WO2001062930A1 - Activated vitronectin as a marker of angiogenesis detected with phage antibodies - Google Patents
Activated vitronectin as a marker of angiogenesis detected with phage antibodies Download PDFInfo
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- WO2001062930A1 WO2001062930A1 PCT/NL2001/000157 NL0100157W WO0162930A1 WO 2001062930 A1 WO2001062930 A1 WO 2001062930A1 NL 0100157 W NL0100157 W NL 0100157W WO 0162930 A1 WO0162930 A1 WO 0162930A1
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- vitronectin
- antibody
- activated
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
Definitions
- the invention relates generally to medicine and the use of antibodies .
- the invention relates to antibody fragments (and derivatives thereof) which have a higher affinity for activated vitronectin than for native vitronectin. Said fragments can be used as markers of angiogenesis and other vitronectin-related diseases.
- Antibodies are proteins that are produced by cells of the immune system, so-called B lymphocytes, in response to encounter with potentially harmful miro-organisms such as bacteria and viruses. Antibodies are capable of binding to the invading micro-organism that elicited their production and aid in their elimination. This activity of the immune system of producing antibodies in response to an invading micro-organism has been exploited in the production of monoclonal antibodies, a technology developed by K ⁇ hler and Milstein (1975) . Monoclonal antibodies are all those immunoglobulin molecules that are produced by the progeny of a single B lymphocyte.
- monoclonal antibodies are obtained by immunizing a mouse with an antigen and fusing the spleen or ly phnode B lymphocytes with an immortalized murine plasma cell line.
- the ensuing hybrid cell lines bear the characteristics of both parental cell types: they are immortal and produce a single species of monoclonal antibody specific for the antigen used to immunize the mouse.
- the advantage of monoclonal antibodies is that they represent a homogeneous population of immunoglobulin molecules with a pre-defined binding specificity.
- monoclonal antibodies have proven invaluable tools in research and diagnostics. Soon after the invention of hybridoma technology, the enormous potential of monoclonal antibodies in human therapy was realized.
- monoclonal antibodies were hypothesized, among others, to be capable of binding to viruses and bacteria and their toxic products facilitati lg their elimination.
- monoclonal antibodies were envisaged to specifically bind to tumor cells to promote their eradication or to bind to soluble molecules produced by cells of the immune system to neutralize their activity in harmful chronic inflammatory conditions and/or in autoimmune disease.
- monoclonal antibodies have been described as magic bullets that could be used in the treatment of a wide variety of human diseases .
- the immunoglobulin variable regions encoding murine antibodies are genetically used to human immunoglobulin constant regions.
- the resulting chimeric antibody still contains >30% murine amino acid sequences.
- Clinical application in humans of chimeric monoclonal antibodies has shown that these proteins retain their immunogenicity in the majority of cases.
- only immunoglobulin variable region sequences relevant for antibody specificity are of murine origin; the constant regions of the immunoglobulin molecule as well as the framework regions of the variable region are of human origin.
- Clinical application of these "humanized" monoclonal antibodies indicates that these molecules are generally more effective and have no or little intrinsic toxicity or immunogenicity.
- One method of obtaining human monoclonal antibodies employs transgenic mice harboring human immunoglobulin loci in combination with conventional hybridoma technology. In these mice, large portions of human immunoglobulin heavy and light chain loci have been inserted in the mouse germ line while the endogenous murine immunoglobulin loci have been silenced by gene knockout. Immunization of these transgenic mice with an antigen results in the production of human antibodies specific for the antigen.
- Human monoclonal antibody-producing cell lines can be obtained from these mice by fusing the spleen cells of immunized mice with plasma cell lines in vitro to obtain immortalized monoclonal antibody- secreting hybridomas .
- phage display libraries an in vi tro, recombinant DNA-based approach that mimics key features of the humoral immune response.
- phage display libraries collections of human antibody heavy and light chain variable region genes are expressed on the surface of bacteriophage particles, either in single chain Fv (scFv) or in Fab format.
- scFv single chain Fv
- Large libraries of antibody fragment -expressing phages typically contain > 10 9 antibody specificities and may be assembled from the immunoglobulin V regions expressed in the B lymphocytes of immunized or non-immunized individuals.
- phage display libraries may be constructed from immunoglobulin variable regions that have been partially assembled in vitro to introduce additional antibody diversity in the library (“ semi-synthetic" libraries) .
- semi-synthetic libraries immunoglobulin variable regions that have been partially assembled in vitro to introduce additional antibody diversity in the library.
- variable regions contain stretches of synthetically-produced, randomized or partially-randomized DNA in those regions of the molecules that are important for antibody specificity.
- specificities not present in natural repertoires may be added to phage display libraries of antibody fragments.
- Recombinant phages expressing antibody fragments of desirable specificities may be selected from a library by one of several methods.
- Target antigens are immobilized on a solid phase and subsequently exposed to a phage library to allow binding of phages expressing antibody fragments specific for the solid phase-bound antigen.
- Non-bound phages are removed by washing and bound phages eluted from the solid phase for infection of Escherichia Coli (E. coli ) bacteria and subsequent propagation. Multiple rounds of selection and propagation are usually required to sufficiently enrich for phages binding specifically to the target antigen.
- Phages may also be selected for binding to complex antigens such as complex mixtures of proteins or whole cells.
- Target antigens are presented in their native configuration, unperturbed by conformational changes that are introduced by immobilizing an antigen to a solid phase.
- the constraints imposed by the natural immune response and the influence of the immunogenicity of the target antigen does not permit the isolation of monoclonal antibodies against any antigen by conventional hybridoma technology. In phage approaches, these factors do not play a role, allowing the isolation of monoclonal antibodies against "difficult" antigens such as autoantigens, carbohydrates and toxic antigens.
- phage display libraries are used in combination with flow cytometry and cell sorting to isolate antibody fragments against molecules expressed on the plasma membrane of subpopulations of eukaryotic cells present in a heterogeneous mixture.
- the heterogeneous mixture of cells is incubated with the phage library allowing phages to bind to the different cell types.
- the cells are stained with fluorochrome- labeled monoclonal antibodies to permit identification of the subpopulation of target cells by immunofluorescence analysis and flow cytometry.
- Target cells and attached phages are collected by flow cytometry and the attached phages are eluted and propagated.
- This method is rapid, independent of the immunogenicity of the target antigen and yields antibody fragments against molecules in their native configuration. Specific antibodies against very small populations of cells in a heterogeneous mixture can be obtained.
- scFv with desirable specificities can be inserted into mammalian expression vectors containing the genes encoding human immunoglobulin constant regions.
- Immunotherapy in cancer is aimed at recruiting the humoral and/or cellular arms of the immune system to eradicate tumor cells.
- approaches has been tested in in vivo and in vi tro systems including unconjugated antibodies, bi-specific antibodies, immunotoxins, radiolabeled monoclonal antibodies, immunoliposomes and cytotoxic T lymphocytes.
- treatment of a large cohort of patients with resected Dukes C colorectal cancer with a murine antibody against the Ep-CAM molecule expressed on colorectal tumor cells has proved very effective.
- a humanized antibody against the HER2/neu receptor that is over-expressed on the tumor cells of 25-30% of patients with breast cancer, was shown to slow the progression of tumor growth and to increase the percentage of patients who experienced tumor shrinkage.
- a chimeric monoclonal antibody against the CD20 antigen was shown to give a response in 48% of patients with relapsed low grade or follicular lymphoma.
- binding of the antibody to the target antigen results in the recruitment of components of the complement system and effector cells of the immune system with Fc receptor for antibody constant regions that act in concert to kill the tumor cell.
- binding of an antibody to a target antigen on the membrane of a cell does not always result in tumor cell killing.
- the chimeric anti-CD20 monoclonal antibody is expressed by B cell tumors but also by non-malignant immature and mature B lymphocytes. Despite this cross-reactivity with non-malignant B cells, treatment with the chimeric anti-CD20 antibody causes few side-effects and the clinical success is considerable.
- the extracellular matrix controls cell survival, cell morphology and tissue organization by supporting cell adhesion. Remodeling of the extracellular matrix is a key process in the development of properly organised blood vessels, tissues and organs. Many extracellular matrix proteins, such as fibronectin and members of the collagen family, and proteases, such as plasmin and metalloproteinases, are involved in the remodeling process.
- remodeling is regulated by members of the "integrin" family of adhesion receptors, which use the extracellular matrix proteins as attachment sites for migration.
- Vitronectin is an important extracellular matrix molecule implicated in the control of cell morphology, proliferation, migration and survival. Because vitronectin is the most important adhesive protein in serum, which is used to culture cells in vi tro, vitronectin is also called "serum spreading factor" . Vitronectin is the primary ligand for ⁇ v ⁇ 3 and ⁇ v ⁇ 5 integrins, and plays a prominent role in angiogenesis and tumor growth (Preissner et al . 1997 and 1998) . Vitronectin is a 75 kDa glycoprotein that is synthesized primarily in the liver, and is present in plasma in a concentration between 200-400 mg/L (for review see Preissner et al .
- vitronectin In plasma, vitronectin is present in a "native" conformation, whereas in serum, vitronectin is present in its “activated” conformation. Activation of vitronectin can be mediated by a complex of thrombin-antithrombin III and occurs during pathological processes, including angiogenesis. When hyperpermeability is induced by angiogenic factors such as vascular endothelial growth factor ("VEGF”), vitronectin is deposited into the extracellular matrix and forms part of a provisional matrix.
- VEGF vascular endothelial growth factor
- activated vitronectin binds, concentrates, and regulates proteins that control the formation of the serine protease plasmin.
- proteins include plasminogen, plasminogen activator inhibitor type l(PAI-l) and the urokinase-type plasminogen activator receptor (uPAR, Kost et al . 1992) .
- PAI-l plasminogen activator inhibitor type l
- uPAR urokinase-type plasminogen activator receptor
- the invention includes the specific (single chain) antibody fragments themselves, the corresponding antibodies containing these fragments, and means and methods for making the antibodies and utilizing the antibodies and fragments in the diagnosis and treatment of disease.
- Diseases to be treated include all diseases involving deposition or presence of activated vitronectin, such as psoriasis, rheumatoid arthritis, artherosclerosis and malignancies.
- the invention further includes a kit of parts that includes the antibodies or their active fragments.
- the invention includes a method of tumor therapy in a subject.
- the method involves administering to the subject anti-activated vitronectin antibodies and/or antibody fragments which interfere with the adhesion and/or migration of tumor-endothelial cells by preventing vitronectin binding to its receptor, thus providing an antibody-mediated, anti-vascular cancer therapy.
- Vitronectin is an important adhesive molecule and, in its activated state as a component of the extracellular matrix, a potential target for tumor therapy. Previous immunohistochemical studies have demonstrated the presence of substantial deposits of vitronectin in most tumors examined and in a number of inflammatory disease states (Carreiras et al . 1996; Loridon-Rosa et al . 1988; Dahlback et al .
- phage display proved to be a powerful tool to generate scFv antibody fragments that discriminate between the activated and inactivated form of a protein.
- vitronectin deposits are found in most tumors examined and in a number of inflammatory diseases by demonstrating that these deposits are indeed formed by activated vitronectin.
- the importance of activated vitronectin as a provisional matrix was supported by the virtual absence of activated vitronectin in normal tissue.
- activated vitronectin was not exclusively localised in the surroundings of blood vessels, but was present in extra-cellular deposits throughout areas in the tumors and in similar regions in focal glomerular sclerosis and lung tissue from a patient with pneumonia.
- vitronectin is thought to play an important role in the adhesion of (tumor) -endothelial cells to a provisional matrix (Carreiras et al . 1999a) and concentrating proteolytic activity on the cell surface and extracellular matrix by trapping key components, like uPA, uPAR, PAI-1 and plasminogen (Waltz et al . 1997) . Furthermore, vitronectin decreases microvascular endothelial cell and tumor cell apoptosis (Isik et al . 19.98; Uhm et al . 1999) .
- vitronectin appears to be prominently localized at sites where angiogenesis and tumor cell migration is thought to play a crucial role in tumor growth.
- vitronectin is normally mainly produced by the liver and that several tumor cell lines also produce vitronectin further indicates that vitronectin plays a role in these conditions (Carreiras et al . 1999b; Tomasini- Johansson et al . 1994; Yasumitsu et al . 1993).
- Activated vitronectin is the ligand for the ⁇ v ⁇ 3 integrin, which is upregulated during the angiogenic process and plays an important role in adhesive interactions during angiogenesis (Brooks et al . 1994b). Inhibiting monoclonal antibodies against molecules involved in the adhesion and migration of tumor-endothelial cells have showed promising results in treating tumor-bearing mice
- the invention provides an antibody fragment comprising at least a CDR3 domain of a heavy chain of an antibody which has a higher affinity for activated vitronectin than for native vitronectin.
- said antibody fragment is a human or humanlike antibody fragment. This renders said fragment more effective and lowers the toxicity or immunogenicity. It is indicated that human or human-like antibody fragments have no or little immunogenicity at all.
- said antibody fragment is obtainable by subtractive phage selections on urea-activated vitronectin in the presence of plasma as a source of native vitronectin.
- an antibody fragment of the invention having a V-region comprising an amino acid sequence of SEQ. I.D. NO. 1, SEQ. I.D. NO. 2, SEQ . I.D. NO. 3, SEQ . I.D. NO. 4 or a functional part, derivative and/or analogue thereof.
- amino acid substitutions can be made in the above mentioned sequences, while retaining at least one property of said sequences in kind, not necessarily in amount.
- the invention also provides an antibody fragment, comprising at least 60 percent, more preferably 75 percent, more preferably 85 percent, most preferably 95 percent homology to an amino acid sequence of SEQ. I.D. NO. 1, SEQ. I.D. NO. 2, SEQ. I.D. NO. 3 , SEQ. I.D. NO. 4 or a functional part, derivative and/or analogue thereof .
- An antibody fragment of the invention is preferably directed against human activated vitronectin.
- said antibody fragment is also capable of binding to activated vitronectin of other species, as for instance chicken activated vitronectin (example 9) .
- an antibody fragment is meant herein a fragment of at least the size of a CDR3 domain, in particular a fragment derived from a CDR3 of a heavy chain. This fragment has at least one same property in kind, not necessarily in amount, as said antibody.
- a higher affinity of a fragment for activated vitronectin than for native vitronectin means herein that said fragment binds more activated vitronectin than native vitronectin when said fragment is exposed to the same excess amount of both kinds of vitronectin. This can be measured in a mixture, comprising equal excess amounts of activated and native vitronectin. This mixture can be provided to a certain amount of the same kind of antibody fragments. After incubation it is possible to determine the amount of bound activated vitronectin and bound native vitronectin. If it is then concluded that said antibody fragments have bound more activated vitronectin than native vitronectin, said antibody fragments are considered to have a higher affinity for activated vitronectin than for native vitronectin.
- a functional part of a protein for instance a functional part of an antibody, is defined herein as a part which has the same kind of properties in kind, not necessarily in amount. Said properties may for instance comprise the capability to bind to a specific antigen.
- a functional derivative of a protein is defined as a protein which has been altered such that the properties of said molecule are essentially the same in kind, not necessarily in amount.
- a derivative can be provided in many ways, for instance through conservative amino acid substitution. For instance, one or more glycine residues may be substituted for alanine, which will not alter much of the properties of said protein.
- a person skilled in the art is well able to generate analogous compounds of a protein, for instance an antibody. This can for instance be done through screening of a peptide library. Such an analogue has essentially the same properties, for instance binding properties, of said protein in kind, not necessarily in amount.
- an anzibody comprising an antibody fragment of the invention.
- said antibody is suitable for being provided to the blood circulation of an individual.
- a target molecule for said antibody can be reached via the blood circulation.
- said antibody must be sufficiently stable to remain in the blood circulation until said target molecule is reached.
- IgG antibodies are very suitable for providing to the blood circulation of a subject.
- the invention provides an antibody of the invention which is an IgG antibody.
- the construction and production of an IgG x antibody comprising an antibody fragment of the invention is described in example 4.
- An antibody or fragment of the invention can, for instance, be used to diagnose a disease state in a subject, for instance through detecting the presence of activated vitronectin in a biological sample taken from said subject.
- the invention provides a pharmaceutical composition comprising an antibody fragment according to the invention.
- Such composition can be used in an anti -vascular therapy, preferably, a cancer therapy.
- the invention therefore also provides the use of an activated form of vitronectin as a target for antibody-mediated anti- vascular cancer therapy.
- a method of cancer therapy comprising administering anti-activated vitronectin antibody fragments or antibodies according to the invention, which interfere with the adhesion and/or migration of tumor-endothelial cells, to a patient. Now that a role of activated vitronectin in the development of certain diseases, for instance cancer, is established, activated vitronectin can be used as a marker to detect said diseases.
- the invention provides a method of detecting the presence of activated vitronectin comprising using an antibody fragment or an antibody of the invention.
- the use of an antibody fragment or an antibody of the invention for the detection of the presence of activated vitronectin is also herewith provided.
- the invention provides a method of diagnosing a disease state in a subject, said method comprising detecting for the presence of activated vitronectin in a biological sample taken from said subject.
- said activated vitronectin is detected with use of an antibody and/or an antibody fragment, more preferably with use of an antibody and/or an antibody fragment of the invention.
- said detection of activated vitronectin comprises detecting the presence of said antibody, antibody fragment, or a part thereof, bound to said vitronectin.
- a lot of techniques are known in the art for detecting the presence of an antibody, for instance bound to activated vitronectin.
- Said antibody may for instance be labeled with a fluorescent or radioactive label.
- unbound antibody can be removed by one or more washing steps. After that, a fluorescent or radioactive signal will reveal the presence of bound antibody and, hence, the presence of activated vitronectin.
- a person skilled in the art can think of many alternative ways to detect the presence of an antibody, for instance bound to activated vitronectin.
- the invention therefore also provides a method of manufacturing an antibody or an antibody fragment directed toward detecting the presence of activated vitronectin.
- Such method can include the use of an apparatus for detecting the presence of activated vitronectin, derived by selection from a library using a phage display method.
- the invention therefore also provides such an apparatus .
- the invention provides an apparatus for detecting the presence of activated vitronectin, using an antibody or an antibody fragment, said antibody or fragment derived by selection from a library comprising urea-treated vitronectin capable of binding to an antibody or antibody fragment of interest , such binding forming a receptor complex and thereby causing a change in a property associated with the binding partner; and a detection means for detecting the measurable change.
- the receptor complex comprises an antibody or an antibody-binding fragment thereof, a peptide or a nucleotide sequence.
- the receptor complex comprises an antibody or an antibody-binding fragment thereof derived by selection from a library using a phage display method.
- the invention further provides an isolated peptide fragment comprising at least a CDR3 domain of a heavy chain of an antibody which has a higher affinity for activated vitronectin than for native vitronectin.
- said peptide fragment is obtainable by subtractive phage selections on urea-activated vitronectin in the presence of plasma as a source of native vitronectin. Mutants of a peptide of the invention can be easily tested for the same binding characteristics in kind, not necessarily in amount, and are therefor also part of the invention.
- Activated vitronectin is the most important adhesive protein in serum. Binding of an antibody fragment of the invention to said vitronectin will therefore affect the capability of said vitronectin to bind to a lot of different targets.
- Said target may for instance be an endothelial cell, preferably a tumor endothelial cell, but may also for instance be platelets.
- the invention provides a use of an antibody or an antibody fragment according to the invention for at least in part changing an adhesion property ⁇ activated vitronectin.
- Said adhesion property preferably comprises the capability to bind to endothelial cells, extracellular matrix proteins, and/or platelets.
- said changing of the capability to bind to extracellular matrix proteins prevents angiogenesis.
- an antibody fragment of the invention provides a method for at least in part inhibiting adhesion of an endothelial cell to activated vitronectin, comprising providing said vitronectin with an antibody fragment, an antibody or a peptide according to the invention.
- Another embodiment of the invention provides a method for at least in part decreasing an interaction of activated vitronectin with collagen and/or endothelial cells, comprising providing said vitronectin with an antibody fragment, an antibody or a peptide according to the invention.
- the invention provides a method for at least in part inducing cell death of an endothelial cell, associated with an extracellular matrix comprising activated vitronectin, comprising providing said matrix with an antibody fragment, an antibody or a peptide according to the invention. Said cell death may comprise apoptosis.
- the invention provides an isolated peptide comprising a vitronectin epitope which is recognized by an antibody fragment, an antibody or a peptide according to the invention.
- Said peptide may for instance be useful for screening a library for their binding properties to said epitope and, hence, for activated vitronectin.
- the vitronectin H2 domain is an important epitope for an antibody fragment of the invention.
- the invention provides a peptide according to the invention, which comprises at least part of the vitronectin H2 domain.
- an antibody fragment of the invention is provided, a person skilled in the art is well capable of generating a nucleic acid molecule encoding said antibody fragment.
- Methods to generate nucleic acid molecules encoding a certain polypeptide, starting from the amino acid sequence of said polypeptide, are known in the art, as well as codon usage in different organisms. Hence this needs no further explanation here.
- the invention provides an isolated or recombinant nucleic acid molecule encoding an antibody fragment, an antibody or a peptide according to the invention, or a functional part, derivative and/or analogue thereof .
- a person skilled in the art is equally well capable of generating a nucleic acid molecule encoding a peptide comprising a vitronectin epitope which is recognized by an antibody fragment, an antibody or a peptide according to the invention, or a functional part, derivative and/or analogue thereof.
- Said nucleic acid molecule is also herewith provided.
- said epitope comprises at least part of the vitronectin H2 domain.
- the artisan is also capable of generating a fusion gene comprising a nucleic acid molecule of the invention.
- Said fusion gene may encode a fusion protein.
- the invention provides a fusion protein comprising a peptide encoded by a nucleic acid molecule of the invention.
- nucleic acid of the invention By at least a functional part of a nucleic acid of the invention is meant a part of said nucleic acid, at least 30 base pairs long, preferably at least 200 base pairs long, comprising at least one expression characteristic (in kind not necessarily in amount) as a nucleic acid of the invention.
- one embodiment of the invention provides a method for at least in part inhibiting platelet aggregation in the presence of vitronectin, comprising providing said vitronectin with an antibody fragment, an antibody or a peptide of the invention.
- an antibody fragment, an antibody or a peptide of the invention is very suitable for the preparation of a medicament.
- a use of an antibody fragment, an antibody or a peptide of the invention for the preparation of a medicament is therefore also provided.
- said medicament is particularly suited for at least one of the above mentioned applications.
- a use of an antibody fragment, an antibody or a peptide of the invention for the preparation of a medicament for at least in part inhibiting platelet aggregation is provided.
- an antibody fragment, an antibody or a peptide of the invention for the preparation of a medicament for at least in part decreasing an interaction of activated vitronectin with collagen and/or endothelial cells.
- Another preferred embodiment provides a use of an antibody fragment, an antibody or a peptide of the invention for the preparation of a medicament for at least in part changing the development of blood vessels.
- Activated vitronectin is an important extracellular matrix protein implicated in the control of cell morphology, proliferation, migration and survival. Activated vitronectin is for instance involved in the development of blood vessels. With the teaching of the present invention it is possible to change said development of blood vessels. This can be done by influencing the remodeling process of the extracellular matrix of an endothelial cell.
- one embodiment provides a method for at least in part changing the development of blood vessels, comprising influencing the remodeling process of the extracellular matrix of an endothelial cell.
- said extracellular matrix is provided with an antibody fragment, an antibody or a peptide according to the invention.
- an antibody fragment with a higher affinity for activated vitronectin than for native vitronectin is provided, the artisan is well capable of designing an antibody fragment, preferably based on a fragment of the invention, with a same binding property in kind, not necessarily in amount.
- Such fragment is capable of at least in part inhibiting binding of an antibody fragment, an antibody or a peptide of the invention, to activated vitronectin.
- inhibiting binding of a molecule to activated vitronectin is meant herein that less molecules will bind to a certain limiting amount of activated vitronectin if an inhibitor is also provided.
- a limiting amount of activated vitronectin is provided with a certain amount of said molecules and an inhibitor, at least a part of said inhibitor will bind to said activated vitronectin.
- the inhibitor-bound activated vitronectin is not capable of binding to said molecules anymore. This results in less amount of said molecules to be bound to vitronectin, as compared to a situation wherein no inhibitor was provided.
- the present invention provides an antibody or functional part, derivative and/or analogue thereof, capable of at least in part inhibiting binding of an antibody fragment, an antibody or a peptide of the invention, to activated vitronectin.
- a phage antibody library cloned in the phagemid pPVT with a size of 4.5 x 10 9 specificities was used.
- Urea treated vitronectin was prepared as described earlier (Yatohgo et al . 1988) .
- Native vitronectin was a gift from Dr K.T. Preissner (Bad Nauheim, Germany) .
- the rabbit polyclonal antibody and the monoclonal antibody 13H1 against vitronectin have been previously described (Preissner et al . 1987) .
- the monoclonal antibody anti- [VSV-G] -peroxidase and anti- [c-myc] -peroxidase were obtained from Roche and anti-CD31 monoclonal antibody EN4 from Monosan.
- the peroxidase-conjugated rabbit anti -mouse polyclonal antibody was obtained from Dako .
- Phage selections were performed as described with minor modifications (De Kruif et al . 1995). Briefly, immunotubes (Gibco BRL) were coated with urea treated, activated vitronectin (10 ⁇ g/ml) overnight at 4°C and blocked with 2% milk powder in PBS (MPBS) for 2 hours. Before the phage were added to the immunotubes, they were blocked for 15 minutes with MPBS containing 50% human pooled plasma to absorb nonspecific binding phages and phages recognizing native vitronectin. The immunotubes were incubated with phages for 2 hours at room temperature.
- MPBS 2% milk powder in PBS
- monoclonal phages were prepared from single ampicillin and tetracycline-resistant colonies of infected XLl-Blue cells. Binding to urea treated vitronectin was tested in an ELISA using a phage antibody detection module (Pharmacia, Inc.) . A monoclonal anti-M13 antibody (Pharmacia, Inc.) was used and the assay was performed according to manufacturer instructions. Nucleotide sequence analysis was determined with the ABI PRISM Dye Terminator Cycle sequencing kit (Perkin Elmer) on an automated sequencer.
- scFv fragments were produced in the E . coli non- suppressor strain SF110.
- Periplasmic preparations of scFv's were purified over a Ni-NTA agarose column (Qiagen) and eluted by imidazole into 2 ml fractions.
- the purified scFv antibody fragments were dialysed against PBS and stored at -20°C until further use.
- ELISA 's scFv antibody fragments (30 ⁇ g/ml) or a control polyclonal anti -vitronectin antibody (5 ⁇ g/ml) were coated overnight on 96 well plates (Nunc Maxisorp) .
- Purified activated or native vitronectin was allowed to bind for 1 hour at room temperature. After washing with PBS/0.1% Tween- 20, bound vitronectin was determined by incubation with murine monoclonal antibody 13H1 which recognizes both native and activated vitronectin (Kost et al . 1992) .
- Phage or scFv's were pre-incubated with plasma or serum for 30 min .
- the pre- blocked phage and scFv's were allowed to bind to coated vitronectin for 1 hour at room temperature. After washing with PBS/0.1% Tween-20, binding was determined by incubation with a peroxidase-conjugated monoclonal anti-M13 antibody (phages) or anti-myc (scFv's) . Staining was performed and measured as described above.
- the ELISA' s were performed in duplicate or triplicate and repeated at least two times.
- the Biacore 2000 system and the reagents including the sensorchips CM5 , surfactant P20 and the amine coupling kit, containing N-hydroxy-succinimide (NHS) , N-ethyl-N'- (dimethanylamino-propyl) carbidiimide (EDC) , ethanolamine hydrochloride and formeate buffer, were from Pharmacia Biosensor AB (Uppsala, Sweden) . The whole procedure was performed according to the manufacturers instructions.
- HUVEC Human Umbilical Vein Endothelial Cells
- Endothelial cells (HUVEC) were brought in suspension with trypsin, trypsin was neutralized in medium containing serum and cells were washed once and resuspended in RPMI -medium containing 1% bovine serum albumin. Cells were counted using a B ⁇ rker-T ⁇ rk chamber and for the adhesion assay 5xl0 4 cells were seeded in 96-well plates in the presence of anti-VN antibodies or a control antibody named L1R3.
- a chromogenic substrate for the ubiquitous lysosomal enzyme, hexosaminidase was used.
- the protocol for the adhesion assay has been described previously (Landegren 1984) . Briefly, urea-treated vitronectin was coated to wells of a 96 well plate overnight (5 ⁇ g/ml) . Plates were blocked with phosphate buffered saline containing 1 M calciumchloride, 1 mM magnesium-chloride (PBS ++ ) and 3% bovine serum albumin (BSA) for 2 hours.
- HUVEC cells were added to the wells (5xl0 4 ) in the presence of the antibodies against activated vitronectin or control antibodies and allowed to adhere for 2 hours at 37°C in a 5% C0 2 humidified incubator.
- the plates were washed four times with PBS ++ /3% BSA.
- Enzyme activity was stopped by addition of 150 ⁇ l 50 mM glycine buffer, pH 10, containing 5 mM EDTA. Absorbance was measured at 405 nm in a microtiter-plate reader (Molecular Devices Corp, Sunnyvale, CA, USA) .
- Annexin-V and propidium iodide were used for apoptosis measurement.
- Urea-treated vitronectin was coated overnight to wells of a 48 well plate (250 ⁇ l , 5 ⁇ g/ml) .
- HUVEC was added (5.10 4 cells/well) and allowed to adhere for 30 minutes.
- the anti-vitronectin antibodies or a control antibody were added with a concentration of 50 ⁇ g/ml and incubated at 37 °C in a 5% C0 2 humidified incubator. At different time points the cells were washed and trypsinised.
- Apoptosis was determined by flow cytometry using the annexin- V-fluos staining kit from Roche.
- PAI-1 was a gift of Dr Pannekoek (Amsterdam, The Netherlands) and uPAR from Dr Meijers (Amsterdam, The Netherlands) .
- a stock solution of 50 ⁇ g/ml TAT was formed by incubation of 20 ⁇ g/ml with 5 times molar excess of ATIII. No binding was observed of VN to ATIII alone, so the excess of ATIII was not removed.
- Human Collagen type I (Sigma) was dissolved in 0.5 M acetic acid (1 mg/ml) and further diluted in PBS. Plasminogen was affinity purified from outdated plasma using lysine-Sepharose (Pharmacia) .
- vitronectin ligands PAI-1, plasminogen, uPAR, TAT and collage 1 "* type I were coated overnight to 96-well plates in PBS (5 g/ml) .
- Phosphatidyl serine (PS, Sigma) was dissolved in methanol at a concentration of 50 ⁇ g/ml and 50 ⁇ l/well was air-dried.
- Coated ligands were blocked with PBS containing 3% BSA and 0.1% Tween-20 (in the case of PS, Tween-20 was omitted) and subsequently incubated with urea-treated vitronectin (0-100 ⁇ g/ml) .
- vitronectin Binding of vitronectin was determined with a polyclonal antibody, followed by peroxidase conjugated swine anti -rabbit IgG and peroxidase substrate (ortho-phenylene- diamine) . The concentration that yielded half-maximal vitronectin binding was calculated. In the competition experiments, this half maximal concentration of vitronectin was used and pre-incubated (30 minutes at 37°C) with increasing amounts of Vnl ⁇ IgG or control IgG (0-100 ⁇ g/ml) . Binding of vitronectin was determined as described above. The effect of VN18 was expressed as percentage of binding of vitronectin to its ligands in the presence of control IgG.
- urea-treated vitronectin (5 ⁇ g/ml) was coated overnight, blocked and incubated with increasing amounts of biotinylated-heparin (0-100 ⁇ g/ml) .
- Heparin binding was determined with peroxidase-conjugated streptavidin in the presence of peroxidase substrate.
- Half- maximal heparin concentrations were used and different concentrations of VN18 or control IgG (0-100 ⁇ g/ml) . Residual heparin binding was expressed as percentage binding of control IgG.
- Vnl CAR - ARRLRLFDY-WGQ (SEQ. I.D. NO. 1)
- Vn7 CAR - DPRGPRSWLDY-WGQ (SEQ. I.D. NO. 2)
- Vnl8 CAR - DDRPRELDS-WGQ (SEQ. I.D. NO. 3)
- Vn26 CAR - ANARAAFDS-WGQ (SEQ. I.D. NO. 4)
- scFv's derived from the monoclonal phage antibodies specifically recognized activated vitronectin scFv's dilutions were used that yielded maximum binding. Since native vitronectin becomes activated upon coating to plastic, a capture ELISA was performed. A rabbit polyclonal anti -vitronectin antibody served as a non-specific control and was coated and used to capture native or activated vitronectin. As can be seen in Figure 2, scFv(Vn7) , scFv(Vnl ⁇ ) and scFv(Vn26) specifically detected activated vitronectin.
- scFv's derived from phage Vnl could not be analyzed, because the anti-VSV monoclonal antibody did not detect scFv(Vnl) .
- a possible explanation could be the presence of a mutation in the sequence encoding the VSV-tag.
- the control polyclonal anti- vitronectin antibody recognized activated and native vitronectin equally well .
- Example 2 Recognition by monoclonal phage antibodies and scFv fragments of activated vitronectin in serum as compared to native vitronectin present in plasma.
- Example 3 Recognition of vitronectin by Vnl8 in normal and tumor tissue sections.
- scFv(Vnl8) was selected to study the presence and distribution of activated vitronectin in human carcinoma of colon, kidney and lung, in other pathological specimens, and in the corresponding normal tissues.
- Activated vitronectin was abundantly present in colon adenocarcinoma ( Figure 4A) , whereas hardly any activated vitronectin could be detected in normal colon ( Figure 4D) .
- the staining pattern of scFv(Vnl ⁇ ) overlapped largely with the pattern of staining with antibodies against CD31 ( Figure 4B) , an endothelial specific marker, indicating the presence of activated vitronectin around blood vessels.
- staining of vitronectin deposits was seen in the extracellular environment, fibrous tissue, where vessels had not yet been formed.
- Example 4 Construction and production of a fully human monoclonal IgG- t /K antibody against activated vitronectin. Construction of eukaryotic expression vectors and production of human monoclonal antibodies (huMabs) has been described in detail elsewhere (Huls et al . 1999) . Briefly, in a two-step cloning procedure, the V H and V L regions encoding a scFv were cloned into the vector pLEADER to add the T-cell receptor ⁇ -chain leader HAVT20 leader peptide sequence and a splice donor site.
- V H and V L regions which contain leader and splice donor sites, were subcloned in the pNUT-C ⁇ l or pNUT-CK expression vectors.
- HuMab Vn7 , Vnl8 and Vn26 were produced by establishing stably transfected BHK21 cells. Cells were maintained at 37°C in a 5% C0 2 humidified incubator in Iscove's modified Dulbecco ' s medium containing 10% FCS, 2 mM glutamine, 100 U/ml penicillin and 100 ⁇ g/ml streptomycin.
- Recombinant antibodies were purified from approximately 2 liters of culture supernatant by protein A sepharose affinity chromatography and run on an SDS-PAGE gel to confirm the stoichiometry and quality of heavy and light chains as described (Huls et al . 1999) . Concentration of purified huMab was determined by spectophotometry at 280 nm.
- Example 5 Inhibition of endothelial cell adhesion to vitronectin.
- scFv's were converted to intact, fully human IgGl monoclonal antibodies by recloning the V H and the V L regions into expression vectors for synthesis of complete IgG x / ⁇ molecules in BHK cells.
- Purified antibodies retained their specificity for activated vitronectin as assessed in ELISA.
- the kinetic association and dissociation rates and affinity constant of the three huMabs were determined by surface plasmon resonance on a BiaCore.
- Association and dissociation constants ranged from 1.5xl0 5 - 4.6x10 s M ⁇ s "1 and 6.8xl0 "4 - 2.9xl0 ⁇ 2 s '1 respectively, yielding affinity constants of 4.5, 9.3 and 92 nM for Vn7 , Vnl8 and Vn26.
- Vitronectin is believed to play a prominent role during angiogenesis (Preissner et al . 1997) .
- Endothelial cells (HUVEC s) were allowed to adhere and after washing, numbers of adhering cells were estimated by adding a chro ogenic substrate for an ubiquitous lysosomal enzyme to the wells (Landegren 1984) .
- Adherence of endothelial cells to vitronectin-coated plates was inhibited with anti -vitronectin antibodies in a dose dependent fashion (Figure 5A) .
- All three anti-vitronectin antibodies inhibited endothelial cell adhesion (Figure 5B) .
- Maximum inhibition of endothelial cell binding was 80-85% at concentrations of anti-vitronectin antibodies of 50 ⁇ g/ml.
- Example 6 Effect of anti-vitronectin antibodies on endothelial cell apoptosis.
- Vitronectin protects endothelial cells from entering an apoptotic pathway (Isik et al . 1998) .
- anti-vitronectin antibodies or a control antibody were added to subconfluent layers of endothelial cells (HUVEC's). After 8 hours, cells were stained with propidium iodide and annexin V and analysed by flow cytometry. In Figure 6 it is shown that the number of apoptotic cells increased as a result of binding of the Vnl8 antibody to vitronectin-coated plates.
- Example 7 Effect of anti-vitronectin antibodies on vitronectin function, mapping of the epitope and cross- reactivity with vitronectin from other species.
- vitronectin is involved in the regulation of protease cascades, such as coagulation and fibrinolysis (Preissner et al . 1997).
- protease cascades such as coagulation and fibrinolysis
- Example 8 Mapping of the epitope on vitronectin that is recognized by Vnl8.
- vitronectin fragments as described by Yoneda et al . (1998) were used. Immunoblot analysis of recombinant vitronectin fragments revealed binding of Vnl8 antibodies to an epitope located in the hemopexin II domain ( Figure 8) . For this, several GST-fusion proteins were purified and let to interact with Vnl8 antibodies. Domains that are present in vitronectin are S
- vitronectin was purified from human and chicken serum according to the method of Yatohgo et al . (1988) .
- An ELISA was performed in which human or chicken vitronectin (10 ⁇ g/ml) were coated onto a 96-wells plate (Costar) , blocked with bovine serum albumin (BSA) . Binding of biotinylated IgG of Vnl8, a control (phage generated) IgG
- Example 10 Effect of VN18 on platelet adhesion and function. Activated vitronectin was sprayed on glass coverslips and the adhesion of isolated bloodplatelets was studied under flow. The isolated platelets were reconstituted in 4% human albumin solution (HAS) to which red blood cells were added, so no plasma proteins were present. Washed platelets (see below) were resuspended in Hepes-Tyrode buffer (10 mM Hepes, 0.145 mM NaCl, 5 mM KCL, 0.5 mM Na 2 HP04 , 1 mM MgS0 4 , pH 7.25) to a concentration of 2x10 s platelets/ ⁇ l .
- Hepes-Tyrode buffer (10 mM Hepes, 0.145 mM NaCl, 5 mM KCL, 0.5 mM Na 2 HP04 , 1 mM MgS0 4 , pH 7.25
- Figure 10 shows that Vnl8 addition inhibited mainly the formation of aggregates, suggesting that Vnl8 is able to reduce the activation of platelets. Since this perfusion experiment was performed in the absence of exogenous vitronectin, Vnl8 must react with vitronectin originating from the platelet intracellular pool. To investigate this, vitronectin expression on the platelet membrane was measured in the FACS after mild stimulation of the platelets with thrombin (0.5
- Platelet rich plasma was obtained from citrated whole blood by centrifugation (10 min at 200xg, 20 °C) .
- One volume of Krebs -Ringer buffer (4 mM KCl , 107 mM NaCl, 20 mM NaHC0 3 , 2 mM Na2 2 S0 4 , 4.76 M citric acid, 14 mM Na-citrate and 5 mM d-glucose, pH 5.0) was added to one volume of PRP. The final pH was approximately 6.
- a platelet pellet was obtained by centrifugation (10 min at 500xg, 20°C), resuspended in Krebs-Ringer buffer (pH 6.0) and washed twice by centrifugation (10 min at 500xg, 20°C) .
- platelets were resuspended in 4% human albumin solution (HAS) (4 mM KCl , 124 mM NaCl, 20 mM NaHC0 3 , 2 mM Na 2 S04, 2.5 mM CaCl 2 , 1.5 mM MgCl 2 , 5 mM d-glucose and 20 U/ml LMWH, pH 7.35) and incubated at 37°C in the absence or presence of epinephrin (lOOnM) or thrombin (0.5 U/ml) .
- HAS human albumin solution
- Figure 1 Schematic representation of the activation of vitronectin and its mediation by a complex of thrombin- antithrombin III that occurs during pathological processes, such as angiogenesis.
- FIG. 3 A) Monoclonal phage antibody (mophabs) and B) scFv recognition of activated vitronectin in serum. Phage and scFv's were allowed to bind to coated urea-treated vitronectin in the absence or presence of plasma or serum. Binding of all mophabs and scFv's could be inhibited by serum, but not by plasma, strongly suggesting that the phages and scFv's also specifically recognized physiologically activated, serum vitronectin, but not native, plasma vitronectin
- FIG. 4 Immuno histochemistry illustrating the presence of activated vitronectin in carcinoma of the colon kidney, lung and other pathological specimens. Also shown are normal specimens of colon, kidney, lung and brorous tissue which indicate little activated vitronectin (see text) .
- Figure 5 A) Binding of endothelial cells to immobilised vitronectin analysed in the presence of various concentrations of anti-vitronectin antibody Vnl ⁇ . B) Binding of endothelial cells to immobilised vitronectin analysed in the presence of three anti-vitronectin antibodies (Vnl , Vnl ⁇ and Vn26) in a concentration of 50 ⁇ g/ml.
- FIG. 7 Binding of the indicated ligands on different sections of vitronectin (- no binding; + binding) .
- the effect of Vnl8 is expressed as percentage inhibition compared to a control IgG (smb against somatomedin B) .
- Known binding epitopes are presented schematically (top drawing) .
- FIG. 8 Immunoblot showing the in vitro interaction of Vnl8 with a set of different purified fusion proteins (GST- vitronectin deletion mutants) .
- S, C, HI and H2 are the different domains in vitronectin (see text and Figure 7) .
- FIG. 10 Left panel. Effect of increasing concentrations of Vnl ⁇ antibody on platelet adhesion. Right panel. Formation of platelet aggregation in the presence (+Vnl8) or in the absence (-Vnl8) of the vitronectin specific IgG Vnl8.
- FIG. 11 FACS analysis on Vnl8 bound to platelets expressing vitronectin after stimulation by thrombin and epinephrin.
- Integrin alpha v beta 3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell 79:1157-64
- Urokinase receptor antagonists inhibit angiogenesis and primary tumor growth in syngeneic mice. Cancer Res 56:2428-33
- Plasmin and plasminogen activator inhibitor type 1 promote cellular motility by regulating the interaction between the urokinase receptor and vitronectin. J Clin Invest 100:58-67
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IL15138901A IL151389A0 (en) | 2000-02-25 | 2001-02-26 | Activated vitronectin as a marker of angiogenesis detected with phage antibodies |
AU2001241264A AU2001241264A1 (en) | 2000-02-25 | 2001-02-26 | Activated vitronectin as a marker of angiogenesis detected with phage antibodies |
JP2001562704A JP2003523770A (en) | 2001-02-26 | 2001-02-26 | Activated vitronectin as a marker of angiogenesis detected by phage antibodies |
CA002401367A CA2401367A1 (en) | 2000-02-25 | 2001-02-26 | Activated vitronectin as a marker of angiogenesis detected with phage antibodies |
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WO2001062930A1 true WO2001062930A1 (en) | 2001-08-30 |
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PCT/NL2001/000157 WO2001062930A1 (en) | 2000-02-25 | 2001-02-26 | Activated vitronectin as a marker of angiogenesis detected with phage antibodies |
Country Status (5)
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AU (1) | AU2001241264A1 (en) |
CA (1) | CA2401367A1 (en) |
IL (1) | IL151389A0 (en) |
NO (1) | NO20024032L (en) |
WO (1) | WO2001062930A1 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001068679A2 (en) * | 2000-03-16 | 2001-09-20 | University Of Pittsburgh | Peptides targeting specifically tumor-derived endothelial cells |
WO2005007674A2 (en) * | 2003-07-11 | 2005-01-27 | Molecular Innovations | Anti-human vitronectin antibody and methods for making the same |
US9988442B2 (en) | 2013-01-23 | 2018-06-05 | Syddansk Universitet | MFAP4 binding antibodies blocking the interaction between MFAP4 and integrin receptors |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998015833A1 (en) * | 1996-10-08 | 1998-04-16 | Universiteit Utrecht | Methods and means for selecting peptides and proteins having specific affinity for a target |
-
2001
- 2001-02-26 WO PCT/NL2001/000157 patent/WO2001062930A1/en not_active Application Discontinuation
- 2001-02-26 AU AU2001241264A patent/AU2001241264A1/en not_active Withdrawn
- 2001-02-26 CA CA002401367A patent/CA2401367A1/en not_active Abandoned
- 2001-02-26 IL IL15138901A patent/IL151389A0/en unknown
-
2002
- 2002-08-23 NO NO20024032A patent/NO20024032L/en not_active Application Discontinuation
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998015833A1 (en) * | 1996-10-08 | 1998-04-16 | Universiteit Utrecht | Methods and means for selecting peptides and proteins having specific affinity for a target |
Non-Patent Citations (5)
Title |
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B. TOMASINI ET AL.: "Conformational states of vitronectin: preferential expression of an antigenic epitope when vitronectin is covalently and noncovalently complexed with thrombin-antithrombin III or treated with urea.", BLOOD, vol. 72, no. 3, September 1988 (1988-09-01), New York, NY, USA, pages 903 - 912, XP000915069 * |
E. HAYMAN ET AL.: "Serum spreading factor (vitronectin) is present at the cell surface and in tissues.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE U.S.A., vol. 80, no. 13, July 1983 (1983-07-01), Washington, DC, USA, pages 4003 - 4007, XP002141489 * |
H. BLOEMENDAL ET AL.: "Targeting tumor vasculature with single chain antibody fragments against the activated form of vitronectin.", PROCEEDINGS OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH, vol. 40, March 1999 (1999-03-01), USA, pages 418 - 419, XP002141487 * |
J. DE KRUIF ET AL.: "Selection and application of human single chain Fv antibody fragments from a semi-synthetic phage antibody display library with designed CDR3 regions.", JOURNAL OF MOLECULAR BIOLOGY, vol. 248, 1995, pages 97 - 105, XP000646544 * |
W. VAN EWIJK ET AL.: "Subtractive isolation of phage-displayed single-chain antibodies to thymic stromal cells by using intact thymic fragments.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE U.S.A., vol. 94, no. 8, 15 April 1997 (1997-04-15), Washington, DC, USA, pages 3903 - 3908, XP002141488 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001068679A2 (en) * | 2000-03-16 | 2001-09-20 | University Of Pittsburgh | Peptides targeting specifically tumor-derived endothelial cells |
WO2001068679A3 (en) * | 2000-03-16 | 2002-05-30 | Univ Pittsburgh | Peptides targeting specifically tumor-derived endothelial cells |
US6974791B2 (en) | 2000-03-16 | 2005-12-13 | The University Of Pittsburgh | Endothelial specific targeting |
WO2005007674A2 (en) * | 2003-07-11 | 2005-01-27 | Molecular Innovations | Anti-human vitronectin antibody and methods for making the same |
WO2005007674A3 (en) * | 2003-07-11 | 2005-09-29 | Molecular Innovations | Anti-human vitronectin antibody and methods for making the same |
US9988442B2 (en) | 2013-01-23 | 2018-06-05 | Syddansk Universitet | MFAP4 binding antibodies blocking the interaction between MFAP4 and integrin receptors |
Also Published As
Publication number | Publication date |
---|---|
AU2001241264A1 (en) | 2001-09-03 |
CA2401367A1 (en) | 2001-08-30 |
IL151389A0 (en) | 2003-04-10 |
NO20024032L (en) | 2002-10-25 |
NO20024032D0 (en) | 2002-08-23 |
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