standard procedure for those of ordinary skill in the art of immunoassay. See, for example, O'Sullivan et al., "Methods for the Preparation of Enzyme-antibody Conjugates for Use in Enzyme Immunoassay," Methods Enzymol. , ed. JJ Langone and H. Van Vunakis, Vol. 73 (Academic Press, New York, New York, 1981), pp. 147-166.

Fixative reagents are required in some assay methods. Immobilization separates the anti-EGFL7 antibody from any EGFL7 that remains free in solution. Anti-EGFL7 antibodies or EGFL7 analogs are typically not made by covalent coupling (eg, using glutaraldehyde cross-linking) by adsorption to a water insoluble matrix or surface (Bennich et al., US 3,720,760) prior to the assay procedure. This is accomplished by solubilization or by insolubilizing the anti-EGFL7 antibody or EGFL7 analog by immunoprecipitation after the assay procedure.

Immunohistochemistry and staining assay procedures can be used to detect the performance of proteins in the sample. Immunohistochemical staining of tissue sections has been shown to be a reliable method for assessing or detecting the presence of proteins in a sample. Immunohistochemistry ("IHC") techniques typically utilize in situ detection and observation of cellular antigens by chromogenic or fluorescent methods. For the preparation of the sample, tissue or cell samples from a mammal (usually a human patient) can be used. Samples may be obtained by a variety of procedures known in the art including, but not limited to, surgical resection, aspiration, or biopsy. The tissue can be fresh or frozen tissue. In one embodiment, the sample is fixed and embedded in paraffin or the like. Tissue samples can be fixed (i.e., preserved) by conventional methods. Those of ordinary skill in the art will appreciate that the choice of fixative is determined by the purpose of histological staining or additional analysis of the sample. Those of ordinary skill in the art will also appreciate that the fixed length depends on the size of the tissue sample and the fixative used.

IHC can be combined with additional techniques such as morphological staining and/or in situ fluorescence hybridization. There are two general methods of IHC, namely direct and indirect verification. The binding of the antibody to the antigen of interest (e.g., EGFL7) is determined directly according to the first assay. This direct assay uses a labeled reagent that can be observed without further antibody interaction, such as a fluorescent label or an enzyme labeled one-stage antibody. In a typical indirect assay, an unconjugated first-level antibody binds to an antigen, and then the labeled secondary antibody binds to the primary antibody. In the case where the secondary antibody is conjugated to the enzyme label, a chromogenic or fluorescent substrate is added to observe the antigen. Signal amplification occurs because several secondary antibodies can react with different epitopes on the primary antibody.

Primary and/or secondary antibodies for immunohistochemistry are typically labeled with a detectable moiety. A variety of markers can be used, which are described elsewhere in the text.

In addition to the sample preparation procedures discussed above, tissue sections may need to be further processed before, during, or after IHC. For example, an epitope-repairing method can be performed, such as heating a tissue sample in a citrate buffer (see, for example, Leong et al . Appl. Immunohistochem. 4(3): 201 (1996)).

After the optional blocking step, the tissue section is exposed to the primary antibody under appropriate conditions for a sufficient period of time to allow the primary antibody to bind to the target protein antigen in the tissue sample. Suitable conditions for achieving this can be determined by routine experimentation. The degree of binding of the antibody to the sample is determined by using any of the detectable labels discussed above. The label is preferably an enzyme label (e.g., HPRO) that catalyzes the chemical change of a chromogenic substrate such as 3,3'-diaminobenzidine chromogen. Preferably, the enzyme label is conjugated to an antibody that specifically binds to the primary antibody (eg, the primary antibody is a rabbit polyclonal antibody and the secondary antibody is a goat anti-rabbit antibody).

The sample thus prepared can be mounted and covered with a cover slip. The slide evaluation is then determined, for example, using a microscope, and staining intensity standards conventionally used in the art can be used.

Other assays known as competition or mezzanine assays have been well established and widely used in the commercial diagnostic industry.

The competition assay is based on the ability of the tracer EGFL7 analog to compete with the test sample EGFL7 for a limited amount of anti-EGFL7 antibody antigen binding site. The anti-EGFL7 antibody is generally insoluble before or after competition, and then the tracer and EGFL7 bound to the anti-EGFL7 antibody are separated from the unbound tracer and EGFL7. This separation can be achieved by decantation (in the case where the binding partner is not dissolved in advance) or by centrifugation (in the case of precipitation of the binding partner after the competition reaction). The amount of test sample EGFL7 is inversely proportional to the amount of bound tracer as measured by the amount of labeling material. A dose response curve of the known amount of EGFL7 was prepared and compared to the test results to quantify the amount of EGFL7 present in the test sample. When an enzyme is used as a detectable marker, such assays are referred to as ELISA systems.

Another competitive assay called the "homogeneous" check does not require phase separation. In this regard, a conjugate of the enzyme and EGFL7 is prepared and used such that when the anti-EGFL7 antibody binds to EGFL7, the presence of the anti-EGFL7 antibody will alter the enzyme activity. In this case, EGFL7 or an immunologically active fragment thereof is conjugated to an enzyme such as a peroxidase via a bifunctional organic bridge. The conjugate for the anti-EGFL7 antibody is selected such that binding of the anti-EGFL7 antibody will inhibit or enhance the enzymatic activity of the label. This method itself has been widely practiced under the name EMIT.

The spatial conjugate is used in a steric hindrance method for homogeneous assay. By conjugated the low molecular weight hapten to the small EGFL7 fragment to synthesize these conjugates, the antibody against the hapten is substantially incapable of binding to the conjugate simultaneously with the anti-EGFL7 antibody. In this assay procedure, EGFL7 present in the test sample will bind to the anti-EGFL7 antibody, thereby binding the anti-hapten to the conjugate, resulting in a change in the characteristics of the conjugate hapten, for example when the hapten is fluorescent Fluorescent changes in the group.

Sandwich assays are especially useful for determining EGFL7 or anti-EGFL7 antibodies. In a continuous sandwich assay, the test sample EGFL7 is adsorbed using a immobilized anti-EGFL7 antibody, such as by removing the test sample by washing, using the bound EGFL7 to adsorb a second, labeled anti-EGFL7 antibody, and then subjecting the bound material to the residue The tracer is separated. The amount of tracer combined has been proportional to the test sample EGFL7. In the "simultaneous" sandwich assay, the test sample is not separated prior to the addition of the labeled anti-EGFL7. A continuous sandwich assay using anti-EGFL7 monoclonal antibody as one antibody and multiple anti-EGFL7 antibodies as another antibody can be used to test EGFL7 in a sample.

The above verification is only an exemplary detection test for EGFL7. Other methods of determining EGFL7 using anti-EGFL7 antibodies, now or later developed, including the bioassays described herein, are included within the scope of the invention.

Manufacturing goods

In another aspect of the invention, an article of manufacture comprising a substance useful for treating, preventing, and/or diagnosing a condition described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container can be formed from a variety of materials such as glass or plastic. The container holds a composition that is alone or in combination with another composition effective to treat, prevent, and/or diagnose the condition, and may have a sterile access port (eg, the container may be an intravenous solution bag or have a stopper pierceable by a hypodermic needle) Vial). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is used to treat a condition selected, such as asthma. Further, the article of manufacture may also comprise (a) a first container comprising the composition, wherein the composition comprises an antibody of the invention; and (b) a second container comprising the composition. In this embodiment of the invention, the article of manufacture may additionally comprise a package insert that should indicate that the first and second antibody compositions are useful for treating a particular condition, such as asthma. Additionally or alternatively, the article of manufacture may additionally comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer Ringer's solution and dextrose solution. It may additionally include other materials that are desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

The following are examples of the methods and compositions of the present invention. It should be appreciated that various other embodiments can be implemented based on the basic description provided above.

Instance Example 1 Manufacture and characterization of anti-EGFL7 monoclonal antibody

Manufacture of monoclonal antibodies Identification and colonization of EGFL7 seeks to discover novel human secretory and transmembrane proteins, particularly those involved in vascular developmental regulation. Details regarding the selection and performance of human EGFL7 are described, for example, in patent application US 2003/0224948 A1 (where EGFL 7 is referred to as PRO1449). Human EGFL7 GenBank The registration number is NM_016215.

Two-week (eight doses) of His6-tagged recombinant human and mouse EGFL7 proteins produced by E. coli diluted with Ribi adjuvant (Corixia, Hamilton, MT) were used to immunize Egfl7 homozygous knockout mice via the footpad (at Genentech produced). B cells were collected from 10 lymph nodes of mice exhibiting high serum valence and made into mouse myeloma cells (X63.Ag8.653; American Type Culture Collection) (ATCC )) Fusion. After 10-14 days, the supernatant was screened for antibody production by direct ELISA using recombinant EGFL7 protein. The subcultured positive group was twice to achieve individual plant properties. For large scale production of purified antibodies, the fusion tumor cells were injected intraperitoneally into BALB/c mice that were pre-sensitized with azepine or cultured in an Integra bioreactor. The ascites fluid or culture supernatant was purified by Protein A affinity chromatography (Pharmacia Fast Protein Liquid Chromatography; Pharmacia, Uppsala, Sweden). Three antibodies designated 4F11, 10G9 and 18F7 in these monoclonal antibodies were selected for further analysis. In January 2006, these individual antibodies were deposited in the ATCC. .

Monoclonal antibodies block HUVEC cell adhesion and migration . EGFL7 coated on culture plates has been shown to promote adhesion of human umbilical vein endothelial cells (HUVEC), but the adhesion is significantly weaker than other cell adhesion molecules such as fibronectin and collagen. Protein (Parker et al, Nature 428: 754-58 (2004)). Therefore, experiments were conducted to determine whether Mab 4F11, 10G9, and 18F7 could block cell adhesion to EGFL7 coated plates. Plates were coated with 5 μg/ml fibronectin (Roche) and recombinant human EGFL7 produced in E. coli. After PBS washing, HUVEC (Cambrex) was coated at a density of 5 × 10 ⁵ /cm ² in EGM2 medium (Cambrex), and centrifuged at 140 g for 5 minutes to synchronize cell attachment, and then incubated. To analyze antibody activity, HUVEG in EGM2 medium was pre-incubated with antibodies at a concentration of 0.5, 5 or 50 μg/ml in 50 mM Tris/125 mM NaCl (pH 8.6) prior to coating. Mab 4F11, 10G9, and 18F7 each blocked cell adhesion to a culture plate coated with human or mouse EGFL7 protein in a concentration-dependent manner and none of the Mabs blocked cell adhesion to the fibronectin-coated plate. Blocking was confirmed to be specific for EGFL7.

It was also tested whether these antibodies could block HUVEC migration on EGFL7 coated plates. Plates were coated with one of 5 μg/ml of protein: BSA (Sgima), collagen (Upstate), fibronectin (Roche), and recombinant human EGFL7 (produced in Genentech in E. coli). After washing with PBS, HUVEC (Cambrex) was coated at a density of 5 x 10 ⁵ /cm ² in EGM2 medium (Cambrex). Cells were allowed to attach for 2 hours and a single layer of pipette was used for scratches. The wells were washed twice with EGM2 and fresh medium containing 50 μg/ml of control Mab or 4F11, 10G9, 18F7 was added separately. A photo of the hole was taken at intervals of 24 hours to monitor the scratch closure. Mab 4F11, 10G9 and 18F7 each blocked cell migration on EGFL7 protein coated plates, but there was no blockage on other protein coated plates. Control Mab has no blocking activity on any protein. These results indicate that all three anti-EGFL7 mabs specifically block the migration of MUCEC on EGFL7.

Interestingly, it was observed that Mab 4F11 blocked the visual modulator. In particular, when antibody stocks were prepared in 50 mM Tris/125 mM NaCl, the antibodies were very effective at blocking HUVEC cell adhesion, but showed minimal efficacy when prepared in PBS. This difference was not observed for the other two Mabs.

Sequence determination of Mab 4F11 and 10G9 using RNeasy A small kit (Qiagen, Germany) extracted total RNA from fusion tumor cells producing mouse anti-human EGFL7 monoclonal antibodies 4F11 and 10G9. The variable light chain (VL) and variable heavy (VH) domains of 4F11 and 10g9 were amplified by RT-PCR using the following degenerate primers: for 4F11: light chain (LC) forward: 5'-GTCAGATATCGTKCTSACMCARTCTCCWGC -3' (SEQ ID NO: 21) heavy chain (HC) forward: 5'-GATCGACGTACGCTCAGATHCARYTGGTGCARTCTGGGATCGACGTACGCTCAGATHCARYTGGTGCARTCTGG-3' (SEQ ID NO: 22) for 10G9: light chain (LC) forward: 5'-GATCGATATCGTGATGACBCARACTCCACT- 3' (SEQ ID NO: 23) heavy chain (HC) forward: 5'-GATCGACGTACGCTGAGGTYCAGCTSCAGCAGTCTGG-3' (SEQ ID NO: 24) for 4F11 and 10G9: light chain reversal: 5'-TTTDAKYTCCAGCTTGGTACC-3' (SEQ ID NO: 25) Heavy chain reverse: 5'-ACAGTGGGCCCTTGGTGGAGGCTGMRGAGACDGTGASHRDRGT-3' (SEQ ID NO: 26).

The forward primer is specific for the VL and VH region N-terminal amino acid sequences of the two antibodies. LC and HC reverse primers were designed to bind to regions in the constant light chain domain (CL) and constant heavy chain domain (CH1), which are highly conserved for both anti-systems and highly conserved across species. The amplified VL was cloned into a pRK mammalian cell expression vector (Shields et al, J. Biol. Chem. 276:659-04 (2000)). The amplified VH is inserted into a pRK mammalian cell expression vector. The polynucleotide sequence of the insert is determined using conventional sequencing methods. The sequences of the 4F11 light and heavy chains (SEQ ID NOS: 1 and 2, respectively) and the 10G9 light and heavy chains (SEQ ID NOS: 3 and 4, respectively) are shown in Figures 1 to 4.

Isotyping and binding affinities Mab 4F11, 10G9 and 18F7 were determined to be homotypic IgG2b using standard methods.

At room temperature, by using Pharmacia BIAcore Surface plasma resonance was performed at 3000 (BIAcore AB, Uppsala, Sweden) to determine the binding affinity of Mab to human and mouse EGFL7 (see, for example, Morton et al, Meth. Enzymol. 295:268-94 (1998)). The anti-EGFL7 antibody was immobilized on a sensor wafer (CM5) via a primary amine group. Activation of carboxymethylation by injecting a mixture of 20 μl of 0.025 MN-hydroxysuccinimide with 0.1 MN-ethyl-N'(dimethylaminopropyl)carbodiimide at 5 μl/min The sensor wafer surface substrate. A solution of 5-10 μl of 10 μg/ml recombinant human or mouse EGFL7 protein in 10 mM sodium acetate (pH 4.5) was injected at 5 μl/min. After coupling, the unoccupied sites on the wafer were blocked by injection of 20 μl of 1 M ethanolamine (pH 8.5). The running buffer was PBS containing 0.05% polysorbate 20. For kinetic measurements, poly-His-labeled EGFL7, which was double-diluted in running buffer, was injected through the flow cell at a flow rate of 30 μl/min for 3 minutes and the combined polyhis The labeled EGFL7 was dissociated for 20 minutes. The binding surface was regenerated by injection of 20 μl of 10 mM glycine hydrochloride (pH 1.5). A flow cell that is activated but does not have a fixed antibody is used as a reference channel. There is no significant non-specific binding of the poly-His-labeled EGFL7 to the No. 1 flow cell. To calculate apparent binding affinities, the 1:1 binding model was used to analyze the data using global fit. At the same time, the association and dissociation rate constants (BIAevaluation software) were fitted. The results of these experiments using a stock solution of Mab in 50 mM Tris/125 mM NaCl are shown in Table 2.

Measurement of EGFL7 epitope recognized by Mab The epitope recognized by the monoclonal antibody was determined, and the EGFL7 region to which each Mab binds was first mapped. 293 cells were transfected with an expression vector containing the full-length EGFL7 or truncated form of the protein as shown in Figure 5. Western blots of cell lysates of transfected cells were then probed with Mab 4F11, 10G9 and 18F7. Each Mab was observed to bind to the EMI portion of EGFL7.

To narrow the range of specific epitopes recognized by each Mab, overlapping polypeptides spanning a portion of the EMI domain were synthesized and tested for their ability to compete with full-length EGFL7 for binding to Mab. For example, the sequence of the polypeptide for Mab 4F11 is as follows: p1 RSPGLAPARPRYA (SEQ ID NO: 27) p2 RPRYACCPGWKRT (SEQ ID NO: 28) p3 GWKRTSGLPGACG (SEQ ID NO: 29)

Mab 4F11 was mixed with EGFL7 protein and 10 times molar excess of polypeptides p1, p2 and p3, and the resulting complex was immunoprecipitated and observed by SDS-PAGE. For Mab 4F11, only p2 was observed to compete with full-length EGFL7 for binding to Mab 4F11. The results indicate that Mab 4F11 recognizes the EGFL7 epitope comprising the sequence CCP.

A similar experiment was performed on the other two Mabs using a polypeptide having the sequence: p4 LTTCDGHRACSTY (SEQ ID NO: 30) p5 RACSTYRTIYRTA (SEQ ID NO: 31) p6 RTAYRRSPGVTPA (SEQ ID NO: 32)

Both Mab 10G9 and 18F7 were determined to recognize the epitope comprising the sequence RTIY (SEQ ID NO 33).

Example 2 Anti-EGFL7 Mab inhibits tumor growth in vivo

In this example, the ability of anti-EGFL7 Mab to inhibit tumor growth in several models was tested in vivo. Mab in PBS was first tested in the Colo205 model (human colorectal cancer) and the A673 model (human rhabdomyosarcoma model). The role of Mab in PBS as a single agent in these models was not observed.

The combination of Mab and/or Mab alone with anti-EGFL7 antibody B20.4.1 (described in WO 2005/012359) was then tested. Antibodies were tested in three models: the Her2 human breast cancer model ("Fo5 model"), the human lung cancer (NSCLC) model ("H1299"), and another human breast cancer model ("MDA-MB231"). Such tumor models have been well established and described, for example, in Lee et al, Clin Cancer Res. 11(16): 6065-74 (2005); Cameron et al, Cancer Cell Int. 5:23 (2005); Finkle et al. Human, Clinical Cancer Res. 10:2499-251 (2004). Each animal was treated with anti-ragweed Mab (control), B20.4 alone, 18F7 alone, or B20.4 with 4F11, 10G9 or 18F7. Briefly, for the H1299 model, 1×10 ⁷ H1299 tumor cells were injected subcutaneously into the flanks of HRLN female nu/nu mice; and for the MDA-MB231 model, 5×10 ⁶ MDA-MB231 tumors were used. The cells were injected subcutaneously into the flanks of HRLN female nu/nu mice. For each model, antibody treatment was initiated when the mean tumor size reached 100 mm ³ (corresponding to day 0 in Figure 6-9). Anti-ragweed control Mab and B20.4 were administered at 10 mg/kg once a week, and 4F11, 10G9 and 18F7 were administered at 10 mg/kg twice a week (in Figures 6, 8 and 9 under x-axis) The arrow indicates).

No effect was observed in the Fo5 model. Significant tumor inhibition was observed in the other two models. As shown in Figures 6-7, in the H1299 model, Mab 4F11 in combination with B20.4.1 was significantly more effective than the control or B20.4.1 alone. As shown in Figure 8, in the MDA-MB231 model, Mab 4F11 or Mab 10G9 in combination with B20.4.1 was more effective than the control or B20.4.1 alone. As shown in Figure 9, in the MDA-MB231 model, M18F7 alone was more effective than the control, and Mab 18F7 in combination with B20.4.1 was significantly more effective than Mab 18F7 alone or B20.184.1 alone.

Interestingly, it was also observed that Mab 4F11 treatment in the H1299 model prevented complete vascular recovery after stopping anti-VEGF therapy (B20.4.1). When treatment was stopped, a significant delay in tumor revascularization was observed in tumors treated with B20.4.1 alone and tumor vascular patterns in tumors treated with B20.4.1 and 4F11. These results clearly indicate that anti-EGFL7 therapy can provide additional or even synergistic effects when combined with anti-VEGF therapies.

The anti-tumor activity of anti-EGFL7 antibodies is also tested using other tumor models available in the art. These models include (but are not limited to): LS174T (colon), BXPC3 (prostate), HCT116 (colon), MV-522 (NSCLC), SKMES (NSCLC), Colon26 (colon), MDA-MB231 (breast), MCF7 (Breast), H1299 (NSCLC), SW620 (colon), LL (lung), Fo5 (breast), 4T1 (breast), HT29 (colon), SW480 (colon), 786-0 (rectal).

The following fusion tumors have been deposited with the American Type Culture Collection , PO Box 1549, Manassas, VA, 20108, USA (ATCC ):

These deposits are carried out in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty). . This ensures that the live deposit can be maintained for 30 years from the date of deposit. These cell lines may be used by the ATCC in accordance with the terms of the Budapest Treaty and are subject to an agreement between Genentech, Inc and the ATCC, which ensures that when a US patent is issued or any US or foreign patent application is made available to the public (regardless of the circumstances) When present, the public uses these cell lines for permanent and unrestricted use and ensures that the US Commissioner of Patents and Trademarks are authorized by the US Commissioner of Patents and Trademarks in accordance with 35 USC § 122 and its committee rules (including The use of these cell lines by persons identified in 37 CFR § 1.14 and for the special reference to 886 OG 638).

The assignee of the present application has agreed that if the deposited cell line is damaged or destroyed when cultured under appropriate conditions, it should be promptly issued to replace it with a sample of the same cell line. The availability of the deposited cell strain should not be construed as a breach of the license granted by any government authority under its patent law to practice the invention.

While the invention has been described in detail herein by reference to the claims

Figure 1 shows the amino acid sequence of the Mab 4F11 light chain variable domain (SEQ ID NO: 1) and HuKI (SEQ ID NO: 17).

Figure 2 shows the amino acid sequence of the Mab 4F11 heavy chain variable domain (SEQ ID NO: 2) and HuIII (SEQ ID NO: 18).

Figure 3 shows the amino acid sequence of the Mab 10G9 light chain variable domain (SEQ ID NO: 3) and HuKI (SEQ ID NO: 17).

Figure 4 shows the amino acid sequence of the Mab 10G9 heavy chain variable domain (SEQ ID NO: 4) and HuIII (SEQ ID NO: 18).

Figure 5 depicts the domain of full length EGFL7 and its truncated form for localization of antibody binding sites. Mab epitope determination by Western blotting and immunoprecipitation.

Figure 6 shows in vivo tumor volumes of a gene transfer mouse model (NSCLC; H1299) transfected with human lung cancer during treatment with an anti-VEGF antibody and an anti-EGFL7 antibody of the invention. Anti-EGFL7 mab pure 4F11 enhances the activity of B20.4.1 (anti-VEGF Mab).

Figure 7 shows the survival rate of an in vivo model of transgenic mice transfected with human lung cancer (NSCLC; H1299) during treatment with an anti-VEGF antibody and an anti-EGFL7 antibody of the invention. The combination of anti-VEGF and anti-EGFL7 produces a survival benefit. H1299 xenograft card-Mal curve.

Figure 8 shows in vivo tumor volumes of a gene transfer mouse model (MDA-MB231) transfected with human breast cancer during treatment with an anti-VEGF antibody and an anti-EGFL7 antibody of the invention. Anti-EGFL7 Mab 4F11 and 10G9 enhance the activity of B20.4.1.

Figure 9 shows in vivo tumor volumes of a gene transfer mouse model (MDA-MB231) transfected with human breast cancer during treatment with an anti-VEGF antibody and the anti-EGFL7 antibody Mab 18F7 of the present invention. Anti-EGFL7 Mab 18F7 enhances the activity of B20.4.1.

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(no component symbol description)

Claims (53)

An anti-epidermal growth factor like 7 (anti-EGFL7) antibody produced by a fusion tumor group selected from the following BCRC registry number: BCRC 960306, BCRC 960307 And BCRC 960308. An anti-EGFL7 antibody comprising the following six complementarity determining regions (CDRs): (a) 4F11 CDR-L1 sequence KASQSVDYDGDSYMS (SEQ ID NO: 5); (b) 4F11 CDR-L2 sequence GASNLES (SEQ ID NO: 6) (c) 4F11 CDR-L3 sequence QQNNEDPYT (SEQ ID NO: 7); (d) 4F11 CDR-H1 sequence TYGMS (SEQ ID NO: 8); (e) 4F11 CDR-H2 sequence WINTHSGVPTYADDFKG (SEQ ID NO: 9 And (f) 4F11 CDR-H3 sequence LGSSA (SEQ ID NO: 10). The anti-EGFL7 antibody of claim 2, wherein the light chain of the antibody comprises the following sequence: DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMSWYQQKPGQPPKLLIYGASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQNNEDPYTFGGGTKVEIKR (SEQ ID NO: 1). An anti-EGFL7 antibody according to claim 2, wherein the heavy chain of the antibody comprises the following sequence: QIQLVQSGPELKKPGETVKISCKASGHTFTTYGMSWVKQAPGKGLKWMGWINTHSGVPTYADDFKGRFAFSLETSASTAHLQINNLKNEDTATYFCARLGSSAVDYWGQG TTVTVSS (SEQ ID NO: 2). An anti-EGFL7 antibody comprising the following six complementarity determining regions (CDRs): (a) 10G9 CDR-L1 sequence RSSQSLVHTNGITYLH (SEQ ID NO: 11); (b) 10G9 CDR-L2 sequence KVSNRFS (SEQ ID NO: 12) (c) 10G9 CDR-L3 sequence SQSTHVPLT (SEQ ID NO: 13); (d) 10G9 CDR-H1 sequence DYYMNSDYYMN (SEQ ID NO: 14); (e) 10G9 CDR-H2 sequence DINPKNGGTTYNQKFKG (SEQ ID NO: 15) And (f) 10G9 CDR-H3 sequence EADYDPIYYAMDY (SEQ ID NO: 34). The anti-EGFL7 antibody of claim 5, wherein the light chain of the antibody comprises the sequence: DIVMTQTPLSLPVSLGDQASISCRSSQSLVHTNGITYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPLTFGAGTKVEIKR (SEQ ID NO: 3). The anti-EGFL7 antibody of claim 5, wherein the heavy chain of the antibody comprises the sequence: EVQLQQSGPELVKPGASVKISCKASGYTFSDYYMNSDYYMNWVKQSHGKSLEWIGDINPKNGGTTYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCAREADYDPIYYAMDYWGQGTTLTVSA (SEQ ID NO: 4). An anti-EGFL7 antibody comprising six complementarity determining regions (CDRs) of an 18F7.1.8 antibody produced by a fusion of BCRC accession number BCRC 960308. An anti-EGFL7 antibody that specifically binds to an EGFL7 polypeptide, wherein the antibody binds to an epitope comprising an amino acid sequence selected from the group consisting of CCP, RTIY (SEQ ID NO 33). An isolated antibody which binds to the same epitope on human EGFL7 as the antibody of any one of claims 1 to 9. An isolated antibody which competes with EGFL7 for binding to an antibody according to any one of claims 1 to 9. The antibody of any one of claims 1 to 11, wherein the antibody is a monoclonal antibody. The antibody of any one of claims 1 to 11, wherein the anti-system is selected from the group consisting of a chimeric antibody, a humanized antibody, an affinity matured antibody, a human antibody, and a bispecific antibody. The antibody of any one of claims 1 to 11, wherein the antibody is an antibody fragment. A pharmaceutical composition comprising the anti-EGFL7 antibody of any one of claims 1 to 14. The pharmaceutical composition of claim 15 which additionally comprises an anti-angiogenic agent. The pharmaceutical composition of claim 16, wherein the anti-angiogenic agent is selected from the group consisting of bevacizumab and ranibizumab. A polynucleotide encoding the antibody of any one of claims 1 to 14. A vector comprising the polynucleotide of claim 18. The vector of claim 19, wherein the vector is a performance vector. A host cell comprising the vector of claim 19 or 20. The host cell of claim 21, wherein the host cell is a prokaryotic cell. The host cell of claim 21, wherein the host cell is a eukaryotic cell. The host cell of claim 21, wherein the host cell is a mammalian cell. A method of making an anti-EGFL7 antibody, the method comprising (a) rendering the vector of claim 20 in a suitable host cell; and (b) recovering the antibody. The method of claim 25, wherein the host cell is a prokaryotic cell. The method of claim 25, wherein the host cell is a eukaryotic cell. Use of an anti-EGFL7 antibody according to any one of claims 1 to 14 or a pharmaceutical composition according to claim 15 for the manufacture of a subject which reduces or inhibits a pathological condition associated with angiogenesis An angiogenic drug. The use of claim 28, wherein the pathological condition is a tumor. The use of claim 29, wherein the tumor is a carcinoma. The use of claim 28, wherein the pathological condition is associated with the eye. The use of claim 31, wherein the pathological condition is intraocular neovascular disease. The use of any one of claims 28 to 30, which additionally comprises administering an anti-angiogenic agent to the subject. The use of claim 33, wherein the anti-angiogenic agent is vascular endothelium An antagonist of long factor (VEGF). The use of claim 34, wherein the antagonist is an anti-VEGF antibody. The use of claim 35, wherein the anti-VEGF antibody is bevacizumab. The use of claim 31 or 32, additionally comprising administering an anti-angiogenic agent to the subject. The use of claim 37, wherein the anti-angiogenic agent is an antagonist of vascular endothelial growth factor (VEGF). The use of claim 38, wherein the antagonist is an anti-VEGF antibody. The use of claim 39, wherein the anti-VEGF antibody is Raney strain monoclonal antibody. The use of claim 33, wherein the anti-angiogenic agent is administered before or after administration of the anti-EGFL7 antibody. The use of claim 33, wherein the anti-angiogenic agent is administered simultaneously with the anti-EGFL7 antibody. Use of an antibody according to any one of claims 1 to 14 or a pharmaceutical composition according to claim 15 or 16 for the manufacture of an anti-angiogenic agent in a subject having a pathological condition associated with angiogenesis A drug that produces the efficacy of a drug. The use of claim 43, wherein the pathological condition is a tumor. The use of claim 44, wherein the tumor is a carcinoma. The use of any one of claims 43 to 45, wherein the anti-angiogenic agent is bevacizumab. The use of any one of claims 43 to 45, which additionally comprises administering a chemotherapeutic agent. The use of claim 43, wherein the pathological condition is associated with the eye. The use of claim 48, wherein the pathological condition is intraocular neovascular disease. The use of claim 48 or 49, wherein the anti-angiogenic agent is Raney strain monoclonal antibody. The use of claim 48 or 49 additionally comprises administering a corticosteroid. The use of claim 48 or 49 additionally includes administration of photodynamic therapy. A fusion tumor selected from the group consisting of BCT 960306, BCRC 960307, and BCRC 960308 deposited under the BCRC registry number below.