CZ20031982A3 - Isolated molecules comprising epitopes containing sulfated compounds, antibodies against such epitopes and their use - Google Patents

Abstract

The present invention provides epitopes present on cancer cells and important in physiological phenomena such as cell rolling, metastasis, and inflammation. Therapeutic and diagnostic methods and compositions using antibodies capable of binding to the epitopes are provided. Methods and compositions according to the present invention can be used in diagnosis of and therapy for such diseases as cancer, including tumor growth and metastasis, leukemia, auto-immune disease, and inflammatory disease.

Description

ISOLATED MOLECULES INCLUDING EPITOPS CONTAINING

SULPHATED COMPOUNDS, ANTIBODIES AGAINST SUCH EPITOPES AND

THEIR USE

FIELD OF THE INVENTION [1.] The present invention relates to epitopes that are present on cells, metastatic cells, leukemic cells and platelets, and which are important in such different physiological phenomena as cell rolling, metastasis, inflammation autoimmune diseases such as idiopathic thrombocytopenic purpura (ITP), adhesion, thrombosis and / or restenosis and aggregation. The present invention relates to therapeutic and diagnostic methods and compositions using antibodies directed against such epitopes. The present invention also relates to the field of tissue targeting and identification, using phage display technology, peptides and polypeptides that specifically bind to target cells. Such peptides and polypeptides are antibodies and antigen-binding fragments thereof, constructs thereof, fragments of both constructs and fragments. More specifically, the peptides and polypeptides may have anti-cancer activity and / or activity against other diseases such as inflammatory diseases, diseases involving abnormal or pathogenic adhesion, thrombosis and / or restenosis, diseases involving abnormal or pathogenic aggregation, autoimmune diseases, cardiovascular diseases such as myocardial infarction, retinoplastic diseases, diseases caused by sulfated tyrosine-protein-dependent protein-drug interactions, and diseased cells in general.

BACKGROUND OF THE INVENTION [2] Tissue selective targeting of therapeutic agents is an emerging discipline in the pharmaceutical industry.

The new targeting-based cancer treatment has been designed to increase the specificity and efficacy of the treatment while reducing toxicity, thereby increasing overall efficacy. Mouse monoclonal antibodies (MAb's) against tumor-associated antigens have been developed in an effort to target toxin, radionucleotide, and chemotherapeutic conjugates to tumors. In addition, differentiation antigens such as CD19, CD20, CD22 and CD25 have been used as tumor-specific targets in the treatment of hematopoietic malignancies. Although this procedure has been extensively studied, it has several limitations. One limitation is the difficulty of isolating suitable monoclonal antibodies that exhibit selective binding. The second limitation is the need for high antibody immunogenicity as a prerequisite for successful antibody isolation. The third limitation is the induction of a patient's immune response against a human anti-mouse antibody (HAMA response), often resulting in a shorter serum half-life of the mouse antibody, preventing re-treatments, thereby reducing the therapeutic value of the antibody. The latter limitation stimulated interest in both the construction of chimeric or humanized monoclonal antibodies of murine origin and the search for human antibodies. Another limitation of this approach is the fact that it allows the isolation of only one kind of antibody against only known and purified antigens. Moreover, this method has so far not been selective since it allows the isolation of antibodies against cell surface markers present on normal as well as malignant cells.

[3.] There are many factors that influence the therapeutic efficacy of monoclonal antibodies (Mab's) for the treatment of cancer. These factors include the specificity of antigen expression on a tumor cell, the level of expression, antigenic heterogeneity, and the availability of tumor mass. Leukemia and lymphomas are generally more sensitive to antibody treatment than solid tumors such as carcinomas. Mab's rapidly bind to leukemic and lymphatic cells in the bloodstream and easily penetrate to malignant cells in lymphatic tissues, making lymphoid tumors an excellent candidate for monoclonal antibody therapy. The ideal system should provide identification of Mabs, which recognizes markers on the cell surface of stem cells that produce malignant cell progeny.

[4] Phage libraries have been used to select random single-chain Fv's (scFv's) that bind to isolated, pre-determined target proteins, such as antibodies, hormones, and receptors. In addition, the use of antibody display libraries in general and phage scFv libraries in particular helps in an alternative way of finding unique molecules for targeting specific, unrecognized and unspecified cell surface substances.

[5] Leukemia, lymphomas and myelomas are tumors that arise in bone marrow and lymphatic tissues and are involved in uncontrolled cell growth. Acute lymphoblastic leukemia ("ALL") is a heterogeneous disease that is defined by specific clinical and immunological features. As with other forms of ALL, the underlying causes of most cases of B-cell ALL ("B-ALL") are unknown, although in many cases the disease is due to the genetic changes in the DNA of one cell that cause the cell it becomes abnormal and continues to multiply. The prognosis of patients affected by B-ALL is significantly worse than the prognosis of patients with other types of leukemia, both in children and adults.

[6.] Acute myeloid leukemia (AML) is a heterogeneous group of neoplasms (neoplasms) with a progenitor cell that normally produces terminally differentiated cells from the myeloid series (erythrocytes, granulocytes, monocytes and platelets). As with other forms of neoplasia, AML is associated with acquired genetic changes that result in the replacement of normally differentiated myeloid cells by relatively undifferentiated blasts that exhibit one or more types of early myeloid differentiation. In general, AML develops in the bone marrow and, to a lesser degree, in secondary hematopoietic organs. AML primarily affects adults with peak incidence between 15 and 40 years of age, but is also known to affect both children and older adults. Almost all AML patients require treatment immediately after diagnosis in order to achieve clinical remission in which abnormal levels of circulating undifferentiated blast cells are not detectable.

[7] To date, a number of monoclonal antibodies have been prepared that induce cytolytic activity against tumor cells. The humanized version of the monoclonal antibody MuMAb4D5, directed to the extracellular domain of the growth factor receptor P185 (HER2), has been approved by the FDA (Food and Drug Administration) and used to treat human breast tumors (US Patents Nos. 5,821,337 and 5,720,954). Upon binding, the antibody is capable of inhibiting the growth of tumor cells that is dependent on the growth factor receptor.

HER2. In addition, a chimeric anti-CD20 antibody that causes rapid depletion of peripheral B cells, including those associated with lymphoma, has recently been approved by the FDA (US Patent No. 5,843,439). Binding of this antibody to target cells results in complement-dependent lysis. This product has recently been approved and is currently being used in the clinical treatment of low-grade B-cell non Hodgkin's lymphoma.

[8] A number of other humanized and chimeric antibodies are under development or are used in clinical trials. In addition, humanized Ig that specifically reacts with CD33 antigen expressed on normal myeloid cells, as well as on most types of myeloid leukemia cells, was conjugated to the anticancer drug calicheamicin, CMA-676 (Sievers et al., Blood Supplement, 308, 504a ( 1997)). This conjugate, known as MYLOTARG®, has recently received FDA approval (Caron et al., Cancer Supplement, 73, 10491056 (1994)). Due to its cytolytic activity, another anti-CD33 antibody (HumM195), which is currently in clinical trials, has been conjugated to several cytotoxic agents, including a gelonin toxin (McGraw et al., Cancer Immunol. Immunother., 1994, 39, 367 374). ) and radioisotopes ¹³¹ I (Caron et al., Blood 83, 1760-1768 (1994)), ⁹⁰ Y (Jurcic et al., Blood Supplement,

92, 613a (1998)) and ²¹³ Bi (Humm et al., Blood Supplement, 38: 231P (1997)).

[9.] The chimeric anti-leukocyte antigen CD-45 (cHuLym3) is in preclinical phase for the treatment of human leukemia and lymphomas (Sun et al., Cancer Immunol.

«·

Immunother., 48, 595-602 (2000)) as a condition for bone marrow transplantation. In in vitro assays, specific cell lysis was observed in an antibody-dependent cell-mediated cytotoxicity (ADCC) assay (Henkart, Immunities,

1, 343-346 (1994); Squier and Cohen, Current Opin. Immunol., 6, 447-452 (1994)).

[12] In contrast to humanization of murine monoclonal antibodies and construction of chimeric antibodies, the use of the phage display method allows isolation of scFv's involving fully human sequences. Recently, a fully human anti-human TGFb2 receptor antibody was developed, based on the scFv clone derived using the phage display method. This scFv, transformed into fully human IgG4, capable of competing for binding to TGFb2 (Thompson et al., J. Immunol. Methods, 227, 17-29 (1999)) has potent antiproliferative activity. This technology, known to those skilled in the art. , is described in more detail in the following publications: Smith, Science, 228, 1315 (1985), Scott et al., Science, 249: 386-390 (1990); Cwirla et al., PNAS, 87, 6378-6382 (1990) Devlin et al., Science, 249, 404-406 (1990); Griffiths et al., EMBO J., 13 (14), 3245-3260 (1994); Bass et al., Proteins, 8, 309-314; (1990);

McCafferty et al., Nature, 348, 552-554 (1990); Nissim et al., EMBO J. 13: 692-698 (1994); US Patent Nos. 5,427,908,

432,018, 5,223,409 and 5,403,484, lib.

Ligand for isolated scFv antibody molecules [11.] Platelets, fibrinogen, GPIb, selectins and PSGL-1 play an important role in a number of pathogenic or disease states, such as abnormal or pathogenic inflammation, abnormal or pathogenic immune responses, autoimmune •

Reactions, metastasis, abnormal or pathogenic adhesion, thrombosis and / or restenosis, and abnormal or pathogenic aggregation. Therefore, antibodies that cross-react with platelets and these molecules may be useful for the diagnosis and treatment of diseases and disorders involving these and other pathogenic conditions.

Platelets [12] Platelets are well characterized components of the blood system and play a number of important roles in hemostasis, thrombosis and / or restenosis and restenosis. Blood vessel damage sets in motion a process known as hemostasis (haemostasis), which is characterized by a number of subsequent processes. The first reaction to injured blood vessels is the adhesion of platelets to the affected area on the inner surface of the blood vessels. The next step is to aggregate many layers of platelets into previously adhered platelets to form a hemostatic cake. This plate cake closes the walls of the vessel. The hemostatic cake is enhanced by deposition of fibrin polymers. The precipitate is dissolved only when the wound is healed.

The importance of platelets in metastasis [13]. Tumor metastasis is perhaps the most important factor limiting the survival of cancer patients. Summary data demonstrate that the ability of tumor cells to interact with host platelets is one of the indispensable determinants of metastasis. Leslie

Oleksowicz, Z.M .: Characterization of Tumor-Induced

Platelet Aggregation: The Role Of Immunorelated GPIb And GPIIb / IIIa Expression By MCF-7 Breast Cancer Cells, Thrombosis Research 79: 261-274 (1995).

[14] The ability of tumor cells to aggregate platelets has been shown to correlate with the metastasis potential of tumor cells, and in rodent models, inhibition of tumor-induced platelet aggregation has been shown to correlate with metastasis suppression. The interaction of tumor cells with platelets has been shown to include membrane-adhered molecules and secretion of agonists. Expression of immunologically related glycoproteins on tumor cell lines has been identified. Platelet immunologically related glycoproteins, GPIb, GPIIb / IIIa, GPIb / IX and the subunit and integrin have been shown to be expressed on the surface of breast cancer cell lines. Oleksowicz, Z.M .: Characterization Of Tumor-Induced Platelet Aggregation: The Role Of Immunorelated GPIb And GPIIb / IIIa Expression By MCF-7 Breast Cancer Cells, Thrombosis Research 79: 261-274 (1995); Kamiyama, M. et al., "Inhibition of platelet GPIIb / IIIa binding to fibrinogen by serum factors: studies of circulating immune complexes and platelet antibodies in patients with hemophilia, immune thrombocytopenic purpura, human immunodeficiency virus-related immune thrombocytopenic purpura, and systemic lupus erythematosus, J. Lab. Clin. Copper. 117 (3): 209-17.

[15] Gasic (JTBGasic et al., Proc. Natl. Acad. Sel. USA 61: 46-52 (1968) and collaborators have demonstrated that antibody-induced thrombocytopenia significantly reduced the number and volume of metastasis produced by intestinal adenocarcinoma CT26, Lewis. lung carcinoma and melanoma B16 Karpatkin, S. et al., "Role of Adhesive Proteins in Platelet Tumor Interaction in Vitro and Metastasis Formation in Vivo, J. Clin. Invest. 81 (4): 1012-9 (1988); P. et al., "The Role of Platelet Membranes" of glycoproteins Ib / IX and Ilb / IIIa, and of platelet alphagranule proteins in platelet aggregation induced by human osteosarcoma cells, Cancer Res. 53 (19): 4695-700 ( In addition, a unique polypeptide chain (60kd) that is closely related to GPIb and corresponds to incomplete or abnormal O-glycosylated and GPIb subunit has been found to be expressed on the surface membrane of HELL cells. Kieffer, N. et al., " Expression of platelet glycoprotein Ib alpha in HEL cells, J. Biol Chem 261 (34): 15854-62 (1986).

GPIb complex [16] Each step in the hemostasis process requires the presence of receptors on the platelet surface. One of the receptors that is important in hemostasis is the glycoprotein complex Ib-IX (also known as CD42). This receptor mediates platelet adhesion (initial attachment) at the site of vascular wall damage by binding to von Willebrand factor (vWF) in the subendothelium. It also plays a critical role in two other platelet functions important in hemostasis (a) in platelet aggregation induced by high force stress in the areas of arterial stenosis and (b) in platelet activation induced by low thrombin concentration.

[17]. The GPIb-IX complex is one of the major components of the outer surface of the platelet plasma membrane. The GPIb-IX complex contains three membrane-bound polypeptides - the 130 kDa α-subunit and the 25 kDa β-subunit of the GPIb chain linked by a disulfide bridge and a non-covalently attached GPIX (22 kDa). All four units are present in equimolar amounts on the membrane of the platelets for efficient cell surface expression and CD42 complex function, resulting in the expression of CD42 complex.

10 .............

suggests that proper assembly of the three subunits into the complex is required for full expression on the plasma membrane. The chain and GPIb consists of three distinct structural domains: (1) globular N-terminal domains containing leucine-rich repeats and flanking sequences with Cys bonds; (2) highly glycosylated mucin-like macroglycopeptide domains; and (3) a membrane-associated terminal region comprising a disulfide bridge to GPIbP and transmembrane and cytoplasmic sequences.

[18]. A number of evidence suggests that the vWF and the thrombin complex GPIb-IX binding domain are located in the globular region, which comprises approximately 300 amino acids at the amino terminus of GPIba. The human platelet complex GPIb-IX is a key membrane receptor mediating both platelet function and their reactivity. Recognition of vWF bound in the subendothelium by GPIb allows platelets to adhere to damaged vessel walls. In addition, binding of vWF to GPIba also induces platelet activation, which may include interaction of the cytoplasmic domain of GPIb-IX with a cytoskeleton or phospholipase A2. In addition, the GPIba contains a high affinity binding site for α-thrombin, which facilitates platelet activation in a manner whose mechanism has not been successfully established.

[19.]. The N-terminal globular domain of GPIBa contains a cluster of negatively charged amino acids. Many evidence suggests that in transfected CHO cells expressing the GPIb-IX complex and in platelet GPIba, three tyrosine residues contained in this domain (Tyr-276, Tyr278 and Tyr-279) are subject to sulfation.

• · • ·

Protein sulphation [20.]. Protein sulfation is a widespread post-translational modification that involves the enzymatic covalent attachment of sulfate, either to the sugar side chain or to the polypeptide backbone. This modification arises in the trans-Golgi compartments and therefore only affects the protein that crosses this compartment. Such proteins include secretory proteins, proteins secreted in granules, and extracellular regions of plasma membrane proteins. To date, it is known that the amino acid residue that is subject to sulfation is tyrosine. J. W. Kehoe et al., Chemistry and Biol., R57-R61 (2000). Other amino acids, such as threonine, may also undergo sulfation, particularly in diseased cells.

[21]. A number of proteins having sulphated tyrosine have been found, but the presence of three or more sulphated tyrosines in a single peptide, as found for GPIb, is not common. GPIba (CD42), which is expressed by platelets and megakaryocytes, mediates platelet attachment and scrolling across the subendothelium by binding to vWF, also containing a number of negative charges on its N-terminal domain. Such a highly acidic and hydrophilic environment is believed to be a prerequisite for sulfation since tyrosyl protein sulfotransferase specifically recognizes and sulfates tyrosines immediately adjacent to acidic amino acid residues. J. R. Bundgaard et al., JBC 272: 2170021705 (1997). Full sulfation of the acidic GPIba region provides a region with an extra negative charge density of 13 negative charges within the 19 amino acid region, which is a suitable candidate site for electrostatic interactions with other proteins.

»[22]. The P-, E-, and L- selectins are a family of adhesion molecules that, among other functions, mediate the rolling of leukocytes along the vascular endothelium. P-selectin is stored in granules and is transported to the surface after activation with thrombin, histamine, phorbol ester and other stimulating molecules. P-Selectin is also expressed on activated endothelial cells. ESelectin is expressed on endothelial cells and LSelectin is expressed on neutrophils, monocytes, cells and B-cells.

[23]. P-Selectin Glycoprotein Ligand-1 (PSGL-1, also called CD 162) is a mucin glycoprotein ligand of P-Selectin, E-Selectin, and L-Selectin. PSGL-1 is a disulfide-linked homodimer having a Paired Basic Amino Acid Converting Enzymes (PACE) cleavage site. Also, PSGL-1 has three potential tyrosine sites for sulfation, followed by approximately 15 decamer repeating sequences that are rich in proline, serine, and threonine. The extracellular portion of PSGL-1 contains three N-linked glycosylation sites and has a number of sialylated, fucosylated O-linked oligosaccharide side chains.

K. L. Moore et al., JBC 118: 445-456 (1992). Most of the N-glycan sites and many of the O-glycan sites are occupied. A subset of these O-glycans are the core-2, sialylated and fucosylated structures that are required for selectin binding. Sulfation of the tyrosine of the amino-terminal portion of PSGL-1 is also necessary for the binding of P-Selectin and LSelectin. In addition, there is an N-terminal propeptide that is probably post-translationally cleaved.

· · «> • ♦ • ···

13..........

[24.]. PSGL-1 has 361 amino acid residues, 267 residues in the extracellular region, 25 residues in the transmembrane region, and 69 residues in the intracellular region in HL60 cells. The sequence encoding PSGL-1 is a single exon, so alternative splicing is not possible. However, PSGL-1 in HL60 cells and in most cell lines has 15 consecutive consensus sequences of 10 amino acid residues present in the extracellular region, but there are also 14 and 16 repeats of this sequence in polymorphonuclear leukocytes, monocytes and many other cell lines. , including most native PSGL-1 leukocytes, form a homodimer linked by cell disulfide bridges. V. Ashshar-Kharghan et al., Blood 97: 33063312 (2001).

[25]. PSGL-1 is expressed on neutrophils as a dimer with an apparent molecular weight of 250 kDa and 160 kDa, while on HL60 the dimeric form is 220 kDa. When analyzed under reducing conditions, each subunit is reduced by half. Molecular weight differences may be due to polymorphism in the molecule due to the presence of a different number of repeating decamer sequences. Leukocyte Typing VI. Edited by T. Kishimoto et al. (1997).

[26]. PSGL-1 is expressed on most blood leukocytes, such as neutrophils, monocytes, leukocytes, B cell subclass and all T-cells, and mediates rolling of neutrophils to P-Selectin. Leukocyte Typing VI. Edited by T. Kishimoto et al. (1997). PSGL-1 can also mediate neutrophil-neutrophil interaction through binding to L-Selectin and thereby mediate inflammation. Snapp, et al., Blood 91 (1): 154-64 (1998).

·· · * ·

[27.]. PSGL-1 mediates leukocyte scrolling on activated endothelium, activated platelets, and other leukocytes and inflammatory sites.

[28.]. Commercially available antibodies against human PGSL-1 and KPL1 were prepared; have been shown to inhibit the interaction between PGSL-1 and P-Selectin and between PGSL-1 and LSelectin. The KPL1 epitope was mapped to the tyrosine sulfation consensus motif of PGSL-1 (YEYLDYD) (Snapp et al., Blood, 91 (1): 154-164 (1998). KPL1 recognizes only this particular epitope and does not cross-react with the sulfated epitopes present on other cells, such as BCLL cells, AML cells, metastatic cells, multiple myeloma cells, and the like.

[29.]. In inflammation, the leukocyte scrolling and interaction between P-Selectin (expressed by activated endothelium and on platelets that can be immobilized at the site of injury) is significant, and PSGL-1 facilitates attachment and scrolling of leukocytes to vessel walls. Ramachadran et al., PNAS, 98 (18): 10166-71 (2001); Afshar-Kharghan, et al., Blood, 97 (10): 3306-7 (2001).

[30]. Cell rolling is also important in metastasis, it is believed that P- and E-Selectin on endothelial cells bind metastatic cells, thereby allowing them to extravasate from the blood stream into surrounding tissues.

[31]. Platelets are also involved in the metastasis process; when metastatic cells enter the bloodstream, multicellular complexes are formed consisting of platelets and leukocytes surrounding the tumor cells. These complexes, which may be referred to as microembolisms, help tumor cells escape the attention of the immune system. Coating of tumor cells with platelets requires expression of R-Selectin by platelets.

[32.]. The action of heparin, an inhibitor of P- and L-Selectin, inhibits the interaction between the tumor cell and the platelet.

Also, treatment of tumor cells with O-sialoprotease, which removes sialylated, fucosylated mucin ligands, inhibits the formation of tumor cell-platelet complexes. In vivo experiments demonstrate that both of these treatments result in greater association of monocytes with circulating tumor cells, suggesting that decreasing platelet binding increases the access of immune cells to circulating tumor cells. Varki and Varki J. Biol. Res. 34 (6): 711-7 (2001).

[33.]. PSGL-1 and GPIb share structural similarity and have mucin-like, strongly glycosylated ligand binding regions. Afshar-Kharghan, et al., Blood 97 (10): 3306-7 (2001).

[34.]. PSGL-1 was found on all leukocytes: neutrophils, monocytes, lymphocytes, activated peripheral T cells, granulocytes, eosinophils, platelets, and some CD34 positive stem cells and a certain subset of B cells. P-Selectin is selectively expressed on activated platelets and on endothelial cells. The interaction between P-Selectin and PSGL-1 promotes the roll of leukocytes on the vascular walls, and abnormal leukocyte accumulation at vascular sites leads to various pathological inflammations. The stereospecific contributions of individual tyrosine sulphates to PSGL-1 are important for the binding of P-Selectin to PSGL-1. The charge is also significant for binding: decreasing NaCl (from 150 to 50 mM) increased binding (· Kd ~ 75 nM). Sulfation of tyrosine to PSGL-1 increases but is not necessarily required for the adhesion of PSGL-1 to β-Selectin. PSGL-1 tyrosine sulphation promotes slower adhesion of scrolling at all intensities of stress and promotes scrolling adhesion at much higher intensities of stress (Rodgers S.D., et al., Biophys J., 81: 2001-9 (2001)).

Fibrinogen [35]. There are two forms of normal human fibrinogen: fibrinogen major and fibrinogen (the primary minor variant), each of which is found in normal individuals. Normal fibrinogen, which is more abundant (constituting ~ 90% of the fibrinogen found in the body), is composed of two identical 55 kDa alpha (a) chains, two identical 95 kDa beta (β) chains and two identical 49.5 kDa gamma (γ) strings. The normal variant fibrinogen, which is a less abundant form (accounting for ~ 10% of the fibrinogen found in the body), is composed of two identical 55 kDa alpha (a) chains, two identical 95 kDa beta (β) chains and one 50.5 kDa gamma prime ( γ ') strings. Both the gamma strand and the gamma prime strand are encoded by the same gene with alternative splicing occurring at the 3 'end. The normal gamma chain consists of 1-411 amino acids. The normal gamma prime chain consists of 427 amino acids: amino acids 1-407 are the same as in the normal gamma chain, and amino acids 408-427 are

VRPEHPAETEYDSLYPEDDL. Normally, this region is occupied by thrombin molecules.

[36.]. Fibrinogen is converted to fibrin by the action of thrombin in the presence of calcium ions, leading to blood coagulation. Fibrin is also a component of thrombi and exudates of acute inflammation.

[37.]. Platelets and molecules (such as fibrinogen, GPIb, selectins and PSGL-1) that play a significant role in cell-cell interactions, cell-matrix interactions, platelet-plate interactions, platelet-cell interactions, platelet-matrix interactions, cell scrolling and adhesion and hemostasis also play a significant role in pathogenic or disease states such as abnormal or pathogenic inflammation, abnormal or pathogenic immune responses, autoimmune reactions, metastasis, abnormal or pathogenic adhesion, thrombosis and / restenosis, abnormal or pathogenic aggregation. Therefore, antibodies that cross-react with platelets and these molecules may be useful in the diagnosis and treatment of diseases and disorders involving these and other pathogenic conditions. Therefore, there is a need to identify general epitopes within or between these molecules and to identify antibodies capable of cross-reacting with them.

[38.]. Antibodies can be provided in many forms, such as fragments, complexes, and multimers. Examples of such antibody fragments include single chain Fv (scFv) fragments and Fab fragments.

[39.] ScFvs have been shown to penetrate tissues and are removed from the blood faster than a full-size antibody because of their smaller size. Adams, G.P., et al., Br. J. Cancer, 1405-1412 (1988); Hudson, P. J., Curr. Opin. Immunol. 11 (5): 548-557 (1999); Wu, A. M., et al., Tumor Targeting 4, 47 (1999). For this reason, scFvs are often used in diagnostics involving radioactive labels such as tumor imaging, allowing for much faster •

removing radioactive labeling from the body. A variety of anti-tumor scFV multimers are currently in preclinical evaluation of in vivo stability and efficacy. Adams, G. P., et al., Br.J. Cancer 77: 1405-1412 (1988); Wu, A.M. et al., Tumor Targeting 4, 47 (1999).

[40.] Single-chain Fv (scFv) fragments are contained in the variable domains of the heavy (V _H ) and light (V _L ) chains of an antibody linked together by a polypeptide linker. The linker is long enough to allow (V _H ) and (V _L ) domains to be folded into a functional Fv domain that allows scFv to recognize and bind its target with similar or increased affinity for the parent antibody. 41. ScFv monomers are typically designed with the C-terminus of the V _H domain attached by a polypeptide linker to the N-terminal V _L residue. Optionally, an inverse orientation is introduced: the C-terminal end of the V _L domain is linked to the N-terminal end of the V _H by a polypeptide linker. Power, B., et al., J. Immun. Meth. 242, 193-204 (2000). The polypeptide linker is typically about twelve amino acid residues in length. If the linker is reduced to about three to seven amino acids, scFvs cannot be folded into a functional Fv domain and instead associate with a second scFv to form a diabody (dimer). Further reducing the linker length to less than three amino acids promotes the association of scFvs to trimers or tetramers depending on the linker length, composition, and Fv domain orientation. BEPowers, PJ Hudson, J. Immuno. Meth. 242 (2000) 193-194.

[42.] It has recently been shown that multivalent antibody fragments, such as dimers, trimers and tetramers, often provide higher affinity for the target compared to the binding of the parent antibody. Higher affinity offers many advantages, including ideal pharmacological kinetics in targeted tumor applications. In addition, when studying P-selectin and its ligand PSGL-1 involved in leukocyte junction and scrolling, experts concluded that cells expressing dimeric forms of PSGL-1 establish more stable scrolling adhesion, based on higher binding affinity. These adhesions are more resistant to force loads and exhibit less variation in rolling speed. Ramachandran, et al. , PNAS, vol. 98 (18): 10166-71 (2001).

[43.] Higher binding affinity of these multivalent forms is desirable in diagnostic and therapeutic regimens. For example, scFv can be used as a blocking agent for target receptor binding, thereby blocking the binding of a "natural ligand." In such cases, it is desirable to have a higher affinity association between the scFv and the receptor so as to reduce the possibility of separation that may allow undesirable binding of the natural ligand to the target. In addition, this high affinity is particularly important when the target receptors are involved in adhesion and rolling, or when the target receptors are on cells that are present at high sheer flow sites such as blood cells. platičky.

[44.] It is an object of the invention to provide isolated epitopes that are present on different molecules and that assist in processes such as cell rolling, adhesion, thrombosis and / or restenosis and aggregation, and which are present on diseased cells such as AML cells, B-CLL cells, multiple myeloma cells, and metastatic cells.

[45.] Another object of the invention is to provide methods using such isolated epitopes to produce antibodies that recognize and cross-react with epitopes that are present on molecules that assist in processes such as cell rolling, inflammation, immune responses, infections, autoimmune reactions, metastasis, adhesion, thrombosis and / or restenosis and aggregation, and which are also present on diseased cells such as AML cells, B-CLL cells, multiple myeloma cells and metastatic cells.

[46.] Other objects of the invention include the use of such antibodies in the development and delivery of medicaments for inhibiting cellular rolling, inflammation, immune response, infection, autoimmune reactions, metastasis, adhesion, thrombosis and / or restenosis and aggregation, and for treating diseases such as is AML, B-CLL, multiple myeloma, metastases, cardiovascular diseases such as myocardial infarction, retinopathic diseases, diseases caused by sulfated tyrosine-dependent protein-protein interactions, or other diseases in which such cellular functions or effects play a significant role.

[47.] The subject of the invention is the use of epitopes and antibodies in methods of diagnosing various individual disease states, such as AML, B-CLL, multiple myeloma, metastasis, or other diseases in which such cellular functions or effects as cellular scrolling inflammation, immune response, infection, autoimmune reaction, metastasis, adhesion, thrombosis and / or restenosis and aggregation play a significant role.

[48.] Another object of the invention is to provide multivalent forms of antibodies, fragments and complexes. More specifically, it is an object of the invention to provide dimers, trimers and tetramers, sometimes referred to herein as diabodies, triabodies, and tetrabodies, respectively.

[49] These and other objects of the invention are further described.

SUMMARY OF THE INVENTION The present invention provides epitopes that are found on ligands and receptors that play important roles in such diverse processes as inflammation, immune response, metastasis, adhesion, thrombosis, restenosis and aggregation. The epitopes of the present invention are also found on leukemic and tumor cells, particularly of leukemic and myeloid origin. Therefore, these epitopes are useful targets for the therapeutic mediation of these processes. Antibodies directed against such epitopes are useful as therapeutic agents against tumors (both as antitumor agents as well as antimetastatic agents), leukemias, autoimmune diseases, inflammatory diseases, cardiovascular diseases such as myocardial infarction, retinopathic diseases and other diseases mediated by abnormal function and diseases caused by sulfated tyrosine-dependent protein-protein interactions. The present invention provides such antibodies, compositions comprising the antibodies, and therapeutic and diagnostic methods using the antibodies.

[51.] The present invention provides an isolated epitope according to Formula I (S) _r

I [(W) _of -P- (Y) _t -P] _{q (I)} in which

W is any amino acid other than aspartate and glutamate

Y is any naturally occurring compound that can be sulfated

P is (A) _m (A) _n (X) u or (X) _u (A) _n (A) _m , or (A) _n (X) _u (A) _m , or (A) _n (A) _m (X) u, or (X) _u (A) _m (A) _n , or (A) _m (X) _u (A) _n

S is a sulfate or sulfated molecule

X is any amino acid except aspartate, glutamate, or tyrosine

A is any negatively charged amino acid or leucine, isoleucine, proline, phenylalanine, serine, or glycine q is 1 to 6 z is 0, 1 or 2 r is 0 or 1 t is 1, 2 or 3 u is 0 to 2 n is Ο to 3 m is 0 to 3, where, if n = 0, then m> 0; where, if m = 0, then n> 0; wherein, if q is 1, r is 1 and if q is> 1, at least one of Y is sulfated; and further wherein the isolated epitope is capable of being bound to an antibody, an antigen-binding fragment thereof, or a complex thereof comprising an antibody or a binding fragment thereof, comprising a first hypervariable region comprising SEQ ID NO: 8 or SEQ ID NO: 20.

[52.] The present invention provides an isolated epitope according to Formula I, wherein the sulfated compound is a peptide or glyco- or lipo-conjugate, or a combination thereof.

[53.] The present invention also provides an isolated epitope of Formula I wherein W is glycine, Y is a tyrosine peptide conjugate or asparagine, serine or threonine glycoconjugate; A is glutamate, γ carboxy glutamate or aspartate; and q is 1, 2 or 3. In a particular embodiment, Y is a peptide-tyrosine conjugate; q is 3; and r is 1.

[54.] The present invention also provides an isolated epitope of Formula II

(II) in which

W is any amino acid other than aspartate and glutamate

Y is any naturally occurring compound that can be sulfated

P is (A) _m (A) _n (X) _u , or (X) _u (A) _n (A) _m , or (A) _n (X) _u (A) _m , or (A) _n (A) ) _m (X) _u , or (X) _u (A) _m (A) _n , or (A) _m (X) _u (A) _n

S is a sulfate or sulfated molecule

X is any amino acid except aspartate, glutamate, or tyrosine

A is any negatively charged amino acid or leucine, isoleucine, proline, phenylalanine, serine, or glycine z is 0, 1 or 2 r is 0 or 1 t is 1, 2 or 3 u is 0 to 2 n is 0 to 3 m is 0 to 3, where, if n = 0, then m> 0; where, if m = 0, then n> 0; wherein, at least one of Y is sulfated; and further wherein the isolated epitope is capable of being bound by the antibody, antigen-binding fragment thereof, or complexes thereof,

comprising an antibody or binding fragment thereof comprising a first hypervariable region comprising SEQ ID NO: 8 or SEQ ID NO: 20.

[55.] The present invention provides an isolated epitope according to Formula II, wherein the sulfated compound is a peptide or glyco- or lipo-conjugate, or combinations thereof.

[56.] The present invention also provides an isolated epitope of Formula II wherein W is glycine, Y is a tyrosine peptide conjugate or asparagine, serine or threonine glycoconjugate; A is glutamate, gamma carboxyglutamate or aspartate, leucine, isoleucine, proline, phenylalanine, serine or glycine. In a particular embodiment, Y is a peptide-conjugate of tyrosine; q is 3; and r is 1.

[57.] The present invention provides an isolated epitope according to Formula III (S) r. (S) _r (S) _R

III (III), (G) _of (X) _u (E) _n (D) _m (Y) _t (X) _u (E) _n (D) _m (Y) _t (X) _u (E) _n ( D) _n, (Y) _t (D) _ni (E) _n (X) _u wherein:

G is glycine

E is glutamate

D is aspartate

Y is tyrosine

S is a sulfate or sulfated compound

X is any amino acid except of the above of is 0, 1 or 2 t is 1, 2 or 3 r is 0 or 1 at is 0 to 2 n is 0 to 3 m is 0 to 3 = o, wherein, at least one of Y is sulfated; then m> 0 / where, if m = 0, then n where, if n > .0 ;; and further,

wherein the isolated epitope is capable of being bound by the antibody, antigen-binding fragment thereof, or complexes thereof, comprising the antibody or binding fragment thereof, comprising a first hypervariable region comprising SEQ ID NO: 8 or SEQ ID NO: 20.

[58.] The present invention provides an isolated epitope of formula III wherein r is 1.

[59.] In any of the above embodiments, Y may be a lipid, carbohydrate, peptide, glycolipid, glycoprotein, lipoprotein and / or lipopolysaccharide molecule.

[60.] The present invention also provides derivatives, homologues, mimetics, and variants of the above-described epitopes and

provides epitopes as described above and having at least one post-translational modification in addition to sulfation.

[61.] The present invention provides compositions comprising one or more of the epitopes described above. Also provided are isolated polynucleotides encoding at least a portion of the epitopes described above.

[62.] The present invention also provides antibodies, antigen-binding fragment thereof, or complexes thereof, comprising at least one antibody or binding fragment thereof, capable of binding or cross-reacting with at least one of the epitopes described above.

[63.] Also provided is a method of preparing an antibody, antigen-binding fragment thereof, or complexes thereof comprising at least one antibody or binding fragment thereof capable of binding or cross-reacting with at least one of the above-described epitopes. The method comprises the steps of: (a) providing a phage display library; (b) providing one of the above-described epitopes; (c) panning the phage expression library to obtain the phage particles! expressing an oligopeptide or polypeptide capable of binding to an isolated epitope; (d) producing the antibody, binding fragment thereof, or complexes thereof, comprising the antibody or binding fragment thereof, comprising a peptide or polypeptide capable of binding to an isolated epitope.

[64.] The present invention also provides antibodies, binding fragments thereof, or complexes thereof, comprising at least one antibody or binding fragment thereof having the scFv antibody fragment binding sequence of SEQ ID NO: 25 (Y1-scFv) and / or with the sequence of SEQ ID NO: 203 (Y17-scFv).

[65.] Also provided are antibodies, binding fragments thereof, or complexes thereof having the ability of a peptide or polypeptide, wherein the peptide or polypeptide has a first hypervariable region comprising SEQ ID NO: 8 (Y1 CDR3) or SEQ ID NO: 20 ( Y17 (CDR3). In these particular embodiments, the peptide or polypeptide has a second hypervariable region comprising SEQ ID NO: 115 and / or a third hypervariable region comprising SEQ ID NO: 114.

[66.] Provided are antibodies, antibody fragments, and antibody complexes that are capable of binding to a peptide or polypeptide epitope having a length of about 3 to 126 amino acid residues and comprising at least one sulfated tyrosine residue and at least two acidic amino acids. In particular embodiments, the epitope further comprises at least one leucine, isoleucine, proline, phenylalanine, serine or glycine residue. In another particular embodiment, one or more of the at least two acidic amino acid residues are replaced by a leucine, isoleucine, proline, phenylalanine, serine, or glycine residue. In other specific embodiments, the epitope comprises a DYD or ΕΥΕ. In particular embodiments, the epitope is DYD or ΕΥΕ. In yet another embodiment, the epitope comprises DYE or EYD.

[67.] In particular embodiments of the invention, the antibodies, antibody fragments, and antibody complexes provided according to the present invention are capable of binding to an epitope on a lipid, carbohydrate, peptide, glycolipid, glycoprotein, lipoprotein and / or molecule. Lipopolysaccharide. Preferably, the antibodies, antigen-binding fragments thereof, or complexes thereof comprising at least one antibody or binding fragment thereof of the present invention are capable of binding to lipid, carbohydrate, peptide epitopes, glycolipid epitopes, glycoprotein epitopes, lipoprotein epitopes, and / or lipolysaccharide epitopes. In many embodiments, the lipid, carbohydrate, peptide, glycolipid, glycoprotein, lipoprotein, and / or lipopolysaccharide molecule comprises at least one sulfated compound.

[68.] The present invention provides antibodies, antigen-binding fragments thereof, or complexes thereof, comprising at least one antibody or binding fragment capable of binding to at least two molecules selected from the group consisting of PSGL-1, fibrinogen (prime), GPIba , heparin, lumican, complement compound 4 (CC4), an inter-alpha inhibitor, and prothrombin, although not necessarily simultaneously. Also, the antibodies, antibody fragments, or complexes of the present invention bind to any analog of these proteins when the receptor epitope is intact.

[69.] In particular preferred embodiments, there are provided antibodies, antigen-binding fragments thereof, or complexes thereof comprising at least one antibody or binding fragment thereof that is capable of binding to at least two molecules selected from the group consisting of PSGL-1, fibrinogen ( prime), GPIba, lumican, complement compound 4, inhibitor, prothrombin and heparin and are capable of binding to diseased cells such as B-CLL cells, AML cells, multiple myeloma cells and metastatic cells. In particular embodiments, there are provided antibodies, antigen-binding fragments thereof, or complexes thereof, comprising at least one antibody or binding fragment thereof capable of binding to each of the molecules selected from the group consisting of PSGL-1, fibrinogen, GPIba, heparin, lumican, complement compound 4, an inter-alpha inhibitor, and prothrombin. In other particular embodiments, there are provided antibodies, antigen-binding fragments thereof, or complexes thereof, comprising at least one antibody or binding fragment thereof capable of binding to each of the molecules selected from the group consisting of PSGL-1, fibrinogen, GPIba, and heparin ; and in further particular embodiments, the antibodies, antigen-binding fragments thereof, or complexes thereof that comprise at least one antibody or binding fragment thereof are also capable of binding to diseased cells such as B-CLL cells, AML cells, multiple myeloma cells, and metastatic cells. .

[70.] The present invention provides antibodies, antigen-binding fragments thereof, or complexes thereof comprising at least one antibody or binding fragment thereof capable of binding to at least two molecules selected from the group consisting of PSGL-1, fibrinogen (prime), heparin, GPIba, lumican, complement compound 4, an inter-alpha inhibitor, and prothrombin, and further capable of binding to an epitope on a carbohydrate and / or lipid molecule comprising at least one sulfated compound.

[71.] The present invention provides antibodies, antigen-binding fragments thereof, or complexes thereof, which:

comprising at least one antibody or binding fragment thereof capable of cross-reacting with one or more epitopes, each epitope comprising one or more sulfated tyrosine residues within a cluster of acidic amino acids. Particular embodiments are antibodies, antigen-binding fragments thereof, or complexes thereof, comprising at least one antibody or binding fragment thereof that is capable of cross-reacting with at least one cell type selected from the group consisting of B-CLL cells, AML cells, multiple myeloma cells and metastatic cells. In another particular embodiment, antibodies, antigen-binding fragments thereof, or complexes thereof are provided that comprise at least one antibody or binding fragment thereof that is capable of cross-reacting with PSGL-1. Preferably, antibodies, antigen-binding fragments thereof, or complexes thereof comprising at least one antibody or binding fragment thereof that are capable of cross-reacting with PSGL-1 bind to an epitope of QATEYEYLDYDFLPETE wherein at least one tyrosine residue is sulfated.

[72.] In another specific embodiment, antibodies, antigen-binding fragments thereof, or complexes thereof are provided that comprise at least one antibody or binding fragment thereof that is capable of cross-reacting with GPIBa. Preferably, antibodies, antigen-binding fragments thereof, or complexes thereof comprising at least one antibody or binding fragment thereof that are capable of cross-reacting with GP1b-α. Bind to the epitope DEGDTDLYDYYPEEDTEGD, wherein at least one tyrosine residue is sulfated, to the epitope TDLYDYYPEEDTE wherein at least one tyrosine residue is sulfated, to the epitope GDEGDTDLYDYYP, wherein at least one tyrosine residue is sulfated,

• ·· »3 · · 44 • 4 4 4 4 44 4 4 4 « «4 • 4 4 4 4 4 4 4 4 · 4 4 4 4 4 • · 4 • 4 • 4 4 4 4 4 4 444 4 · 4

to the YDYYPEE epitope wherein at least one tyrosine residue is sulfated, and / or to the TDLYDYYP epitope wherein at least one tyrosine residue is sulfated, [73.] Still other of these embodiments are antibodies, antigen-binding fragments thereof, or complexes thereof, containing at least one antibody or binding fragment thereof that is capable of cross-reacting with fibrinogen γ prime. Preferably, the antibodies, antigen-binding fragments thereof, or complexes thereof comprising at least one antibody or binding fragment thereof that are capable of cross-reacting with fibrinogen γ prime bind to the epitope EPHAETEYDSLYPED, wherein at least one tyrosine residue is sulfated.

[74.] In yet another embodiment, antibodies, antigen-binding fragments thereof, or complexes thereof are provided that comprise at least one antibody or binding fragment thereof that is capable of cross-reacting with heparin.

[75.] In yet another embodiment, provided are antibodies, antigen-binding fragments thereof, or complexes thereof, comprising at least one antibody or binding fragment thereof that is capable of cross-reacting with complement compound 4,

CC4). Preferably, antibodies, antigen-binding fragments thereof, or complexes thereof that contain at least one antibody or binding fragment thereof that are capable of cross-reacting with CC4 bind to an epitope

MEANEDYEDYEYDELPAK, wherein at least one tyrosine residue is sulfated.

[76.] The present invention provides antibodies, antigen-binding fragments thereof, or complexes thereof, comprising at least one antibody or binding fragment thereof capable of binding to the fragments, analogs, variants, and mimetics of the above proteins as long as the epitope is basically intact.

[77.] The present invention provides antibodies, antigen-binding fragments thereof, or complexes thereof, comprising at least one antibody or binding fragment thereof capable of cross-reacting with at least one cell type selected from the group consisting of B-CLL cells, AML cells , multiple myeloma cells, and metastatic cells.

[78.] The present invention provides antibodies, antigen-binding fragments thereof, or complexes thereof, comprising at least one antibody or binding fragment thereof that is capable of inhibiting cell rolling; inhibit inflammation; inhibit autoimmune diseases; inhibit thrombosis; inhibit restenosis; inhibit metastasis; inhibit the growth and / or replication of tumor cells; increase tumor cell mortality; inhibit the growth and / or replication of leukemia cells; increase the rate of leukemia cell mortality; to increase the sensitivity of tumor cells to damage by anti-cancer agents; to increase the sensitivity of leukemia cells to damage by antileukemic agents; inhibit the increase in the number of tumor cells in a patient having a tumor; reducing the number of tumor cells in a patient having a tumor; inhibit the increase in the number of leukemia cells in a patient having leukemia; inhibit the formation of cell-cell, cell-matrix, platelet-matrix, platelet-platelet and / or cell-platelet complexes; inhibit cell-cell adhesion, cell-matrix adhesion;

platelet-matrix, platelet-platelet and / or cell-platelet; aggregation.

[79.] Pharmaceutical compositions are provided comprising the antibodies, antigen-binding fragments thereof, or complexes thereof, comprising at least one antibody or binding fragment thereof of the present invention in amounts effective to inhibit, treat, ameliorate their effects, or prevent diseases and / or or conditions of interest.

[80.] The present invention provides the use of the antibodies, antigen-binding fragments thereof, or complexes thereof of the present invention in a method of preparing a medicament for inhibiting, treating, ameliorating their effects, or preventing diseases and / or conditions of interest.

[81.] The present invention provides antibodies, antigen-binding fragments thereof, or complexes thereof for use as medicaments to inhibit, treat, ameliorate their effects, or prevent diseases and / or conditions of interest.

[82.] The present invention provides a method of inhibiting, treating, ameliorating the effects, or preventing diseases and / or conditions of interest, comprising administering to a patient in need thereof a pharmaceutical composition comprising an effective amount of an antibody, antigen-binding fragment thereof, or complexes thereof comprising at least one antibody or binding fragment thereof of the present invention.

[83.] Antibodies, antigen-binding fragments thereof, or complexes thereof that comprise at least one antibody or binding fragment thereof of the present invention can be complexed or bound with different agents.

[84.] The present invention provides antibodies, antigen-binding fragments thereof, or complexes thereof, comprising at least one antibody or binding fragment thereof, bound to an agent selected from the group consisting of anticancer, antimetastatic, antileukemic, disease control, anti-adherence, antithrombotic agents. , antirestenotic, anti-autoimmune, anti-aggregation, antibacterial, antiviral and anti-inflammatory agents.

[85.] The present invention provides antibodies, antigen-binding fragments thereof, or complexes thereof, comprising at least one antibody or binding fragment thereof, bound or complexed with one or more toxins, radioisotopes, and pharmaceutical agents.

[86.] The present invention provides antibodies, antigen-binding fragments thereof, or complexes thereof, comprising at least one antibody or binding fragment thereof, bound or complexed to a vehicle or carrier capable of being coupled or complexed to more than one agent. Examples of such vehicles and carriers include dextran, lipophilic polymers, hydrophilic polymers, HPMA, and liposomes.

[87.] The present invention also provides antibodies, antigen-binding fragments thereof, or complexes thereof, comprising at least one antibody or binding fragment thereof, bound or complexed to a radioactive isotope or other imaging agent. Also provided is a diagnostic kit comprising the antibodies, antigen-binding fragments thereof, or complexes thereof, comprising at least one antibody or binding fragment thereof of the present invention.

[88.] The present invention provides an isolated epitope comprising the amino acid sequence GPIba - Tyr 276 to Glu 282, wherein at least one of amino acids 276, 278 and 279 is sulfated. In a preferred embodiment, the epitope further comprises amino acids GPIba-283-285.

[89.] The present invention also provides antibodies, antigen-binding fragments thereof, or complexes thereof, comprising at least one antibody or binding fragment thereof, which is capable of binding to an epitope comprising the amino acid sequence of GPIba Tyr 276 to Glu 282, wherein at least one of amino acids 276, 278, and 279 is sulfated, wherein the binding intensity is increased if the epitope further comprises amino acids GPIba - 283-285.

[90.] Also provided is an isolated N-terminal GPIba peptide having an apparent molecular weight of about 40 kDa, said peptide comprising an epitope having the sequence YDYYPEE, wherein at least one of the tyrosine residues in the epitope is sulfated, and an isolated GPIba peptide consisting of amino acids. 1 to 282, wherein at least one of amino acids 276, 278, and 279 is sulfated.

[91.] The present invention also provides polyclonal antibodies, antibody fragments or antibody complexes that cross-react with the variable light chain of the human monoclonal antibody scFv Y1.

In particular embodiments, polyclonal antibodies, antibody fragments, or antibody complexes cross-react with a peptide encoded by the NdeEcoR1 variable light chain restriction fragment of the human monoclonal antibody Y-1. Also provided are diagnostic kits comprising such polyclonal antibodies.

Definition:

[92.] Antibodies (Ab's), or immunoglobulins (Ig's) are protein molecules that bind to an antigen. They consist of units of four polypeptide chains (2 light and 2 heavy) interconnected by disulfide bridges. Each of the chains has a constant and variable region. They can be divided into five classes, IgG, IgM, IgA, IgD and IgE, based on their heavy chain. The IgG class comprises several subclasses including, but not limited to, IgG 1, IgG ₂ , IgG ₃ and IgG ₄ . Immunoglobulins are produced in vivo by B lymphocytes and recognize specific foreign antigenic determinants and facilitate antigen clearing.

[93.] Antibodies can be produced and used in many forms, including antibody complexes. As used herein, the term "antibody complex or antibody complexes" means a complex of one or more antibodies with another antibody, or with a fragment or fragments of antibodies, or a complex of two or more antibody fragments. Examples of antibody fragments include Fv fragments,

F (ab ') 2, F (ab'), Fc, and Fd.

The term Fv, as used in the specification and claims, means a molecule that is made up of a human antibody heavy chain variable region and a human antibody light chain variable region, which may be the same or different, and in which it is variable. a heavy chain region joined, bound, fused or covalently attached, associated with a light chain variable region.

The Fv may be a single chain Fv (scFv), or a disulfide bridge stabilized Fv (dsFv). ScFvs form part of the variable domains of each of the antibody heavy and light chains and are linked by a flexible amino acid spacer or linker. The linker may be branched or unbranched. Preferably, the linker is from 0 to 15 amino acid residues, most preferably the linker is (Gly4Ser) 3.

[95.] The Fv molecule itself consists of a first chain and a second chain, each chain comprising a first, a second and a third hypervariable region. Hypervariable loops within the variable region (domain) of the light and heavy chains are called Complementary Determining Regions (CDRs).

There are CDR1 regions in each heavy and light chain,

CDR2 and CDR3. These regions form an antigen binding site and can be specifically modified to achieve increased binding activity. Naturally, the most variable of these regions is the heavy chain CDR3 region. The CDR3 region is considered to be the most exposed region of the Ig molecule and, as shown and demonstrated herein, is the site primarily responsible for the observed selective and / or specific binding characteristics.

* [96.] A fragment of an Fv molecule is defined as any molecule smaller than the original Fv that still retains the selective and / or specific binding characteristics of the original Fv. Examples of such fragments include, but are not limited to (1) a minibody that contains a heavy chain fragment of only Fv, (2) a microbody that contains a small fractionation unit from the heavy chain variable region (PCT Application No. PCT / IL99 / 00581), (3) similar antibodies comprising a light chain fragment and (4) similar antibodies comprising a functional unit from a light chain variable region.

[97.] As used herein, the "Fab fragment" is a monovalent antigen-derived fragment derived from an immunoglobulin. The Fab fragment consists of a light chain and part of a heavy chain.

[98.] F (ab ') 2 fragment is a bivalent immunoglobulin antigen-binding fragment obtained by pepsin digestion. It contains both light chains and part of both heavy chains.

[99.] The Fc fragment is the non-antigen-binding portion of an immunoglobulin. It comprises the carboxy-terminal portion of the heavy chains and Fc receptor binding sites.

[100.] The Fd fragment is the variable region and the first constant region of an immunoglobulin heavy chain.

[101.] Polyclonal antibodies are the product of an immune response consisting of a number of distinct B cells. Monoclonal antibodies are derived from a single cell.

• · ·

[102.] According to the present invention, a cassette used for polypeptides refers to a sequence of consecutive amino acids that serves as a reading frame, is considered as one unit and is treated as such. At one or both ends, amino acids can be exchanged, inserted, deleted, or attached. Similarly, stretches may be interchanged, inserted, deleted or attached at one or both ends.

[103.] As used herein, "epitope" refers to an antigenic determinant or antigenic site that interacts with an antibody, antibody fragment, or antibody complex, or a complex comprising a binding fragment or T cell receptor thereof. The term epitope is used interchangeably with the terms ligand, domain, and binding region.

[104.] Selectivity is defined herein as the ability of a targeting molecule to select and bind a T cell of one type or cell stage from a mixture of cell types or cell stages, all cell types or cell stages that may be specific to the targeting molecule.

[105.] The term "affinity" is used herein as a measure of the binding potency (association constant) between a receptor (ie, one binding site on an antibody) and a ligand (ie, an antigenic determinant). The strength of the sum of all non-covalent interactions between one antigen binding site on an antibody and one epitope is the affinity of the antibody for that epitope. Low affinity antibodies bind antigen weakly and tend to dissociate easily, while high affinity antibodies bind antigen more tightly and remain in binding for longer.

The term "avidity" differs from affinity because it expresses the valency of the antigen-antibody interaction.

[106.] Specificity of antigen-antibody interaction: Although the antigen-antibody response is specific, in some cases an antibody elicited by one antigen may cross-react with another unrelated antigen. Such cross-reactions occur when two different antigens carry homologous or similar epitopes, or their anchor region, or when antibodies specific for one epitope bind to an unrelated epitope having similar chemical properties.

[107.] Platelets are disk-like cytoplasmic megakaryocyte fragments that originate in the bone marrow sinus and then circulate in peripheral blood. Platelets have several functions including a major role in blood clotting.

The plate contains granules and peripherally pure protoplasm in the central portion, but no distinct nucleus. [108] Agglutination here means the process by which suspended bacteria, cells, disks, and other particles of similar size adhere to form clumps. The process is similar to precipitation, but the particles are larger and more in suspension than in solution.

[109.] Aggregation refers to the aggregation of platelets induced in vitro, and thrombin and collagen, as part of a gradual mechanism leading to the formation of clots or hemostatic blood cakes.

[110.] A conservative amino acid substitution is defined herein as a substitution of one or two amino acids.

in the amino acid composition of a peptide, polypeptide or protein, or a fragment thereof. Substitution refers to amino acids that generally have similar properties (such as acidity, basicity, aromaticity, size, positive or negative charge, polarity, non-polarity), in such a way that substitution does not substantially alter the characteristics of the peptide, polypeptide or protein (i.e. isoelectric point, affinity, avidity, conformation, solubility) or activity. Typical substitutions that may be made for such conservative amino acid substitutions may be within the following amino acid groups:

(i) glycine (G), alanine (A), valine (V), leucine (L) and isoleucine (I) (ii) aspartic acid (D) and glutamic acid (E) (iii) alanine (A), serine (S) and threonine (T) (iv) histidine (H), lysine (K) and arginine (R) (v) asparagine (N) and glutamine (Q) (vi) phenylalanine (F), tyrosine (Y) and tryptophan (W).

[111.] Conservative amino acid substitutions can be made in regions flanking the hypervariable regions primarily responsible for selective and / or specific binding characteristics in the molecule, as well as in other parts of the molecule, such as a heavy chain variable portion cassette. Additionally or alternatively, modifications may be accompanied by the reconstruction of full-length antibody molecules, diabodies (dimers), triabodies (trimers) and / or tetrabodies (tetramers), or minibodies or microbodies.

[112.] Phagemid is defined as a phage particle that carries plasmid DNA. Phagemids are plasmid vectors designed to contain an origin of replication from filamentous phage, such as M13 or fd. Because it carries plasmid DNA, the phagemid particle does not have enough space to contain the complete complement of the phage genome.

A component that is missing in the phage genome is essential information for packaging the phage particle. In order to propagate the phage, it is necessary to cultivate the desired phage particles with a helper phage strain that complements the missing folding information.

[113.] A promoter is a region of DNA to which RNA polymerase binds and initiates transcription.

[114.] A phage display library (also called a phage peptide / antibody library, phage library, or peptide / antibody library) contains a large population of phage (generally 10 ⁸ - 10 ⁹ ) in which each phage particle expresses a different peptide or antibody. polypeptide sequence. The peptide or polypeptide fragments may be constructed of variable length. These display peptides or polypeptides can be derived, but not limited to, from the light or heavy chains of human antibodies.

[115.] The pharmaceutical composition relates to a composition comprising a peptide or polypeptide of the invention and a pharmaceutically acceptable carrier, excipient or diluent.

[116.] A pharmaceutical agent refers to a substance that is useful in the prophylactic treatment or diagnosis of mammals, including, without limitation, human, bovine, horse, swine, mouse, dog, cat, or any other warm-blooded animal. The pharmaceutical agent is selected from the group consisting of a radioisotope, a toxin, an oligonucleotide.

a recombinant protein, antibody fragment, and anti-tumor agent. Examples of such pharmaceutical agents include, without limitation, antiviral agents including acyclovir, ganciclovir, zidovudine; thrombosis / restenosis agents including cilostazole, dalteparin sodium, reviparin sodium and aspirin; anti-inflammatory agents including zaltoprofen, pranoprofen, droxicam, acetyl salicylic 17, diclofenac, ibuprofen, dexibuprofen, sulindac, naproxen, amtolmetin, celecoxib, indomethacin, rofecoxib and nimesulfide; anti-autoimmune agents including leflunomide, denileukin diftitox, subreum, WinRho SDF, defibrotide, and cyclophosphamide; and antiadhesive / anti-aggregating agents including limaprost, clorcromen and hyaluronic acid.

[117.] An anti-leukemic agent is a substance with anti-leukemic activity. For example, antileukemic agents include those that inhibit or inhibit the growth of leukemic or immature preleukemic cells, agents that kill leukemic or preleukemic cells, agents that increase the sensitivity of leukemic or preleukemic cells to other antileukemic agents, and agents that inhibit leukemic metastasis cells. According to the present invention, the anti-leukemic agent may also be a substance with anti-angiogenic activity that prevents, inhibits, retards or stops the vascularization of tumors.

[118.] A gene expression pattern can be studied by analyzing the amount of gene product produced under different conditions, at certain times, in different tissues, and the like. An over-expressed gene is considered to be one whose amount of gene product is higher than that found in a normal control, ie a healthy control.

[119.] The cell may express a protein having a binding site (or epitope) for a particular antibody on its surface, but the binding site may exist in a hidden (cryptic) form (i.e., may be spherically hindered or blocked, or lack the necessary for binding with an antibody) in a cell in a stage that can be called the first stage (stage I). For example, stage I may be a normal, healthy, not a diseased status. If the epitope exists in cryptic form, it is not recognized by a particular antibody, ie the antibody does not bind to that epitope or to a given cell in stage I. However, the epitope may be detected, for example, by undergoing its own modification, or molecules, or by its region undergoing a conformational change. Examples of modifications include changes in folding, changes in post-translational modifications, changes in phospholipidization, changes in sulfation, changes in glycosylation, and the like. Such modifications may occur when the cell enters a different stage, which may be called the second stage (stage II). Examples of the second stage include activation, proliferation, transformation or malignant status. After modification, the epitope may be exposed and the antibody may bind.

[120.] Peptide mimetics are small molecules, peptides, polypeptides, lipids, polysaccharides or conjugates thereof that have the same functional effect or activity as other entities, such as antibodies.

• · · · · · · · · ·.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows endoprotease cleavage sites on the GPIb α chain.

FIG. 2 depicts Western blotting showing binding of Y1 and Y17 to platelets under reduced and non-reduced conditions.

FIG. 3 shows an overview of the reactivity of Y1 with various preparations of GC platelets and cell membrane fraction

KG-1.

FIG. 4 shows Western blotting demonstrating that cleavage of platelet GPIb by a sialoglycoprotein endoprotease disrupts binding of both Y1 and Y17.

FIG. 5 depicts Western blotting demonstrating that Y1 and Y17 similarly bind glycocalicin fragments after digestion with O-sialoglycoprotein endoprotease.

FIG. 6 shows the results of FACS analysis demonstrating that specific proteolysis of GPIb abrogates Y1 binding to platelets.

FIG. 7 depicts Western blotting demonstrating that Y1 binds to the N-terminal (His-1 to Glu-282) fragment of platelet GPIba after digestion with mocarhagine.

FIG. 8 shows Western blotting showing binding of Y1 and Ϋ17 to glycocalicin after mocarhagine digestion.

FIG. 9 depicts Western blotting showing binding of Y1 and Y17 to platelets.

FIG. 10 depicts Western blotting demonstrating that Y1 and Y17 bind glycocalicin similarly after digestion with Ficin.

FIG. 11 depicts Western blotting demonstrating that Y1 reacts with the larger fragment resulting from cleavage of GPIbaa by cathepsin G.

FIG. 12 depicts Western blotting demonstrating that Y1 and Y17 react with a larger fragment resulting from cleavage of GPIbaa by cathepsin G.

FIG. 13 shows a Western blotting demonstrating that cleavage of glycocalicin by mocarhagine and cathepsin G abrogates Y1 binding.

FIG. 14 depicts Western blotting demonstrating binding of Y1 and Y17 to the lysate of washed mocarhagine and cathepsin G cleaved plates.

FIG. 15 is a graph illustrating the inhibition of agglutination of washed plates by Y1-scFv.

FIG. 16 is a graph illustrating the inhibition of platelet aggregation in platelet rich plasma by Y1-scFv.

[137 washed] FIG. 17 is a graph illustrating platelet induction by Y1-IgG.

agglutination [138. inserts in

Giant. 18 is a graph illustrating the induction of platelet rich plasma aggregation by Y1-IgG.

FIG. 19 provides ELISA results.

FIG. 20 depicts Western blotting illustrating the specificity of binding of Y1 and α-CD42 (N1-19) to their ligands.

FIG. 21 shows Western blot reactivity of Y1 with Y1-ligand on purified KG-1 cell membrane using immunoprecipitation and RP-HPLC.

FIG. 22 depicts Western blotting showing the effect of O-sialoglycoprotein endopeptidase cleavage on Y1 binding.

FIG. 23 depicts Western blotting showing the effect of aryl sulfatase cleavage on Y1 binding to RP-HPLC purified KG-1 cell lysate and heparin-BSA.

FIG. 24 depicts the immunoprecipitation scheme used in Y1 binding specificity analysis.

FIG. 25 shows Western blotting comparing the binding of Y1 and anti-CD-162 antibody to cells obtained from AML patients and normal blood.

FIG. 26 depicts the results of FACS analysis showing the ability of KPL1, PL1, and PL2 to compete with Y1 for binding.

9 [147.] FIG. 27 depicts the results of FACS analysis that demonstrate the specificity of Y1 binding.

FIG. 28 also reports the results of FACS analysis that demonstrate the specificity of Y1 binding.

FIG. 29 is a graph illustrating% inhibition of Y1 binding in the presence of various peptides.

[150.] Giant. 30 is chart introducing mass : liver mice from different treatment groups. [151.] Giant. 31 is chart introducing % cells MOLT v bone marrow mice from differently treated groups. [152.] Giant. 32 is chart introducing % cells MOLT v blood

mice from different treatment groups.

FIG. 33 is a Western blot showing the effect of aryl sulfatase and mocarhagine cleavage on Y1 binding.

FIG. 34 is a graph showing liver weight (mean +/- SEM) of mice at day 35.

FIG. 35 is a graph illustrating the effect of treatment on survival.

FIG. 36 is a graph showing the% incidence of leukemia in the different treatment groups.

% of KG-1 cells KG-1 cells [157.] FIG. 37 is a graph depicting blood in various treatment groups.

[158.] Fig. 38 is a graph illustrating% in bone marrow of experimental animals.

FIG. 39 is a graph illustrating the pharmacokinetics of TCA-precipitable radioactivity in the plasma of mice following intravenous injection of ¹²⁵ I-CONY1. The sequence CONY1 is shown in SEQ ID NO: 204.

FIG. 40 is a graph illustrating the specific radioactivity of various organs / tissues of mice following intravenous injection of ¹²⁵ I-CONY1.

FIG. 41 is a graph illustrating the distribution of radioactivity in various organs / tissues of mice following intravenous injection of ¹²⁵ I-CONY1.

FIG. 42 is a graph showing the Superdex 75 profile for Y1-cys-kak.

FIG. 43 relates to the size of the dimers compared to the monomer under reducing and non-reducing conditions.

[164.] FIG.44 shows a FACS analysis showing the level of binding of an IgG-Y1 molecule as compared to scFv-Y1 binding.

FIG. 45 shows Western blots showing binding of Y1 and other antibodies to native human platelet-derived glycocalicin and to recombinant glycocalicin produced in E. coli.

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · [166.] FIG. 46 shows a comparison of the binding between the dimer Y1, Y1-scFv (CONY1) and Y1-IgG.

FIG. 47 shows a comparison of the bond between the sulfide bridge Y1 dimer and Y1-scFv (CONY1).

FIG. 48 provides the amino acid and nucleotide sequences of the heavy and light Y1-IgG chains.

The open reading frame (ORF) of the nucleotide sequence Y1-HC (SEQ ID NO: 205), the amino acid sequence Y1-HC (SEQ ID NO: 206), the ORF nucleotide sequence Y1-LC (SEQ ID NO: 207) are shown. ), and the amino acid sequence Y1-LC (SEQ ID NO: 208).

FIG. 49 provides the amino acid sequence of TMI (SEQ ID NO: 209).

FIG. 50 provides the amino acid and nucleotide sequences of the Y16 scFv (SEQ ID NO: 210).

FIG. 51 provides the amino acid sequence of the Y1 Biotag (SEQ ID NO: 211).

FIG. 52 provides the amino acid sequence of Ycys-KAK scFv (SEQ ID NO: 212).

DETAILED DESCRIPTION OF THE INVENTION In the present invention, whole cells have been used to select specific antibodies that recognize the surface determinants of leukemic cells whose specific receptor has not been known or characterized. In addition, a multistage biopanning procedure was employed in which phage was selected by panning on more than one cell type. This procedure is a significant improvement over the prior art methods in which the selection of antigen-specific phage antibodies depends largely on biopanning against the immobilized single antigen, and thus limited selection is performed using whole cells as targets.

[174.] Certain epitopes that have been identified by this multi-step procedure are characterized by the presence of sulfated compounds such as sulfated tyrosine residue or sulfated carbohydrate or lipid compound, preferably within a cluster of two or more acidic amino acids, are found on ligands and receptors that they play an important role in such diverse processes as inflammation, immune reactions, infections, autoimmune reactions, metastasis, adhesion, thrombosis and / or restenosis, cellular scrolling and aggregation. Such epitopes are also found on diseased cells such as B-CLL cells, AML cells, multiple myeloma cells and metastatic cells. Knowledgeable epitopes are useful targets for therapeutic treatment of these processes and for diagnostic purposes.

[175.] Although these epitopes have variable primary amino acid sequences, antibodies against such sulfated epitopes are often capable of binding or cross-reacting with more than one such epitope on more than one molecule, although not necessarily simultaneous binding. Such antibodies are useful as therapeutic agents against tumors (both as anti-tumor agents as well as antimetastatic agents), leukemias, autoimmune diseases, viral diseases, diseases that include abnormal aggregation, diseases that include abnormal adhesion, heart attacks, cardiovascular diseases, and inflammatory diseases disease.

[176.] The human scFv Y1 antibody was isolated from a human antibody phage expression library that was used to screen fixed human platelets to identify platelet-binding antibodies. A number of clones (different scFv antibodies) were isolated and characterized. One of these clones, designated Y1, has been found to unexpectedly bind to leukemia cells obtained from AML patients and from patients having certain other types of leukemias. Panning performed on fixed platelets isolated another clone, Y17, which was found to bind to human blood.

[177.] Proteins extracted from human platelets were analyzed by Western Blotting on SDS-PAGE using Y1 scFv antibody and Y17-scFv antibody to identify epitopes to which platelet surface antibodies bind. Using this method, the epitope on the Y1 scFv and Y17-scFv plates was determined to be glycocalicin, one of the subunits of the CD42 complex.

[178.] Glycocalicin extracellular fragment obtained from human platelets was purified from activated platelets. It was cleaved by various proteases, such as ficin, mocarhagine, cathepsin G, so as to precisely locate the epitope for binding to Y1 on the glycocalicin molecule. Western blotting analysis was performed using antibody Y1 as a detection means. In addition, commercially available anti-glycocalicin antibodies (antibodies known to be known to be Y1-binding epitopes to glycocalicin) were used. They bind to different epitopes of glycocalicin) in the Y1 antibody competition binding assay.

[179.] Based on the results, the amino acids glycocalicin 272-285 are considered to play an important role in the binding of Y1 to glycocalicin. In addition, since the recombinant N-terminal glycocalicin polypeptide (amino acids 1 to 340 and 1 to 480) was not detectable by the Y1 antibody, it is concluded that binding of Y1 to its epitope is dependent on post-translational modifications such as glycosylations or sulfations that are modifications which are not known to occur in E. coli.

[180.] To verify this hypothesis, purified glycocalicin was treated with enzymes (glycosidases) that remove N- and O-linked sugar compounds from proteins, and enzymes (sulfatases) that remove sulfate compounds from proteins. The binding of antibody Y1 to glycocalicin or fragments derived from glycocalicin was not affected by glycosidases. This result indicates that sulfated groups are essential for the binding of Y1 to glycocalicin.

[181.] To further validate these results, sulfated and non-sulfated synthetic peptides were prepared based on the identified epitope (amino acids 272-285 glycocalicin) and used to determine the binding specificity of Y1 to glycocalicin in their presence (ELISA). The sulfated peptides inhibited binding of Y1 to glycocalicin by several fold more than the corresponding non-sulfated peptides, suggesting that sulfation is essential for binding.

[182.] From the above experimental results, it can be concluded that the Y1 antibody epitope in glycocalicin is located between amino acids 272 to 285, where a cluster of negatively charged amino acids is found.

[183.] In parallel, binding of Y1 to KG-1 cells (a human cell line obtained from an AML patient), various human plasma derived proteins, and blood samples of patients with primary leukemia was studied.

[184.] Y1 antibody was found to bind with relatively low affinity to two human plasma derived proteins, one of ~ 50 kD, identified as fibrinogen-gamma prime, and the other of ~ 80 kD, which was identified as complement component 4 (CC4) and human lumican. Said proteins contain sulfated tyrosine residues in combination with a region of negatively charged amino acids.

[185.] PSGL-1 has been identified as a Y1 ligand on KG-1 cells, which is a receptor for E, L- and Pselectins, based on competition assays (in which binding of Y1 to KG-1 cells occurred in the presence of various commercially available anti-PSGL-1 antibodies) and after numerous experiments using sulfated and non-sulfated synthetic peptides derived from the N-terminal site of PSGL-1, the N-terminal site of PSGL-1 contains sulfated tyrosine residues accompanied by a negatively charged amino acid region.

[186.] Although Y1 binds to several molecules, such as platelet glycocalicin, fibrinogen-gamma prime, a component of Complement 4 from human plasma and the PSGL-1 molecule on KG-1 cells, its affinity for primary leukemic cells obtained from either AML or multiple myeloma (MM) patients is many times higher compared to the previously described epitopes. In addition, the fact that a commercially available anti-PSGL-1 antibody (KPL1) does not recognize all (7 out of 12) patients of primary leukemia cells in blood samples obtained from patients, whereas Y1 specifically and selectively recognizes them, indicates that on primary leukemia cells there are other epitopes for the Y1 antibody that differ from epitopes on KG-1 cells.

Examples of sulfated epitopes of the present invention include those depicted by Formulas I, II, and III as well as derivatives, homologs, mimetics, and variants thereof.

Formula (I):

(S) r [(W) _z -P-COt-P] q

W is any amino acid other than aspartate and glutamate

Y is any naturally occurring compound that can be sulfated

P is (A) _m (A) _n (X) _u , or (X) _u (A) _n (A) _m , or (A) _n (X) _u (A) _m , or (A) _n (A) ) _m (X) _u , or (X) _u (A) _m (A) _n , or (A) _m (X) _u (A) _n

S is a sulfate or sulfated molecule

X is any amino acid except aspartate, glutamate, or tyrosines

A is any negatively charged amino acid or leucine, isoleucine, proline, phenylalanine, serine, or glycine q is 1 to 6 z is 0, 1 or 2 r is 0 or 1 t is 1, 2 or 3 u is 0 to 2 n is 0 to 3 m is 0 to 3, wherein, if n = 0, then m> 0; where, if m = 0, then n> 0; where, if q is 1, r is 1 and if q is>

1, at least one of Y is sulfated; and further wherein the isolated epitope is capable of being bound to an antibody, an antigen-binding fragment thereof, or a complex thereof comprising an antibody or a binding fragment thereof, comprising a first hypervariable region comprising SEQ ID NO: 8 or SEQ ID NO: 20.

[188]. A preferred epitope is an epitope of Formula I wherein W is glycine, Y is a tyrosine peptide conjugate or asparagine, serine or threonine peptide conjugate; A is glutamate, γ carboxy-glutamate or aspartate; and q is 1, 2, or 3. In particular embodiments, Y is a tyrosine peptide conjugate; q is 3; and r is 1.

Formula II:

(S) r (S) _r (S) _r III (W) _from -P-COr-P- (Y) _t -P- (Y) tP (II) in which

W is any amino acid other than aspartate and glutamate

Y is any naturally occurring compound that can be sulfated

P is (A) _m (A) _n (X) _u , or (X) _u (A) _n (A) _m , or (A) _n (X) _u (A) _m , or (A) _n (A) ) _m (X) _u , or (X) _u (A) _m (A) _n , or (A) _m (X) _u (A) _n

S is a sulfate or sulfated molecule

X is any amino acid except aspartate, glutamate, or tyrosine

A is any negatively charged amino acid or leucine, isoleucine, proline, phenylalanine, serine, or glycine z is 0, 1 or 2 r is 0 or 1 is 1, 2 or 3 is 0 to 2 is 0 to 3 is 0 to 3, where, if n = 0, then m> 0; where, if m = 0, then n> 0; wherein at least one of Y is sulfated; and further wherein the isolated epitope is capable of being bound to an antibody, an antigen-binding fragment thereof, or a complex thereof comprising an antibody or a binding fragment thereof, comprising a first hypervariable region comprising SEQ ID NO: 8 or SEQ ID NO: 20.

[189.] A preferred epitope is an epitope of Formula II wherein W is glycine, Y is a tyrosine peptide conjugate or asparagine, serine or threonine glycoconjugate; A is glutamate, γ carboxy glutamate or aspartate, leucine, isoleucine, phenylalanine, serine or glycine. In particular embodiments, Y is a peptide conjugate of tyrosine; q is 3; and r is 1.

·· ·· · · ·

4 ···· ··· · «» «« «« * «* * * *

Formula III:

(S) _r (S) _r (S) _r

III (G) _of (X) u (E) _n (D) r (Y) t (X) u (E) n (P) inOOtGOu (E) n (D) _m (Y) t (D) _m ( E) _n (X) u (III), in which

G is glycine

E is glutamate

D is aspartate

Y is tyrosine

S is a sulfate or sulfated molecule

X is any amino acid except the above z is 0, 1 or 2 t is 1, 2 or 3 r is 0 or 1 u is 0 to 2 n is 0 to 3 m is 0 to 3, wherein at least one of Y is sulfated ; where, if n 0, then m> 0; where, if m = 0, then n> 0; and further wherein the isolated epitope is capable of being bound to an antibody, an antigen-binding fragment thereof, or a complex thereof comprising an antibody or a binding fragment thereof, comprising a first hypervariable region comprising SEQ ID NO: 8 or SEQ ID NO: 20.

[190.] A preferred epitope is an epitope of formula III wherein r is 1.

[191.] The sulfated compound in any of the formulas may also be a peptido, glyco- or lipoconjugate. Y may comprise a lipid and / or carbohydrate molecule. Epitopes may have at least one other post-translational modification in addition to sulfation.

[192.] Such epitopes are found on such diverse molecules as GPIb and PSGL-1, and are found on certain diseased cells such as B-CLL cells, AML cells, multiple myeloma cells and metastatic cells. The sulfation of tyrosine and / or other compounds is particularly important for binding to these epitopes. Human proteins known to contain sulphated tyrosine include the following proteins:

Peptide

Thrombomodulin (408-426)

Human GPIba (269-287)

Human Heparin Cofactor 11 (56-75)

Human fibrinogen γ '(408-427) α-2-Antiplasmin

Sequence

ECPEGY1LDDGFICTDIDE

DEGDTDLYDYYPEEDTEGD

GEEDDDYLDLEEDDDY1DIVD

VRPEHPAETEYDSLYPEDOL

PPMEEDYPQFGSP • · • ·

Cholecystokinin (CCK) α, -2-Choriogonadotropin

Complement C4

PSGL-1

Factor VIII (716-731)

Lumican

Production and Selection Yl

RISDRDYMGWMDF

CHCSTCYYHKS-COOH

MEANEDYEDYEYDELPAK

QATEYEYLDYDFLPET

GDYYEDSYEDISAYLL

GYYDYDFPL [193.] One example of an antibody of the present invention that binds to epitopes of formulas I to III is a fully human antibody Y1. Selection, production, and initial characteristics of Y1 are described in detail in US Patent Applications Nos. 09/751 181 and No. 60/258 948. Briefly, a phage display library expressing scFv antibody fragments was used to obtain and produce targeting molecules, and flow cytometry. , in particular fluorescent activated cell sorting (FACS), has been used to identify and isolate specific phage clones whose peptide or polypeptide recognizes target cells. The phage expression library used was constructed from peripheral blood lymphocytes of non-immunized human donors.

[194.] Phage clones were selected and identified by a multi-step procedure known as biopanning.

Biopanning was performed by incubating phages expressing ligand variants (phage expression (display) library) with targets, removing unbound phages by a washing technique, and specifically eluting the bound phages. The eluted phages were eventually amplified before being taken to the next binding cycles and eventual amplification enriched the pool of specific

sequences in favor of phage clones carrying antibody fragments that showed the best binding to the target. After several cycles, individual phage clones were characterized and sequences of peptides expressed by clones were sequenced by sequencing of the corresponding phage virion DNA.

[195.] According to the present invention, platelets were screened against unidentified epitopes in the initial stages of biopanning, followed by clone selection performed with the desired target cell (eg, BCLL cells, AML cells, multiple myeloma cells and metastatic cells) whose target cell surface markers are known.

[196.] Malignant and diseased blood cells (eg, leukemia and lymphoma) are characterized as immature cells that express cell surface proteins normally found on partially differentiated hematopoietic progenitors.

For this reason, platelets are not an interesting source for identifying surface markers of immature cells that are expressed on diseased or malignant blood cells.

[197.] A Y1-producing scFv clone was selected that binds to platelets and myelogenous leukemia cells, particularly AML cells. The Y1 scFv has the sequence of SEQ ID NO: 25. The binding characteristics of Y1 can be primarily attributed to the CDR3 region having the sequence of SEQ ID NO: 8. Complete Y1-IgG antibodies were also prepared.

[198.] A second scFv clone, Y17, has also been selected that binds to platelets and cell lines derived from human myelogenous leukemia cells, particularly cells

AML. Y17-scFv has the sequence of SEQ ID NO: 203. The binding characteristics of Y17 can be primarily attributed to the heavy chain CDR3 region of SEQ ID NO: 20.

Preparation of Antibodies The CDRs of the present invention may also be inserted into antibody cassettes. According to the present invention, the cassette used for polypeptides refers to a sequence of consecutive amino acids that serves as a reading frame, is considered as one unit and is treated as such. Amino acids may be exchanged, inserted, deleted, or attached at one or both ends. Similarly, stretches may be interchanged, inserted, deleted or attached at one or both ends.

[200.] The amino acid sequence of the cassette may appear to be invariant, while the interchanged, inserted, or linked sequences can be highly variable. The cassette may be comprised of a number of domains, each containing a critical function for the final construct.

[201.] The hypervariable regions of the antibodies of the invention form an antigen binding site for the antibodies of the present invention. The antigen binding site is complementary to the structure of the epitopes to which the antibodies bind and is therefore referred to as complementarity-determining regions,

CDRs). There are three CDRs on each antibody light and heavy chain, each located on loops that connect the β chains of the V _H and V _L domains.

[202.] A cassette according to a particular embodiment of the present invention comprises, from the N-terminus, region 1 reading frame (FR1), CDR1, region 2 reading frame (FR2), CDR2, and region 3 reading frame (FR3).

[203.] In an embodiment of the invention, it is possible to interchange different regions within the cassette. For example, in a cassette, the hypervariable CDR2 and CDR1 regions may be interchanged or modified by non-conservative or preferably conservative amino acid substitutions. More specifically, CDR2 and CDR1 cassette regions consisting of a sequence of consecutive amino acids selected from the group consisting of SEQ ID NOs: 30-113, or a fragment thereof, may be replaced by SEQ ID NOs: 115 and 114, respectively. More specifically, CDR2 and CDR1 cassette regions consisting of a sequence of consecutive amino acids selected from the group consisting of SEQ ID NOs: 30-32, 35, 37-39, 41, 43, 45, 46, 48, 51, 54, 57 , 59-68, 70, 71, 76-85, 87, 89-92, 94, 97, 99, 103, 106, 112 and 113, or a fragment thereof, may be replaced by SEQ ID NOs: 115 and 114, respectively. .

[204.] In a preferred embodiment of the invention, the peptide or polypeptide comprises a heavy and a light chain, each chain comprising first, second and third hypervariable regions which are the CDR3, CDR2 and CDR1 regions, respectively. Binding selectivity and specificity are determined in particular by the CDR3 region of the chain, probably the CDR3 region of the light chain, and preferably the CDR3 region of the heavy chain, and secondarily the CDR2 and CDR1 regions of the light chain, preferably the heavy chain. Binding selectivity and specificity may be secondarily affected by regions lining upstream and downstream of the first, second and / or third hypervariable regions.

[205.] In a preferred embodiment, the CDR3 region of the peptide or polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NOS: 8-24.

In a more preferred embodiment, the heavy chain CDR3 region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-24, CDR2 has an amino acid sequence identical to SEQ ID NO: 115, and CDR1 region has an amino acid sequence identical to SEQ ID NO: 114 [207.] In a most preferred embodiment of the invention, the CDR3 region has an amino acid sequence identical to SEQ ID NO: 8.

[208.] A preferred embodiment of the invention is a scFv with a CDR3 sequence identical to SEQ ID NO: 8 and a complete scFv sequence identical to SEQ ID NO: 25.

[209.] In a most preferred embodiment of the invention, the CDR3, CDR2, and CDR1 regions have the amino acid sequences of SEQ ID NOs: 8, 115, and 114, respectively.

[210.] In another embodiment of the invention, the Fv peptide comprises a variable heavy chain CDR1 and CDR2 region, which itself comprises a cassette with an amino acid sequence selected from the group consisting of SEQ ID NO: 30113; a CDR3 region, preferably a variable heavy chain, having an amino acid sequence selected from the group consisting of SEQ ID NOS: 8-24; an upstream region flanking the CDR3 region having the amino acid sequence of SEQ ID NO: 117; a downstream region flanking the CDR3 region having the amino acid sequence of SEQ ID NO: 116; spacer of 0-20

..... ........

amino acid residues of sequence SEQ ID NO: 123 or 124; a light chain variable region having the sequence of SEQ ID NO: 7.

Similarly, in another embodiment, the upstream flanking CDR2 region has the amino acid sequence of SEQ ID NO: 119; the downstream flanking CDR2 region has the amino acid sequence of SEQ ID NO: 118, the upstream flanking CDR1 region has the amino acid sequence of SEQ ID NO: 121 and the upstream flanking CDR1 region has the amino acid sequence of SEQ ID NO: 120.

[212.] In a preferred embodiment of the invention there is provided a peptide or polypeptide, wherein the second and third hypervariable regions are CDR2 and CDR1 hypervariable regions, respectively, and wherein the amino acid sequence for CDR3 is SEQ ID NO: 8, the amino acid sequence for CDR2 is SEQ ID NO: 115, the amino acid sequence for CDR1 is SEQ ID NO: 114, and wherein the upstream region of CDR3 has the amino acid sequence of SEQ ID NO: 117, upstream region of CDR3 has the amino acid sequence of SEQ ID NO: 116, the upstream flanking CDR2 region has the amino acid sequence of SEQ ID NO: 119, the upstream flanking CDR2 region has the amino acid sequence of SEQ ID NO: 118, the upstream flanking CDR1 region has the amino acid sequence of SEQ ID NO: 121 and the flanking region The downstream CDR1 has the amino acid sequence of SEQ ID NO: 120.

[213.]. Another embodiment of the invention provides an Fv molecule comprising a first chain having first, second and third hypervariable regions, and a second chain having first second and third hypervariable regions wherein one of the first hypervariable regions of the first chain has a sequence selected from the group consisting of of SEQ ID NO: 824, and wherein one of the hypervariable regions of the second chain has a sequence selected from the group consisting of SEQ ID NOs: 1-6 and 125-202, and wherein the first, second and third hypervariable regions are CDR3, CDR2, and A CDR1 region, respectively, wherein the Fv is a scFv or dsFv having one or more identifier sequences.

[214.] Another embodiment of the invention provides a peptide or polypeptide (i) wherein each of the first and second chains comprises a first hypervariable region selected from the group consisting of SEQ ID NOS: 8-24; or (ii) wherein the first hypervariable regions of the first and second chains are identical and are selected from the group consisting of SEQ ID NOs: 8-24, or (iii) wherein the first hypervariable region of the first chain is selected from the group consisting of SEQ ID NOs. 8-24 and the first hypervariable region of the second chain is selected from the group consisting of SEQ ID NOS: 1-6 and 125-202; or wherein the first hypervariable region of the first chain is selected from the group consisting of SEQ ID NOS: 1-6 and 125-202 and the first hypervariable region of the second chain is selected from the group consisting of SEQ ID NO: 824.

Another embodiment of the invention provides a peptide or polypeptide in which the second and third hypervariable regions of the first chain are SEQ ID NOS: 114 and 115, respectively, [215.] With respect to all amino acid sequences of the 25 amino acid residues described and detailed herein ( eg CDR regions, CDR flanking regions) should be understood and taken into account as further embodiments of the invention that these amino acid sequences include within their scope sequences with one or two amino acid substitutions, and that the substitutions are preferably conservative amino acid substitutions. With respect to all amino acid sequences of> 25 amino acid residues described and detailed herein, it is to be understood and taken as a further embodiment of the invention that these amino acid sequences include within their scope sequences with 90% sequence similarity to the original sequence ( Altschul et al., Nucleic Acids Res., 25, 3389-3402, (1997)). Similar or homologous amino acids are defined as non-identical amino acids that exhibit similar properties, such as acidity, alkalinity, aromaticity, size, positive or negative charge, polarity, and non-polarity.

[216.] The percentage of amino acid similarity or homology or sequence similarity is determined by comparing the amino acid sequences of two different peptides or polypeptides. The two sequences are aligned, usually using one of a series of computer programs designed for this purpose, and the amino acid residues at each position are compared. Thus, the amino acid identity or homology is determined. An algorithm is then used to determine the percentage of amino acid similarity. In general, it is preferred to compare amino acid sequences due to the considerably increased sensitivity of detecting slight correlations between peptide, polypeptide, or protein molecules. Protein alignment may take into account the presence of conservative amino acid substitutions, while mismatch may provide a positive score if non-identical amino acids have similar physical and / or chemical properties (Altschul et al., Nucleicides Res., 25, 3389-3402 ( 1997).

[217.] In an embodiment of the invention, the three hypervariable regions of each of the light and heavy chains may be interchanged between two chains and between three hypervariable sites within and / or between the chains.

Polyclonal Antibodies to V _L (derived from Y1) The DNA fragment encoding the V _L (variable light chain) domain of a human antibody was cloned by PCR from Y1 clone (an identical fragment can be obtained from any Nissim I library clone (Nissim et al. , EMBO J. 13 (3): 692-698 (1994)), or even from the human genome using the same methodology) with the following synthetic oligonucleotide primers: oligo 5 ' -WdeI (TTTCATATGGAGCTGACTCAGGACCCTGCT) and 3'EcoR1 oligo (TTTGAATTCCTATTTTGCTTTTGCGGC). After amplification by polymerase chain reaction (PCR conditions: 94 ° 1 ', 56 °', 72 ° 2'x 30, then 65 ° 5 '), the obtained DNA fragment was digested with the restriction enzymes NdeI and FcoRI and cloned into the restriction enzyme sites NdeI and FcoRI a pre-cleaved plasmid which is an IPTG inducible expression vector used for prokaryotic expression of recombinant proteins in E. coli. E. coli cells were transformed with the ligation mixture and positive clones were selected by PCR amplification using the above oligonucleotide primers. Cells carrying this plasmid were grown and induced for expression by IPTG. After induction of IPTG, bacterial cells were harvested by centrifugation from a 1-liter culture, inclusion bodies were Q4-Q, Q-Q, Q-Q, Q-Q, Q-Q, Q-Q, Q-Q, Q-Q

Isolated and solubilized in guanidine-HCl + DTT, refolding was performed by dilution in a buffer containing Tris-Arginine-EDTA. After refolding at 5-10 ° for 48 hours, the protein containing solution was dialyzed and concentrated against 20 mM glycine, pH 9. The dialyzed protein containing solution was repeatedly purified using an HiTrapQ ion exchange column and eluted with a NaCl gradient. The major peak was analyzed by SDS PAGE and gel filtration. From the original 1 liter culture was obtained at least 10 mg of purified V _L.

[219.] Rabbits were immunized in _L (400 mg) in the presence of CFA (complete Freund's adjuvant), then in _L (200 mg) in the presence of IFA (incomplete Freund's adjuvant) at 2-4 week intervals. The titers obtained were low (1:50 - 1: 100) probably due to the high homology between human and rabbit V _L chains.

Polyclonal antibodies to scFv antibodies [220.] Two individual scFv clones (Y1 and N14), obtained from the Nissim I antibody phage expression library (Nissim et al., Antibody fragments from a single poison display library and immunochemical reagents, EMBO J. 13) (3): 692698 (1994)) were separately cultured. Following IPTG induction, cultures were grown at 22 ° C for 16 hours.

The scFv antibody fragments were harvested from the periplasm of bacterial cells and purified on a Protein A-Sepharose column. All bacterial cloning culture procedures, induction procedure, scFv antibody fragment harvesting, and antibody fragment purification were performed according to: Harrison JL, Williams SC, Winter G, and Nissim A. Methods Enzymol. 267: 83-109 (1996). Essentially, any two or more individual clones from the antibody phage expression library of I may be selected so that rabbit polyclonal antibodies that recognize any single scFv antibody present in the Nissim library, or as any one, can be prepared. IgG or a fragment thereof, provided that it contains the same V _L chain or fragment thereof.

[221.] Rabbits were immunized with 400 mg in a 1: 1 mixture of purified scFv antibody fragments in the presence of complete Freund's adjuvant and then 200 mg of this mixture in the presence of incomplete Freund's adjuvant at 2-4 week intervals.

[222.] For detection of scFv antibodies binding to cells by flow cytometry (FACS) or various protein fractions on SDS-PAGE (Western blotting analysis), polyclonal anti-scFv antibodies were used directly from serum from immunized rabbits or after column purification. Protein A-Sepharose.

Characterization of Y1 platelet binding sites [223.] Circulating platelets are cytoplasmic particles released from the peripheral portion of megakaryocytes. Platelets can play an important role in hemostasis. After vascular damage, the platelets adhere to the surface of the damaged tissue and join together (cohesion). This sequence of processes is formed rapidly to form a structured mass (commonly called a platelet plug or thrombus) at the site of vascular injury. The phenomenon of cohesion, also known as aggregation, can be initiated in vitro by a variety of agents or agonists such as: collagen, adenosine diphosphate (ADP) epinephrine, serotonin, and ristocetin. The aggregation is one of the ₇₃ ........ ₇₃ ........ .

from a series of in vitro assays used to measure platelet function.

[224.] Many evidence in the prior art suggests that a cluster of negatively charged amino acids between Asp269 and Asp287 in GPIba is important for the binding of von

Willebrand Factor (vWF) to platelets, which in turn mediates platelet adhesion to damaged blood vessels, platelet aggregation due to high stress in arterial stenosis, and platelet activation induced by low thrombin concentrations Ward, CM, et al., Biochemistry 35 (15): 4929 39 (1996). The interaction of vWF with GPIb is dependent on activation or conformational change in vWF structure when bound to matrix or exposed to force. This process is mimicked by in vitro specific modulators that bind to vWF, such as ristocetin and botrocetin.

Reactivity of Yl to platelet cell extract [225.] Immunoblotting and endoprotease digestion were used to identify the epitope for Yl on the platelet surface membrane. The endoprotease cleavage sites on the GPIba molecule are shown in FIG. 1.

Western blotting [226.] Y1-scFv was selected from phage antibody library by biopanning on human platelets and was found to bind to fixed and washed human platelets. Characterization of Y1 was performed by ELISA and analysis

FACS.

[227.] To characterize the epitope on the membrane of the platelets to which Y1 binds, platelet surface proteins were separated by SDS-PAGE (both under reducing and non-reducing conditions) and immunoblotting with Y1-labeled biotin was performed. The results of this experiment demonstrate that Y1 reacts with a protein of 135 kDa under reducing conditions and with a protein of 160 kDa under non-reducing conditions. These molecular weights correspond to platelet GPIba, which has a molecular weight of 135 kDa under reducing conditions. Under non-reducing conditions, the disulfide bonded GPIba chain to GPIbP has a molecular weight of 160 kDa. (Fig. 2).

[228.] The GPIb chain is bound by a disulfide bridge to the GPIbp chain to form the platelet membrane protein GPIb. Monoclonal antibodies, MCA466S (Serotec) and SC7071 (Santa Cruz) are known to bind respectively to the C-terminal fragment of GPIba and to the N-terminus of GPIba, and have also been found to react with the same fragments with which and non-reducing conditions react Y1 (SC was used only under reducing conditions). These results further confirm that Y1 binds to the platelet surface protein GPIba.

[229.] Further analysis of the semi-purified fragment of GPIb (glycocalicin) by Western analysis confirmed that Y1 actually binds to the alpha subunit of the GPIb complex.

[230.] Western analysis of recombinant GPIb expressed in E. coli showed that GPIb expressed in E. coli did not react with Y1. For this reason, post-translational modifications that do not occur in E.coli appear to be necessary for Y1 binding. Neither N- or O-glycanases act on «

be aryl _{Π Γ} · · · · ·

5 shows binding of Y1 to KG-1 cells. However, Y1 binding can be inactivated (eliminated) by the action of ligands with sulfatases or proteases (Fig. 3).

Localization of the Y1 epitope site on the GPIba glycocalicin (GC) fragment [231.] For further localization of the Y1 binding site, specific endoproteases with known cleavage sites were used to cleave the GPIb and the fragments were tested for Y1 binding.

Effect of O-Sialoglycoprotein Endoprotease on Y1 Binding to Platelet GPIba [232.] The O-sialoglycoprotein endoprotease from Pasteurella haemolytica (Cedarlan CLE 100) selectively cleaves human platelet GPIb and specifically cleaves only sialylated, O-linked glycine-containing proteins. Oialoglycoprotein endoprotease does not cleave N-linked glycoproteins or non-glycosylated proteins. This enzyme is reported to cleave GPIb, which is strongly O-glycosylated but not GPIIb-IIIa or other platelet receptors. GPIba was cleaved by the O-sialoglycoprotein endoprotease to further determine the binding of Y1 to the molecule.

[233.] Immunoblots (Figs. 4 and 5) and FACS analysis (Fig. 6) show that incubation of washed plates with 0sialoglycoprotein endoprotease abolishes Y1 binding, as well as binding of monoclonal antibody MCA466S (Serotec), which is directed against GPIba. The endoprotease did not alter the binding of a monoclonal antibody (anti-CD61) directed against • · · · ·

• · · ····· · · · · · · · · · ·

GPIIb / IIIa (Fig. 4). These results provide further evidence that the Y1 receptor on platelet membranes is GPIba.

GPIb cleavage by mocarhagine - epitope mapping for Yl [234.] Mocarhagine (Sigma L4515a) is a cobra venom metalloproteinase that cleaves platelet GPIba specifically at one site between Glu-282 and Asp-283 residues to produce two stable products: ~ 45 kDa The N-terminal fragment (His-1 to Glu-282) that is released into the supernatant, and the membrane-bound ~ 95 kDa C-terminal fragment.

[235.] The washed plates were treated with mocarhagine and the plate lysates were transferred to nitrocellulose after electrophoresis on SDS polyacrylamide gels. Western blot analysis of mocarhagine-treated washed lysates with Y1 demonstrates a loss of band corresponding to GPIba (135 kDa), and binding of Y1 to the N-terminal ~ 45 kDa fragment. The monoclonal antibody (MCA466S) directed against the C-terminal fragment of GPIba reacted with the ~ 95 kDa C-terminal fragment and the monoclonal antibody (SC7071) directed against the N-terminal fragment of GPIba reacted with the same ~ 45 kDa fragment that was recognized by Y1 (Fig. 7).

[236.] The action of mocarhagine on glycocalicin (a soluble, extracellular fragment of GPIba) yielded results similar to those found in washed plates showing the binding of Y1 and the monoclonal antibody SC7071 to the ~ 45 kDa N-terminal fragment as a cleavage product of GPIba (Figs.). 8). These results demonstrate that the epitope for Y1 is contained within the sequence His-1 to Glu-282.

• ·

Characterization of Y17 Clone - Binding to GPIb [237.] Y17, the second human scFv antibody fragment of the invention, selected in the same way as Y1, was characterized using the methods used to characterize Y1. [see Example 17]. Briefly, Y17 was selected from the phage antibody library by biopanning on human platelets. Y17 characterization was performed using ELISA and FACS analysis. Y17 was found to bind to both fixed and washed human platelets. To further characterize the Y17-binding receptor on platelet membranes, platelet proteins were separated by SDS-PAGE and immunoblotted with Y17-labeled biotin under reducing and non-reducing conditions. The results showed that Y17 reacts with a protein having an apparent molecular weight of 135 kDa under reducing conditions and with a protein having an apparent molecular weight of ~ 160 kDa under non-reducing conditions. The results obtained correspond to platelet GPIba, which under reducing conditions has a molecular weight of 135 kDa and under non-reducing conditions has a molecular weight of 160 kDa, and consists of a chain of GPIba linked by a disulfide bridge to GPIba. The monoclonal antibody MCA466S (Serotec), directed against the C-terminal fragment of GPIba, and the monoclonal antibody S.C.7071 (Santa Cruz), which recognizes the N-terminus of GPIba, react with the same bands as Y17 under reducing and non-reducing conditions (Fig. 2).

[238.] Western blots suggest that Y1 and Y17 similarly bind to platelet lysates (Fig. 9).

• ·

[239.] Y1 and Y17 also similarly bind to glycocalicin after cleavage of glycocalicin by O-sialoglycoprotein endoprotease or ficin (Figs. 5 and 10).

[240.] FACS analysis showed that Y1 and Y17 have similar binding profiles to platelets and KG-1 cells. In addition, both do not bind to Paradise and T2 cells. In contrast, TMI (SEQ ID NO: 209), Y16 (SEQ ID NO: 210) and Y45 do not bind to any of the above human cell lines.

[241.] These results demonstrate that Y1 and Y17, two monoclonal antibody fragments of the present invention, recognize an epitope on different cells, and that this epitope is not recognized by any of the other monoclonal antibodies tested.

GPIb cleavage by cathepsin G - epitope mapping for Yl [2421.] Cathepsin G (Sigma C4428), a serine protease of neutrophils, cleaves glycocalicin at the first cleavage site between Leu-275 and Tyr-276 and at the second cleavage site between Val-296 and Lys-297 The action of cathepsin G on glycocalicin provides two N-terminal fragments: a small N-terminal fragment of 42 kDa (His-1 to Leu-275) and a large N-terminal fragment of 45 kDa (His-1 to Val-296), corresponding to ~ 95 kDa C -terminal fragments. (Fig. 1).

[243.] Glycocalicin and glycocalicin fragments generated by cathepsin G digestion were electrophoresed on SDS-polyacrylamide gels and transferred to nitrocellulose for Western analysis. In immunoblots, Y1 bound to the larger N-terminal fragment (His-1 to Val-296) but not to the smaller N-terminal fragment (His-1 to Leu). · · · · · ··········

275), or to the C-terminal fragment. Similarly, the commercially available monoclonal antibody SZ2 (Immunotech 0719), known to recognize an epitope on GPIba between residues Tyr-276 and Glu-282, also only reacts with the larger N-terminal fragment (Figures 11 and 12).

[244.] In addition, the monoclonal antibody S.C.7071, which is known to recognize an epitope between His-1 and Leu275, bound to both N-terminal fragments. Y1 does not bind to the His-1 to Leu-275 fragment that is bound by S.C.7071. These results demonstrate that the epitope for Y1 is located between the first and second cathepsin G cleavage sites that are within the sequence Tyr-276 to Val-296, or more likely between amino acids ~ 276 to 282.

Effect of partial synthetic GPIb peptides on Y1 binding to purified glycocalicin and washed platelets (WP) [245.] An ELISA assay was performed to determine the effect of synthetic GPIb-derived peptides on Y1 binding to purified glycocalicin. In addition, FACS analysis was performed using washed plates. Competitive FACS binding analysis was used to determine the significance of sulfated tyrosines within the GPIb binding site for Y1. Yl-scFv at 1 μρ was pre-incubated with different 2.5 and 200 μΜ peptides. After this preincubation for 30 minutes at room temperature, the mixture was added to a tube containing ~ 10 ⁷ washed plates and binding of Y1 to the washed plates was determined using a polyclonal rabbit anti-scFv-PE antibody. The inhibitory effect of the peptides compared to control binding (Y1 alone) was determined by measuring the residual binding of Y1 to the washed plates. The peptides and results are described in Tables 1 and 3, respectively, and are similar to those found using identical peptides in the ELISA (Table 2). In both assays, the control level of Y1 binding was determined as follows. The MaxiSorp ™ polystyrene microtiter plate was coated with (a) purified glycocalicin or (b) washed plates. After extensive washing, Yl was added at 0.5 µg / well. The plate was then incubated with rabbit anti-scFv followed by the addition of anti rabbit-HRP (worse radish peroxidase, horseradish peroxidase)) and HRP substrate. The level of anti-rabbit-HRP binding was measured by the intensity of the color produced, and the level of anti-rabbitHRP binding correlated with the level of anti-Y1-scFv binding and the level of Y1 binding. It was measured by optical density at A ₄ o5- Each sample was tested in duplicate and the average was calculated.

[246.] The effect of synthetic GPIba peptides on Y1 binding to purified glycocalicin was determined by mixing different concentrations of peptides with a constant amount of Y1. After preincubation for 30 minutes at room temperature, the mixture was added to a Maxisorp polystyrene microtiter plate coated with purified glycocalicin as described for the Y1 binding assay in the absence of peptides. The inhibitory effect of the peptides was determined by measuring the residual binding of Y1 to glycocalicin using rabbit anti Y1-scFv and anti rabbit-HRP antibodies as described for determining Y1 binding in the absence of peptides. The experiment was performed with four peptides representing different subgroups from sequences 268-285 and a control peptide. Each peptide was tested at different concentrations of 200 μΜ, 25 μΜ, 2.5 μΜ and 0.5 μΜ.

[247.] Five peptides are shown in Table 1 below:

Table 1

Name of the peptide Characterization Sequence EGR negative control peptide REEGRQHFFLLEGRSSYS P-l residues 268-285 GPIba GDEGDTDLYDYYPEEDTE P-1-S residues 268-285 GPIba GDEGDTDLY * DY * Y * PEEDTE P-2-S residues 273-285 GPIba TDLY * DY * Y * PEEDTE P-3-S residues 268-280 GPIba GDEGDTDLY * DY * Y * P

Y * is identical to Y, i.e. tyrosine, but which is sulfated.

[248.] The results obtained in these assays are shown in Tables 2 and 3 below.

Table 2

Effect of Synthetic GPIba Peptides on Yl Binding to Glycocalicin, 0.25 µg / well Yl

Residual Yl bond (% of base) Concentration of peptides 200 μΜ 2 5 μΜ 2,5 μΜ 0,5 μΜ

EGR 85 89 100 ALIGN! 121 P-l 61 71 94 WHAT what P-1-S 0 25 62 89 P-2-S 15 Dec 52 78 P-3-S 21 67 80

[249.] These results clearly demonstrate that the inhibitory effect of the sulfated tyrosine-containing peptides is significantly higher than that observed for the unsulfated peptides. The effect is dose dependent and peptides containing longer (upstream) flanking sequences have a higher inhibitory effect than peptides with C 'extension (downstream) flanking sequences. The results clearly support the conclusion that sulfated tyrosine is required for binding of Y1 to GPIba, and that upstream and downstream of the sulfated region increases Y1 binding to GPIba.

Table 3

The effect of synthetic GPIba peptides on Y1 binding to washed plates as determined by comparative FACS analysis.

Residual Yl bond (% of base) (geometric diameter) Concentration of peptides 200 μΜ 2,5 μΜ EGR 119 96 P-l 87 106 P-1-S 5 41 P-2-S 7 61 P-3-S 26 82 Control-no peptide 114

· G g g [[[[[[[ 250.] These results further support the hypothesis that sulfated tyrosine residues within a specific region are important for the recognition of Y1 on GPIb. In summary, analysis of N-terminal peptide fragments of proteolysis by mocarhagine and cathepsin G indicates that the amino acid sequence Tyr276 to Glu-282 in GPIba is or contains a significant epitope for Y1 binding (Fig. 3). Further characterization suggests that in addition to residues 276-282 (sulfated anionic sequence) of glycocalicin, amino acids upstream of positions 283-285 are involved in the Y1 recognition site.

Biological activity of Y1-scFv, Y17-scFv and IgG Y1 on platelet function [251]. Localization experiments suggest that the Y1 binding site is located at the alpha-thrombin and vWF binding sites that are important for platelet aggregation. For this reason, binding of Y1-scFv, Y17-scFv, and Y1-IgG to washed platelets and platelet-rich plasma was studied to determine the effects of binding on platelet aggregation.

Effect of Y1-scFv and Y17-scFv on washed platelets (W.P.) [252.] Aggregation is determined in PRP due to the presence of thrombotic agents, while agglutination is determined in washed platelets. The effect of Y1 (scFv) on the agglutination of washed plates was tested at various concentrations of Y1. Plates were preincubated with Y1-scFv, Y17-scFv, Y16scFv, or control TM-1 scFv for 4 min at 37 ° C prior to exposure to ristocetin, which is an inducer of agglutination and platelet aggregation.

[253.] The results of these studies are shown in Table 4. and in FIG. 15. Preincubation of 25 μς / γηΐ Yl-scFv plates inhibited ristocetin-induced washed platelet agglutination. At a Yl concentration of 12.5 µg / ml, only partial platelet agglutination was observed. No platelet agglutination was observed at a Yl concentration of 4 μρ / ml. The results indicated that the inhibitory activity of Y1 on the agglutination of washed platelets is dose-dependent. Incubation of washed plates with scFv TMI, as a negative control, had no effect on ristocetin-induced platelet agglutination. Neither Y17 nor Y16, which are other scFv clones selected from the same phage expression library using the same multi-step procedure used for Y1 selection, significantly inhibited agglutination of washed plates.

Table 4

ScFv concentration % inhibition % agglutination TM-1 scFv of 25 gg / ml 10 90 Yl-scFv of 25 µg / ml 77 23 Y1-scFv of 12.5 pg / ml 33 67 Y1-scFv 4 gg / ml 8 92 Y17-scFv 25 μρ / γηΐ 15 Dec 85 Y16-scFv 38 μρ / πιΐ 14 86

★ 100% agglutination is calibrated based on ristocetin treatment

Effect of Y1-scFv and Y17-scFv on platelet-rich-plasma (PRP) aggregation [254.] The effect of Y1 (scFv) on platelet-rich-plasma (PRP) aggregation was tested at

different concentrations of Y1. PRP was pre-incubated with YlscFv, Y17-scFv, or control TM-1 scFv for 4 minutes at 37 ° C prior to exposure to ristocetin, which is an inducer of agglutination and aggregation. A reversible inhibitory effect was observed when scFv was added to PRP prior to ristocetin addition in a dose-dependent manner.

[255.] The results of these studies are shown in Table 5 and Figure 2. 16. Yl inhibited ~ 80% of platelet aggregation induced by ristocetin in platelet rich plasma at a final concentration of 50 μρ / πι, and this aggregation was noted in the first 4 minutes. No significant inhibition of platelet aggregation was observed at a Yl concentration of 25 μρ / πιΐ. Y17 did not inhibit platelet aggregation. Incubation of washed plates with 50 µg / ml TM1-scFv negative control had no effect on ristocetin-induced platelet aggregation (Table 5).

[256.] Comparison between washed platelets and PRP indicates that (1) scFv-inhib has an inhibitory effect on platelet aggregation and agglutination induced by ristocetin; (2) the effect is dose-dependent; (3) a higher inhibitory effect is observed in washed platelets compared to PRP; (4) a reversible inhibitory effect was detected in PRP; (5) neither the TMI nor the Y16-scFv antibody fragment showed effect; and (6) Y17 is a negative control in this assay.

Table 5

ScFv concentration % inhibition % aggregation TM1-scFv 50 pg / ml 0 100 ALIGN! Y1-scFv 50 pg / ml 80 20 May Y1-scFv 25 pg / ml 13 87

• ·

Y17-scFv 38 pg / ml

100 * 100% agglutination is calibrated based on ristocetin treatment

Effect of Y1-IgG on Agglutination of Washed Plates (Washed

Platelets, W.P.) [257.] Due to its natural structure, full-length IgG Y1 has two GPIba binding sites and one Fc receptor binding site. It is likely that when total IgG Y1 binds to two GPIba molecules, it activates platelets and induces platelet agglutination. In addition, since platelets have an Fc receptor, Y1 IgG can induce platelet agglutination by binding to GPIba and the Fc receptor, thereby producing platelet agglutination by binding each IgG Y1 to three platelets. Therefore, the effect of IgG Y1 on washed platelet aggregation was tested at various concentrations of Y1 IgG in the presence or absence of ristocetin. Induction of platelet aggregation with Y1 IgG was monitored for 4 minutes at 37 ° C, followed by the addition of ristocetin.

[258.] The results are shown in Table 6 and FIG. 17 without agonists. Yl-IgG alone at a final concentration of 50 gg / ml induced platelet agglutination of ~ 39% compared to normal platelet agglutination. The induction of platelet agglutination by Y1-IgG was tested for 4 minutes at 37 ° C, followed by the addition of ristocetin. No further effect on platelet agglutination was observed after the addition of ristocetin: normal platelet agglutination was observed. However, no agglutination was detected when the plates were incubated with 1 µg / ml Y1.

· · · · · · · · · · · · · · · · · · · · · · • · [259.] If a commercial monoclonal antibody against GPIba (CD42) (Pharmigen), which inhibits platelet agglutination, or a control human IgG-Lambda () was used in the same manner as described above. Sigma), no reduction in agglutination was found

Table 6

Q. O inhibition O, O agglutination Concentration Without WITH Without WITH antibodies IgG ristocetin ristocetin ristocetin ristocetin Y1-IgG 50 gg / ml 61 5 39 95 Y1-IgG 25 μg / ml 65 5 35 95 Y1-IgG 12.5 μg / ml 62 5 38 95 Y1-IgG 3.5 μg / ml 66 14 34 86 Y1-IgG 1 μg / ml 92 7 8 93 Mice anti-human CD42 IgG 20 μg / ml 99.5 100 ALIGN! 0.5 75 Checklist human IgG 20 μg / ml 99, 5 25 0.5 75 Control activation ristocetin 0 100 ALIGN!

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·· ·· · ·· ·· ·

Effect of Y1-IgG on platelet-rich plasma aggregation (Platelet-Rich-Plasma, PRP) [260.] The effect of Y1-IgG on platelet-rich plasma aggregation was tested at various concentrations of Y1-IgG in the presence or absence of ristocetin. The induction of platelet aggregation with Y1-IgG was tested for 4 minutes at 37 ° C, followed by the addition of ristocetin.

[261.] The results are shown in Table 7 and FIG. 18. No effect on platelet aggregation was observed after addition of ristocetin: normal platelet aggregation was observed. Yl-IgG at a final concentration of 50 µg / ml induced platelet aggregation in platelet-rich plasma prior to the addition of ristocetin. Yl-IgG at 25 μρ / πιΐ only partially induced platelet aggregation prior to the addition of ristocetin.

No inhibition of platelet aggregation was observed at Yl-IgG concentrations of 10 µg / ml, 4 µg / ml, or 1 µg / ml. Commercially available monoclonal antibodies against GPIba (Pharmigen), which inhibit platelet aggregation at concentrations of 20 μς / πιΐ, did not induce platelet aggregation. Control human IgG-Lambda (Sigma) at the same concentration as Y1-IgG also did not induce platelet aggregation.

Table 7

O. O inhibition O, 0 agglutination Concentration Without WITH Without WITH IgG ristocetin ristocetin ristocetin ristocetin Y1-IgG 64 0 36 100 ALIGN!

· · · «« «« «

50 μρ / μ Y1-IgG 2 5 μρ / ιηΐ 75 8 25 92 Y1-IgG 10 μρ / ιηΐ 93 10 7 90 Y1-IgG 4 μg / ml 98 5 2 95 Y1-IgG 1 μg / ml 95.5 0.5 0.5 99, 5 Human IgG anti-CD42 20 μg / ml 99.5 0.5 0.5 99, 5 Control activation ristocetin 0 100 ALIGN!

Identification of Plasma Soluble Y1 Ligands and Cell Lines [262.] Antibodies to GPIba (CD42b) recognize platelet lysate and glycocalicin but not KG-1 cell lysate (Y1 positive binding myeloid line) or Raji cell lysate (negative B cell line) for Y1 binding (concentrations at which KG-1 cells are positive for Y1 binding). Conversely, Y1 recognizes both glycocalicin, platelet lysate and KG-1 cells, but not extracts from RAJ1 cells. The negative control of scFv-181 did not recognize any of the relevant proteins (Fig. 20).

[262.] The uniqueness of Y1 cross-reactivity was further demonstrated in a comparative study between Y1 and SZ2 (Mab versus the sulfated GPIb region). Unlike SZ2, Y1 binds

not only to GPIb but also to plasma proteins and to myeloid-derived cell extracts as described below.

Ligands Y1 in human plasma.

[264.] Two proteins in both normal and leukemia patients showed immunoreactivity with Y1. The first one was designated as H β-ligand 1, which has a molecular weight under reducing conditions of ~ 50 kDa and> 300 kDa under non-reducing conditions and which disappears completely from the serum after coagulation; and (2) H β-ligand 2 having a molecular weight of both reducing and non-reducing conditions of 8080 kDa and which remains in serum after coagulation. After purification using Q-Sepharose reverse phase 2D gel electrophoresis (RP-HPLC) and peptide mapping, a ~ 50kDa ligand was identified as a normal gamma chain variant of human fibrinogen. The VRPEHPAETEYDSLYPEDDL sequence is present only in fibrinogen gamma 'but not in the abundant form of fibrinogen gamma, it is similar to the anionic region of GPIb containing sulfated tyrosine.

Most likely, this is a Y1 binding site. The ~ 80 kDa protein was identified as complement compound 4 (CC4) and lumican. As mentioned above, it contains sulfated tyrosine residues accompanied by a negatively charged amino acid stretch.

Binding of Y1 to Primary Leukemia Cells [265.] FACS analysis suggests that Y1 selectively binds to leukemia cells but not to normal blood cells in both normal blood samples and normal cells in leukemic blood samples. Summary • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · The results of this patient analysis are shown in the following tables.

Table 8: Results in Yl patients

Disease Number positive % positive multiple myeloma 16/16 100% AML 60/78 80% B-leukemia 29/43 67%

Table 9: B-Leukemia

Type Source Number positive % positive % negative Pre-B- -ALL BM 3/3 100 ALIGN! 0 B-ALL MB 3/9 33 67 B-CLL PB 17/23 74 26 B-lymphoma PB 5/8 62 38

BM = Bone Marrow

PB = Peripheral Blood

Y1 epitope characteristics on myeloid cells (KG-1) [266.] Approximately 25 billion KG-1 cells were prepared for purification of the Y1 epitope from KG-1 cell membranes. KG-1 membrane preparations were found to contain at least 2 subunits that bind to the Y1: -HOkDa subunit and the ~ 120 kDa subunit. Y1 also binds to a subunit of ~ 220 kD, which may be a dimer of the -110 kDa subunit. Purification of the Y1 epitope was performed by immunoprecipitation with Y1 and reverse phase HPLC (RP-HPLC). A 2: 1 pool (pool) was used for Western blotting with scFv Y1 and 40: 1 was used for silver staining (Fig. 21).

[267.] Y1 ligand was further characterized using the enzymatic action of proteases, glycanases and sulfatases; Western blotting with Y1, anti-CD42 antibodies, anti-CD162 antibodies and 181, immunoprecipitation using Y1 and antiCD162 antibodies; FACS analysis using Y1 and anti-CD162 antibodies; and sequencing.

[268.] The table below summarizes the biochemical experiments that were performed to characterize and localize the Y1 binding site on KG-1 cells.

Western blot analysis with Y1 on SDS-PAGE reducing gels.

Table 10

Substrate Action Conditions Reactivity with Yl Showing on Illustration RP-HPLC KG-1 0- 30'pr Reactivity Giant. 22nd membrane sialoglycoprotein Deň: 32 ° C only fractions endopeptidase with form 120 kDa RP-HPLC KG-1 0- 4 hours None Giant. 22nd membrane sialoglycoprotein at 37 ° C reactivity fractions endopeptidase RP-HPLC KG-1 aryl sulfatase 18 hrs. None Giant. 23 membrane at 22 ° C reactivity fractions

RP-HPLC KG-1 membrane fractions mocarhagin 7 'at Deň: 32 ° C None reactivity Giant. 33 Glycocalicin (GC) 0- sialoglycoprotein endopeptidase 30'pr Deň: 32 ° C Increased custody Giant. 22nd Heparin-BSA aryl sulfatase 18 hrs. at 22 ° C He binds himself to Yl like without action Giant. 23

[269.] In summary, after treatment with endopeptidases, the signal for Y1 is cleaved and cannot be detected (Fig. 22). More likely, the fragment containing the Y1 binding site is found at the N-terminus and is too small to be determined under the conditions used in the above experiments. Similarly, after mocarhagine treatment, the Y1 signal was cleaved and could not be detected (Fig. 33), demonstrating that the Y1 epitope is found at the N'-terminus of the ligand. In addition, the results obtained with aryl sulfatase, which removes sulfate compounds from proteins (in KG-1 cell extract) but not sugar compounds (in heparin), further support our hypothesis that sulfate is required for Y1 recognition (Fig. 23). . Surprisingly, the O-sialoglycoprotein endopeptidase increased the signal for Y1 in the GC (glycocalicin) cleavage product. It is believed that after such treatment on the Y1 binding site, this site is now located at the C-terminus and is better exposed for binding to Y1.

Correlation between Y1 and KPL1 against PSGL-1: analysis

Western Blotting [270.] Binding of scFv Y1 and commercially available anti-PSGL-1 monoclonal antibody (KPL1) to immunoprecipitated (IP) membrane proteins was determined.

KPL1, derived from KG-1 cells. Raji cell lysate was used as a negative control for Y1 and KPL1.

[271.] Membrane fractions from KG-1 cells were immunoprecipitated with KPL1. The immunoprecipitated (IP) fraction was further immunoprecipitated with either scFv Y1 or KPL1. Non-precipitated (eluted) fractions were analyzed by Western blotting using either scFv Y1 or KPL1.

[272.] Both immunoprecipitation schemes and results are shown in Figure 24. KPL1 does not recognize glycocalicin. However, both scFv Y1 and KPL1 recognize membrane proteins on KG-1 cells.

[273.] Lysates from cell lines and primary white cell blood cells were immunoprecipitated with anti-CD162 antibodies and centrifuged to obtain supernatant and eluate. Western blot analysis of proteins present in the eluate and supernatant was performed using scFv Y1 antibody and anti-CD162 antibody. Membrane preparations from KG-1 cells contain two subunits (-110 kDa and -120 kDa) to which the anti-CD162 antibody (PSGL1) binds. In contrast, membrane preparations from normal white cell blood cells have only a smaller pd-unit. Membrane preparations from AML patients have only a larger subunit.

ScFv Y1 binds to a different species found in the supernatant after immunoprecipitation and to which anti-CD 162 antibodies do not bind (Fig. 25).

• · · · · · · · · · · ·

FACS analysis [274.] Binding of Y1 (both scFv and IgG forms) to KG-1 cells in the presence of anti-PSGL-1 (anti-CD162) (KPL1) antibodies was determined in a competitive binding assay using FACS analysis. Various commercially available anti-PSGL-1 antibodies, KPL1 (an antibody that identifies sulfated N-terminal domain PSGL1), PL1 (an antibody that identifies the non-sulfated N-terminal domain of PSGL-1) and PL2 (an antibody that identifies the non-sulfated internal domain of the PSGL-1 receptor). Only KPL1 completely inhibits Y1 binding to KG-1 cells, while PL1 inhibits partially. No inhibition of binding was found in the presence of the PL2 antibody (Fig. 26).

Paradise cells did not bind to KPL1 antibodies. Similarly, complete IgG Y1 at different concentrations inhibits KPL1 antibody binding to KG-1 cells in a dose-dependent manner (FIG.

27). Similarly, KPL1 inhibits full-length binding of IgG Y1 to KG-1 cells in a dose-dependent manner (FIG.

28).

Correlation between Y1 and KPL1 in Binding to Primary Leukemia Cells [275.] The analysis of binding of scFv Y1 antibodies and anti-CD162 antibodies to diseased cells further illustrates that scFv has binding characteristics different from those of anti-CD162 antibodies. In particular, analysis of FACS binding of Y1 and anti-CD162 antibody to pre-B-ALL, HCL, AML, B-ALL, B-CLL, unclassified leukemia, B-PLL, multiple myeloma cells from human patients showed that the two antibodies have different binding profiles (Table 11). Y1 binds to leukemia cells in 10 out of 12 samples. In contrast, only 5 out of 12 samples bound anti-CD162 antibody. Therefore, it can be concluded that in leukemic cells scFv Y1 binds to a ligand other than the ligand recognized by the anti-CD162 antibody.

Table 11: Leukemia samples - anti-CD162 vs Y1 antibody analysis

Reactions with leukemia cells Patient # Disease ScFV Yl Anti-CD162 42291 Pre-B-ALL + - 42299 HCL - - 42311 AML + + 42321 B-ALL - - 42323 B-CLL + - 42325 unclassified + - 42332 B-CLL + - 42352 B-PLL + + / - 42330 AML + 42334 MM + - 42366 AML + + 42370 AML / ALL + + / -

[276.] In summary, the Y1 binding domains containing sulphated tyrosine in GPIba, fibronectin ('and PSGL-1 are DEGDTDLYDYYPEEDTEGD (amino acids 269-287), EHPAETEYDSLYPED (amino acids 411-427) and QATEYELDYDFLPETE (amino acids 411-427)) Interestingly, blood samples that are positive with both scFv Y1 and anti-CD162 antibody were obtained from patients with AML, while the blood binding sites with higher affinity for Y1 are more likely to be expressed on primary leukemic cells. B cells were negative with anti-CD162.

»♦ · · ·

Analysis of Binding of Sulphated Peptides to Y1 [277.] An ELISA competitive binding assay was used to determine the significance of the presence and position of sulphated tyrosines for peptide binding to Y1.

[278.] Glycocalicin was immobilized on a Maxisorb plate.

ScFv Y1 was pre-incubated with the test peptide for 10 minutes at three different concentrations (1, 10, and 100 μΜ) to perform dose-response observation (Table 12). After preincubation, the mixture (Y1 + peptide) was added to the plate and binding of scFv Y1 was determined using a polyclonal rabbit anti-V _L antibody that recognizes the V _L chain of scFv Y1, with a decrease in scFv Y1 binding to glycocalicin compared to control binding. In mixtures in which the scFv Y1 peptide did not bind, no change in binding of scFv Y1 to glycocalicin was observed compared to that of the control.

[279.] The experiment was performed twice and the results are shown in an ELISA plot (Figure 29). Peptides derived from fibrinogen showed no inhibition of Y1 binding, regardless of sulphation. Unsulfated peptides from PSGL-1 did not inhibit Y1 binding to glycocalicin. All sulfated peptides derived from PSGL1 inhibited Y1 binding to glycocalicin. P-YYY * and PYY * Y * peptides were the best inhibitors, followed by P-Y * YY *, then P-Y Y * Y, then P-Y * Y * Y and PY * YY. Unsulfated glycocalicin-derived peptides did not inhibit Y1 binding to glycocalicin, but a glycocalicin-derived peptide having the same three-sulfate sulfated sequence (G-Y * Y * Y *) did not inhibit binding, with an activity similar to that of P-Y Y * Y.

· É 280 280 280 280 280 280 280 280 280 280 280 280 280 280 280 280 280 280 [280 Therefore, it is apparent that not every sulfated peptide binds to scFv Y1 to the same extent. These results also significantly demonstrate that only one sulfated tyrosine is necessary for binding, as can be seen with PY * YY and P-YY Y * peptides. In addition, it can be observed that the amino acid context of the sulfated tyrosines has an effect on Y1 binding. For example, P-Y * YY (containing one sulfated tyrosine in the EY * E sequence) only inhibits binding activity at a concentration of 100 μΜ. In contrast, P-YYY * (containing one sulfated tyrosine in the DY * D sequence) effectively inhibits binding at a concentration of 1 μΜ.

Table 12

Sulphated peptides

Name Source peptide Sequence #aa MW (molecule- lová mass) Sulfation F-YY fibrinogen (string Ί ') VRPEHPAETEYESLYPEDDL 20 May 2389 F- y * y * fibrinogen (string γ ') VRPEHPAETEY * ESLY * PEDDL 20 May 2549 sulfato- vaný p-yyy PSGL-1 N-end QATEYEYLDYDFLPETE 17 2126 - P- y * yy PSGL-1 N-end QATEY * EYLDYDFLPETE 17 2206 sulfato- vaný P- Y * Y * Y PSGL-1 N-end QATEY * EY * LDYDFLPETE 17 2286 sulfato- vaný

· · · · · · · · · · · · · · · · · · · ·

P- * * Y YY PSGL-1 N-end QATEY * EYLDY * DFLPETE 17 2286 sulfato- vaný P- PSGL-1 QATEYEY * LDYDFLPETE 17 2286 sulfato- YY * Y N-end sincere P- PSGL-1 QATEYEY * LDY * DFLPETE 17 2286 sulfato- YY * Y * N-end sincere P- PSGL-1 QATEYEYLDY * DFLPETE 17 2286 sulfato- YYY * N-end sincere G-YYY GPIba GDEGDTDLYDYYPEEDTE 18 2126 - G- GPIba GDEGDTDLY * DY * Y * PEEDTE 18 2366 sulfato- Y * Y * Y sincere

Y * = sulfated tyrosine

Hypothesis / Conclusions [281.] (1) Y1 is similar to L-selectin, which recognizes both sulfated protein and sugar compounds, and is distinct from P-selectin, which recognizes only sulfated proteins. For this reason, it can compete for binding with both proteins.

[282.] (2) Variation in sulfation during cell differentiation and growth may affect Y1 binding. For this reason, Y1 can compete with both P- and L-selectins for binding to their sulfated ligands.

In vivo models for determining the efficacy of leukemia-specific antibodies [283.] Two human models of leukemia in immunodeficient mice have been developed as well as assay systems.

• ·

The human cell lines used were MOLT4 cells obtained from a T cell leukemia patient and KG1 cells obtained from an AML patient. Antibodies specific for the corresponding human antigens on each of the cell lines were used to identify and quantify malignant cellular engraftment.

T-ALL Model (MOLT4) [285.] The in vivo mouse model for T-ALL uses a mouse (Jackson Laboratories) to indicate MOLT4 cells obtained from a patient with T cell leukemia.

[286.] In one experiment, mice were pre-treated with 100 mg / kg Cytoxene (CTX, injectable cyclophosphamide, Mead Johnson) and injected 2x10 ⁷ MOLT-4 cells / mouse 5 days after cyclophosphamide treatment. From day 5 after injection of MOLT-4 cells, mice were injected sequentially three times a week with iv anticancer agent or PBS (as a negative control). At week 35, animals were bled and animals were sacrificed, and their liver was excised and weighed. The livers of untreated animals, i.e. animals treated with PBS and MOLT-4 cells, showed very massive tumor growth, and their size was increased 23-fold compared to control PBS uninfected animals. There were five groups in this experiment:

Table 13

Group not injected with MOLT-4 cells treated with PBS

2. Control group injected with PBS-treated MOLT-4 cells.

101 ·

3. Group injected with MOLT-4 cells, treated with Y1 scFv (CONY 1), 75 gg / mouse

4. Group injected with MOLT-4 cells, treated with CONY 1 scFv-Doxorubicin, 75 µg / mouse

5. Doxorubicin-treated MOLT-4 cells injected with 0.1 mg / kg.

Number of mice Inoculation Treatment 5 PBS only - 9 MOLT-4 - 9 MOLT-4 CONY-Dox (2.5 mg / kg) 9 MOLT-4 CONY-Dox (2.5 mg / kg) 8 MOLT-4 Single Dox (0.1 mg / kg)

[287.] Mice started to die 32 days after cell inoculation and surviving mice were sacrificed at this time. Bone marrow cells were analyzed by flow cytometry using anti-human CD44-FITC and YlBiotin / SAV-PE antibodies. Blood samples from several animals were monitored for platelet and white blood cell counts. The liver was weighed and examined for tumor. Other organs were also examined for tumor.

[288.] Results are shown (Figures 30, 31 and 32). Massive tumor growth (white nodules) was observed in the liver of all mice injected with MOLT-4 cells.

[289.] The percentage of MOLT-4 cells found in bone marrow was very low (Fig. 31).

[290.] In summary, these results demonstrate the suitability of using the MOLT-4 model as a model for hepatic leukemia cell metastases.

102 * [291.] In parallel, parts of the liver were collected for histological examination and cells were harvested for FACS analysis. Survival of a group of treated animals was compared to a control group of untreated mice.

[292.] At day 35, the liver was weighed, weights are shown (Fig. 30). As shown, liver size in tumor-infected mice, as a PBS-treated negative control, is nearly tripled compared to PBS control and mice not injected with MOLT-4 cells. The liver weights of mice treated with a low dose of Doxorubicin were similar to those of mice infected with tumor and treated with PBS. On the other hand, treatment with CONY1-scFv and CONY1-scFv-Doxorubicin conjugate significantly inhibited tumor growth in the liver (much lower liver weight).

[293.] SCID / MOLT-4

1.

2.

3.

.

5.

6.

In the second experiment, an identical protocol was used on 6 groups of animals:

Group not injected with MOLT-4 cells, PBS control

MOLT-4 injected group treated with PBS MOLT-4 cell injected group with CONY 1 scFv treated, 75 μg / mouse

MOLT-4 cells treated with non-specific scFv antibody obtained from the Nissim I library, 75 μρ / πτ / έ MOLT-4 treated group, treated with Y1-IgG, 5 µg / mouse

Group injected with MOLT-4 cells treated with non-specific human IgG, 5 µg / mouse.

103

[294.] Results are presented (Fig. 34), demonstrating that treatment with either CONY1-scFv or Y1 IgG inhibited tumor growth (based on liver weights), while little or no effect was observed in animals treated with non-specific antibody molecules. .

[295.] Survival was determined in mice from three groups treated continuously and the results are shown (Fig. 35). As shown, survival of only CONY1-scFv-treated mice was prolonged.

AML KG-1 Model [296.] The in vivo mouse model for human AML uses a SCID / NOD mouse (Jackson Laboratories) inoculated with KG-1 cells obtained from the human AML cell line.

[297.] In the first experiment, NOD / SCID mice were pre-treated with 100 mg / kg CYTOXAN ®. Four days after CYTOXAN® injection, KG-1 cells were injected intravenously into the tail vein into six groups of mice (Table 14, Groups 2 and 5-9). One group of mice (Table 14, Group 1) was injected with PBS alone (control). Starting on day 14 after KG-1 cell injection, mice were treated with: CONY 1, Doxorubicin, CONY 1-Doxorubicin conjugate, or MYLOTARG®. MYLOTARG® is a monoclonal antibody (anti-CD33) chemically conjugated to calcheamicin (recently approved by the FDA for the treatment of AML in patients over 60 years of age or after the first AML relapse). Mice were treated one or three times per week for three weeks. One group (group 2) of mice injected with KG-1 cells was left untreated (Table 14). Two additional groups of mice (groups 3 and 4) were injected with KG-1 cells pre-incubated with CONY 1 or

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181-scFv (negative, non-specific control antibody) in serum-free RPIM containing 1% BSA (bovine serum albumin) at 4 ° C. Antibodies were used at concentrations of 0.25 mg scFv / 10 ⁸ cells (75 µg / mouse). Prior to mouse injection, pre-incubated KG-1 cells were washed and resuspended in RPMI. KG-1 cells in RPMI were inoculated to mice at a concentration of 75 µg scFv / 0.2 ml RPMI per mouse. Group of mice 3 was inoculated with KG-1 + CONY1, and group of mice 4 was inoculated with KG-1 + 181-scFv. (Table 14). This treatment (groups 3 and 4) was performed one day after inoculation of groups 1-2 and 5-9, i.e. 5 days after CYTOXAN® injection.

Table 14

# mice Group # Inoculation Treatment 9 1 PBS - 11 2 KG-1 - 9 3 KG-1 + Yl - 9 4 KG-1 + 181 - 8 5 KG-1 75 μρ / mouse (2.5 mg / kg) CONY 1 3 times a week 9 6 KG-1 0.1 mg / kg Doxorubicin, 3 times weekly 10 7 KG-1 5 mg / kg Doxorubicin, once a week 11 8 KG-1 75 μg / mouse (2.5 mg / kg) CONY 1- Doxorubicin, 3 times a week 9 9 KG-1 0.2 mg / kg MYLOTARG®, once a week

[298.] Mice were sacrificed from day 60 to day 65 after cell injection. Bone marrow and blood samples were analyzed by flow cytometry using mouse anti-human antibody

105

CD34-FITC (IQP 144F) (or anti-CD44-FITC (MCA89F,

Serotec) and Y1-Biotin / SAV-PE. Mouse IgG1-FITC (IQP191-F) was used as an isotype control and mouse IgG2a-FITC (MCA929F, Serotec) was used as a negative control. Flow cytometry was performed using a FACS Calibur system and CellQwest software, Becton Dickinson.

[299.] Results are shown (Figures 36 and 37). Nine out of ten mice injected with KG-1 cells and treated with Doxorubicin only at 5 mg / kg (Group 7) died within three weeks after initiation of treatment.

[300.] The bone marrow of mice injected with KG-1 cells and untreated (Group 2) contained on average 30% of KG-1 cells in the bone marrow cell population. All mice in this group developed leukemia.

[301.] In summary, almost all mice injected with KG-1 cells developed leukemia at an average of 30% of KG-1 cells in bone marrow (as determined by FACS analysis). In general, engraftment of KG-1 cells was localized to the bone marrow. Less than 10% of KG-1 cells were found in the blood. In one case, a tumor in the peritoneum wall was observed.

[302.] Mice injected with KG-1 cells and treated with Doxorubicin alone at a dose of 0.1 mg / kg (Group 6) had a statistically significantly (p <0.05) lower percentage cell content compared to Group 2 KG-1 in bone marrow.

[303.] Mice injected with KG-1 cells and treated with CONY1-Doxorubicin conjugate (Group 5) had a lower percentage of KG-1 cells compared to Group 2.

106 in bone marrow (16.3% versus 30.4%, respectively). However, this difference was not found to be statistically significant. During the experiment CONY1-Doxorubicin was found to be contaminated with lipopolysaccharides (LPS). Therefore, optimal CONY1Doxorubicin concentrations could not be used and treatment was discontinued before the end of the experiment.

[304.] Mice injected in vitro with KG-1 cells preincubated with CONY1 or 181-scFv (Groups 3 and 4, respectively) had a significantly lower percentage of KG-1 cells in their bone marrow.

[305.] Bone marrow of both mice injected with PBS only (negative control) and mice injected with KG-1 cells and treated with MYLOTARG® (Group 9) was free of KG-1 cells. These results demonstrate that this in vivo model is suitable as a model for AML.

[306.] The overall percentage of KG-1 cells found in the bloodstream of the various groups was overall very low with high variability within the groups. It should be noted that one mouse treated with MYLOTARG® showed a high percentage of KG-1 cells in the blood but not in the bone marrow.

[307.] Identification of human leukemic cells (KG-1 cells originating) in the bone marrow and bloodstream of the mouse was performed by FACS analysis using commercially available anti-human CD34 or CD44 antibodies, in parallel with the Y1 scFv antibodies.

[308.] On the first day of the analysis, there was a significant difference in mice injected with KG-1 cells only (Group 2). Which had a high percentage of KG-1 cells in the bone marrow compared to the group of mice treated with CONY1-Doxorubicin (Group 8). On day 3 of the analysis, the situation was reversed: mice from Group 8 had a higher percentage of KG-1 cells in their bone marrow compared to group of mice 2. This situation could be due to the following: A) poor physical condition selection on day 1 experiment, B) proliferation of KG-1 cells in Group 8 mice during the days after cessation of treatment, and C) the number of mice in each group was too small to obtain statistically significant results.

Pharmacokinetics of CONY1 in Mice [309.] CONY1 scFv was labeled with ¹²⁵ I-Bolton Hunter reagent (to lysine). The labeling reaction was carried out at 4 °

C in borate buffer (pH 9.2) with ¹²⁵ I-Bolton Hunter reagent, then ¹²⁵ I-CONY1 was purified on a PD-10 chromatography column. The radioactive protein was then admixed with unlabeled CONYl to obtain a CONYl 75 μρ / πιΐ solution containing 2.5 x 10 ⁶ CPM / ml in saline.

[310.] Male Balb-C mice were pretreated with 0.5 ml / mouse 0.9% NaI intraperitoneal injection. After two hours, mice were injected intravenously with 0.2 ml of CONY-1 labeled solution such that a final dose of ¹²⁵ ICONY1 was 15 μρ (5 x 10 ⁵ CPM) per mouse.

[311.] At various times after injection, blood was collected into EDTA, mice were sacrificed, and tissues were excised. Samples and tissues were collected at 5, 15, and 30 minutes and at 1, 2, 4, 8, 24 hours after injection. 2-4 mice were used per time interval. Plasma was separated and either in it

The radioactivity of gamma irradiation was read in 108 or subjected to trichloroacetic acid (TCA) precipitation. After centrifugation, TCA precipitates were subjected to gamma radioactivity determination. Liver, lung, kidney, spleen and bone marrow samples were weighed for gamma radioactivity. TCA precipitated plasma radioactivity was plotted against time and a two-dimensional kinetic model was employed. Total organ / tissue radioactivity and specific radioactivity were determined. The results are shown (Fig. 39, and 41).

[312.] A comparison of blood and plasma radioactivity values suggests that virtually all CONY-1 was located in plasma and did not adhere to erythrocytes. Plasma radioactivity values were similar to TCA values of precipitates, suggesting that they were associated with undegraded protein. Giant. 39 shows plasma CONY-1 levels at various timepoints after administration. Values were statistically evaluated in a two-dimensional model and the blood clearance half-lives obtained were 35 and 190 minutes, respectively.

[313.] The distribution of radioactivity in different tissues at certain time points after administration are reported as specific and total radioactivity, respectively (Figs. 40 and 41). In most tissues, there was no specific accumulation of radioactivity, as is apparent from a comparison of their specific activity to blood activity. Slightly lower activities were observed in the kidneys at 4 o'clock and in bone marrow at 4 and 8 o'clock; this is most likely associated with the excretion of degradation products.

109

[314.] The results indicate that in mice CONY-1 is secreted with a half-life of 35 minutes. The secondary excretion rate is less important and may result from the presence of some polymeric forms of injected material. There is no major specific CONY-1 intake in the tissues except for a slight increase in bone marrow.

Production of Antibodies and Fragments Antibodies, fragments thereof, constructs thereof, peptides, polypeptides, proteins, and fragments and constructs thereof can be produced in either prokaryotic or eukaryotic expression systems. Methods for producing antibodies and fragments thereof in prokaryotic and eukaryotic systems are well known in the art.

[316.] The eukaryotic cell system as defined and discussed herein relates to an expression system for producing peptides or polypeptides by genetic engineering methods in which the host cell is a eukaryote. The eukaryotic expression system may be a mammalian system and the peptide or polypeptide produced in the mammalian expression system is substantially free of mammalian contaminants after purification. Other examples of suitable eukaryotic expression systems include yeast expression systems.

[317.] A preferred prokaryotic system for producing the peptide or polypeptides of the invention uses E. coli as the host for the expression vector. After purification, the peptide or polypeptide produced in E. coli is substantially free of contaminating proteins from E. coli. The use of prokaryotic expression systems may result in •

110

to add a methionine residue to the N-terminus of some or all of the sequences provided in accordance with the present invention. Removal of the N-terminal methionine residue after production of the peptide or polypeptide to allow expression of the complete peptide or polypeptide may be accomplished by methods known in the art, one example of which is to use an aminopeptidase from Aeromonas under suitable conditions (US Patent No. 5 763 215).

Types of antibody fragments and constructs.

[318.] The present invention provides a peptide or polypeptide comprising an antibody or antigen-binding fragment thereof, a construct thereof, or a construct from a fragment. Antibodies of the present invention include IgG, IgA, IgD, IgE, or IgM antibodies. The IgG class presents several subclasses of IgG including IgG _{LZ 2,} IgG 3 and IgG [319.] Antibody fragments according to the present invention include Fv, scFv, dsFv, Fab, Fab _2, and Fd. Smaller antibody fragments, such as Fv fragments, are also included within the term fragments as long as they retain the binding characteristics of the parent antibody or larger fragment. Examples of such fragments may be (1) minibodies that contain a heavy chain fragment from Fv only, (2) microbodies that contain a small fractionation unit from the antibody heavy chain variable region (PCT Application No. PCT / IL99 / 00581), (3) similar antibodies comprising a light chain fragment and (4) similar antibodies comprising a functional unit from a light chain variable region. Constructs include, for example, multimers such as diabodies, triabodies and tetrabodies. Expression • ·

111 "antibody, binding fragment or complex comprising an antibody or binding fragment thereof" is meant to include all of these molecules, as well as derivatives and homologs thereof, mimetics and variants thereof, unless otherwise specified or otherwise defined based on context and / or knowledge in the art.

[320.] Once an antibody, fragment, or construct having the desired binding capabilities has been selected and / or engineered, it is easy with the knowledge of one skilled in the art and using the disclosure provided herein to construct constructs and fragments that retain the characteristics of the parent antibody. For example, whole antibody molecules, Fv fragments, Fab fragments, Fab2 fragments, dimers, trimers, and other constructs can be prepared that retain the desired characteristics of the original selected and engineered antibody, fragment or construct thereof.

[321.] If amino acid substitution is desired while maintaining antibody or fragment characteristics, it is within the skill of the art to make conservative amino acid substitutions. Modifications such as conjugation of antibodies or fragments thereof to pharmaceutical or diagnostic agents can also be made without altering their binding characteristics. Other modifications, such as those that result in the production of more stable antibodies or fragments, may be made without altering its specificity. For example, peptoid modifications, semipeptoid modifications, cyclic peptide modifications, N-terminal modifications, C-terminal modifications, peptide bond modifications, modifications can be performed

112 spine and residue modification. It is easy with the ability of one skilled in the art and using the disclosure provided herein to test modified antibodies or fragments thereof and to determine whether their binding characteristics have been altered.

[322.] Similarly, it is within the ability and within the skill of one of ordinary skill in the art to utilize the disclosure provided herein to vary the binding characteristics of an antibody, fragment, or construct thereof to obtain a molecule with more desirable characteristics. For example, once an antibody having the desired properties is identified, random or directed mutagenesis can be used to generate antibody variants and these variants can be screened for the desired characteristics.

[323.] The antibodies and fragments of the present invention may have an identifier tag that can be inserted into or attached to them for preparation and identification and for diagnosis.

The identifier sequence may later be removed from the molecule. Examples of suitable identifier sequences include: AU1, AU5, BTag, c-myc, FLAG, Glu-Glu, HA, His6, HSV, HTTPHH, IRS, KT3, Protein C, S-TAG®, T7, V5, VSV-G ( Jarvik and Telmer, Ann Rev. Gen., 32, 601-618 (1998).

A preferred identifier sequence is c-myc or KAK.

Multimeric Antibodies [324] The present invention provides a peptide or polypeptide of Y1 and Y17 comprising a scFv molecule. The scFv used herein is defined as a molecule that is created.

113 from a human antibody heavy chain variable region and a human antibody light chain variable region, which may be the same or different, and in which the heavy chain variable region is joined, bound, fused or covalently linked, or associated with the light chain variable region.

[325.] The Y1 and X17 construct of a scFv can be a multimer (eg, a dimer, a trimer, a tetramer, and the like) of scFv molecules that include one or more hypervariable domains of an Y1 or Y17 antibody. All scFv-derived constructs and fragments retain enhanced binding characteristics such that they selectively and / or specifically bind to a target cell in favor of other cells. Binding selectivity and / or specificity is primarily determined by hypervariable regions.

[326.] Hypervariable loops within the variable domains of the light and heavy chains are called Complementary Determining Regions (CDRs). In each of the heavy and light chains, there are CDR1, CDR2, and CDR3 regions.

The most variable of these regions is the heavy chain CDR3 region. The CDR3 region is considered to be the most exposed region of the Ig molecule and, as shown herein, is the site primarily responsible for the observed selective and / or specific binding characteristics.

[327.] The peptide Y1 and Y17 of the present invention can be frameworked to be folded into multivalent Fv forms. The multimeric forms Y1 and Y17 were designed to improve binding affinity and specificity and to increase their half-life in blood.

114

[328.] Multivalent forms of scFvs have also been produced by other experts. One approach connects two scFvs using linkers. Another approach for coupling involves the use of disulfide bonds between two scFvs. The simplest approach for preparing dimeric or trimeric Fvs is disclosed by Hollinger et al., PNAS, 90, 6444-6448 (1993) and A. Kortt, et al., Protein Eng., 10, 423-433 (1977). One method was designed such that scFv dimers are prepared by adding a sequence of FOS and JUN protein regions to form a leucine zipper between them at the C-terminus of the scFv. Kostelny S.A. et al., J. Immunol. 1992 Mar 1; 148 (5): 1547-53. De Kruif, J. et al., J. Biol. Chem. 1996 Mar 29; 271 (13): 7630-4. Another method was designed to prepare tetramers by adding a sequence encoding streptavidin to the C-terminus of the scFv. Streptavidin is composed of four subunits, and when scFv-streptavidin is folded, 4 subunits conform to form a tetramer. Kipriyanov SM et al., Hum. Antibodies Hybridomas, 1995; 6 (3): 93-101. In another method for preparing dimers, trimers, and tetramers, free cysteine is introduced into the protein of interest. A peptide-based cross linker with a variable number of (2-4) maleimide groups was used to cross-link the free cysteines of the desired protein. Cochran J.R. et al., Immunity, 2000 Mar; 12 (3) 24150.

[329.] In this system, a phage library (as described above) has been designed to express scFvs that can fold into a monovalent form of the Fv region of an antibody. In addition, as discussed above, the construct is suitable for bacterial expression. The genetically engineered scFv fragments contain the heavy and light chain variable regions attached to the continuously coded

115

flexible peptide spacer of 15 amino acids. The preferred spacer is (Gly4Ser) 3. The overall length of this spacer with amino acid components provides a bulky spacer that allows the V _H and V _L regions to fold into a functional Fv domain that provides effective binding to the target.

[330.] The present invention is directed to Y1 and X17 multimers prepared by methods known in the art. A preferred method for preparing multimers, especially dimers, involves the use of cysteine residues to form disulfide bridges between monomers. In this embodiment, dimers are formed by adding cysteine to the carboxyl terminus of the scFv (referred to as Y1-cys scFv or Y1 dimer) to facilitate dimer formation. After the DNA construct (see Example 2D and 6D) was prepared and used for transfection, Y1 dimers were expressed in a production vector and refolded in vitro. Protein was analyzed by SDS-PAGE, HPLC and FACS. None of these first attempts suggested dimer formation.

For this reason, the procedure was repeated and this time two-liter fermentation batches of antibodies were run. After Y1-cys expression in E.coli strain BL21, protein refolding was performed in arginine. After refolding, the protein was dialyzed and purified by Q-Sepharose and gel filtration (Sephadex 75). Two peaks were detected by SDS-PAGE (unreduced) and gel filtration. The peak corresponding products were individually collected and analyzed by FACS. Binding of monomer and dimer to Jurkat cells was controlled by FACS. Dimer binding required only 1/100 of the amount of monomeric antibody for the same level of staining, indicating that the dimer had higher avidity. The conditions for refolding the dimer were determined and a material containing> 90% dimers (mg amount) was obtained after subsequent dialysis, chromatography and gel filtration steps.

116

The purified dimer was characterized by gel filtration and SDS-PAGE analysis under oxidation conditions. The binding capacity of the dimers was confirmed by a radoreceptor assay, ELISA and FACS analysis.

[331.] In vitro binding competition experiments on KG-1 cells were performed to compare the binding of scFv monomer (also referred to as CONY1) and dimer Y1. In addition, these experiments also compared full-length Y1 IgG binding with scFv Y1 monomers. To conduct the study, Y1 IgG was labeled with biotin. The study revealed competition between Yl IgG and YlBiotin IgG. Competition was not found between irrelevant human IgG and labeled Y1 IgG. Partial competition was observed between Yl scFv (5 μρ and 10 μρ) and Yl IgG-Biotin (50 ng). Studies also showed that 1 ng of IgGY1-FITC bound KG-1 cells (serum free) to the same extent as 1 µg of scFv-FITC, but in the presence of serum most of the Y1 IgG binding was blocked. Studies have also shown that the binding of dimer Y1 is at least 20-fold higher than that of the scFv monomer as determined by radioreceptor assay, ELISA, or FACS analysis.

[332.] In another embodiment of the invention, in addition to cysteine, lysine-alanine-lysine (referred to herein as Y1-cys-KAK scFv) was added to the carboxyl terminus. The amino acid sequence of this scFv construct is shown below and is also SEQ ID NO: 212.

MEVQLVESGG GVVRPGGSLR LSCAASGFTF DDYGMSWVRQ

APGKGLEWVS GINWNGGSTG 60

61YADSVKGRFT ISRDNAKNSL YLQMNSLRAE DTAVYYCARM

RAPVIWGQGT LVTVSRGGGG 120

117 to * to to · to to to to to to to · to · to · to · to · to · to · to · to · to · to · to · to · to · to · to · to · to · to · · ··· ·· ·

121 SGGGGSGGGG SSELTQDPAV SVALGQTVRI TCQGDSLRSY

YASWYQQKPG QAPVLVIYGK 180

181 NNRPSGIPDR FSGSSSGNTA SLTITGAQAE DEADYYCNSR

DSSGNHVVFG GGTKLTVLGG 240

241 GGCKAK [333.] Y1-cys-KAK was prepared in bacteria using the vector λ-pL. Expression in the λ-pL vector was induced by raising the temperature to 42 ° C. Inclusion antibodies were obtained from induced cultures and were semi-purified with aqueous solutions to remove undesirable soluble proteins. The inclusion antibodies were solubilized in guanidine, reduced by DTT, and left in vitro in a solution based on arginine / oxidized glutathione for refolding. After refolding, the protein was dialyzed and concentrated by tangential flow filtration against a buffer containing urea / phosphate buffer. The protein was re-purified and concentrated by ion chromatography on an SP-column.

HPLC was performed to obtain the dimer profile after refolding and purification from the Superdex 75 gel filtration column. In Figure 42, the first peak for the Yl-cys-KAK (dimer) is left (~ 10.8 minutes) and the following peak is for the monomer (~ 12). minutes). According to the protein size markers traveling on the same column, the dimer has approximately 52 kDa and the monomer 26 kDa. The balance between dimer and monomer can be altered by adjusting the refolding conditions (oxidized agent concentration and protein concentration in the refolding buffer). Dimer and monomer were separated by Superdex 75 column chromatography.

* «C r · r r r r r r r

118 ......... ·· * [334.] An ELISA assay was performed to detect the binding differences between monomer (CONY1 scFv also referred to as Yl-ΚΑΚ) and er-cys KAK dimer (cysteine dimer) to GPIb antigen (glycocalicin), derived from platelets. Polyclonal anti-single chain antibody and / or novel polyclonal anti-V _L (derived from rabbits) and anti-rabbit HRP antibodies were used to detect binding to GPIb. The dimer was approximately 100 times more active than the monomer. For example, 12.8 μς / πιΐ monomer was used to achieve 0.8 OD units compared to just 0.1 μρ / ιηΐ dimer. See Figure 19.

[335.] In addition, FACS binding analysis to KG-1 cells showed that the dimer is more sensitive than the monomer when a two- or three-step binding assay was performed. Dimers directly labeled with FITC showed a slight superiority (10x less material used) over the monomer. A radioreceptor assay on KG-1 cells in which dimers were used as competitors showed that the dimer was 30 times more potent than the monomer.

[336.] Varying spacer lengths is another preferred method of forming dimers, trimers, and tetramers (often referred to in the art as diabodies, triabodies, and tetrabodies, respectively). Dimers are formed under conditions where the spacer linking the two variable scFv chains is generally truncated. This truncated spacer prevents the variable chains of the same molecule from folding into the functional Fv domain. Instead, the domains are forced to pair with the complementary domains of other molecules to form two binding domains. In a preferred method, a spacer of only 5 amino acids (Gly ₄ Ser) was used to construct the diabody. This dimer can be formed from two identical scFvs, or from two different populations of scFvs, and a different scFv population.

It may retain selective and / or specific increased binding activity of the parent scFv (s), and / or exhibit increased binding strength or affinity.

[337.] Similarly, triabodies are generated under conditions where the spacer linking the two scFv variable chains is generally truncated to less than 5 amino acid residues, preventing the variable chains of the same molecule from folding into the functional Fv domain. Instead, three separate scFv molecules associate to form a trimer. In a preferred embodiment, the triabodies are obtained by completely removing the flexible spacer. The triabody may be formed from three identical scFvs or two or three different scFv populations and retains selective and / or specific increased scFv (s) binding binding activity and / or exhibits increased binding potency or affinity.

Similarly, tetrabodies are generated under conditions where the spacer linkage of two scFv variable chains is generally truncated to less than 5 amino acid residues, preventing two variable chains from the same molecule from folding into the functional Fv domain. Instead, four separate scFv molecules associate to form tetramers. Tetrabodies can be generated from four identical scFvs or 1-4 individual units from different scFv populations and should retain selective and / or specific increased scFv (s) binding activity and / or exhibit increased binding strength or affinity.

[339.] Whether a spacer where generally is less than 5 amino acid residues will produce triabodies or tetrabodies depends on the amino acid

120 sequences of particular scFv (s) in the mixture and under the reaction conditions.

[340.] In a preferred embodiment, the tetramers are formed through the biotin / streptavidin association. A new fermentation construct has been created which is capable of being enzymatically labeled with biotin (referred to herein as Y1-biotag or Y1-B). The sequence that is the substrate for BirA was added to the C-terminus of Y1. The BirA enzyme attaches biotin to a lysine residue within the sequence. The Y1-biotag was cloned and expressed in E.coli. Antibody inclusion material was isolated and refolded. The purity of the folded protein was> 95% and> 100 mg was obtained from a 1 liter culture (small scale, non-optimized conditions). The molecular weight of this form was found to be similar to scFv by HPLC, SDS-PAGE and mass spectroscopy. Y1-biotag was found to be the most consistent reagent for FACS analysis. However, when binding of Y1-biotag and KG-1 cells in the presence of serum was studied, high concentrations (10-fold more) were required compared to comparable binding in the absence of serum. However, this construct offered the advantage of specific biotinylation where the binding site of the molecule remains intact. In addition, each molecule is labeled with only one biotin, i.e. each molecule receives one biotin at the carboxyl terminus.

[341.] Limited labeling with one biotin per molecule at the desired position allowed the production of streptavidin tetramers. Tetramers were generated by incubating Y1-B with streptavidin-PE.

[342.] FACS analysis indicated that tetramers generated from Y1-biotag and Streptavidin-PE were in the absence of serum 100

121-100 times more sensitive than Y1 scFv monomers. Ylbiotag tetramers with streptavidin-PE specifically bind to a single cell line (KG-1) that is Y1-reactive. The difference of this reaction from background binding was very high and provided a high sensitivity for detecting small amounts of receptor.

Evaluation of FACS of whole normal blood with Y1BSAV tetramers indicated that no highly reactive population was present. Monocytes and granulocytes were positive to a small extent. Tetramers were at least 100-fold more reactive in cell lines that had positive results, such as KG-1 cells.

[343.] Then, the tetramers were incubated with cell samples. A low dose of Y1 tetramers (5 ng) binds well with the cell line (KG-1) and provides a 10 to 20 fold higher response than previously observed with other antibody forms of Y1. A minor response was found when negative cell lines with different doses of tetramers were tested.

Conjugates for Diagnostic and Pharmaceutical Purposes The antibodies and binding fragments thereof of the present invention can be associated, combined, fused or bound with various pharmaceutical agents such as drugs, toxins and radioactive isotopes, optionally with a pharmaceutically active carrier, to form compositions. containing drug-peptide compositions, fusion components or conjugates having activity against disease or cancer. Such conjugates and fusion components may also be used for diagnostic purposes.

122 [345.] Examples of suitable carriers suitable for use in the invention include dextran, HPMA (lipophilic polymer) or any other polymer. In another embodiment, modified liposomes, such as liposomes bearing scFv Y1 molecules, for example Doxil, which is a commercially available liposome preparation containing large amounts of Doxorubicin, may be used. Such liposomes can be prepared to contain one or more desired pharmaceutical agents and can be mixed with the antibodies of the present invention to provide a high drug to antibody ratio.

[346.] In another embodiment, the binding between the antibody or fragment thereof and the pharmaceutical agent can be direct. A direct bond between two or more adjacent molecules can be formed through chemical bonding between elements or groups of elements in the molecules. For example, the chemical bond may be an ionic bond, a covalent bond, a hydrophobic bond, a hydrophilic bond, an electrostatic bond, or a hydrogen bond. The bonds may be through a group, for example, amino, carboxy, amido, hydroxy, and peptide and disulfide linkages. Preferably, the direct binding may be a protease resistant binding.

[347.] The bond between the peptide and the pharmaceutical agent, or between the peptide and the carrier, or between the carrier and the pharmaceutical agent can be via a linker compound. As used herein, a linker compound is defined herein as a compound that joins two or more compounds together. The linker may have a straight or branched chain. The branched chain linker may be comprised of two-branched, three-branched or four-branched compounds.

··· · ··· · · · · ····· · · ·

123 ····· ·· ··· ·· ·

Linker compounds useful in the invention include compounds selected from the group consisting of carboxylic acids, maleimido hydrazides, PDPH, carboxylic acid hydrazides, and small peptides.

[348.] More specific examples of linker compounds useful in the present invention include:

a. dicarboxylic compounds such as succinic acid, glutaric acid, and adipic acid;

b. maleimido hydrazides such as N- [ε-maleimido-caproic acid hydrazide], 4- [N-maleimidomethyl] cyclohexane-1-carboxylic hydrazide, and N- [K-maleimidoundecanoic acid hydrazide];

c. PDPH linkers such as (3- [2-pyridyldithio] propionyl hydrazide) conjugated to a sulfhydryl reactive protein; and

d. carboxylic acid hydrazides selected from 2-5 carbon atoms [349.] Also suitable are direct bonds using small peptide linkers. For example, direct binding between free sugar, for example the Doxorubicin anticancer agent, and the scFv can be made using small peptides. Examples of small peptides include AU1, AU5, Btag, cmyc, FLAG, Glu-Glu, HA, His6, HSV, HTTPHH, IRS, KT3,

Protein C, S-TAG (R), T7, V5, VSV-G.

[350.] Antibodies and fragments thereof of the present invention may be attached, conjugated, complexed or otherwise associated with imaging agents (also called indicative markers) such as radioisotopes, and these conjugates may be useful for: · · · · · · · · · · ·

124 ··· »· ·· ··· Diagnostics and imaging purposes. Kits comprising such radioisotope-antibody conjugates (or antibody fragments) are provided.

[351.] Examples of radioisotopes useful for diagnostics include indium ^her, indium ^{113, 99 m} Rhenium, ¹⁰⁵ Rhenium, ¹⁰¹ Rhenium, ^{99 m} techneciurn, tellurium ^{121 m, 122 m} tellurium, tellurium ^{125 m, 165} Thulium, ¹⁶⁷ Thulium, ¹⁶⁸ Thulium, ¹²³ Iodine, ¹²⁶ iodine, ¹³¹ iodine, ¹³³ iodine, ^81m krypton, ³³ xenon, ⁹⁰ yttrium, ²¹³ bismuth, ⁷⁷ bromine, ¹⁸ fluorine, ⁹⁵ ruthenium, ⁹⁷ ruthenium, ¹⁰³ ruthenium, ¹⁰⁵ ruthenium, ¹⁰⁷ mercury, ²⁰³ mercury, ⁶⁷ gallium and ⁶⁸ gallium. Preferred radioactive isotopes are opaque isotopes in X (X-rays) or paramagnetic ions.

[352.] The indicative marker molecule may also be a fluorescent marker molecule. Examples of fluorescent marker molecules include fluorescein, phycoerythrin, or rhodamine, or modifications or conjugates thereof.

[353.] Antibodies or fragments thereof conjugated to indicative markers can be used to diagnose or monitor disease states. Such monitoring may be performed in vivo, in vitro, or ex vivo. When monitoring is performed in vivo or ex vivo, the imaging agent is preferably physiologically acceptable, ie, such that it does not harm the patient to an unacceptable extent. Acceptable levels of injury can be determined by clinicians using criteria such as the severity of the disease and the availability of other alternatives.

··· · · ··· · ··

[354.] The present invention provides a diagnostic kit for in vitro analysis of treatment efficacy before, during and after treatment, comprising an imaging agent consisting of a peptide of the invention associated with an indicative marker molecule. The invention further provides a method of using an imaging agent for diagnostic localization and imaging of a tumor, particularly a tumor, comprising the following steps:

a) contacting the cells with the composition,

(b) measuring cell - bound radioactivity; and

c) tumor visualization.

[355.] Examples of suitable imaging agents include fluorescent dyes such as FITC, PE and the like, fluorescent proteins such as green fluorescent proteins. Other examples include radioactive molecules and enzymes that react with the substrate to produce a discernible change, such as a discoloration.

[357.] In one example, the imaging agent in the kit is a fluorescent dye, such as FITC, and the kit allows analysis of the efficacy of treating tumors, more particularly blood cancer, ie, leukemia, lymphoma, and myeloma. FACS analysis is used to determine the percentage of cells stained with a fluorescent agent and the intensity of staining at each stage of the disease, ie after diagnosis, during treatment, during remission and during relapse.

[358.] The antibodies and fragments thereof of the present invention may be bound, conjugated to or otherwise associated with anti-cancer agents, anti-leukemic agents.

126

agents, antimetastatic agents, anti-inflammatory agents, antithrombotic agents, antirestenotic agents, anti-aggregation agents, anti-autoimmune agents, anti-adhesion agents, anti-cardiovascular agents, or other anti-disease agents and pharmaceutical agents. Pharmaceutical agent refers to an agent suitable for prophylactic treatment or diagnosis in mammals including, but not limited to, human, bovine, horse, swine, mouse, dog, cat, or any other warm-blooded animal.

Examples of such pharmaceutical agents include, but are not limited to, antiviral agents including acyclovir, ganciclovir, zidovudine; thrombosis / restenosis agents including cilostazole, dalteparin sodium, reviparin sodium and aspirin; anti-inflammatory agents including zaltoprofen, pranoprofen, droxicam, acetyl salicylic 17, diclofenac, ibuprofen, dexibuprofen, sulindac, naproxen, amtolmetin, celecoxib, indomethacin, rofecoxib and nimesulfide; anti-autoimmune agents including leflunomide, denileukin diftitox, subreum, WinRho SDF, defibrotide, and cyclophosphamide; and antiadhesive / anti-aggregating agents including limaprost, clorcromen and hyaluronic acid.

[359.] Other exemplary pharmaceutical agents include Doxorubicin (adriamycin), morpholinodoxorubicin, methoxymorpholinyldoxorubicin, cis-platin, taxol, calicheamicin, vincristine, cytarabine (Ara-C), cyclophosphamide, prednisone, daunorububinin, morpholinodine, morpholinodine, morpholinodine, morpholinodine, morpholinodine , interferon alfa,

127 hydroxyurea, temozolomide, thalidomide and bleomycin and derivatives and combinations thereof.

[360. ] An anticancer agent is a substance with anticancer activity. For example, anti-cancer agents include those that inhibit or arrest the growth of cancerous or immature precancerous cells, agents that kill cancerous or precancerous cells, agents that increase the sensitivity of cancerous or precancerous cells to other anti-cancer agents, and agents that inhibit cancer metastasis. cells. According to the present invention, the anti-cancer agent may also be a substance with anti-angiogenic activity that prevents, inhibits, retards or stops the vascularization of tumors.

[361.] Inhibiting cancer cell growth includes, for example, (i) preventing cancer or metastatic growth, (ii) reducing cancer or metastatic growth, (iii) completely preventing the cancer cell growth or metastatic process while keeping the cell intact and alive or (iv) killing the cancer cell.

[362.] An anti-leukemic agent is a substance with anti-leukemic activity. For example, antileukemic agents include those that inhibit or inhibit the growth of leukemic or immature preleukemic cells, agents that kill leukemic or preleukemic cells, agents that increase the sensitivity of leukemic or preleukemic cells to other antileukemic agents, and agents that inhibit leukemic metastasis cells. According to the present invention, the anti-leukemic agent may also be a substance with anti-angiogenic activity that prevents, inhibits, retards or stops the vascularization of tumors.

128 [363.] Inhibition of leukemic cell growth includes, for example, i) preventing leukemic or metastatic growth, (ii) reducing leukemic or metastatic growth, (iii) completely preventing the leukemic cell growth or metastatic process while keeping the cell intact and viable or (iv) killing the leukemia cell.

[364.] Examples of anti-disease, anti-cancer, and anti-leukemia agents to which the antibodies and fragments thereof of the present invention can be suitably bound include toxins, radioisotopes, and pharmaceutical agents.

[365.] Examples of toxins include gelonin, Pseudomonas exotoxin (PE), PE40, PE38, diphtheria toxin, ricin, or modifications or derivatives thereof.

[366.] Examples of radioisotopes include gamma-emitters, positron-emitters and x-ray emitters that can be used for localization and / or therapy, and beta-emitters and alpha-emitters that can be used for treatment.

[367.] More specific examples of therapeutic radioisotopes include ^{1: L1} indium, indium ^{113, 99 m} rheniurn, ¹⁰⁵ Rhenium, ¹⁰¹ Rhenium, ^{99 m} technetium, tellurium ^{121 m, 122 m} tellurium, tellurium ^{125 m, 165} Thulium, ¹⁶⁷ Thulium, ¹⁶⁸ Thulium, ¹²³ Iodine, ¹²⁶ iodine, ¹³¹ iodine, ¹³³ iodine, ^81m krypton, ³³ xenon, ⁹⁰ yttrium, ²¹³ bismuth, ⁷⁷ bromine, ¹⁸ fluorine, ⁹⁵ ruthenium, ⁹⁷ ruthenium, ¹⁰³ ruthenium, ¹⁰⁵ ruthenium, ¹⁰⁷ mercury, ²⁰³ mercury, ⁶⁷ gallium and ⁶⁸ gallium .

129 [368.] Non-limiting examples of anti-cancer and anti-leukemic agents include Doxorubicin (adriamycin), morpholinodoxorubicin, methoxymorpholinyldoxorubicin, cisplatin, taxol, calicheamicin, vincristine, cytarabine (AraC), cyclophununinicin, daunorinolorinoline, daunorinoloricin, daunorinoloricin, daunorinin, interferon alpha, hydroxyurea, temozolomide, thalidomide and bleomycin and derivatives and combinations thereof.

Pharmaceutical Compositions The antibodies, constructs, conjugates and fragments of the present invention can be administered to a patient in need thereof through a suitable method. Exemplary methods include intravenous, intramuscular, subcutaneous, topical, intratracheal, intrathecal, intraperitoneal, intralymphatic, nasal, sublingual, oral, rectal, vaginal, respiratory, buccal, intradermal, transdermal, or intrapleural routes of administration.

For intravenous administration, the composition is preferably formulated so that the amount administered to the patient is an effective amount of from about 0.1 mg to about 1000 mg in the desired composition. More preferably, the amount administered will range from about 1 mg to about 500 mg in the desired composition. The compositions of the invention are effective over a wide dosage range and depend on such factors as the specificity of the disease being treated, the half-life of the peptide or polypeptide pharmaceutical composition in the patient, the physical and chemical characteristics of the pharmaceutical agent and the pharmaceutical composition, the route of administration. * · · ··· · ··········································· ·· · · ·

130 specific features of the patient being treated or diagnosed, as well as other parameters that the treating physician believes are important.

[371.] The composition for oral administration may be in any suitable form. Examples include tablets, liquids, emulsions, suspensions, syrups, pills, capsules or capsules. Methods for preparing pharmaceutical compositions are well known in the art. See, e.g., Remington, The Science and Practice of Pharmacy, Alfonso R. Gennaro (Ed.), Lippincott, Williams & Wilkins (pub).

[372.] The pharmaceutical composition may also be formulated to facilitate timed, maintenance, pulsed, or continuous release. The pharmaceutical composition may also be administered as a device such as a timed, sustained, pulsed, or continuous release device.

[373.] The pharmaceutical composition for topical administration may be in any suitable form such as creams, ointments, lotions, patches, solutions, suspensions and gels.

[374.] A composition comprising antibody fragments prepared according to the invention may comprise conventional pharmaceutically acceptable diluents, excipients, carriers, and the like. Tablets, pills, capsules and capsules may contain conventional excipients such as lactose, starch and magnesium stearate. Suppositories may contain excipients such as waxes and glycerol. Injectable solutions contain sterile pyrogen-free media, such as saline, and may contain buffering agents, stabilizing agents, or stabilizing agents. · · · · ···················

131 preservatives. Conventional enteric coating agents may also be used.

[375.] The following examples are presented for understanding the invention, but are not intended and should not be construed as limiting the scope of the invention. Although specific reagents and reaction conditions are described, modifications may be made to the scope of the invention. The following examples are therefore provided to further illustrate the invention.

EXAMPLES

Example 1: Preparation of Plates

1.1 Preparation of washed plates [376.] Acid-citrate-dextrose (ACD) plate concentrate was obtained from a blood bank, the plates were isolated, washed once in a buffer containing ACD and saline 1: 7 . After each wash, the plates were centrifuged at 800 xg for 10 minutes and resuspended in Tyrodes solution (2 mM MgCl ₂ , 137 mM NaCl, 2.68 mM KCl, 3 mM NaH ₂ PO ₄ , 0.1% glucose, 5 mM Hepes and 0.35% albumin, pH 7.35) and cell number was determined.

1.2 Preparation of platelet-rich plasma [377.] Blood was collected in a tube containing 3.8% sodium citrate. Platelet-rich plasma was prepared by centrifugation at 250 x g for 10 minutes.

Example 2: Platelet aggregation Aggregation of platelets Aggregation of platelets Aggregation of platelets Aggregation of platelets

[378.] To study platelet aggregation, platelet rich plasma (PRP) and washed plates were agitated at 500 rpm and 37 ° C in platelet rich plasma (PRP). Lumiagregometer for whole blood (Lumiaggregometer Chronolog, Havertown, PA). The difference in light transmission when passing through platelet suspensions and suspending medium was taken as 100% aggregation. The effect of Y1 on platelet aggregation was evaluated by adding various concentrations of Y1 before the agonist addition and the effect was recorded for four minutes.

Example 3: Platelet treatment with endoproteases

3.1 Platelet digestion with Mocarhagine [379.] For digestion with Mocarhagin, 10 ⁸ washed plates in TS buffer (0.01 M Tris, 0.15 M sodium chloride, pH 7.4) containing 1 mM calcium chloride were treated with 12 µg / ml. ml of mocarhagine (final concentration) for 1 hour at 22 ° C and cleavage was stopped by adding EDTA to a concentration of 0.01 M.

3.2 Cleavage of glycocalicin by Cathepsin G [380.] 10 ⁸ washed plates in TS buffer containing 1 mM calcium chloride were incubated with 1.8 μρ / ιηΐ cathepsin G (final concentration) for 4 hours at 22 °

C and cleavage was stopped by adding PMSF to a concentration of 1 mM.

[381.] 10 ⁶ plates in 0.1 M Tris pH 7.4 buffer containing 0.2% albumin and protease inhibitors (10 μΜ leupeptin, 0.24 mM PMSF) were incubated with 0.14 mg / ml. ·

9 9

133

O-sialoglycoprotein endoprotease (final concentration) for 45 minutes at 37 ° C and cleavage was stopped by addition of buffer sample and boiling. The buffer sample used contained 3% SDS, 12% glycerol, 50 mM Tris.HCl, 2% β-mercaptoethanol, and 0.03% bromophenol blue.

Example 4: Cleavage of glycocalicin by endoproteases

Glycocalicin digestion with Mocarhagine [382.] For mocarhagine cleavage, glycocalicin in TS buffer containing 1 mM calcium chloride was incubated with 10 µg / ml mocarhagine (final concentration) for 1 hour at 22 ° C and cleavage was terminated by adding EDTA to 0 , 01 M.

Glycocalicin cleavage with Cathepsin G [383.] For cathepsin G cleavage, glycocalicin in TS buffer containing 1 mM calcium chloride was incubated with 3.4 gg / ml cathepsin G (final concentration) for 4 hours at 22 ° C and cleavage was terminated by addition PMSF to a concentration of 1 mM.

Glycocalicin O-sialoglycoprotein endoprotease [384.] Glycocalicin in 0.05 M Tris buffer pH 7.4 was incubated with 1.2 mg / ml O-sialoglycoprotein endoprotease (final concentration) for 15 min at 37 ° C and digestion was terminated by addition of sample buffer (as described in Example 3 (YH) and boiling).

· · * · * * * * * * * * * * * *

134 ··· · · ····· · ·

Example 5: Construction of full-length Y1 IgG [385.] Full IgG molecules have several advantages over Fv forms, including a longer half-life in vivo and the potential for inducing cellular responses in vivo, such as ADCC-mediated or CDC (complement dependent cytotoxicity) responses; Tomlinson Current Opinions of Immunology, 5, 83-89 (1993)). By the molecular cloning procedure described below, Y1 Fv regions were converted to full-length IgG1 molecules. Construction of Y1-IgG1 was performed by fusion of the cDNAs in the following order: The Sek-IgG light and heavy chain sequences are shown in FIG. The open reading frame (ORF) of the nucleotide sequence Y1-HC (SEQ ID NO: 205), the amino acid sequence Y1-HC (SEQ ID NO: 206), the ORF of the nucleotide sequence Y1-LC (SEQ. And the amino acid sequence of Y1-LC (SEQ ID NO: 208).

[386.] Leader sequence compatible with mammalian expression system: An interchangeable system has been developed allowing the appropriate insertion of the elements required for a full length IgG molecule. The following complementary double-stranded oligonucleotides encoding the putative leader sequence were synthesized and annealed and ligated to the XhoI site of the mammalian expression vector pBJ-2 (under the control of the SRcc5 promoter).

5'-TCGACCTCATCACCATGGCCTGGGCTCTGCTGCTCCTCACCCTCCTCACTCA

GGACACAGGGTCCTGGGCCGAT a

5'-GATCGATTGCACCAGCTGGATATCGGCCCAGGACCCTGTGTCCTGAGTGAG

GAGGGTGAGGAGCAGCAGAGCCCAGGCCATGGTGATGAGG. Two Kozak elements were incorporated upstream of the ATG initiation codon.

135

In addition, an internal FcoRV site was introduced between the putative leader sequence cleavage site and an XhoI site so as to allow subcloning of the variable regions. This modified vector was designated pBJ-3.

[387.] The V _L coding sequence derived from the Y1-scFv cDNA sequence was inserted between the leader coding sequence and the light chain constant region coding sequence. Similarly, the V _H coding sequence derived from the cDNA sequence for Y1-scFv was inserted between the leader coding sequence and the heavy chain constant region coding sequence. This was done by PCR amplification of the pHEN-Y1 vector encoding the original Y1 to obtain the V _L and V _H regions respectively.

Oligonucleotides [388.] Oligonucleotides 5'-TTTGATATCCAGCTGGTGGAGTCTGGGGGA (sense) and

5'-GCTGACCTAGGACGGTCAGCTTGGT (anti-sense) was used for PCR reaction to amplify V _L The expected cDNA product of ~ 350 bp was purified, sequenced and digested with EcoRV and AvrII restriction enzymes. The same procedure was performed to amplify and purify the V _H region cDNA using sense and anti-sense oligonucleotides:

[389.]

5'-GGGATATCCAGCTG (C / G) (A / T) GGAGTCGGGC

5'-GGACTCGAGACGGTGACCAGGGTACCTTG, respectively.

··· ·· ·· ···

136 [390.] Constant regions: The λ3 (CLIS) constant region and CH1-CH3 heavy chain constant regions, derived for IgG1 cDNAs, were individually synthesized as follows:

[391.] For the CL-13 constant region, RTPCR was performed on mRNA extracted from a pool of normal peripheral B cells (CD19 + cells) in combination with the sense oligonucleotide 5'-CCGTCCTAGGTCAGCCCAAGGCTGC and the anti-sense oligonucleotide 5'-TTTGCGGCCGCTCATGAACATTCTGTAGGG. The expected PCR product (~ 400) was purified, sequenced, and digested with the restriction enzymes AvrII and NotI.

[392.] For the IgG1 constant region (γ chain), a human B cell clone (CMV clone - clone # 40), immortalized with BTG, was selected for PCR amplification. This clone was found to secrete IgG1 against human CMV and was also found to induce an ADCC response in in vitro assays. Oligonucleotides were synthesized for the CH1-CH3 cDNA regions

5'-ACCGCTCGAGTGC (T / C) TCCACCAAGGGCCCATC (G / C) GTCTTC (sense) and

5'-TTTGCGGCCGCTCATTTACCC (A / G) GAGACAGGGAGAGGCT (anti-sense) and were used for PCR amplification. As described for the cDNA sequence encoding CL, the expected PCR product (~ 1500 bp) was purified, sequenced, and digested with the restriction enzymes AvrII and NotI.

[393.] For the final expression vector, a triple ligation procedure was performed using the pBJ-3 vector pre-digested with EcoRV-Notl, cDNA for the variable regions digested.

137

EcoRV-AvrII, and constant region cDNA cleaved by AvrIIIN1. The final heavy and light chain expression vector was named as pBJ-Y1-HC and pBJ-Y1-LC, respectively.

[394.] Another vector, pBJ-Y1-LP, was constructed based on pBJ-Y1-LC to allow double selection based on the puromycin resistant gene (PAC). In this vector, the neomycin resistance gene of plasmid pBJ-Y1-LC was replaced with a ~ 1600 bp fragment encoding the PAC gene (from the pMCCZP vector).

[395.] The open reading frame (ORF) for both Y1-HC and Y1-LC and the amino acid sequences encoded by them are set forth in SEQ ID NOS: 205 to 208.

[396.] The leader sequence is underlined. Each of the encoded V _H and V _L regions is underlined, followed by either the IgG1 constant region sequence (for the heavy chain) or the λ3 constant region sequence (for the light chain).

[397.] Expression of Y1 heavy and light chains in cells

CHO.

The vectors pBJ-Y1-HC and pBJ-Y1-LC were individually used for transfection and selection of stable cells that express heavy and light chains. After selection for G418 and cell growth, the secreted protein in the supernatant was analyzed for IgG1 expression using a sandwich EIA assay and Western blotting analysis as described below.

138 [398.] Sandwich EIA Assay: Mouse anti-human IgG1 Fc (Sigma) was pre-bound to wells of a 96-well plate. The supernatant from the above procedure was added to the wells and the presence of the IgG1 heavy chain was detected by biotinylated goat anti-γ chain specific antibody (Sigma), streptavidin-HRP and substrate. ELISA plate reader noted staining at A405 · [399.] Western blotting analysis: The supernatant from the above cells was plated on 12.5% SDS-PAGE. Expression of each chain was detected with (a) a goat anti-human IgG-HPR antibody (H + L; Sigma Cat # A8667) for heavy chain detection and (b) a biotinylated goat anti-human λ3 chain antibody (Southern Biotechnology Association, Cat # 2070-08) for light chain detection.

[400.] The expression of both chains was confirmed by the above assays and then co-transfected to obtain full-length IgG-IgG1.

IgG-Y1 expression and purification [401.] Cell Culture and Transfection: CHO cells were cultured in F-12 medium with 10% fetal calf serum and 40 µg / ml gentamicin at 37 ° C in 5% CO 2 One day before transfection, 0.8-1x10 ⁶ cells were plated on 90 mm plates. Cultures were co-transfected with 10 µg of light and heavy chain DNA by FUGene (Roche) transfection reagent technique. After two days of growth in nonselective medium, cells were cultured for 10-12 days in F-12 medium containing 550 µg / ml neomycin and 3 µg / ml puromycin. Cells were trypsinized and cloned by limiting dilution of 0.5 cells / well in Costar well plates. Individually

139 colonies were harvested, grown in six-well dishes and transferred to bottles.

[402.] Determination of heavy and light chain secretion: A sandwich ELISA was used to determine the antibody secreted into the supernatant of transfected CHO cells. The following reagents were used to determine antibody concentration: monoclonal anti-human IgG1 (Fc) (Sigma) as bound antibody, biotin conjugate with goat anti-human IgG antibody (γ-chain specific) as detector (Sigma) and pure human IgG1, lambda (Sigma) as standard. In the ELISA, production rates ranged from 3-4 μρ / πιΐ.

[403.] Mab Production and Purification: Cells were grown in spinning flasks to a final concentration of 12x10 ⁸ cells per flask in F-12 medium with 10% fetal calf serum supplemented with neomycin and puromycin. For production, cells were cultured in the same medium but with 2% fetal calf serum for an additional two days.

[404.] The secreted antibody was purified on a G-Sepharose column (Pharmacia). Binding was performed in 20 mM sodium phosphate buffer, pH 7.0; elution was performed in 0.1M glycine buffer, pH 2.5-3.0. The amount of purified antibody was determined by UV absorbance, the purity was analyzed by SDS-PAGE. Under non-denaturing conditions, the total IgG antibody had an expected molecular weight of 160 kD. In denatured gels, both the heavy and light chains had the expected molecular weights of 55 and 28 kD, respectively.

140 [405.] Binding of full-length IgG-Y1 molecule: Binding experiments were performed to determine the level of binding of the IgG-Y1 molecule as compared to the level of binding of the scFv-molekuly molecule. A double staining method was used in which 5 ng of IgG-Y1 reacted with both Raji cells (negative control, Figures 44-47a and Jurkat cells (Y1 positive cells, Figures 44-47b and 44-47c). goat PE-labeled anti-human IgG (Figures 44-47c) Similarly, 1 μς scFv-ΥΙ reacted with Jurkat cells (Figures 44-47b) and rabbit PE-labeled anti-scFv was used for detection. Y1 and scFv-ΥΙ bind Jurkat cells with approximately 10 ^{3 *} times more scFv-moleku molecules than IgG-Y1 molecules to detect a similar level.

Example 6: Preparation of Fab and F (ab ') ₂ fragments derived from whole IgG antibody Y1

Cell culture and transient transfection CHO cells were cultured in F-12 medium with 10% fetal calf serum and 40 µg / ml gentamicin at 37 ° C in 5% CO ₂ . One day before transfection, 1, 5x10 ⁶ cells were plated on 90 mm plates. The cultures were cotransfected with 10 µg of DNA encoding the variable light and variable heavy chains of antibody Y1, for each chain separately in a eukaryotic expression system. Transfections were performed using the FUGene transfection reagent technique (Roche).

[407.] After two days of growth in non-selective medium, cells were cultured for 10-12 days in F-12 medium containing 550 µg / ml neomycin and 3 µg / ml puromycin. Cells were trypsinized and cloned by limiting dilution of 0.5 cells / well in Costar-96 well plastic plates.

141

Individual colonies were harvested, grown in six-well dishes and transferred to flasks for further selection (to determine expression level and antibody secretion into growth medium).

Cell culture and long-term transfection:

[408. CHO cells were cultured in F-12 medium with 10% fetal calf serum and 40 µg / ml gentamicin at 37 ° C in 5% CO ₂ . One day before transfection, 0.8-1x10 ⁶ cells were plated on 90 mm plates. The cultures were transfected with 10 µg of DNA encoding the variable light and variable heavy chains of the Y1 antibody cloned under the CMV (cytomegalovirus) promoter, and the dhfr gene under the sv40 promoter. Transfection was performed using the FUGene transfection reagent technique (Roche). After two days of growth in nonselective medium, cells were cultured in medium containing 100 nM - 5 μΜ methotrexate (MTX) and dialyzed fetal calf serum to select clones (after limiting dilution) that express elevated levels of total Y1 antibody.

Determination of heavy and light chain secretion:

[409.] A sandwich ELISA was used to determine the concentration of antibody secreted into the supernatant of transfected CHO cells. The following reagents were used to quantify antibody concentration: monoclonal anti-human IgG1 (Fc) antibody (Sigma) as bound antibody, biotin conjugate with goat anti-human IgG antibody (specific for the γ-chain) as detector (Sigma) and pure human IgG1 , lambda (Sigma) as standard.

142

Mab production and purification from cells:

Cells were grown in rotating flasks to a final concentration of 1-2x10 ⁸ cells per flask in F-12 medium with 10% fetal calf serum supplemented with neomycin and puromycin (as above). For antibody production, cells were cultured in the same medium but with 2% fetal calf serum for an additional two days.

[411.] The secreted antibody was purified on a G-Sepharose column (Pharmacia) and a QSepharose ion exchange column (Pharmacia). Binding was performed in 20 mM sodium phosphate buffer, pH 7.0, while elution was performed in 0.1 M glycine buffer, pH 2.5-3.0. The amount of purified antibody was determined by UV absorbance and ELISA, while purity was analyzed by SDS-PAGE and HPLC. Under non-denaturing conditions, the total IgG antibody had an expected molecular weight of 160 kD. In denatured gels, both the heavy and light chains had the expected molecular weights of 55 and 28 kD, respectively.

Fragmentation of Y1 IgG to Fab and F (ab ') 2:

[412.] An IgG molecule is composed of two identical light chains and two identical heavy chains. These chains are interconnected into folds (domains) by a combination of non-covalent interactions and covalent bonds (disulfide bridges). The light chain consists of one variable domain and one constant domain. The heavy chain consists of a variable domain (V _H ) and three separate constant domains (CH 1, 2 and 3). The hinge region between the first constant domain of the heavy chain (CH1) and the second constant domain of the heavy chain (CH2) is easily

143 accessible for proteolytic attack of enzymes. The cleavage at this point produces Fab and (Fab ') 2 fragments and a Fc portion. The Fc portion of the molecule retains the ability of the molecule to bind antigen, but has low non-specific binding. The Fab proportion is most suitable for situations where the ability to bind antigen without effector functions is desired.

Fab and F (ab ') ₂ fragments are used in vivo as diagnostic and therapeutic agents. To prepare tumor-specific chemotherapeutic anti-cancer agents instead of damaging all cells, the agents can be bound to antibodies that bind to the cell surface antigens of tumors. Using Fab or F (ab ') ₂ instead of intact IgG offers a number of advantages:

[414.] (1) Fragments can more easily pass through capillaries and diffuse to tissue surfaces.

[415.] (2) Fragments that are not bound in the conjugate will be removed more rapidly than intact unbound IgG; hence a greater proportion of the agent fragments of the therapeutic agent reaches the target region.

Detailed procedure for Fab preparation:

[416.] 1 mg of purified Y1 antibody was loaded onto a 2 ml column of immobilized Ficin in 2 mg / ml cysteine HCl cleavage buffer (to prepare F (ab ') ₂ fragments) and 20 mg / ml (to prepare Fab fragments), at 37 ° C for 5 hours (for Fab) and 20 hours (F (ab ') ₂ ) The reaction was terminated by elution of the digestion products with 4 ml of Immunopure Binding buffer. Separation of Fab or F (ab') ₂ fragments from uncleaved IgG and Fc fragments were performed using a Protein A coupled column and binding buffer.

Fab or F (ab ') ₂ fragments were contained in the flow. Fractions corresponding to peaks and containing fragments were collected by absorbance reading at 280 nm. The fragments were concentrated and dialyzed against PBS using a 10,000 Dalton molecular weight interrupter. Protein regeneration, purity, and characteristics were determined using absorbance at 280 nm, gel electrophoresis, and HPLC.

Preparation of cell extract (Lysate) [417.] 2x10 ⁶ cells were harvested and centrifuged in a microcentrifuge (1300 rpm, 4 ° C, 5 minutes).

For washing, 0.5-1 ml PBS + pi was added to the pellet and the sample was mixed gently. The mixture was centrifuged as above. The wash with 0.5-1 ml PBS + pi was repeated and the mixture centrifuged as above.

The pellet cells were resuspended in lysis buffer (200 μ1 / 20χ10 ⁶ pellet cells). The lysis buffer used was 50 mM Tris pH 7.4, 1 mm PMSF 1% NP-40, and 1 mM EDTA, although other suitable lysis buffers may also be used. The suspensions were incubated for approximately 60 minutes on ice, then centrifuged (3000 rpm, 4 ° C, 5 min). The supernatant was collected and aliquoted.

Preparation of crude membrane fraction and membrane protein extraction.

[418.] Twenty volumes of homogenization buffer was added to one volume of thickened cells. Used

145 the homogenization buffer was 2% (w / v) Tween 20, 1 mM MgSO ₄ , 2 mM CaCl ₂ , 150 mM NaCl, and 25 mM Tris-HCl, pH 7.4. The following protease inhibitors were also added: 1 mM PMSF, 5 µg / ml leupeptin and 5 µg / ml aprotonin. Cells were homogenized using three to five interventions in a Plotter-Elvehjem rotary teflon piston (Ultra-Torex) homogenizer. The sample was kept cold during homogenization, then stirred for 1 hour in an ice bath. Then the sample was again subjected to several additional impulses of the homogenizer, then centrifuged at 3000 g for 30 minutes at 4 ° C. The supernatant was collected and centrifuged at 45,000 g (19,000 rpm, ss-34 rotor) for 1 hour. at 4 ° C. The supernatant from the centrifugation at 45,000 g was discarded. A solution of 50 mM Tris, 7.4, 1 mM EDTA, 1% NP-40 and protease inhibitors was added to the pellet and then the dissolved pellet was placed on ice for one hour.

HPLC Fractionation Purification of Membranes [419.7.] An RPC column (Pharmacia Type PLRP-S 300 A) was used for fractionation purification of membranes. The buffers used were: (A) 20 mM Tris, 8.0 and (B) 60% propanol in DDW. The flow rate was 1 ml / min, except for the wash step during which the wash rate was 2 ml / min. The whole procedure was carried out at ambient temperature.

[420.] Elution sample after immunoprecipitation (IP) of Jurkat or KG-1 membrane fractions was added to sample buffer (62 mM Tris, pH 6.8, 10% glycerol, 3% SDS, 720 mM mercaptoethanol) diluted 1: 4 with Buffer A.

146 [421.] The sample was applied to the column and the flowing liquid was collected. The column was washed with Buffer A until the optical density of the flowing liquid had dropped to zero.

[422.] Proteins were eluted from the column as follows: 5 minutes with 80% A, then 40 minutes gradient of 80-0% buffer A and 20-100% buffer B. The second gradient was applied to convert the wash liquid composition to 80% And this composition was used to wash the column for 5 additional minutes. The eluted liquid was collected in a fractionation collector into 1 ml fractions.

[423.] Samples were evaporated to small volumes in a Speed-Vac and then analyzed using SDS-PAGE (polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate) and Western blotting.

Purification of normal human plasma on Q Sepharose columns [424.] 5 ml of normal human fresh frozen plasma was diluted 1:10 with starting buffer and filtered through a filter (0.45 μπι syringe filter, Sartorius, cat #

16555). The starting buffer was 20 mM Tris-HCl, pH 8.0 containing protease inhibitors (5 gg / ml leupeptin, 5 gg / ml aprotinin and 1 mM PMSF).

[425.] A 5 ml HiTrap Q Sepharose column (Amersham Pharmacia, cat # 17-1154-01) was connected to a P-1 peristaltic pump (Amersham Pharmacia). The column was washed at a flow rate of approximately 4 ml / minute as recommended by the manufacturer. The diluted filtered plasma was applied to the column and the flowing liquid was collected.

• · · · ·

147

The column was washed with 20 volumes of 0.3M NaCl solution in the starting buffer. Proteins were eluted at NaCl concentrations in 0.6 M, 0.8 M, and 1.0 M starting buffer. Elution volumes were 50, 20, and 20 ml, respectively. The whole procedure was carried out at 4 ° C.

[426.] Eluted fractions were subjected to SDS-PAGE analysis in duplicate gels followed by Western blotting analysis in which biotinylated antibody Y1 and antibody 181 were used as a negative control. Fibrinogen γ 'was found in the elution fraction containing 0.6 M NaCl. The elution fraction of 1.0M NaCl contained Complement Compound 4 (CC4), lumican, prothrombin, and an inter-alpha inhibitor.

Purification of normal human plasma on an HPLC column [427.] Normal human plasma purified on Q-Sepharose was mixed with urea (to a final concentration of at least 8 M), DTT (to a final concentration of 30 mM) and TFA (to a final concentration of 0.1%). ).

The purified plasma solution was applied to a 3 ml RPC column (Amersham Pharmacia) and the flowing liquid was collected. The column was washed with buffer A until the optical current density dropped to zero. Proteins were eluted from the column as follows: 5 minutes with 90% buffer A, then 40 minutes with a gradient of 90 to 0% buffer A and 10 to 100% buffer B. The second gradient was then used to transfer the flowing composition into 90% buffer A and this composition was used to continue the column wash for an additional 5 minutes. The buffers used were (A) 0.1% TFA in DDW and (B) 80% CAN and 0.1% TFA in DDW. The flow rate was 1 ml / minute.

147 ·· * ** ........

except for the washing step during which the flow rate was 2 ml / minute.

[429.] The eluted liquid was collected in a fraction collector into 1 ml fractions. The whole procedure was carried out at ambient temperature.

[430.] Samples were evaporated to small volumes in a Speed-Vac and then analyzed using SDS-PAGE and Western blotting.

Western blotting analysis of Y1 receptor splicing on a post-blot filter [431.] The nitrocellulose membrane was blocked using 5% skim milk for one hour at room temperature. The membrane was then washed 3 times for 5 minutes each time with 0.05% Tween 20 in PBS at room temperature. The membrane was incubated with 5 μρ / πιΐ Yl-biotin (or 181-biotin) in 2% skim milk in PBS for 1 hour at room temperature. Then, the membrane was washed 3 times for 5 minutes each time with cold 0.05% Tween 20 in PBS in a cold room (about 4 to about 10 ° C). Then, the membrane was incubated in a cold room with SAV-HRP (streptavidin-HRP) in 2% skim milk, diluted 1: 1000 with 0.05% Tween (to a final concentration of 1 g / ml). Dilution was performed at room temperature (approximately 25 ° C), then the diluted SAV-HRP was cooled on ice for 10-15 minutes before use. Incubation was carried out for 1 hour with gentle agitation. After incubation with SAV-HRP, the membrane was washed as above. Then, the membrane was incubated with Super Signa! mixture (Pierce) for 5 minutes according to

149 and then the excess solution was dried. The membrane was used for exposure to an X-ray film (Fuji) and the film was developed.

Example 7: Prokaryotic expression of recombinant glycocalicin (GC) [432.] A DNA fragment encoding glycocalicin (GC, amino acid 1 to amino acid 493 from GPIba) was cloned into a prokaryotic vector cassette, inducible by IPTG. E. coli cells (BL21 DE3) carrying the newly constructed plasmid were grown at 37 ° C to an O.D. 0.7-0.8, then cultured at 37 ° C for 3 hours in the presence of IPTG for induction.

[433.] SDS-polyacrylamide gel loaded with either partially purified glycocalicin, obtained from human platelets (GC) or loaded with cell lysates of E. coli (total), obtained from induced and non-induced cells was analyzed. Western blot analysis with biotinylated Y1-scFv, a polyclonal rabbit anti-human glycocalicin antibody, a commercially available murine anti-human CD42 monoclonal antibody (SZ2 Immunotech, PM640 Serotec, HIP1 Pharmigen, AN51 DAKO), and a polyclonal anti-N-terminal antibody GPIBa (Sc-7071, Santa Cruz).

[434.] Polyclonal antibodies recognized both recombinant glycocalicin derived from bacteria and natural glycocalicin derived from human platelets. Y1-scFv and commercially available antibodies recognized natural glycocalicin derived from human platelets, but

150 «·· ·· 4 ·

did not recognize bacteria-produced recombinant platelet-derived glycocalicin. Giant. 45.

[435.] The procaryotic system (e.g., E. coli) lacks post-translational modification mechanisms, such as glycosylation and sulfation mechanisms. For this reason, the fact that ΥΙ-scFv does not recognize the bacteria-produced recombinant platelet-derived glycocalicin supports the conclusion that post-translational modifications, such as glycosylation and sulfation, are necessary for the binding of sc-scFv to glycocalicin.

FACS protocol for blood / bone marrow samples [436.] The samples were provided by hospitals. Patient samples of 30 μΐ / tube. Add 5 μΐ / tube CD33-APC (for AML), or CD19-APC (for B-CLL), or CD38-APC (for multiple myeloma). Add 5 μΐ / tube CD45-PerCp and 5 μΐ scFv-nebo, or control scFv-N31, or CD162-PE (KPL1). The tubes are incubated for 30 minutes at 4 ° C with slow stirring. Wash with 2 ml PBS and centrifuge for 5 minutes at 1200 rpm. Discard the supernatant.

[437.] For the one-stage test, continue with the ski step:

[438.] Add 500 μΐ of BD Lysine diluted 1:10 ddH ₂ O (300 μΐ per patient sample). Vortex at high speed and incubate for 12 minutes at 4 ° C. Wash as above. Discard the supernatant. Add 500 μΐ PBS. Samples are read by FACS using the obtained blood sample settings according to international standards.

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151

[439.] For two or more assays, the working buffer is PBS + 1% BSA + 0.05% sodium azide. Incubate and wash as above.

[440.] Lysis of red blood cell cells is the final step of the assay, followed by resuspension with 500 μΐ PBS.

Example 8: Construction of triabodies [441. ] The vector pHEN-Yl, Yl original encoding was amplified using PCR individually both for V _L and V _H region PCR reaction for amplifying the V _L used .byly 5'AACTCGAGTGAGCTGACACAGGACCCT sense oligonucleotide and antisense oligonucleotide product 5'TTTGTCGACTCATTTCTTTTTTGCGGCCGCACC The expected ~ 350 bp cDNA was purified, sequenced, and digested with XhoI and NotI.

[442.] The same procedure was used to amplify the V _{H region} (using the sense oligonucleotide 5'ATGAAATACCTATTGCCTACGG and the anti-sense oligonucleotide 5'AACTCGAGACGGTGACCAGGGTACC). The PCR product for V _H was digested with the restriction enzymes NcoI and XhoI. Three times ligation was performed into the pHEN vector, pre-digested with NcoI-NotI. The final vector was designated pTria-Y1.

[443.] After transformation of E. coli, a number of clones were removed for further analysis, which included DNA sequencing, protein expression, and extraction from the periplasmic space of the bacteria. SDS-PAGE under reducing conditions and Western blotting were performed to confirm the size of the Y1 triabodies.

152

Example 9: Construction of diabodies The above vector pTria-Y1 was linearized with the restriction enzyme XhoI and synthetic complementary double-stranded oligonucleotides (5'-TCGAGAGGTGGAGGCGGT and 5'TCGAACCGCCTCCACCTC) were pre-annealed and ligated to the heavy XhoI site between the Y1-site. -Light chains. This new vector was designated as pDia-Y1. As described for triabodies, DNA sequence determination and protein expression were performed.

Example 10: Expression and purification of diabodies and triabodies.

[445.] Expression in E. coli was essentially as described for scFv-ΥΙ. However, purification of the Y1 antibody diabodies and triabodies from the periplasm of transformed E. coli cells was different. The scFv Y1 monomeric form can be purified on a Protein A-bound Sepharose affinity column. However, the multimeric Y1 forms are inefficiently purified by this procedure. For this reason, periplasmic proteins extracted from bacteria were precipitated overnight with 60% ammonium sulfate, resuspended in H ₂ O, and loaded onto a size exclusion gel Sephacryl200 (Pharmacia) column, previously equilibrated with 0.1xPBS. Fractions were collected and analyzed by HPLC and individual fractions containing either dimeric or trimeric forms were concentrated for FITC labeling and FACS analysis.

Example 11: Binding of Y1 diabodies and triabodies to cells [446.] FACS analysis was performed on Jurkat cells using a three step staining procedure ("three step")

153 ........

staining procedure). First, crude extracts or purified unlabeled scFvs are stained, then murine anti-myc antibodies and finally FITC- or PE-conjugated anti-mouse antibodies. FACS analysis requires 5-8x10 ⁵ cells resuspended in PBS + 1% BSA. Binding was performed for 1 hour at 4 ° C. After each step, cells were washed and resuspended in PBS + 1% BSA. After the final staining step, cells were fixed by resuspending in PBS, 1% BSA, 2% formaldehyde and then read by FACS (BectonDickinson).

[447.] Y1-scFv binding was compared to diabodies and triabodies binding. In this analysis, the binding profile of all three forms was very similar, suggesting that the above modifications in the molecule do not alter, obscure, or interfere with the apparent binding affinity of Y1 to their ligand.

Example 12: Study of S-S affinity of Y1-dimer compared to CONY1 and Y1-IgG using a Radioreceptor Binding Assay (RRA) on KG-1 cells.

[448.] The assay system involved the use of radioactive ligands that were prepared by iodizing with ¹²⁵ L using chloramine T to construct a Y1-IgG or Bolton-Hunter (CONY1) reagent. The test tubes contained 5x10 ⁶ KG-1 cells per 0.2 ml and a labeled isotope indicator with varying amounts of unlabeled competitor in PBS + 0.1% BSA, pH 7.4. After one hour of incubation with shaking at 4 ° C, the cells were thoroughly washed with ice buffer and used for radioactivity evaluation.

[449.] In the RRA Radioreceptor Binding Assay, 2 ng / ¹²⁵ I-Y1-IgG tube was used and competition was performed.

154 ..........

with each of the three molecules. The results obtained are shown in Figure 46. The results shown in this figure show that the affinity of the SS dimer Y1 was twice the affinity of the complete Y1 antibody and 30 times the affinity of CONY1. The approximate Y-IgG affinity estimate in this experiment is 2x10 ^-8 M.

The corresponding affinity of the dimer is therefore 4x10 ⁸ M.

[450.] In the second RRA assay using labeled CONY1, 100 ng / tube of ¹²⁵ I-Y1-IgG was used and competition was performed with each of the three molecules. The results obtained are shown in Figure 47. The results shown in this figure show that the affinity of the SS dimer Y1 was 20 times higher than that of CONY1. The approximate affinity estimation of CONY1 in this experiment is 10 ⁶ M. The corresponding dimer affinity is therefore 5x10 ^-8 M.

Example 13: Production of Y1-cys-KAK (cysteine dimer) [451.] One liter of λρα-yl-cys-KAK bacterial culture was induced at 42 ° C for 2-3 hours. The culture was centrifuged at 5000 rpm for 30 minutes. The pellet was resuspended in 180 ml TE (50 mM Tris-HCl pH 7.4, 20 mM EDTA). 8 ml of lysozyme (from a 5 mg / ml stock solution) was added and incubated for 1 hour. Next, 20 ml of 5M NaCl and 25 ml of 25% Triton were added and incubated for an additional hour. The mixture was centrifuged at 13,000 rpm for 60 minutes at 4 ° C. The supernatant was discarded. The pellet was resuspended in TE with a tissue homogenizer. This procedure was repeated 3-4 times until the light gray color of the inclusion bodies (pellet). The inclusion bodies were solubilized in 6M guanidine-HCl,

0.1M Tris, pH 7.4, 2mM EDTA (1.5 grams inclusion bodies in 10ml solubilization buffer gave ~ 10mg / ml.

soluble protein). Such a sample was first incubated for at least 4 hours. Protein concentration was measured and adjusted to a concentration of 10 mg / ml. DTT was added to a final concentration of 65 mM and incubated overnight at room temperature. Refolding was initiated by diluting (drop by drop) 10 ml of protein to a solution containing 0.5 M arginine, 0.1 M Tris, pH 8, 2 mM EDTA, 0.9 mM GSSG. The refolding solution was incubated at ~ 10 ° C for 48 hours. The protein-containing refolding solution was dialyzed against a buffer containing 25 mM phosphate buffer, pH 6, 100 mM urea, and concentrated to 500 ml. The concentrated / dialyzed solution was bound to an SP-Sepharose column and the protein was eluted with a NaCl gradient (up to 1M).

Example 14: GC (Glycocalicin) ELISA [452.] 100 μΐ of purified glycocalicin was incubated overnight at 4 ° C in 96-well Maxisorp plates. The plate was washed with PBTS (PBS + 0.05% Tween) 3 times, then 200 ml PBTS-milk (PBTS + 2% skim milk) for 1 hour at room temperature. The plate was washed with PBTS and monomer or dimer (100 ml) in PBTS-milk was added at various concentrations for 1 hour at room temperature. Then, the plate was washed and anti-V _L polyclonal antibody (obtained from Y1-derived V _L rabbits immunized) (diluted 1: 100 in PBTS-milk) was added for one hour. The plate was washed and anti-rabbit HPR was added for another one hour. The plate was washed 5 times and 100 μΐ of TMB substrate was added for approximately 15 minutes, then 100 μΐ 0.5 H2SO4 was added to stop the reaction. Optical density was measured at 450 nm on an ELISA reader.

[453.] Post-translational modifications, such as glycosylation and sulfation, are necessary for binding of scFv and commercially available antibodies to GC. The procaryotic (?. Coli) system lacks the mechanisms of posttranslational modifications such as glycosylation and sulfation.

Example 15: Preparation of tetramers from Y1 [454.] A construct was designed in which the following LNDIFEAQKIEWHE sequence was added to the C-terminus of Y1 by PCR and cloning into an IPTG-inducible expression cassette.

The clone was named Y1-biotag. This sequence is a substrate for the BirA enzyme, an enzyme that, in the presence of free biotin, is capable of covalently attaching biotin to the lysine (K) residue (Science. 1996 Oct 4; 274 (5284): 94-6). , Altman, JD et al. The construct was produced as inclusion bodies in BL21 bacterial cells. Refolding was performed as described above. The inclusion bodies were solubilized by guanidine-DTT. Refolding was performed by dilution in a buffer containing arginine-Tris-EDTA. Dialysis and concentration was performed followed by HiTrapQ ion exchange purification.

[455.] The purified Y1-biotag scFv was incubated with BirA (provided by Avidity) and biotin according to the manufacturer's recommendations. The biotinylated Y1-biotag was analyzed by the HABA assay (which determines the amount of biotin per molecule) and was shown to contain approximately> 0.8 biotin residues / molecule.

[456.] The biotinylated Y1-biotag was incubated with Streptavidin-PE (Phycoerytrin) to form complexes and was

157 used in FACS experiments using KG-1 cells (Y1 positive). Binding sensitivity was increased at least 100-fold due to increased avidity. Streptavidin can bind up to 4 biotinylated Y1-biotag molecules.

[457.] The sequence of the Y1-biotag is as follows and is SEQ ID NO: 211:

MEVQLVESGGGWRPGGSLR LSCAASGFTF DDYGMSWVRQ

APGKGLEWVS GINWNGGSTG YADSVKGRFT ISRDNAKNSL

YLQMNSLRAE DTAVYYCARMRAPVIWGQGT LVTVSRGGGG

121 SGGGGSGGGG SSELTQDPAV SVALGQTVRI TCQGDSLRSY

161 YASWYQQKPG QAPVLVIYGK NNRPSGIPDR FSGSSSGNTA

201 SLTITGAQAE DEADYYCNSR DSSGNNWFG GGTKLTVLGG

241 GGLNDIFEAQ KIEWHE

Example 16: Characterization of Y17 activity [458.] O-sialoglycoprotein endoprotease enzyme from Pastor! haemolytica, which selectively cleaves GPIb from human platelets, was used to determine the specificity of Y17 binding to GPIba. The O-sialoglycoprotein endoprotease specifically cleaved only proteins containing sialylated, O-linked glycans, and did not cleave N-linked glycoproteins and non-glycosylated proteins. This enzyme is known to cleave GPIb, which is strongly O-glycosylated but does not cleave GPIIbIIa or other receptors on platelets. Incubation of washed plates with O-sialoglycoprotein endoprotease that cleaves GPIb breaks the binding to GPIb in Y17, as well as that of the monoclonal antibody (MCA466S-serotec) directed against GPIb as shown by immunoblots and FACS analysis. The endoprotease did not alter the binding of the monoclonal antibody (anti CD61) directed against GPIIb / IIIa (Fig. 4).

Example 17: Identification of epitopes for Y17 on platelet

GPIb [459.] An interesting approach for identifying Y1 epitopes on platelet GPIb is to use the enzyme endoprotease, whose cleavage sites on platelet GPTb are fully characterized.

Figure 17.1: Effect of mocarhagine on Y1 epitope mapping [460.] Mocarhagine, a cobra snake venom metalloproteinase, cleaves platelet GPIba specifically at one site between residues Glu-282 and Asp-283, creating two stable products, the 45 kDa N-terminal fragment (His -1 to Glu-282), which is found in the supernatant, and a membrane-bound 100kDa C-terminal fragment.

[461.] Washed plates were treated with mocarhagine and the plate lysate was separated on SDS-polyacrylamide gels and transferred to nitrocellulose. Analysis of the washed, mocarhagine-treated plates in the Western blot with Y1 results in a loss of band corresponding to GPIb (135 kDa) and binding of Y17 to the N-terminal 45 kDa cleavage product. The monoclonal antibody MCA466S, directed against the C-terminal fragment of GPIba, reacted with the 100kDa C-terminal fragment, while the monoclonal antibody S.C.7071, which recognizes the N-terminus of GPIba, reacted with the same 45kDa N-terminal fragment that was recognized by Y17 (FIG. 14).

[462.] The effect of mocarhagine on glycocalicin gave results similar to those found in washed plates and showed binding of Y1 and the monoclonal antibody S.C.7071 to the 45 kDa N-terminal cleavage product of GPIba.

159 ..........

(Fig. 8). Based on these results, it can be concluded that the Y1 epitope is contained within the sequence His-1 to Glu-282.

17.2: Effect of cathepsin G on epitope mapping for Y17.

[463.] Cathepsin G, a serine protease from neutrophils, cleaved glycocalicin between residues Leu-275 and Tyr-276 and in the second cleavage site between residues Val-296 and Lys-297. Glycocalicin treated with cathepsin G provides two N-terminal fragments, a small 42kDa fragment (His-1 to Leu-275) and a large 45kDa N-terminal fragment (His-1 to Val-296), in addition to ~ 95kDa C- end fragment. Glycocalicin and fragments of glycocalicin, generated by its cleavage by cathepsin G, were separated on SDS polyacrylamide gels and transferred to nitrocellulose. Y17 bound to the larger fragment (His-1 to Val-296) but not to the smaller fragment (His-1 to Leu-275). In addition, the monoclonal antibody S.C.7071, which recognizes an epitope within His-1 to Leu-275, blots both fragments (Fig. 12). Analysis of N-terminal peptide fragments from proteolysis by mocarhagine and cathepsin G confirmed that the GBP1ba amino acid sequence Tyr-276 to Glu-282 is an important recognition motif for Y17 binding.

Example 18: Effect of Y17-scFv on vWF-dependent agglutination [464.] The effect of Y17-scFv on vWF-dependent platelet agglutination was tested at various concentrations of Y17. In contrast to Y1, Y17 at a final concentration of 10, 25 or 50 µg / ml did not show vWF-dependent agglutination of washed platelets induced by ristocetin. Analysis of N-terminal peptide fragments from proteolysis by mocarhagine and cathepsin G confirmed that the amino acid sequence

160 • ·

• · · · · · · · ·

GBPIba Tyr-276 to Glu-282 is an important recognition motif for Y17 and Y1 binding. Since Y17 does not inhibit platelet aggregation, Y1 and Y17 appear not to bind to the same sequences but to the overlapping sequences.

Claims (322)

An isolated epitope according to Formula I (S) _r I [fWX-CY-P) _t -P] _q in which W is any amino acid other than aspartate and glutamate Y is any naturally occurring compound that can be sulfated is (A) _m (A) _n (X) u, or (X) _u (A) _n (A) _m , or (A) _n (X) _u ( A) _m , or (A) _n (A) _m (X) _u , or (X) _u (A) _m (A) _n , or (A) _m (X) _u (A) _n S is a sulfate or sulfated molecule X is any amino acid except aspartate, glutamate, or tyrosines A is any negatively charged amino acid or leucine, isoleucine, proline, phenylalanine, serine, or glycine q is 1 to 6 z is 0, 1 or 2 r is 0 or 1 • * 162 t is 1, 2 or 3 at is 0 to 2 n is 0 to 3 m is 0 to 3, where, if n = 0, > 0; where, if q at least one of Y is then m> 0; where, if m = 0, then n is 1, r is 1 and if q is> 1, sulfated; and further, where isolated the epitope is capable of binding to an antibody, antigen-binding fragment thereof, or complex thereof comprising an antibody or binding fragment thereof comprising a first hypervariable region comprising SEQ ID NO: 8 or SEQ ID NO: 20. The isolated epitope of claim 1, wherein the sulfated compound is a peptido, glyco- or lipoconjugate. An isolated epitope of formula I wherein: W is glycine, Y is a tyrosine peptide conjugate or an asparagine, serine or threonine glyco-conjugate; A is glutamate, γ carboxy glutamate or aspartate; and q is 1, 2 or 3. The isolated epitope of claim 3, wherein: * Y is a tyrosine peptide conjugate; q is 3; and r is 1. An isolated epitope according to Formula II ( ^S ) r (S) _r (S) _r III (ww-γVP-cn-β-cYw, _TT) . in which W is any amino acid other than aspartate and glutamate Y is any naturally occurring compound that can be sulfated P is (A) _m (A) _n (X) _u , or (X) _u (A) _n (A) _m , or (A) _n (X) _u (A) _m , or (A) _n (A) ) _m (X) _u , or (X) _u (A) _m (A) _n , or (A) _m (X) _u (A) _n S is a sulfate or sulfated molecule X is any amino acid except aspartate, glutamate, or tyrosine A is any negatively charged amino acid or leucine., Isoleucine, proline, phenylalanine, serine, or glycine z is 0, 1 or 2 164 • r is 0 or 1 t is 1, 2 or 3 u is 0 to 2 n is 0 to 3 m is 0 to 3, where, if n = 0, then m> 0; where, if m = 0, then n> 0; wherein, at least one of Y is sulfated; and further wherein the isolated epitope is capable of being bound by the antibody, antigen-binding fragment thereof, or complexes thereof, comprising the antibody or binding fragment thereof, comprising a first hypervariable region comprising SEQ ID NO: 8 or SEQ ID NO: 20. The isolated epitope of claim 5, wherein the sulfated compound is a peptido, glyco- or lipoconjugate. The isolated epitope of claim 5, wherein W is glycine Y is a peptide-conjugate of tyrosine or a glyco-conjugate of asparagine, lilac or threonine A is glutamate, γ carboxy glutamate or aspartate, leucine, isoleucine, proline, phenylalanine, serine or glycine. 165 ··· ············································· The isolated epitope of claim 7, wherein Y is a tyrosine peptide conjugate; q is 3; a r j e 1. An isolated epitope of formula III (S) r (S) r (S) _r (G) _from (X) _u (E) _n (D) _m (Y) _t (X) u (E) n (D) _m CY) t (X) _u (E) _n (D) _m CY) _t (D) _n (E) _n (X) _u ⁽¹ 11) in which: G is glycine E is glutamate D is aspartate Y is tyrosine S is a sulfate or sulfated compound X is any amino acid except for the above z is 0, 1 or 2 t is 1, 2 or 3 r is 0 or 1 u is 0 to 2 166 n is Ο to 3 m is 0 to 3 wherein, at least one of Y is sulfated; where, if n = 0, then m> 0; where, if m = 0, then n> 0; and further wherein the isolated epitope is capable of being bound by the antibody, antigen-binding fragment thereof, or complexes thereof, comprising the antibody or binding fragment thereof, comprising a first hypervariable region comprising SEQ ID NO: 8 or SEQ ID NO: 20. The isolated epitope of claim 9, wherein r is 1. The isolated epitope of any one of claims 1 to 8, wherein the naturally occurring compound Y, which is capable of being sulfated, comprises a lipid, carbohydrate, peptide, glycolipid, glycoprotein, lipoprotein and / or lipopolysaccharide molecule. The isolated epitope homolog or mimetic of any one of claims 1 to 10. The isolated epitope of any one of claims 1 to 10, wherein the isolated epitope comprises at least one post-translational modification in addition to sulfation. A composition comprising an isolated epitope according to any one of claims 1 to 10. • · ·· · ·· · 167 ··························· 15. The composition of claim 14, further comprising an upstream or upstream region capable of increasing epitope binding capacity. The composition of claim 15, wherein the upstream or upstream region is near epitopes. An isolated polynucleotide encoding at least a portion of an isolated epitope according to any one of claims 1 to 10. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, capable of binding or cross-reacting with the isolated epitope of claim 1. The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, capable of binding or cross-reacting with the isolated epitope of claim 5. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, capable of binding or cross-reacting with an isolated epitope according to claim 9. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, capable of binding or cross-reacting with an isolated epitope according to any one of claims 2 to 4, 6 to 8, or 10 to 13. A method of preparing an antibody, antigen-binding fragment thereof, or complex thereof comprising 168 at least one antibody, or binding fragment thereof, capable of binding or cross-reacting with an isolated epitope according to any one of claims 1 to 13, comprising the steps of: (a) providing a phage expression library; (b) providing an isolated epitope according to any one of claims 1 to 13; (c) panning the phage expression library to obtain phage particles expressing an oligopeptide or polypeptide capable of binding to an isolated epitope; (d) producing an antibody, binding fragment thereof, or complex thereof comprising an antibody or binding fragment thereof, comprising a peptide or polypeptide capable of binding to an isolated epitope. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof having the binding capabilities of an scFv antibody fragment of SEQ ID NO: 25 or SEQ ID NO: 203. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof having a binding peptide or polypeptide, wherein the peptide or polypeptide comprises a first hypervariable region comprising SEQ ID NO: 8 or SEQ ID NO: 20. The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof according to any one of claims 23 to 24, wherein the peptide or polypeptide further has a second hypervariable. 169 · region containing SEQ ID NO: 115 and / or third a hypervariable region comprising SEQ ID NO: 114. 26. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, capable of binding to a peptide or polypeptide epitope having a length of about 3 to 126 amino acid residues and comprising at least two acidic amino acids and at least one sulfated tyrosine residue. The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 26, wherein the epitope further comprises a proline, leucine, isoleucine, serine, glycine, or phenylalanine residue. The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 23 to 27, wherein the antibody or antigen-binding fragment thereof is further capable of binding to an epitope on a lipid molecule, carbohydrate, peptides, glycolipid, glycoprotein, lipoprotein and / or lipopolysaccharide. The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 28, wherein the epitope on the lipid, carbohydrate, peptide, glycolipid, glycoprotein, lipoprotein and / or lipopolysaccharide molecule comprises at least one sulfated compound. • · · An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, which is capable of binding to at least two different molecules selected from the group consisting of PSGL-1, fibrinogen γ ', GPIba, heparin, lumican , complement compound 4 (CC4), an inter-alpha inhibitor and prothrombin. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, which is capable of binding to at least two different molecules selected from the group consisting of PSGL-1, fibrinogen γ ', GPIba, heparin, lumican , complement compound 4 (CC4), an inter-alpha inhibitor and prothrombin, and capable of binding to at least one cell type selected from the group consisting of cells B-CLL, AML cells, multiple myeloma cells, and metastatic cells. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 31, which is capable of binding to each molecule selected from PSGL-1, fibrinogen γ ', GPIba, and heparin. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 32, capable of binding to each molecule selected from PSGL-1, fibrinogen γ ', GPIba, and heparin, and capable of binding to a molecule. at least one cell type selected from the group consisting of B-CLL cells, AML cells, multiple myeloma cells and metastatic cells. • · · · · · 171 34. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof capable of binding to at least two different molecules selected from the group consisting of PSGL-1, fibrinogen γ ', GPIba, heparin, lumican , complement compounds 4 (CC4), an inter-alpha inhibitor and prothrombin, and further capable of binding to a lipid, carbohydrate, peptide, glycolipid, glycoprotein, lipoprotein, and / or lipopolysaccharide molecule. The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 34, wherein the epitope on the lipid, carbohydrate, peptide, glycolipid, glycoprotein, lipoprotein and / or lipopolysaccharide molecule comprises at least one sulfated compound. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof capable of cross-reacting with one or more epitopes, each epitope comprising one or more sulfated tyrosine residues and at least one cluster of two or more; more acidic amino acids. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 36, which is capable of cross-reacting with PSGL-1. 38. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 37, which binds to said antibody. 172 to QATEYEYLDYDFLPETE, wherein at least one tyrosine residue is sulfated. The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 36, which is capable of cross-reacting with GPIba. The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 36, which binds to DEGDTDLYDYYPEEDTEGD, wherein at least one tyrosine residue is sulfated. The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 39, which binds TDLYDYYPEEDTE, wherein at least one tyrosine residue is sulfated. The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 39, which binds to DEGDTDLYDYYP, wherein at least one tyrosine residue is sulfated. The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 39, which binds to YDYYPEE, wherein at least one tyrosine residue is sulfated. 44. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody, or 173 a binding fragment thereof, according to claim 39, which binds to TDLYDYYP, wherein at least one tyrosine residue is sulfated. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 36, which is capable of cross-reacting with fibrinogen γ '. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 45, which binds to EPHAETEYDSLYPED, wherein at least one tyrosine residue is sulfated. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 36, which is capable of cross-reacting with heparin. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 36, which is capable of cross-reacting with complement compound 4 (CC4). The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 48, which binds to MEANEDYEDYEYDELPAK, wherein at least one tyrosine residue is sulfated. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 36, which is capable of 174 cross-reactions with at least one cell type selected from the group consisting of B-CLL cells, AML cells, multiple myeloma cells and metastatic cells. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, which is capable of inhibiting cellular scrolling. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, which is capable of inhibiting inflammation. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, which is capable of inhibiting autoimmune disease. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, which is capable of inhibiting thrombosis. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, which is capable of inhibiting restenosis. 56. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or antibody 175, or a binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, which is capable of inhibiting metastasis. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, which is capable of inhibiting the growth and / or replication of tumor cells. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, which is capable of increasing tumor cell mortality. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, which is capable of inhibiting the growth and / or replication of leukemic cells. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, which is capable of increasing the rate of mortality of leukemic cells. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, which is capable of increasing the susceptibility of diseased cells to damage by agents against the disease. 176 • · An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, which is capable of increasing the sensitivity of tumor cells to damage by anticancer agents. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, which is capable of increasing the susceptibility of leukemic cells to leukemia damage. 6.4. The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, which is capable of inhibiting an increase in the number of tumor cells in a patient having cancer. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, which is capable of reducing the number of tumor cells in a patient having cancer. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, which is capable of inhibiting an increase in the number of leukemic cells in a patient having leukemia. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 18. 177 20, 23 to 25, which are capable of reducing the number of leukemia cells in a patient having leukemia. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or cell-cell binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, which is capable of inhibiting the formation of a cell-cell, cell- matrix, platelet-matrix, platelet-platelet and / or cell-platelet. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, which is capable of inhibiting cell-cell, cell-matrix, platelet-matrix adhesion, platelet-platelet and / or cell-platelet. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, which is capable of inhibiting cell-cell, cell-matrix, platelet-matrix aggregation, platelet-platelet and / or cell-platelet. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, bound or complexed with an agent selected from the group consisting of anti-cancer, antimetastatic, antileukemic substances, anti-disease, anti-adhesion, antithrombotic, 178 antiresthenotic, anti-autoimmune, anti-aggregation, antibacterial, antiviral and anti-inflammatory agents. 72. The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 71, wherein the agent is an antiviral agent selected from the group consisting of acyclovir, ganciclovir and zidovudine. The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 71, wherein the agent is an antithrombotic / antirestenotic agent selected from the group consisting of cilostazol, dalteparin sodium, reviparin sodium and aspirin. 74. The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 71, wherein the agent is an anti-inflammatory agent selected from the group consisting of zaltoprofen, pranoprofen, droxicam, acetyl salicylic 17, diclofenac, ibuprofen, dexibuprofen, sulindac, naproxen, amtolmetin, celecoxib, indomethacin, rofecoxib and nimesulide. 75. The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof according to claim 71, wherein the agent is an anti-autoimmune agent selected from the group consisting of leflunomide, denileukin diftitox, subrea, WinRho SDF, defibrotide and cyclophosphamide. . • · 179 · ···················· The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 71, wherein the agent is an anti-adhesion / anti-aggregation agent selected from the group consisting of limaprost, clorcromen and hyaluronic acid. 77. The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 71, wherein the agent is a substance selected from the group consisting of toxins, radioisotopes, and pharmaceutical agents. 78. The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 77, wherein the toxin is selected from the group consisting of gelonin, Pseudomonas exotoxin (PE), PE40, PE38, a diphtheria toxin, ricin and their modifications and derivatives. 79. The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof according to claim 77, wherein the radioisotope is selected from the group consisting of gamma-emitters, positron emitters, x-ray emitters, beta-emitters and alpha-emitters. . 80. The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 77, wherein the radioisotope is selected from the group consisting of ⁿⁱ indium, ¹¹³ indium, ^99m rheniurn, ¹⁰⁵ rhenium, ¹⁰¹ rhenium , technetium ^{99 m, 121 m} tellurium, tellurium ^{122 m, 125 m} tellurium, ¹⁶⁵ thulium, ¹⁶⁷ thulium, 180 ¹⁶⁸ thulium, ¹²³ iodine, ¹²⁶ iodine, ¹³¹ iodine, ¹³³ iodine, ^81m krypton, ³³ xenon, ⁹⁰ yttrium, ²¹³ bismuth, ⁷⁷ bromine, ¹⁸ fluorine, ⁹⁵ ruthenium, ⁹⁷ ruthenium, ¹⁰³ ruthenium, ¹⁰⁵ ruthenium, ¹⁰⁷ mercury, ²⁰³ mercury, ⁶⁷ gallium and ⁶⁸ gallium. The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 78, wherein the pharmaceutical agent is selected from the group consisting of Doxorubicin (adriamycin), morpholinodoxorubicin, methoxymorpholinyldoxorubicin, cis-platin, taxol. , calicheamicin, vincristine, cytarabine (Ara-C), cyclophosphamide, prednisone, daunorubicin, morpholinodaunorubicin, methoxymorpholinyldaunorubicin, idarubicin, fludarabine, chlorambucil, interferon alpha, hydroxyurea, thomolide and thomolide derivatives, thomolomide and thomolomide, thomolomide and thomolomide, thomolomide and thomolomide derivatives. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, bound or complexed to a vehicle or carrier capable of being attached or complexed with more than one substance. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof according to any one of claims 18 to 20, 23 to 25, wherein the vehicle or carrier is selected from the group consisting of dextran, lipophilic polymers, hydrophilic polymers, HPMA and liposomes. 181 · «9 9 9 9 · 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 í * · · · · · 9 · An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, bound or complexed to a radioactive isotope or other imaging agent. 85. A diagnostic kit comprising the antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof according to claim 84. 86. A pharmaceutical composition comprising an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, in an amount effective to inhibit cell scrolling. 87. A pharmaceutical composition comprising an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, in an amount effective to inhibit inflammation. . 88. A pharmaceutical composition comprising an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, in an amount effective to inhibit autoimmune disease. 89. A pharmaceutical composition comprising the antibody, antigen-binding fragment thereof, or 182 a complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, in an amount effective to inhibit thrombosis. 90. A pharmaceutical composition comprising an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, in an amount effective to inhibit restenosis. . 91. A pharmaceutical composition comprising an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, in an amount effective to inhibit metastasis. . 92. A pharmaceutical composition comprising an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, in an amount effective to inhibit growth and / or tumor cell replication. 93. A pharmaceutical composition comprising an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, in an amount effective to increase mortality. tumor cells. 94. A pharmaceutical composition comprising an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody, or 183 • ··· ·························· A binding fragment thereof according to any one of claims 18 to 20, 23 to 25, in an amount effective to inhibit the growth and / or replication of leukemic cells. 95. A pharmaceutical composition comprising an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, in an amount effective to increase rate. leukemia cell mortality. 96. A pharmaceutical composition comprising the antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, in an amount effective to increase sensitivity. diseased cells to be damaged by agents against the disease. 97. A pharmaceutical composition comprising an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, in an amount effective to increase sensitivity. tumor cells to be damaged by anticancer agents. 98. A pharmaceutical composition comprising an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, in an amount effective to increase sensitivity. leukemic cells to be damaged by antileukemic agents 184 99. A pharmaceutical composition comprising an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, in an amount effective to inhibit the increase. number of tumor cells in a patient having cancer. 100. A pharmaceutical composition comprising an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, in an amount effective to reduce the number. tumor cells in a patient having cancer. 101. A pharmaceutical composition comprising an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, in an amount effective to inhibit the increase. number of leukemic cells in a patient having leukemia. 102. A pharmaceutical composition comprising an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, in an amount effective to reduce the number. leukemia cells in a patient having leukemia. 103. A pharmaceutical composition comprising an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, in an amount effective to inhibit aggregation cell185 ··· ·· ·· ··· cell, cell-matrix, platelet-matrix, platelet-platelet and / or cell-platelet. 104. A pharmaceutical composition comprising an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, in an amount effective to inhibit formation a cell-cell complex, cell-matrix, platelet-matrix, platelet-plate and / or cell-platelet. 105. A pharmaceutical composition comprising an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, in an amount effective to inhibit adhesion. cell-cell, cell-matrix, platelet-matrix, platelet-platelet and / or cell-platelet. 106. A pharmaceutical composition comprising an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, bound or complexed with an agent selected from the group consisting of anti-cancer, anti-metastatic, anti-leukemic, anti-disease, anti-adhesion, anti-thrombotic, anti-esthenotic, anti-autoimmune, anti-aggregation, anti-bacterial, anti-viral and anti-inflammatory agents. 107. A pharmaceutical composition comprising an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody, or 186 a binding fragment thereof, according to claim 106, wherein the agent is an antiviral selected from the group consisting of acyclovir, ganciclovir and zidovudine. 108. A pharmaceutical composition comprising an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof according to claim 106, wherein the agent is an antithrombotic / antiresthenotic agent selected from the group consisting of cilostazol, dalteparin sodium, reviparin sodium and aspirin. 109. A pharmaceutical composition comprising an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof according to claim 106, wherein the agent is an anti-inflammatory agent selected from the group consisting of zaltoprofen, pranoprofen, droxicam, acetyl salicylic 17, diclofenac, ibuprofen, dexibuprofen, sulindac, naproxen, amtolmetin, celecoxib, indomethacin, rofecoxib and nimesulide. 110. A pharmaceutical composition comprising the antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof according to claim 106, wherein the agent is an anti-immune agent selected from the group consisting of leflunomide, denileukin diftitox , subrea, WinRho, SDF, defibrotide and cyclophosphamide. 111. A pharmaceutical composition comprising an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody, or 187 a binding fragment thereof, according to claim 106, wherein the agent is an anti-adhesion / anti-aggregating agent selected from the group consisting of limaprost, clorcromen, and hyaluronic acid. 112. The pharmaceutical composition of claim 106, wherein the agent is selected from the group consisting of toxins, radioisotopes, and pharmaceutical agents. 113. The pharmaceutical composition of claim 106 wherein the toxin is selected from the group consisting of gelonin, Pseudomonas exotoxin (PE), PE40, PE38, ricin, and modifications and derivatives thereof. 114. The pharmaceutical composition of claim 106 wherein the radioisotope is selected from the group consisting of gamma-emitters, positron-emitters, x-ray emitters, beta-emitters and alpha-emitters. 115. The pharmaceutical composition of claim 106, wherein the radioisotope is selected from the group consisting of indium ^111, indium ^{113, 99 m} rheniurn, ¹⁰⁵ Rhenium, ¹⁰¹ Rhenium, "technetium, tellurium ^{121 m, 122 m} tellurium, tellurium ^{125R, 165} thulium , ¹⁶⁷ thulium, ¹⁶⁸ thulium, ¹²³ iodine, ¹²⁶ iodine, ¹³¹ iodine, ¹³³ iodine, ⁸¹ⁱⁿ krypton, ³³ xenon, ⁹⁰ yttrium, ²¹³ bismuth, ⁷⁷ bromine, ¹⁸ fluorine, ⁹⁵ ruthenium, ⁹⁷ ruthenium, ¹⁰³ ruthenium, ¹⁰⁵ ruthenium, ¹⁰⁷ mercury, ²⁰³ mercury, ⁶⁷ gallium and ⁶⁸ gallium. 116. The pharmaceutical composition of claim 106, wherein the pharmaceutical agent is selected from the group consisting of Doxorubicin (adriamycin), morpholinodoxorubicin, methoxymorpholinyldoxorubicin, cisplatin, taxol, calicheamicin, vincristine, cytarabine. 188 (Ara-C), cyclophosphamide, prednisone, daunorubicin, morpholinodaunorubicin, methoxymorpholinyldaunorubicin, idarubicin, fludarabine, chlorambucil, interferon alpha, hydroxyurea, temozolomide, thalidomide and bleomycin and derivatives and combinations thereof. 117. A pharmaceutical composition comprising an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, bound or complexed to a vehicle, or a carrier capable of being attached or complexed with more than one agent. 118. The pharmaceutical composition of claim 117, wherein the vehicle or carrier is selected from the group consisting of dextran, lipophilic polymers, hydrophilic polymers, HPMA, and liposomes. 119. Use of an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for the preparation of a medicament that is capable of inhibiting cellular scrolling. 120. The use of an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for the preparation of a medicament capable of inhibiting inflammation. 189 121. Use of an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for the preparation of a medicament that is capable of inhibiting autoimmune disease. 122. The use of an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for the preparation of a medicament capable of inhibiting restenosis. 123. Use of an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for the preparation of a medicament capable of inhibiting thrombosis. 124. The use of an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for the preparation of a medicament capable of inhibiting metastasis. 125. Use of an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for the preparation of a medicament capable of inhibiting the growth and / or replication of tumor cells. 126. The use of an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of: 190 of claims 18 to 20, 23 to 25, for the preparation of a medicament capable of increasing the rate of mortality of tumor cells. 127. The use of an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for the preparation of a medicament capable of inhibiting the growth and / or replication of leukemic cells. 128. Use of an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for the manufacture of a medicament capable of increasing the mortality rate of leukemic cells. 129. The use of an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for the preparation of a medicament capable of increasing the susceptibility of diseased cells to substance damage. against disease. 130. Use of an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for the preparation of a medicament capable of increasing the sensitivity of tumor cells to anticancer injury substances. 131. Use of an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for the preparation of a medicament which is 191 capable of increasing the sensitivity of leukemic cells to damage by antileukemic agents. 132. The use of an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for the manufacture of a medicament capable of inhibiting an increase in tumor cell numbers in a patient. having cancer. 133. The use of an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for the manufacture of a medicament capable of reducing the number of tumor cells in a patient having cancer. 134. The use of an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for the manufacture of a medicament capable of inhibiting an increase in leukemia cell counts in a patient. having leukemia. 135. Use of an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for the manufacture of a medicament capable of reducing the number of leukemic cells in a patient having leukemia. 136. Use of an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one 192 an antibody or binding fragment thereof according to any one of claims 18 to 20, 23 to 25, for the preparation of a medicament that is capable of inhibiting the formation of a cell-cell, cell-matrix, platelet-matrix, platelet-platelet and / or cell-platelet complex. 137. The use of an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for the preparation of a medicament capable of inhibiting cell-cell aggregation, cell -matrix, platelet-matrix, platelet-platelet and / or cell-platelet. 138. Use of an antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for the preparation of a medicament capable of inhibiting cell-cell adhesion, cell -matrix, platelet-matrix, platelet-platelet and / or cell-platelet. 139. The method of any one of claims 119 to 138, wherein the antibody or binding fragment thereof, or a complex thereof comprising at least one antibody or binding fragment thereof, is bound or complexed with an agent selected from the group consisting of anti-cancer, antimetastatic agents. , anti-leukemic substances, anti-disease substances, anti-adhesion, anti-thrombotic, anti-esthenotic, anti-autoimmune, anti-aggregation, anti-bacterial, anti-viral and anti-inflammatory substances. 193 140. The method of claim 139, wherein the agent is an antiviral selected from the group consisting of acyclovir, ganciclovir and zidovudine. 141. The method of claim 139, wherein the antithrombotic / antirestenotic agent is selected from the group consisting of cilostazol, dalteparin sodium, reviparin sodium and aspirin. 142. The method of claim 139, wherein the agent is an anti-inflammatory agent selected from the group consisting of zaltoprofen, pranoprofen, droxicam, acetyl salicylic 17, diclofenac, ibuprofen, dexibuprofen, sulindac, naproxen, amtolmetin, recoxacin, indecoxibin, indecoxibin, indecoxibin, indecoxibin, indecoxib nimesulide. 143. The method of claim 139, wherein the agent is an anti-autoimmune agent selected from the group consisting of leflunomide, denileukin diftitox, subrea, WinRho SDF, defibrotide, and cyclophosphamide. 144. The method of claim 139, wherein the agent is an anti-adhesion / anti-aggregating agent selected from the group consisting of limaprost, clorcromen, and hyaluronic acid. 145. The method of claim 139, wherein the agent is a substance selected from the group consisting of toxins, radioisotopes, and pharmaceutical agents. 146. The method of claim 139, wherein the toxin is selected from the group consisting of gelonin, 194 Pseudomonas exotoxin (PE), PE40, PE38, diphtheria toxin, ricin and their modifications and derivatives. 147. The method of claim 139, wherein the radioisotope is selected from the group consisting of gamma-emitters, positron-emitters, x-ray emitters, beta-emitters, and alpha-emitters. 148. The method of claim 139, wherein the radioisotope is selected from the group consisting of ^Lu indium, indium ^{113, 99 m} rheniurn, ¹⁰⁵ Rhenium, ¹⁰¹ Rhenium, ^{99 m} techneciurn, tellurium ^{121 m, 122 m} tellurium, tellurium ^{125 meters, 165} thulium, ¹⁶⁷ thulium, ¹⁶⁸ thulium, ¹²³ iodine, ¹²⁶ iodine, ¹³¹ iodine, ¹³³ iodine, ^81m krypton, 33, 95, xenon, ⁹⁰ yttrium, ^zinc bismuth, bromine, ¹⁰ fluorine, ^S3 ruthenium, ruthenium, ¹⁰³ ruthenium, ¹⁰⁵ ruthenium, ¹⁰⁷ mercury, ²⁰³ mercury, gallium and ⁶⁸ gallium. 149. The method of claim 139, wherein the pharmaceutical is selected from the group consisting of Doxorubicin (adriamycin), morpholinodoxorubicin, methoxymorpholinyldoxorubicin, cisplatin, taxol, calicheamicin, vincristine, cytarabine (Ara-C), cyclophosphamide, prednisone, daunorubicin, morpholinodaunorubicin, methoxymorpholinyldaunorubicin, idarubicin, fludarabine, chlorambucil, interferon alpha, hydroxyurea, temozolomide, thalidomide and bleomycin, and derivatives and combinations thereof. 150. The method of any one of claims 119 to 138, wherein the antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25 is bound. or complexed 195 with a vehicle or carrier capable of being attached or complexed with more than one substance. 151. The method of claim 150, wherein the vehicle or carrier is selected from the group consisting of dextran, lipophilic polymers, hydrophilic polymers, HPMA, and liposomes. 152. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for use as a medicament that is capable of inhibiting cellular scrolling. 153. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for use as a medicament that is capable of inhibiting inflammation. 154. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for use as a medicament capable of inhibiting autoimmune disease. 155. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for use as a medicament capable of inhibiting restenosis. 196 ··· · · ···· · ····· · · ··· 156. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for use as a medicament capable of inhibiting thrombosis. 157. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for use as a medicament capable of inhibiting metastasis. 158. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for use as a medicament that is capable of inhibiting tumor growth and / or replication cells. 159. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for use as a medicament capable of increasing the rate of mortality of tumor cells. 160. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for use as a medicament capable of inhibiting the growth and / or replication of leukemic cells. 161. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 18. 197 · ··· · ··· · ··················· 20, 23-25, for use as a medicament which is capable of increasing the rate of mortality of leukemic cells. 162. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for use as a medicament capable of increasing the susceptibility of diseased cells to substance damage against disease. 163. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for use as a medicament capable of increasing the susceptibility of diseased cells to anti-cancer damage. substances. 164. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for use as a medicament capable of increasing the susceptibility of leukemic cells to antileukemic damage substances. 165. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for use as a medicament capable of inhibiting an increase in tumor cell numbers in a patient having cancer. 166. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody, or 198 A binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, or a binding fragment thereof. , for use as a medicament that is capable of reducing the number of tumor cells in a patient having cancer. 167. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for use as a medicament capable of inhibiting an increase in the number of leukemic cells in a patient. having cancer. 168. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for use as a medicament capable of reducing the number of leukemia cells in a patient having leukemia. 169. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for use as a medicament that is capable of inhibiting cell-cell complex formation, cell-matrix, platelet-matrix, platelet-platelet and / or cell-platelet. 170. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for use as a medicament capable of inhibiting cell-cell aggregation, cell -matrix, plateletamatrix, platelet-platelet and / or cell-platelet. 199 * · «c c c c c c c 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 · »·» 171. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 18 to 20, 23 to 25, for use as a medicament capable of inhibiting cell-cell adhesion, cell -matrix, plateletamatrix, platelet-platelet and / or cell-platelet. 172. The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 152 to 171, wherein the antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or antibody thereof. the binding fragment, are bound or complexed with an agent selected from the group consisting of anti-cancer, antimetastatic, antileukemic, anti-disease, anti-adherence, antithrombotic, antirestenotic, anti-autoimmune, anti-aggregate, antibacterial, antiviral and anti-inflammatory agents. 173. The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 172, wherein the agent is an antiviral agent selected from the group consisting of acyclovir, ganciclovir and zidovudine. 174. The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 172, wherein the agent is an antithrombotic / antirestenotic agent selected from the group consisting of cilostazol, dalteparin sodium, reviparin sodium and aspirin. 200 175. The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 172, wherein the agent is an anti-inflammatory agent selected from the group consisting of zaltoprofen, pranoprofen, droxicam, acetyl salicylic 17, diclofenac, ibuprofen, dexibuprofen, sulindac, naproxen, amtolmetin, celecoxib, indomethacin, rofecoxib and nimesulide. 176. The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 172, wherein the agent is an anti-autoimmune agent selected from the group consisting of leflunomide, denileukin diftitox, subrea, WinRho SDF, defibrotide and cyclophosphamide. . 177. The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 172, wherein the agent is an anti-adhesion / anti-aggregation agent selected from the group consisting of limaprost, clorcromen and hyaluronic acid. 178. The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 172, wherein the agent is a substance selected from the group consisting of toxins, radioisotopes, and pharmaceutical agents. 179. The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 172, wherein the toxin is selected from the group consisting of gelonin, Pseudomonas. ································ 201 ............. exotoxin (PE), PE40, PE38, diphtheria toxin, ricin and modifications and derivatives thereof. 180. The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 172, wherein the radioisotope is selected from the group consisting of gamma-emitters, positron emitters, x-ray emitters, beta-emitters and alpha-emitters. . 181. The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 172, wherein the radioisotope is selected from the group consisting of ¹¹¹ indium, ¹¹³ indium, ^99m rheniurn, ¹⁰⁵ rhenium, ¹⁰¹ rhenium , ^{99 m} Tc, ^{121 m} tellurium, ^{122 m} tellurium, ^{125 m} tellurium, ¹⁶⁵ thulium, ¹⁶⁷ thulium, ¹⁶⁸ thulium, ¹²³ iodine, ¹²⁶ iodine, ¹³¹ iodine, ¹³³ iodine, ^{81 m} krypton, ³³ xenon, ⁹⁰ yttrium, ²¹³ Bi, ⁷⁷ Br, ¹⁸ fluorine, ⁹⁵ ruthenium, ⁹⁷ ruthenium, ¹⁰³ ruthenium, ¹⁰⁵ ruthenium, ¹⁰⁷ mercury, ²⁰³ mercury, ⁶⁷ gallium and ⁶⁸ gallium. ¹⁸² 182. The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 172, wherein the pharmaceutical is selected from the group consisting of Doxorubicin (adriamycin), morpholinodoxorubicin, methoxymorpholinyldoxorubicin, cis-platin, taxol. , calicheamicin, vincristine, cytarabine (Ara-C), cyclophosphamide, prednisone, daunorubicin, morpholinodaunorubicin, methoxymorpholinyldaunorubicin, idarubin.cin, fludarabine, chlorambucil, interferon alpha, hydroxyurea, thalidosomide and thernozolomide derivatives thereof. 202 * 183. The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to any one of claims 152 to 171, wherein the antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or antibody thereof. the binding fragment is bound or complexed with a vehicle or carrier that is capable of being attached or complexed with more than one agent. 184. The antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof, according to claim 183, wherein the vehicle or carrier is selected from the group consisting of dextran, lipophilic polymers, hydrophilic polymers, HPMA, and liposomes. 185. An isolated epitope comprising the amino acid sequence of GPIba from Tyr-276 to Glu-282, wherein at least one of amino acids 276, 278, and 279 is sulfated. 186. The isolated epitope of claim 185, further comprising amino acids from GPIba 283-285. 187. An antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof capable of binding the epitope of 185, wherein binding is increased if the epitope of claim 185 further comprises amino acids from GPIba 283-285. 188. An isolated N-terminal peptide of GPIba having an apparent molecular weight of about 40 kDa, which contains a molecular weight of about 40 kDa. · · · ··· ····· ·· ··· ·· · A 203 epitope having the sequence YDYYPEE, wherein at least one tyrosine residue in the epitope is sulfated. 189. An isolated GPIba peptide consisting of amino acids 1 to 282, wherein at least one of amino acids 276, 278, and 279 is sulfated. 190. An antibody multimer comprising at least the first and second antigen binding fragments, wherein at least the first or second antigen binding fragments, or both, are capable of cross-reacting with an epitope of Formula I: (S) _r I [(W) z - P - (Y) _t - P] q _(I) , in which W is any amino acid other than aspartate and glutamate Y is any naturally occurring compound that can be sulfated P is (A) _m (A) _m (X) _u , or (X) _u (A) _n (A) _m , or (A) _n (X) _u (A) _m , or (A) _n (A) ) _m (X) _u , or (X) _u (A) _m (A) _n , or (A) _m (X) _u (A) _n S is a sulfate or sulfated molecule X is any amino acid except aspartate, glutamate, or tyrosine 204 A is any negatively charged amino acid or leucine, isoleucine, proline, phenylalanine, serine, or glycine q is 1 to 6 z is 0, 1 or 2 r is 0 or 1 t is 1, 2 or 3 u is 0 to 2 n is 0-3 m is 0-3 where, if n = 0, then m> 0; where, if m = 0, then n> 0; where, if q is 1, r is 1 and if q is> 1, at least one of Y is sulfated. 191. The antibody multimer of claim 190, wherein the first or second antigen binding fragment, or both, binds cross-reactively with an epitope in which W is glycine, Y is a peptido-conjugate of tyrosines or a glyco-conjugate of asparagine, serine or threonine; A is glutamate, γ carboxy glutamate or aspartate; • q is 1, 2, or 3. 192. The antibody multimer of claim 190, wherein the first or second antigen-binding fragment or both bind cross-reactively with an epitope in which Y is a tyrosine peptide conjugate; q is 3; r is 1. 193. An antibody multimer comprising at least the first and second antigen binding fragments, wherein at least the first or second antigen binding fragments, or both, are capable of cross-reacting with the epitope of Formula II: (SX (S) _r (SX ™ III (W)) P- (YX-P- (YX-P- (YX-P _(II)) in which W is any amino acid other than aspartate and glutamate Y is any naturally occurring compound that can be sulfated P is (A) _m (A) _n (X) _u , or (X) _u (A) _n (A) _m , or (A) _n (X) _u (A) _m , or (A) _n (A) ) _m (X) _u , or (X) _u (A) _m (A) _n , or (A) _m (X) _u (A) _n S is a sulphate or sulphated molecule • · X is any amino acid except aspartate, glutamate, or tyrosine A is any negatively charged amino acid or leucine, isoleucine, proline, phenylalanine, serine, or glycine z is 0, 1 or 2 r is 0 or 1 t is 1, 2 or 3 u is 0 to 2 n is 0 to 3 m is 0 to 3 wherein, if n = 0, then m> 0; where, if m = 0, then n> 0; wherein, at least one of Y is sulfated. 194. The antibody multimer of claim 193, wherein the first or second antigen-binding fragment or both bind cross-reactively with an epitope in which W is glycine, Y is a tyrosine peptide conjugate or an asparagine, serine or threonine glyco-conjugate; A is glutamate, γ carboxy glutamate or aspartate, leucine, isoleucine ,. proline, phenylalanine, serine or glycine. 207 195. The antibody multimer of claim 193, wherein the first or second antigen-binding fragment or both bind cross-reactively with an epitope wherein: Y is a tyrosine peptide conjugate; q is 3; a r j e 1. 196. An antibody multimer comprising at least a first and a second antigen-binding fragment, wherein at least the first or second antigen-binding fragment, or both, are capable of cross-reacting with an epitope of formula III: (S) r (S) r (S) _r (GWCEinO ^ COt (X) _u (E) _n (D) _m (Y) _t (X) _u (E) _n (D) _m (Y) _t (P) ) _m (E) _n (X) _u ^{(111 J} < in which: G is glycine E is glutamate D is aspartate Y is tyrosine S is a sulfate or sulfated compound X is any amino acid except the above 208 z is 0, 1 or 2 t is 1, 2 or 3 r is 0 or 1 u is 0 to 2 n is 0 to 3 m is 0 to 3 wherein, at least one of Y is sulfated; where, if n = 0, then m> 0; where, if m = 0, then n> 0. 197. The antibody multimer of claim 196, wherein the first or second antigen-binding fragment or both bind cross-reactively with an epitope wherein r is 1. 198. The antibody multimer of claim 190, 193 or 196, wherein the multimer is a dimer, a trimer or a tetramer. 199. The antibody multimer of claim 198, wherein the multimer is a dimer. 200. The dimer of claim 199, wherein at least one of the antigen binding fragments is selected from the scFv fragments of Y1 and Y17. 201. The dimer of claim 199, wherein the first and second antigen binding fragments are linked by a disulfide bridge. 209 202. The dimer of claim 201, wherein the first and second fragments are Y1-CysKAK. 203. The dimer of claim 199, wherein the first and second antigen binding fragments are linked by a polypeptide linker of 5 to 20 amino acids. 204. The dimer of claim 203, wherein the polypeptide linker comprises 5 amino acids. 205. The dimer of claim 204, wherein the polypeptide linker is Gly4Ser. 206. The antibody multimer of claim 198, wherein the multimer is a trimer. 207. The trimmer of claim 206, comprising three antigen binding fragments, wherein at least one of the antigen binding fragments is a sc-scFv or Y17-scFv fragment. 208. The trimer of claim 207, wherein the antigen-binding fragments are linked by a polypeptide linker. 209. The trimer of claim 208, wherein the polypeptide linker comprises 1 to 5 amino acids. 210. The antibody multimer of claim 198, wherein the multimer is a tetramer. 211. The tetramer of claim 210, comprising four antigen binding fragments, wherein at least one of the antigen binding fragments is a Y1 scFv fragment or a Y17 scFv fragment. 210 • to ··· this * 212. The tetramer of claim 21, wherein the antigen-binding fragments are linked by a polypeptide linker. 213. The tetramer of claim 212, wherein the polypeptide linker comprises 1 to 5 amino acids. 214. The tetramer of claim 210, wherein the four antigen-binding fragments form a complex through association with streptavidin-biotin. 215. The antibody multimer of claim 198, comprising identical antigen-binding fragments. 216. The antibody multimer of claim 198, wherein at least the first and second antigen binding fragments comprise a first hypervariable region comprising SEQ ID NO: 8. 217. The antibody multimer of claim 198, wherein at least the first and second antigen binding fragments, or both, comprise a first hypervariable region comprising SEQ ID NO: 20. 218. The antibody multimer of claim 216 or 217, wherein at least the first and second antigen binding fragments, or both, have a second hypervariable region comprising SEQ ID NO: 115 and / or a third hypervariable region comprising SEQ ID NO: 114. 219. The antibody multimer of any one of claims 190, 193, 196, 216 and 217, wherein the multimer is capable of binding to at least two different molecules selected from the group consisting of PSGL-1, fibrinogen γ ', GPIba, heparin, · · · 211 lumican, complement compound 4 (CC4), an inter-alpha inhibitor and prothrombin. 220. The antibody multimer of any one of claims 190, 193, 196, 216, and 217, wherein the multimer is capable of binding to at least two different molecules selected from the group consisting of PSGL-1, fibrinogen γ ', GPIba, heparin, lumican, complement compound 4. (CC4), an inter-alpha inhibitor and prothrombin, and is capable of binding to at least one cell type selected from the group consisting of B-CLL cells, AML cells, multiple myeloma cells, and metastatic cells. 221. An antibody dimer comprising a first and a second antigen binding fragment, wherein the first or second antigen binding fragment, or both, comprise a hypervariable region comprising the amino acid sequence of SEQ ID NO: 8 [Y1 CDR3]. 222. An antibody dimer comprising first and second antigen binding fragments, wherein the first or second antigen binding fragment, or both, comprise a hypervariable region comprising the amino acid sequence of SEQ ID NO: 20 [Y17 CDR3]. 223. The antibody dimer of claim 221 or 222, wherein the first or second antigen binding fragment, or both, further comprise a second hypervariable region comprising the amino acid sequence of SEQ ID NO: 115 and / or a third hypervariable region comprising SEQ ID NO: 114. 224. An antibody multimer comprising a first or second antigen binding fragment, wherein the first or second 212 Antigen-binding fragment · Binding fragment • Antigen binding fragment capable of cross-reacting with two or more epitopes comprising one or more sulfated tyrosine residues and at least one cluster of two or more acidic amino acids. 225. The antibody multimer of claim 224 which is capable of cross-reacting with PSGL-1. 226. The antibody multimer of claim 224, which binds to the sequence QATEYEYLDYDFLPETE, wherein at least one tyrosine residue is sulfated. 227. The antibody multimer of claim 224, which is capable of cross-reacting with GPIba. 228. The antibody multimer of claim 224, which binds to the sequence DEGDTDLYDYYPEEDTEGD, wherein at least one tyrosine residue is sulfated. 229. The antibody multimer of claim 224, which binds to a TDLYDYYPEEDTE sequence wherein at least one tyrosine residue is sulfated. 230. The antibody multimer of claim 224, which binds to a DEGDTDLYDYYP sequence wherein at least one tyrosine residue is sulfated. 231. The antibody multimer of claim 224, which binds to a YDYYPEE sequence wherein at least one tyrosine residue is sulfated. 213 ·· ··· · · · · · · · · · · · · · · · · · · · · · · · 9 · «e · · · · · · Λ · · »* * * * * * * 232. The antibody multimer of claim 224, which binds to the sequence TDLYDYYP wherein at least one tyrosine residue is sulfated. 233. The antibody multimer of claim 224 which is capable of cross-reacting with fibrinogen γ '. 234. The antibody multimer of claim 233, which binds to an EPHAETEYDSLYPED sequence wherein at least one tyrosine residue is sulfated. 235. The antibody multimer of claim 224, which is capable of cross-reacting with heparin. 236. The antibody multimer of claim 224, which is capable of cross-reacting with complement compound 4 (CC4). 237. The antibody multimer of claim 224, which is capable of cross-reacting with at least one cell selected from the group consisting of B-CLL cells, AML cells, multiple myeloma cells, and metastatic cells. 238. A pharmaceutical composition comprising the antibody multimer of any one of claims 1, 4, 7, 27 and 28. 239. The pharmaceutical composition of claim 238 comprising the antibody multimer in an amount effective to increase cancer cell mortality, or to increase the sensitivity of the tumor cells to damage by an anticancer agent. 214 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 240. The pharmaceutical composition of claim 238 comprising the antibody multimer in an amount effective to inhibit the growth and / or replication of leukemic cells. 241. The pharmaceutical composition of claim 238 comprising the antibody multimer in an amount effective to inhibit abnormal cell-cell, cell-matrix, platelet-matrix, platelet-platelet and / or platelet-cell adhesion. 242. The pharmaceutical composition of claim 238, comprising the antibody multimer in an amount effective to increase the susceptibility of diseased cells to disease agents. 243. The pharmaceutical composition of claim 238 comprising an antibody multimer in an amount effective to increase leukemia cell mortality, or to increase the sensitivity of leukemia cells to damage by anti-leukemic agents. 244. A pharmaceutical composition comprising an antibody multimer according to any one of claims 190, 193, 196, 216, and 217 wherein the multimer is bound or complexed with an agent selected from the group consisting of anticancer, antimetastatic, antileukemic, disease control, anti-adherence agents. , antithrombotic, antirestenotic, antiautoimmune, antiplatelet, antibacterial, antiviral and anti-inflammatory agents. 215 245. The pharmaceutical composition of claim 244 wherein the agent is selected from the group consisting of toxins, radioisotopes, and pharmaceutical agents. 246. The pharmaceutical composition of claim 244, wherein the agent is an antiviral agent selected from the group consisting of acyclovir, ganciclovir and zidovudine. 247. The pharmaceutical composition of claim 244 wherein the agent is an antithrombotic / antiresthenotic agent selected from the group consisting of cilostazol, dalteparin sodium, reviparin sodium, and aspirin. 248. The pharmaceutical composition of claim 244 wherein the agent is an anti-inflammatory agent selected from the group consisting of zaltoprofen, pranoprofen, droxicam, acetyl salicylic 17, diclofenac, ibuprofen, dexibuprofen, sulindac, naproxen, amtolmetin, indecoxibu, indecoxibu, celecoxibu and nimesulide. 249. The pharmaceutical composition of claim 244, wherein the agent is an anti-autoimmune agent selected from the group consisting of leflunomide, denileukin diftitox, subrea, WinRho SDF, defibrotide, and cyclophosphamide. 250. The pharmaceutical composition of claim 244, wherein the agent is an anti-stick / anti-aggregating agent selected from the group consisting of limaprost, clorcromen, and hyaluronic acid. • · · · · · · · · · · ... · · .... • ····· · ... · ... 216 ............. 251. The pharmaceutical composition of claim 245 wherein the radioisotope is selected from the group consisting of gamma-emitters, positron-emitters, x-ray emitters, beta-emitters and alpha-emitters. 252. The pharmaceutical composition of claim 251 wherein the radioisotope is selected from the group consisting of ^{11 L} of indium, indium ^{113, 99 m} rheniurn, ¹⁰⁵ Rhenium, ¹⁰¹ Rhenium, ^{99 m} Tc, ^{121 m} tellurium, tellurium ^{122 m, 125 m} tellurium, ¹⁶⁵ thulium, ¹⁶⁷ thulium, ¹⁶⁸ thulium, ¹²³ iodine, ¹²⁶ iodine, ¹³¹ iodine, ¹³³ iodine, ^81m krypton, ³³ xenon, ⁹⁰ yttrium, ²¹³ bismuth, ⁷⁷ bromine, ¹⁸ fluorine, ⁹⁵ ruthenium, ⁹⁷ ruthenium, ¹⁰³ ruthenium, ¹⁰⁵ ruthenium , ¹⁰⁷ mercury, ²⁰³ mercury, ⁶⁷ gallium and ⁶⁸ gallium. 253. The pharmaceutical composition of claim 245, wherein the agent is a pharmaceutical selected from the group consisting of Doxorubicin (adriamycin), morpholinodoxorubicin, methoxymorpholinyldoxorubicin, cisplatin, taxol, calicheamicin, vincristine, cytarabine (Ara-C), cyclophosphamide. daunorubicin, morpholinodaunorubicin, methoxymorpholinyldaunorubicin, idarubicin, fludarabine, chlorambucil, interferon alpha, hydroxyurea, temozolomide, thalidomide and bleomycin, and derivatives and combinations thereof. 254. The pharmaceutical agent of claim 244, wherein the agent is bound or complexed with a vehicle or carrier capable of being coupled or complexed with more than one agent. 255. The pharmaceutical composition of claim 254, wherein the vehicle or carrier is selected from the group consisting of: 217 a group consisting of dextran, lipophilic polymers, hydrophilic polymers, HPMA, and liposomes. 256. The pharmaceutical composition of claim 238 comprising the antibody multimer in an amount effective to inhibit cell rolling. 257. The pharmaceutical composition of claim 238 comprising the antibody multimer in an amount effective to inhibit inflammation. 258. The pharmaceutical composition of claim 238 comprising the antibody multimer in an amount effective to inhibit autoimmune diseases. 259. The pharmaceutical composition of claim 238 comprising the antibody multimer in an amount effective to inhibit thrombosis. 260. The pharmaceutical composition of claim 238 comprising the antibody multimer in an amount effective to inhibit restenosis. 261. The pharmaceutical composition of claim 238 in an amount effective to inhibit metastasis. 262. The pharmaceutical composition of claim 238 comprising an antibody multimer in an amount effective to inhibit the growth and / or replication of tumor cells, to increase tumor cell mortality, or to increase the sensitivity of tumor cells to damage by anticancer agents. • · 218 ....... 263. The pharmaceutical composition of claim 238 comprising an antibody multimer in an amount effective to inhibit the growth and / or replication of leukemic cells, to increase the rate of leukemic cell mortality, or to increase the susceptibility of leukemic cells to antileukemic agent injury. 264. The pharmaceutical composition of claim 238, comprising the antibody multimer in an amount effective to increase the susceptibility of diseased cells to disease agents. 265. The pharmaceutical composition of claim 238 comprising the antibody multimer in an amount effective to inhibit cell-cell aggregation, cell-matrix, platelet-matrix, platelet-platelet and / or cell platelet, adhesion or complex formation. 266. The pharmaceutical composition of claim 238 which is bound or complexed with an agent selected from the group consisting of anticancer, antimetastatic, antileukemic, anti-disease, antiadhesive, antithrombotic, antirestenotic, antiautoimmune, antiplatelet, antibacterial, antiviral and antiviral agents. substances. 267. The pharmaceutical composition of claim 266, wherein the agent is an antiviral agent selected from the group consisting of acyclovir, ganciclovir and zidovudine. 268. The pharmaceutical composition of claim 238, wherein the antibody multimer is bound; 219 ............ complexed with a vehicle or carrier capable of being attached or complexed with more than one agent. 269. The pharmaceutical composition of claim 238, wherein the vehicle or carrier is selected from the group consisting of dextran, lipophilic polymers, hydrophilic polymers, HPMA, and liposomes. 270. The use of an antibody multimer according to any one of claims 1, 4, 7, 27 and 28 for the preparation of a medicament that inhibits cell rolling. 271. The use of an antibody multimer according to any one of claims 190, 193, 196 and 216 to 218 for the preparation of a medicament that inhibits inflammation. 272. The use of an antibody multimer according to any one of claims 1, 4, 7, 27 and 28 for the preparation of a medicament that inhibits autoimmune disease. 273. The use of an antibody multimer according to any one of claims 190, 193, 196 and 216 to 218 for the preparation of a medicament that inhibits restenosis. 274. The use of an antibody multimer according to any one of claims 190, 193, 196 and 216 to 218 for the preparation of a medicament that inhibits thrombosis. 275. The use of an antibody multimer according to any one of claims 190, 193, 196 and 216 to 218 for the preparation of a medicament that inhibits metastasis. • · * · 220 276. The use of an antibody multimer according to any one of claims 190, 193, 196 and 216 to 218 for the preparation of a medicament that inhibits the growth and / or replication of tumor cells. 277. The use of an antibody multimer according to any one of claims 190, 193, 196 and 216 to 218 for the preparation of a medicament that increases the rate of cancer cell mortality. 278. The use of an antibody multimer according to any one of claims 190, 193, 196 and 216 to 218 for the preparation of a medicament that inhibits the growth and / or replication of leukemic cells. 279. The use of an antibody multimer according to any one of claims 190, 193, 196 and 216 to 218 for the preparation of a medicament which increases the rate of leukemia cell mortality. 280. The use of an antibody multimer according to any one of claims 190, 193, 196 and 216 to 218 for the preparation of a medicament that increases the susceptibility of diseased cells to damage by agents against the disease. 281. The use of an antibody multimer according to any one of claims 190, 193, 196 and 216 to 218 for the preparation of a medicament that increases the sensitivity of tumor cells to damage by anticancer agents. 282. The use of an antibody multimer according to any one of claims 190, 193, 196 and 216 to 218 for the use of an antibody multimer. The preparation of a medicament which increases the sensitivity of leukemic cells to damage by antileukemic agents 283. The use of an antibody multimer according to any one of claims 190, 193, 196 and 216 to 218 for the preparation of a medicament that inhibits an increase in the number of tumor cells in a patient having cancer. 284. The use of an antibody multimer according to any one of claims 190, 193, 196 and 216 to 218 for the preparation of a medicament that reduces the number of tumor cells in a patient having cancer. 285. The use of an antibody multimer according to any one of claims 190, 193, 196 and 216 to 218 for the manufacture of a medicament that inhibits an increase in the number of leukemia cells in a patient having leukemia. 286. The use of an antibody multimer according to any one of claims 190, 193, 196 and 216 to 218 for the preparation of a medicament that reduces the number of leukemic cells in a patient having leukemia 287. The use of an antibody multimer according to any one of claims 190, 193, 196 and 216 to 218 for the preparation of a medicament that inhibits the formation of a cell-cell, cell-matrix, platelet-matrix, platelet-platelet and / or cell-platelet complex. 288. The use of an antibody multimer according to any one of claims 190, 193, 196 and 216 to 218 for the preparation of a medicament that inhibits cell-cell aggregation, cell-matrix, platelet-matrix, platelet-platelet and / or cell-platelet. 289. The use of an antibody multimer according to any one of claims 190, 193, 196 and 216 to 218 for the preparation of a medicament that inhibits cell-cell, cell-matrix, platelet-matrix, platelet-platelet and / or cell platelet adhesion. 290. The method of any one of claims 270 to 289, wherein the antibody multimer is bound or complexed with an agent selected from the group consisting of anticancer, antimetastatic, antileukemic, anti-disease, anti-stick, anti-thrombotic, anti-esthenotic, anti-autoimmune, anti-aggregation, anti-bacterial, and anti-inflammatory agents. 291. The method of claim 290, wherein the agent is an antiviral agent selected from the group consisting of acyclovir, ganciclovir and zidovudine. 292. The method of claim 290, wherein the agent is an antithrombotic / antirestenotic agent selected from the group consisting of cilostazol, dalteparin sodium, reviparin sodium, and aspirin. 293. The method of claim 290, wherein the agent is an anti-inflammatory agent selected from the group consisting of zaltoprofen, pranoprofen, droxicam, acetyl salicylic 17, diclofenac, ibuprofen, dexibuprofen, sulindac, naproxen, amtolmetin, celofoxin, indomethacinib, indomethacinib. 223 294. The method of claim 290, wherein the agent is an anti-autoimmune agent selected from the group consisting of leflunomide, denileukin diftitox, subrea, WinRho SDF, defibrotide, and cyclophosphamide. 295. The method of claim 290, wherein the agent is an anti-stick / anti-aggregating agent selected from the group consisting of limaprost, clorcromen, and hyaluronic acid. 296. The method of claim 290, wherein the agent is a substance selected from the group consisting of toxins, radioisotopes, and pharmaceutical agents. 297. The method of claim 290, wherein the toxin is selected from the group consisting of gelonin, Pseudomonas exotoxin (PE), PE40, PE38, diphtheria toxin, ricin, and modifications and derivatives thereof. 298. The method of claim 290, wherein the radioisotope is selected from the group consisting of gamma-emitters, positron emitters, x-ray emitters, beta-emitters and alpha-emitters. ²⁹⁹ 299. The method of claim 290, wherein the radioisotope is selected from the group consisting of ⁱⁿ indium, ¹¹³ indium, ^99m rheniurn, ¹⁰⁵ rhenium, ¹⁰¹ rhenium, ^99m technetium, ^121m tellurium, ^122m tellurium, ^125m tellurium, ¹⁶⁵ thulium, ¹⁶⁷ thulium, ¹⁶⁸ thulium, ¹²³ iodine, ¹²⁶ iodine, ¹³¹ iodine, ¹³³ iodine, ^81m krypton, ³³ xenon, ⁹⁰ yttrium, ²¹³ bismuth, ⁷⁷ bromine, ¹⁸ fluorine, ⁹⁵ ruthenium, ⁹⁷ ruthenium, ¹⁰³ ruthenium, ¹⁰⁵ ruthenium, ¹⁰⁷ mercury, ²⁰³ mercury , ⁶⁷ gallium and ⁶⁸ gallium. 224 300. The method of claim 290, wherein the pharmaceutical agent is selected from the group consisting of Doxorubicin (adriamycin), morpholinodoxorubicin, methoxymorpholinyldoxorubicin, cisplatin, taxol, calicheamicin, vincristine, cytarabine (Ara-C), cyclophosphamide, prednisicin, munororinin, munororin, munororin, munoricin , methoxymorpholinyldaunorubicin, idarubicin, fludarabine, chlorambucil, interferon alpha, hydroxyurea, temozolomide, thalidomide and bleomycin, and derivatives and combinations thereof. 301. The method of any one of claims 270 to 289, wherein the antibody, antigen-binding fragment thereof, or complex thereof comprising at least one antibody or binding fragment thereof is bound or complexed with a vehicle or carrier capable of being attached or complexed. with more than one substance. 302. The method of claim 301, wherein the vehicle or carrier is selected from the group consisting of dextran, lipophilic polymers, hydrophilic polymers, HPMA, and liposomes. 303. The antibody multimer of any one of claims 190, 193, 196 and 216 to 218 for use as a medicament for inhibiting cellular scrolling. 304. The antibody multimer of any one of claims 190, 193, 196 and 216 to 218 for use as a medicament for inhibiting inflammation. 225 • ·········· 305. The antibody multimer of any one of claims 190, 193, 196 and 216 to 218 for use as a medicament for inhibiting autoimmune diseases. 306. The antibody multimer of any one of claims 190, 193, 196 and 216 to 218 for use as a medicament for inhibiting restenosis. 307. The antibody multimer of any one of claims 190, 193, 196 and 216 to 218 for use as a medicament for inhibiting thrombosis. 308. The antibody multimer of any one of claims 190, 193, 196 and 216 to 218 for use as a medicament for inhibiting metastasis. 309. The antibody multimer according to any one of claims 190, 193, 196 and 216 to 218 for use as a medicament for inhibiting the growth and / or replication of tumor cells. 310. The antibody multimer of any one of claims 190, 193, 196 and 216 to 218 for use as a medicament for increasing the rate of cancer cell mortality. 311. The antibody multimer of any one of claims 190, 193, 196 and 216 to 218 for use as a medicament for inhibiting the growth and / or replication of leukemia cells. 312. The antibody multimer according to any one of claims 190, 193, 196 and 216 to 218 for use as a medicament for increasing the rate of leukemia cell mortality 226 313. An antibody multimer according to any one of claims 190, 193, 196 and 216 to 218 for use as a medicament for increasing the susceptibility of diseased cells to disease agents. 314. An antibody multimer according to any one of claims 190, 193, 196 and 216 to 218 for use as a medicament to increase the sensitivity of tumor cells to damage by anticancer agents. 315. An antibody multimer according to any one of claims 190, 193, 196 and 216 to 218 for use as a medicament to increase the sensitivity of leukemic cells to damage by anti-leukemic agents. 316. The antibody multimer of any one of claims 190, 193, 196 and 216 to 218 for use as a medicament for inhibiting an increase in the number of tumor cells in a patient having cancer. 317. The antibody multimer of any one of claims 190, 193, 196 and 216 to 218 for use as a medicament for reducing the number of tumor cells in a patient having cancer. 318. A polyclonal antibody, antibody fragment, or complex that cross-reacts with the variable light chain of the human monoclonal antibody scFv Y1. 319. The polyclonal antibody, antibody fragment or complex of claim 318, which cross-reacts with the NdeI-EcoRI variable light chain restriction fragment of the human monoclonal antibody Y-1. 227 320. A diagnostic kit comprising the antibody, antibody fragment or complex of any one of claims 318 to 319. 321. A composition comprising the antibody, antibody fragment or complex of any one of claims 318 to 319 conjugated to Doxorubicin. 322. A composition comprising the antibody, antibody fragment or complex of any one of claims 318 to 319 and a pharmaceutically acceptable carrier selected from the group consisting of dextran, HPMA, hydrophilic polymers, lipophilic polymers, and liposomes. 323. A composition comprising the antibody, antibody fragment or complex of any one of claims 318 to 319 mixed with Doxorubicin-bearing liposomes.