ES2365004T3 - HUMANIZED ANTIBODIES DERIVED FROM DD-3B6 / 22, SPECIFIC FOR THE FRAGMENT OF FIBRINE D-DAY. - Google Patents

Abstract

A deimmunized antibody or deimmunized antibody fragment specific for an epitope in the human D-dimer that recognizes cross-linked fibrin but not fibrinogen, wherein one or more amino acid residues in the variable domain (v) of said antibody or antibody fragment mutate to eliminate or reduce the association of peptide fragments of said v domain with MHC class II molecules, wherein said antibody or antibody fragment comprises a combination of v domains of the H and L chain comprising a combination of amino acid sequences selected from the group consisting of: SEQ ID NO: 1 / SEQ ID NO: 4, SEQ ID NO: 2 / SEQ ID NO: 4, SEQ ID NO: 3 / SEQ ID NO: 4, SEQ ID NO: 1 / SEQ ID NO : 6, SEQ ID NO: 2 / SEQ ID NO: 6, SEQ ID NO: 2 / SEQ ID NO: 5, SEQ ID NO: 3 / SEQ ID NO: 5, SEQ ID NO: 3 / SEQ ID NO: 6 and SEQ ID NO: 1 / SEQ ID NO: 5.

Description

FIELD OF THE INVENTION

The present invention relates generally to carrier molecules derived from a species or strain of animal or bird and that are substantially non-immunogenic that are exposed to an immune system of a species

or strain of another animal creature or bird. More particularly, the present invention provides deimmunized immunoreactive molecules and even more particularly deimmunized antibodies for use in diagnostic and therapeutic applications.

BACKGROUND OF THE INVENTION

Bibliographic details of the publications named in this specification are collected at the end of the description.

No reference is made to any prior art in this specification, and should not be taken as, a knowledge

or any form of suggestion that this prior art is part of the common general knowledge in any country.

Fibrinogen is a large protein molecule that normally circulates in the blood plasma in a dissolved state. In the presence of thrombin, fibrinogen molecules form polymers such as long strands or networks called fibrin, which is the primary ingredient in blood clots.

After plasmin digestion, the fibrinogen forms fragments designated A-E. Fragments D and E are the predominant fragments and there is approximately twice as much D as of E. Fibrinogen has a trinodular form where fragment E is a central component and fragment D is a terminal component.

Plasmin digestions of fibrin and fibrinogen can be differentiated from one another using polyacrylamide gel electrophoresis (PAGE). The crosslinking of fibrin with Factor XIIIa forms the dimers of fragment D called dimers D. Factor XIIIa is an enzyme that introduces covalent bonds between adjacent monomers in fibrin (Budzynski et al., Blood 54 (4): 794-804, 1979 ). Factor XIIIa is activated by catalyzed removal of thrombin from a peptide from a precursor in plasma and in blood platelets. The D-dimer is a molecule of approximately 189,000 daltons consisting essentially of two functional groups of fragment D derived from different fibrin molecules covalently linked by cross-linked bonds between the remnants of γ chain of fibrinogen. Fibrinogen itself comprises six chains with two copies of an α, β, and γ chain.

Another complex (DD) E is formed by the degradation of crosslinked human fibrin plasmin and comprises a combination of two D fragments and fragment E.

Other crosslinked derivatives are described by Graeff and Halfer (Graeff and Halfer, "Detection and Relevance of Cross-Linked Fibrin Derivatives in Blood", Seminars in Thrombosis and Hemostatis 8 (1), 1982) and include high molecular weight crosslinked derivatives such as DY, YY, XD, XY, DXD and YXD.

Normal homeostasis or blood clotting involves maintaining intravascular constituents in a liquid phase or suspension while concomitantly allowing local deposition of solid phase blood components in areas of vessel damage. In health, it is assumed, but never experimentally demonstrated, that there is a balance between a low-grade intravascular deposition of fibrin and its removal by fibrinolysis or cellular phagocytosis.

Early clinical observations reveal that some of several patients develop signs of hemorrhage and massive bruising and have prolonged coagulation times and thrombocytopenia. In post-morten, in some cases, fibrin thrombi is demonstrated in the microvasculature. The diffuse nature of these thrombi gives rise to disseminated intravascular coagulation (DIC) also known as consumption coagulopathy. Subsequently, thrombins are associated with conditions such as deep vein thrombosis (DVT) and pulmonary embolism (PE).

Conditions such as DIC, DVT and PE involve activation of the coagulation system that results in platelet consumption, thrombin generation, fibrin deposition and secondary fibrinolysis. The net biological effect of this process reflects a balance between fibrin deposition and fibrin removal. The resulting clinical manifestations can be hemorrhage, when the depletion of the predominant factors predominates, or the damage of ischemic tissue, due to the effects of vascular occlusion among other conditions.

DIC, DVT and PE have been reported as a secondary phenomenon a wide variety of disorders, particularly those accompanied by a combination of shock, acidosis and hypoxemia. The well-recognized clinical associations are sepsis, main trauma, malignancy and obstetric disorders. Recently, DVT has been recognized as a particular problem during prolonged air travel or other prolonged immobility. In any event, activation of the coagulation sequence results in the consumption of coagulation protein and platelets, which leads to fibrin deposits in the microcirulation.

Ideally, a definitive diagnosis of conditions such as DIC, DVT and PE requires direct demonstration of diffuse fibrin deposition. The practical difficulty in obtaining multiple evidence of direct biopsies to differentiate between the formation of localized and generalized fibrin has led to the development of indirect tests that are replaced as diagnostic endpoints. However, these tests are not specific for intravascular fibrin deposition syndrome. Its specificity is further reduced by the action of other enzymes that, although not capable of converting fibrinogen to fibrin, can cause thrombin-like alterations in the other coagulation factors involved in thrombosis. All indirect tests are based on the principle that thrombin is only the enzyme (snake venoms excluded) capable of converting fibrinogen to fibrin in mammals.

Also, apart from paracoagulation tests that detect the presence of circulating soluble fibrin monomer complexes, none of the more specific thrombin tests is readily available or is useful for immediate clinical application in the diagnosis of these conditions associated with fibrin. These tests include the FPA (fibrinopeptide A) test where FPA is measured by a specific RIA procedure, fibrin monomer assays, fibrinogen gel exclusion chromatographs and tests for FPB (fibrinopeptide B) or increased thrombin FPB.

Tests without biochemical specificity for thrombin action include tests for prothrombin time (PT), thromboplastin time (A PTT) and thrombin coagulation time (TCT). Although frequently used in practice, it should be recognized that the information obtained from these tests is not specific in nature, which acts as a measure of a depletion of the coagulation factor independent of the etiology.

Coagulation factor assays have been found to be relatively non-specific and these include tests for cofactors V and VIII as well as tests for fibrinogen levels.

The tests for fibrin-fibrinogen degradation products have not been approved because they are specific for the action of plasmin in fibrin and can provide positive results where there has been fibrinolysis without the action of previous thrombin in the fibrinogen molecule. These tests include fragments D and E.

Tests for thrombin-mediated platelet interaction or release have been found to be non-specific in nature. These include platelet count, platelet survival and platelet release tests.

The use of radiolabelled fibrinogen has also been attempted in relation to the identification of coagulation factors but is found to be time consuming and difficult to perform.

Thus, the efficiency of a diagnostic test is based on its ability to indicate the presence or absence of the disease. There are well-recognized essential principles for studies that determine the effectiveness of a diagnostic test that allows the four indices of sensitivity, specificity, positive predictive value and negative predictive value to be determined. The first requirement is the adoption of an appropriate standard for diagnosis. Ideally, this standard should be slightly greater than a clinical definition and should be as specific as possible for the disease entity. An inherent difficulty in relation to DVT and PE in particular is that many of the routinely available laboratory tests also lack diagnostic specificity. A low platelet count supports the likelihood of these conditions but may occur as an isolated finding secondary to the infection. Similar limitations apply to many of the coagulation assays. Hypofibrinogenemia does not distinguish between primary fibrinolysis, due to the action of plasmin or elastases and secondary fibrinolysis follows the thrombin-mediated conversion of fibrinogen to fibrin. Alternatively, sensitivity tests of thrombin tests are available but are obvious antecedents of their clinical use. An example is the FPA test that, although specific for the action of thrombin, is exquisitely sensitive and can detect localized intravascular coagulation that produces a positive result in uncomplicated venous thrombosis. This clinical significance of a high FPA level, even with a positive paracoagulation test, is then the fact, particularly if platelet count, global coagulation tests and fibrinogen level are normal.

For these reasons, sensitivity, specificity and predictive values cannot be determined in standard form. The clinical presentation of these disorders is complex and unpredictable. The application of the tests available for diagnosis are, therefore, better considered in relation to the different clinical syndromes of intravascular coagulation.

The murine 3B6 monoclonal antibody is described (US Patent No. 4,758,524). This antibody is specific for the D-dimer and represents the first coagulation specific antibody. The ability to use this antibody, however, in humans as a systemic diagnostic agent is limited due to the immunogenicity of the molecule. There remains a need, therefore, to modify the 3B6 antibody to reduce its immunogenicity in non-murine and human animals.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise, the word "comprises", or variations such as "comprising" or "comprising", shall be understood to imply the inclusion of an indicated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

The articles "a" and "a" are used here to refer to one or more than one (ie at least one) grammatical object of the article. For example, "an element" means an element or more than one element.

In the work conducted in the present invention, deimmunization technology is used to reduce the immunogenicity of the 3B6 antibody. This has allowed the development of a diagnostic procedure for thrombosis imaging for use in humans. Additionally, the deimmunized form of antibody 3B6 allows its as an objective clotting agent to deliver clot dissolution or clotting growth prevention agent such as anti-coagulants at the site of a clot. The deimmunized molecules of the present invention therefore act as carriers of therapeutic and diagnostic agents at a target site such as a clot. The molecules also have their own diagnostic or therapeutic properties. The development of a deimmunized form of the 3B6 antibody has application for a range of conditions such as DVT and PE. Additionally, 3B6 deimmunized antibodies can be used in combination with computer-aided imaging techniques or computer-assisted topographic nuclear medicine such as CT, MRI or ultrasound.

The present invention therefore provides a carrier molecule in general in the form of an immunoreactive molecule and in particular a chimeric and / or mutated monoclonal antibody relative to a progenitor molecule such that it exhibits reduced capacity for immunogenicity in a target host, just like a human. The process of chimerism or mutation is referred to herein as deimmunization. In a particularly preferred embodiment, the immunoreactive molecule such as the monoclonal antibody is humanized to reduce its immunogenicity in humans. Deimmunization can be conducted in different forms but in a preferred embodiment, one or more amino acids in the variable region (v) of a mutant monoclonal antibody (eg substituted) to reduce the recognition of MHC II from peptides derived from this region. In other words, the deimmunization process is aimed at reducing an immune response mediated by the T cell epitope for the antibody. The most preferred antibody of the present invention is a deimmunized form of murine 3B6 monoclonal antibody that exhibits specificity for the D-dimer. The generation of a deimmunized form of 3B6 allows the inter-alia development of a target systemic clotting agent for blood clots in humans. This allows its use as an image forming agent and as a vehicle for supplying the clot dissolution or clot growth prevention agent such as at the site of a clot.

The deimmunized antibody therefore acts alone or as a carrier for a range of therapeutic and / or diagnostic agents.

In accordance with the foregoing, one aspect of the present invention provides a variant of an immunoreactive molecule comprising a portion that has specificity for cross-linked fibrin derivatives and whose portion is derived from an immunoreactive molecule that can be obtained from an animal creature or bird where the variant exhibits reduced immunogenicity in another animal creature or bird of the same or different species.

Preferably, the immunoreactive molecule is a variant monoclonal antibody comprising a portion that has specificity for cross-linked fibrin derivatives.

More preferably, the monoclonal antibody is a variant of a murine-derived monoclonal antibody that has specificity for the human-derived D-dimer and other derivatives of cross-linked fibrin and without reactivity with fibrinogen or fibrinogen degradation products including D and E fragments in where the variant murine-derived monoclonal antibody is substantially non-immunogenic in a human.

Preferably, the antibody is a deimmunized antibody molecule that has specificity for an epitope recognized by monoclonal antibody 3B6 and which comprises at least one of the complementarity determining regions (CDRs) of the variable domain derivatives of monoclonal antibody 3B6 and Remaining parts of immunoglobulin remaining from the deimmunized antibody molecule are derived from an immunoglobulin or an analogue thereof from the host for which the antibody is deimmunized.

The present invention therefore provides a deimmunized antibody molecule that has specificity for an epitope recognized by monoclonal antibody 3B6 wherein at least one of the complementarity determining regions (CDRs) of the variable domain of said deimmunized antibody is derived. of the 3B6 monoclonal antibody wherein one or more amino acids in a variable region of said 3B6 antibody mutates to reduce the MHC class II recognition of peptides derived from this region.

The present invention further provides a variant of 3B6 monoclonal antibody of deimmunized murine for use in humans comprising one or more amino acid mutations in region v of antibody 3B6 designated to remove or reduce peptide fragments of region v associated with the molecule MHC class II.

Deimmunized immunoreactive molecules are useful alone or as carriers of diagnostic and / or therapeutic agents at a target site such as a blood clot.

In accordance with the foregoing, the present invention contemplates a method for detecting a blood clot in a human patient by introducing into the patient an immunized form of murine 3B6 monoclonal antibody or an antigen-binding fragment thereof labeled with a reporter molecule. which allows the dissemination of the labeled antibody through the circulatory system and then submit the patient to report the means of molecule detection to identify the location of the antibody in a clot.

In an alternative embodiment, the deimmunized form of 3B6 is not labeled but a second antibody is labeled has anti-immunoglobulin specificity. This antibody forms a complex labeled with the first antibody mentioned.

As a carrier, the deimmunized port can deliver any clot binding molecule at the site of a clot as well as other diagnostic or therapeutic agents.

The present invention therefore contemplates the use of a deimmunized murine monoclonal antibody specific for the D-dimer or other cross-linked fibrin derivatives in the manufacture of the clotting agent.

Yet another aspect of the present invention contemplates a method for facilitating the dissolution or removal of a blood clot in a human, said method comprising administering to said human a clot solution

or effective amount of clot growth prevention of a variant murine-derived monoclonal antibody that has specificity for human-derived D-dimer and other cross-linked fibrin derivatives and without reactivity with fibrinogen or fibrinogen degradation products including D and E fragments wherein said variant murine-derived monoclonal antibody is substantially non-immunogenic in a human wherein said monoclonal antibody additionally comprises a clot dissolution or fused, bound or otherwise associated growth clotting agent associated therewith.

Yet another aspect of the present invention is directed to the use of a variant murine-derived monoclonal antibody that has specificity for the human-derived D-dimer and other cross-linked fibrin derivatives and without reactivity with fibrinogen or fibrinogen degradation products including fragments D and E wherein said variant murine derived monoclonal antibody is substantially non-immunogenic in a human and said antibody further comprises a clot dissolution or clotting growth prevention agent fused, bound or otherwise adhered thereto in the manufacture of a medicine for dissolving a blood clot in a human.

A preferred molecule is a 3B6 monoclonal antibody of variant deimmunized murine for use in humans and comprising a combination of light and heavy chain v regions comprising the amino acid sequences encoded by the nucleotide sequences selected from SEQ ID NO: 7 / SEQ ID NO: 10, SEQ ID NO: 8 / SEQ ID NO: 10, SEQ ID NO: 9 / SEQ ID NO: 10, SEQ ID NO: 7 / SEQ ID N0: 12, SEQ ID NO: 8 / SEQ ID NO: 12, SEQ ID NO: 8 / SEQ ID NO: 11, SEQ ID NO: 9 / SEQ ID NO: 11, SEQ ID NO: 9 / SEQ ID NO: 12 and SEQ ID NO: 7 / SEQ ID NO: eleven

or combinations of amino acid sequences encoded by nucleotide sequences that have at least 70% similarity in one or both amino acid sequences in each of the pairs listed above.

or the nucleotide sequences capable of hybridizing under conditions of low requirement in one or both nucleotide sequences or their forms of complementarity in each of the pairs listed above.

5

10

fifteen

twenty

25

30

35

The variant 3B6 deimmunized antibody for use preferably comprises a combination of light and heavy chain v regions comprising the amino acid sequences selected from SEQ ID NO: 1 / SEQ ID NO: 4, SEQ ID NO: 2 / SEQ ID NO: 4 , SEQ ID NO: 3 / SEQ ID NO: 4, SEQ ID NO: 1 / SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 2 / SEQ ID NO: 5, SEQ ID NO: 3 / SEQ ID NO: 5, SEQ ID NO: 3 / SEQ ID NO: 6 and SEQ ID NO: 1 / SEQ ID NO: 5,

Another preferred variant of 3B6 comprises a combination of light and heavy chain v regions selected from VHv5 / VKv1, VHv6 / VKv1 VHv7 / VKv1, VHv5 / VKv7, VHv6 / VKv7, VHv6 / VKv4, VHv7 / VKv4, VHv7 / VHv7 / VHv7 / VHv7 / VKv4.

Another aspect of the present invention contemplates a method for detecting a blood clot in a human patient by introducing into the patient an immunized form of murine 3B6 monoclonal antibody or an antigen binding fragment thereof labeled with a reporter molecule that allows the dissemination of the labeled antibody through the circulatory system and then subject the patient to a computer-assisted tomographic nuclear medicine scan to visualize the clot.

Yet another aspect of the present invention contemplates a method for detecting a blood clot in a human patient by introducing into the patient an immunized form of murine monoclonal antibody 3B6 or an antigen binding fragment thereof labeled with a reporter molecule that allows dissemination of the labeled antibody through the circulatory system and then subjecting said patient to flat clot imaging to visualize the clot.

The deimmunized immunoreactive molecule of the present invention is also useful for anchoring an anti-coagulant at a particular site. This aspect therefore provides specific tissue anchoring of a therapeutic or diagnostic agent to, for example, a clot. Additionally, the immunoreactive molecule can be engineered to have multiple specificities. For example, a bispecific deimmunized antibody is contemplated which comprises a specificity for a clot and another specificity at a site of a clot (for example in a cellular receptor). This allows the antibody to remain at the clot site.

In an alternative, a multistage treatment is contemplated wherein, for example, the deimmunized 3B6 or other interactive molecule conjugated with an anti-coagulant is administered to the target clot and forms a complex and then a second antibody directed to the first antibody and / or the anti-coagulant is administered to improve or monitor the first complex.

The deimmunized immunointeractive molecule can also be used to determine the kinetics of clot dissipation or clot disappearance. This is useful for even predicting the onset or early disappearance of clots and, therefore, helps determine the kinetics when the second treatments such as anti-coagulant treatments are initiated.

The present invention further provides conjugates comprising deimmunized immunoreactive molecules and therapeutic and / or imaging tags. Examples of imaging tags include MRI, ultrasound and CT tags. Examples of therapeutic labels include radioactive isotopes, anti-coagulation agents and cytokines.

A summary of sequence identifiers used through the object specification is provided in Table 1.

TABLE 1 Summary of Sequence Identifiers

SEQUENCE ID NO:

DESCRIPTION

one

3B6DIVHv5 amino acid

2

3B6DIVHv6 amino acid

3

3B6DIVHv7 amino acid

4

3B6DIVKv1 amino acid

(continuation)

SEQUENCE ID NO:

DESCRIPTION

5

3B6DNKv4 amino acid

6

Amino acid 3B6DIVKv7

7

3B6DIVHv5 nucleotide sequence that codify

8

3B6DIVHv6 nucleotide sequence that codify

9

3B6DIVHv7 nucleotide sequence that codify

10

3B6DNKv1 nucleotide sequence that codify

eleven

3B6DNKv4 nucleotide sequence that codify

12

3B6DNKv7 nucleotide sequence that codify

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a representation showing (A) a scheme of monoclonal antibody 3B6; (B) a photograph of 3B6 binding to blood clots (magnification x4,2000).

Figure 2 is a schematic representation of a 3B6 antibody labeled with a nuclear tag (99mTc).

Figure 3A is a diagrammatic representation showing the administration of 3B6 99mTc to the circulatory system of a human.

10 Figure 3B is a photographic representation showing the visualization of blood clots in the front of the thighs as radiation of 99mTc concentrates at the site of the clot.

Figures 4A, 4B and 4C are graphical representations showing the dimer d capture assay using 3B6 deimmunized monoclonal antibodies.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is partly predicted in the application of biochemical techniques to present an immunoreactive molecule derived from an animal creature or bird substantially non-immunogenic in another animal creature or bird of the same species or of different species. The biochemical process is referred to herein as "deimmunization." The reference here to "disinmunization" includes processes such as complementarity determining region graft (CDR), "reconformation" with respect to a region of molecule structure

20 immunoreactive and variable region mutation (v), all aimed at reducing the immunogenicity of an immunoreactive molecule in a particular host. In the present case, the preferred immunoreactive molecule is an antibody such as a monoclonal or polyclonal antibody. In a more preferred embodiment, the immunoreactive molecule is a monoclonal antibody, derived from an animal or bird creature and exhibiting reduced immunogenicity in another animal or bird creature of the same or different species.

The present invention relates generally to carrier molecules derived from a species or strain of animal or bird and that are substantially non-immunogenic when exposed to an immune system of a species or strain of another animal or bird creature. The carrier molecules may exhibit useful diagnostic or therapeutic properties per se or may be used to deliver active compounds (eg anticoagulant agents, radioactive isotopes) at a target site. In general, carrier molecules are immunoreactive molecules. More particularly, the present invention is directed to a deimmunized form that includes a non-murine mammalian form of a murine derived monoclonal antibody substantially incapable of inducing an immune response against itself in a non-murine animal and in particular a human. Even more particularly, the present invention provides a deimmunized form of murine monoclonal antibody 3B6 in such a way that it is incapable of, or exhibits reduced ability to induce a substantial immune response against itself or its derivatives when administered to a human. Deimmunized immunoreactive molecules and in particular the antibodies of the present invention have a range of useful diagnostic and therapeutic applications such as in the detection of blood clots in the circulatory system of a human and as the target agent of a clot to deliver a solution. of the clot or clot growth prevention agent such as an anticoagulant. The deimmunized form that includes humanized forms of the subject monoclonal antibody are particularly useful in the diagnosis and treatment of conditions such as deep vein thrombosis and pulmonary embolism. The molecules of the present invention are particularly useful as agents for delivering the active compounds at a target site. Such active compounds include clot binding molecule.

In accordance with the foregoing, one aspect of the present invention provides a variant of an immunoreactive molecule, said variant comprises a portion that has specificity for crosslinked fibrin derivatives and whose portion is derived from an immunoreactive molecule that can be obtained from an animal creature or bird where said variant exhibits reduced immunogenicity in another animal or bird creature of the same or different species.

As indicated above, the preferred form of the immunoreactive molecule is an antibody and in particular a monoclonal antibody.

In accordance with the foregoing, another aspect of the present invention provides a variant monoclonal antibody that comprises a portion that has specificity for cross-linked fibrin derivatives and whose portion is derived from a monoclonal antibody that can be obtained from a first animal or bird creature in wherein said variant exhibits reduced immunogenicity in a second animal or bird creature of the same or different species.

In a particularly preferred embodiment, a monoclonal antibody of a murine animal is obtained and de-immunized with respect to another murine animal or a different species of animal such as a human. The murine monoclonal antibody arises in the murine animal in the non-denatured D-dimer that is derived from fibrinogen. The last molecule is digested by plasmin and generates a range of the fragments designated fragments A to E. Crosslinking fibrin with Factor XIIIa forms dimers of fragment D referred to as "dimer D". The D-dimer is a molecule of approximately 189,000 daltons and comprises two functional groups of the D-fragment linked by cross-linked bonds between the remnants of the γ chain of fibrinogen.

In accordance with the foregoing, another aspect of the present invention is directed to a variant murine derived monoclonal antibody that has specificity for the D-dimer derived from human and other cross-linked fibrin derivatives and without reactivity with fibrinogen or fibrinogen degradation products including of fragments D and E wherein said variant murine-derived monoclonal antibody is substantially non-immunogenic in a human.

the reference to "substantially non-immunogenic" includes reduced immunogenicity compared to the parent antibody, that is to say an antibody before exposure to the deimmunization processes. The term "immunogenicity" includes an ability to elicit, induce or otherwise facilitate the T-cell and / or humoral mediated response in a host animal. Particularly suitable immunogenic criteria include the ability for amino acid sequences derived from a variable region (v) of an antibody that interacts with the MHC class II molecule thereby stimulating and facilitating a T-cell mediated response that includes a humoral response assisted by T cell. The immunoreactive molecule and in particular a monoclonal antibody contemplated by the present invention includes reference to a clotting target agent.

The preferred murine-derived monoclonal antibody is referred to herein as 3B6 monoclonal antibody which is described in US Patent No. 4,758,524.

In accordance with the foregoing, in a particularly preferred embodiment, the present invention provides a deimmunized form of monoclonal antibody 3B6 wherein said deimmunized 3B6 is substantially non-immunogenic in humans.

Again, "substantially non-immunogenic" in this context means a reduced ability of the 3B6 deimmunized monoclonal antibody to induce or facilitate an immune response against itself (after initial or subsequent administration) in a human compared to murine 3B6 monoclonal antibody. , before the deimmunization.

Although the preferred invention is particularly directed to a deimmunized form of 3B6 with respect to humans, the present invention extends to this antibody or other antibody with a similar specificity for the D-dimer and / or other derivatives of deimulated crosslinked fibrin for any other species of animal or bird.

Reference herein to other cross-linked fibrin derivatives include, for example, in addition to the dimer D, derivatives of the dimer D and a complex comprising fragments D and E. The latter includes (DD) E and is formed by plasmin degradation of the crosslinked human fibrin and comprises a combination of two D fragments and the E fragment. Other crosslinked derivatives include DY, YY, XD, XY, DXD and YXD wherein the letters represent fibrinogen fragments formed after degradation by plasmin where X and Y are different and are selected from fragments A to C and E. Preferably, the deimmunized antibody of the subject invention is derived from an antibody specific for D-dimer and other cross-linked fibrin derivatives that do not cross-react with fibrinogen. , fibrinogen degradation products including fragment D and fragment E. Preferably, the clones that produce the antibody are selected using solution phase tion of the D-dimer molecules unlike the immobilized D-dimer.

Preferably, the deimmunized antibody exhibits an affinity for its target antigen that is similar to the affinity exhibited by murine 3B6 monoclonal antibody.

"Affinity" in relation to the interaction between an individual antigen bound site in an antigen binding molecule and its corresponding site in the antigen includes the resistance of this interaction.

"Antibody" means a protein from the immunoglobulin family that is capable of combining, interacting or otherwise associated with an antigen. An antibody is, therefore, an antigen binding molecule. An "antibody" is an example of an immunoreactive molecule and includes a monoclonal or polyclonal antibody. Preferred immunoreactive molecules of the present invention are monoclonal antibodies. An antibody includes parts thereof that include Fab portions and antigen binding determinants.

The term "antigen" used herein in its broadest sense refers to a substance that is capable of reacting in and / or inducing an immune response. Reference to an "antigen" includes an antigenic determinant or epitope. The antigen in the current context is presented as the immunoreactive molecule and, more particularly, a monoclonal antibody.

Any molecule that has binding affinity for a target antigen is referred to as an "antigen binding molecule." It will be understood that this term extends to immunoglobulins (for example polyclonal or monoclonal antibodies), immunoglobulin fragments and non-immunoglobulin-derived protein structures that exhibit antigen binding activity. The terms "antibody" and "antigen binding molecules" include deimmunized forms of these molecules.

That part of an antigenic molecule against which a particular immune response is directed is referred to as an "antigenic determinant" or "epitope" and includes a hapten. Typically, in an animal, antigens present in several or even many antigenic determinants simultaneously. A "hapten" is a substance that can combine specificity with an antibody but cannot or only poorly induces an immune response unless it is linked to a carrier. A hapten typically comprises a single antigen or epitope.

As indicated above, although the preferred antibodies of the present invention are deimmunized forms of murine monoclonal antibodies for use in humans, the subject invention extends to antibodies from any source and is deimmunized for use in any host. Examples of animal and bird sources and hosts include humans, primates, farm animals (e.g. sheep, cows, horses, pigs, donkeys), animals for laboratory tests (e.g. mice, rabbits, guinea pigs, hamsters), animals companion (for example dogs, cats), poultry (for example chickens, ducks, geese, turkeys) and game birds (for example pheasants). Deimmunized antibodies or part thereof can also be generated in non-animal tissues such as plants. Plants are particularly useful as a source of single chain antibodies.

Another aspect of the present invention contemplates a method for generating a deimmunized monoclonal antibody that has specificity for antigenic determinants in the human D-dimer or other derivatives of cross-linked fibrin, said method comprises:

(i)

obtain a crosslinked fibrin derivative or extract that contains the same from a human;

(ii)

generating an antibody in a non-human animal specific for said cross-linked fibrin derivative but which does not cross-react with fragment D; Y

(iii) subjecting said non-human derivative antibody to deimmunization means.

The crosslinked fibrin derivative can be derived from any antigenic extract that includes degradation mediated by plasmin of fibrin clots or by the simultaneous action of thrombin, Factor XIIIa and plasmin in the fibrinogen with formation of transient clot and subsequent clot lysis. In the latter method, fibrinogen is converted to fibrin by the action of thrombin and Factor XIIIa and subsequently digested with plasmin. Of course, it will be appreciated that the fibrin derivative or extract containing the same can be obtained from an animal source other than human. The antigenic source is conveniently from a biological sample.

A sample that can be extracted, not treated, treated, diluted or concentrated from an animal is included in the term "biological sample".

The above method to obtain the crude antigenic fraction is represented in an in vitro method. A suitable in vivo method includes obtaining serum or other body fluid containing the crosslinked fibrin derivative of an animal that includes human and subjecting the body fluid to a PAGE process where the substantially pure crosslinked fibrin derivative is isolated.

Alternatively, cross-linked fibrin derivatives can be purified from serum obtained from patients suffering from severe thrombotic disorders based on a technique using gel filtration in combination with ion exchange chromatography as described by Willner et al., Biochemistry 21: 2687 -2692, 1982.

The antigen (i.e. the D-dimer or other cross-linked fibrin derivative) can be separated from the biological sample by any suitable means. For example, the separation can take advantage of any one or more of the surface loading properties of antigen, size, density, biological activity and its affinity during another entity (for example another protein or chemical compound to which it binds or another form is associated). Thus, for example, the separation of the antigen from the biological fluid can be achieved by one or more ultra-centrifugation, ion exchange chromatography (for example anion exchange chromatography, cation exchange chromatography), electrophoresis (for example electrophoresis of polyacrylamide gel, isoelectric focusing), size separation (for example, gel filtration, ultrafiltration) and affinity-mediated separation (for example immunoaffinity separation that includes, but is not limited to, separation of magnetic globules such as separation Dynabead ™, immunochromatography, immuno-precipitation). The selection of the separation techniques employed may depend on the biological activity or physical properties of the particular antigen.

Preferably, the separation of the antigen from the biological fluid preserves the conformational epitopes present on the surface of the antigen and, thus, adequately avoids techniques that cause the denaturation of the antigen. Persons skilled in the art will recognize the importance of maintaining or imitating as much as possible of the peculiar physiological conditions in the antigen (for example the biological fluid from which they are obtained) to ensure that the antigenic determinants or sites active in the antigen , which are exposed to the animal, are structurally identical to that of the native antigen. This ensures the emergence of appropriate antibodies in the immunized animal that would recognize the native antigen. In a preferred embodiment of this type, the antigen is separated from the biological fluid using any one or more affinity separation, gel filtration and ultrafiltration.

Immunization and subsequent production of monoclonal antibodies can be carried out using standard protocols as described for example by Köhler and Milstein (Nature 256: 495-499, 1975; Köhler and Milstein, Eur. J. Immunol. 6 (7) : 511-519, 1976), Coligan et al. (Current Protocols in Immunology, John Wiley & Sons, Inc., 1991-1997) or Toyama et al. ("Monoclonal Antibody, Experiment Manual", published by Kodansha Scientific, 1987). Essentially, an animal is immunized with a biological fluid that contains antigen or fraction thereof by standard methods to produce cells that produce antibodies, particularly somatic cells that produce antibodies (for example B lymphocytes). These cells can then be removed from the immunized animal for immortalization. The antigen may need to be first associated with a larger molecule. The latter is any typically high molecular weight substance in which a poorly immunogenic or non-immunogenic substance (for example a hapten) binds naturally or artificially to improve its immunogenicity.

Immortalization of antibody producing cells can be carried out using methods, which are well known in the art. For example, immortalization can be achieved by the transformation method using the Epstein-Barr virus (EBV) (Kozbor et al., Methods in Enzymology 121: 140, 1986). In a preferred embodiment, the cells that produce antibody using the cell fusion method (described in Coligan et al., 1991-1997, supra), are widely used for the production of monoclonal antibodies. In this method, the cells that produce the somatic antibody with the potential to produce antibodies, particularly B cells, fuse with a myeloma cell line. These somatic cells can be derived from the lymph nodes, spleen and peripheral blood of primed animals, preferably rodent animals such as mice and rats. In the exemplary embodiment of this invention mouse spleen cells are used. It would be possible, however, to use cells from rats, rabbits, sheep or goats, or cells from other animal species instead.

Specialized myeloma cell lines have developed from lymphocytic tumors for use in fusion procedures that produce hybridoma (Köhler and Milstein, 1976, supra; Shulman et al., Nature 276: 269-270, 1978; Volk et al., J Virol. 42 (1): 220-227, 1982). These cell lines have been developed for at least three reasons. The first is to facilitate the selection of fused hybridomas of myeloma cells that self-propagate indefinitely in a similar manner or do not fuse. Usually, this is done by using myelomas with enzyme deficiencies that are unable to grow in certain selective media that support hybridoma growth. The second reason arises from the inherent ability of lymphocytic tumor cells to produce their own antibodies. To eliminate the production of tumor cell antibodies by hybridomas, myeloma cell lines unable to produce endogenous heavy or light immunoglobulin chains are used. A third reason for the selection of these cell lines is because of their suitability and efficiency for fusion.

Many myeloma cell lines can be used for the production of fused cell hybrids, including, for example, P3X63-Ag8, P3X63-AG8.653, P3 / NS1-Ag4-1 (NS-1), Sp2 / 0-Ag14 and S194 / 5.XXO.Bu.1. The P3X63-Ag8 and NS-1 cell lines have been described Köhler and Milstein (1976, supra). Shulman et al. (1978, supra) develops the Sp2 / 0-Ag14 cell line. Line S194 / 5.XXO.Bu.1 is reported by Trowbridge (J. Exp. Med. 148 (1): 313-323, 1978).

Methods for generating hybrids of lymph node or spleen cells that produce antibody usually involve mixing somatic cells with myelomaoma cells in a 10: 1 ratio (although the ratio may vary from about 20: 1 to about 1: 1), respectively. , in the presence of an agent or agents (chemical, viral or electrical) that promotes the fusion of cell membranes. Fusion methods have been described in (Köhler and Milstein, 1975, supra; Köhler and Milstein, 1976, supra; Gefter et al., Somatic Cell Genet. 3: 231-236, 1977; Volk et al., 1982, supra ). The agents that promote fusion used by those researchers are Sendai virus and polyethylene glycol (PEG).

Because fusion procedures produce viable hybrids at a very low frequency (for example when using spleens from a somatic cell source, only one hybrid is obtained approximately every 1x105 spleen cells), it is preferable to have a means to select the hybrids fused cell counts of the remaining non-fused cells, particularly the non-fused myeloma cells. A means is also necessary to detect hybridomas that produce antibodies among others that result from fused cell hybrids. In general, the selection of fused cell hybrids is carried out by culturing the cells in medium that supports the growth of hybridomas but that prevents the growth of non-fused myeloma cells, which will normally be indefinitely divided. The somatic cells used in the fusion do not maintain long-term viability in in vitro culture and therefore do not have a problem. In the example of the present invention, myeloma cells lacking hypoxanthine phosphoribosyl transferase (HPRT-negative) are used. Selection against these cells is made in hypoxanthine / aminopterin / thymidine (HAT) medium, a medium in which fused cell hybrids survive due to the HPRT-positive genotype of spleen cells. It is also possible to use myeloma cells with different genetic deficiencies (drug sensitivities, etc.) that can be selected against the medium supports the growth of genotypically competent hybrids.

It takes several weeks to selectively cultivate the fused cell hybrids. Early in this period of time, it is necessary to identify those hybrids that produce the desired antibody, since they can be subsequently cloned and propagated. In general, about 10% of the hybrids obtained produce the desired antibody, although a range of about 1 to about 30% is not common. Detection of the hybrids that produce antibody can be achieved by any one of several standard test methods, which includes enzyme-linked radioimmunoassay and immunoassay techniques, for example, as described in Kennet et al. ((eds) Monoclonal Antibodies and Hybridomas: A New Dimension in Biological Analyzes, pp. 376-384, Plenum Press, New York, 1980). In a particularly preferred embodiment, an enzyme-linked immunosorbent assay (ELISA) is developed to select clones that produce antibody using the solution phase D-dimer.

Once desired fused cell hybrids have been selected and cloned into cell lines that produce individual antibodies, each cell line can be propagated in two standard forms. A suspension hybridoma cells can be injected into a histocompatible animal. The injected animal will then develop tumors that secrete the specific monoclonal antibody produced by the fused cell hybrid. The animal's body fluids, such as serum or ascites fluid, can be used to provide monoclonal antibodies in high concentration. Alternatively, individual cell lines can be propagated in vitro in laboratory vessels for culture. The culture medium containing high concentrations of a single specific monoclonal antibody can be harvested by decantation, filtration or centrifugation, and subsequently purified.

Cell lines are tested for their specificity to detect the antigen of interest by any suitable immunodetection means. For example, cell lines can be divided into aliquots in a number of

5

10

fifteen

twenty

25

30

35

wells are incubated and the supernatant of each well is analyzed by enzyme-linked immunoabsorbent assay (ELISA), indirect fluorescence antibody technique, or the like. Cell lines that produce a monoclonal antibody capable of recognizing the target antigen but that does not recognize recognized target epitopes are then identified and then cultured directly in vitro or injected into a histocompatible animal to form tumors and to produce, collect and purify the required antibodies.

Thus, the present invention provides in a first stage monoclonal antibodies that specifically interact with the D-dimer or other cross-linked fibrin derivative.

As indicated above, non-animal cells such as plant, yeast and / or microbial cells can typically be used to typically generate single chain antibodies. In this embodiment, such cells are engineered to express nucleic acid molecules that encode an antibody chain.

The monoclonal antibody is then subjected to deimmunization media. Such a process can take any of a number of forms that include the preparation of chimeric antibodies having the same or similar specificity as the monoclonal antibodies prepared in accordance with the present invention. Chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, of the constant and variable region immunoglobulin genes that belong to different species. Thus, according to the present invention, once a hybridoma that produces the desired monoclonal antibody is obtained, techniques are used to produce interspecific monoclonal antibodies in which the binding region of a species is combined with a non-binding region in the antibody of another species (Liu et al., Proc. Natl. Acad. Sci. USA 84: 3439-3443, 1987). For example, non-human monoclonal antibody CDRs (for example murine) can be grafted onto a human antibody, such as the "humanizing" murine antibody (European Patent Publication No. 0 239 400; Jones et al., Nature 321: 522-525, 1986; Verhoeyen et al., Science

239: 1534-1536, 1988; Riechmann et al., Nature 332: 323-327, 1988). In this case, the deimmunizing process is specific to humans. More particularly, CDRs can be grafted into a variable region of human antibody with or without human constant regions. The non-human antibody provides that the CDRs are typically referred to as the "donor" and the human antibody provides the structure that is typically referred to as the "recipient." The constant regions need not be present, but if they are, they can be substantially identical to the human immunoglobulin constant regions, that is at least about 85-90%, preferably about 95% or more identical. Therefore, all parts of a humanized antibody, except possibly the CDRs, are substantially identical to the corresponding parts of the natural human immunoglobulin sequences. Thus, a "humanized antibody" is an antibody comprising a humanized heavy chain or humanized light chain immunoglobulin. A donor antibody is said to be "humanized," through the "humanization" process, because the resulting humanized antibody is expected to bind to the same antigen as the donor antibody that provides the CDRs. The reference here to "humanized" includes reference to an deimmunized antibody to a particular host, in this case, a human host.

It will be understood that deimmunized antibodies may have conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions. Example conservative substitutions can be made in accordance with Table 2.

TABLE 2

ORIGINAL WASTE

EXAMPLE SUBSTITUTIONS

To

Be

Arg

Lys

Asn

Gln, His

Asp

Glu

Cys

Be

Gln

Asn

(continuation)

ORIGINAL WASTE

EXAMPLE SUBSTITUTIONS

Glu

Asp

Gly

Pro

His

Asn, Gln

Ile

Leu, Val

Leu

Ile, Val

Lys

Arg, Gln, Glu

Met

Leu, Ile

Phe

Met, Leu, Tyr

Be

Thr

Thr

Be

Trp

Tyr

Tyr

Trp, Phe

Val

Ile, Leu

Example methods are described which can be used to produce deimmunized antibodies according to the present invention, for example, in Richmann et al., 1988, supra; US Patent Nos. 6,056,957, 6,180,370 and 6,180,377 and Chothia et al., J. Mol. Biol. 196: 901, 1987.

Thus, in one embodiment, the present invention contemplates a deimmunized antibody molecule that has specificity for an epitope recognized by the 3B6 monoclonal antibody wherein at least one or at least two or at least three or at least four or by at least five of the determining regions of

Complementarity (CDR) of the variable domain of said deimmunized antibody is derived from said monoclonal antibody 3B6 and the remaining immunoglobulin derived parts of the deimmunized antibody molecule are derived from an immunoglobulin or an analogue thereof from the host for which the antibody.

This aspect of the present invention involves the manipulation of the structure region of a non-human antibody.

Preferably, the deimmunized antibody is a humanized form of murine 3B6.

A preferred deimmunization process is referred to herein as a variable region graft (v) and results in a chimeric antibody. The resulting antibody comprises one or more amino acid substitutions within the v region when compared to the current antibody (eg murine). The reason for making changes in region v is to improve the potential for an immune response induced in the intended host (for example a

20 human). The basis for deinmunization is predicted in part on the assumption that a substantive immune response for an introduced antibody requires a T-cell mediated response. Activation for the T cell response is the presentation of processed peptides emanating from the antibody introduced into the antibody. surface of antigen presenting cells (APC). APCs present such peptides in association with the surface of the MHC class II molecule. The deimmunization method, therefore, is based on:

(I) predict peptide sequences capable of associating with MHC class II molecules; and (ii) change strategic residues to eliminate the ability of the peptide to associate with the MHC class molecule

II.

In accordance with the foregoing, another aspect of the present invention provides a variant of 3B6 monoclonal antibody of deimmunized murine for use in humans, said variant comprises one or more amino acid mutations in region v of said 3B6 antibody to remove or reduce fragments peptide of said v region associated with the MHC class II molecule.

One or more amino acid substitutions, additions and / or deletions or one or more nucleotide substitutions, additions and / or deletions are encompassed within the term "mutation" or "mutations".

In a particularly preferred embodiment, the deimmunized antibodies are generated by cotransfection of different combinations of three deimmunized H chain genes and three deimmunized L chain genes. The resulting variants are derived from different combinations encoded by the L chain and H chain genes. Preferred H chains are Hv5, Hv6 and Hv7. These are referred to herein as 3B6DIVHv5 (SEQ ID NO: 1), 3B6DIVHv6 (SEQ ID NO: 2) and 3B6DIVHv7 (SEQ ID NO: 3). Preferred L chains Kv1, Kv4 and Kv7. These are referred to herein as 3B6DIVKv1 (SEQ ID NO: 4), 3B6DIVKv4 (SEQ ID NO: 5) and 3B6DIVKv7 (SEQ ID NO: 6). Particularly useful combinations include VHv5 / VKv1, VHv6 / VKv1, VHv7NKv1, VHvS / VKv7, VHv6 / VKv7, VHv6 / VKv4, VHv7 / VKv4, VHv7 / VKv7 and VHv5 / VKv4. The sequence identifier numbers (SEQ ID NOS :) in parentheses represent the amino acid sequences of the particular chains. The corresponding nucleotide sequences encoding each of SEQ ID NOS: 1-6 are represented by SEQ ID NOS: 7-12.

All such combinations of the H and L chains are also encompassed by the present invention.

In accordance with the foregoing, the present invention provides a 3B6 variant monoclonal antibody of deimmunized murine for use in humans, said variant comprises a combination of light and heavy chain v regions comprising the amino acid sequences encoded by the nucleotide sequences selected from SEQ ID NO: 7 / SEQ ID NO: 10, SEQ ID NO: 8 / SEQ ID NO: 10, SEQ ID NO: 9 / SEQ ID NO: 10, SEQ ID NO: 7 / SEQ ID NO: 12, SEQ ID NO: 8 / SEQ ID NO: 12, SEQ ID NO: 8 / SEQ ID NO: 11, SEQ ID NO: 9 / SEQ ID NO: 11, SEQ ID NO: 9 / SEQ ID NO: 12 and SEQ ID NO : 7 / SEQ ID NO: 11.

All such combinations of H and L chains are also encompassed by the present invention.

In accordance with the foregoing, the present invention provides a 3B6 variant monoclonal antibody of deimmunized murine for use in humans, said variant comprises a combination of light and heavy chain v regions comprising the amino acid sequences selected from SEQ ID NO: 1 / SEQ ID NO: 4, SEQ ID NO: 2 / SEQ ID NO: 4, SEQ ID NO: 3 / SEQ ID NO: 4, SEQ ID NO: 1 / SEQ ID NO: 6, SEQ ID NO: 2 / SEQ ID NO: 6, SEQ ID NO: 2 / SEQ ID NO: 5, SEQ ID NO: 3 / SEQ ID NO: 5, SEQ ID NO: 3 / SEQ ID NO: 6 and SEQ ID NO: 1 / SEQ ID NO: 5

More particularly, the present invention provides a variant of a 3B6 monoclonal antibody of deimmunized murine for use in humans, said variant comprising a combination of light and heavy chain v regions selected from VHv5WKv1, VHv6 / VKv1, VHv7 / VKv1 VHv5 / VKv7, VHv6 / VKv7, VHv6 / VKv4, VHv7 / VKv4, VHv7 / VKv7 and VHv5 / VKv4.

The term "similarity" as used herein includes exact identity between the sequences compared at the nucleotide or amino acid level. Where there is no identity at the nucleotide level, "similarity" includes differences between the sequences that result in different amino acids that nonetheless relate to each other at the structural, functional, biochemical and / or conformational levels. Where there is no identity at the amino acid level, "similarity" includes amino acids that nonetheless relate to each other at the structural, functional, biochemical and / or conformational levels. In a particularly preferred embodiment, nucleotide and sequence comparisons are made at the level of identity as opposed to similarity.

Terms used to describe the sequence relationships between two or more polynucleotides or polypeptides include "reference sequence", "comparison window", "sequence similarity", "sequence identity", "sequence similarity percentage", " percentage of sequence identity "," substantially similar "and" substantial identity ". A "reference sequence" is at least 12 but often 15 to 18 and often at least 25 or less, such as 30 monomer units, including nucleotides and amino acid residues, in length. Because two polynucleotides can each comprise (1) a sequence (i.e. only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, Sequence comparisons between two (or more) polynucleotides are typically developed by comparing the sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of typically 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (ie spaces) of approximately 20% or less when compared to the reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. Optimal sequence alignment to align a comparison window can be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA ) or by inspection and the best alignment (ie resulting from the higher homology percentage over the comparison window) is generated by any of the various methods selected. Reference can also be made to the BLAST family of programs, for example, described by Altschul et al. (Nucl. Acids Res. 25: 3389-3402. 1997). A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al. ("Current Protocols in Molecular Biology" John Wiley & Sons Inc, 1994-1998, Chapter 15).

The terms "sequence similarity" and "sequence identity" as used herein refers to the degree that the sequences are identical or functionally or structurally similar on a nucleotide base by nucleotide or an amino acid base by amino acid on a comparison window. . Thus, a "sequence identity percentage", for example, is calculated by comparing two optimally aligned sequences on the comparison window, determining the number of positions in which the identical nucleic acid base (for example A, T, C , G, I) or the identical amino acid residue (for example Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occur in both sequences to produce the number of matching positions, which divides the number of matching positions by the total number of positions in the comparison window (that is, the size of the window), and multiply the result by 100 to produce the percent sequence identity. For the purposes of the present invention, "sequence identity" shall be understood to mean the "match percentage" calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco , California, USA) using default standards as used in the reference manual that accompanies the software. Similar comments apply in relation to sequence similarity.

Mutations and derivatives contemplated by the present invention include redundant mutations in nucleotide sequences that do not result in a change in amino acid sequence. The reference here to a low requirement includes and covers at least about 0 to at least about 15% v / v of formamide and at least about 1 M to at least about 2 M of salt for hybridization, and at least about 1 M to at least about 2 M of salt for washing conditions. In general, the low requirement is approximately 25-30 ° C to approximately 42 ° C. The temperature can be altered and the higher temperatures used to replace formamide and / or to give conditions of alternative requirement. The alternative requirement conditions can be applied when necessary, such as medium requirement, which includes and covers at least about 16% v / v at least about 30% v / v formamide and at least about 0.5 M to at least about 0.9 M of salt for hybridization, and at least about 0.5 M to at least about 0.9 M of salt for washing conditions, or high demand, which includes and covers at least about 31% v / goes at least about 50% v / v of formamide and at least about 0.01 M to at least about 0.15 M of salt for hybridization, and at least about 0.01 M to at least about 0.15 M of salt for conditions washing In general, washing is carried out Tm = 69.3 + 0.41 (G + C)% (Marmur and Doty, J. Mol. Biol. 5: 109, 1962). However, the Tm of a duplex DNA is reduced by 1 ° C with each increment of 1% in the number of mismatched base pairs (Bonner and Laskey, J. Mol. Biol. 5: 109, 1962). Formamide is optional under these hybridization conditions. In accordance with the foregoing, particularly preferred levels of demand are defined as follows: the low requirement is 6 x SSC buffer, 0.1% w / v SDS at 25-42 ° C; a moderate requirement is 2 x SSC buffer, 0.1% w / v SDS at a temperature in the range of 20 ° C to 65 ° C; The high requirement is 0.1 x SSC buffer, 0.1% w / v SDS at a temperature of at least 65 ° C.

As used herein, the term "CDR" includes CDR structural loops that cover three light chain regions and three heavy chain regions in the variable portion of an antibody structure region that bridges β strains in the binding portion of the molecule These loops have characteristic canonical structures (Chothia et al., J. Mol. Biol. 227: 799, 1992; Kabat et al., "Sequences of Proteins of Immunological Interest", U.S. Department of Health and Human Services, 1983).

An immunoglobulin heavy or light chain variable region, which is disrupted by three hypervariable regions, also referred to as CDRs, is referred to herein as a "structure region." The extent of the structure region and the CDRs have been precisely defined (see, for example, Krebber et al., J. Immunol. Methods 201 (1): 35-55, 19). The sequences of the structure regions of different heavy or light chains are relatively conserved within a species. As used herein, a "human structure region" is a region of structure that is substantially identical (approximately 85% or more, usually 90-95% or more) to the structure region of a naturally occurring human immunoglobulin. The structure region of an antibody, which is the combined structure regions of the light and heavy constituent chains, serves to position and align the CDRs. CDRs are primarily responsible for the binding of an epitope of an antigen.

As used herein, the term "heavy chain variable region" means a polypeptide that is approximately 110 to 125 amino acid residues in length, the amino acid sequence of which corresponds to that of a heavy chain of a monoclonal antibody of the invention, starting from the amino acid residue of Terminal amino (Terminal N) of the heavy chain. Similarly, the term "light chain variable region" means a polypeptide that is approximately 95 to 130 amino acid residues in length, the amino acid sequence of which corresponds to that of a light chain of a monoclonal antibody of the invention. , starting from the amino acid residue of Terminal N of the light chain. The full-length immunoglobulin "light chains" (approximately 25 Kd or 214 amino acids) are encoded by a variable region gene in the NH2 terminal (approximately 110 amino acids) and a constant region gene κ or λ in the COOH terminal. The full length immunoglobulin "heavy chains" (approximately 50 Kd or 446 amino acids), are similarly encoded by a variable region gene (approximately 116 amino acids) and one of the other constant region genes mentioned above, for example γ ( encoding approximately 330 amino acids).

The term "immunogenicity" is used here in its broadest sense to include property to prevent an immune response within an organism. The immunogenicity typically depends partly on the size of the substance in question, and partly unlike what the host molecules are. It is generally considered that highly conserved proteins tend to have low immunogenicity.

The term "immunoglobulin" is used herein to refer to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. Recognized immunoglobulin genes include the constant regions κ, λ, α, γ (IgG1, IgG2, IgG3, IgG4), δ, ε and µ, as well as a myriad of immunoglobulin variable region genes. One form of immunoglobulin constitutes the basic structural unit of an antibody. This form is a tetramer and consists of two identical pairs of immunoglobulin chains, each pair has a heavy chain and a light chain. In each pair, heavy and light chain variable regions are together responsible for binding to an antigen, and constant regions are responsible for antibody effector functions. In addition to antibodies, immunoglobulins can exist in a variety of other forms, including, for example, Fv, Fab, Fab ’and (Fab’) 2.

The reference here to "immunointeractive" includes the reference to any interaction, reaction, or other form of association between molecules and in particular where one of the molecules is, or mimics, a component of the immune system. An "immunoreactive molecule" includes an antibody, antibody fragment, synthetic antibody or a T-cell associated binding molecule (TABM).

"Isolated" means material that is substantially or essentially free of components that normally accompany it in its native state.

A sample of biological fluid that is isolated from, or derived from, a particular source of the host is described as "obtained from".

The invention also contemplates the use and generation of monoclonal antibody fragments produced by the method of the present invention which include, for example, Fv, Fab, Fab ’and F (ab’) 2 fragments. Such fragments can be prepared by standard methods for example as described by Coligan et al. (1991-1997, supra).

The present invention also contemplates recombinant or synthetic antigen binding molecules with the same or similar specificity as the monoclonal antibodies of the invention. Antigen-binding molecules of this type may comprise a synthetic stabilized Fb fragment. Example fragments of this type include single stranded Fv fragments (sFv, often called scFv) in which a peptide linker is used to bridge Terminal N or Terminal C of a VH domain with terminal C or terminal N, respectively, of a VL domain. ScFv lacks all constant parts of complete antibodies and is not able to activate complement. Suitable peptide linkers for binding to the VH and VL domains are those that allow the VH and VL domains to fold into a single polypeptide chain having an antigen binding site with a three-dimensional structure similar to that of the binding site a complete antibody antigen from which the Fv fragment is derived. The linkers having the desired properties can be obtained by the method described in US Patent No. 4,946,778. However, in some cases a linker is absent. ScFvs can be prepared, for example, according to the methods highlighted in Krebber et al. (1997, supra). Alternatively, they can be prepared by the methods described in US Patent No. 5,091,513, European Patent No. 239,400 or articles by Winter and Milstein (Nature 349: 293, 1991) and Plückthun et al. (In Antibody engineering: A practical approach 203-252, 1996).

Alternatively, the synthetic stabilized Fv fragment comprises a disulfide stabilized Fb (dsFv) in which the cysteine residues are introduced into the VH and VL domains such that in the fully folded Fb molecule the two residues will form a disulfide bond. Suitable methods for producing dsFv are described, for example, in (Glockshuber et al., Biochem. 29: 1363-1367, 1990; Reiter et al., J. Biol. Chem. 269: 18327-18331, 1994; Reiter et al., Biochem. 33: 5451-5459, 1994; Reiter et al., Cancer Res. 54: 2714-2718, 1994; Webber et al., Mol. Immunol. 32: 249-258, 1995).

Also contemplated as recombinant or synthetic antigen binding molecules are single variable domain domains (called dAbs), for example, as described in (Ward et al., Nature 341: 544-546, 1989; Hamers-Casterman et al. , Nature 363: 446-448, 1993; Davies & Riechmann, FEBSLett. 339: 285-290, 1994).

Alternatively, the recombinant or synthetic antigen binding molecule may comprise a "mini-body." In this regard, minibodies are small versions of complete antibodies, which encode in a single chain the essential elements of a complete antibody. Suitably, the mini-body is comprised of the VH and VL domains of a native antibody fused in the pivot region and the CH3 domain of the immunoglobulin molecule, for example, is described in US Patent No. 5,837,821.

In an alternate embodiment, the recombinant or synthetic antigen binding molecule may comprise protein structures, derived from immunoglobulin. For example, reference may be made to Ku & Schutz (Proc. Natl. Acad. Sci. USA 92: 6552-6556, 1995) which describes a b562 four-helices protein cytochrome that has two random loops to create the determining regions of Complementarity (CDR), which have been selected for antigen binding.

The recombinant or synthetic antigen binding molecule can be multivalent (i.e. it has more than one antigen binding site). Such multivalent molecules may be specific for one or more antigens. Multivalent molecules of this type can be prepared by dimerization of two antibody fragments through a cysteinyl-containing peptide, for example as described by (Adams et al., Cancer Res. 53: 40264034, 1993; Cumber et al. , J Immunol. 149: 120-126, 1992;). Alternatively, dimerization can be facilitated by fusion of the antibody fragments for amphiphilic helices that naturally dimerize (Plünckthun, Biochem. 31: 1579-1584, 1992) or by the use of domains (such as leucine zippers jun and fos) that preferentially heterodimerize (Kostelny et al., J. Immunol. 148: 1547-1553, 1992). In the further embodiment, a multi-stage process is employed such as by first administering an deimmunized antibody and then an anti-antibody with, for example, a reporter molecule.

The present invention further encompasses chemical analogs of the amino acids in the variant antibodies. The use of chemical amino acid analogs is useful inter alia to stabilize the molecules when administered to a subject. The amino acid analogs contemplated herein include, but are not limited to, side chain modifications, incorporation of unnatural amino acids and / or their derivatives during the synthesis of peptide, polypeptide or protein and the use of crosslinkers and other methods that impose restrictions. conformations in the proteinaceous molecule or its analogues.

Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by the reduction of NaBH4; amidation with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulfonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH4.

The guanidine group of arginine residues can be modified by forming heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group can be modified by activating carbodiimide through the formation of Oacilisourea followed by subsequent derivation, for example, with a corresponding amide.

Sulfihydrile groups can be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; oxidation of perforic acid with cysteic acid; formation of disulfides mixed with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercury derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulfonic acid, phenylmercury chloride, 2-chloromercuri4-nitrophenol and other mercurials; carbomoylation with cyanate in alkaline pH.

Tryptophan residues can be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulfenyl halides. Tyrosine residues, on the other hand, can be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

The modification of the imidazole ring of a histidine residue can be carried out by alkylation with derivatives of iodoacetic acid or N-carbethoxylation with diethyl pyrcarbonate.

Examples for incorporating amino acids and unnatural derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine. , norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and / or the D-isomers of amino acids. A list of unnatural amino acids, contemplated here is shown in Table 3.

TABLE 3

Unconventional Unconventional code Amino acid code amino acid α-aminobutyric acid Abu LN-methylalanine Nmala α-amino-α-methylbutyrate Mgabu LN-methylaginine Nmarg aminocyclopropane-Cpro LN-methylasparagine Nmasn carboxylate LN-methylaspartic acid Nmasp aminoisobutylamine aminoibyl ammonium ammonium nibyl ammonium ammonium ammonium ammonium nibyl ammonium ammonium nibyl ammonium ammonium nibyl ammonium ammonium ammonium ammonium nibyl ammonium ammonium ammonia methylglutamine Nmgln -Norb LN-carboxylate methylglutamic acid Nmglu cyclohexylalanine LN-Chexa L-Nmetilhistidina Nmhis ciclopentilalanina cPen LN-metilisolleucina Nmile D-alanine Dal methylleucine Nmleu D-LN-LN-arginine Darg methyllysine Nmlys D-aspartic acid methylmethionine Nmmet DASP LN-D -cysteine Dcys LN-methylnorleucine Nmnle D-glutamine Dgln LN-methylnorvaline Nmnva D-glutamic acid Dglu LN-methylornitine Nmom D-histidine Dhis LN-methylphenylalanine Nmphe D-isoleucine Dile LN-methylproline Nmple-Dine Nmpro-Nerserine Nmpro-Nerserine Nmpro-Nerserine Nmpro-Nerserine Nmpro-Nerserine Nmpro-Nerserine Nmpro-Nerserine Nmpro-Nerserine lysine Dlys LN-methyltreonin Nmthr D-methionine Dmet LN-methyltriptophen Nmtrp D-ortinine Dom LN-meti ltyrosine Nmtyr D-phenylalanine Dphe L-N-methylvaline Nmval D-proline Dpro L-N-methyl ethyl glycine Nmetg

(continued) (continued) 5

Non-conventional

Code  Non-conventional Code

amino acid

amino acid

D-serine

Dser L-N-methyl-t-butylglycine Nmtbug

D-threonine

Dthr L-Norleucine Nle

D-tryptophan

Dtrp L-Norvaline Nva

D-tyrosine

Dtyr α-methyl-aminoisobutyrate Maib

D-valine

Dval α-methyl-γ-aminobutyrate Mgabu

D-α-methylalanine

Dmala α-methylcyclohexylalanine Mchexa

D-α-methylaginine

Dmarg α-methylcyclopentylalanine Mcpen

D-α-methylasparagine

Dmasn α-methyl-a-naphthylalanine Manap

D-α-methylaspartate

Dmasp α-methylpenicillamine Mpen

D-α-methylcysteine

Dmcys N- (4-aminobutyl) glycine Nglu

D-α methylglutamine

Dmgln N- (2-aminoethyl) glycine Naeg

D-α-methylhistidine

Dmhis N- (3-aminopropyl) glycine Nom

D-α methylisoleucine

Tell me N-amino-α-methylbutyrate Nmaabu

D-α-methylleucine

Dmleu α-naphthylalanine Anap

D-α-Methylillin

Dmlys N-Benzylglycine Nphe

D-α-methylmethionine

Dmmet N- (2-carbamylethyl) glycine Ngln

D-α-methylortinin

Dmom N- (carbamylmethyl) glycine Nasn

D-α methylphenylalanine

Dmphe N- (2-carboxyethyl) glycine Nglu

D-α-methylproline

Dmpro N- (carboxymethyl) glycine Nasp

D-α-methylserine

Dmser N-Cyclobutylglycine Ncbut

D-α-methyltreonine

Dmthr N-Cycloheptylglycine Nchep

D-α-methyltriptophan

Dmtrp N-Cyclohexylglycine Nchex

D-α-methyltyrosine

Dmty N-Cyclodecylglycine Ncdec

D-α-methylvaline

Dmval N-Cyclododecylglycine Ncdod

D-N-Methylalanine

Dnmala N-Cyclooctylglycine Ncoct

D-N-methylaginine

Dnmarg N-Cyclopropylglycine Ncpro

Non-conventional

Code  Non-conventional Code

amino acid

amino acid

D-N-Methylasparagine

Dnmasn N-Cycloundecylglycine Ncund

D-N-Methylaspartate

Dnmasp N- (2,2-diphenylethyl) glycine Nbhm

D-N-methylcysteine

Dnmcys N- (3,3-diphenylpropyl) glycine Nbhe

D-N-methylglutamine

Dnmgln N- (3-guanidinopropyl) glycine Narg

D-N-methylglutamate

Dnmglu N- (1-hydroxyethyl) glycine Nthr

D-N-methylhistidine

Dnmhis N- (hydroxyethyl)) glycine Nser

D-N-methylisoleucine

Dnmile N- (imidazolylethyl)) glycine Nhis

D-N-methylleucine

Dnmleu N- (3-indolyliethyl) glycine Nhtrp

D-N-Methylillin

Dnmlys N-methyl-γ-animobutyrate Nmgabu

N-methylcyclohexylalanine

Nmchexa D-N-methylmethionine Dnmmet

D-N-methylortinin

Dnmorn N-methylcyclopentyl alanine Nmcpen

N-methylglycine

Nala D-N-methylphenylalanine Dnmphe

N-Methylaminoisobutyrate

Nmaib D-N-methylproline Dnmpro

N- (1-methylpropyl) glycine

Nile D-N-methylserine Dnmser

N- (2-methylpropyl) glycine

Nleu D-N-methyltreonine Dnmthr

D-N-methyltriptophan

Dnmtrp N- (1-methylethyl) glycine Nval

D-N-methyltyrosine

Dnmtyr N-Methyla-Naphthylalanine Nmanap

D-N-methylvaline

Dnmval N-methylpenicillamine Nmpen

Γ-aminobutyric acid

Gabu N- (p-hydroxyphenyl) glycine Nhtyr

L-t-Butylglycine

Tbug N- (thiomethyl) glycine Ncys

L-ethylglycine

Etg penicillamine Pen

L-homophenylalanine

Hphe L-α-Methylalanine Bad

L-α-methylaginine

Marg L-α-Methylasparagine Masn

L-α-Methylaspartate

More P L-α-methyl-t-butylglycine Mtbug

L-α-methylcysteine

Mcys L-Methyl Glycine Metg

L-α-methylglutamine

Mgln L-α-methylglutamate Mglu

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Non-conventional

Code  Non-conventional Code

amino acid

amino acid

L-α-methylhistidine

Mhis L-α-methylhomophenylalanine Mhphe

L-α-methylisoleucine

Mile N- (2-methylthioethyl) glycine Nmet

L-α-methylleucine

Mleu L-α-Methylillin Mlys

L-α-methylmethionine

Mmet L-α-methylnorleucine Mnle

L-α-methylnorvaline

Mnva L-α-methylortinin Morn

L-α-methylphenylalanine

Mphe L-α-methylproline Mpro

L-α-methylserine

Mser L-α-methyltreonine Mthr

L-α-methyltriptophan

Mtrp L-α-methyltyrosine Mtyr

L-α-methylvaline

Mval L-N-methylhomophenylalanine Nmhphe

N- (N- (2,2-diphenylethyl)

Nnbhm N- (N- (3,3-diphenylpropyl) Nnbhe

carbamylmethyl) glycine

carbamylmethyl) glycine

1-carboxy-1- (2,2-diphenyl-Nmbc

ethylamino)

cyclopropane

Crosslinkers can be used, for example, to stabilize 3D conformations, using homofunctional crosslinkers such as bifunctional imido esters having spacer groups (CH2) n with n = 1 an = 6, glutaraldehyde, N-hydroxysuccinimide esters and heterobifunctional reagents containing usually the amino reactive functional group such as N-hydroxysuccinimide and another group specific reactive functional group such as the maleimido or dithio (SH) or carbodiimide (COOH) functional group. Additionally, the peptides can be conformationally restricted by and, for example, the incorporation of Cα and Nα-methylamino acids, introduction of double bonds between Cα and Cβ amino acid atoms and the formation of cyclic or analogous peptides by introducing covalent bonds such as forming a amide bond between terminal N and C, between two side chains or between a side chain and terminal N or C.

In a monoclonal antibody obtained above, deimmunization can be identified by any number of means including the steps of:

(to)

coating a surface with an antigen selected from the crosslinked fibrin derivative or the extract containing the same or the fibrinogen degradation product;

(b)

contacting the antigen in step (a) with the monoclonal antibodies derived from the crosslinked fibrin derivative prepared as described above; Y

(C)

subjecting the complex formed in step (b) in an amplification signal stage.

Suitably, in step (a), a well plate can be used in which cross-linked fibrin derivatives such as D-dimer and / or fibrinogen degradation product (preferably obtained from a process where fibrinogen suitably digested with thrombin to obtain fragment D, fragment E and optionally fragments X and Y) is applied to individual wells. Subsequently, the monoclonal antibodies derived from a crosslinked fibrin derivative are then added to each well. An appropriate signal amplification stage that can be applied is an EIA stage where an appropriate enzyme conjugate can be coupled with the complex and the subsequently added substrate. Alternatively, RIA, FIA, agglutination, adhesion or chemiluminescence can be used as appropriate signal amplification steps.

The purpose of the screening procedure described above is to ensure that the cells tested produce specific antibody for the relevant cross-linked fibrin derivative, but not for the D fragment.

There should be minimal reaction with fibrinogen or fibrinogen degradation products and a positive reaction with the derivative. The term "minimum" does not include reactivity but extends to baseline levels as compared to an antibody directed to fibrinogen per se. Subsequently, a minimal reaction includes suboptimal reactivity compared to a specific fibrinogen antibody.

The present invention also includes within its scope an assay to detect bound fibrin derivatives that include the steps of:

(one)

contacting a specific monoclonal antibody to the crosslinked fibrin derivatives but not the D fragment with a biological sample suspected of containing an antigen derived from a crosslinked fibrin derivative or comprising a crosslinked fibrin derivative per se; Y

(2)

subjecting the complex formed in step (1) in an amplification signal stage.

In the aforementioned assay, the crosslinked fibrin derivative is suitable for the dimer D, D2E or any other high molecular weight derivative as described above. The monoclonal antibody is prepared as previously described that is relevant to the particular cross-linked fibrin derivative being tested.

The presence of the cross-linked fibrin derivative can be used as a diagnostic aid suitable for pre-thrombotic, thrombotic or other conditions that involve fibrin formation and lysis.

The deimmunized monoclonal antibody of the present invention is particularly useful for imaging blood clots as well as for objectifying blood clots in order to bring the clot into contact with enzymes or other chemical agents capable of dissolving, completely or partially, the clot

With respect to clot imaging, a reporter molecule adheres to the deimmunized monoclonal antibody or to an antibody that has specificity for the deimmunized antibody or a portion or conjugate thereof and is then introduced to a host, such as a human. By detecting the reporter molecule, blood clots can be visualized. A particularly useful form of reporter molecule is a nuclear tag. Nuclear tags contemplated for use in the present invention include but are not limited to a bifunctional metal ion chelate. The chelate can adhere to the antibody itself or multiple chelates can adhere to the protein by means of dendrimers. Particularly preferred are nuclear labels 99mTc, 18F, 64Cu, 67Ga, 68Ga, 77Br, 97Ru, 111In, 123I, 124I, 131I and 188Re. The most preferred nuclear tag is 99mTc. Preferably, the host is a human and, therefore, it is necessary for the murine 3B6 monoclonal antibody to be deimmunized.

Alternative forms of immunoscintigraphy can be obtained using isotopes such as a 68Ga or 124I isotope or other PET isotopes. Such technology can be described as "immuno-PET." The technology has advantages over gamma camera screen printing and can provide high resolution images of blood clots especially in less pleasant body areas to conventional diagnostic means such as lungs or small clots in the calf or pelvis.

In accordance with the foregoing, the present invention provides a conjugate molecule comprising an immunoreactive deimmunized molecule such as an deimmunized antibody and one or both of an imaging label or a therapeutic agent.

Preferred imaging tags are MRI-, ultrasound-and / or CT-type tags such as but not limited to 99mTc, 18F, 64Cu, 67Ga 68Ga, 77Br, 97Ru, 111In, 123I, 124I, 131I and 188Re.

Therapeutic labels include cytokines, anticoagulant agents, wound repair agents and anti-infection agents.

Another aspect of the present invention contemplates a method for detecting a blood clot in a human patient, said method comprising introducing into said patient an immunized form of murine monoclonal antibody 3B6 or an antigen binding fragment thereof labeled with a reporter molecule. which allows the dissemination of the labeled antibody through the circulatory system and then submit said patient to report the molecule detection means to identify the location of the antibody in a clot.

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Preferably, the reporter molecule is a nuclear tag.

Preferably, the nuclear tag is 99mTc, 18F, 64Cu, 67Ga, 68Ga, 77Br, 97Ru, 111In, 123I, 124I, 131I and 188Re.

Preferably, the nuclear tag is 99mTc.

The present invention further contemplates the use of a deimmunized murine monoclonal antibody specific for the D-dimer or other cross-linked fibrin derivative in the manufacture of the clotting agent.

Preferably, the murine monoclonal antibody is 3B6 or a homologue thereof.

Preferably, the clot imaging tag is for use in humans.

The same antibody can also carry multiple tags such as multiple anti-coagulant agents and / or reporter molecules. Alternatively, or additionally, multiple antibodies can each be administered on a different label.

The target clot antibody present alone or in combination with other imaging protocols can be used. One such protocol is flat image formation such as but not limited to CT, MRI or ultrasound.

According to the foregoing, another aspect of the present invention contemplates a method for detecting a blood clot in a human patient, said method comprising introducing into said patient a deimmunized form of murine monoclonal antibody 3B6 or an antigen binding fragment of the same labeled with a reporter molecule that allows the dissemination of the labeled antibody through the circulatory system and then subject said patient to flat clot imaging.

Preferably, the flat image formation is MRI or CT scan. Ultrasound can be used in the imaging process.

According to the foregoing, another aspect of the present invention contemplates a method for detecting a blood clot in a human patient, said method comprising introducing into said patient a deimmunized form of murine monoclonal antibody 3B6 or an antigen binding fragment of the same labeled with a reporter molecule that allows the dissemination of the labeled antibody through the circulatory system and then subject said patient to a computer-assisted tomographic nuclear medicine scan to visualize the clot.

Preferably, the reporter molecule is a nuclear tag.

Preferably, the nuclear tag is 99mTc, 18F, 64Cu, 67Ga, 68Ga, 77Br, 97Ru, 111In, 123I, 124I, 131I and 188Re.

Preferably, the nuclear tag is 99mTc.

The clotting agents of the present invention are also useful as therapeutic agents. In particular, the clotting target agents are fused, bound or otherwise associated with a clot dissolution or clot growth prevention agent such as an anticoagulant molecule. In accordance with the foregoing, another aspect of the present invention contemplates a method for facilitating the dissolution or removal of a blood clot in a human, said method comprising administering to said human a clot solution or effective amount of clot growth prevention. of a variant murine-derived monoclonal antibody that has specificity for the human-derived D-dimer and other derivatives of cross-linked fibrin and without reactivity with fibrinogen or fibrinogen degradation products including fragments D and E wherein said murine-derived monoclonal antibody variant is substantially non-immunogenic in a human wherein said monoclonal antibody additionally comprises a clot dissolution or growth clotting agent of fused, bound or otherwise associated clots.

Yet another aspect of the present invention is directed to the use of a variant murine-derived monoclonal antibody that has specificity for the human-derived D-dimer and other cross-linked fibrin derivatives and without reactivity with fibrinogen or fibrinogen degradation products including fragments D and E wherein said variant murine-derived monoclonal antibody is substantially non-immunogenic in a human and said antibody further comprises a clot dissolution or clotting growth prevention agent fused, bound or otherwise adhered thereto in the manufacture of a medicine for the dissolution of a blood clot in a human.

In an alternative embodiment, multiple deimmunized antibodies can be used. In one example, a 3B6 deimmunized antibody is administered alone and then the deimmunized anti-immunoglobulin antibodies each carry out an agent such as a therapeutic or diagnostic agent that will target a complex of a 3B6 clot. Still another alternative is engineered antibodies with multiple (eg bi-) specificities. In this case, one specificity may be in the clot and another in the clot site (for example in a cell receptor). This can also be done using multiple antibodies.

The present invention further contemplates compositions comprising the clotting target agents of the present invention and one or more pharmaceutically acceptable carriers and / or diluents.

Pharmaceutical forms suitable for injectable use include sterile aqueous solutions as well as lyophilized forms of antibody preparations together with stabilizing agents such as sugar, proteins or other compounds or molecules that facilitate the radiolabeled process. This can be stable under manufacturing and storage conditions and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a dilution medium or solvent comprising, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), their suitable mixtures and vegetable oils. Appropriate fluidity can be maintained, for example, by the use of surfactants. Prevention of the action of microorganisms can be brought about by various anti-bacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thymerosal and the like. In many cases, it may be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by use in the compositions of delayed absorption agents, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with the active ingredient and optionally other active ingredients as required, followed by filtered sterilization or other appropriate sterilization means.

Pharmaceutically acceptable carriers and / or diluents include any and all solvents, dispersion media, coating, antibacterial agents and antifungal agents, agents that delay absorption and isotonic agents and the like. The use of such means and agents for the pharmaceutical active substances is well known in the art and except for any conventional means or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Complementary active ingredients can also be incorporated into the compositions.

The clotting agents of the present invention are useful for the diagnosis and / or treatment of thrombin-associated conditions such as DVT, PE and DIC.

Yet another aspect of the present invention contemplates a method for treating a subject with fibrin-associated cancer. In this embodiment, antibodies to the epitope of dimer D can be used to deliver cytotoxic agents such as an isotope that emits β or γ or combinations thereof. Such isotopes include but are not limited to 131I, yttrium-90, rhenium-186, rhenium-188, lutetium-117 and copper-67. Fibrin associated with a cancer includes an encapsulated fibrin tumor.

The deimmunized immunoreactive molecules of the present invention are, therefore, carriers for any clot binding agents or clot dissolving agents or for any agents having useful therapeutic or diagnostic properties. The deimmunized immunoreactive molecules of the present invention are also useful for determining the kinetics of clot dissolution, dissipation and / or disappearance. Once this information is available, the clot dissolution or imaging agents can be administered very quickly.

The present invention is further described by the following non-limiting Examples.

EXAMPLE 1

Cell fusion and hybrid selection

The spleens are aseptically removed from 2 immunized mice killed by cervical dislocation three days after an injection of the dimer D. Previously, the mice have been immunized with three injections of fibrin lysate digested with thrombin of proteolytic enzymes and plasmin as reported in the reference of Graeff and Hafter mentioned above. Two spleens are placed in a 60 mm Petri dish (Falcon, 3001, Oxnard, Calif.) Containing 5 ml of complete medium (85% RPMI 1640, 15% w / v fetal calf serum, 100 IU / ml of penicillin, 100 µg / ml of streptomycin and 2 X 10-3 M of glutamine; Gibco, Grand Island, NY). A cell suspension is prepared by decapsulating the spleen with 2 x 18 gauge needles attached to 3 ml disposable syringes with the last cm with the tip tilted at an angle of 60 °. The cellular suspicion is then aspirated into a 10 ml syringe provided with a 22 gauge needle and ejected with moderate pressure. This operation is carried out twice before filtering the cells in a 2001 Falcon tube through a fine mesh stainless steel sieve to remove residues and large groups of cells.

The cell suspension is allowed to stand for 5 minutes at room temperature to allow small groups and membrane fragments to decant before transferring the cell suspension to a fresh Falcon 2001 tube. The cells are centrifuged at 350G for 5 minutes at room temperature and the supernatant is decanted from the first cell in a fresh tube and rotated at 700G for five minutes to give a second cell and the two cells are grouped and resuspended in 5 ml of complete medium. The spleen white blood cells (SWBC) are then counted and their viability is estimated by Turks and Trypan blue strains, respectively, and 100 x 106 viable SWBCs are placed in separate Falcon 2001 tubes in a total volume of 5 ml of complete medium. The NS-1 myeloma cells to be used for fusion, are washed once by centrifugation at 380G for 15 minutes at room temperature and adjusted to 5 x 10 6 viable cells / ml in complete medium.

Twenty-five x 106 NS-1 and 100 x 105 SWBC immune are mixed and rotated 350G for 5 minutes at room temperature. The supernatant is decanted, the remaining medium is carefully removed with a Pasteur pipette and 2 ml of a 42% w / v solution of polyethylene glycol (PEG, MW1540) (Baker Chemical Co., New Jersey). In RPMI 1640 containing 15% v / v dimethyl sulfoxide (DMSO) at 37 ° C it is added with a 5 ml disposable glass pipette (Corning Glass, Coming, NY) and the cells are resuspended with the same pipette at 5 ml for 30 seconds with the help of electric pipetting machine (Pipet-aid Drummond Scientific Co., Broomall, PA). The PEG cell suspension is allowed to stand for an additional 30 seconds at room temperature before adding 5 ml of complete medium, in the form of drops, with a Pasteur pipette, for a period of 90 seconds with constant agitation of the tube, sufficient to ensure the complete mixture with the viscous PEG solution. 5 ml of complete medium is immediately added and mixed by inversion and the cell suspension is allowed to stand for 150 seconds at room temperature before centrifugation at 350G for 5 minutes at room temperature. The supernatant is decanted and the cell globules are gently resuspended in 5 ml of complete medium using a 5 ml pipette with electric pipettor; Extreme care is taken not to break all cell groups. Using a Tridak repeater (Bellco Glass Inc., Vineland, NJ), 0.05 ml of the cell suspension is added to each 4 Costar well of 24-well plates (Costar 3524, Cambridge, Mass.) Containing 1 x 106 BALB / c of normal SWBC mouse as feeder cells in 1 ml of complete medium containing 10-4 M of Hypoxanthine (Sigma), 4 x 10-7 M of Aminopterin (Sigma), 1.6 x 10-5 M of Thymidine (Sigma) and 4 x 10-5 M of 2-Mercaptoethanol (HAT medium), hereinafter referred to as first fusion plates.

The first melting plates are then placed in a humidified atmosphere at 95% of 5% CO2 at 37 ° C. The cells are first loaded on days 5 or 7 and thereafter when necessary, with 0.5 ml of fresh HAT. In general, on day 10, 0.5 ml of the medium is removed for the detection test of each well that shows hybridoma growth and 0.5 ml of fresh HAT medium is replaced. A number of the strongest growth wells are selected for maintenance at the base of the detection assay. The selected wells are allowed to grow in confluence in the original well (1st well), then each is divided to the mutad and transferred to a fresh well (2nd well) of a 24-well Costar plate (2nd plate). The wells are checked daily and expanded in a second, third or fourth well of the second Costar plate when necessary. From days 14-28, cells are loaded with HT medium. When there is growth in at least two wells of the second plate, the supernatant of a well of each clone is selected for re-detection and a number of clotypes that produce specific antibodies are selected from the results of the second detection assay to produce the monoclonal antibody that secretes cell lines by limiting dilution.

EXAMPLE 2

Cloning of hybridomas

A second well of each selected clone is resuspended and the number of viable cells per well is estimated by the exclusion of Trypan blue. Immediately before plating each clone, relevant series of dilutions are made in HT medium or complete medium (if the cells are older than 28 days post fusion) to give a frequency of 0.5 cells / 0.05 ml. This volume is then added with a Tridak Repeater to each well of a 96-well flat round bottom tissue culture plate (Flow Laboratories, Mississauga, Ontario, Canada) (LD plate) containing 1 x 105 cells loaded with spleen from Normal mouse in 0.1 ml of HT or complete medium. The LD plates are then placed in an atmosphere of 95% air, 5% CO2, humidified at 37 ° C and detected for clonal growth 7-10 days later. From each positive growth well, 0.1 ml of supernatant is removed for detection and these wells are loaded for the first time with 0.1-0.15 ml of HT medium or complete medium. At the base of the LD detection assay, a minimum of two of the clones that produce "better" specific antibodies are finally selected for expansion in mass culture.

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Alternatively, if a large amount of Mab is desired, female BALB / c mice are given an intraperitoneal injection of 0.5 ml 2, 5, 10, 14, tetramethylpentadecane (Pristane, Aldrich Chemical Corp., Milwaukee, Wisonsin ) 14 days before the injection of 2 x 106 viable hybridoma cells and ascites fluids are collected from mice 12-14 days after the injection of the cells. The ascites fluid is clarified by centrifugation and the MAb is recovered by precipitation with 45% ammonium sulfate and stored at 4 ° C or -70 ° C in phosphate buffered saline (PBS) solution containing 0.01% azide of sodium.

EXAMPLE 3

Monoclonal antibody detection assay

The wells of a 96-well U bottom microtest plate (Disposable Products Pty. Ltd., Adelaide, South Australia) are covered by adding 50 µl of D-dimer (5 µg / ml) or Fibrinogen degradation products (5 µg / ml in PBS for one hour at room temperature (25 ° C) Excess antigen is removed by inverting and shaking the plate and the plate is then washed three times with PBS containing 0.05% w / v Tween 20 (Sigma Chemical Corp., St Louis, Missouri.) Clones that secrete MAb in dimer D or fibrinogen degradation products are then detected by adding 50 µl of the tissue culture supernatant to each well and incubating for one hour at room temperature Unbound MAb is removed by inversion and shaking and the plate is washed three times with PBS / Tween One hundred µl of a 1/1000 dilution of peroxidase-conjugated rabbit anti-mouse immunoglobulin (Dakopatts, Copenhagen, Denmark) in PBS / Tween is added and allowed to incubate an additional hour at room temperature. The plate is inverted again and washed three times with PBS / Tween and 100 µl of the activated substrate (immediately before use, 10 µl of 3% hydrogen peroxide solution is added to 10 ml of a substrate solution containing 50 mM citrate, 2.5 mM 0-tolidine dihydrochloride (0-tolidine, Sigma Chemical Co., recrystallized from diluted HCl) 0.025 mM EDTA pH 4.5) is added to each well. The color reaction is stopped after 10 minutes by the addition of 50 µl of 3M HCl which causes a color change from blue to yellow and the absorbance is recorded at 450 nm in a Titertek multiskan.

EXAMPLE 4

3B6 variable region sequence identification

The murine hybridoma 3B6 is propagated in RPMI 1640 medium supplemented with 15% w / v fetal calf serum. Total RNA is prepared from 107 hybridoma cells. VH and VK cDNA is prepared using reverse transcriptase and the mouse IgG constant region primers and the mouse κ constant region. The first strain cDNA is amplified by PCR using a variety of mouse signal sequence primers (6 sets for VH and 7 sets for VK). The amplified DNAs are gel purified and cloned into the pGem® T Easy vector (Promega). The VH and VK clones obtained are detected for inserts of the expected size by PCR and the DNA sequence of the selected clones determined by the dideoxy chain termination method. The productive VH and VK genes are identified by sequence analysis. The location of the complementarity determining regions (CDR) is determined with reference to other antibody sequences (43). The 3B6 VH can be assigned to subgroup IA of mouse heavy chains. The 3B6 VK can be assigned to subgroup I of mouse κ chains.

EXAMPLE 5

Analysis of 3B6 (v) variable region sequences with potential T cell epitopes

The 3B6 VH and VK sequences are analyzed for the presence of potential T cell epitopes using the procedures previously described (Carr et al., International Patent Publication No. WO 98/52976). Peptides identified as potential T-cell epitopes (MHC class II binding peptide) are modified on silica and the modified sequence is re-analyzed to ensure the loss of potential MHC class II binding and verify that the MHC class II binding motifs that They have not been generated in the surrounding sequence. Alternatively, the sequence is modified to convert the MHC class II binding motif into one found in a human germ line. Only, in general, conservative amino acid substitutions are tested and substitutions made with respect to the general antibody structure. A number of variant sequences is compiled for VH and VK, each containing different numbers of substitutions.

EXAMPLE 6

Designated sequences of variable regions 3B6 variants (v) with reduced numbers of potential T cell epitopes

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The heavy and light regions (v) designated according to the scheme of Example 5 are constructed in vitro by the described overlapping PCR recombination method (Daugherty et al., Nucleic Acids Research 19: 24712476, 1991). Cloned murine VH and VK genes are used as templates for mutagenesis of the structure regions in the required humanized sequences. Sets of mutagenic primer pairs that cover regions to be altered are synthesized. Adjacent primers include 15 bp of the homologous sequence. A first round of PCR using these primers produces 5 to 8 fragments of overlapping DNA that span the gene from the assigned region (v). The VH-PCR1 and VK-PCR1 vectors (Orlandi et al., Proc. Natl. Acad. Sci. USA 86: 38333837, 1989) are used as templates to introduce the 5 'flanking sequence that includes the leader signal peptide, the leading intron and murine immunoglobulin promoter, and the 3 'flanking sequence that includes the site of division and intron sequences, into two additional overlapping fragments. The DNA fragments are combined in a second round of PCR using external flanking primers to obtain the PCR products of the required full length. These PCR products are cloned into the pUC19 vector for the determination of the DNA sequence. Clones containing the expected sequence alterations are selected and the complete DNA sequence is confirmed to be correct for each desired VH and VK. Light and heavy chain genes are transferred to the pSVgpt and pSV hyg expression vectors with human IgG1 or the κ constant regions as described (Tempest et al., Biotechnology 9: 266-271, 1991). The VH-PCR1 and VK-PCR1 vectors (Orlandi et al., 1989, supra) are used as templates to introduce the 5 'flanking sequence that includes the leader signal peptide, the leader intron and the murine immunoglobulin promoter, and the 3 'flanking sequence that includes the division site and intron sequences.

EXAMPLE 7

Expression and purification of 3B6 variant antibodies

The 3B6 light and heavy chain expression vectors variants are co-transfected in different combinations by electroporation in NS0, a mouse myeloma that does not produce immunoglobulin, obtained from the European Collection of Animal Cell Cultures, Porton, U.K. (ECACC No. 85110505). Colonies expressing the gpt gene are selected in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 0.8 µg / ml of mycophenolic acid and 250 µg / ml of xanthine. The production of a human antibody by transfected cell clones is measured by ELISA for human IgG (48). Cell lines that secrete the antibody and expand are selected. Variant 3B6 antibodies are purified using Prosep®-A (Bioprocessing Ltd, Conset, U.K.).

EXAMPLE 8

Functional test of 3B6 variant antibodies

Variant antibodies for binding of the D-dimer are tested using ELISA-based assays extensively as described in Example 3. The binding specificity is confirmed using the human fibrinogen assay. In a preferred embodiment, however, dimer D is used in the solution phase. In this assay, 3B6 antibodies are coated on the ELISA plate in 0.5 µg / well, to capture the D-dimer in the solution. D-dimer is applied at 10 µg / ml (500 ng / well) and double dilutions. The revealing antibody is HRPO conjugated mouse monoclonal anti-D (Dimertest EIA Tag; Agen) and the results are developed by the OPD substrate and read at 492 nm. The 3B6 deimmunized antibodies are compared with the murine and chimeric 3B6 antibodies and the previously conducted deimmunized 3B6 antibody DIVH1 / DIVK1. The results are shown in Figures 4A, 4B and 4C. The use of the dimer D of the solution phase proves to be better than the solid phase of the dimer D in the selection of clones and is a preferred aspect of the present invention.

EXAMPLE 9

Thrombus visualization using 3B6-99mTc

The 3B6 monoclonal antibody of mice and deimmunized form for humans is depicted in Figure 1A and exhibits specificity for fibrin which is a major part of blood clots (Figure 1B). The concept of clot imaging is developed by labeling the 3B6 monoclonal antibody with a nuclear tag, in this case, 99mTc (Figure 2). Administration of labeled 3B6 deimmunized monoclonal antibody in humans (Figure 3A). Visualization of clots in the circulatory system such as blood clots in the front of the thighs (Figure 3B) occurs by binding the monoclonal antibody to fibrin resulting in the concentration of radiation at the site of the clot.

EXAMPLE 10

Thrombus visualization using 3B6-99mTc

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Pulmonary emboli (0.1-0.5 g) are created in anesthetized dogs by embolization of developed thrombi made by infusion of thrombin and human fibrinogen through balloon catheters placed in the femoral veins. The purified Fab ’fragments (0.35 mg) of a chimeric derivative (human / murine) of a fibrin specific monoclonal antibody are labeled with a 15 mCi of 99mTc. One hour after embolization, the preparation of the radiolabeled antibody is injected through a peripheral intravenous catheter. Eight hours after antibody injection, imaging scans are developed to visualize the emboli.

99mTc labeled antibody fragments extracted from the circulation with a t1 / 2 of one hour for both subjects. In subject 1, two small plungers are visible in the lower right lobe (combined mass, 0.187 g). The blood clot / radioactivity ratio is 38: 1. In subject 2, an embolism in the right lower lobe (mass, 0.449 g) is visible. The blood clot / radioactivity ratio is 27: 2. A small embolism (0.091 g) in the right ventricle of the subject 1. The blood clot / radioactivity ratio is 45: 1. No adverse effects of antibody or methodology administration are noted. of exploration.

Infusion of radiolabelled anti-fibrin antibody fragments followed by images that produce plunger images, even relatively small emboli on the periphery of the lung. The images are reliable and require minimal training for interpretation. The technique can be used for images of deep vein thrombi in the same set. This agent is well tolerated by subjects. There is no need to hold your breath or heart synchronization. This use is not nephrotoxic intravenous contrast dye. The radiation dose is similar to the dose used for ventilation / perfusion scans. This technology can simplify and clarify the diagnosis of PE and DVT, using technology available in most medical centers.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications different from those specifically described. The invention also includes all stages, features, compositions and the compounds named or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said stages or features.

BIBLIOGRAPHY

Adams et al., Cancer Res. 53: 4026-4034, 1993.

Altschul et al., Nucl. Acids Res. 25: 3389-3402. 1997

Ausubel et al., "Current Protocols in Molecular Biology" John Wiley & Sons Inc, 1994-1998, Chapter 15. Budzynski et al., Blood 54 (4), 1979.

Carr et al. (International Patent Publication No. WO 98/52976).

Chothia et al., J. Mol. Biol. 196: 901, 1987.

Chothia et al., J. Mol. Biol. 227: 799, 1992. Chou et al. (U.S. Patent No. 6,056,957).

Coligan et al., Current Protocols in Imnunology, John Wiley & Sons, Inc., 1991-1997.

Cumber et al., J. Immunol. 149: 120-126, 1992.

Daugherty et al., Nucleic Acids Research 19: 2471-2476, 1991.

Davies & Riechmann, FEBS Lett. 339: 285-290, 1994.

European Patent Publication No. 0 239 400.

Gefter et al., Somatic Cell Genet. 3: 231-236, 1977.

Glockshuber et al., Biochem. 29: 1363-1367, 1990.

Graeff and Halfer, "Detection and Relevance of Reticulated Fibrin Derivatives in Blood", Seminars in Thrombosis and Hemostatis 8 (1), 1982.

Hamers-Casterman et al., Nature 363: 446-448, 1993. Jones et al., Nature 321: 522-525, 1986.

Kabat et al., "Sequences of Proteins of Immunological Interest", U.S. Department of Health and Human Services, 1983.

Kennet et al. (eds) Monoclonal Antibodies and Hybridomas: A New Dimension in Biological Analyzes, pp. 376-384, Plenum Press, New York, 1980. Kohler and Milstein, Eur. J. Immunol. 6 (7): 511-519, 1976. Kohler and Milstein, Nature 256: 495-499, 1975. Kostelny et al., J. Immunol. 148: 1547-1553, 1992. Kozbor et al., Methods in Enzymology 121: 140, 1986. Krebber et al., J. Immunol. Methods 201 (1): 35-55, 1997. Ku & Schutz, Proc. Natl Acad. Sci. USA 92: 6552-6556, 1995. Liu et al., Proc. Natl Acad. Sci. USA 84: 3439-3443, 1987. Morgan et al. (U.S. Patent No. 6,180,377). Orlandi et al., Proc. Natl Acad. Sci. USA 86: 3833-3837, 1989. Plückthun et al., In Antibody engineering: A practical approach 203-252, 1996. Plünckthun, Biochem. 31: 1579-1584, 1992. Queen et al. (U.S. Patent No. 6,180,370). Reiter et al., Biochem. 33: 5451-5459, 1994. Reiter et al., Cancer Res. 54: 2714-2718, 1994. Reiter et al., J. Biol. Chem. 269: 18327-18331, 1994. Riechmann et al., Nature 332: 323-327, 1988. Shulman et al., Nature 276: 269-270, 1978; Tempest et al., Biotechnology 9: 266-271, 1991. Toyama et al., "Monoclonal Antibody, Experiment Manual", published by Kodansha Scientific, 1987. Trowbridge, J. Exp. Med. 148 (1): 313-323, 1978. Verhoeyen et al., Science 239: 1534-1536, 1988. Volk et al., J. Virol. 42 (1): 220-227, 1982. Ward et al., Nature 341: 544-546, 1989. Webber et al., Mol. Immunol 32: 249-258, 1995. Willner et al., Biochemistry 21: 2687-2692, 1982. Winter and Milstein, Nature 349: 293, 1991, SEQUENCE LIST

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Claims (21)

one.

A deimmunized antibody or deimmunized antibody fragment specific for an epitope in the human D-dimer that recognizes cross-linked fibrin but not fibrinogen, wherein one or more amino acid residues in the variable domain (v) of said antibody or antibody fragment mutate to eliminate or reduce the association of peptide fragments of said v domain with MHC class II molecules, wherein said antibody or antibody fragment comprises a combination of v domains of the H and L chain comprising a combination of amino acid sequences selected from the group consisting of:

SEQ ID NO: 1 / SEQ ID NO: 4, SEQ ID NO: 2 / SEQ ID NO: 4, SEQ ID NO: 4, SEQ ID NO: 1 / SEQ ID NO: 6, SEQ ID NO: 2 / SEQ ID NO: 6, SEQ ID NO: 2 / SEQ ID NO: 5, SEQ ID NO: 3 / SEQ ID NO: 5, SEQ ID NO: 3 / SEQ ID NO: 6 and SEQ ID NO: 1 / SEQ ID NO: 5.

2.

The deimmunized antibody or antibody fragment of claim 1 wherein said antibody or fragment is specific for an epitope recognized by the murine anti-fibrin monoclonal 3B6 antibody.

3.

Use of the antibody or antibody fragment of claim 1 or 2 wherein said antibody is labeled with a reporter molecule for the manufacture of an imaging agent to detect a blood clot in a human patient where in the introduction of said antibody in said patient said labeled antibody is allowed to spread through the circulatory system and where the patient undergoes an imaging protocol to visualize the clot.

Four.

The use of claim 3 wherein said imaging protocol is MRI, CT scan, ultrasound or computer-assisted tomographic nuclear medicine scan that includes gamma range camera or PET.

5.

The use of claim 3 wherein said reporter molecule is a nuclear tag.

6.

The use of claim 5 wherein said nuclear tag is 99mTc, 18F, 64Cu, 67Ga, 68Ga, 77Br, 97Ru, 111In, 123I, 124I, 131I or 188Re.

7.

The use of claim 6 wherein said nuclear tag is 99mTc.

8.

Use of an antibody or antibody fragment of claim 1 or 2 for the manufacture of a medicament to facilitate the dissolution, prevention of growth or removal of a blood clot in a human wherein said antibody further comprises a solution of the clot or agent of prevention of growth of fused clots, attached or otherwise associated with this.

9.

A conjugate comprising an antibody or antibody fragment of claim 1 or 2 and one or both of an imaging tag and / or a therapeutic agent.

10.

The conjugate of claim 9 wherein said imaging label is an MRI-, ultrasound-, CT-, or nuclear medicine (including gamma camera and PET type labeling.

eleven.

The conjugate of claim 10 wherein said imaging label is selected from 99mTc, 18F, 64Cu, 67Ga, 68Ga, 77Br, 97Ru, 111In, 123I, 124I, 131I and 188Re.

12.

The conjugate of claim 11 wherein said image forming label is 99mTc.

13.

The conjugate of claim 9 wherein said therapeutic agent is an anticoagulation agent or a cytokine.

14.

The use of any one of claims 3 or 5 to 7 wherein the imaging protocol is a computer-assisted or flat tomographic nuclear medicine scan.

fifteen.

The antibody or antibody fragment of claim 1 or 2 wherein said antibody is labeled with a reporter molecule for in vivo use to detect a blood clot in a human patient wherein at the introduction of said antibody into said patient said labeled antibody It is allowed to disseminate through the circulatory system and where the patient undergoes an imaging protocol to visualize the clot.

16.

The antibody or antibody fragment of claim 15 wherein said imaging protocol is MRI, CT scan, ultrasound or scanning of computer-assisted tomographic nuclear medicine that includes gamma camera or PET screen.

17. The antibody or antibody fragment of claim 15 wherein said reporter molecule is a nuclear tag.

18.

The antibody or antibody fragment of claim 17 wherein said nuclear tag is 99mTc. 18F, 64Cu, 67Ga, 68Ga, 77Br, 97Ru, 111In, 123I, 124I, 131I or 188Re.

19.

The antibody or antibody fragment of claim 18 wherein said nuclear tag is 99mTc.

twenty.

An antibody or antibody fragment of claim 1 or 2 for in vivo use to facilitate dissolution,

10 prevention of growth or removal of a blood clot in a human wherein said antibody additionally comprises a clot dissolution or growth clotting agent of fused, bound or otherwise associated clots. 21. The antibody or antibody fragment of any one of claims 15 or 17 to 19 wherein the Imaging protocol is a computer-assisted tomographic nuclear medicine scan or flat 15.