HK1089189A1 - CHIMERIC AND HUMANIZED ANTIBODIES TO α5β1 INTEGRIN THAT MODULATE ANGIOGENESIS - Google Patents

Description

Chimeric and humanized antibodies to alpha 5 beta 1 integrin that modulate angiogenesis

Technical Field

The present invention provides chimeric and humanized antibodies that specifically recognize α 5 β 1 integrin and methods of using the antibodies to reduce or inhibit tissue angiogenesis. The invention also provides methods of determining therapeutically acceptable dosages of the antibodies and pharmaceutical compositions containing the antibodies.

Background

Angiogenesis is a process of neovascularization. Angiogenesis, also known as neovascularization, occurs during normal embryogenesis and development, as well as during wound healing and placental development in fully developed bodies. In addition, angiogenesis occurs in a variety of pathological conditions, including ocular diseases such as diabetic retinopathy and macular degeneration caused by neovascularization; diseases associated with tissue inflammation such as rheumatoid arthritis and inflammatory bowel disease; and cancer, in which angiogenesis within a tumor in its growth state provides oxygen and nutrients to tumor cells, and tumor cells can also metastasize to other sites throughout the body via new blood vessels. Since millions of people worldwide are affected by these diseases, considerable effort has been devoted to studying the mechanisms involved in angiogenesis in order to find ways to detect and inhibit this unwanted angiogenesis.

Angiogenesis may be induced by stimulation with one or more known growth factors, and may occur including other unknown factors. Endothelial cells are a layer of cells that overlies the endothelium of mature blood vessels and are normally non-proliferating. However, if given appropriate stimulation, endothelial cells activate and begin to proliferate, migrating into avascular tissue to form new blood vessels. In some cases, precursor cells can be activated to differentiate into endothelial cells, which form new blood vessels.

The vessels are encapsulated by the extracellular matrix. In addition to stimulation by growth factors, angiogenesis is dependent on the interaction between endothelial cells and the extracellular matrix. Growth factor activation of endothelial cells, migration of endothelial cells into the extracellular matrix, and interaction of endothelial cells with the extracellular matrix are dependent on the cell surface receptor expressed by the endothelial cells. These cell surface receptors, including growth factor receptors and integrins, can interact specifically with specific molecules.

Under pathological conditions, such as age-related macular degeneration and diabetic retinopathy, the ability of the retina to acquire oxygen is reduced, thereby resulting in hypoxia and stimulation of the secretion of angiogenic growth factors, such as Vascular Endothelial Growth Factor (VEGF). This secretion can induce abnormal proliferation and migration of endothelial cells into ocular tissues, leading to vascularization of ocular tissues, induction of corneal scarring, retinal detachment, and accumulation of choroidal fluid, all of which can irreversibly affect vision, leading to blindness.

Angiogenesis is also associated with the progression and exacerbation of inflammatory diseases including psoriasis, rheumatoid arthritis, osteoarthritis, inflammatory bowel diseases such as ulcerative colitis and segmental colitis. For example, in inflammatory joint diseases, lymphocyte influx into the area surrounding the joint stimulates the formation of new blood vessels in the synovial membrane of the joint, which provide a pathway for leukocyte influx that promotes cartilage and bone damage in the joint. The same effect is produced in the intestine by neovascularization that occurs in inflammatory bowel disease.

Capillary growth into the atherosclerotic plaque of the coronary arteries is another pathological condition associated with growth factor-induced angiogenesis. Excessive blood flow into the newly vascularized plaque can lead to rupture and bleeding of the blood-containing plaque, releasing blood clots, and ultimately leading to coronary thrombosis.

Since angiogenesis is involved in the development of various diseases such as cancer, ocular diseases and inflammatory diseases, attempts have been made to find a specific method for inhibiting angiogenesis as a therapeutic method for these diseases. For cancer patients, this treatment has substantial advantages over existing treatments such as chemotherapy, which not only kills or damages tumor target cells, but also kills normally proliferating cells in the patient, such as blood cells, epithelial cells, and transitional epithelial cells in the intestinal lumen. This non-specific killing by the chemotherapeutic agent can produce side effects that are at least uncomfortable and often result in unacceptable morbidity or mortality. Indeed, the toxic side effects associated with cancer therapy often limit patients to receive such therapy.

Summary of The Invention

The present invention provides therapeutic chimeric and humanized antibodies against α 5 β 1 integrin; methods of purifying these antibodies and methods of using these antibodies to treat conditions comprising undesirable tissue angiogenesis are provided.

In one embodiment, the invention includes a nucleic acid encoding a chimeric or humanized anti- α 5 β 1 integrin antibody polypeptide having 65%, preferably greater than 75%, more preferably greater than 85%, 90%, 95%, 97%, or 99% sequence identity to one or more amino acid sequences selected from the group consisting of seq id no: SEQ ID NOS: 1-12, 16, 18, 20, 22, 25-26, 28, 31-32. More preferably, the nucleic acid encodes a chimeric or humanized anti- α 5 β 1 integrin antibody polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 2-6, 8-12, 16, 18, 20, 22, 25-26, 28, 31-32. The peptides encoded by these nucleic acids are single chain antibodies or fabs, which contain several peptides bound by disulfide bonds in addition to the Fab or antibody.

The invention also includes polypeptides having 65%, preferably more than 75%, more preferably more than 85%, 90%, 95%, 97% or 99% sequence identity to one or more amino acid sequences selected from the group consisting of: SEQ ID NOS: 1-12, 16, 18, 20, 22, 25-26, 28, 31-32. More preferably, the nucleic acid encodes a polypeptide comprising one or more amino acid sequences selected from the group consisting of: SEQ ID NOS: 2-6, 8-12, 16, 18, 20, 22, 25-26, 28, 31-32. These peptides include chimeric, human and humanized antibodies and Fab fragments.

In another embodiment, the invention includes a chimeric anti- α 5 β 1 integrin antibody. These antibodies comprise a first polypeptide from a first source having 65%, preferably more than 75%, more preferably more than 85%, 90%, 95%, 97% or 99% sequence identity to an amino acid sequence selected from the group consisting of: SEQ ID NOS: 1. 7, 16, 18, 20, 22, a second polypeptide of a second origin having 65%, preferably more than 75%, more preferably more than 85%, 90%, 95%, 97% or 99% sequence identity to the constant region of the antibody of the second origin, wherein the first and second polypeptides form a protein complex immunoreactive with α 5 β 1 integrin. In a preferred embodiment, the second source of constant regions is human IgG. In another preferred embodiment, the constant region is human IgG 4.

In other preferred embodiments, the chimeric antibody comprises a first polypeptide of a first origin and a second polypeptide of a second origin, wherein the first polypeptide comprises one or more amino acid sequences selected from the group consisting of: SEQ ID NOS: 1. 7, 16, 18, 20, 22, a second polypeptide comprising a constant region sequence of an antibody of a second origin, the first polypeptide and the second polypeptide forming a protein complex immunoreactive with α 5 β 1 integrin.

In a most preferred embodiment, the invention includes a chimeric anti- α 5 β 1 integrin antibody comprising a heavy chain amino acid sequence of SEQ ID NO: 25 and the light chain amino acid sequence SEQ ID NO: 26.

in another embodiment, the invention includes a polypeptide encoding the chimeric anti- α 5 β 1 integrin antibody heavy chain variable region sequence of SEQ ID NO: 19, and a nucleic acid encoding a chimeric anti- α 5 β 1 integrin antibody heavy chain variable region sequence of SEQ ID NO: 21.

In another embodiment, the invention includes a Fab fragment of a chimeric anti- α 5 β 1 integrin antibody comprising the heavy chain amino acid sequence of SEQ ID NO: 25 and the light chain amino acid sequence SEQ ID NO: 26. in a most preferred embodiment, the Fab fragment comprises the heavy chain amino acid sequence SEQ ID NO: 28 and the light chain amino acid sequence SEQ ID NO: 26.

in another preferred embodiment, the invention includes a humanized antibody derived from a chimeric anti- α 5 β 1 integrin antibody comprising the heavy chain amino acid sequence of SEQ ID NO: 25 and the light chain amino acid sequence SEQ ID NO: 26. in a most preferred embodiment, the humanized antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 28 and the light chain amino acid sequence SEQ ID NO: 26.

in other embodiments, the invention includes an expression vector encoding one or more nucleic acids selected from the group consisting of: SEQ ID NOS: 15. 17, 19, 21, 23, 24, 27, 29, 30. In a preferred embodiment, the expression vector comprises SEQ ID NOS: 19 and 21.

In another embodiment, the invention includes a cell transformed with an expression vector comprising one or more nucleic acids selected from the group consisting of: SEQ ID NOS: 15. 17, 19, 21, 23, 24, 27, 29, 30. In a preferred embodiment, the expression vector comprises SEQ ID NOS: 19 and 21.

In another embodiment, the invention encompasses pharmaceutical compositions comprising chimeric or humanized anti- α 5 β 1 integrin antibodies described herein. In certain embodiments, these compositions may contain drugs that promote uptake or localization of therapeutic ingredients, reduce inflammation, or provide local symptom relief.

In one aspect of this embodiment, the pharmaceutical composition comprises a topical cream that can be applied directly to the damaged tissue. In another aspect, the pharmaceutical composition is an eye drop that can be used directly to injure the eye. In yet another aspect, the pharmaceutical composition is an injectable formulation that can be administered systemically to treat a tissue injury in one or both eyes, or to inhibit angiogenesis in a tumor tissue.

In another embodiment, the invention includes a method of controlling vascularization of damaged tissue. These methods comprise the application of single or multiple doses of chimeric or humanized anti- α 5 β 1 integrin antibodies to damaged tissue, wherein the damage to the tissue may be the result of physical or chemical injury, or disease.

In another embodiment, the invention includes a method of administering a therapeutic antibody, the method comprising: there is provided a medicament comprising a therapeutic antibody, wherein the therapeutic antibody comprises a heavy chain variable region comprising a sequence selected from the group consisting of seq id no: SEQ ID NOS: 2-6, 16, 20, the light chain variable region is independently selected from the group consisting of: NOS: 8-12, 18, 22; and administering the therapeutic antibody into the damaged tissue. In this embodiment of the invention, the damaged tissue responds to the injury by increasing blood supply through neovascularization, which is inhibited by the therapeutic antibody. In one aspect, the method comprises injecting a therapeutic antibody into a diseased or injured eye of a patient having both eyes affected by intravitreal injection; or injected into one eye by intravitreal injection to treat both eyes.

In other embodiments, the invention encompasses methods of purifying anti- α 5 β 1 integrin antibodies. The method comprises adsorbing the antibody to an antibody affinity matrix bound to a substrate and eluting the antibody from the antibody affinity matrix bound to the substrate with an eluent having a pH of about 3.0 to 5.5. The method further comprises recovering the purified antibody. Antibodies that can be purified by this method include those that comprise at least two CDR regions independently selected from the group consisting of amino acid sequences SEQ ID NOS: 1-12, 16, 18, 20 and 22. Preferably, one of the selected CDRs is from V_LChain, the other from V_HAnd (3) a chain.

In certain aspects of this purification method, the pH of the eluate is about 3.3 to about 5.5. In other aspects, the pH of the eluent is about 3.5-5.5. In still other aspects, the pH of the eluate is about 3.5-4.2. In a further aspect, the pH of the eluent is about 4.2 to about 5.5.

Another embodiment of the invention encompasses methods for evaluating the physiological effects (e.g., anti-angiogenic properties) modulated by humanized anti- α 5 β 1 integrin antibodies, including antibodies and Fab fragments. The method comprises providing a biopsy sample capable of producing a regenerated blood vessel; creating lesions in living tissue sufficient to cause choroidal neovascularization; administering single or multiple doses of or a humanized anti- α 5 β 1 integrin antibody to living tissue; and monitoring the condition of revascularization of the living tissue after administration. In a preferred embodiment, the evaluation method uses eye tissue as the living tissue. In certain embodiments, the macula of the eye is used. The ocular tissue used in the evaluation method may also be that of a live primate (e.g., cynomologous monkey).

In another embodiment, the method of evaluation comprises intravitreal injection of a chimeric or humanized anti- α 5 β 1 integrin antibody. In one aspect of the invention, both eyes of the patient are injured and injection of the antibody into one eye brings the antibody into contact with the injured tissue in both eyes.

Another aspect of the method of assessing physiological effects includes contacting living tissue with a laser to create a lesion. The laser intensity is about 300-700 milliwatts and the exposure time is no more than 0.1 second, preferably less than 0.05 second, and most preferably less than about 0.01 second. The lesion diameter should be less than 200 μm, preferably less than 100 μm, more preferably between about 50 and 100 μm, and most preferably between about 75 and 25 μm in diameter.

Certain aspects of the method include a monitoring step comprising periodically taking photographs while treating the injury with single or multiple doses of the humanized anti- α 5 β 1 integrin antibody. In another aspect, the monitoring step further comprises indirectly examining the posterior chamber of the eye with an ophthalmoscope and examining the anterior segment of the eye with a biomicroscope. In another aspect, the method includes a monitoring step comprising intravenous injection of fluorescein dye followed by examination of the biopsy by fluorescein angiography.

A method of assessing the physiological effects modulated by a chimeric or humanized anti- α 5 β 1 integrin antibody, wherein the chimeric or humanized anti- α 5 β 1 integrin antibody comprises a heavy chain variable region comprising a sequence having 65%, preferably more than 75%, more preferably more than 85%, 90%, 95%, 97% or 99% sequence identity to an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NOS: 1-6, 16, 20, the light chain variable region is selected individually and comprises a sequence having 65%, preferably more than 75%, more preferably more than 85%, 90%, 95%, 97% or 99% sequence identity to an amino acid sequence selected from the group consisting of: NOS: 8-12, 18 and 22. Most preferably, the humanized anti- α 5 β 1 integrin antibody comprises a heavy chain variable region comprising a sequence selected from the group consisting of seq id no: SEQ ID NOS: 1-6, 16, 20, the light chain variable region is independently selected from the group consisting of: NOS: 8-12, 18 and 22.

A method of evaluating the physiological effects modulated by a chimeric or humanized anti- α 5 β 1 integrin antibody, wherein the chimeric or humanized anti- α 5 β 1 integrin antibody comprises a heavy chain variable region comprising a sequence selected from the group consisting of seq id nos: SEQ ID NOS: 2-6, 16, 20, the light chain variable region is independently selected from the group consisting of: NOS: 8-12, 18 and 22.

Brief Description of Drawings

FIG. 1 depicts the heavy chain variable region (V) of the murine anti-. alpha.5-. beta.1 integrin antibody (IIA1) and 5 murine humanized antibodies (1.0-5.0)_H) And light chain variable region (V)_L) The amino acid sequence of (SEQ ID NOS: 1-12).

FIG. 2 depicts the amino acid sequences (SEQ ID NOS: 1-12) in which the highlighted portions represent the amino acid residues of 5 humanized antibody sequences that were substituted relative to their murine antibody (IIA 1).

Fig. 3 illustrates: (A) IIA1V_HThe nucleic acid sequence (SEQ ID NO: 13) and the amino acid sequence (SEQ ID NO: 46) of (A); (B) IIA1V_LThe nucleic acid sequence (SEQ ID NO: 14) and the amino acid sequence (SEQ ID NO: 47).

Fig. 4 illustrates: (A) antibody 200-4V_HThe nucleic acid sequence (SEQ ID NO: 15) and the amino acid sequence (SEQ ID NO: 16); (B) antibody 200-4V_LThe nucleic acid sequence (SEQ ID NO: 17) and the amino acid sequence (SEQ ID NO: 18) of (A).

Fig. 5 illustrates: (A) M200V_HThe nucleic acid sequence (SEQ ID NO: 19) and the amino acid sequence (SEQ ID NO: 20) of (SEQ ID NO: 19); (B) M200V_LThe nucleic acid sequence (SEQ ID NO: 21) and the amino acid sequence (SEQ ID NO: 22) of (A).

FIG. 6 depicts the structure of plasmid p200-M-H expressing the M200 heavy chain.

FIG. 7 depicts the structure of plasmid p200-M-L expressing the M200 light chain.

FIG. 8 depicts a single plasmid p200-M expressing the heavy and light chains of M200.

FIG. 9 depicts the complete DNA sequences of the M200 heavy and light chains (SEQ ID NOS: 23-24).

FIG. 10 depicts the complete amino acid sequence of the M200 heavy and light chains (SEQ ID NOS: 25-26).

FIG. 11 depicts the complete DNA sequence and the complete amino acid sequence of the F200 heavy chain (SEQ ID NOS: 27-28).

FIG. 12 depicts the complete DNA sequences of the huM200 heavy and light chains (SEQ ID NOS: 29-30).

FIG. 13 depicts the complete amino acid sequence of the huM200 heavy and light chains (SEQ ID NOS: 31-32).

Figure 14 depicts the inhibitory effect of M200 as a potential inhibitor on endothelial cell growth, including the antiproliferative properties of anti-vegfa mab, HuMV 833.

FIG. 15 depicts the results showing that M200 inhibits VEGF-induced cell proliferation and that anti-idiotype monoclonal antibodies inhibit M200 activity.

The results depicted in fig. 16 show that: (A) m200-induced cell death was observed by annexin staining; (B) annexin stained cells were quantitatively detected by flow cytometry.

FIG. 17 depicts the results showing that M200 induces an increase in the number of deaths of HUVEC cells in a proliferative state, while having no effect on the number of deaths of aged HUVEC cells.

FIG. l8 depicts the results of an in vitro microtubule formation assay analyzing the inhibition of angiogenesis by F200.

Figure 19 depicts fluorescein contrast images of laser-induced damage in primate eyes on day 20 after treatment with (a) control (rituxan) and (B) M200.

FIG. 20 depicts fluorescein contrast images of laser-induced damage in the left and right eyes of primates at day 13 after treatment with (A) control (left eye) and (B) M200 (right eye).

FIG. 21 depicts fluorescein contrast images of laser-induced damage in the left and right eyes of primates at day 20 after treatment with (A) control (left eye) and (B) M200 (right eye).

FIG. 22 depicts fluorescein contrast images of laser-induced damage in the left and right eyes of primates at day 27 after treatment with (A) control (left eye) and (B) M200 (right eye).

FIG. 23 depicts fluorescein contrast images of laser-induced damage in the left and right eyes of primates at day 13 after treatment with (A) control (left eye) and (B) F200 (right eye).

FIG. 24 depicts fluorescein contrast images of laser-induced damage in the left and right eyes of primates at day 20 after treatment with (A) control (left eye) and (B) F200 (right eye).

FIG. 25 depicts fluorescein contrast images of laser-induced damage in the left and right eyes of primates at day 27 after treatment with (A) control (left eye) and (B) F200 (right eye).

FIG. 26 depicts the results of an ELISA competition binding assay comparing two humanized versions of murine antibody IIA1, chimeric antibody M200(200-4EOS) and M200: huM200-G4 and huM200-G2m 3G.

Detailed Description

I. Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide the skilled artisan with a general definition of a number of terms used in the present invention: singleton et al, Dictionary of microbiology and Molecular Biology (Dictionary of microbiology and Molecular Biology) (second edition, 1994); cambridge scientific Dictionary (the Cambridge Dictionary of Science and Technology) (Walker, eds., 1988); the Glossary of Genetics, 5 th edition, R.Rieger et al (eds.), Springer Verlag (1991); and Hale & Marham, Harper Collins Biology dictionary (The Harper Collins dictionary) (1991). The following terms used herein have the meanings described in the above documents unless otherwise specified.

The term "antibody" as used herein includes immunoglobulin molecules that are immunoreactive with a particular antigen, i.e., includes polyclonal antibodies as well as monoclonal antibodies. The term also includes genetically engineered antibodies such as chimeric antibodies (e.g., humanized murine antibodies) and heterologous covalent antibodies (e.g., bispecific antibodies). The term "antibody" also includes antigen-binding regions of antibodies, including fragments that have antigen-binding function (e.g., Fab ', F (ab')₂Fab, Fv and rIgG). See Pierce catalog and handbook, 1994-1995(Pierce Chemical Co., Rockford, IL) and Kuby, J., Immunology, 3 rd edition, W.H.Freeman&Co., NewYork (1998). The term also refers to recombinant single chain Fv fragments (scFv). The term "antibody" also includes bivalent or bispecific molecules, diabodies, triabodies and tetrabodies. Bivalent and bispecific molecules are described in Kostelny et al, (1992) J Immunol 148: 1547, Pack and Pluckthun (1992) Biochemistry 31: 1579, Hollinger et al, 1993, supra, Gruber et al, (1994) J Immunol: 5368, Zhu et al, (1997) Protein Sci 6: 781, Hu et al, (1996) Cancer Res.56: 3055, Adams et al, 1993) cancer Res.53: 4026 and McCartney et al, (1995) Protein Eng.8: 301.

antibodies that immunoreact with a particular antigen can be prepared by recombinant methods, such as screening recombinant antibody phage libraries or similar vector libraries, see Huse et al, Science 246: 1275-1281 (1989); ward et al, Nature 341: 544-546 (1989); and Vaughan et al, Nature Biotech.14: 309, 314(1996), or immunizing an animal with an antigen or DNA encoding an antigen.

Generally, immunoglobulins comprise both a heavy chain and a light chain. Both heavy and light chains have constant and variable regions (regions also referred to as domains). The light and heavy chain variable regions contain 4 "framework" regions separated by 3 hypervariable regions, which are also referred to as "complementarity determining regions" or "CDRs". The extent to which the framework regions and CDRs are encompassed is well defined. The framework sequences of different light or heavy chains are well conserved within the same species. The framework regions of the antibody, i.e., the framework regions of the light and heavy chains, combine to position and align the CDRs in three dimensions.

The CDRs are primarily responsible for binding epitopes of the antigen. The CDRs in one chain are usually divided into CDRs 1, CDRs 2 and CDRs 3, arranged in order from the N-terminus, and the chain is also usually used to define the location of a particular CDR. Thus, V_HCDR3 refers to the CDR located in the variable region of an antibody heavy chain, whereas V_LCDR1 refers to CDR1 from which the variable region of an antibody light chain is derived.

“V_H"refers to the variable region of an antibody immunoglobulin heavy chain, including the heavy chain of an Fv, scFv, or Fab. ' V_L"refers to the variable region of an antibody immunoglobulin light chain, including the light chain of an Fv, scFv, dsFv, or Fab.

The term "single chain Fv" or "scFv" refers to an antibody in which the variable regions of the heavy and light chains of a conventional diabody are combined to form one chain. Generally, a linker peptide is inserted between the two chains to ensure proper folding and formation of the active binding site

"chimeric antibody" refers to (a) an immunoglobulin molecule in which the constant region or a portion thereof is altered, replaced, or exchanged such that the antigen binding site (variable region) is linked to a constant region of a different class, function and/or species, or completely different molecule, such as an enzyme, toxin, hormone, growth factor, drug, etc., to confer new properties to the chimeric antibody; or (b) an immunoglobulin molecule in which the variable region or a portion thereof is altered, replaced, or exchanged with a variable region having a different antigenic specificity.

"humanized antibody" refers to an immunoglobulin molecule that contains very little sequence derived from a non-human immunoglobulin. Humanized antibodies comprise human immunoglobulins (recipient antibody) in which residues from a Complementarity Determining Region (CDR) of the recipient are replaced by residues from a CDR (donor antibody) of a non-human species, such as mouse, rat or rabbit, having the desired specificity, affinity, and function. In some cases, residues on the Fv framework region of the human immunoglobulin are replaced by residues of a corresponding non-human animal immunoglobulin. Humanized antibodies also comprise residues that are neither present on the recipient antibody nor on the donor CDR or framework region sequences. Typically, a humanized antibody comprises nearly all of the sequences of at least one, and typically two, variable regions, wherein all or nearly all of the sequences of the corresponding CDR regions and all or nearly all of the sequences of the Framework Regions (FRs) of a non-human immunoglobulin are those of a human immunoglobulin consensus sequence. The humanized antibody may also comprise at least a portion of the sequence of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin constant region (Jones et al, Nature 321: 522-525 (1986); Riechmann et al, Nature 332: 323-329 (1988); and Presta, curr. Op. Structure. biol. 2: 593-596 (1992)). Humanization can be accomplished using the methods described by Winter and co-workers (Jones et al, Nature 321: 522-525 (1986); Riechmann et al, Nature 332: 323-327 (1988); Verhoeyen et al, Science 239: 1534-1536(1988)), using the CDRs or CDR sequences of the human antibody in place of the corresponding sequences in the rodent. Thus, such humanized antibodies are chimeric antibodies (U.S. Pat. No.4,816,567) in which a portion of the entire variable region of human origin is replaced by the corresponding sequence of a non-human species.

An "epitope" or "antigenic determinant" refers to the site at which an antibody binds to an antigen. Epitopes can be formed from contiguous amino acids or noncontiguous amino acids on a protein that undergoes tertiary folding. Epitopes formed by consecutive amino acids are generally retained when treated with denaturants, whereas epitopes formed by tertiary folding are generally lost when treated with denaturants. Epitopes generally comprise at least 3, more usually 5, or 8-10 amino acids with a unique spatial configuration. Methods for determining spatial configuration of epitopes include X-ray crystallography and two-dimensional nuclear magnetic resonance. See "guidance for Epitope Mapping in molecular biology Methods" (Epitope Mapping Protocols in Methods in molecular biology), volume 66, compiled by Glenn E. Morris (1996).

"pH-sensitive anti- α 5 β 1 integrin antibody" refers to an antibody that specifically recognizes α 5 β 1 integrin, and that precipitates from solution when immunopurification is performed under neutral or alkaline pH conditions using α 5 β 1 integrin as a ligand. The pH-sensitive anti- α 5 β 1 integrin antibody generally comprises any V from that described in FIG. 1, respectively_HOr V_LTwo or more CDR sequences of the sequence.

"angiogenesis" and "neovascularization" refer to the formation of new blood vessels, typically in response to a stimulus, injury, or disease. For purposes of the present invention, the term "injury" and its cognate terms include stimuli, disease or other event that results in a tissue response, including angiogenesis. Angiogenesis may also occur during tumor formation and metastasis, as well as during embryonic development, growth and development in higher animals.

The term "identical" or percent "identity," when used to compare two or more nucleic acid or polypeptide sequences, means that the two or more sequences or subsequences are the same, or that a specified percentage of amino acid residues or nucleotides thereof are the same (i.e., about 60% identity, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over a specified region, when the two or more sequences are compared and aligned to determine the maximum correspondence over a comparison window or designated region), and that identity can be determined using the BLAST or BLAST2.0 sequence comparison algorithm, with default parameters described below, or by manual alignment and visual inspection (see NCBI http:// www.ncbi.nlm.nih.gov/BLAST/etc.). Such sequences are considered to be "substantially identical". This term also refers to, or can also be used to define, the complement of the test sequence. The term also includes sequences containing deletion and/or insertion mutations, as well as natural polymorphic or allelic mutants and artificial mutants. Good algorithms may perform crack compensation, etc., as described below. A region where identity exists preferably contains at least 25 amino acids or nucleotides, more preferably 50 to 100 amino acids or nucleotides.

In general, when comparing sequences, one sequence is used as a reference sequence, and a sequence to be detected is compared therewith. When sequence comparison is carried out by using a sequence comparison algorithm, a sequence to be detected and a reference sequence are input into a computer, a matched subsequence is designed if necessary, and parameters of a sequence algorithm program are set. Preferably, default program parameters are used, or custom parameters are used. Based on the program parameters, the sequence comparison algorithm calculates the percent sequence identity of the test sequence relative to the reference sequence.

The term "comparison window" as used herein refers to a segment having a number of consecutive positions, typically about 20 to 600, typically about 50 to 200, and more typically about 100 to 150, wherein a sequence is compared to the same number of consecutive positions on a reference sequence after optimal alignment of the two sequences. Methods of sequence alignment for comparison are well known in the art. Optimal alignment for sequence comparison can be performed by a variety of methods, such as the local homology algorithm of Smith & Waterman, adv. 482 (1981); homology alignment algorithm of Needleman & Wunsch, j.mol.biol.48: 443 (1970); method for searching similarity of Pearson & Lipman, proc.nat' 1.acad.sci.usa 85: 2444 (1988); and Computer programming of these algorithms (GAP, BESTFIT, FASTA and TFASTA within the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment followed by visual inspection (see Current protocols Molecular Biology, eds., 1995 supplement).

Preferred algorithms suitable for determining sequence percent identity and sequence similarity include the BLAST and BLAST2.0 algorithms described in Altschul et al, Nuc. acids Res.25: 3389-: 403-. BLAST and BLAST2.0 can be used to determine the sequence percent identity of the nucleic acids and proteins of the invention using the parameters described herein. Software to perform BLAST analyses can be downloaded free of charge from the national center for Biotechnology information (http:// www.ncbi.nlm.nih.gov /). This algorithm first identifies high scoring sequence pairs (HSPs) by identifying short words of length W within the query sequence, which may match or satisfy some positive scoring threshold T when aligned with words of the same length within the database sequence. T is referred to as the neighbor score threshold (Altschul et al, supra). These initial neighborhood target words (word hits) serve as seeds for initiating searches to find longer HSPs containing them. The target word extends in both directions along each sequence until the cumulative alignment score increases. The cumulative scores of nucleotide sequences are calculated using the parameters M (reward score for pairs of matching residues); always > 0) and N (penalty score for mismatching residues); always < 0 ═ for amino acid sequences, a scoring matrix (probabilistic matrix) is used to calculate the cumulative scores, the extension of the word of interest (word hit) in each direction stops when the cumulative alignment score decreases from a maximum value due to the number X, when the cumulative score reaches 0 or below 0 due to the accumulation of one or more negative-scoring residue alignments, or when the end of the sequence is reached, the BLAST algorithm parameters W, T and X determine the sensitivity and speed of alignment, the BLAST N program (for nucleic acid sequences) uses default parameter lengths (W) of 11, the expectation values (E) of 10, M ═ 5, N ═ 4, and the two strands are compared, the default parameter number for the BLASTP program is 3, the expectation (E) is 10, the BLOSUM62 scoring matrix (see Henikoff & Henikoff, proc. natl. acad. sci. usa 89: 10915(1989)) is arranged (B) to be 50, the expectation (E) is 10, M-5, N-4, and the two strands are compared.

The BLAST algorithm can also be used to perform statistical analysis of the similarity between two sequences (see Karlin and Altschul, Proc. Nat' l. Acad. Sci. USA 90: 5873-5787 (1993)). One similarity algorithm provided by BLAST is the smallest sum probability (P (N)), which calculates the likelihood that two nucleotide or amino acid sequences will match. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability calculated for the test nucleic acid when compared to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001. The log value may be a large negative number such as 5, 10, 20, 30, 40, 70, 90, 110, 150, 170, etc.

Two nucleic acid sequences or polypeptide sequences are substantially identical, meaning that the polypeptide encoded by the first nucleic acid is cross-immunoreactive with an antibody directed against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, e.g., two polypeptides differ only by conservative substitutions. Two nucleic acid sequences are substantially identical also means that two molecules or their complements can hybridize to each other under stringent conditions, as described below. The other meaning that two nucleic acid sequences are substantially identical is that the two sequences can be amplified using the same primers.

The terms "free", "purified" or "biologically pure" mean a material that is substantially free of components with which it normally accompanies in nature. Purity and homogeneity are generally determined by analytical chemical techniques such as polyacrylamide gel electrophoresis or high pressure liquid chromatography. The protein or nucleic acid present as the major component in the preparation is substantially purified. Isolated nucleic acids isolated from certain open reading frames flanking a gene may encode a protein other than the protein encoded by the gene. The term "purified" in certain embodiments means that the nucleic acid or protein exhibits substantially one band in the electrophoresis gel. This means that the nucleic acid or protein is at least 85% pure, preferably at least 95% pure, and most preferably at least 99% pure. "purifying" in certain embodiments refers to removing at least one impurity from the composition to be purified. In this sense, a homogeneous compound, i.e. a compound with a purity of 100%, does not require purification.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. This term may be used to refer to amino acid polymers in which one or more amino acid residues are synthetic chemical mimetics of their corresponding natural amino acids, as well as to refer to natural amino acid polymers, those containing modified residues, and non-natural amino acid polymers.

The term "amino acid" refers to both natural and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to natural amino acids. Natural amino acids refer to the amino acids encoded by the genetic code as well as those amino acids that are later modified, such as hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds having the same basic chemical structure as a natural amino acid, e.g., an alpha carbon atom to which a hydroxyl, carboxyl, amino, and R group are attached, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but still retain the same basic chemical structure as a natural amino acid. Amino acid mimetics refers to compounds that differ in chemical structure from a natural amino acid, but that function similarly to a natural amino acid.

In this context, amino acids may be represented by the three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Commission on biological Nomenclature, which is well known. Nucleotides, however, may be represented by the well-known single-letter symbols.

"conservatively modified mutants" may be used both in the context of amino acid sequences and in the context of nucleic acid sequences. Conservatively modified mutants refer to those nucleic acids which encode identical or substantially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence which is substantially identical or related to the natural contiguous sequence, for a particular nucleic acid sequence. Due to the degeneracy of the genetic code, there may be a plurality of functionally identical nucleic acids encoding the same protein. For example, the codons GCA, GCC, GCG and GCU all encode alanine. Thus, at the codon position encoding alanine, the codon can be replaced with another codon as described above without altering the polypeptide encoded thereby. Such nucleic acid mutations are referred to as "silent mutations," which are conservatively modified mutations. Each nucleic acid sequence herein that encodes a polypeptide also includes silent mutants of that nucleic acid. Those skilled in the art will appreciate that in certain instances codons in the nucleic acid (except AUG and TGG, because AUG is the only codon encoding methionine and TGG is the only codon encoding tryptophan) may be modified to produce a functionally identical molecule. Accordingly, silent mutants of nucleic acids encoding polypeptides are used to describe only the sequences associated with the expression product and not the actual probe sequence.

With respect to amino acid sequences, those skilled in the art will appreciate that substitution, deletion, or insertion of a single residue on a nucleic acid, peptide, polypeptide, or protein to result in a single amino acid or a small number of amino acid changes, additions, or deletions is also a conservative modified mutant in which a modification results in the substitution of one amino acid with another amino acid of similar chemical identity. The generation of a list of functionally similar amino acid conservative substitutions is well known in the art. Such conservatively modified mutants are also included within, and do not exclude, the polymorphic mutants, interspecies homologs, and alleles of the invention. Typical conservative substitutions include: 1) alanine (a), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine (C), methionine (M) (see Creighton, Proteins (1984)).

A "label" or "detectable motif" is a composition that is detectable spectrophotometrically, photochemically, biochemically, immunochemically, chemically, and by other physical means. For example, useful labels include fluorescein dyes, electron-dense reagents, enzymes (such as those commonly used in ELISA), biotin, digoxigenin, heparin, and proteins or other molecules that can be detected, such as by incorporating a radioactive label into the peptide, or for detecting antibodies that specifically react with the peptide. The radioisotope may be 3H, 14C, 32P, 35S or 125I.

In some cases, particularly with anti- α 5 β 1 integrin antibodies, radioisotopes may be used as the toxicity motif, as described below. The label may be inserted at any position within the antibody. Any method known in the art for attaching an antibody to a label can be used, including those described in the following references: hunter et al, Nature, 144: 945 (1962); david et al, Biochemistry, 13: 1014 (1974); pain et al, j.immunol.meth, 40: 219 (1981); and Nygren, j.histochem.andcechem., 30: 407(1982). The half-life of the radiolabeled peptide or radiolabeled antibody composition may be extended by adding a substance that stabilizes the radiolabeled peptide or antibody and prevents degradation thereof. Any substance and mixture thereof that is capable of stabilizing a radiolabeled peptide or antibody may be used, including those described in U.S. Pat. No.5,961,955.

By "antibody affinity matrix" is meant any material that is capable of preferentially binding antibodies. Antibody affinity matrices include polypeptides, polysaccharides, fatty acids, lipids, nucleic acids, including aptamers, or conjugates of these (e.g., glycoproteins, lipoproteins, glycolipids). In some cases, macromolecules such as multiprotein complexes, biofilms, or viruses may serve as antibody affinity matrix materials. Other examples of antibody affinity matrix materials are protein a, protein G, lectins and Fc receptors.

"protein A" refers to a highly stable surface receptor produced by Staphylococcus aureus that binds to the Fc region of immunoglobulins, particularly IgG, from a variety of species (Boyle, M.D.P. and K.J.Reis. bacterial Fc receptors, Biotechnology 5: 697-one 703 (1987)). One Protein A molecule can bind at least 2 molecules of IgG simultaneously (Sj ö quist, J., Meloun, B., and Hjelm, H.) (Protein A isolated from Staphylococcus aureus after digestion of lysostaphin) (Protein A isolated from Staphylococcus aureus and Eur J Biochem 29: 572-578 (1972)).

"protein G" refers to a cell surface associated protein of streptococcal origin with high affinity for IgG. It contains 3 highly homologous IgG binding regions (see Lian et al, 1992, Journal of mol. biol. 228: 1219-.

The term "recombinant" when used in reference to a cell, nucleic acid, protein or vector refers to a modified cell, nucleic acid, protein or vector, such as a cell into which a foreign nucleic acid or protein has been introduced, from which a native nucleic acid or protein has been altered, or from which a modified cell has been derived. Thus, a recombinant cell may express a gene that is not present in its native (non-recombinant) cell, or abnormally express, under-express, or not express a native gene at all. The term "recombinant nucleic acid" as used herein refers to a nucleic acid formed in vitro, typically by alteration of the nucleic acid, e.g., using polymerases and endonucleases, to a form that does not occur in nature. In this way, different nucleic acids can be linked. Thus, linear free nucleic acids or expression vectors formed by ligating DNA otherwise unrelated in vitro are considered recombinants for the purposes of the present invention. It will be appreciated that a recombinant nucleic acid, once formed and newly introduced into a host cell or host organism, may replicate in a non-recombinant manner, i.e., it utilizes mechanisms within the host cell rather than an in vitro mode of operation. However, once such nucleic acids are produced by recombination, subsequent replication is considered to be non-recombinant, but recombination is still considered for the purposes of the present invention. Likewise, "recombinant protein" refers to a protein prepared using recombinant techniques, such as by expression of a recombinant nucleic acid as described above.

The term "heterologous" when used in reference to a nucleic acid means that the nucleic acid contains at least two or more subsequences that are not normally found in the natural context. For example, nucleic acids are typically prepared by recombinant techniques, i.e., by rearranging two or more sequences from unrelated genes to form a nucleic acid with a new function, e.g., a promoter from one gene and a coding region from another gene. Likewise, a heterologous protein also refers to two or more subsequences that are not normally found in the relationship between these sequences in nature (e.g., a fusion protein).

A "promoter" is defined as a nucleic acid control sequence that directs the transcription of a nucleic acid. As described herein, a promoter comprises essential nucleic acid sequences near the transcription start site, such as a type II polymerase promoter, a TATA element. Promoters may also contain distal enhancer or repressor elements that are up to several thousand base pairs away from the transcription start site. A "constitutive" promoter is one that is active under normal environmental and developmental conditions. An "inducible" promoter refers to a promoter that is activated under environmental or developmental regulation. The term "operably linked" promoter refers to a nucleic acid expression control sequence (e.g., promoter, transcription factor binding site) and a second nucleic acid sequence that are functionally related, wherein the expression control sequence directs the transcription of the corresponding nucleic acid sequence of the second sequence.

An "expression vector" is a nucleic acid construct, either recombinantly produced or synthesized, containing a series of specific nucleic acid elements that enable transcription of a particular nucleic acid in a host cell. The expression vector may be a plasmid, a portion of a virus, or a nucleic acid fragment. Generally, an expression vector comprises a nucleic acid that can be transcribed, operably linked to a promoter.

The term "specifically (or selectively) binds to" an antibody or is "specifically (or selectively) immunoreactive with" a protein or peptide refers to the binding reaction that determines the presence of the protein within a population of heterologous proteins and other biomolecules. Thus, under the conditions of the immunoassay set forth, the binding activity of a specific antibody to a particular protein sequence is at least two-fold that of the background, typically 10-fold to 100-fold over the background.

Specific binding to an antibody under such conditions requires that the antibody selected be specific for a particular protein. For example, antibodies directed against a particular protein, polymorphic mutant, allele, ortholog, conservatively modified mutant, or splice mutant, or a portion thereof, may be selected from those polyclonal antibodies that specifically immunoreact with the α 5 β 1 integrin only and not with other proteins. There are many immunoassay methods available for screening antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are commonly used to screen for Antibodies specifically immunoreactive with a protein (see the immunoassay methods and assay conditions for determining a specific immunoreaction as described in Harlow & Lane, Antibodies, A Laboratory Manual (1988)).

"cancer cells", "transformed cells" or "transformants" in tissue culture means that the cells are either spontaneous or induced to undergo phenotypic changes such that uptake of new genetic material is no longer required. Although transformation is achieved by transfection of a transforming virus and insertion of new genomic DNA, or by uptake of exogenous DNA, transformation may occur spontaneously or upon exposure to a carcinogen, thereby mutating an endogenous gene to produce transformed cells. Transformation is associated with alterations in phenotype, such as immortalization of the cell, abnormal growth regulation, changes in non-morphology and/or the development of a malignant phenotype (Freshney, handbook of Animal cell Culture Basic technology (third edition, 1994)).

Introduction II

The present invention provides an anti- α 5 β 1 integrin humanized antibody and a chimeric antibody having superior characteristics to those of conventional anti- α 5 β 1 integrin antibodies. The invention also provides pharmaceutical compositions containing the novel antibodies and improved methods of treating diseases and tissue damage exacerbated by angiogenesis.

The humanized and chimeric antibodies of the invention have a longer half-life and are less antigenic than existing antibodies when used in humans. These improvements are illustrated graphically in Table 2, which includes changes to the framework and constant regions of the murine anti- α 5 β 1 integrin (IIA1) antibody to humanize it.

There are at least three advantages of humanized antibodies for use in human therapy. First, it can better interact with the human immune system, such as by Complement Dependent Cytotoxicity (CDC) or antibody dependent cytotoxicity (ADCC), to more efficiently destroy target cells. Second, the human immune system does not recognize these antibodies as foreign proteins. Third, it has the same half-life as natural antibodies in the human body in the blood circulation, and thus can reduce the dose and the number of times of administration.

Structurally, humanized antibodies typically contain constant regions and Framework Regions (FRs) of human origin and Complementarity Determining Regions (CDRs) derived from animal anti- α 5 β 1 integrin antibodies.

Structurally, chimeric antibodies typically contain variable regions derived from animal anti- α 5 β 1 integrin antibodies and constant regions derived from humans.

Functionally, both chimeric and humanized anti- α 5 β 1 integrin antibodies can specifically recognize α 5 β 1 integrin and block the binding of α 5 β 1 integrin to its antibody.

Provided herein are various methods of making humanized and chimeric anti- α 5 β 1 integrin antibodies. "humanized" antibodies are generally chimeric or mutated monoclonal antibodies of mouse, rat, hamster, rabbit or other species of antibody origin, containing constant and/or variable regions of human origin or other particular alterations. Methods for making "humanized" and "chimeric" anti- α 5 β 1 integrin antibodies are well known to those skilled in the art and are described in the references and patents cited herein.

Preparation of recombinant chimeric and humanized anti- α 5 β 1 integrin antibodies and Fab fragments thereof

The antibody of the present invention can be produced by immunizing an animal with α 5 β 1 integrin or a peptide derived therefrom to produce an anti- α 5 β 1 integrin antibody. The antibody-expressing lymphoid tissue is then isolated and the nucleic acids encoding the heavy and light chains of the anti- α 5 β 1 integrin antibody are purified. The purified nucleic acid is then processed by recombinant means (using methods well known in the art) to produce a chimeric, human-derived nucleic acid encoding a polypeptide that specifically recognizes the α 5 β 1 integrinAcylated, single-chain, Fab or Fab₂Nucleic acid of an antibody.

The nucleic acid produced by recombinant means is then used to prepare anti- α 5 β 1 integrin antibody-secreting cells. Monoclonal antibodies produced by these cells inhibit or block the binding of α 5 β 1 integrin to its receptor, resulting in the inhibition of angiogenesis in sensitive tissues.

A. Anti-alpha 5 beta integrin antibody-producing cell and production of anti-alpha 5 beta integrin antibody-encoding nucleic acid

To prepare recombinant anti- α 5 β 1 integrin chimeric and humanized antibodies, it is first necessary to isolate nucleic acids encoding non-human anti- α 5 β 1 integrin antibodies. Animals such as mice are usually immunized with α 5 β 1 integrin or the antigenic peptide from which it is derived, and the mice are generally immunized twice, with about 50mg of antibody protein per mouse being injected intraperitoneally. Serum is isolated from the immunized mice and the antibody activity is detected in a host system expressing the polypeptide by immunohistological or immunocytological methods, or the expressed polypeptide is analyzed for antibody activity by ELISA. If immunohistological methods are used, the antibody activity of the invention is characterized by the use of a biotin-labeled anti-mouse immunoglobulin followed by the addition of avidin-peroxidase and chromogenic peroxidase substrates. Such agents are commercially available, for example, from Zymed Corp, San Francisco, Calif. Mice in which the activity of the antibody of the present invention was detectable in serum were sacrificed 3 days later, and their spleens were taken for fusion and hybridoma preparation. Positive supernatants from such hybridomas can be identified by methods well known to those skilled in the art, such as Western blot hybridization techniques.

The nucleic acid encoding the antibody chain of interest is then isolated by PCR amplification of the heavy and light chain genes using hybridoma mRNA or splenocyte mRNA as a template [ Huse et al, Science 246: 1276(1989)]. Nucleic acids encoding antibodies and intrabodies can be isolated from murine monoclonal antibody hybridomas using this method [ Richardson j.h. et al, Proc Natl Acad Sci USA 92: 3137-3141 (1995); biocca s. et al, Biochem and biophysis res Comm, 197: 422 and 427 (1993); mhashilkar, a.m. et al, EMBO J14: 1542-1551(1995)]. These hybridomas are a reliable source of known reagents for the construction of antibodies, and are particularly useful if their epitope reactivity and affinity are known. Methods for isolating nucleic acids from free cells are further described in the following references: clackson, T, et al, Nature 352: 624-; barbas, c.f., et al, supra; marks, j.d., et al, supra; barbas, c.f. et al, Proc Natl Acad Sci USA 88: 7978-7982(1991) (human peripheral blood lymphocytes).

B. Preparation of recombinant antibodies

Humanized non-human (e.g., murine) antibodies are immunoglobulins, immunoglobulin chains or fragments thereof (e.g., Fv, Fab ', F (ab')₂Or other antigen binding sequence of an antibody) that contains few non-human immunoglobulin sequences. Humanized antibodies comprise human immunoglobulins in which residues from a Complementarity Determining Region (CDR) of the acceptor are replaced by residues from a CDR of a non-human species (donor antibody), such as mouse, rat or rabbit antibody, having the desired specificity, affinity and function. In some instances, Fv framework residues on a human immunoglobulin are replaced by residues of a corresponding non-human antibody. Humanized antibodies also contain some residues that are neither present on the recipient antibody nor on the donor CDR or framework region sequences. In general, a humanized antibody will comprise substantially all of the sequences of at least one variable region, and typically two variable regions, in which all or a substantial portion of the CDR regions correspond to those of a non-human immunoglobulin and all or a substantial portion of the FR regions correspond to those of a human immunoglobulin consensus sequence. The humanized antibody may also comprise at least a portion of the constant region (Fc) of an immunoglobulin, typically of human origin [ Jones et al, Nature, 321: 522-525 (1986); riechmann et al, Nature, 332: 323-329 (1988); and Presta, curr, op.struct.biol., 2: 593-596(1992)]。

Methods for making humanized antibodies are well known in the art and are described in detail elsewhere. See Queen et al, U.S. patent Nos: 5,530,101; 5,585,089; 5,693,761; 5,693,762; 6,180,370 (all of which are incorporated herein by reference in their entirety). A number of methods well known in the art can be used to humanize antibodies including CDR-grafting (EP 239,400; PCT patent publication WO 91/09967; U.S. Pat. Nos.5,225,539; 5,530,101 and 5,585,089), surface modification or resurfacing (EP 592,106; EP519,596; Padlan, mol. Immunol., 28: 489-.

There are many methods available for the production of recombinant chimeric antibodies. For example, different regions of an antibody can be rearranged by connecting them together via disulfide bonds between proteins to form a chimeric antibody (Konieczny et al, Haematologica, 14 (1): 95-99, 1981). The recombinant DNA technology can be used for constructing fusion genes of the coding DNA sequences of the mouse antibody light chain variable region and the heavy chain variable region and the coding DNA sequences of the human antibody light chain constant region and the heavy chain constant region. See Morrison et al, proc.natl.acad.sci.usa, 81 (21): 6851 6855, 1984; morrison, Science 229: 1202-1207 (1985); oi et al, BioTechniques 4: 214-221 (1986); gilles et al, j. immunol. methods 125: 191-202 (1989); U.S. patent nos.5,807,715; 4,816,567 and 4,816,397, which are incorporated herein by reference in their entirety.

DNA sequences encoding the antigen binding or Complementarity Determining Regions (CDRs) of murine monoclonal antibodies can be grafted onto DNA sequences encoding the framework regions of the heavy and light chains of human antibodies using molecular techniques (Jones et al, Nature, 321 (6069): 522-525, 1986; Riechmann et al, Nature, 332 (6162): 323-327, 1988). The expressed recombinant product is called a "reshaped" antibody or humanized antibody comprising the framework regions of the light or heavy chain of a human antibody and the antigen recognition region CDRs of a murine monoclonal antibody.

Other methods for preparing humanized antibodies are described in U.S. Pat. No.5,639,641, which is incorporated herein by reference. The method produces a humanized rodent antibody by a resurfacing technique, which has improved therapeutic efficacy due to the presence of a human surface sequence in the variable region of the antibody. In this method: (1) positioning a set of antibody heavy and light chain variable region domains such that the surface of the heavy and light chain variable region framework is in an exposed position, wherein at least 98% of all variable regions are positioned in the same position; (2) a set of heavy and light chain variable region framework surface exposed amino acid residues defined as of rodent antibody (or fragment thereof) origin; (3) identifying a set of heavy and light chain variable region framework surface exposed amino acid residues having the highest identity to a set of rodent surface exposed amino acid residues; (4) replacing the set of heavy and light chain variable region framework-exposed amino acid residues defined in step (2) with the set of heavy and light chain variable region framework surface-exposed amino acid residues identified in step (3), except those within any atom 5 Å of any residue of the complementarity determining region of the rodent antibody; and (5) preparing a humanized rodent antibody having binding specificity.

A similar method for making humanized antibodies is described in U.S. patent nos.5,693,762; 5,693,761; 5,585,089 and 5,530,101, all of which are incorporated herein by reference. These methods involve the preparation of humanized immunoglobulins having one or more donor immunoglobulin Complementarity Determining Regions (CDRs) and possibly other amino acids and an acceptor human immunoglobulin framework region. Generally, each humanized immunoglobulin chain comprises, in addition to the CDRs, amino acids of the framework of the donor immunoglobulin that interact with the CDRs to affect their binding affinity, and such amino acid or amino acids are, as predicted by molecular modeling, located very close to or within about 3 Å of the CDRs of the donor immunoglobulin. Using U.S. patent nos.5,693,762; 5,693,761; any one of the alignment standards described in U.S. Pat. Nos.5,585,089 and 5,530,101, combinations of these standards, or all of these standards may be designed for both the heavy and light chains. When joined into a complete antibody, the humanized immunoglobulin is substantially antibody-free in humans, but has the same affinity as the original antigen as the donor immunoglobulin.

Other methods for preparing humanized antibodies are described in U.S. Pat. Nos.5,565,332 and 5,733,743, which are incorporated herein by reference. This approach combines the concept of humanized antibodies with phage libraries, which are also described in detail herein. In a general sense, the method utilizes the antigen binding site sequence of an antibody or population of antibodies against an antigen of interest. Thus, for a single rodent antibody, sequences comprising part of the sequence of the antigen-binding site of the antibody can be combined with different sequences of a human antibody to form the complete antigen-binding site.

The antigen binding site formed by this method is different from that prepared by the CDR method, and only a part of the sequence of the original rodent antibody may be contacted with the antigen in the same manner. The selected human sequence may differ in sequence from the antigen containing the original binding site and may otherwise be contacted therewith. However, the restriction due to the binding of part of the original sequence to the antigen, the shape of the antigen and its antigen binding site may allow new contacts of the human sequence to the same region or epitope of the antigen. This method is therefore also referred to as "epitope blot screening" (EIS).

Antibodies having a partially human antibody sequence can be selected by a treatment starting with an animal antibody. Such antibodies have sufficient sequence identity to human antibodies for therapeutic use either directly or after alteration of several key residues. Differences in sequence between rodent and human antibodies within selected antibodies can be minimized by site-specific mutation of individual residues or by replacing those different residues with residues from the human sequence by CDR grafting of the entire loop. However, antibodies having fully human sequences may also be prepared. Thus EIS provides a method for producing partially human or fully human antibodies that bind to the same epitope as an animal or a partially human antibody. In EIS, a group of antibody fragments are displayed on the surface of filamentous phage, and genes encoding the fragments having antigen-binding activity can be found by screening the phage for binding to the antigen.

Other methods of preparation that can be used for the humanized antibodies of the present invention are described in U.S. Pat. Nos.5,750,078; 5,502,167, respectively; 5,705,154, respectively; 5,770,403, respectively; 5,698,417, respectively; 5,693,493, respectively; 5,558,864; 4,935,496 and 4,816,567, both of which are incorporated herein by reference.

Techniques for making single chain antibodies (U.S. Pat. No.4,946,778) are also suitable for making single chain humanized antibodies to α 5 β 1 integrin.

C. Expression of recombinant chimeric or humanized antibodies

The resulting antibodies can be expressed by one or more vectors containing antibody-encoding nucleic acids.

The nucleic acid fragments encoding the heavy and light chains of the antibody are preferably located within one transcription unit, each encoding nucleic acid being translated under the control of an IRES sequence. The vectors contain chemical linkers as described in WO 93/64701, which linkers carry targeting motifs (e.g.ligands for cell surface receptors) and nucleic acid binding motifs (e.g.polylysine), viral vectors (e.g.DNA or RNA viral vectors), fusion proteins as described in PCT/US 95/02140(WO 95/22618) which contain target motifs (e.g.antibodies specific for target cells) and nucleic acid binding motifs (e.g.protamine), plasmids, phages and the like. The vector may be chromosomal, nonchromosomal or synthetic.

Preferred vectors comprise a viral vector, a fusion protein and a chemical linker. Retroviral vectors include moloney murine leukemia virus. DNA viral vectors are preferred. These vectors include poxvirus vectors such as orthopoxvirus or avipox virus (avipox) vectors, herpes virus vectors such as the herpes simplex virus type I (HSV) vector [ Geller, a.i. et al, j.neurochem, 64: 487 (1995); lim, f, et al, "DNA cloning: mammalian Systems (DNA Cloning: Mammarian Systems), eds. D.Glover, (Oxford Univ.Press, Oxford, England) (1995); geller, a.i. et al, Proc natl.acad.sci.: U.S. a.90: 7603 (1993); geller, a.i. et al, Proc natl.acad.sci USA 87: 1149(1990), adenovirus vectors [ LeGal LaSalle et al, Science, 259: 988 (1993); davidson et al, nat. Genet 3: 219 (1993); yang et al, J.Virol.69: 2004(1995) and adeno-associated viral vectors [ Kaplitt, M.G. et al, nat. Genet.8: 148(1994)].

Poxvirus vectors can introduce genes into the cytoplasm. Fowlpox vector transduction can only produce short-term expression of the nucleic acid. Adenovirus vectors, adeno-associated virus vectors, and Herpes Simplex Virus (HSV) vectors are preferred for introducing the nucleic acid into nerve cells. Transduction with adenovirus produces shorter expression (about 2 months) than adeno-associated virus (about 4 months), which in turn produces shorter expression than HSV vectors. The choice of which vector will depend on the target cell and the disease to be treated. Vector introduction can be carried out using standard methods, such as infection, transfection, transduction or transformation. Examples of gene transfer patterns include naked DNA, CaPO₄Precipitation, DEAE dextran, electroporation, protoplast fusion, lipofectin, cell microinjection, and viral vectors.

The vector may also be targeted to any target cell of interest, such as a glioma cell. For example, stereotactic (stereotaxic) injection can be used to transfer vectors (e.g., adenovirus, HSV) to a desired site. In addition, particles can be infused into the ventricle via intraventricular (icv) Infusion using a micro-vacuum pump Infusion System such as the Synchromed Infusion System. A method designed based on the principles of dilatory flow, known as convection, has also been shown to effectively transfer macromolecules into large areas of the brain, and can be used to transfer vectors to target cell sites (Bobo et al, Proc. Natl. Acad. Sci. USA 91: 2076. sup. 2080 (1994); Morrison et al, am. J. Physiol.266: 292. sup. 305 (1994)). Other methods that may be used include catheters, intravenous, parenteral, intraperitoneal and subcutaneous injections, as well as oral or other known modes of administration.

D. Isolation and characterization of recombinant antibodies

The term "antibody" as used herein includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments such as F (ab')₂And Fab proteolytic fragments. In addition, the complete genetically engineered antibody or the whole genetically engineered antibodyFragments, such as chimeric antibodies, Fv fragments, single chain antibodies, and the like, as well as synthetic antigen-binding peptides and polypeptides. Non-human antibodies are humanized by grafting non-human CDRs onto human framework and constant regions, or by inserting intact non-human variable regions (which can also be covered with human-like surfaces by replacing exposed residues, creating a "surface-modified" antibody). In some cases, humanized antibodies contain residues within the framework regions of the variable regions of human origin that retain some non-human origin to increase the correct binding capacity of the antibody. The antibody can prolong the biological half-life after being humanized and modified, and the capability of generating adverse immune response after being administered to a human body is reduced. Other methods of making or screening antibodies useful herein include contacting lymphocytes with α 5 β 1 integrin proteins or peptides in vitro, screening phage antibody display libraries or similar vector libraries (e.g., by using immobilized or labeled α 5 β 1 integrin proteins or peptides).

E. Affinity purification

Affinity purification of antibody libraries or sera provides practitioners in the art with a more uniform reagent. Methods for the preparation of affinity chromatography columns using antibody affinity matrices to enrich for antibody-. alpha.5. beta.1 integrin antibodies are well known in the art and commercially available (antibody shop, c/o Statens Serum institute, Artillerive j 5, Bldg. P2, DK-2300 Copenhagen S). Briefly, an antibody affinity matrix is attached to an affinity support (e.g., CNBR Sepharose (R), Pharmacia Biotech). The antibody-containing mixture is then passed through an affinity matrix to bind the antibody to the matrix. The bound antibody is then released by methods well known in the art to yield a concentrated antibody library. The enriched antibody library is then subjected to further immunological analysis, some of which are described herein by way of example. Although the antibody affinity matrix used for isolating the antibody of the present invention is not designed as a matrix specifically recognizing the anti- α 5 β 1 integrin antibody of the present invention, it is not limited to the use of the affinity matrix for purifying antibodies, since the antibody is expressed as a recombinant protein, which is monoclonal in nature in the expression system.

The isolated antibody, α 5 β 1 integrin antibody, a mutant of α, e.g., 5 β 1 integrin, can be compared to a second protein using the competitive binding immunoassay described above. For comparison, the concentration range analyzed for both proteins was broad, and the amount required to inhibit 50% of the binding of antisera to the immobilized protein. A second protein is considered to be capable of specifically binding to an antibody to α 5 β 1 integrin if the amount required for the second protein to inhibit 50% binding is less than 10 times the amount required for α 5 β 1 integrin to inhibit 50% binding.

Purification of pH-sensitive antibodies

Certain antibodies of the invention have the property of being readily precipitated when subjected to affinity purification at neutral or basic pH. In order to solve this problem, another aspect of the present invention provides a method for purifying a pH-sensitive antibody comprising an amino acid sequence shown in FIGS. 1 to 5, 10, 11 and 13 and a chimeric antibody comprising a murine variable region or having 80% or more sequence identity to the murine variable region or a chimeric antibody having 80% or more sequence identity to the CDR of the antibody shown in FIGS. 1 to 5. The method generally comprises affinity chromatography of the antibody using a chromatography column, such as an ion exchange chromatography column, containing a bound antibody affinity matrix, followed by elution of the antibody at a pH of about 3.0 to 5.5, preferably 3.3 to 5.5, most preferably 3.5 to 4.2 or 4.2 to 5.5. Lower pH conditions in this range are more suitable for small scale purification, while pH of about 4.2 or higher is more suitable for large scale operations. Purification within this range results in a product that is not or only minimally agglutinated, and preferably substantially free of agglutination.

Affinity chromatography is a well known method of isolating or purifying substances such as antibodies or other biologically active macromolecules in the art. Affinity chromatography generally begins with the passage of an antibody-containing solution through a chromatographic column containing one or more ligands that specifically bind to antibodies immobilized within the column. This combination can extract the antibody from solution by ligand affinity reaction. After binding is complete, the antibody can be recovered by eluting the column.

This aspect of the invention therefore includes a method for purifying an anti- α 5 β 1 integrin antibody using an antibody affinity matrix bound to a substrate, wherein the improvement comprises eluting the antibody from the antibody affinity matrix bound to the substrate using an eluent having a pH of about 3.0 to about 5.5.

More particularly, this aspect of the invention includes a method of purifying an anti- α 5 β 1 integrin antibody, the method comprising: (a) adsorbing the antibody to an antibody affinity matrix bound to a substrate; and (b) eluting the antibody from the antibody affinity matrix bound to the substrate with an eluent having a pH of about 3.0 to 5.5. In certain embodiments, the method further comprises step (c): recovering the purified antibody.

However, when the antibody is to be further purified or processed, a special recovery step is not necessarily required in this regard.

The purification process involves adsorption of the antibody onto an antibody affinity matrix which is bound to a substrate. Various forms of antibody affinity matrices may be used. As long as the antibody affinity matrix has the ability to bind the antibody to be purified. For example, naturally derived antibody affinity matrices, antibody affinity matrices prepared using recombinant DNA techniques, modified forms of antibody affinity matrices, fragments of such matrices that retain the ability to bind purified antibodies may be used. Common materials that can be used as antibody affinity matrices include polypeptides, polysaccharides, fatty acids, lipids, aptamers, glycoproteins, lipoproteins, glycolipids, polyprotein complexes, biofilms, viruses, protein a, protein G, lectins, Fc receptors, and the like.

The antibody affinity matrix may be attached to the solid phase or support by ordinary interactions (e.g., non-specific, ionic, hydrophobic/hydrophilic interactions), specific interactions (e.g., antigen-antibody interactions), or covalent bonding between the ligand and the solid phase. Alternatively, an intermediate compound or a spacer may be attached to the solid phase, and then the antibody affinity matrix is immobilized to the solid phase by being attached to the spacer. The pad itself may be a ligand (i.e. a second ligand) with specific affinity for the free antibody affinity matrix.

The antibody affinity matrix may be attached to a variety of substrates or supports. Generally, ion exchange or coupling resins (e.g., CNBr activated) can be used for this purpose. Antibodies can also be adsorbed to an antibody affinity matrix bound to a substrate by other methods. Preferred is a column method in which the antibody is adsorbed to a column using a suitable buffer. Common buffers and operating conditions are well known in the art.

The antibody may be eluted from the antibody affinity matrix bound to the substrate using conventional methods, such as elution of the antibody from a chromatography column using a buffer. To minimize precipitation, the pH sensitive anti- α 5 β 1 integrin antibody is preferably eluted with a buffer containing 0.1M glycine at pH 3.5. To minimize degradation and/or denaturation, the temperature of the buffer is preferably maintained below 10 ℃, preferably 4 ℃ or below 4 ℃. For the same reason, the exposure time of the antibody to acidic pH should be minimized. This can be achieved by adding a predetermined amount of an alkaline solution to the antibody eluent. The alkaline solution is preferably a buffer, more preferably a volatile alkaline buffer, most preferably aqueous ammonia.

Elution of the antibody from the antibody affinity matrix bound to the substrate can be carried out by various methods well known in the art. For example, if a chromatography column is used, the fraction eluted from the column is collected and the presence or absence of protein is judged based on the absorbance of the fraction. If an antibody of known specificity is purified, it can be determined whether the antibody is contained in a fraction collected from the column by an immunoassay technique such as Radioimmunoassay (RIA) or enzyme-linked immunoassay (EIA).

The purification process of the invention may be carried out at any convenient temperature, provided that such temperature does not cause substantial degradation of the purified antibody or a detrimental effect on the binding of the antibody affinity matrix to the substrate. The preferred temperature is room temperature. If desired, the antibody eluted from the antibody affinity matrix chromatography column may be recovered by a variety of methods well known in the art.

Affinity assays

Affinity assays allow one skilled in the art to identify antibodies that specifically recognize one or more epitopes of α 5 β 1 integrin. The binding of an antibody is considered specific if the following occurs: 1) its binding activity reaches a threshold level, and/or 2) the antibody does not significantly cross-react with the relevant polypeptide molecule. First, if the antibodies herein have a binding affinity (K) for an α 5 β 1 integrin polypeptide, peptide, or epitope_a) Up to 10⁶mol^-1Or higher, preferably 10⁷mol^-1Or higher, more preferably 10⁸mol^-1Or higher, most preferably 10⁹mol^-1Or higher, the binding of the antibody is specific. The binding affinity of an antibody is readily determined by one skilled in the art by Scatchard analysis (Scatchard, Ann. NY Acad. Sci.51: 660-72, 1949) or by surface plasmon resonance using BIAcore.

Second, antibody binding can be considered specific if it does not significantly cross-react with the relevant polypeptide. An antibody is considered to not significantly cross-react with a polypeptide molecule of interest if only the α 5 β 1 integrin polypeptide is detected and no known related polypeptide is detected by standard western blot analysis (Ausubel et al, supra). Known examples of related polypeptides are orthologs, proteins from the same species as the members of the integrin protein family, polypeptides as shown in FIG. 1, mutant α 5 β 1 integrin polypeptides, and the like. Alternatively, antibodies can be used to "screen" known related polypeptides to isolate a panel of polypeptides that specifically bind to α 5 β 1 integrin. For example, an antibody to a human α 5 β 1 integrin polypeptide is adsorbed to a polypeptide of interest that is attached to a soluble substrate; an antibody specific for the human α 5 β 1 integrin polypeptide is flowed through the matrix under suitable buffer conditions. This screen allows the isolation of polyclonal and monoclonal Antibodies that are not cross-reactive with the relevant polypeptide (Antibodies: A Laboratory Manual, Harlow and Lane, eds.), Cold Spring Harbor Laboratory Press, 1988, Current Protocols in immunology, Cooligan et al, National Institutes of Health, John Wileyand Sons, Inc., 1995). Methods for screening and isolating specific antibodies are well known in the art (see basic Immunology, compiled by Paul), Raven Press, 1993; getzoff et al, adv.in Immunol.43: 1-98, 1988; monoclonal antibodies: principles and practices (MonoclonalAntibodies: Principles and Practice), Goding, J.W, (eds.), Academic Press Ltd., 1996, Benjamin et al, Ann.Rev.Immunol.2: 67-101, 1984). Typical examples of such analytical methods include: parallel immunoelectrophoresis, radioimmunoassay, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), dot blot or western blot analysis, inhibitory or competitive assay, and sandwich assay. For an overview of immunological and immunological methods, see Basic and clinical immunology (Basic and clinical immunology), (eds. & Terr, 7 th edition, 1991).

Method for detecting angiogenic regulatory effect

The present invention provides methods for evaluating the physiological regulatory effects produced by humanized anti- α 5 β 1 integrin antibodies. As a threshold issue, these methods can be used to screen compositions containing the antibodies of the present invention to determine safe and effective therapeutic doses. Some of these methods are useful for monitoring the progression of the disease during treatment and for adjusting the dosage administered to achieve optimal clinical efficacy.

These methods include providing a living tissue suitable for analysis or treatment, i.e., where tissue damage, including immortalization, occurs as a poor event of choroidal neovascularization, which if inhibited or prevented, would improve patient prognosis and/or aid in repair of damaged tissue. Typical tissues suitable for treatment or study include tumors and ocular tissues, particularly macular degeneration of the eye. The term "tumor" is used herein in a broad sense to refer to any new pathological tissue proliferation. For the purposes of the present invention, angiogenesis is part of the tumor characteristics. Tumors may be benign, such as hemangiomas, gliomas, teratomas, and the like, and malignant, such as adenocarcinomas, sarcomas, glioblastomas, astrocytomas, neuroblastomas, retinoblastomas, and the like. The term "tumor" is used generically to refer to benign or malignant tumors, while the term "cancer" is used generically to refer to malignant tumors, whether metastatic or non-metastatic. Malignancies that can be diagnosed using the methods of the present invention include carcinomas such as lung, breast, prostate, cervical, pancreatic, colon and ovarian carcinomas, sarcomas such as osteosarcoma and kaposi's sarcoma, provided that such tumors are at least partially characterized by angiogenesis associated with α 5 β 1 expression leading to neovascularization. For research, these tissues can be isolated from readily available resources by methods well known to those skilled in the art.

When the therapeutic effect of the antibody of the present invention is examined using a biopsy, it is first necessary to cause tissue damage to promote choroidal neovascularization. Tissue damage may be caused by any suitable means, including mechanical, chemical, or biological means. Examples of mechanical injuries are cutting, puncturing or clamping. Chemical means include administering a drug to the tissue to cause it to necrose, to die or to lose cell-to-cell contact. The biological means includes treatment with an infectious agent such as a virus, a bacterium or a virus. The preferred method of causing damage is by laser. Any laser that can cause tissue damage can be used, CO₂Gas laser is a preferred type, the most preferred type being the OcuLight GL (532 nm) laser coagulator with IRISmedical ® portable slit lamp adapter. Other laser sources that provide a laser of about 300 to 700 milliwatts may be used, with the laser induced damage typically being less than 200 μm in diameter, preferably less than 100 μm, more preferably between 50 and 100 μm in diameter, and most preferably between about 75 and 25 μm in diameter. In general, the laser irradiates the tissue for a period of time within 1 second, typically less than 0.5 second, more preferably less than 0.1 second, and most preferably less than 0.05 second.

The antibodies used herein are chimeric or humanized anti- α 5 β 1 integrin antibodies. The antibody comprises a heavy chain variable region having 65%, preferably more than 75%, more preferably more than 85%, 90%, 95%, 97% or 99% sequence identity to an amino acid sequence selected from the group consisting of: SEQ ID nos.: 1-6, 16 and 20 and an independently selected light chain variable region having 65%, preferably more than 75%, more preferably more than 85%, 90%, 95%, 97% or 99% sequence identity to an amino acid sequence selected from the group consisting of seq id no: SEQ ID nos.: 7-12, 18 and 22. Most preferred chimeric or humanized anti- α 5 β 1 integrin antibodies comprise a heavy chain variable region of SEQ ID No.: 2-6, 16, 20, 25, 28 and 31 and a light chain variable region having a sequence selected from the group consisting of seq id no: SEQ ID nos.: 8-12, 18, 22, 26 and 32.

The antibodies of the invention may be administered by different routes, such as intravenous injection, oral administration or directly to the area to be treated, such as directly into a malignant tumor; dripping into the pathologically changed eye; or injected into the synovial membrane of the joint.

The dosage of therapeutic antibody required will depend on whether the administration is for diagnostic or therapeutic purposes, particularly for individual subjects. Methods for determining effective doses of drugs for diagnostic or therapeutic purposes are well known in the art and include phase I, II, and III clinical trials. Using the method of the invention

The effective dose can be determined, for example, by determining the dose to be administered to the subject, and the progression of the disease can be recorded as an index for evaluating the neovascularization inhibitory function.

The total dose of drug administered to the patient may be a single dose, delivered to the patient in the form of a bolus or over a relatively short period of time, or multiple doses of drug may be administered to the patient over a relatively long period of time using a step-wise dosing regimen. As discussed above, one skilled in the art will appreciate that the concentration of drug required to achieve an effective amount of drug to the angiogenic domain associated with α 5 β 1 integrin expression will depend on a variety of factors including the age of the patient, general health, route of administration, number of administrations, and the nature of the drug. Based on these factors, one skilled in the art can adjust the dosage to effectively interfere with the binding of α 5 β 1 integrin to its ligand and thereby reduce or inhibit angiogenesis.

Monitoring the clinical progression of a disease is another aspect of the invention. Monitoring may be carried out by suitable methods well known in the art. Preferred methods include microscopy, nuclear magnetic resonance and X-ray. For eye tissue, the posterior chamber of the eye can be examined by indirect ophthalmoscopy, and the anterior half of the eye can be examined by biomicroscopy. A preferred method of examining the extent of choroidal neovascularization is by intravenous injection of a fluorescent dye followed by examination of the biopsies by fluorescein angiography.

A preferred method of screening for anti- α 5 β 1 integrin antibodies effective to inhibit or prevent neovascularization causes damage to the retina of an animal by applying the anti- α 5 β 1 integrin antibody to the damaged area and observing neovascularization in the damaged tissue as compared to a control. This method is discussed in detail in example 6 below. These experiments gave surprising results and found that the use of anti- α 5 β 1 integrin antibodies in one eye can treat both eye injuries. Indicating that newly formed blood vessels in the damaged tissue are "leaky", and that instillation of antibodies in one eye will enter the blood system and reach the other eye. This result was obtained whether the treatment was with whole antibody or Fab fragment. These results indicate that this is a novel method of treating ocular injury by systemic administration, such as intravenous administration, of the therapeutic anti- α 5 β 1 integrin antibodies of the invention.

Therapeutic use

Another embodiment of the invention encompasses pharmaceutical compositions comprising the therapeutic antibodies described herein. These compositions contain factors that enhance the uptake or localization of therapeutic agents, reduce inflammatory responses, or provide local release of drugs.

The antibodies of the invention or pharmaceutical compositions comprising the antibodies, which reduce or inhibit angiogenesis associated with α 5 β 1 integrin expression, are useful for treating any pathological condition characterized, at least in part, by angiogenesis. One skilled in the art will know of various routes of administration, including oral administration or administration by parenteral routes, such as intravenous injection, intramuscular injection, subcutaneous injection, intracameral administration, intracapsular administration, intrasynovial administration, intraperitoneal injection, intracisternal administration, or passive or enhanced absorption through the skin using a transdermal patch or by penetration of dermal ions for isotonicity. Alternatively, the antibody may be administered by injection, intubation, by suppository, oral or topical formulation, the latter being passively absorbed, e.g. by direct application of an ointment or powder containing the antibody or active ingredient, which powder may be administered by intranasal spray or inhalation. The antibody may also be administered by means of a topical spray, in which case one of the components of the composition may be a suitable propellant, if desired. If desired, the pharmaceutical composition may be incorporated into liposomes, microspheres or other polymeric matrices (Gregoriadis Liposome Technology, Vol.1 (CRC Press, Boca Raton, Fla.1984), incorporated herein by reference). Liposomes are composed of phospholipids or other lipids, are a non-toxic physiologically metabolizable carrier, and are relatively simple to prepare and use.

Angiogenesis associated with α 5 β 1 integrin expression may occur locally, such as in the retina of patients with diabetic retinopathy, and may occur systemically, such as in patients with rheumatoid arthritis or malignant metastases. Since the area of angiogenesis may be local or systemic, one skilled in the art should select a particular route and method of administration for use of the therapeutic antibody of the invention based on this factor, which is at least one of the factors to be considered.

For example, the angiogenesis area related to the expression of alpha 5 beta 1 integrin in a patient with diabetic retinopathy is limited to the retina, and then the medicament can be prepared into a medicament composition preparation convenient for eye dropping and can be directly dropped into eyes. For metastatic cancer patients, the pharmaceutical composition containing the drug should be prepared into a preparation which can be injected intravenously, orally or be distributed to the whole body by other routes. Thus, the antibodies of the invention may be administered by different routes, e.g., intravenous injection, oral administration, or directly to the site in need of treatment, e.g., directly into a malignant tumor; instilling the solution into the eye that produces the pathological change; or injected into the synovial membrane of the joint.

The therapeutic antibody should be administered in an effective amount, which is an amount sufficient to interfere with the specific binding of α 5 β 1 integrin to its ligand in vivo. Generally, the pharmaceutical antagonist will be administered in an amount of about 0.0001 to about 100 mg/kg body weight, but may be adjusted for specific use. Based on the results of the above clinical efficacy tests, the skilled artisan should be able to determine effective dosage ranges for different diseases. When the drug is administered topically, the amount administered can be adjusted accordingly.

A preferred method of administration of the antibodies of the invention is by injection, intradermal injection, intravenous injection or direct injection into the damaged joint or tissue. For example, where damage to retinal tissue occurs, a therapeutic antibody of the invention may be injected intravitreally into the damaged eye. A surprising result of the present invention is that treatment of one eye produces a beneficial clinical effect in both eyes (assuming both eyes are damaged). The newly formed blood vessels appear to be "leaky," and antibodies used in the first eye can enter the bloodstream and then reach the second eye. When treating an ocular condition in this manner, the dosage is preferably less than 5 μ M, more preferably between 0.5 and 2 μ M, and most preferably between 0.1 and 1.0 μ M. As indicated above, the treatment may take the form of multiple doses, applied to one area over a period of time. The dosage of each may be the same in a multiple dose regimen or may be determined and used individually. This result also indicates that other methods of treating a lesion associated with neovascularization can also be used to systemically administer (e.g., intravenously) an effective amount of a therapeutic antibody, wherein neovascularization occurring in the damaged tissue is inhibited or prevented

All documents and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were individually incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Examples

The scope of application of the invention is very broad in view of the above description. The following examples are therefore provided for illustrative purposes and are not meant to limit the invention in any way. One skilled in the art will readily recognize that many non-standard parameters may be altered to produce substantially similar results.

Example 1-construction of M200 chimeric antibodies starting from murine IIA1 anti- α 5 β 1 integrin antibody

This example describes the process of construction of chimeric antibody M200.

IIA1 and starting DNA sequences for the 200-4VH and VL regions

Cloning of the heavy chain variable region (V) of murine anti-human α 5 β 1 integrin antibody IIA1(Pharmingen, San Diego CA) from the IIA1 hybridoma cDNA_H) And light chain variable region (V)_L) The sequence, after sequencing, was part of the starting construct for the 200-4 antibody. FIG. 3 shows IIA1V_H(SEQ ID NO: 13) and V_L(SEQ ID NO: 14) region. In the construction of the 200-4 murine/human chimeric IgG4 antibody derived from IIA1, the silent XhoI restriction enzyme site (CTCGAG) (SEQ ID NO: 33) was introduced into IIA1V_HAnd V_LFramework 4 region (b). 200-4V containing these silencing XhoI sites_H(SEQ ID NO: 15) and V_L(SEQ ID NO: 17) the DNA sequences were found in the expression construct DEF38 IIA 1/human G4 chimera and NEF5IIA1/K chimera, as shown in FIG. 4. These 200-4V_HAnd V_LThe sequence was used as the starting sequence for all subsequent recombinant DNA manipulations.

Design of M200 VH and VL small exons

Expression of 200-4V in plasmid DEF38 IIA 1/human G4 chimera and NEF5IIA1/K chimera_HAnd V_LThe region is directly linked to its adjacent constant region via a silent XhoI site, without an intron in the middle. In order to make these variable regions compatible with antibody expression vectors derived from genomic DNA, it is necessary to design "mini-exons" that regenerate functional donor splice sites at the 3' end of the variable regions. The V of IIA1 is found by sequence comparison_HAnd V_LThe murine JH4 and JK1 segments were used separately; therefore, the small exon is designed to be in V_HAnd V_LThe last amino acid of the region was followed by the addition of a native murine JH4 and JK1 donor splice site. In addition, the XhoI site was removed, restoring the backbone 4 sequence seen in the original IIA1 hybridoma. Restriction sites flanking the minor exon are as follows: 5 'and 3' XbaI sites (TCTAGA) of the VH small exon (SEQ ID NO: 34), 5 'MluI (ACGCGT) (SEQ ID NO: 35) and 3' XbaI (TCTAGA) (SEQ ID NO: 34) of the VL small exon.

Recombinant antibody variable regions occasionally contain unwanted other mRNA splice sites that result in the production of other forms of spliced mRNA. Theoretically, this site is present in the murine variable region, but is only activated in heterologously expressed cells and/or in the presence of new receptor sites derived from chimeric constant regions. These unwanted extra splice sites can be removed by taking advantage of the degeneracy of the codon to remove the potential extra splice sites but keeping the coding amino acid sequence unchanged. To detect M200V_HAnd V_LAny redundant splice sites within the mini-exon that are active are initially designed by the splice site prediction program of the university of Danish technology center (http:// www.cbs.dtu.dk/services/NetGene2/) analysisAnd (4) columns. The correct donor splice sites within two 200-M small exons have been identified; however, V also at CDR3_HV of minor exon and CDR1_LPotential additional donor splice sites were detected on the small exons. To eliminate the possibility of these splice sites being used, single base pair silent mutations were added within the small exon. In design V_HWhen the silent codon GGT is mutated to GGA at valine 29; in design V_LThe silent codon GTA was mutated to GTC at glycine 100(Kabat numbering). Both silent mutations can eliminate potential secondary splice donor signals on the V gene.

FIG. 5 shows the final design M200V_HAnd V_LSmall exons (SEQ ID NOS: 19, 21), containing flanking restriction sites, murine donor splice sites, 200-4 XhoI sites and potentially other donor splice sites, were removed.

M200 V_HConstruction of Small exons and plasmid p200-M-H

M200V shown in FIG. 5A_HThe small exon of (2) is constructed by using a 200-4 expression plasmid DEF38 IIAI/human G4 chimera as a starting sequence through a PCR mutagenesis technology. Briefly, 200-4V was amplified from the DEF38 IIA 1/human G4 chimera using primers #110 (5'-TTTTCTAGACCACCATGGCTGTCCTGGGGCTGCTT-3') (SEQ ID NO: 36) and #104 (5'-TTTTCTAGAGGTTGTGAGGACTCACCTGAGGAGACGGTGACTGAGGT-3') (SEQ ID NO: 37)_HRegion, primer #110 and 200-4V_HThe 5' end of the signal sequence is complementary, carrying a Kozak sequence and an XbaI site, primer #104 and 200-4V_HThe 3' end of (A) is complementary and carries an XbaI site. The 469 bp PCR fragment was cloned into the pCR4Blunt-TOPO vector (Invitrogen) and the plasmid p200M-VH-2.1 was established by DNA sequencing. This intermediate plasmid was then subjected to secondary PCR mutagenesis to remove potential aberrant splice sites in CDR3 and to generate V_HThe 3' end of the coding region was added to a murine JH4 donor splice site. Two complementary primers #111 (5'-TGGAACTTACTACGGAATGACTACGACGGGG-3') (SEQ ID NO: 38) and #112 (5'-CCCCGTCGTAGTCATTCCGTAGTAAGTTCCA-3') (SEQ ID NO: 38)ID NO: 39) for mixing M200V_HThe codon for glycine 100(Kabat numbering) in CDR3 was mutated from GGT to GGA. Primers #110 and #112 were used to amplify a 395bp fragment at the 5 'end of the M200 VH exon minor by PCR, and a 101bp fragment at the 3' end of the VH exon minor was amplified by PCR using primers #111 and #113 (5'-TTTTCTAGAGGCCATTCTTACCTGAGGAGACGGTGACTGAGGT-3') (SEQ ID NO: 40). The two PCR products were purified by 1.5% low melting agarose gel electrophoresis, mixed and subjected to the final PCR reaction using primers #110 and # 113. The 465 bp product from the last PCR reaction was purified, digested with XbaI and cloned into XbaI and shrimp alkaline phosphatase treated vector pHuHCg4. D. The resulting plasmid p200-M-H (FIG. 6) was DNA sequenced to ensure 200-M V between the two XbaI sites_HThe correctness of the small exon sequence and the correctness of the orientation of the XbaI-XbaI insert.

M200 V_LConstruction of Small exons and plasmid p200-M-L

M200V shown in FIG. 5B_LThe small exon of (2) is constructed by PCR mutagenesis technology by taking 200-4 expression plasmid NEF5IIA1/K as a starting sequence. Amplification of 200-4V from plasmid NEF5IIA1/K using primers #101 (5'-TTTACGCGTCCACCATGGATTTTCAGGTGCAGATT-3') (SEQ ID NO: 41) and #102 (5'-TTTTCTAGATTAGGAAAGTGCACTTACGTTTGATTTCCAGCTTGGTGCC-3') (SEQ ID NO: 42)_LRegion, primer #101 complementary to the 5' end of the signal sequence, carrying a Kozak sequence and a MluI site, primer #102 with 200-4V_LThe 3' end of (A) is complementary and carries an XbaI site. A432 bp PCR fragment was cloned into the pCR4Blunt-TOPO vector (Invitrogen) and the plasmid p200M-VL-3.3 was established by DNA sequencing. This intermediate plasmid was then subjected to secondary PCR mutagenesis to remove potential aberrant splice sites in CDR1 and to generate V_LThe 3' end of the coding region was added with a murine JK1 donor splice site. Two complementary primers #114 (5'-TGCCAGTTCAAGTGTCAGTTCCAATTACTTG-3') (SEQ ID NO: 43) and #115 (5'-CAAGTAATTGGAACTGACACTTGAACTGGCA-3') (SEQ ID NO: 44) were used to amplify V_LCodon for valine 29(Kabat numbering) in CDR1Mutation from GTC to GTA. Primers #110 and #112 were used to amplify a 395bp fragment at the 5 ' end of the M200 VH exon minor by PCR, and V was amplified by PCR using primers #114 and #116 (5'-TTTTCTAGACTTTGGATTCTACTTACGTTTGATTTCCAGCTTGGTGCC-3') (SEQ ID NO: 45)_LA 280bp fragment at the 3' end of the minor exon. The two PCR products were purified by 1.5% low melting agarose gel electrophoresis and mixed for the final PCR using primers #101 and # 116. The 431 bp product from the final PCR reaction was purified and digested with MluI and XbaI and cloned into MluI and XbaI digested light chain expression vector pHuCkappa.rgpt.dE. The resulting plasmid p200-M-L (FIG. 7) was DNA sequenced to ensure V between MluI and XbaI sites_LCorrectness of small exon sequences.

The final expression plasmid p200-M is constructed by using the plasmids p200-M-H and p200-M-L

To construct a single plasmid expressing M200, p200-M-H and p200-M-L were digested with EcoRI, and the EcoRI fragment carrying the complete IgG4 heavy chain gene from p200-M-H was ligated to EcoRI-linearized p200-M-L to form plasmid p200-M (FIG. 8). Endotoxin-Free Plasmid p200-M was prepared in large scale from 2.5 l E.coli cultures using the endo-Free Plasmid Maxi-prep Kit (Qiagen). The correctness of the plasmid structure was confirmed by restriction map of the restriction enzymes BamHI, XbaI and FspI. M200V determination by DNA sequencing_H、V_LCorrectness of the entire coding regions for C and C4. The full-length DNA sequences of M200 heavy chain (SEQ ID NO: 23) and M200 light chain (SEQ ID NO: 24) are shown in FIG. 9. The corresponding amino acid sequences of the M200 heavy chain (SEQ ID NO: 25) and M200 light chain (SEQ ID NO: 26) are shown in FIG. 10.

Example 2 preparation of Fab fragment F200 from M200

This example describes the process of making Fab fragment F200.

Fab fragments were prepared from M200 IgG starting material by enzymatic digestion. The starting IgG was buffer exchanged into a solution containing 20mM sodium phosphate, 20mM N-acetylcysteine, pH 7.0. Soluble papain was added and the mixture was vortexed at 37 ℃ for 4 hours. The mixture was digested and passed through a protein a chromatography column to remove Fc fragments and undigested IgG. 10mM sodium tetrathionate was added and incubated at room temperature for 20 minutes. The resulting solution was buffer exchanged into a solution containing 20mM sodium phosphate, 100mM sodium chloride, pH 7.4 to give a F200 solution.

The DNA sequence and amino acid sequence of the F200 light chain is identical to that of the M200 light chain, since it is a Fab fragment. The complete heavy chain DNA sequence (SEQ ID NO: 27) and amino acid sequence (SEQ ID NO: 28) of F200 are shown in FIG. 11.

Example 3 inhibition of endothelial cell proliferation by M200 in vitro

This example describes the effect of the M200 antibody on endothelial cell proliferation activity. M200 is a highly specific, blocking monoclonal antibody against α 5 β 1 integrin.

HUVECs were seeded into 96-well plates at a density of 5000 cells per well, with different antibodies (M100, M200, anti-VEGF antibody or control IgG) at the concentrations shown in FIG. 14 in the culture medium. The plates were first pretreated with 10. mu.g/mL fibronectin or 0.1% poly-L-lysine (PLL) and blocked with 2% heat-denatured BSA. Cells were cultured in serum-free defined medium containing about 2ng/ml VEGF, bFGF, or both. Total cell viability was determined 4 days after inoculation by the tetrazolium salt MTT (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyl-tetrazolium bromide + + +) assay (see Wasserman & Tventman, "determination of radiosensitivity of murine solid tumor cells by colorimetric microtiter Method (MTT)", Use of assay in diagnostic thermo sensitivity of cell suspensions, Int J radio on biological proteins 15 (3): 699-702 (1988); Romikin, JC, Verkoelen, CF, Schroeder, FH, "Use of MTT assay in vitro cultured human prostate cancer cell lines: determination of assay conditions, evaluation of hormone-stimulated growth effects and drug-induced cytostatic and cytotoxic effects" (T assay of hormone-induced growth effects and drug-induced growth effects) and cytoxic effects), prodate 12 (1): 99-110(1988)). Background values were subtracted from the data and compared to no antibody controls. Each data was taken from 3 replicate wells and the data shown represents 3 independent experiments.

As shown in FIG. 14, M200 inhibited HUVEC growth under PLL and fibronectin (0.40 nM; maximum inhibition 80%) in a dose-dependent manner, whereas control IgG did not. In addition, M100 (murine antibody from which M200 was derived) also exhibited similar cytostatic capacity.

Importantly, as shown in FIG. 14, the high affinity, functionally blocking anti-VEGF monoclonal antibody HuMV833 (K) under various assay conditions (0.40 nM; maximum inhibition 80%)_D＝5.84×10^-11nM) produced insignificant HUVEC growth inhibitory effects. Treatment of cells with M200 and HuMV833 together did not increase the inhibitory effect.

According to the data shown in FIG. 15A, a higher concentration of VEGF (50ng/ml) was included in the HUVEC proliferation assay on fibronectin as described above. As shown in fig. 15A, VEGF-stimulated HUVEC proliferation on fibronectin was inhibited by M200 to the same extent as HuMV 833. Thus, the M200-mediated cytostatic effect was evident, even up to the level of the growth-stimulating factor VEGF (50 ng/ml).

Two high affinity antibodies directed against the idiotypic region of M200 blocked the binding of M200 to α 5 β 1 integrin. The effect of these two anti-idiotype mAbs (10. mu.g/ml) on the growth inhibition of M200-dependent HUVEC was evaluated in the HUVEC proliferation assay described above. Both mAbs reduced the inhibition of HUVEC proliferation by M200 (1. mu.g/ml). As shown in fig. 15B, the inhibitory activity of M200 was completely reversed by the anti-idiotypic mAb to M200.

Taken together, these results indicate that the mechanism of action of M200 to inhibit HUVEC proliferation overlaps somewhat with that of the anti-VEGF antibody HuMV833, but there is also a different counterpart.

Example 4 Effect of M200 on the viability of endothelial cells

This example describes the effect of the M200 antibody on endothelial cell viability.

Antibodies against a particular integrin can induce cell death both in vitro and in vivo. Recently, it was found that the function-blocking α 5 β 1mAb promotes apoptosis of cultured human endothelial cells as a result of annexin V staining, cascade enzyme 3 cleavage and DNA fragmentation.

Similarly, growth of HUVEC cells exposed to M200 or HuMV833 was analyzed using annexin V staining. HUVECs grown in serum-free medium (containing VEGF and bFGF except where noted) were grown in the presence of M200 (10. mu.g/ml), HuMV833 (10. mu.g/ml) or an oncogene inhibitor (5. mu.M positive control). Any cell death was observed under a fluorescent microscope by staining with annexin V-alexa488 (green) and Hoechst 33258 (blue) (FIG. 16A is a fluorescent micrograph). As a parallel experiment, cells were analyzed for cell death by flow cytometry 16 hours after seeding (fig. 16B).

As shown in 16A and 16B, the number of annexin V staining was significantly increased in cells treated with M200, which was not the case in cells treated with HuMV 833. Thus, M200, unlike HuMV833, promotes endothelial cell death.

In addition, the effect of M200 on senescent cells with a weak proliferative capacity was compared. HUVEC were grown in serum and growth factor containing medium (middle row), grown to confluence (left row) or serum and growth factor were removed after logarithmic growth phase (right row), respectively, after inoculation. Cells grown under different conditions were divided into bright groups, one untreated, one treated with M200 (10. mu.g/ml) or an oncogene inhibitor (5. mu.M) for 16 hours, and then stained with annexin V-alexa 488.

As shown in FIG. 17, M200 induced death of dividing HUVEC cells, but not HUVECs that senesced due to contact inhibition or lack of growth factor stimulation. These results indicate that M200 specifically promotes the death of proliferating endothelial cells.

Example 5-inhibition of microtubule formation in vitro by F200

This example describes a microtubule formation assay to demonstrate the angiogenesis inhibitory effect of F200 in vitro. The single cell suspension of HUVEC was mixed with a mixture of human serum and growth factors into a fibrin clot (prepared with fibrinogen and a-thrombin) (0.01 mg/ml rTGF-alpha and 0.1mg/ml VEGF and HGF were added to the medium used in the experiment shown in FIG. 18A; 0.1mg/ml VEGF was added to the medium used in the experiment shown in FIG. 18B; and 0.1mg/ml HGF was added to the medium used in the experiment shown in FIG. 18C). The detection antibody was added to the medium at the concentrations shown in the figure. After 96 hours, individual HUVECs began to migrate, contact other cells and matrix, form cell bundles, and finally form three-dimensional tubular structures. Microtubule formation was analyzed 6 days later by 4% formaldehyde fixation and Alexa 488-phalloidin staining. As can be seen from the graph shown in fig. 18 and the mean fluorescence intensity data, the presence of F200 can significantly inhibit the formation of microtubules. Inhibition of microtubule formation is also observed in the presence of the growth factors VEGF, HGF and mixtures of these factors with rtfa.

Example 6-M200 and F200 inhibit CNV in the eyes of primates in vivo

This example describes the effect of M200 and F200Fab on blood vessel formation in the eye of primates after laser stimulation of the macula. Background literature reporting studies of animal models of choroidal neovascularization include: ryan, "establishment of a Model for testing Subretinal Neovascularization after macular degeneration of The ellipse" (The Development of experimental Model of The Experimental neurological of The cardiovascular in The Disform), Transactions of The American Ophthalmological Society77: 707 and 745 (1979); s.j.ryan, "subretinal neovascularization: natural development history of the test model "(Subretin Neovascularation: Natural His)tory of an Experimental Model)，Archivesof Ophthalmology100: 1804-; tolentino et al, "Angiography Using Fluorescentized Anti-Vascular endothelial growth Factor antibodies and dextran in an Experimental Choroidal Neovascularization model" (Angiography of Fluorescentized Anti-Vascular endothelial growth Factor and dextran), Archives of Ophthematology118：78-84(2000)。

A. Design of experiments

The 8 monkeys were divided into treatment groups as shown in the following table and treated as follows.

Group of	N	Treatment (left eye)	Treatment (Right eye)
				1	2	Untreated	Buffer (50. mu.l)
2	2	M200(1μM；50μL)	M200(1μM；50μL)
				3	2	F200(1μM；50μL)	F200(1μM；50μL)
4	1	Control (Rituxan 1. mu.M; 50. mu.L)	M200(1μM；50μL)
				5	1	Control (Rituxan 1. mu.M; 50. mu.L)	F200(1μM；50μL)

M200 and F200 were dissolved in carrier buffer for administration. Rituxan was used as a control. On day 1, Choroidal Neovascularization (CNV) was induced by laser irradiation of the macula of both eyes of each animal as described below. All animals were treated with M200, F200 or control at the doses indicated in the table, once weekly for 4 consecutive weeks. The first day of administration was designated as day 1. The animals were observed for changes in clinical performance, body weight and other indicators using standard methods. All animals were sacrificed at 28 days.

Laser-induced Choroidal Neovascularization (CNV)

Animals were fasted overnight before laser irradiation and dosing. Animals were sedated with ketamine hydrochloride (intramuscular injection until onset), followed by intravenous injection of ketamine and sedation (after onset), followed by laser irradiation and administration.

Choroidal Neovascularization (CNV) can be induced by laser irradiation of the macula of both eyes. Injury was caused by irradiation of the macula with a laser [ OcuLight GL (532 nm) laser coagulator with IRIS Medical ® portable slit lamp adapter ] in a standard 9-point grid pattern. The laser spot for the right eye may be reflected to the site of the left eye. The approximate parameters of the laser settings are as follows: spot size: 50-100 μm; power of the laser: 300-700 milliwatts; irradiation time: 0.1 second. The parameters irradiated by each animal were recorded while laser irradiation was performed. Photographs were taken using a TRC-50EX Ret ina camera with a digital CCD camera and/or a SL-4Ed slit lamp.

Dosage to be administered

The intravitreal injection of immunoglobulin (test group) or control was administered to each eye. The drug was injected immediately after laser irradiation on the first day. Mydriatic drug (1% tropicamide) was instilled in the eyes before administration. The eye is rinsed with a dilute antibiotic solution (5% povidone iodine solution or the like) and then the antibiotic is washed away with 0.9% sterile saline (or the like) and two drops of local anesthetic (proparacaine or equivalent) are dropped into the eye. During the operation, an eyelid dilator is inserted to open the eyelid and retract the eyeball. The syringe needle passed through the sclera and plateau approximately 4mm back from the limbus. The needle is directed back through the lens into the middle of the vitreous. The test article was slowly injected into the vitreous. Forceps are used to grasp the conjunctiva around the needle before it is withdrawn. Forceps are used to hold the conjunctiva during or shortly after withdrawal of the needle. The eyelid dilator is then removed. Immediately after dosing, the eyes were examined with an indirect ophthalmoscope to see any apparent post-dose problems. Topical antibiotics (Tobrex) administered and immediately one day after administration^®Or an equivalent drug) is administered to each eye. The animals were returned to their cages after being fully awake.

The drugs were administered once weekly according to the dosing schedule set forth in the following table:

group of	Animal number (M/F)	Test piece (left eye)	Dosage form	Test piece (Right eye)	Dosage form	Volume of administration (L/eye)
							1	1/1	Is free of	NA	Buffer solution	0	50
2	1/1	M200	300g	M200	300g	50
							3	1/1	F200	100g	F200	100g	50
4	1/0	Control	100g	M200	300g	50
							5	1/0	Control	100g	F200	100g	50

The amount of drug administered to each eye is given in grams as indicated in the table. Assuming an average volume of 2ml for one eye, the dose per eye is about 150 μ g/ml M200 and about 50 μ g/ml F200. In both cases, the molar concentration of M200 or F200 was l μ M.

D. Monitoring inhibition of angiogenesis

An indirect ophthalmoscope is used to examine the posterior chamber and a biomicroscope is used to examine the anterior half of the eye. Eyes were scored using standard methods (Robert b.hackett and t.o.mcdonald.1996, dermotoxicy, 5 th edition, ed.b.marzulli and h.i.maibach eds., HemispherePublishing corp., Washington, d.c.).

Fluorescein angiography was performed before lesion formation, 5, 12, 19 and 26 days after lesion formation and at the beginning of treatment. Animals were administered ketamine and diazepam (approximately 10mg/kg ketamine and 0.5mg/kg diazepam, i.v.) to keep them quiet. The eyelid expanders retract the eyelids. In the presence of fluorescenceBefore the pigment dye, the animals were placed in an ophthalmic chair and kept in head position while taking pictures. The photographs were taken with a fundus camera (TRC-50EX Retina camera). Using TOPCON IMAGEnet^TMThe system captures an image. Fluorescein dye (10% fluorescein sodium, ca. 0.1mL/kg) was injected via the cephalic vein or saphenous vein. Color and black-and-white photographs were taken at several time points after dye injection, including arterial, early and several late arteriovenous phases to monitor fluorescein leakage due to CNV injury. The unmodified image is transferred to disk for storage and transport.

Alternatively, the eyes may be photographed (TRC-50EX Retina camera and/or SL-4ED slit lamp with digital CCD camera). Animals may be sedated with ketamine hydrochloride prior to taking a photograph and a few drops of mydriatic solution (typically 1% tropicamide) are dropped into the eyes for examination.

E. Results

The presence of CNV at day 13 and day 20 is clearly indicated by analysis of the fluorescein angiography images of these several groups. The control group also knows that CNV was present at day 28 (e.g., groups 1, 4 (left eye) and 5 (left eye)). While eyes treated with M200 and F200 had significantly reduced CNV (e.g., 2, 3,4 groups (right eye) and 5 groups (left eye)). As shown in fig. 19, the M200 treated eyes had fewer CNV symptoms at day 20 compared to the control group.

FIGS. 22-25 show the effect of M200 and F200 on CNV in the right eye of each monkey at days 13, 20 and 27 and the effect of controls on CNV in the left eye of the same monkey. The CNV was significantly reduced in eyes treated with M200 or F200 compared to untreated eyes. Whereas the degree of CNV reduction in the F200 treated eyes was more pronounced. It is estimated that this apparent difference is due to the entry of M200 into the untreated left eye via the bloodstream. Treatment of the right eye with M200 also inhibited CNV in the left eye, resulting in insignificant differences between eyes. In contrast, the difference in CNV inhibitory effect between the eyes of the same animal when M200 was not leaked into the untreated eyes was more pronounced.

Example 7 binding affinities of M200, F200 and humanized mutants

A. Kinetic analysis by surface plasmon resonance

The affinity of AAB1/B2Fc to IIA1, M200 or F200 was analyzed using BIAcore 3000 and 2000(BIAcore, Sweden). IIA1, M200, or F200 were immobilized on a Pioneer F1 chip using a standard amine coupling kit (BIAcore). Surface plasmon resonance was measured at a flow rate of 50ul/min at 24 ℃. AAB1/B2Fc (binding phase) was injected within 180 seconds. The dissociation was monitored over the following 3 hours. The binding kinetics parameters were calculated using the BIAevaluation program for the data obtained at 5 concentrations (320nM, 160nM, 80nM, 40nM, 20 nM). Reactions generated by the reference surface and buffer control were removed using a dual reference. The K can be obtained by simultaneously analyzing the binding phase and the dissociation phase of the series of concentration induction spectrums_D. K of M200_DIs 0.367. + -. 0.132nM, K of F200_DWas 0.332. + -. 0.065 nM.

B. Determination of affinity of HuM200 by competitive ELISA assay

The binding affinity of HuM200 relative to IIA1 and M200 was determined by ELISA competition binding assays.

A96-well ELISA plate (Nunc-Immuno Maxisorp plate, NalgeNunc, Naperville, IL) was coated with 100. mu.l of 0.2M sodium carbonate-sodium bicarbonate buffer (pH 9.4) containing 1.0. mu.g/ml of soluble recombinant human α 5 β 1 integrin-Fc fusion protein per well overnight at 4 ℃. After washing with washing Buffer (PBS containing 0.1% Tween 20), the cells were blocked with 200. mu.l Superblock Blocking Buffer (Pierce) for 30 minutes, and then washed with washing Buffer. A mixture of biotin-labeled murine IIA1 (0.1. mu.g/test) and competing antibodies (duplicate wells, 3-fold serial dilutions of competing antibodies, starting at 5mg/ml) in ELISA buffer (PBS containing 1% BSA and 0.1% Tween 20) was added to the ELISA plates in a total volume of 100. mu.l per well. The ELISA plate was incubated at room temperature for 1 hour and then washed with wash buffer. Mu.l of ELISA buffer containing 1/1,000 diluted HRP-linked streptavidin (Pierce, Rockford, IL) was added to each well. After incubation at room temperature for 30 minutes, the cells were washed with washing buffer and 100. mu.l of TMB substrate was added. Absorbance at 450nm was measured using a VERSAmax microplate reader (Molecular Devices, Menlo Park, Calif.). The final concentration of competing antibody in the reaction was plotted against the absorbance value at 450 nm.

HuM200 contains the heavy and light chain amino acid sequences shown in FIG. 13 (SEQ ID NOS: 31 and 32). HuM200 (also referred to as HuM200-G4) contains the constant region of IgG 4. The second humanized version of M200, HuM200-g2M3G, contained the same variable regions as HuM200, but contained the constant regions of IgG 2.

As shown in FIG. 26, both versions of the humanized M200 antibody HuM200-G4 and HuM200-G2M3G have substantially the same binding affinity curves as M200. Furthermore, the IC50 values of HuM200-G4 and HuM200-G2m3G were 131.8. mu.g/ml and 102.8. mu.g/ml, respectively. These values are associated with the IC of M200₅₀The value (106.3. mu.g/ml) is comparable, but slightly higher than the IC of IIA1₅₀Value (79.1. mu.g/ml).

Sequence listing

<110> Protein Design laboratories, Inc. (Protein Design Labs, Inc.)

V. Lanmakris south (Ramakrishan, Vanitha)

D. Palls (Powers, David)

D.E. Johnson (Johnson, Dale E)

U.Jeffry (Jeffry, Ursula)

Bohansca (Bhaskar, Vinay)

<120> chimeric and humanized antibodies to α 5 β 1 integrin that modulate angiogenesis

<130>05882.0178.00PC00

<140>PCT/US2003/038172

<141>2003-11-26

<150>60/429,743

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Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu

85 90 95

Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His

100 105 110

Gln Tyr Leu Arg Ser Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu

115 120 125

Ile Lys

130

<210>19

<211>459

<212>DNA

<213> Artificial

<220>

<223> chimeric antibody

<400>19

tctagaccac catggctgtc ctggggctgc ttctctgcct ggtgactttc ccaagctgtg 60

tcctgtccca ggtgcagctg aaggagtcag gacctggcct ggtggcgccc tcacagagcc 120

tgtccatcac atgcaccatc tcagggttct cattaaccga ctatggtgtt cactgggttc 180

gccagcctcc aggaaagggt ctggagtggc tggtagtgat ttggagtgat ggaagctcaa 240

cctataattc agctctcaaa tccagaatga ccatcaggaa ggacaactcc aagagccaag 300

ttttcttaat aatgaacagt ctccaaactg atgactcagc catgtactac tgtgccagac 360

atggaactta ctacggaatg actacgacgg gggatgcttt ggactactgg ggtcaaggaa 420

cctcagtcac cgtctcctca ggtaagaatg gcctctaga 459

<210>20

<211>143

<212>PRT

<213> Artificial

<220>

<223> chimeric antibody

<400>20

Met Ala Val Leu Gly Leu Leu Leu Cys Leu Val Thr Phe Pro Ser Cys

1 5 10 15

Val Leu Ser Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala

20 25 30

Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Ile Ser Gly Phe Ser Leu

35 40 45

Thr Asp Tyr Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu

50 55 60

Glu Trp Leu Val Val Ile Trp Ser Asp Gly Ser Ser Thr Tyr Asn Ser

65 70 75 80

Ala Leu Lys Ser Arg Met Thr Ile Arg Lys Asp Asn Ser Lys Ser Gln

85 90 95

Val Phe Leu Ile Met Asn Ser Leu Gln Thr Asp Asp Ser Ala Met Tyr

100 105 110

Tyr Cys Ala Arg His Gly Thr Tyr Tyr Gly Met Thr Thr Thr Gly Asp

115 120 125

Ala Leu Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

130 135 140

<210>21

<211>425

<212>DNA

<213> Artificial

<220>

<223> chimeric antibody

<400>21

acgcgtccac catggatttt caggtgcaga ttttcagctt cctgctaatc agtgcctcag 60

tcataatgtc cagaggacaa attgttctca cccagtctcc agcaatcatg tctgcatctc 120

taggggaacg ggtcaccatg acctgcactg ccagttcaag tgtcagttcc aattacttgc 180

actggtacca gcagaagcca ggatccgccc ccaatctctg gatttatagc acatccaacc 240

tggcttctgg agtcccagct cgtttcagtg gcagtgggtc tgggacctct tactctctca 300

caatcagcag catggaggct gaagatgctg ccacttatta ctgccaccag tatcttcgtt 360

ccccaccgac gttcggtgga ggcaccaagc tggaaatcaa acgtaagtag aatccaaagt 420

ctaga 425

<210>22

<211>130

<212>PRT

<213> Artificial

<220>

<223> chimeric antibody

<400>22

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Met Ser Arg Gly Gln Ile Val Leu Thr Gln Ser Pro Ala Ile

20 25 30

Met Ser Ala Ser Leu Gly Glu Arg ValThr Met Thr Cys Thr Ala Ser

35 40 45

Ser Ser Val Ser Ser Asn Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly

50 55 60

Ser Ala Pro Asn Leu Trp Ile Tyr Ser Thr Ser Asn Leu Ala Ser Cly

65 70 75 80

Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu

85 90 95

ThrIle Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His

l00 105 110

Gln Tyr Leu Arg Ser Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu

115 120 125

Ile Lys

130

<210>23

<211>1353

<212>DNA

<213> Artificial

<220>

<223> chimeric antibody

<400>23

caggtgcagc tgaaggagtc aggacctggc ctggtggcgc cctcacagag cctgtccatc 60

acatgcacca tctcagggtt ctcattaacc gactatggtg ttcactgggt tcgccagcct 120

ccaggaaagg gtctggagtg gctggtagtg atttggagtg atggaagctc aacctataat 180

tcagctctca aatccagaat gaccatcagg aaggacaact ccaagagcca agttttctta 240

ataatgaaca gtctccaaac tgatgactca gccatgtact actgtgccag acatggaact 300

tactacggaa tgactacgac gggggatgct ttggactact ggggtcaagg aacctcagtc 360

accgtctcct cagcttccac caagggccca tccgtcttcc ccctggcgcc ctgctccagg 420

agcacctccg agagcacagc cgccctgggc tgcctggtca aggactactt ccccgaaccg 480

gtgacggtgt cgtggaactc aggcgccctg accagcggcg tgcacacctt cccggctgtc 540

ctacagtcct caggactcta ctccctcagc agcgtggtga ccgtgccctc cagcagcttg 600

ggcacgaaga cctacacctg caacgtagat cacaagccca gcaacaccaa ggtggacaag 660

agagttgagt ccaaatatgg tcccccatgc ccatcatgcc cagcacctga gttcctgggg 720

ggaccatcag tcttcctgtt ccccccaaaa cccaaggaca ctctcatgat ctcccggacc 780

cctgaggtca cgtgcgtggt ggtggacgtg agccaggaag accccgaggt ccagttcaac 840

tggtacgtgg atggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagttc 900

aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaacggc 960

aaggagtaca agtgcaaggt ctccaacaaa ggcctcccgt cctccatcga gaaaaccatc 1020

tccaaagcca aagggcagcc ccgagagcca caggtgtaca ccctgccccc atcccaggag 1080

gagatgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta ccccagcgac 1140

atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1200

gtgctggact ccgacggctc cttcttcctc tacagcaggc taaccgtgga caagagcagg 1260

tggcaggagg ggaatgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1320

acacagaaga gcctctccct gtctctgggt aaa 1353

<210>24

<211>645

<212>DNA

<213> Artificial

<220>

<223> chimeric antibody

<400>24

caaattgttc tcacccagtc tccagcaatc atgtctgcat ctctagggga acgggtcacc 60

atgacctgca ctgccagttc aagtgtaagt tccaattact tgcactggta ccagcagaag 120

ccaggatccg cccccaatct ctggatttat agcacatcca acctggcttc tggagtccca 180

gctcgtttca gtggcagtgg gtctgggacc tcttactctc tcacaatcag cagcatggag 240

gctgaagatg ctgccactta ttactgccac cagtatcttc gttccccacc gacgttcggt 300

ggaggcacca agctggaaat caaacgaact gtggctgcac catctgtctt catcttcccg 360

ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc 420

tatcccagag aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 480

caggagagtg tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg 540

acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600

ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgt 645

<210>25

<211>451

<212>PRT

<213> Artificial

<220>

<223> chimeric antibody

<400>25

Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Ile Ser Gly Phe Ser Leu Thr Asp Tyr

20 25 30

Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Val Val Ile Trp Ser Asp Gly Ser Ser Thr Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Met Thr Ile Arg Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Ile Met Asn Ser Leu Gln Thr Asp Asp Ser Ala Met Tyr Tyr Cys Ala

85 90 95

Arg His Gly Thr Tyr Tyr Gly Met Thr Thr Thr Gly Asp Ala Leu Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu

130 135 140

Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn

195 200 205

Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser

210 215 220

Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln

260 265 270

Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Leu Gly Lys

450

<210>26

<211>215

<212>PRT

<213> Artificial

<220>

<223> chimeric antibody

<400>26

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val Ser Ser Asn

20 25 30

Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Ser Ala Pro Asn Leu Trp

35 40 45

Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu

65 70 75 80

Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Tyr Leu Arg Ser Pro

85 90 95

Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210>27

<211>696

<212>DNA

<213> Artificial

<220>

<223> chimeric antibody

<400>27

caggtgcagc tgaaggagtc aggacctggc ctggtggcgc cctcacagag cctgtccatc 60

acatgcacca tctcagggtt ctcattaacc gactatggtg ttcactgggt tcgccagcct 120

ccaggaaagg gtctggagtg gctggtagtg atttggagtg atggaagctc aacctataat 180

tcagctctca aatccagaat gaccatcagg aaggacaact ccaagagcca agttttctta 240

ataatgaaca gtctccaaac tgatgactca gccatgtact actgtgccag acatggaact 300

tactacggaa tgactacgac gggggatgct ttggactact ggggtcaagg aacctcagtc 360

accgtctcct cagcttccac caagggccca tccgtcttcc ccctggcgcc ctgctccagg 420

agcacctccg agagcacagc cgccctgggc tgcctggtca aggactactt ccccgaaccg 480

gtgacggtgt cgtggaactc aggcgccctg accagcggcg tgcacacctt cccggctgtc 540

ctacagtcct caggactcta ctccctcagc agcgtggtga ccgtgccctc cagcagcttg 600

ggcacgaaga cctacacctg caacgtagat cacaagccca gcaacaccaa ggtggacaag 660

agagttgagt ccaaatatgg tcccccatgc ccatca 696

<210>28

<211>232

<212>PRT

<213> Artificial

<220>

<223> chimeric antibody

<400>28

Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Ile Ser Gly Phe Ser Leu Thr Asp Tyr

20 25 30

Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Val Val Ile Trp Ser Asp Gly Ser Ser Thr Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Met Thr Ile Arg Lys Asp Ash Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Ile Met Asn Ser Leu Gln Thr Asp Asp Ser Ala Met Tyr Tyr Cys Ala

85 90 95

Arg His Gly Thr Tyr Tyr Gly Met Thr Thr Thr Gly Asp Ala Leu Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu

130 135 140

Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn

195 200 205

Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser

210 215 220

Lys Tyr Gly Pro Pro Cys Pro Ser

225 230

<210>29

<211>1353

<212>DNA

<213> Artificial

<220>

<223> chimeric antibody

<400>29

gaggtgcagc tggtggagtc aggaggaggc ctggtgcagc ccggaggaag cctgagactg 60

tcatgcgccg cctcagggtt ctcattaacc gactatggtg ttcactgggt tcgccaggcc 120

ccaggaaagg gtctggagtg gctggtggtg atttggagtg atggaagctc aacctataat 180

tcagctctca aatccagaat gaccatctca aaggacaacg ccaagaacac cgtgtactta 240

cagatgaaca gtctcagagc tgaggacacc gccgtgtact actgtgccag acatggaact 300

tactacggaa tgactacgac gggggatgct ttggactact ggggtcaagg aaccctggtc 360

accgtctcct cagcttccac caagggccca tccgtcttcc ccctggcgcc ctgctccagg 420

agcacctccg agagcacagc cgccctgggc tgcctggtca aggactactt ccccgaaccg 480

gtgacggtgt cgtggaactc aggcgccctg accagcggcg tgcacacctt cccggctgtc 540

ctacagtcct caggactcta ctccctcagc agcgtggtga ccgtgccctc cagcagcttg 600

ggcacgaaga cctacacctg caacgtagat cacaagccca gcaacaccaa ggtggacaag 660

agagttgagt ccaaatatgg tcccccatgc ccatcatgcc cagcacctga gttcctgggg 720

ggaccatcag tcttcctgtt ccccccaaaa cccaaggaca ctctcatgat ctcccggacc 780

cctgaggtca cgtgcgtggt ggtggacgtg agccaggaag accccgaggt ccagttcaac 840

tggtacgtgg atggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagttc 900

aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaacggc 960

aaggagtaca agtgcaaggt ctccaacaaa ggcctcccgt cctccatcga gaaaaccatc 1020

tccaaagcca aagggcagcc ccgagagcca caggtgtaca ccctgccccc atcccaggag 1080

gagatgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta ccccagcgac 1140

atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1200

gtgctggact ccgacggctc cttcttcctc tacagcaggc taaccgtgga caagagcagg 1260

tggcaggagg ggaatgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1320

acacagaaga gcctctccct gtctctgggt aaa 1353

<210>30

<211>645

<212>DNA

<213> Artificial

<220>

<223> chimeric antibody

<400>30

gaaattgttc tcacccagtc tccagcaacc ctctctctct ctccggggga acgggctacc 60

ctctcctgca ctgccagttc aagtgtcagt tccaattact tgcactggta ccagcagaag 120

ccaggacagg ccccccgtct cctcatttat agcacatcca acctggcttc tggagtccca 180

gctcgtttca gtggcagtgg gtctgggacc tcttacaccc tcacaatcag cagcctcgag 240

ccagaagatt tcgccgtcta ttactgccac cagtatcttc gttccccacc gacgttcggt 300

ggaggcacca aggtcgaaat caaacgaact gtggctgcac catctgtctt catcttcccg 360

ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc 420

tatcccagag aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 480

caggagagtg tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg 540

acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600

ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgt 645

<210>31

<211>451

<212>PRT

<213> Artificial

<220>

<223> chimeric antibody

<400>31

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Thr Asp Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Val Val Ile Trp Ser Asp Gly Ser Ser Thr Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Met Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg His Gly Thr Tyr Tyr Gly Met Thr Thr Thr Gly Asp Ala Leu Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu

130 135 140

Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn

195 200 205

Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser

210 215 220

Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln

260 265 270

Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Leu Gly Lys

450

<210>32

<211>215

<212>PRT

<213> Artificial

<220>

<223> chimeric antibody

<400>32

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

l 5 10 15

Glu Arg Ala Thr Leu Ser Cys Thr Ala Ser Ser Ser Val Ser Ser Asn

20 25 30

Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Ser Tyr Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Tyr Leu Arg Ser Pro

85 90 95

Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210>33

<211>6

<212>DNA

<213> Artificial

<220>

<223> oligonucleotide

<400>33

ctcgag 6

<210>34

<211>6

<212>DNA

<213> Artificial

<220>

<223> oligonucleotide

<400>34

tctaga 6

<210>35

<211>6

<212>DNA

<213> Artificial

<220>

<223> oligonucleotide

<400>35

acgcgt 6

<210>36

<211>35

<212>DNA

<213> Artificial

<220>

<223> oligonucleotide

<400>36

ttttctagac caccatggct gtcctggggc tgctt 35

<210>37

<211>47

<212>DNA

<213> Artificial

<220>

<223> oligonucleotide

<400>37

ttttctagag gttgtgagga ctcacctgag gagacggtga ctgaggt 47

<210>38

<211>31

<212>DNA

<213> Artificial

<220>

<223> oligonucleotide

<400>38

tggaacttac tacggaatga ctacgacggg g 31

<210>39

<211>31

<212>DNA

<213> Artificial

<220>

<223> oligonucleotide

<400>39

ccccgtcgta gtcattccgt agtaagttcc a 31

<210>40

<211>43

<212>DNA

<213> Artificial

<220>

<223> oligonucleotide

<400>40

ttttctagag gccattctta cctgaggaga cggtgactga ggt 43

<210>41

<211>35

<212>DNA

<213> Artificial

<220>

<223> oligonucleotide

<400>41

tttacgcgtc caccatggat tttcaggtgc agatt 35

<210>42

<211>49

<212>DNA

<213> Artificial

<220>

<223> oligonucleotide

<400>42

ttttctagat taggaaagtg cacttacgtt tgatttccag cttggtgcc 49

<210>43

<211>31

<212>DNA

<213> Artificial

<220>

<223> oligonucleotide

<400>43

tgccagttca agtgtcagtt ccaattactt g 31

<210>44

<211>31

<212>DNA

<213> Artificial

<220>

<223> oligonucleotide

<400>44

caagtaattg gaactgacac ttgaactggc a 31

<210>45

<211>48

<212>DNA

<213> Artificial

<220>

<223> oligonucleotide

<400>45

ttttctagac tttggattct acttacgttt gatttccagc ttggtgcc 48

<210>46

<211>143

<212>PRT

<213> mouse (mus musculus)

<400>46

Met Ala Val Leu Gly Leu Leu Leu Cys Leu Val Thr Phe Pro Ser Cys

1 5 10 15

Val Leu Ser Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala

20 25 30

Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Ile Ser Gly Phe Ser Leu

35 40 45

Thr Asp Tyr Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu

50 55 60

Glu Trp Leu Val Val Ile Trp Ser Asp Gly Ser Ser Thr Tyr Ash Ser

65 70 75 80

Ala Leu Lys Ser Arg Met Thr Ile Arg Lys Asp Asn Ser Lys Ser Gln

85 90 95

Val Phe Leu Ile Met Asn Ser Leu Gln Thr Asp Asp Ser Ala Met Tyr

100 105 110

Tyr Cys Ala Arg His Gly Thr Tyr Tyr Gly Met Thr Thr Thr Gly Asp

115 120 125

Ala Leu Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

130 135 140

<210>47

<211>130

<212>PRT

<213> mouse (mus musculus)

<400>47

Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Met Ser Arg Gly Gln Ile Val Leu Thr Gln Ser Pro Ala Ile

20 25 30

Met Ser Ala Ser Leu Gly Glu Arg Val Thr Met Thr Cys Thr Ala Ser

35 40 45

Ser Ser Val Ser Ser Asn Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly

50 55 60

Ser Ala Pro Asn Leu Trp Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly

65 70 75 80

Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu

85 90 95

Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His

100 105 110

Gln Tyr Leu Arg Ser Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu

115 120 125

Ile Lys

130

Claims (42)

1.A chimeric anti- α 5 β 1 integrin antibody, comprising;

(a) comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 7; and

(b) comprising the constant region sequence of an antibody of a second origin, said second origin of constant region sequence being human IgG.

2. The antibody of claim 1, wherein the second source of constant region sequences is human IgG2 or human IgG 4.

3. The antibody of claim 1, wherein the second source of constant region sequence is human IgG 4.

4. A chimeric anti- α 5 β 1 integrin antibody, which antibody;

comprising a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NO: 1. 16 and 20, and the light chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 7. 18 and 22; and

wherein the antibody inhibits VEGF-stimulated neovascularization.

5. The antibody of claim 4, wherein said heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 1, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 7.

6. The antibody of claim 4, wherein said heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 16, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 18.

7. The antibody of claim 4, wherein said heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 20, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 22.

8. An anti- α 5 β 1 integrin antibody, which antibody;

comprising a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NO: 25 and 28, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 26.

9. The antibody of claim 8, wherein said heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 25, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 26.

10. The antibody of claim 8, wherein said heavy chain variable region comprises the amino acid sequence of SEQ id no: 28, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 26.

11. A humanized anti- α 5 β 1 integrin antibody, which antibody;

comprising a heavy chain variable region having a heavy chain variable region corresponding to SEQ ID NO: 1, and the light chain variable region comprises an amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO: 7 has an amino acid sequence having at least 75% identity.

12. The antibody of claim 11, wherein said heavy chain variable region comprises the amino acid sequence of SEQ id no: 2, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 7.

13. The antibody of claim 11, wherein said heavy chain variable region comprises the amino acid sequence of SEQ id no: 3, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 9.

14. The antibody of claim 11, wherein said heavy chain variable region comprises the amino acid sequence of SEQ id no: 4, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 10.

15. The antibody of claim 11, wherein said heavy chain variable region comprises the amino acid sequence of SEQ id no: 5, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 11.

16. The antibody of claim 11, wherein said heavy chain variable region comprises the amino acid sequence of SEQ id no: 6, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 12.

17. A humanized anti- α 5 β 1 integrin antibody which:

comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 32.

18. A chimeric, or humanized, anti- α 5 β 1 integrin antibody, which is a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12, 16, 18, 20, 22, 25-26, 28, 31-32.

19. A pharmaceutical composition comprising an anti- α 5 β 1 integrin antibody of any one of claims 1-18.

20. Use of an antibody according to any one of claims 1 to 18 for the manufacture of a medicament for reducing or inhibiting angiogenesis.

21. The use of claim 20, wherein the medicament is for the treatment of ocular diseases associated with angiogenesis.

22. The use as claimed in claim 21 wherein said ocular disease is selected from diabetic retinopathy, macular degeneration, and ocular tissue vascularization.

23. Use of an antibody according to any one of claims 1 to 18 for the manufacture of a medicament for the treatment of a growth factor-related eye disease.

24. The use as claimed in claim 23, wherein said ocular disease is selected from diabetic retinopathy, macular degeneration, and ocular tissue vascularization.

25. The use of claim 24, wherein said growth factor is VEGF.

26. Use of an antibody according to any one of claims 1 to 18 for the manufacture of a medicament for the treatment of cancer.

27. The use of claim 25, wherein the cancer is selected from the group consisting of lung cancer, breast cancer, prostate cancer, cervical cancer, pancreatic cancer, colon cancer, and ovarian cancer.

28. Use of an antibody according to any one of claims 1 to 18 for the manufacture of a medicament for reducing or inhibiting endothelial cell proliferation.

29. Use of an antibody according to any one of claims 1 to 18 for the preparation of a medicament for inducing death of proliferating endothelial cells.

30. A polypeptide comprising one or more amino acid sequences selected from the group consisting of: SEQ ID NOS: 1-12, 16, 18, 20, 22, 25, 26, 28, 31, and 32.

31. A nucleic acid encoding the polypeptide of claim 30.

32. The nucleic acid of claim 31, wherein the nucleic acid is selected from the group consisting of: SEQ ID NO: 15. 17, 19, 21, 23, 24, 27, 29 and 30.

33. A vector comprising the nucleic acid of claim 31 or 32.

34. A host cell comprising the vector of claim 33.

35. A method of purifying a pH-sensitive anti- α 5 β 1 integrin antibody, the method comprising:

(a) adsorbing an antibody according to any one of claims 1 to 18 to an antibody affinity matrix bound to a substrate; and

(b) the antibody is eluted from the substrate-bound antibody affinity matrix with an eluent having a pH of about 3.0 to 5.5.

36. The method of claim 35, further comprising the step of:

(c) recovering the purified antibody.

37. The method of claim 36, further comprising the step of neutralizing the eluted solution containing the eluted antibody with a basic solution, wherein the pH of the neutralizing solution is between about 6.0 and 8.0.

38. The method of claim 35 wherein the pH of the eluent is about 3.3-5.5.

39. The method of claim 35 wherein the pH of the eluent is about 3.5 to about 5.5.

40. The method of claim 35 wherein the pH of the eluent is about 3.5-4.2.

41. The method of claim 35 wherein the pH of the eluent is about 4.2-5.5.

42. The method of claim 35, wherein the antibody affinity matrix is selected from the group consisting of: polypeptides, polysaccharides, fatty acids, lipids, aptamers, glycoproteins, lipoproteins, glycolipids, polyprotein complexes, biological membranes, viruses, protein a, protein G, lectins, and Fc receptors.