MXPA06000253A - Specific human antibodies. - Google Patents

Abstract

The present invention provides antibodies that bind an epitope of PSGL-1 comprising the motif D-X-Y-D, wherein X represents any amino acid or the covalent linkage between D and Y, and Y is sulfated, which antibody can be complexed with one or more copies of an agent. The antibodies of the invention can be used in a method of inducing antibody-dependent cell cytotoxicity and/or stimulating natural killer (NK) cells or T cells. In addition, by administering these antibodies to a patient in need thereof, a method of inducing cell death is provided. A method of preventing infection by a virus (e.g., HIV) by administering to a patient in need thereof an antibody of the present invention is also provided. The present invention also provides a method of introducing an agent into a cell that expresses sulfated PSGL-1 by coupling or complexing an agent to an antibody of the present invention and administering the complex to the cell. Finally, the present invention provides methods of diagnosis, prognosis and staging using the present antibodies.

Description

SPECIFIC HUMAN ANTIBODIES FIELD OF THE INVENTION The present invention relates to antibodies that bind to particular epitopes that are present in cells, such as cancer cells, metastatic cells, leukemia cells, leukocytes and platelets, and which are important in diverse physiological phenomena such as cell coiling, metastasis , inflammation and autoimmune diseases. More particularly, the antibodies can have anticancer activity, anti-etastatic activity, anti-leukemia activity, antiviral activity, anti-infection activity, and / or activity against other diseases, such as inflammatory diseases, autoimmune diseases, cardiovascular diseases such as heart attack, myocardium, retinopathic diseases, and diseases produced by protein-protein interactions that depend on sulfated tyrosine. In addition, the antibodies of the present invention can be used as a target agent to direct a therapeutic compound to a specific cell or site within the body.

BACKGROUND OF THE INVENTION Leukemia, lymphoma and myeloma are cancers that originate in the bone marrow and lymphatic tissues and participate in the uncontrolled growth of cells. Acute lymphoblastic leukemia (ALL) is a heterogeneous disease defined by specific clinical and immunological characteristics. Like other forms of ALL, the definitive cause of most ALL cases of B cells (B-ALL) is unknown; although, in many cases, the disease derives from genetic alterations acquired in the DNA of a single cell, which produce abnormalities and continuous multiplication. The prognosis for patients affected by B-ALL is significantly worse for patients with other leukemias, both in children and as. Chronic lymphocytic leukemia (CLL), one of whose examples is CLL of B cells (B-CLL), is a slow progressing form of leukemia, characterized by an increased number of lymphocytes. Acute myelogenous leukemia (AML) is a heterogeneous group of neoplasms that have a progenitor cell that, under normal conditions, gives rise to differentiated cells at the ends of the myeloid series (erythrocytes, granulocytes, monocytes and platelets). As in other forms of neoplasia, AML is associated with acquired genetic alterations that result in the replacement of myeloid cells normally differentiated with relatively undifferentiated blasts, which present one or more types of early myeloid differentiation. AML usually evolves in the bone marrow and, to a lesser extent, in the secondary hematopoietic organs. AML mainly affects as and has incidence peaks between the ages of 15-40, but it is also known to affect both children and older as. Almost all patients with AML require treatment immediately after diagnosis to achieve clinical remission, where there is no evidence of abnormal levels of undifferentiated blasts cells in circulation.

Ligand for Isolated scFv Antibody Molecules Platelets, fibrinogens, GPIb, selectins, and PSGL-1 (Ligand 1 P-Selectin Glycoprotein) each play an important role in various pathogenic conditions or disease states, such as abnormal or pathogenic inflammation, abnormal or pathogenic immune reactions, reactions autoimmune, metastasis, abnormal or pathogenic adhesion, thrombosis and / or restenosis, and abnormal or pathogenic aggregation. Therefore, antibodies that bind or cross-react with platelets and with these molecules would be useful in the diagnosis and treatment of diseases and disorders involving these and other pathogenic conditions.

Platelets Platelets are well-characterized components of the blood system and play important roles in hemostasis, thrombosis and / or restenosis. Blood vessel injury sets in motion a process called hemostasis, which is characterized by a series of sequential events. The initial reaction to the injured blood vessels is the adhesion of the platelets to the affected region on the inner surface of the vessel. The next step is the aggregation of many layers of platelets to the platelets initially adhered, forming a stopper and sealing the vessel wall. The hemostatic plug is made more resistant by the deposition of fibrin polymers. The clot or plug degrades only when the lesion has been repaired.

Circulating platelets are cytoplasmic particles released from the periphery of megakaryocytes. Platelets play an important role in hemostasis. When a vascular lesion occurs, the platelets adhere to the surfaces of the injured tissue and attach to each other (cohesion). This sequence of events occurs rapidly, forming a structureless mass (usually called platelet plug or thrombus) at the site of the vascular lesion. The phenomenon of cohesion, also called aggregation, can be initiated in vitro with a variety of substances, or agonists, such as collagen, adenosine diphosphate (ADP), epinephrine, serotonin, and ristocetin. Aggregation is one of the numerous in vitro tests performed as a measure of platelet function.

Importance of Platelets in Metastasis Metastasis of tumors is perhaps the most important factor limiting the survival of cancer patients. The accumulated data indicate that the ability of tumor cells to interact with host platelets represents one of the essential determinants of metastasis (Ole so icz, Thrombosis Res. 79: 261-74 (1995)). When metastatic cancer cells enter the bloodstream, multicellular complexes composed of platelets and leukocytes lining the tumor cells form. These complexes, which can be called microemboli, help tumor cells evade the immune system. The coating of tumor cells by platelets requires the expression of P-selectin by platelets.

It has been demonstrated that the ability of tumor cells to aggregate platelets correlates with the potential for metastasis of tumor cells and that inhibition of tumor-induced platelet aggregation has been shown to correlate with the suppression of metastasis in rodent models. It has been shown that the interaction of tumor cells with platelets involves molecules of adhesion to the membrane and secretion of agonists. The expression of immuno-related platelet glycoproteins has been identified in tumor cell lines. It has been shown that the immuno-related glycoproteins of platelets, GPIb, GPIIb / IIIa, GPIb / IX and the integrin subunit av are expressed on the surface of breast tumor cell lines (Oleksowicz, (1995), supra; Kaniyama et al, J. Lab. Clin. Med. 117 (3): 209-17 (1991)).

Gasic et al (PNAS 61: 46-52 (1968)) demonstrated that antibody-induced thrombocytopenia markedly reduced the number and volume of metastases produced by CT26 colon adenocarcinoma, Lewis lung carcinoma, and B16 melanoma (Karpatkin et al. al, J. Clin. Invest. 81 (4): 1012-19 (1988); Clezardin et al, Cancer Res. 53 (19): 4695-700 (1993)).

In addition, it was found that a single polypeptide chain (60kd) is expressed on the surface membrane of HEL cells that is closely related to GPIb and corresponds to an O-glycosylated GPIb subunit in incomplete or abnormal form (Kieffer et al, J. Biol. Chem. 261 (34): 15854-62 (1986)).

GPIb complex Each step in the process of hemostasis requires the presence of receptors on the surface of the platelets. One of the receptors that is important in hemostasis is the glycoprotein Ib-IX complex (also called CD42). This receptor mediates the adhesion (initial binding) of platelets to the blood vessel wall at sites of injury by binding von Willebrand factor (vWF) to the subendothelium. It also fulfills crucial functions in two other functions of platelets important in hemostasis: (a) platelet aggregation induced by high cut in regions of arterial stenosis and (b) platelet activation induced by low concentrations of thrombin.

The GPIb-IX complex is one of the main components of the outer surface of the plasma membrane of platelets. This complex comprises three membrane-bound polypeptides: a disulfide-linked 130 kDa α-chain and a 25 kDa β-chain of GPIb and a GPIX (22 kDa) associated in non-covalent form. All subunits are presented in equimolar amounts at the platelet membrane for efficient cell surface expression and the function of the CD42 complex, which indicates that the correct union of the three subunits in a complex is required for complete expression in the plasma membrane. The a chain of GPIb comprises three distinguishable structural domains: (1) a globular N-terminus peptide domain containing repeated leucine-rich sequences and Cys binding flank sequences; (2) a macroglicopeptide domain similar to highly glycosylated mucin; and (3) an end region of C associated with the membrane containing the disulfide bridge to GPIba and transmembrane and cytoplasmic sequences.

Several lines of evidence indicate that vWF and the thrombin binding domain of GPIb-IX complex reside in a globular region encompassing approximately 300 amino acids at the amino terminus of GPIba. As a human platelet, the GPIb-IX complex is a key receptor of the membrane that mediates both the function and the reactivity of the platelet, the vWF recognition of subendothelial binding by GPIb allows platelets to adhere to the injured blood vessels. In addition, the binding of vWF to GPIba also induces the activation of platelets, which may involve the interaction of a cytoplasmic domain of GPIb-IX with the cytoskeleton or phospholipase A2. In addition, GPIba contains a high affinity binding site for a-tombin, which facilitates the activation of platelets by an even worse defined mechanism.

The globular domain of the N-terminus of GPIba contains a group of negatively charged amino acids. Several lines of evidence indicate that in transfected CHO cells expressing the GPIb-IX complex and in platelet GPIba, the three tyrosine residues contained in this domain (Tyr-276, Tyr-278 and Tyr-279) undergo sulphation.

Protein Sulfation Protein sulfation is a broad post-translational modification consisting of the enzymatic covalent attachment of sulfate, to sugar side chains or to the polypeptide backbone. This modification occurs in the trans-Golgi compartment. Sulfated proteins include secretory proteins, proteins with white granules, and the extracellular regions of plasma membrane proteins. Tyrosine is an amino acid residue that is currently known to undergo sulfation. Kehoe et al, Chem. Biol. 1: R57-61 (2000). Other amino acids, for example threonine, can also undergo sulfation, particularly in diseased cells. It has been discovered that numerous proteins are sulphated with tyrosine, but the presence of three or more sulfated tyrosines in a single polypeptide, found in GPIb, is not common. GPIba (CD42), expressed by platelets and megakaryocytes and mediating the binding of platelets and coiling in the subendothelium through binding with vWF, also contains numerous negative charges in its domain of the N-terminus. It is thought that this medium Highly acidic and hydrophilic is a prerequisite for sulphation, because tyrosylprotein sulfotransferase specifically recognizes and sulfata tyrosines adjacent to amino acid residues (Bundgaard et al, J. Biol. Chem. 272: 21700-05 (1997)). The complete sulfation of the acid region of GPIba gives a region with a remarkable negative charge density, 13 negative charges within a stretch of 19 amino acids, and is a candidate site for electrostatic interaction with other proteins.

It is also thought that sulphated N-terminal tyrosines influence the function of CC-chemokine receptors, such as CCR5, which serve as co-receptors with the seven transmembrane-related segment receptor (7TMS) for the entry of immunodeficiency virus human and simian (HIV-1, HIV-2, and SIV) in target cells. For example, it is thought that sulphated N-terminal tyrosines contribute to the binding of CCR5 to the MlP-la, MlP-lß and HIV-1 gpl20 / CD4 complexes and to the ability of HIV-1 to enter cells that express CCR5 and CD4. CXCR4, another important HIV-1 co-receptor, is also sulfated (Farzan et al, Cell 196 (5): 1667-76 (1999)). Tyrosine sulfation plays a less significant role in the entry of HIV-1 that depends on CXCR4 than in the input that depends on CCR5; therefore, a possible function of the tyrosine sulfation in the XCC-Iquimiocin family is demonstrated and it qualifies more under a general difference in the use of HIV-1 of CCR5 and CXCR4 (Farzan et al, J. Biol .. Chem 277 (33): 219, 484-89 (2002)).

Selectins and PSGL-1 The P-, E- and L-Selectins are members of a family of adhesion molecules that, among other functions, mediate the winding of leukocytes over the vascular endothelium. P-Selectin is stored as granules in platelets and is transported to the surface after activation by thrombin, histamine, phorbol ester, or other stimulatory molecules. P-Selectin is also expressed in activated endothelial cells. E-Selectin is expressed in endothelial cells and L-Selectin is expressed in neutrophils, monocytes, T cells and B cells.

PSGL-1 (also called CDl62) is a mucin glycoprotein ligand for P-Selectin, E-Selectin and L-Selectin that shares structural similarity with GPIb (Afshar-Kharghan et al (2001), supra). PSGL-1 is a disulfide-linked homodimer that has a PACE cleavage site (Enzymes that Convert Basic Amino Acids to Pairs). PSGL-1 also has three potential tyrosine sulfation sites followed by repeats of 10-16 decamers that are high in proline, serine, and threonine. The extracellular portion of PSGL-1 contains three N-linked glycosylation sites and has numerous branches of sialylated, fucosylated O-linked oligosaccharides (Moore et al, J. Biol. Chem. 118: 445-56 (1992)). Most of the N-glycan sites and many of the O-glycan sites are occupied. The structures of the O-glycan of PSGL-1 from human HL-60 cells have been determined. The subsets of these O-glycan are sialylated and fucosylated core 2 structures that are required for binding to selectins. Tyrosine sulfation of an amino-terminal region of PSGL-1 is also required for binding to P-Selectin and L-Selectin. In addition, there is an N-end propeptide that probably breaks after translation.

PSGL-1 has 361 residues in 1HL60 cells, with an extracellular region of 267 residues, a trans-embryonic region of 25 residues and an intracellular region of 69 residues and forms an hourly or heterodimer bound to disulfide at the cell surface (Afshar -Kharghan et al, Blood 97: 3306-12 (2001)). The sequence encoding PSGL-1 is in a single axon, so an alternative splice should not be possible. However, PSGL-1 in HL60 cells and in most cell lines, has 15 consecutive repeats of a 10-residue consensus sequence present in the extracellular region, although there are 14 and 16 repeats of this sequence in the polymorphonuclear leukocytes, monocytes, and several other cell lines, which include most of the native leukocytes.

PSGL-1 is expressed in neutrophils as a dimer, with apparent molecular weights of 250 kDa and 160 kDa, while in HL60 the dimeric form is approximately 220 kDa. When analyzed under reduced conditions, each subunit is reduced by half. The differences in molecular mass can be due to polymorphisms of the molecule caused by the presence of different numbers of repeats of decamers (Leukocyte Typing VI, edited by T. Kishi oto et al (1997)).

Most leukocytes, such as neutrophils, monocytes, leukocytes, subsets of B cells, and all T cells express PSGL-1 (Kishimoto et al (1997), supra). PSGL-1 mediates the winding of leukocytes in the activated endothelium, in activated platelets, and in other leukocytes and inflammatory sites and mediates the neutrophil coiling in P-Selectin. PSGL-1 can also mediate neutrophil-neutrophil interactions through L-Selectin binding, thereby mediating inflammation (Snapp et al, Blood 91 (1): 154-64 (1998)).

The winding of leukocytes is important in inflammation, and the interaction between P-Selectin (expressed by the activated endothelium and in platelets, which can be immobilized in sites of injury) and PSGL-1 is instrumental in binding and rolling leukocytes in the walls of vessels (Ramachandran et al, PNAS 98 (18): 10166-71 (2001); Afshar-Kharghan et al (2001), supra). Cell coiling is also important in metastasis and it is believed that P- and E-Selectin in endothelial cells binds to metastatic cells, thus facilitating extravasation from the bloodstream into the surrounding tissues.

Therefore, PSGL-1 has been found in all leukocytes: neutrophils, monocytes, lymphocytes, activated peripheral T cells, granulocytes, eosinophils, platelets and in some CD34 positive stem cells. P-Selectin is actively expressed in activated platelets and endothelial cells. The interaction between P-Selectin and PSGL-1 promotes the coiling of leukocytes in the walls of the vessels, and the abnormal accumulation of leukocytes in vascular sites derives in different pathological inflammations. Stereo-specific contributions of individual tyrosine sulfates in PSGL-1 are important for the binding of P-Selectin to PSGL-1. Loading is also important for binding: the reduction of NaCl (150 to 50 mM) improved the binding (Kd 75 nM). Tyrosine sulfation in PSGL-1 improves, but is not ultimately required for the adhesion of PSGL-1 in P-Selectin. The tyrosine sulfation of PSGL-1 supports the slower winding adhesion at all cutting speeds and supports winding adhesion at much higher cutting speeds (Rodgers et al, Biophys, J. 81: 2001-09 (2001)). In addition, it has been suggested that the expression of PSGL-1 in platelets is 25-100 times lower than that of leukocytes (Frenette et al, J. Exp. Med. 191 (8): 1413-22 (2001)).

It has been shown that a monoclonal antibody commercially available for human PSGL-1, KPL1 inhibits the interactions between PSGL-1 and P-Selectin and between PSGL-1 and L-selectin. The epitope of KPL1 was mapped to the tyrosine sulfation region of PSGL-1 (YEYLDYD) (SEQ ID NO: 1) (Snapp et al, Blood 91 (1): 154-64 (1998)).

Pretreatment of tumor cells with O-sialoglycoprotease, which removes sialylated, fucosylated mucin ligands, also inhibited the formation of the tumor cell-platelet complex. In vivo experiments indicate that any of these treatments leads to a greater association of monocytes with circulating tumor cells, which suggests that the reduction of platelet binding increases access by immune cells to tumor cells (Varki and Varki, Bras. J. Biol. 34 (6): 1711-17 (2001)).

Fibrinogen There are two forms of normal fibrinogen: (?) Normal and? premium, each of which is found in normal individuals. Normal fibrinogen, which is the most abundant form (approximately 90% of the total fibrinogen that is in the body), is composed of two identical 55 kDa α chains, two identical 95 kDa ß chains and two ß chains. of 419.5 kDa identical. The normal variant of fibrinogen, which is the least abundant form (approximately 10% of the fibrinogen in the body), is composed of two identical 55 kDa strands, two identical 95 kDa strands, a strand of ? of 49.5 kDa and a variant of the? chain? premium of 150.5 kDa. The gamma and gamma-prime chains are both encoded by the same gene, with an alternative splice occurring at the 3 'end. The normal gamma chain is composed of amino acids 1-411 and the normal variant of the gamma prime chain is composed of 427 amino acids, of which amino acids 1-407 are the same as those of the normal gamma chain and amino acids 408-427 are VRPEHPAETEYDSLYPEDDL (SEQ ID N ° 2). This region is normally occupied with thrombin molecules.

Fibrinogen is converted to fibrin by the action of thrombin in the presence of ionized calcium to produce blood coagulation. Fibrin is also a component of thrombi, and acute inflammatory exudates.

Accordingly, an object of the invention is to provide different antibodies or polypeptides that bind to sulfated PSGL-1 and methods of using them.

Specifically, an object of the invention is to provide methods of activating ADCC or of stimulating natural killer (NK) cells or T cells by administering the antibodies of the present invention.

Another specific objective of the invention is to provide a method of inducing cell death.

Yet another specific objective of the invention is to provide a method of preventing infection by a virus, such as HIV, which comprises administering to a patient in need thereof an antibody of the present invention.

Another specific objective of the invention is to provide a method of introducing an agent into a sulphated PSGL-1 expressing cell having the following steps: coupling or forming agent complexes with an antibody described herein and administering the antibody-agent pair or complex to the cell.

Finally, a specific objective of the present invention is to provide diagnostic, prognostic and phase determination methods using the present antibodies.

EXTRACT OF THE INVENTION The present invention provides antibodies or polypeptides that bind to an epitope of PSGL-1 comprising the motif DXYD (SEQ ID No. 3), wherein X represents any amino acid or covalent bond between D and Y, and Y is sulphlated, whose antibody can be coupled or complexed with several copies of an agent selected from the group consisting of anti-cancer, anti-leukemia, anti-metastasis, anti-neoplastic, anti-disease, anti-adhesion, anti-thrombosis, anti-restenosis agents , anti-autoimmune, anti-aggregation, antibacterial, antiviral and anti-inflammatory.

The antibodies of the invention can be used in a method of inducing cellular cytotoxicity that depends on the antibody and / or stimulating natural killer (NK) cells or T cells. Furthermore, by administering these antibodies to a patient in need thereof, a method of induce cell death. A method of preventing infection by a virus (e.g., HIV) by administering to a patient in need thereof an antibody of the present invention is also provided. The present invention also provides a method of introducing an agent into a cell expressing sulfated PSGL-1 by coupling or forming a complex of an agent with an antibody of the present invention and administering the antibody-agent pair or complex to the cell. The present invention further provides a method of identifying, isolating and purifying tumor cell markers. Finally, the present invention provides diagnostic, prognostic and phase determination methods using the present antibodies.

BRIEF DESCRIPTION OF THE DRAWINGS The invention is described herein in more detail, by way of example only, and in no way as limitation, with reference to the accompanying drawings which are described below, wherein: Figure 1 shows a Western blot analysis of a lysate of partially purified AML-R1 cells before and after passage through the affinity column of Yl-IgG.

Figure 2 shows that, from three tyrosines of the sulfated tyrosine motif of the purified protein, tyrosines 2 and 3 are sulfated.

Figure 3 shows ADCC mediated by Yl-IgG (20 μg / ml) (percentage of cytotoxicity) in primary B-CLL samples.

Figure 4 shows ADCC mediated by Yl-IgG (percentage of cytotoxicity) by PBMC against AML cells.

Figure 5 shows increased ADCC (percentage of cytotoxicity) in ML-2 cells as a function of Yl-IgG concentration.

Figure 6 shows ADCC (percentage of cytotoxicity) by PBMC against ML-2 according to the competition between Yl-IgG and KPL-1.

Figure 7A shows the analysis of ADCC mediated by Yl-IgG (percentage of cytotoxicity) by natural killer cells from normal donors and from B-CLL patients against ML2 cells.

Figure 7B shows the participation of 1CD14 + cells (monocytes) in ADCC against white M12.

Figure 8 shows the expression of CD69 (an early activation marker) in NK cells mediated by Yl.

Figure 9 shows the apoptotic effect of Yl-IgG in mononuclear cells (CD19 +, CD5 +) of B-CLL patients by FACS analysis.

Figure 10 shows the analysis of ADCC activity (percentage of cytotoxicity) against the mononuclear cells of human B-CLL patients, ie primary human B-CLL cells (KBC115 and KBC116 cells) mediated by Yl-IgG and Rituximab The figure. 11 shows the analysis of CDC activity (percentage of lysis) against mononuclear cells from human B-CLL patients (KBC156, KBC159, KBC160, KBC166 and RAJI cells) mediated by Yl-IgG, Rituximab, and Campath® in the presence and absence of patients' plasma.

Figure 12 shows the scheme of the reaction for the preparation of the antibody bound to morpholino-doxorubicin.

Figure 13 shows the cytotoxicity of an antibody-agent complex, namely complexes of Yl-morpholinodaunorubicin (Yl-M-DNR) and Yl-morpholinodoxorubicin (Yl-M-Dox) in blood cells of spine and AML.

Figure 14 shows the cytotoxicity of the antibody-agent complex Yl-M-DNR against AML samples from 2 patients (phase M4 and M5) and against CD34 + cells.

Figure 15 shows the cytotoxicity of different complexes of Yl as the percentage of control in B-ALL cells.

Figure 16A shows the binding of Yl scFv to KU812 cells and Figure 16B shows the surface expression of GPIb in sulfate-deprived KU812 cells.

Figure 17A shows inhibitory effects of DLYDYYPE slulphated peptides on the binding of Yl-scFv to platelets. The peptides are disclosed in SEQ ID N ° 5, 4, 9, 10, 8, 7 and 6, respectively in order of appearance from left to right. Figure 17B shows the effects of substitution mutant peptides in the binding assay of Yl-scFv platelet. The peptides are disclosed in SEQ ID Nos. 16, 15, 11, 13, 14, 12 and 16-19, respectively in order of appearance from left to right.

Figure 18A shows the effects of mutant peptides on the inhibition of Yl-scFv binding to purified glycocalicin. Figure 18B shows the binding of YlscFv to peptides covalently coupled to CovaLink® Plates by ELISA. The peptides of Figures 18A and B are disclosed in SEQ ID Nos. 5, 29-34, 15, 11, 13, 14, 12 and 16-19, respectively in order of appearance from left to right.

Figure 19 shows the binding of Yl to sulfated, immobilized peptides derived from PSGL-1.

Figure 20 shows the percentage of binding activity of Yl to unsulfated PSGL-1 and sulfated PSGL-1 in the first, second and third positions.

Figure 21 shows some potential Yl binding motifs that are highly acidic and have sulfated tyrosines (SEQ ID N ° 22, 20 and 23-25, respectively in order of appearance).

Figure 22 shows the recognition of small cell lung carcinoma (SCLC) lysed by Yl.

Figure 23 shows endocytosis of Yl in primary AML cells.

Figure 24 shows endocytosis of Yl in primary AML cells.

Figure 25 shows the analysis of the binding of Yl to healthy CD34 + stem cells.

Figure 26 shows the analysis of the binding of Yl to healthy CD34 + stem cells.

Figure 27 shows the internalization of Yl in primary AML cells at 37 ° C.

Figure 28 shows the visualization of Yl staining in primary AML cells after scraping the membrane-bound protein by treatment with acids.

Figure 29 shows the visualization of Yl staining in primary AML cells after scraping the membrane bound protein by treatment with pronase.

Figure 30 shows the visualization of Yl staining in primary AML cells after acid treatment, pre-incubation of sucrose at 4 ° C (Figure 30A) and at 37 ° C (Figure 30B).

Figure 31 shows that Yl-scFv effectively inhibits the binding of activated human platelets to ML2 cells.

Figure 32 shows the effect of Yl-scFv (10 μg / ml) on the winding of ML2 cells in rh-P-Selectin immobilized at low density (0.2 μg / ml).

Figure 33 shows the effect of Yl-scFv (10 μg / ml) on the winding of ML2 cells in rh-P-Selectin immobilized at high density (1.0 μg / ml).

Figure 34 shows the effect of Yl-IgG (1 μg / ml) on the winding of ML2 cells in immobilized rh-P-Selectin (1.0 μg / ml) at different shear deformation forces.

Figure 35 shows the effect of increasing concentrations of Yl-scFv on the coil of human neutrophils in rh-P-Selectin immobilized at high density (1.0 μg / ml).

Figure 36 shows the effect of Yl-IgG on the coiling of human neutrophils in rh-P-Selectin immobilized at high density (1.0 μg / ml).

DETAILED DESCRIPTION OF THE INVENTION Antibodies (Abs), or immunoglobulins (Igs), are protein molecules that bind to an antigen. Each unit of functional binding of natural antibodies is composed of units of four polypeptide chains (two heavy and two light) linked by disulfide bonds. Each of the chains has a constant and variable region. The natural antibodies can be divided into several classes including IgG, IgM, IgA, IgD and IgE, based on their heavy chain component. The IgG class comprises several subclasses that include, in non-limited form, IgGi, IgG2, IgG3 and IgG. Immunoglobulins are produced in vivo with B lymphocytes, and each of these molecules recognizes a particular foreign antigenic determinant and facilitates the clearance of that antigen.

The antibodies can be produced and used in many forms, including antibody complexes. As used herein, the term "antibody complex" or "antibody complexes" is used to indicate a complex of one or more antibodies with another antibody or with a fragment or fragments of antibody, or a complex of two or more fragments of antibody. Examples of antibody fragments include Fv, Fab, F (ab ") 2, Fe and Fd fragments. Accordingly, an antibody according to the present invention comprises a complex of an antibody or a fragment thereof.

As used herein in the specification and in the claims, an Fv is defined as a molecule that is composed of a variable region of a heavy chain of a human antibody and a variable region of a light chain of a human antibody, which can be the same or different, and where the variable region of the heavy chain connects, binds, fuses or covalently binds or associates with the variable region of the light chain. The Fv can be a single chain Fv (scFv) or a disulfide stabilized Fv (dsFv). A scFv is composed of the variable domains of each of the heavy and light chains of an antibody, linked by a flexible amino acid polypeptide spacer or a linker. The linker can be branched or unbranched. Preferably, the linker has 0-15 amino acid residues, and more preferably the linker is (Gly4Ser) 3 (SEQ ID No. 26).

The Fv molecule, itself, is composed of a first chain and a second chain, each chain having a first, second and third hypervariable region. The hypervariable loops within the variable domains of the light and heavy chains are called Complementary Determining Regions (CDR). There are the CDR1, CDR2 and CDR3 regions of each of the heavy and light chains. It is believed that these regions form the antigen binding site and can be specifically modified to give the enhanced binding activity. The most variable of these regions is the CDR3 region of the heavy chain. It is understood that the CDR3 region is the most exposed region of the Ig molecule and, as shown and provided herein, is the site primarily responsible for the selective and / or specific binding characteristics observed.

A fragment of an Fv molecule is defined as any molecule smaller than the original Fv that still maintains the selective and / or specific binding characteristics of the original Fv. Examples of such fragments include, but are not limited to, an antibody, comprising a fragment of the heavy chain only of the Fv, (2) an icrobody, comprising a small fractional unit of the variable region of the heavy chain of the antibody ( International Application No. PCT / IL99 / 00581), (3) similar bodies having a fragment of the light chain, and (4) similar bodies having a functional unit of a light chain variable region. It should be appreciated that a fragment of an Fv molecule can be a substantially circular or loopy polypeptide.

As used herein, the term "Fab fragment" is a monovalent antigen binding fragment of an immunoglobulin. A Fab fragment is composed of the light chain and part of the heavy chain.

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

A Fe fragment is a portion that does not bind to an antigen of an immunoglobulin. It contains the carboxy-terminus portion of heavy chains and the binding sites for the Fe receptor.

An Fd fragment is the variable region and the first constant region of the heavy chain of an immunoglobulin.

Polyclonal antibodies are the product of an immune response and are formed by numerous different B cells. Monoclonal antibodies are obtained from a clonal B cell.

A cassette, applied to polypeptides and defined in the present invention, refers to a given sequence of consecutive amino acids that serves as a framework and is considered a single unit and is handled as such. The amino acids can be replaced, inserted, removed or attached to one or both ends. Similarly, amino acid stretches can be replaced, inserted, removed or joined to one or both ends.

The term "epitope" is used herein to denote the antigenic determinant the recognition site or antigen site that interacts with an antibody, antibody fragment, antibody complex or a complex having a binding fragment thereof or T cell receptor. The term epitope is used interchangeably here with the terms ligand, domain, and binding region.

Selectivity here is defined as the ability of a white molecule to choose and join an entity or cellular state from a mixture of entities or entity states, all entities or entity states can be specific to the target molecule.

The term "affinity" as used herein is a measure of the resistance of the binding (association constant) between a binding molecule (eg, a binding site in an antibody) and a ligand (eg, antigenic determinant) . The resistance of the total sum of non-covalent interactions between a single antigen binding site in an antibody and a single epitope is the affinity of the antibody for that epitope. The low affinity antibodies bind to the antigen weakly and tend to dissociate easily, while the high affinity antibodies bind to the antigen more tightly and remain together longer. The term "avidity" differs from affinity, because the former reflects the valence of the antigen-antibody interaction.

Specificity of the antibody-antigen interaction: Although the antigen-antibody reaction is specific, in some cases the antibodies produced by an antigen can cross-react with another unrelated antigen. These cross-reactions occur if two different antigens share a homologous or similar structure, epitope or an anchor region thereof, or if antibodies specific for an epitope bind to an unrelated epitope that has a conformation of similar structure or chemical properties.

A platelet is a cytoplasmic fragment similar to a disc of a megakaryocyte that spreads into the medulla and then circulates in the peripheral bloodstream. Platelets have several physiological functions that include the main function in the formation of clots. A platelet contains granules located in the center and peripheral clear protoplasm, but it has no defined nucleus.

Agglutination as used herein means the process by which bacteria, cells, disks or other suspended particles of similar size are made to adhere and form masses. The process is similar to precipitation but the particles are larger and are in suspension instead of being in solution.

The term aggregation means an agglutination of platelets induced in vitro, and trorabine and collagen, as part of a sequential mechanism that results in the formation of a thrombus or hemostatic plug.

The substitution of conservative amino acids is defined as a change in the composition of the amino acid by changing one or two amino acids of a peptide, polypeptide or protein, or a fragment thereof. The substitution is of amino acids with generally similar properties (eg, acids, basic, aromatic, size, positive or negative charge, polarity, non-polarity) such that the substitutions do not substantially alter the characteristics of the peptide, polypeptide or protein ( for example, charge, isoelectric point, affinity, avidity, conformation, solubility) or its activity. Typical substitutions that can be made for that conservative amino acid substitution can be between the following amino acid groups: Glycine (G), alanine (A), valine (V), leucine (L) and isoleucine (I) Aspartic acid (D) and glutamic acid (E) Alanine (A), serine (S) and threonine (T) Histidine (H), lysine (K) and arginine (R) Asparagine (N) and glutamine (Q) Phenylalanine (F), tyrosine (Y) and tryptophan (W) Conservative amino acid substitutions can be made, for example, in regions flanking the hypervariable regions primarily responsible for the selective and / or specific binding characteristics of the molecule, as well as other parts of the molecule, for example a heavy chain cassette. variable. Additionally or alternatively, the modification can be achieved by reconstructing the molecules to form full size antibodies, diabodies (dimers), triabodies (trimers), and / or tetrabodies (tetramers) or to form minibodies or microbodies.

A phagemid is defined as a phage particle that carries the plasmid DNA. Phagemids are plasmid vectors designed to contain an origin of replication from a filamentous phage, such as mi3 or fd. As it carries the plasmid DNA, the phagemid particle does not have enough space to contain the complete complement of the phage genome. The missing component of the phage genome is essential information for packaging the phage particle. To propagate the phage, therefore, it is necessary to cultivate the desired phage particles together with a helper phage strain that complements the missing packaging information.

A promoter is a region of DNA in which RNA polymerase binds and initiates transcription.

A phage display library (also referred to as a peptide / phage antibody library, phage library, or peptide / antibody library) comprises a large population of phages (108 or more), each phage particle having a peptide or peptide sequence. different polypeptide. These peptide or polypeptide fragments can be constructed so that the presented peptide or polypeptide is of variable length can be obtained, but need not be, of heavy or light chains of human antibodies.

A "pharmaceutical composition" refers to a formulation comprising an antibody or peptide or polypeptide of the invention and a pharmaceutically acceptable carrier, excipient or diluent thereof, or an antibody-pharmaceutical agent (antibody-agent) complex and a carrier, excipient or pharmaceutically acceptable diluent thereof.

An agent in the context of the present invention is useful in the treatment of active disease, prophylactic treatment, or diagnosis of a mammal that includes, but is not limited to, a human, bovine, equine, porcine, murine, canine, feline, or any other warm-blooded animal. The agent is selected from the group of radioisotope, toxin, oligonucleotide, recombinant protein, antibody fragment, anticancer agents, anti-leukemic agents, antineoplastic, anti-age, anti-adhesion, anti-thrombosis, anti-restenosis, anti-autoimmune, anti-aggregation, antibacterial, antiviral, and anti-inflammatory. Other examples of such agents include, but are not limited to, antiviral agents that include acyclovir, ganciclovir and zidovudine; anti-thrombosis / restenosis agents including cilostazol, sodium dalteparin, sodium reviparin, and aspirin; anti-inflammatory agents including zaltoprofen, pranoprofen, droxicam, acetylsalicylic 17, diclofenac, ibuprofen, dexibuprofen, sulindac, naproxen, tol toline, celecoxib, indomethacin, rofecoxib, and nimesulid; anti-autoimmune agents that include leflunomide, denileucine, diftitox, subreo, WinRho SDF, defibrotide, and cyclophosphamide; and anti-adhesion / anti-aggregation agents including limaprost, chlorchromen and hyaluronic acid.

An anti-leukemia agent is an agent with anti-leukemia activity. For example, anti-leukemia agents include agents that inhibit or stop the growth of immature leukemic or preleukemic cells, agents that eliminate leukemic or preleukemic cells, agents that increase the susceptibility of leukemic or preleukemic cells to other anti-leukemia agents, and agents that inhibit metastasis. of leukemic cells. In the present invention, an anti-leukemia agent can also be an agent with anti-angiogenic activity that prevents, inhibits, retards or stops the vascularization of tumors.

The expression pattern of a gene can be studied by analyzing the amount of the gene product produced in different conditions, at specific times, in different tissues, etc. A gene is considered to be "overexpressed" when the amount of gene product is higher than that found in a normal control, for example a non-diseased control.

A given cell can express on its surface a protein that has a binding site (or epitope) for an antibody, but that binding site can exist in a cryptic form (e.g., sterically hindered or blocked, or lacking the characteristics necessary for antibody binding) in the cell in a state, which can be termed a first phase (phase I). Phase I may be, for example, a non-diseased, healthy, normal state. When the epitope exists in cryptic form, it is not recognized by the given antibody, ie, there is no binding of the antibody to this epitope or to the cell given in phase I. However, the epitope can be exposed for example, suffering itself modifications, or being unblocked because the neighboring or associated molecules change or because a region undergoes a conformational change. Examples of modifications include changes in folding, changes in post-translational modifications, changes in phospholipidation, changes in sulfation, changes in glycosylation, and the like. These modifications can occur when the cell enters a different state, which can be called a second phase (phase II). Examples of second states, or phases, include activation, proliferation, transformation, or in a malignant state. Upon modification, the epitope can then be exposed and the antibody can be bound.

Peptide mimetics (peptide mimics) are molecules (eg antibodies) that no longer contain any peptide bonds, ie amide bonds, between amino acids; however, in the context of the present invention, the term "mimetic peptide" includes molecules that no longer have a completely peptide character, such as pseudo-peptides, semi-peptides and peptoids. Whether fully or partially non-peptides, the peptide mimetics according to this invention provide a spatial arrangement of reactive chemical groups that closely resembles the three-dimensional arrangement of active groups in the peptide on which the peptide mimetic is based. These molecules include small molecules, lipids, polysaccharides or conjugates of them.

Phagemids are plasmid vectors designed to contain an origin of replication from a filamentous phage, such as M13 or fd.

There is a broad spectrum of diseases comprising diseased, altered or otherwise modified cells expressing cell-specific and / or disease-specific ligands on their surfaces. These ligands can be used to effect the recognition, selection, diagnosis and treatment of specific diseases through the recognition, selection, diagnosis and treatment of each individual cell. The present invention provides peptides or polypeptides comprising an Fv molecule, a construction thereof, a fragment thereof, a construction of a fragment thereof, or a fragment of a construct, all of which have improved binding characteristics. These binding characteristics allow the peptide or polypeptide molecule to bind selectively and / or specifically to a target cell in favor of other cells, the specificity and / or selectivity of the binding is determined primarily by a first hypervariable region. The Fv can be a scFv or a dsFv.

The Fv molecule described above can be used to have a white diseased cell. The target cell can be, for example, a cancer cell. Examples of cancer types that are amenable to diagnosis and / or treatment by specific target include, but are not limited to, carcinoma, sercoma, leukemia, adenoma, lymphoma, myeloma, blastoma, inoma and melanoma. Leukemia, lymphoma, and myeloma are cancers that originate in the bone marrow and lymphatic tissues and are involved in the uncontrolled growth of cells.

Antibodies that bind to PSGL-1 and / or GPIb were identified using a phage display library and were disclosed in US Patent Applications No. 10 / 032,423; 10 / 032,037; 10 / 029,988; 10 / 029,926; 09 / 751,181; 10 / 189,032; and 60 / 258,948 and International Applications No. PCT / US01 / 49442 and PCT / US01 / 49440. Specific examples of antibodies disclosed in these applications include antibodies Y1, Y17 and L32. These antibodies were isolated from the germ line (DP32) and found to bind specifically to an epitope, found in hematopoietic cell proteins, which is sulfated on N-terminal tyrosine and thought to be involved in the migration of cells, for example the metastasis of cells.

Sulfated epitopes that bind to Y1 / Y17 / Y32 are characterized by the presence of sulfated groups, such as sulfated tyrosine residues or sulfated carbohydrate or lipid groups, preferably within a group of two or more acidic amino acids, which are ligands and receptors that fulfill important functions in various processes such as inflammation, immune reactions, infection, autoimmune reactions, metastasis, adhesion, thrombosis and / or restenosis, cell coiling, and aggregation. These epitopes are also found in diseased cells, such as T-ALL cells, leukemia B cells, B-CLL cells, AML cells, multiple myeloma cells, and metastatic cells. These epitopes are useful targets for the therapeutic mediation of these processes (as well as target agents) and for diagnostic procedures.

Furthermore, it was found that these binding antibodies of the present invention are dependent on the cell's development phase (the AML subtype is classified based on the French-American-British system using the observed morphology with routine processing and cytochemical staining). ): the antibodies bind to AML cells that are subtype M3 or higher, but not to cells of the MO or Ml subtype. In addition, the antibodies may or may not bind to cells of the M2 subtype. Accordingly, the antibodies of the present invention do not bind healthy, normal bone marrow (e.g., to CD34 + cells). It is thought that these differences are based on alterations in the expression and / or sulfation of PSGL-1, as well as possible conformational changes in PSGL-1 that expose a slightly different epitope.

In particular, it has been discovered that KU812 cells, a line of human chronic myeloid leukemia cells expressing low levels of GlPb, binds to antibody Yl. After the growth of KU812 cells in sodium chlorate, which inhibits sulfation but not the expression of the GPIb protein, the binding of Yl to the cells was reduced by 50%. In addition, it has been discovered that tyrosine-sulfated peptides based on amino acids 273 to 285 of GPIb competitively inhibit the binding of antibody Yl to platelets, while unsulfated peptides do not inhibit the binding of antibody Yl to platelets.

The invention comprises or employing an antibody or a fragment thereof that recognizes and binds to an epitope comprising a sulphated tyrosine motif. That motif comprises a peptide sequence that is rich in acidic residues (aspartame and glutamate) and contains at least one tyrosine. The recognition and binding depend at least in part on at least one of the tyrosines that are sulfated. One of those antibodies is Yl or a fragment of it. Although Y1 is the antibody mentioned in the embodiments described herein, it is not to be understood that it limits the invention to embodiments employing Y1. The invention includes embodiments that use other antibodies that bind to an epitope comprising a tyrosine sulphated motif, including but not limited to antibodies related to Yl and fragments thereof that maintain the binding specificity.

In accordance with the present invention, there is provided an antibody or a fragment thereof that binds to an epitope comprising a tyrosine sulphated motif, wherein the binding depends on at least one tyrosine of the subject being sulfated. In one embodiment, the antibody mediates cell-dependent cytotoxicity. In a preferred embodiment, the antibody is Yl or a related antibody, or a fragment thereof.

The invention further provides an agent that complexes with (e.g., combines, fuses or binds) that antibody or fragment thereof. Between 1 and 16 molecules of agents, or more, can bind to each antibody. The antibody has four disulfide bonds in the hinge region that can be selectively reduced to eight thiol groups. Using a linker that can covalently bind thiol functions and transport an agent molecule, up to eight agent molecules can be bound to the antibody. Using a linker that reacts simulatively with thiol functions but carries n agent molecules, up to 8n agent molecules can be bound to the antibody. In one embodiment only the heavy chains complex with the agent or only the light chains complex with the agent, while in a more preferred embodiment, each antibody chain complexed with 2 copies of the agent and each light chain complexed with 2 copies of the agent.

Those skilled in the art know that an even greater number of agent molecules can be bound to the antibody using intermediate drug carriers such as natural (e.g., dextran) or synthetic polymers (e.g., HPMA) as well as liposomes (e.g., antibody-linker-carrier-agent). The agents can also bind directly or indirectly to free amino groups of the antibody. For example, the agents can be linked to e- or α-amino acid groups through a linker. Generally an agent is attached to a linker directly, or first to a carrier, which is then attached to a linker. The linker-agent or linker-carrier-agent complex then binds to the antibody. The antibody-agent complex can be internalized by a tumor cell, where the agent causes the death of the cell. In one embodiment, the ligation of the antibody-agent complex can be broken within the cell, for example by acid cleavage or enzyme cleavage. In a preferred embodiment, the antibody is Yl or a related antibody, or a fragment thereof.

The invention further provides a composition for treating a disease comprising Yl or a Yl-agent complex.

In one embodiment, the present invention provides methods of inducing or activating ADCC by administering the antibodies of the present invention. Accordingly, these antibodies can activate ADCC and / or stimulate natural killer (NK) cells (e.g., CD56 +) ΔT cells, and / or monocytes, which can lead to cell lysis. Generally, after administration of an antibody comprising a Fe region or portion of the antibody, said antibody binds to a Fe receptor (FcR) in effector cells, eg, NK cells, which trigger the release of perforin and granzyme B and / or the induction of Fas B expression, which then results in apoptosis. The binding of FasL, expressed in effector cells to the Fas receptor on the surface of the target cell, can induce apoptosis of the target cell through the activation of the signal transduction pathway of the Fas receptor. In one embodiment, the antibody of the invention induces the expression of FasL in effector cells. Different factors can affect ADCC, which include the type of effector cells involved, the cytokines (IL-2 and G-CSF, for example), the incubation time, the number of receptors present on the surface of the cells, and the affinity of the antibody.

In yet another embodiment, a method of inducing cell death is provided by administering to a patient in need thereof an antibody of the present invention coupled or complexed with an agent, wherein the pair or antibody-agent complex enters the cell by internalization and the antibody-agent conjugate or complex is broken, releasing the agent. Internalization can take place by any suitable means, for example, by endocytosis or by phagocytosis. The invention therefore provides a means of treating a disease (for example, treating may include improving the effects of a disease, preventing a disease, or inhibiting the progress of a disease) in a patient.

Specifically, an antibody is used to introduce an agent into a cell. The antibody binds to proteins that are preferentially expressed on the surface of diseased cells, such as proteins with sulphated tyrosine residues. In a preferred embodiment, the agent is a toxin such as doxorubicin, morpholino-doxorubicin, or morpholino-daunorubicin. In a more preferred embodiment, the toxin is bound to the antibody through an adipic acid linker or a [N-e-Maleimidocaproic acid] hydrazide linker. The adipic acid linker has been used to bind the amino groups, while the N- [aleimidocaproic acid] hydrazide linker has been used to bind both the amino groups and the SH groups of the disulfide bonds reduced (through the maleimido group to form a CS bond). In addition, a hydrazone bond is formed between the drug and the N- [meleimidocaproic acid] hydrazide linker.

After the antibody-agent complex binds to the cell surface protein, the cell internalizes the complex. The enzymes that are inside the cell then break the ligation of the antibody-toxin and the toxin acts on the cell to cause its death. In another embodiment, the invention provides a composition for treating a disease comprising said antibody-toxin conjugate.

Another embodiment provides an analogous method for introducing a non-toxic agent into a cell. The non-toxic agent can be used to change the behavior or activity of the cell, for example by activating or repressing directly or indirectly the activity of a specific gene.

In another embodiment, the present invention provides a method of preventing infection by a virus comprising administering to a patient in need thereof an antibody as herein. Thus, a means of treating a disease is accomplished by administering an antibody that blocks the infection. The cell expresses on its surface a protein containing an epitope containing the sulphated tyrosine motif which is recognized by the antibody and which is also necessary for infection by the infectious agent. The antibody binds to the protein, thus blocking the infection. Proteins known to bind the preferred antibody through the epitope containing the sulfated tyrosine motif include a fibrinogen chain, a GPIb-a chain, the C4 complement and PSGL-1. Proteins that are believed to bind the preferred antibody through an epitope containing the sulfated tyrosine motif include CCR5 and CXCR4. Any of CCR5 and CXCR4 can function as a necessary co-receptor for HIV infection. In a preferred embodiment, the antibody can be used to block infection by an HIV strain. Preferably the antibody is Yl.

Finally, a method for introducing an agent into a sulphated PSGL-1 expressing cell having the following steps is provided: coupling or forming a complex of the agent with an antibody described herein and administering the pair or antibody-agent complex to the cell.

The antibody of the present invention binds to PSGL-1-sulphated. Although the cells involved in inflammation, such as monocytes, neutrophils, and lymphocytes, are recruited mainly by the four molecules, PSGL-1, P-Selectin, VLA-4 and VCAM-1 in the inflammatory processes of diseases such as atherosclerosis ( Huo and Ley, Acta Physiol Scand., 173: 35-43 (2001), Libby, Sci. Am. May: 418-55 (2002), Wang et al, J. Am. Coll. Cardiol. 38: 1577- 582 (2001)). Antibody interference with any of these core molecules may suggest a potential role for the antibody in canceling related diseases. Specifically, P-selectin controls the binding and winding of the cells. In addition, the interactions of P-selectin-PSGL-1 • activate numerous other molecules in cells that are integrally connected with tumorigenesis (when it refers to malignant cells) and inflammatory responses (when referring to white blood cells) (Shebuski and Kilgore, J. Pharmacol., Ther. 300: 729-735 (2002)). Based on this interpretation of the ability of P-selectin to regulate cellular processes, it is evident that the improved scFv selectivity of the antibody to sulfated PSGL-1 can make it a superior molecule to treat a variety of malignant and inflammatory diseases. In addition, malignant disease models have shown that the binding of P-selectin to malignant cells requires the sulfation of PSGL-1 (Ma and Geng, J. Immunol., 168: 1690-1696 (2002)). This requirement is similar to that for antibody binding. Therefore, it can be expected that the antibody can cancel the facilitation of P-selectin from the progression of the malignant disease.

Preferably, the antibody of the present invention binds to an epitope present in at least one cell type involved in inflammation or tumorigenesis, which includes T-ALL cells, AML cells, Pre-B-All cells, B-leukemia, B-CLL cells, multiple myeloma cells and metastatic cells. More preferably, the antibody of the present invention can bind to epitopes on a molecule of lipid, carbohydrate, peptide, glycolipid, glycoprotein, lipoprotein, and / or lipopolysaccharide. These epitopes preferably have at least one sulphated group. Alternatively, but also preferably, the antibody of the present invention cross-reacts with two or more epitopes, each epitope having one or more tyrosine residues, and at least one group of two or more acidic amino acids, one of whose examples is PSGL -1.

These antibodies, antigen-binding fragment or complex thereof, of the present invention can be internalized in a cell after binding to PSGL-1 on the surface of the cell. That internalization can occur through endocytosis as an active process, which depends on the manner, time and temperature. For example, Yl is internalized specifically in the cells of patients with AML through PSGL-1.

The antibody of the present invention binds to proteins that have tyrosine sulfation sites. Those proteins include PSGL-1, GPIb, α-2-antiplasmin; aminopeptidase B; chemokine CC receptors such as CCR2, CCR5, CCR3, CXCR3, CXCR4, CCR8, CCR2b and CXCI; seven-segment transmembrane receptors (7TMS); coagulation factors such as factor V, VIII and IX; Gamma fibrinogen chain; Heparin II cofactor; secret grains such as secretgram I and II; vitronectin, amyloid precursor, a-2-antiplasmin; cholecystokinin; a-choriogonadotropin; C4 complement; dermatan sufaieproteiglican; fibrinectin; and castrina. In a preferred embodiment, the antibody of the present invention binds to chemokine CC receptors such as CCR5, CXCR4, CXCI and CCR2b. As mentioned above, sulphated tyrosines can contribute to the binding of CCR5 to MlP-la, MlPβ, and HIV-1 gpl20 / CD4 and to the ability of HIV-1 to enter cells expressing 1CCR5 and CD4.

Antibodies, peptides, polypeptides, proteins, and fragments and constructions thereof can be produced in prokaryotic or eukaryotic expression systems. Methods for producing antibodies and fragments in prokaryotic and eukaryotic systems are known in the art.

A system of eukaryotic cells, defined in the present invention and discussed, refers to an expression system for producing peptides or polypeptides by genetic engineering methods, wherein the host cell is a eukaryote. A eukaryotic expression system can be a mammalian system, and the peptide or polypeptide produced in the mammalian expression system, after purification, is preferably substantially free of mammalian contaminants. Other examples of a useful eukaryotic expression system include yeast expression systems.

A preferred prokaryotic system for the production of the peptide or polypeptide of the invention uses E. coli as the host for the expression vector. The peptide or polypeptide produced in the E. coli system, after purification, is substantially free of proteins that contaminate E. coli. The use of a prokaryotic expression system can result in the addition of a methionine residue to the N-terminus of some or all of the sequences provided in the present invention. Removal of the methionine residue from the N-terminus, after production of the peptide or polypeptide to allow full expression of the peptide or polypeptide, can be performed as is known in the art, an example is the use of Aeromonas aminopeptidase under suitable conditions ( United States Patent No. 5,763,215).

The antibodies and polypeptides of the present invention can form complexes, eg, associate, combine, fuse or bind with different pharmaceutical agents, such as drugs, toxins, and radioactive isotopes and optionally, with a pharmaceutically effective carrier, to form peptide compositions. drug comprising an antibody / polypeptide and a pharmaceutical agent having an anti-disease and / or anti-cancer activity. These compositions can also be used for diagnostic purposes.

For example, conjugation or complexing of anthracyclines to antibodies is generally known in the art (Dubowchik &Walker, Pharmacol. &Thera., 83: 167-123 (1999), Trail et al, Cancer Immunol. Immunother. 52: 328-337 (2003)). Such conjugation can be by direct conjugation or through linkers, such as linkers that can be broken with acid or linkers that can be cleaved with enzyme and can involve the use of intermediate carriers such as dextran and synthetic polymers. The anthracyclines have complexed with the antibodies of the present invention through (1) amino groups (pH 8) to produce a drug: antibody ratio of 4: 1 (in which case two drug molecules bind to the chain heavy and two to the light chain); and (2) disulfide ligatures to produce a drug: antibody ratio between 4: 1 and 8: 1 depending on the method used. As is known in the art, the ratio of antibody to drug can, for example, be doubled, tripled or quadrupled, etc., using a linker of two, three, four, etc. branches. One skilled in the art can make chemical modifications to the antibody, linker, carrier and / or drug to make the reactions more convenient for the purposes of preparing a conjugate.

In one embodiment of the present invention, the two ligatures of the Fe region were reduced with mercaptoethylane and then reacted with the drug ligature at pH 7, which results in a drug: antibody ratio of 4: 1 (in which case the four drugs join in the heavy chains). In another embodiment, the four disulfide bonds of the hinge region were reduced with DTT (pH 7) and then reacted with the drug linker, which results in a drug: antibody ratio of 7: 1 to 8: 1 (in whose case 5 or 6 of the drug molecules bind to the heavy chains and one or two of the drug molecules bind to the light chains.

Examples of carriers useful in the invention include dextran, HPMA (a hydrophilic polymer), or any other polymer, such as a hydrophilic polymer, as well as derivatives, combinations and modifications thereof. Alternatively, decorated liposomes, also called immunoliposomes, may be used, such as liposomes decorated with scFv Yl molecules, such as Doxil, a commercially available liposome containing large amounts of doxorubicin. These liposomes can be prepared to contain one or more desired agents and mixed with the antibodies of the present invention to provide a high drug to antibody ratio.

Alternatively, the binding between the antibody or polypeptide and the agent can be a direct binding. A direct link between two or more neighboring molecules can be produced through a chemical bond between elements or groups of elements in the molecules. The chemical bond can be, for example, an ionic bond, a covalent bond, a hydrophobic bond, a hydrophilic bond, an electrostatic bond, or a hydrogen bond. The bonds can be, for example, amide, carbon sulfide, peptide and / or disulfide bonds. To bind the antibody to the agent or linker, amine, carboxy, hydroxyl, thiol and ester functional groups, which are known in the art to form covalent bonds, can be used.

The binding between the peptide and the agent or between the peptide and the carrier, or between the carrier and the agent can be through a binding compound. As used herein, a linker compound is defined as a compound that joins two or more groups. The linker can be straight or branched chain. A branched linker compound can be comprised of a double branch, a triple branch, or a quadruple or more branched branched compound. The binder compounds useful in the present invention include those which are selected from the group having dicarboxylic acids, maleimido hydrazides, PDPH, carboxylic acid hydrazides and small peptides.

More specific examples of useful linker compounds, according to the present invention, include: (a) dicarboxylic acids such as succinic acid, glutaric acid, and adipic acid; (b) maleimido hydrazides such as N- [maleimidocaproic acid] hydrazide, 4- [N-maleimidomethyl] cyclohexane-1-carboxylhydrazide, and N- [maleimidoundecanoic acid] hydrazide, (c) (3- [2-pyridylthio] propionyl hydrazide ) derivatives, combinations, modifications and analogues thereof; and (d) carboxylic acid hydrazides which are selected from 2-5 carbon atoms.

Binding through direct coupling using small peptide linkers is also useful. For example, direct coupling between free sugar, for example, of the anti-cancer drug doxorubicin and mon scFv can be performed using small peptides. Examples of small peptides include AU1, AU5, BTag, c-myc, FLAG, Glu-Glu, HA, His6 (SEQ ID No. 27), HSV, HTTPHH (SEQ ID No. 28), IRS, KT3, Protein C, S-TAG®, T7, V5, VSV-G and KAK. The antibodies and polypeptides of the present invention can be joined, conjugated, complexed or otherwise associated with imaging agents (also reporter markers), such as radiosotopes, and these conjugates can be used for diagnostic and imaging purposes. . Cases having radioisotope-antibody (or fragment) complexes are provided.

Examples of radioisotopes useful for diagnosis include xllindium, indium, 99mrenium, 105renium, 101renium, 99mtecnetium, 121mtelurio, 122ratelurio, 125mtelurio, 165tulio, 167tulio, 168tulio, Iodine, Iodine, Iodine, Iodine, 81mkrypton, 33xenone, 90ithium, bismuth, bromine, fluorine, ruthenium, ruthenium, ruthenium, 105Rothenius, 107mercury, 203mercury, 67galio and 68galio. The preferred radioactive isotopes are opaque to X-rays whatever any suitable paramagnetic ion.

The indicator marker molecule can also be a fluorescent marker molecule. Examples of fluorescent marker molecules include fluorescein, phycoerythrin, or rhodamine, or modifications or conjugates thereof.

Antibodies and polypeptides that are conjugated with indicator markers can be used to diagnose, predict, or monitor disease states. Generally, those methods include providing a sample of at least one cell of a patient and determining whether the antibody or a fragment thereof of the present invention binds to the patient's cell, thus indicating that the patient is at risk or has the disease . That monitoring can be done in vivo, in vitro, or ex vivo. When monitoring or diagnosis is performed in vivo or ex vivo, the imaging agent is preferably physiologically acceptable in that it does not harm the patient to an unacceptable level. Acceptable levels of harm can be determined by clinicians using criteria such as the severity of the disease and the availability of other options.

With respect to cancer, determining the phases of a disease in a patient usually consists in determining the classification of the disease based on the size, type, location and invasiveness of the tumor. A cancer classification system based on the characteristics of the tumor is the "TNM Classification of Malignant Tumors" (6th edition) (LH Sobin, Ed.), Which is incorporated herein by reference and which classifies cancer phases in T categories. , N and M where T describes the primary tumor according to its size and location, N describes the regional lymph nodes, and M describes distant metastases. In addition, the numbers I, II, III and IV are used to indicate the phases and each number refers to a possible combination of TNM factors. For example, a Phase I breast cancer is defined with the TMN group: TI, NO, MO which means: TI: the tumor is 2 cm or less in diameter, NO: non-regional lymph node metastasis, MO: no metastasis distant. Another system is used to determine phases of AML, with classification subtypes based on the French-American-British system using the morphology observed under routine processing and cytochemical staining.

In addition, a proposed phase determination or classification of the World Health Organization recently proposed for neoplastic diseases of the hematopoietic and lymphoid tissues includes (specifically for AML) traditional FAB type categories of disease, as well as additional disease types that correlate with specific cytogenetic findings and AML associated with myelodysplasia. Others have also proposed proposed pathological classifications. For example, a specific proposal for AML includes types of disease that correlate with specific cytogenetic translocations and that can be recognized reliably by morphological and immunophenotyping evaluation and that incorporate the importance of associated myelodysplastic changes. This system would be supported by cytogenetic or molecular genetic studies and could be expanded as new recognizable clinicopathological entities are described (Arber, Am. J. Clin. Pathol. 115 (4): 552-60 (2001)).

The present invention provides a diagnostic kit for the in vitro analysis of the effectiveness of the treatment before, during or after treatment, having an imaging agent having a peptide of the invention linked to a marker molecule, or an agent of image formation. The invention provides a method of using the imaging agent for the diagnosis, localization and imaging of a cancer, more specifically a tumor, having the following steps: (a) bringing the cells into contact with the composition; (b) measuring the radioactivity bound to the cells; and therefore (c) visualize the tumor.

Examples of suitable imaging agents include fluorescent dyes, such as FITC, PE and the like, and fluorescent proteins, such as green fluorescent proteins. Other examples include radioactive molecules and enzymes that react with a substrate to produce a recognizable change, such as a color change.In one example, the cassette imaging agent is a fluorescent dye, such as FITC, and the kit provides analysis of the efficacy of the treatment of cancers, more specifically of blood-related cancers, for example leukemia, lymphoma and myeloma The FACS analysis is used to determine the percentage of cells stained by the imaging agent and the intensity of the staining at each stage of the disease, for example at diagnosis, during treatment, during remission and during relapse.

The antibodies and polypeptides of the present invention can be bound, conjugated or otherwise associated with anticancer agents, antineoplastic agents, antiviral agents, anti-metastasis agents, anti-inflammatory agents, anti-thrombosis agents, anti-restenosis agents, anti-aggregation agents, anti-autoimmune agents. , anti-adhesion agents, anti-cardiovascular disease agents, pharmaceutical agents, or other anti-disease agents. An agent refers to an agent that is useful in the prophylactic treatment or diagnosis of a mammal that includes, but is not limited to, a human, bovine, equine, porcine, murine, canine, feline, or any other warm-blooded animal. .

Examples of such agents include, but are not limited to, antiviral agents including acyclovir, ganciclovir and zidovudine; anti-thrombosis / restenosis agents including cilostazol, sodium dalteparin, sodium reviparin, and aspirin; anti-inflammatory agents including zaltoprofen, pranoprofen, droxicam, acetylsalicylic 17, diclofenac, ibuprofen, dexibuprofen, slulindac, sulindac, naproxen, antolmethin, celecoxib, indomethacin, rofecoxib, and nimesulid; anti-autoimmune agents including leflunomide, denileucine diftitox, subreo, WinRho SDF, defibrotide, and cyclophosphamide; and anti-adhesion / anti-aggregation agents including limaprost, chlorchromen and hyaluronic acid.

Examples of pharmaceutical agents include anthracyclines such as doxorubicin (adriamycin), daunorubicin, idarubicin, detorubicin, carminomycin, epirubicin, esorubicin, morpholinodoxorubicin, morpholinodaunorubicin, methoxymorpholinyldoxorubicin, methoxyorfolinodaunorubicin and methoxymorpholinyldoxorubicin and substituted derivatives, combinations and modifications thereof. Other examples of pharmaceutical agents include cis-platinum, taxol, calichea icine, vincristine, cytarabine (Ara-C), cyclophosphamide, prednisone, fludarabine, idarubicin, chlorambucil, interferon alpha, hydroxyurea, temozolomide, thalidomide and bleomycin and derivatives, combinations and modifications from them.

An anti-cancer agent is an agent with anticancer activity. For example, anticancer agents include agents that inhibit or stop the growth of immature cancer or precancerous cells, agents that kill cancer or precancerous cells, agents that increase the susceptibility of cancer or precancerous cells to other anticancer agents, and agents that inhibit cancer. metastasis of cancer cells. In the present invention, an anti-cancer agent can also be an agent with anti-angiogenic activity that prevents, inhibits, retards or stops the vascularization of tumors.

Inhibition of the growth of a cancer cell includes, for example, (i) prevention of cancerous or metastatic growth, (ii) slowing of cancerous or metastatic growth, (iii) total prevention of the growth process of the cancer cell or metastatic process, while leaving the cell intact and alive, (iv) interference from the contact of cancer cells with micromedia, (v) elimination of the cancer cell. For example, an antibody can effect the elimination of a cancer cell by binding it to the cancer cell and stimulation of T cells or natural killer cells to eliminate the bound cell by the antibody-dependent cell cytotoxicity.

An anti-leukemia agent is an agent with anti-leukemia activity. For example, anti-leukemia agents include agents that inhibit or stop the growth of leukemic or immature preeleukemic cells, agents that eliminate leukemic or preleukemic cells, agents that increase the susceptibility of leukemic or preleukemic cells to other anti-leukemia agents, and agents that inhibit the metastasis of leukemic cells. In the present invention, an anti-leukemia agent can also be an agent with anti-angiogenic activity that prevents, inhibits, retards or stops the vascularization of tumors.

Inhibition of the growth of a leukemia cell includes, for example, (i) prevention of leukemic or metastatic growth, (ii) leukemia of leukemic or metastatic growth, (iii) total prevention of the growth process of the leukemia cell or the metastatic process, while leaving the cell intact and alive, (iv) contact interference of cancer cells with the micro-environment, or (v) elimination of the leukemia cell.

Examples of anti-disease, anti-cancer, and anti-leukemic agents to which antibodies and fragments of the present invention can be usefully attached include toxins, radioisotopes, and pharmaceutical compositions. Examples of toxins include gelonin, Pseudomonas exotoxin (PE), PE40, PE38, diphtheria toxin, ricin, or derivatives, combinations and modifications thereof.

Examples of radioisotopes include gamma radiation emitters, positron emitters, and X-ray emitters that can be used for localization and / or therapy, and gamma radiation emitters and alpha radiation emitters that can be used for therapy. The radioisotopes that were previously described as useful for diagnosis are also useful for therapeutics.

Non-limiting examples of anti-cancer or anti-leukemia agents include anthracyclines such as doxorubicin such as doxorubicin (adriamycin), daunorubicin, idarubicin, detorubicin, carminomycin, epirubicin, esububicin, morpholinodoxorubicin, morpholinodaunorubicin, oteximorpholinyldoxorubicin, methoxymorpholinodaunorubic and methoxymorpholinyldoxorubicin and substituted derivatives, combinations and modifications of them. Examples of pharmaceutical agents include cis-platinum, taxol, calicheamicin, vincristine, cytarabine (Ara-C), cyclophosphamide, prednisone, daunorubicin, idarubicin, fludarabine, chlorambucil, interferon alpha, hydroxyurea, temozolomide, thalidomide, and bleomycin, and derivatives, combinations and modifications of them.

In one embodiment, the pharmaceutical compositions of the present invention have an antibody or polypeptide of the present invention and a pharmaceutically acceptable carrier. The antibody or polypeptide may be present in an amount effective to inhibit cell coiling, inflammation, autoimmune disease, metastasis, growth and / or replication of tumor cells or leukemia cells or increase the number of tumor cells in a patient who has tumor cells or leukemia in a patient who has leukemia. Alternatively, the antibody or polypeptide may be present in an amount effective to increase the mortality of tumor cells or leukemia cells. Also alternatively, the antibody or polypeptide may be present in an amount effective to alter the susceptibility of diseased cells to damage by anti-disease agents, from tumor cells to damage by anti-cancer agents, or from leukemia cells to damage by anti-leukemia agents. Also alternatively, the antibody or polypeptide may be present in an amount effective to reduce the number of tumor cells in a patient having tumor or leukemia cells in a patient having leukemia. Still alternatively, the antibody or polypeptide may be present in an amount effective to inhibit restenosis. The antibody or polypeptide may also be present in an amount effective to inhibit HIV entry. Alternatively, the antibody or polypeptide can be used as a target agent to direct a therapeutic agent to a specific cell or site.

The antibodies and polypeptides of the present invention can be administered to patients in need thereof by any suitable method. Examples of methods include intravenous, intramuscular, subcutaneous, topical, intratracheal, intrathecal, intraperitoneal, intralymphatic, nasal, sublingual, oral, rectal, vaginal, respiratory, buccal, intradermal, transdermal, or intrapleural administration.

For intravenous administration, the formulation is preferably prepared such that the amount administered to the patient is an effective amount from 0.1 mg to 1000 mg of the desired composition. More preferably, the amount administered is in the range of 1 mg to 500 mg of the desired composition. The compositions of the invention are effective in a wide range of dosage and depend on factors such as the details of the disease to be treated, the half-life of the peptide, or the pharmaceutical composition based on the polypeptide in the patient's body, the characteristics physical and chemical agents of any agent that complexes with the antibody or fragment thereof and of the pharmaceutical composition, the manner of administration of the pharmaceutical composition, the details of the patient to be treated or diagnosed, as well as other parameters that the treating physician considers important.

The pharmaceutical composition for oral administration can be in any suitable form. Examples include tablets, liquids, emulsions, suspensions, syrups, pills, and capsules. Methods of manufacturing pharmaceutical compositions are known in the art (See, for example, Remington, The Science and Practice of Pharmacy, Alfonso R. Gennaro (Ed.) Lippincott, Williams &Wilkins (pub)).

The pharmaceutical composition can also be formulated so as to facilitate timed, sustained, boosted or continuous release. The pharmaceutical composition can also be administered in a device, such as a timed, sustained, driven or continuous release device.

The pharmaceutical composition for topical administration may be in any suitable form, such as creams, ointments, lotions, patches, solutions, suspensions, lyophilisates and gels. The compositions having antibodies, constructs, conjugates and fragments of the present invention may comprise conventional pharmaceutically acceptable diluents, excipients, carriers, and the like. The tablets, troches and capsules may include conventional excipients such as lactose, starch and magnesium stearate. Suppositories can include excipients such as waxes and glycerol. Injectable solutions include sterile pyrogen-free media such as saline and may include buffering agents, stabilizing agents or preservatives. Conventional enteric coatings can also be used.

The antibodies and polypeptides of the present invention and the pharmaceutical compositions thereof, can be used in methods of ameliorating the effects of a disease, preventing a disease, or inhibiting the progress of a disease in patients in need thereof. Those methods include inhibiting cell coiling, inflammation, autoimmune disease, metastasis, growth and / or replication of tumor cells or leukemia cells, or increasing the number of tumor cells in a patient who has cells of tumor or leukemia in a patient who has leukemia. In addition, these methods include increasing the mortality rate of tumor cells or leukemia cells, altering the susceptibility of diseased cells to damage by anti-disease agents, of tumor cells to damage by anti-cancer agents, or of the leukemia cells to damage by anti-cancer agents. These methods also include reducing the number of tumor cells in a patient having tumor or leukemia cells in a patient who has leukemia. These methods also include inhibiting or reducing the entry of HIV into the cells. These methods also include preventing or inhibiting cardiovascular diseases such as restenosis.

The present invention further provides a method of making a medicament for the treatment of different disease states such as, for example, AML, T-ALL, B-leukemia, B-CLL, Pre-B-ALL, multiple myeloma, metastasis, infection with HIV, cardiovascular diseases, or other diseases in which cellular functions or actions such as cell coil, inflammation, immune reactions, infection, autoimmune reactions, metastasis, fulfill a significant function. That medicament comprises the antibodies and the polypeptides of the present invention.

In one embodiment, the invention provides a method of diagnosing cancer in a person by testing the ability of Yl to specifically bind to a tissue sample and compare the binding of Yl to the binding by a control antibody such as KPL-1. In one embodiment, the method comprises isolating samples from blood cells or solid tissue of the person, incubating the cells with an antibody or a fragment thereof that recognizes an epitope containing sulfated tyrosine motifs ("the experimental antibody"), washing I read bound antibody in non-specific form, and compare the results with those of a corresponding staining procedure performed with a reference standard such as a control antibody with the known binding activity. A control antibody is one that recognizes an epitope that contains the unsulfated form of the tyrosine motif or antigens that contain it. The presence of tumor cells is indicated when the binding of the experimental antibody is substantially greater than the binding by the control, determined by the resistance of the staining. The staining procedure can be performed by standard methods. For example, the first antibody can be visualized using secondary antibodies that recognize the first antibody and that are conjugated to an enzyme substrate that produces a color reaction when activated by the enzyme. Alternatively, the presence of tumor cells is indicated when both the experimental antibody and the control antibody bind the cells, but the cells internalize Yll and do not internalize the control antibody. In one embodiment, the cancer is a solid tumor. In another embodiment, the cancer lies a tumor carried by the blood. In a preferred embodiment, the experimental antibody is Yl or a fragment thereof, or a related antibody or a fragment thereof. In another preferred embodiment, the control antibody is KPL1.

In another embodiment, the invention provides a method of diagnosing a cancer which comprises detecting samples of blood cells or other tissue for the presence of tumor cells. Western blot analyzes were performed on samples of cells using Yl or a fragment thereof, or a related antibody or fragment thereof. Yl binding can be observed by labeling Yl with a detectable label, or using standard methods that employ a detectable antihuman antibody. The presence of tumor cells is indicated when the binding of Yl is substantially greater than the binding by the control, where the control is as defined above. The presence of tumor cells is indicated when the binding of Yl is substantially greater than the binding by the control.

In another embodiment, the invention provides a method of identifying tumor protein markers transported by blood or solids by preparing a cell lysate and purifying the lysate by passing it through an affinity column. The affinity column incorporates Yl or a fragment thereof, or a related antibody or a fragment thereof. In one embodiment, the cell lysate is obtained from a sample of primary tissue extracted from a human. In another embodiment, the cell lysate is obtained from a tumor cell line. In another embodiment, the tumor cell line can be an immortalized cell line.

In another embodiment, the invention provides a method of monitoring the phase of a cancer transported by blood comprising isolating white blood cells from a patient with a cancer carried by the blood, incubating the cells with Y1, determining the level of Y1 binding. in relation to the reference standard.

As an alternative approach to the therapeutic target of tyrosine-sulfated epitopes present in proteins, such as GPIb and PSGL-1, a small inorganic chemical entity can be identified by detecting an appropriate combinatorial library. Such an entity can have numerous advantages with respect to a therapeutic agent based on scFv or IgG. For example, an inorganic chemical entity can be administered orally and have an improved biosecurity profile, which includes cross-reactive immunoreactivity. It can provide improved selectivity towards the target, particularly following the rational drug design to optimize an initially selected compound. Other advantages include lower production costs, a longer duration and a less complicated regulatory approval process.

Since numerous embodiments of the epitope of the invention have been identified, for example in GPIb and PSGL-1, a ligand-driven approach can be taken to identify inorganic guanic entities, which have very narrow specificity, or alternatively, target more of a sulfated tyrosine epitope for disease states such as reperfusion injury consists of more than one distinguishable target each carrying that epitope. The ligand-driven approach significantly shortens the detection process to identify targets for therapeutic intervention, and allows simultaneous validation of the target with optimization, which can be done with a series of focused libraries.

A library of specialized inorganic chemical entities can be designed and developed for white sulphated tyrosine epitopes by first analyzing the three-dimensional interaction between an antibody such as Yl and its known targets such as sulfated residues Tyr-276 and Asp-277 of GPIb. Chemical libraries composed of entities that mimic the binding site of Y1 and that provide enhanced affinity to the target can be developed by designing combinatorial libraries assisted by computer.

Throughout this application, reference has been made to different publications, patents and patent applications. The teachings and disclosures of these publications, patents and patent applications in their entirety are incorporated by reference in this patent application to more fully describe the state of the art to which the present invention pertains.

EXAMPLES The following examples are given to help understand the invention but are not intended and should not be construed as limiting its scope in any way. Although specific reagents and reaction conditions are described, modifications may be made that are encompassed by the scope of the invention. The following examples, accordingly, are given to further illustrate the invention.

EXAMPLE 1: Identification of the Yl ligand of primary AML cells (R1198-3) 1. 1 Primary AML cells (M4 phase) were extracted from a patient and lysed. The lysate was subjected to purification comprising affinity chromatography on a Yl-IgG column (see Figure 1). The isolated protein was digested with endoproteinase. Asp-N and the resulting peptide sequence was determined using mass spectrometry. The sequence was identical to the amino acid sequence of the N-terminus of published human PSGL-1. These results indicate that the primary AML cells in phase 4 express PSGL-1 that bind by Yl-IgG. It was also determined that the purified protein was sulphited in tyrosines 2 and 3 of the recognition motif of Yl (see Figure 2). Internal controls were used to verify the specificity of the immunomodulatory effects of Yl, for example no induction of mouse interleukin 6 secretion was detected.

EXAMPLE 2: Cell cytotoxicity that depends on the antibody 2.1 Effect of Yl-IgG: Studies to determine if Yl-IgG is capable of mediating antibody-dependent cell cytotoxicity (ADCC) showed that this antibody mediates the cytotoxicity of effector cells from different target cells, including ML2 (a cell line derived from AML which served as a target in our model system) and B-CLL cells from clinical samples of patients. Yl-IgG binds to these cell types through CD162 (PSGL-1), a molecule that is substantially absent in healthy B cells and early phase AML.

The population of effector cells participating in ADCC of Yl-IgG has been defined. For Yl-IgG to mediate ADCC, natural killer (NK) cells (CD56 +),? DT cells and monocytes (CD14 +) are required, but helper T cells (CD4 +) and cytotoxic T cells (CD8 +) are not required. This was confirmed with donor cells from both healthy subjects and patients with B-CLL.

Furthermore, even in the absence of target cells, Yl-IgG mediates the activation of different types of effector cells, as measured by the appearance of an early activation marker (CD69 +), the secretion of cytokines, such as TNFa and TNF? and the induction of FasL. Hybridization (XL) of Yl-IgG with secondary anti-human Fe antibodies demonstrated that an apotopic mechanism also contributes to the elimination of cells.

The activity of Yl-IgG towards primary B-CLL cells in vitro was compared with that of two commercially available antibodies that are widely used today for the treatment of different diseases: Rituximab (which binds to CD20) and Campath (which binds to CD52). While the mechanism of action of Rituximab against B-CLL is unclear, its cytotoxic effects against CD20-positive malignant B cells may involve one or more of complement-dependent cytotoxicity (CDC), ADCC and induction of apoptosis. The cytotoxic effects of Campath against CD52-positive malignant B cells, as well as normal B and T cells, involve CDC, ADCC and induction of apoptosis. The administration of Campath is associated with complete ablation of all mature B and T cells, which results in severe haematological toxicity.

For the ADCC experiments, the mononuclear and target effector cells were separated in FICOLL®. The target cells were then labeled with PKH26, which stably incorporated a fluorescent dye into the lipid regions of the cell membranes. The cells were then washed and incubated with effector cells at different Effector-White ratios.

(E: T), in the absence or presence of different concentrations of Yl-IgG or control antibodies for 24 hours. The dead cells were stained with TOPRO® (Molecular Probes, Inc., Eugene, OR) and analyzed by FACS in confined white cells.

For the CDC experiments, mononuclear cells from patients with B-CLL were separated in FICOLL®. The cells were incubated with or without Yl-IgG or control antibodies for 24 hours in the presence or absence of the patient's plasma. The apoptotic cells were then stained with A exin-PI and analyzed by FACS.

To evaluate the activation of the effector cells, mononuclear cells were separated from healthy donors in FICOLL®. The cells were incubated with or without Yl-IgG or control antibodies for 24 hours. FACS analysis with the aCD69 antibody (an early activation marker) was performed for different types of effector cells. The secretion of cytokines such as TNFa and IFN? it was measured by ELISA.

For the apoptosis experiments, the mononuclear cells of patients with B-CLL were separated in FICOLL®. The cells were incubated in the presence or absence of Yl-IgG or control antibodies for 10 minutes at 37 ° C. Then anti-human Fe antibodies were added and incubated for 4-24 hours at 37 ° C. The diseased cells (CD19 +, CD5 +) were then stained for the apoptotic markers, Amexin-TOPRO® and analyzed by FACS.

Comparative studies with Yl-IgG and Rituximab indicate that Yl-IgG is superior to Rituximab with respect to the mediation of ADCC and the induction of apoptosis with B-CLL cells. Unlike Campath®, it was found that Yl-IgG is unable to mediate CDC against primary B-CLL cells. These in vitro results indicate that Yl-IgG may be useful as a therapeutic agent for the treatment of B-CLL based on its ADCC activity. 2. 1.1 ADCC in B primary chronic lymphocytic leukemia (B-CLL): To determine whether Yl-IgG mediates aDCC in primary B-CLL, B-CLL cells from different patients were co-incubated for 24 hours with PBMC effector cells at different effector / target relationships. The analysis of thirteen different clinical samples of B-CLL indicated that Yl-IgG mediated the cytotoxicity of the effector cells in all cases (Figure 3) with an average level of cell lysis of 21,4%. Four of the thirteen samples (30%) presented more than 30% lysis, while only two of the thirteen samples (15%) presented less than 10% lysis. In some cases, a high level of lysis was observed even at a low E: ratio, for example KBC171, which presented 62% lysis, while in other cases, a low level of lysis was observed even at a high ratio of E: T, for example KBC104, which presented only 7% lysis. The variation can be attributed to the effector cell samples obtained from different healthy donors, as well as to differences between the B-CLL samples. 2. 1.2 ADCC mediated by Yl-IgG with PBMC against acute myeloid leukemia cells: PBMCs also performed ADCC against a patient's primary AML cells using variable ratios of PBMC: AML (10, 20 or 40) in the presence of 10 or 20 μg / ml of Yl-IgG. For example, at a cell ratio of 10: 1, 17.9% of the AML cells died in the absence of the antibody and 6% died in the presence of human IgG. In the presence of 10 and 20 μg / ml of Yl-IgG, 14.2% and 17.6% of the AML cells died, respectively (Figure 4). The level of cytotoxicity increased with the increase in the concentration of Yl-IgG (10 or 20 μg / ml). Human IgG did not induce ADCC. A similar result was obtained for a primary AML sample (data not shown).

ML-2 cells offer a good model for ADCC since Yl-IgG binds without undergoing detectable internalization. to. ADCC of ML2 increases with the concentration of Yl- gG: After 24 hours incubation, the cytotoxicity was higher in the presence of Yl-IgG than in its absence at four different effector (PBMC) ratios to different targets (5: 1, 10: 1, 20: 1, 40: 1) (Figure 5). This effect decreased or was absent when the anti-mouse PSGL-1 antibody KPLl was replaced by Yl-IgG and further decreased when human IgG (which binds to the Fe receptor in the effector cells) was used instead of Yl- IgG A concentration of Yl-IgG as low as 5 μg / ml could induce ADCC when the ratio of target effector was 40: 1. b. Competition between Yl-IgG and KPL-1: Yl-IgG (20 and 50 μg / ml) induced ADCC against ML2 cells at effector: target ratios of 20: 1 and 40: 1. The anti-mouse PSGL antibody KPL-1 alone did not induce ADCC, and could consequently be used as a competitor for ADCC induced by the binding of Yl. KPL-1 partially inhibited ADCC induced by Yl-IgG (Figure 6), and for example, while 74.1% cytotoxicity was observed after 48 hours of incubation in the presence of Yl-IgG (20 μg / ml; of effector: white 20: 1), the additional presence of KPL1 (20 μg / ml) resulted in only 58.8% cytotoxicity. Therefore, ADCC induced by Yl-IgG of ML2 involves the binding of PSGL-1 by Yl-IgG. c. Involvement of Natural Killer cells,? DT and Monocytes in ADCC mediated by Yl-IgG: Positively selected effector cells were analyzed for their ability to effect ADCC mediated by Yl-IgG from ML2 or B-CLL target cells. Natural killer (NK) cells (CD56 +), dT cells and cytotoxic T cells (CD8 +) from normal donors and cells from patients with B-CLL were isolated using commercially available magnetic beads. As shown in Figure 7A, NK cells from both normal donors and patients with B-CLL (KCS samples of Figure 7A) are capable of. perform ADCC on ML2 and B-CLL targets (KCS samples of Figure 7A), which derives at 13% at 68% lysis compared to the control. Also,? DT cells were shown to mediate ADCC of ML2 cells. In contrast, cytotoxic T cells do not appear to participate in Yl-IgG mediated cytotoxicity.

Negative selection of PBMC-specific cell populations showed that CD14 + cells (monocytes) also participate in ADCC against white ML2, in addition to NK cells (CD56 +) and Δd + T cells (Figure 17B). All of the effector cells that participated in the Yl-IgG-mediated toxicity express the Fe receptor, CD16. d. Activation of NK cells by Yl: Yl mediates ADCC with natural killer cells, measured by the expression of CD69. The effector cells from six healthy donors were incubated for 24 hours at 37 ° C in the presence of Yl-IgG or human IgG or a murine anti-CD62 antibody (KPLI, PL1 or PL2) or in the absence of any antibody (control) . The FACS analysis was then carried out and the expression of the early activation marker CD69 in natural killer cells (NK) (CD56 +) was determined. As shown in Figure 8, activation of NK cells by Yl-IgG was measured with the six donor cells. In contrast, no effect could be detected either by human IgG or by anti-CD162 mouse antibodies KPL1, PL1 or PL2. Preliminary studies have shown the induction of FasL expression in effector cells after incubation with Yl-IgG (no data shown). and. Apoptosis induced by Yl-IgG Mononuclear cells (CD19 +, CD5 +) from patients with B-CLL incubated in the presence of Yl-IgG presented 5% apoptosis within 24 hours, as assessed by FACS analysis (Figure 9). The addition of secondary antibodies that cross-link with Yl-IgG produced an additional 50% of apoptosis within 24 hours (Figure 9).

These results suggest that cross-linking of an antibody directed to a PSGL-1 triggers signals for apoptosis of primary B-CLL cells. This implies that PSGL-1 can be a target for inducing apoptosis in patients with B-CLL in vivo, where the cross-linking effect can be mediated by the Fe receptor that transports cells, eg monocytes, CD56 + NK cells and cells ? d + T.

The effects of apoptosis and cross-linking described above can be inhibited using the anti-PSGL-1 KPL1 antibody (data not shown). This antibody alone does not induce apoptosis. This confirms that the apoptotic signal is mediated through an epitope in PSGLL-1.

F. The effect of ADCC of Yl-IgG on B-CLL in relation to Rituximab The percentage of cell death induced by the Yl-IgG antibody in two samples of patients with primary human B-CLL was significantly higher than that obtained by Ritluximab. Figure 10 shows that Yl induced 25% to 35% cytotoxicity with respect to the control compared to only 10% to 13% induced by Rituximab. The saturation of receptor molecules in the target cells was achieved at 10 μg / ml of the Yl-IgG antibody but not with the same concentration of Rituximab.

Taken together, the results suggest that Yl-IgG is a promising candidate as a therapeutic agent in the treatment of B-CLL, since its cytotoxic and apoptotic effects appear to be mediated through the specific recognition of a sulfated epitope of PSGL-1 expressed in these sick cells g. Analysis of the CDC effect of Yl-IgG, Rituximab and Campath on B-CLL Mononuclear cells from patients with B-CLL were incubated with Yl-IgG, Rituximab or Campath® in the presence and absence of 25% of patients' plasma . As shown in Figure 11, only Campath® mediated the cytotoxicidass of the primary B-CLL cells through CDC. Neither Rituximab nor Yl-IgG induced cytotoxicity through complement fixation.

These in vitro results indicate that Yl-IgG may be useful as a therapeutic agent for the treatment of B-CLL based on its ADCC activity.

EXAMPLE 3: Derivative of Yl-IgG-M-Daunorubicin 3. 1 Preparation of the Yl-IgG-M-Daunorubicin Derivative: Antibody-toxin conjugates were prepared such as orfolino-doxorubicin-Yl-IgG (Figure 13) and M-daunorubicin-antibody conjugates (see below). Daunorubicin was modified, bound to one of two different linkers, and then bound to the antibody through the free amino groups of the antibody or through the reduced disulfide bonds of the antibody. The term M-DNR-LIGATOR refers to both (6-Maleidocaproyl) hydrazone of Morphlinyldaunorubicin acetate and M-DNR-AES. to. Preparation of 3 'acetate-Deamin-3' - (4-morpholinyl) daunorubicin (M-DNR-Ac) Triethylamine was added to the moon solution of daunorubicin hydrochloride in dry dimethylformamide, under argon, followed by bis (2-iodoethyl) ether. The reaction mixture was protected from light and stirred for 36 hours at room temperature.

The resulting aqueous mixture was extracted with methylene chloride. The organic phase was dried over anhydrous sodium sulfate, filtered through celite and evaporated to dryness. The crude product was purified by silica gel column chromatography and the relevant fractions were mixed together and evaporated to give the free base of M-DNR as a red oil, which was found to be 98% pure (by HPLC) . The yield was 55%.

After the reaction with acetic acid, the resulting free base was isolated as its solid acetate salt followed by lyophilization. M-DNR-Ac is stable for at least 12 months under argon at -20 ° C. b. Preparation of Morpholinyldaunorubicin acetate (6-Meleimidocaproyl) hydrazone 6-Maleimidocaproylhydrazide was added to a solution of M-DNR-Ac in dry methanol, under argon, followed by trifluoroacetic acid. The clear solution was protected from light and stirred for 24 hours at room temperature.

The ethanolic solution was evaporated to dryness under reduced pressure at 25 ° C, which resulted in a red oily residue, which was dissolved in dry methanol. To this solution, dry ether was added and the precipitated red solid was isolated by centrifugation. The crude crystalline product, which had a purity of 98% and a yield of 88%, was obtained after three triturations with dry ether, dried under vacuum, and kept under argon at -20 ° C. (6-Maleimidocaproyl) hydrazone of Morphillinyldaunorubicin acetate is stable for at least 4 months under argon at -20 ° C. c. Preparation of Morpholinodaunorubicin adipic acid monohydrazone N-hydroxysuccinimide ester (M-DNR-AES) 1. Preparation of morpholinodaunorubicin adipic acid monohydrazone The following were combined and stirred at room temperature under Ar while protected from light for 1 hour: morpholinodaunorubicin acetate salt, dry MeOH, freshly prepared methanolic solution (hydrochloride material solutions of hydrazidoadipic acid and Et3N and TFA).

The solvent was removed under reduced pressure and the resulting residue was dissolved in NHOAc: AN and injected onto a semi-preparative HPLC column. The mixture was washed and concentrated under isocratic conditions. The desired product was collected after 4.5 minutes and concentrated C-18 Sep Pak cartridge. The product was eluted, lyophilized and stored at -20 ° C under Ar. The product was obtained as a red solid with 80% yield and 95% purity. 2. Preparation of Morpholinodaunorubicin adipic acid monohydrazone N-hydroxysuccinimide ester Hydroxysuccinimide in dry THF and DCC in dry THF were added to the monohydrazone of morpholinodaunorubicin adipic acid in dry THF. The clear red solution was stirred for 24 hours at room temperature under Ar. The end of the reaction was determined by analytical HPLC and the solvent was removed. The glacial solid was then dissolved in buffer solution. (N-methylmorpholinium acetate / AN) and filtered through cotton.

The product was isolated by RP-HPLC, diluted with two volumes of n-methylmorpholinium acetate solution and loaded onto a sep-Pak (900 mg). The product was eluted and lyophilized to obtain a powder with 37% yield and 97.4% purity. d. Preparation of M-DNR-Yl-gG Conjugate M-DNR-LIGATOR in dry DMF was added to the MAb solution at a molar ratio of M-DNR-LIGATOR / Yl-IgG of 23. The mixture was stirred gently overnight at room temperature under argon, then centrifuged. The supernatant was filtered through SPIN-X tubes (Costar) and shaken with Bio-beads SM-2 (Biorad) for 1 hour at room temperature. The mixture was allowed to stand for 10 minutes. The supernatant was passed through columns of PD-10 (Pharmacia) that had been balanced with PBS. The conjugate was eluted with PBS and the fractions containing protein were combined. The purified conjugate was sterilized by filtration with SPIN-X. The conjugate solutions were frozen and stored at -70 ° C. The conjugate product was obtained with 45% -50% yield and had the following characteristics: 5% aggregation, 2%. -5% absorbed, M-DNR derivatives bound non-covalently; the average molecular ratio of drug to antibody is 4. and. Preparation of conjugate of M-DNR-YlIgG (through the reduction of the S-S bonds in the Fe region) Yl-IgG in a buffer composed of NaCl, MES and EDTA was added to a solution of cysteamine hydrochloride (Merck) in the same buffer. The mixture was incubated at 37 ° C for 1.5 hours under argon. The reaction mixture was loaded on a PD-10 column (Sephadex G-25, Pharmacia) that had been balanced with PBS / EDTA. The reduced protein was eluted with PBS / EDTA. The fractions with the highest protein concentrations were columbinated and stored at 4 ° C. The molecular ratio of the free sulfhydryl groups to the antibody was at least 3.5. (6-Maleimidocaproyl) hydrazone of morphlinyldaunorubicin acetate was diluted in DMF and added to the solution of the reduced protein and incubated for 30 minutes at 4 ° C. The reaction mixture was passed through a PD-10 column pre-equilibrated with PBS, then eluted with PBS. The protein fractions were combined, then sterilized by filtration of SPIN-X (Costar). The purified conjugate was aliquoted and stored at -70 ° C. The conjugate product was obtained with a 50% yield and had the following characteristics: less than 55 of aggregates; l% -2% of free M-DNR derivatives; the average molecular ratio of drug to antibody is 4.

F. Preparation of conjugate of gG-Yl-M-DNR (by reduction of S-S bonds) IgG-Yl was reduced by passing it through a column of PD-10 (Sephadex, -25M, Pharmacia) in EDTA and eluting in PBS / EDTA. Fractions containing protein were mixed. The molecular ratio of the free sulfhydryl groups to the antibody was at least 6. (6-Maleimidocaproyl) hydrazone of Morphlinyldaunorubicin acetate in dry dimethylformamide was diluted in DMF and added to the reduced protein. The solution was incubated for 30 minutes at 4 ° C. The conjugated protein was purified on a PD-10 column in PBS and the protein fractions were sterilized by filtration with SPIN-X (Corning Life Sciences). The conjugate was frozen and stored at -70 ° C. The yield was 50% in relation to the original antibody. The final product contained less than 55 of aggregates, free M-DNR was between 0-2% and the average molecular ratio of drug to antibody was 7. 3. 2 Cytotoxicity of the Yl-IgG-M-DNR Derivative (Figures 12-14): The specific cytotoxic effect of Yl-drug conjugates was evaluated in semisolid clonogenic assays (METHOCULT®, Ste Cell Technologies Inc., Vancouver BC, Canada). The cells (CD34 + stem cells from spinal blood samples or patients with primary AML) were incubated with free or conjugated drugs at concentrations of up to 10"6M for 1 hour at 37 ° C. The cells were washed, germinated in METHOCULT® and grew for 10-12 days, after which the colonies were counted, bovine (B) -IgG-M-DNR was used as non-specific conjugate control.

The results (Figure 13) showed a limited effect of the Yl-IgG-M-DNR conjugate on the blood samples from the spine. That is, non-target cells (healthy CD34 + stem cells) incubated in the presence of 1 μM Yl-IgG-M-DNR survived at least as well as control cells and cells incubated in the presence of 1 μM b-IgG-M -DNR or 0.1 μM M-DNR (ie, a non-lethal dose of the free drug). Therefore, no significant effect was observed in any of the spine blood samples.

In contrast, in the AML sample (M7 megakaryocytic leukemia), Yl-IgG M-DNR was three times more inhibitory of colony growth, in relation to the nonspecific b-IgG-M-DNR conjugate at the same concentration (Figure 13). That is, 1 μM Yl-IgG-M-DNR reduced the viability of the primary AML cells by 140% relative to the control (white cells incubated alone), while 0.1 μM M-DNR and 1 μM b- IgG-M-DNR each reduced the viability of the primary AML cells to 80% relative to the control.

The specificity of the Yl-IgG-M-DNR conjugate was also demonstrated in a similar experiment that showed that after incubation with 1 μM Yl-IgG-M-DNR, the formation of colonies from AML-M4 and AML- cells M5 (obtained from patients) was inhibited at 60% and 35% respectively, relative to the control (Figure 14). In contrast, all AML-M4 cells and 78% of AML-M5 cells gave rise to colonies after incubation with 1 μM b-IgG-M-DNR. Cells incubated with 1 μM M-DNR did not give rise to colonies, confirming that the cells were sensitive to the drug.

The B-ALL cells, which do not express an Yl epitope (evaluated for failing to bind to Yl), are not sensitive to Yl-IgG-M-DNR and therefore gave rise to colonies after incubation with 1 μM Yl-IgG-M-DNR at the same index as the control (Figure 15).

EXAMPLE 4: Recognition motif and sulfation of Yl To evaluate the potential effect of tyrosine sulfation on the binding of Yl-scFv to GPIb, the human chronic myeloid leukemia cell line KU812 expressing GPIb grew in a sulfate-free medium in the presence of 100 mM sodium chlorate to inhibit sulfation. In the employed condition, tyrosine sulfation was inhibited up to 95% without affecting protein synthesis or other modifications after translation. As shown in Figure 16A, the binding of Yl-scFv to KU812 cells was reduced by 50% after growth with sodium chlorate, compared to control cells that grew in a complete medium containing no sodium chlorate. Under the same conditions, the surface expression of GPIb in sulfate-stripped cells did not change, as indicated by the FACS analysis using the mouse anti-GPIb monoclonal antibody AK2-FITC (Figure 16B).

To further assess whether the modification of tyrosine sulfate plays a role in the binding of Yl-scFv to PGIb, different synthetic peptides based on residues 273-285 of GPIb were evaluated for the ability to inhibit the binding of Yl-scFv to platelets.

Briefly, peptides were synthesized by the solid phase methodology using a multiple peptide synthesizer ABIMED AMS-422, and as necessary, tyrosine sulfate was incorporated using an FMOC salt try sodium salt. The synthetic peptides were purified using a Lichrosorb RP-18 column. For the binding assay of Yl-scFv platelet, a mixture of synthetic peptide (2.5, 25 or 200 μM) and Yl-scFv (10 μg) was incubated and washed. After washing, the platelets were incubated with anti scFv labeled with R-phycoerythrin, washed and analyzed by FACS.

As shown in Figure 17A, the peptide DLYSDYSYSPE (SEQ ID No. 4) (comprising 3 sulfated tyrosines) at 25 μM effectively inhibited the binding of Yl-scFv to the platelets, while the corresponding non-sulphated control DLYDYYPE (SEQ. ID No. 5) had no effect even at 200 μM. In addition, each of the peptides DLYSDYYPE (SEQ ID No. 6), DLYSDYSYPE (SEQ ID No. 7) and DLYSDYYSPE (SEQ ID No. 8) (sulfated in the first, in the first and in the second and in the first and in the third tyrosines, respectively) at 25 μM effectively inhibited the binding of Yl-scFv. The peptides DLYDYSYSPE (SEQ ID No. 8) and DLYDYYSPE (SEQ ID No. 19) (sulphated in the second and third, and in the third tyrosines, respectively) had no effect on the binding of Yl-scFv even at 200 μM (Figure 17A). These results clearly demonstrate that the sulfation of Tyr-276 in GPIb, to say the "first" position of tyrosine, is important for significant competition and consequently for the binding of Yl-scFv to GPIb.

To evaluate whether other amino acids within the region of residues 273-285 of GPIb contribute to the binding of Yl-scFv, substitution mutant peptides based on the peptide DLYSDYYPE (SEQ ID No. 6) were tested in the binding assay of Yl-scFv platelets (Figure 17B). When Sulphated Tyr-276 was replaced with a negatively charged Glu residue, the mutant peptide DLEDYYPE (SEQ ID No. 11) did not exert any substantial inhibition on the binding of Yl-scFv to the platelets, unlike DLYSDYYPE (SEQ ID. No. 6). Similarly, the mutant peptides DLYSEYYPE (SEQ ID No. 12), DLYSNYYPE (SEQ ID No. 13) and DLYSAYYPE (SEQ ID No. 14), which have Asp-277 replaced by Glu, and Ala respectively were substantially incapable of inhibit the binding of Yl-scFv to platelets. On the other hand, the replacement of Leu-275 by Ala (DAYSDYYPE) (SEQ ID No. 15) and different replacements of amino acids 278 to 280 [DLYSDFYPE (SEQ ID No. 16), DLYSDAYPE (SEQ ID No. 17), DLYSDYYAE (SEQ ID No. 18) and DLYSDYYPA (SEQ ID No. 19)] gave mutant peptides that inhibited all Yl-scFv binding to platelets, in a manner substantially identical to that of DLYSDYYPE (SEQ ID No. 6) (Figure 17B).

To validate the platelet binding inhibition assay, the mutant peptides were also tested for the inhibition of Yl-scFv binding to purified glycocalicin (Figure 18A). Briefly, glycocalicin immobilized on microtiter plates was incubated with Yl-scFv (5 μg / ml) and peptide (25 μM). After washing, bound Yl-scFv was detected using anti-scFv polyclonal (generated by immunization of a rabbit with a mixture of scFv) and anti-rabbit IgG antibody conjugated to horseradish peroxidase and reading the absorbance at 450 nm in a reader of ELISA.

The results obtained in the inhibition test of glycocalicin binding (Figure 18A) indicated that both sulphated Tyr-276 and the adjacent Asp-277 residue of GPIb are important for the binding of Yl-scFv, thus confirming the results obtained in the inhibition assay of platelet binding (Figures 17A and B). Another confirmation was obtained by evaluating by ELISA the direct binding of Yl-scFv to peptides covalently coupled to CovaLink Plates (Figure 18B). This study indicated that mutant peptides derived from GPIb having replacements of sulfated Tyr-276 or Asp-277 were substantially unable to direct binding by Yl-scFv, unlike mutant peptides that have replacements at positions 275, 278, 279 or 1280, or that have unsulfated Tyr-278 or Tyr-279, all of which were substantially able to direct binding by Yl-scFv .

Analogous direct binding experiments using synthetic peptides based on residues 42-58 of PSGL-1 confirmed that tyrosine sulfation of PSGL-1 is important for the binding of Yl-scFv (see Figure 19). Specifically, sulfation of the third tyrosine position in the sequence of PSGL-1 QATEYEYLDYDFLPETE (SEQ ID No. 20) results in 100% binding activity of Yl-scFv relative to the corresponding non-sulphated control peptide (cf. Figure 20). In contrast, sulfation of the same linear peptide in the second tyrosine position confers only 40% binding in relation to the control, and sulfation in the first tyrosine position confers a binding activity substantially not distinguishable from the control.

Taken together, the results obtained using synthetic peptides based on GPIb and PSGL-1 indicate that the sequence YSD, which is within the motif DXYSD (SEQ ID No. 21), where X represents any amino acid and Y? represents sulfated tyrosine, it is important for the binding of Yl to its epitope.

Several proteins are known that contain the YSD sequence found within the motif DXYSD (SEQ ID No. 21) and / or within a highly acidic medium (Figure 21). It is believed that this acidic medium is significant for the tyrosine sulfation in vivo. It is predicted that Yl is able to bind to those proteins in this sulphated sequence, as shown for GPIb and PSGL-1. 4. 2 Yl binds to solid tumor antigens while KPL-1 does not: Western blot analysis of a small cell lung carcinoma cell line lysate (SCLC) was performed to compare the binding of Yl and the anti-cancer antibody -PSGL-1 of commercially available KPL1 mouse (Figure 22). A single broad band was observed in the Yl spot, whereas no band was observed in the KPL1 spot. This indicates that PSGL-1 may serve as a target for immunotherapy in patients with SCLC using Yl.

EXAMPLE 5: Yl-IgG mediated endocytosis through PSGL-1 PSGL-1 is highly expressed in blood cells of patients with AML. The following results indicate that Yl specifically recognizes and internalizes into tumor cells expressing PSGL-1. Commercially available KPL-1 (anti-PSGL-1) binds to tumor cells but is not internalized. This indicates that PSGL-1 can serve as a target for immunotherapy in patients with AML using Yl. 5. 1 Confocal Microscope Studies: White blood cells were isolated from patients on a FICOLL® gradient. The cells were incubated for different periods of time, at 37 ° C in the presence of fluorescent anti-PSGL-1 antibodies (Yl or commercial antibodies KPL1 and PL1). The location of fluorescent antibodies was determined in living cells visualizing the cells by confocal microscope.

Figures 23 and 24 show live cells from patients with AML visualized by confocal microscope after incubation of the cells at 37 ° C for 2 hours with Yl-PE (left), KPLI-PE (center) and Yl-IgG-FITC (right). The cells were explored in three-dimensional form (X, Y and Z planes) and the photographs presented here were taken from the center of the sphere with respect to the Z plane. As shown, after incubation Yl-IgG was present in the inside the cells (but not in the nucleus), whereas KPLI was present in the membranes of the cells and was not internalized. 5. 1 Fluorescence Microscope Studies: The white blood cells of patients were isolated in a FICOLL gradient. The cells were incubated for different periods of time, at 37 ° C, in the presence of Yl-IgG. For the detection of Yl-IgG, the cells were fixed, permeabilized and then stained with anti-human antibodies (Fe) labeled with rhodamine. The cells were visualized by fluorescence microscope.

Figures 25 and 26 show live CD34 + cells (from healthy bone marrow and healthy spinal blood, respectively) visualized with a Confocal Microscope after incubation of the cells at 37 ° C for two hours with Yl-IgG-FITC (left) and KPL1-PE (right) and with anti-CD34-PE or FITC. As shown, Yl-IgG did not bind to normal CD34 + stem cells. In contrast, normal cells labeled with KPL-1-PE that include CD34 + cells, evidenced by double staining of some cells in the lower right panel. 5. 3 Monitoring of Endocytosis: The binding of the cell surface of Yl-IgG was detected after incubation of the cells in the presence of the antibody, at 4 ° C with 0.1% NaN3 (which inhibits the active process of internalization) . Incubation at 37 ° C for 10 minutes-2 hours (without NaN3) led to coronation and patching and internalized staining. Similar photographs were obtained with different AML samples by means of a confocal microscope or by means of a fluorescence microscope.

Figure 27 shows four individual cells from a patient sample with AML incubated with unlabeled Yl-IgG for different periods of time, at 37 ° C or at 4 ° C. As shown, cell surface binding of Yl-IgG was detected after incubation of the cells in the presence of the antibody, at 4 ° C with 0.1% NaN3. Incubation of Yl-IgG at 37 ° C for 10 minutes - 2 hours (without NaN3) led to coronation and patching and internalized staining. Internalization increased over time. No internalization was observed in cells maintained at 4 ° C. Yl-IgG was detected with anti-human antibodies (Fe) labeled with rhodamine. The visualization of the cells was carried out by means of a fluorescence microscope. 5. 4 Acid Scraping: The treatment of the cells with 50 mM Glycine (pH 2.5) resulted in the removal of the cell surface binding of Yl-IgG and allowed the detection of internalized Yl-IgG.

Figure 28 shows cells from patients with AML who were incubated with unlabeled Yl-IgG for 1 hour at 37 ° C. The cells shown in the lower row were then incubated at room temperature for 5 minutes with 50 mM glycine, at pH 2.5 to remove Yl-IgG bound to the surface. As shown, the upper panel represents the crowning and patching of the internalized Yl-IgG surface. In the lower panel, only internalized Yl-IgG was detected. 5. 5 Pronasa: The removal of cell surface proteins by the proteolytic enzyme, pronase, also resulted in the removal of cell surface binding of Yl-IgG and allowed the detection of internalized Yl-IgG.

Figure 29 shows cells from patients with AML who were incubated with unlabeled Yl-IgG for 1 hour at 37 ° C. The cells shown in the lower row were then incubated at room temperature for 60 minutes with 1 mg / ml pronase to remove Yl-IgG bound to the surface. As shown, the top panel represents the coronation and patching of the cell surface and internalized Yl-IgG. In the lower panel, only internalized Yl-IgG is detected. Note that the uropods were removed with pronase, which implies that the uropods formed by the crosslinking of Yl-IgG of CD162 are formed on the outer surface of the cell surface. 5. 6 Endocytosis mediated by coated holes: receptor-mediated endocytosis can occur through coated pits. Coated hole-mediated endocytosis can be blocked by incubating the cells with 0.45M sucrose for 15 minutes at 317 ° C before incubation with Yl-IgG. This method inhibited the endocytosis of Yl-IgG without affecting the binding of Yl-IgG to the cell surface.

Figure 30 shows cells from patients with AML who were incubated with an unlabeled Yl-IgG for 1 hour at 4 ° C (Fig. 30A) or at 37 ° C (Figure 30B). The cells shown in the middle row were then incubated at room temperature for 5 minutes with 50 mM glycine at pH 2.5 to remove the Yl-IgG bound to the surface. As shown (FIG. 30A), the upper panel represents staining of the cell surface of Yl-IgG. Yl-IgG bound to the surface was removed by acid washing (central panel). At 37 ° C (Figure 30B) coronation and patching and internalization of Yl-IgG was observed (upper panel). The acid wash removed Yl-IgG bound to the cell surface and only the internalized antibody could be detected (central panel).

The blogging of endocytosis mediated by coated holes (lower rows) was obtained by incubating the cells with 0.45 M Sucrose for 15 minutes at 37 ° C before incubation with Yl-IgG. As shown in the lower panels, treatment of the cell with 0.45M sucrose did not affect the binding of Yl-IgG to the cells at 4 ° C (Figure 30A) but inhibited internalization at 37 ° C (Figure 30B).

EXAMPLE 6: Inhibition of Yl-IgG from Leukocyte-Platelet Interactions The adherence of leukocytes to vascular surfaces leads to organ damage in different disorders, including reperfusion injury, seizure, mesenteric and peripheral vascular disease, organ transplantation and circulatory shock. Reperfusion injury is associated with the adhesion of leukocytes to the vascular endothelium in the ischemic zone, presumably in part due to the activation of platelets and the endothelium by thrombin and cytokines, which makes their surfaces adhesive to leukocytes. The main initiator of reperfusion injury is the interaction between von Willbrand factor (vWF) and the platelet GPIb receptor. Cardiac patients who are treated with thrombolytic agents such as tissue plasminogen activator and streptokinase to relieve coronary artery obstruction may still suffer from myocardial necrosis due to reperfusion injury. Therefore, drugs are needed that are capable of reducing the adherence of leukocytes to vascular surfaces and that can be administered in conjunction with thrombolytic agents to improve the outcome of cardiovascular disorders.

Since Yl (both scFv and full IgG) binds to sulfated molecules distinguishable in platelets (ie GPIb) and leukocytes (ie PSGL-1), this antibody has potential as a therapeutic agent to inhibit different cell-cell interactions.

Figure 31 shows that Yl-scFv effectively inhibits the binding of activated human platelets to ML2 cells (a cell line derived from human AML which expresses PSGL-1). Optimal inhibition was obtained when the antibody was incubated simultaneously with both platelets and ML2 cells, while partial inhibition was obtained when the antibody was initially incubated with platelets or ML2 cells, followed by removal of the unbound antibody and the aggregate of the remaining cell type (Figure 31).

Figure 31 also shows that the murine KPLI antibody (directed against the N-terminal domain of human PSGL-1, but not dependent on tyrosine sulfation) was also effective in inhibiting the binding of activated platelets to ML2, but inhibition was less than that exercised by Yl-scFv. This could be due to the fact that KPL1 does not recognize an epitope present in both cell types, as Yl-scFv does. No inhibition was observed with the murine antibody, the PL2 antibody which is also directed against human PSGL-1 (not shown).

EXAMPLE 7: Inhibition of Yl-IgG of Cell Wrap in Immobilized P-Selectin under Flow Conditions For the preparation of a ligand coated substrate, recombinant human P-Selectin (rh) (R & D Systems, Minneapoils, MN) was diluted in 0.2-1.0 μg / ml in a coating medium (PBS supplemented with 20 mM bicarbonate, pH 8.5) and adsorbed immediately on a polystyrene plate overnight at 4 ° C, followed by washing with PBS containing 2 μg / ml human serum albumin (Calbiochem) at 4 ° C for 1 hour.

For laminar flow assays, a polystyrene plate on purified ligand was immobilized and ligated into a parallel plate laminar flow chamber previously described (Lawrence &Springer, Cell 65, 859-873 (1991)). Human neutrophils (isolated from anti-coagulated blood by dextran sedimentation and density separation on FICOLL) or ML-2 cells were washed in an H / H medium (Hanks balanced salt solution)., 10 mM HEPES, resuspended in a cell binding medium (H / H medium supplemented with 2 mM CaCl 2) at 2 x 10 6 cells / ml, and perfused at room temperature through the flow chamber at a rate that generates wall shear stress at the desired flow rate, were generated with an automated syringe pump (Harvard Apparatus, Natick, MA). Upon reaching the top side of the test adhesive substrate, the flow velocity was raised to generate a shear stress of 1 dyn / cm2 and all cell interactions were visualized in two different visual fields (each of 0.17 mm2 surface area). ) using an objective lOx an inverted phase contrast microscope (Diaphot 300, Nikon Inc., Tokyo, Japan). An imaging system was used for the analysis of the instantaneous leukocyte velocities, WSCAN-Array-3 (Galai, Migdal-Ha 'emek, Israel) described above (Dwir et al, J. Biol. Chem. 275, 118682 -18691 (2000)).

The accumulation of the leukocyte winding in the test fields was determined by computerized cell movement drift. The frequency of cell winding was defined as the number of cells in the cell flow that initiated the persistent winding in the adhesive substrate that lasted at least 3 s after the initial ligation. Cells were incubated with antibodies at different concentrations and were prefused to the flow chamber with a binding medium containing the same concentration of the antibody. The cell winding was analyzed after washing the reagent ("wash") or in the presence of the reagent.

For image analysis, an imaging system was developed for the quantitative analysis of instantaneous cell winding speeds in different adhesive substrates. The video frame images comprising 768 x 574 pixels (with a pixel size of 1.15 μm using an lOx objective), were digitized using a Matrox Pulsar frame grabber (Matrox Graphics Inc., Dorval, Quebec, Canada) , and the images were explored and processed using the WSCAN-Array-3 image formation software (Galai, Migdal-Ha 'emek, Israel), which runs on an Atlas pentium MMX-200 workstation. Cell movements were identified from images dragged at intervals of 0.02 s. The program output provided the coordinates of the center point of each cell in successive interleaved fields separated by 0.02 s.

A computer program for the analysis of cell movement was developed in collaboration with the laboratory of Professor David Malah (Faculty of Electrical Engineering, Technion, Haifa, Israel). The software runs under Matlab 5.2 l and compares instantaneous positions of individual cells in successive video images for a period of up to 5 seconds. Individual cell ligatures persistently rolling in the ligand-coated field or moving through it in a spasmodic motion were determined according to changes in instantaneous cellular velocities in the flow direction. A winding pause was defined as a drop in instantaneous velocity to below 20 μm / s at shear stresses of 1-1.75 dyn / cm2. This threshold value of the speed gave moon optimal correlation between pause analysis performed on representative cells by computerized system and manually, directly from the video monitor. The pitch distances between successive passages of an individual winding cell were averaged to give the average pitch distance of a given winding cell.

Figure 32 shows the effect of Yl-scFv (10 μg / ml) in an ML2 cell in coil in immobilized rH-P-selectin at low density (0.2 μg / ml). The analysis showed that at the cutting force of 1 dyn / cm 2, the number of cells in winding per field was totally eradicated in the presence of Yl-scFv. No effect was obtained when equal amounts of scFv-N06 (negative control) were used.

Figure 33 shows the effect of Yl-scFv (10 μg / ml) in winding of ML2 cells in immobilized rh-P-selectin at high density (1.0 μg / ml) at different shear forces. The analysis showed that at the cutoff strengths of 1, 5 and 10 dyn / cm 2, the number of winding cells per field was inhibited by 83%, 98% and 100%, respectively in the presence of Yl-scFv. No effect was obtained with the negative control, N06, nor when the cells were washed after incubation with Yl-scFv and then tested (Yl wash).

Figure 34 shows the effect of Yl-IgG (1 μg / ml) in winding of ML2 cells in immobilized rH-P-selectin (1 μg / ml) at different shear force forces. The analysis showed that at the cutting force of 1 dyn / cm2, the cell winding was inhibited by 89% and that at the cutting forces of 5 and 10 dyn / cm2 of 100% cell winding. When the cells were washed after incubation with Yl-IgG and then assayed (washing with Yl-IgG), the cell coil was inhibited by 46%, 48% and 54% at shear forces of 1.5. and 10 dyn / cm2, respectively. The murine anti-PSGL-1 antibody KPL1 was also capable of 100% inhibition of cell winding at all cutting forces.

Figure 35 shows the effect of increasing Yl-scFv concentrations on the coiling of human neutrophils in high density immobilized P-selectin rh (1.0 μg / ml). The analysis showed that at a cutting force of 1 dyn / cm 2, Yl-scFv at 1, 5 and 10 μg / ml inhibited the number of neutrophils in winding by 20%, 81% and 100%, respectively.

Figure 36 shows the effect of Yl-IgG on the neutrophil coil in immobilized rh-P-selectin at high density (1.0 μg / ml). The analysis showed that at a cutting force of 1 dyn / cm 2, the number of neutrophils in winding per field was completely eradicated (100% inhibition) in the presence of Yl-IgG (1 μg / ml). Similar results were obtained with KPL-1.

Example 8: Detection of the Library of Inorganic Compounds A synthetic sulfated peptide (sulfated at a specific tyrosine residue given within the known amino acid sequence of the peptide) obtained from a specific receptor (protein) can be prepared with a biotin label. (biotinylated) coupled to the synthetic peptide through a linker such as caproic acid. Control peptides using the same synthetic peptide can be prepared without sulfation and without the biotin label ("B"). In addition, synthetic sulphated peptides obtained from other unrelated proteins can be prepared without having the biotin ("C") label as additional controls.

The preceding biotinylated peptide ("A") can be coupled to magnetic beads coated with strepavidin and the excess unbound biotinylated peptide is then washed. The biotin-estretavidin peptide conjugate ("D") can be detected against a small chemical entity library in the presence of a large excess of unsulfated control peptide ("B") under physiological conditions (37 ° C, pH 1 , 0-1,, concentration of salts, conductivity, etc.) for molecules that bind to "A". The coupled magnetic beads were then washed twice with buffer, each time centrifuged to remove excess unbound molecules. The molecules attached to the magnetic beads ("E") can be eluted, chemically identified and prepared in a larger quantity for further detection.

The confirmation of the binding to biotinylated sulphated peptides by the selected chemical compounds ("E") can be carried out by any detection process. This process includes competition with unrelated sulfated peptides that are biotinylated (process 1) or competition with an antibody or fragments thereof (eg scFv) that binds specifically to the biotinylated peptide, "A" (process 2). 8. 1 Detection of Re by competition with unrelated biotinylated sulphated peptides (Process 1) To ensure that the compounds specifically bind to "A", a second round of detection was performed. The biotin-streptavidin peptide conjugate ("D") can be detected again with the selected "E" compounds in the presence of a large excess of unrelated biotinylated sulphated peptides, "C". The tube was then centrifuged, the biotin-peptide stretavidin conjugate was attached to magnetic beads, washed twice with buffer and centrifuged each time to remove excess unbound molecules. The compounds that were attached to the magnetic beads can be eluted for chemical identification. Larger amounts of the chemical compound can be prepared for other studies, for example validation of selective binding to "A" and efficacy assay in vitro and in vivo. 8. 2 New detection by competition with a specific anti-sulfated scFv antibody (Process 2) Compounds with preferred binding affinity to "A" can be detected again by competing the binding of the biotin-streptavidin peptide conjugate ("D") with each of the selected "E" compounds in the presence of an excess of the antibody scFv specifies that it recognizes and is specifically linked to "A". Chemical compounds that are specifically inhibited from binding to "A" by the scFv antibody can be prepared for other studies, such as the validation of "A" selective binding and the in vitro and in vivo efficacy assay.

The invention has been described with reference to specific examples, materials and data. As one skilled in the art will appreciate, there may be alternative means to use or prepare different aspects of the invention. It can be interpreted that these alternative means are included within the object and spirit of the present invention defined in the following claims.

Claims (85)

1. CLAIMS 1. An antibody or a fragment thereof that binds to an epitope comprising the D-X-Y-D motif, wherein X represents any amino acid or the covalent bond between D and Y, and Y is sulfated. 2. The antibody or a fragment thereof according to claim 1, wherein the antibody or fragment thereof forms complexes with an agent selected from the group consisting of anticancer, anti-leukemic, anti-metastasis, anti-neoplastic, anti-disease, anti-adhesion agents, anti-thrombosis, anti-restenosis, anti-autoimmune, anti-aggregation, antibacterial, antiviral, and anti-inflammatory. 3. The antibody or fragment thereof according to claim 2, wherein the antibody or fragment thereof forms complexes with the agent through free amino groups. 4. The antibody or fragment thereof according to claim 2, wherein the antibody or fragment thereof forms complexes with between 1 and 16 copies of the agent. 5. The antibody or fragment thereof according to claim 4, wherein the antibody or fragment thereof is an IgG, each heavy chain of the antibody or fragment thereof forms complexes with 3 copies of the agent, and each light chain forms complexes with 1 copy of the agent. 6. The antibody or fragment thereof according to claim 2, wherein the agent is an antiviral agent selected from the group consisting of acyclovir, ganciclovir and zidovudine. 7. The antibody or fragment thereof according to claim 2, wherein the agent is an anti-thrombosis / anti-restenosis agent is selected from the group consisting of cilostazol, sodium dalteparin, sodium reviparin and aspirin. 8. The antibody or fragment thereof according to claim 2, wherein the agent is an anti-inflammatory agent selected from the group consisting of zaltoprofen, pranoprofen, droxica, acetylsalicylic 17, diclofenac, ibuprofen, dexibuprofen, sulindac, naproxen, amtolmetin , celecoxib, indomethacin, rofecoxib, and nimesulid. 9. The antibody or fragment thereof according to claim 2, wherein the agent is an anti-autoimmune agent that is selected from the group consisting of leflunomide, denileucine diftitox, subreo, WinRho SDF, defibrotide, and cyclophosphamide. 10. The antibody or fragment thereof according to claim 2, wherein the agent is an anti-adhesion / anti-aggregation agent that is selected from the group consisting of limaprost, chlorchromen, and hyaluronic acid. 11. The antibody or fragment thereof according to claim 2, wherein the agent is selected from the group consisting of toxin, radioisotope, imaging agent and pharmaceutical agent. 12. The antibody or fragment thereof according to claim 11, wherein the toxin is selected from the group consisting of gelonin, Pseudomonas exotoxin (PE), PE40, PE38, ricin, and modifications and derivatives thereof. 13. The antibody or fragment thereof according to claim 11, wherein the radioisotope is selected from the group consisting of gamma radiation emitters, positron emitters, X-ray emitters, beta radiation emitters, and alpha radiation emitters. 14 The antibody or fragment thereof according to claim 11, wherein the radioisotope is selected from the group consisting of 11: Lindium, Indium, 99mrenium, 105renium, 101renium, 99mtecnetium, 121mtelurium, 122mtelurium, 125mtelurium, 165tulium, 167tulium, 168tulium, Iodine, Iodine, Iodine, Iodine, 81mkrypton, 33xenone, 90ithium, 213bismuth, 77bromo, 18fluor, 95rutenium, ruthenium, ruthenium, ruthenium, mercury, mercury, gallium and 68 gallium. fifteen . The antibody or fragment thereof according to claim 11, wherein the pharmaceutical agent is anthracycline. 16. The antibody or fragment thereof according to claim 15, wherein the anthracycline is selected from the group consisting of doxorubicin, daunorubicin, idarubicin, detorubicin, carminomycin, epirubicin, esorubicin, morpholinodoxorubicin, morpholinodaunorubicin and methoxymorpholinyldoxorubicin. 17. The antibody or fragment thereof according to claim 11, wherein the pharmaceutical agent is selected from the group consisting of cis-platinum, taxol, calicheamicin, vincristine, cytarabine (Ara-C), cyclophosphamide, prednisone, fludarabine, chlorambucil, interferon alfa, hydroxyurea, temozolomide, thalidomide and bleomycin, and derivatives and combinations thereof. 18. The antibody or fragment thereof according to claim 2, wherein the antibody or fragment thereof forms complexes with a carrier or carrier that can complex with more than one agent. 19. The antibody or fragment thereof according to claim 18, wherein the carrier or carrier is selected from the group consisting of dextran, lipophilic polymers, HPMA, and liposomes, and derivatives and modifications thereof. 20. A pharmaceutical composition comprising an antibody or fragment thereof according to claim 1 and a pharmaceutically acceptable carrier. 21. A kit for diagnosis, prognosis and phase determination comprising an antibody or fragment thereof according to claim 1 and an imaging agent. 22. The diagnosis, prognosis or phase determination kit according to claim 21, wherein the imaging agent is a radioisotope. 23. A method to induce antibody-dependent cell cytotoxicity (ADCC) which comprises administering to a patient in need thereof the pharmaceutical composition according to claim 20. 24. A method of stimulating a natural killer cell (NK) or a T cell comprising administering to a patient in need thereof the pharmaceutical composition according to claim 20. 25. A method of inducing cell death which comprises administering to a patient in need thereof an antibody or fragment thereof according to claim 2, wherein the antibody or fragment thereof that forms complexes with the agent enters the cell and the antibody or the fragment thereof is broken from the agent, thus releasing the agent. 26. A method of treating HIV which comprises administering to a patient in need thereof a pharmaceutical composition according to claim 20. 27. The method according to claim 26, wherein the administration prevents the entry of HIV. 28. The method of introducing an agent into a cell expressing an epitope comprising the motif DXYD, wherein X represents any amino acid or the covalent bond between D and Y, and Y is sulphated, wherein the method comprises the following steps: forming a complex of the agent with an antibody or fragment thereof according to claim 1, and administering to the cell the antibody or fragment thereof that complexed with the agent. 29. The method according to claim 28, wherein the method treats a disease in a patient in need thereof. 30. The method according to claim 28, wherein the method treats the winding of cells in a patient who needs it. 31. The method according to claim 28, wherein the method treats inflammation in a patient in need thereof. 32. The method according to claim 28, wherein the method treats the autoimmune disease in a patient who needs it. 33. The method according to claim 28, wherein the method treats metastasis in a patient in need thereof. 34. The method according to claim 28, wherein the method treats the growth and / or replication of tumor cells in a patient in need thereof. 35. The method according to claim 28, wherein the method increases the mortality rate of the tumor cells in a patient in need thereof. 36. The method according to claim 28, wherein the method inhibits the growth and / or replication of leukemia cells in a patient in need thereof. 37. The method according to claim 28, wherein the method increases the mortality rate of leukemia cells in a patient in need thereof.38. The method according to claim 28, wherein the method alters the susceptibility of diseased cells to damage by anti-disease agents in a patient in need thereof. 39. The method according to claim 28, wherein the method increases the susceptibility of tumor cells to damage by anticancer agents in a patient in need thereof. 40. The method according to claim 28, wherein the method increases the susceptibility of leukemia cells to damage by anti-leukemia cells in a patient in need thereof. 41. The method according to claim 28, wherein the method inhibits the increase in the number of cells in a patient in need thereof. 42. The method according to claim 28, wherein the method reduces the number of tumor cells in a patient who needs it. 43. The method according to claim 28, wherein the method inhibits the increase in the number of leukemia cells in a patient having leukemia. 44. The method according to claim 28, wherein the method reduces the number of leukemia cells in a patient who has leukemia. 45. The method according to claim 28, wherein the method inhibits the aggregation of platelets in a patient in need thereof. 46. The method according to claim 28, wherein the method inhibits restenosis in a patient in need thereof. 47. A method of monitoring a tumor cell in a patient comprising: providing a tumor cell of the patient and incubating the tumor cell with an antibody or fragment thereof according to claim 1, thereby determining the cell phase of the tumor cell. tumor. 48. The method according to claim 47, wherein the method further comprises: determining the specific binding of the antibody or the fragment thereof in relation to a reference standard. 49. The method according to claim 47, wherein the antibody or fragment thereof is Yl. 50. A method of isolating a tumor-specific antigen comprising: obtaining a sample from a cell, lysing the cell, identifying a protein ligand of an antibody or a fragment thereof according to claim 1, and purifying the protein ligand by the lysate of cells through an affinity column comprising the antibody or fragment thereof. 51. The method according to claim 50, wherein the method further comprises sequencing the protein ligand, thus identifying the tumor specific antigen. 52. The method according to claim 50, wherein the antibody or fragment thereof is Yl. 53. The method according to claim 50, wherein the cells are obtained from a tumor of a human being. 54. The method according to claim 53, wherein the tumor is a solid tumor. 55. The method according to claim 54, wherein the tumor is a small cell lung carcinoma. 56. The method according to claim 53, wherein the tumor is a tumor carried by the blood. 57. The method according to claim 56, wherein the tumor is leukemia. 58. A method of diagnosing, predicting, determining phases of a disease in a patient comprising: providing a sample comprising a patient's cell and determining whether the antibody or fragment thereof according to claim 1 is attached to the patient's cell , thus indicating that the patient is at risk or has the disease. 59. The method according to claim 58, wherein the method further comprises determining the specific binding of the antibody or fragment thereof and comparing the specific binding of the antibody or fragment thereof to the cell in relation to a reference standard. 60. The method according to claim 58, wherein the Western blot analysis is used to determine whether the antibody or fragment thereof according to claim 1 binds to the patient's cell. 61. The method according to claim 58, wherein the disease is a cancer. 62. The method according to claim 61, wherein the cancer is a solid tumor. 63. The method according to claim 62, wherein the cancer is small cell lung carcinoma. 64. The method according to claim 61.1 wherein the cancer is a tumor carried by the blood. 65. The method according to claim 64, wherein the cancer is leukemia. 66. The method according to claim 58, wherein the antibody or fragment thereof is Yl. 67. A method of purging tumor cells from a patient comprising: providing a sample containing patient cells and incubating the patient's cells with an antibody or fragment thereof according to claim 1. 68. The method according to claim 67, wherein the purge occurs ex vivo. 69. A process for forming an anthracycline-agent complex comprising providing an anthracycline, reacting an adipic acid with anthracycline, generating an active ester of the adipic acid anthracycline, and reacting the adipic acid anthracycline with a polypeptide to form a complex of anthracycline-agent. 70. The method according to claim 69, wherein the anthracycline is the anthracycline which is selected from the group consisting of doxorubicin, daunorubicin, idarubicin, detorubicin, carminomycin, epirubicin, esorubicin, morpholinodoxorubicin, morpholinodaunorubicin, and methoxymorpholinyldoxorubicin. 71. The method according to claim 69, wherein the polypeptide is an antibody or a fragment thereof. 72. A complex produced according to the process of claim 6. 73. The complex according to claim 72, wherein the anthracycline is doxorubicin, daunorubicin, idarubicin, detorubicin, carminomycin, epirubicin, esorubicin, morpholinodoxorubicin, morpholinodaunorubicin, and methoxymorpholinyldoxorubicin. 74. The complex according to claim 72, wherein the polypeptide is an antibody or a fragment thereof. 75. An antibody or fragment thereof that binds to an epitope comprising the sequence YD, wherein YD is located within the DXYD motif or within an acidic medium, and wherein X is any amino acid or the covalent bond between D and Y , and where Y is sulphated. 76. A pharmaceutical composition comprising an antibody or a fragment thereof according to claim 75 and a pharmaceutically acceptable carrier. 77. A diagnostic, prognostic, or phase determination kit comprising an antibody or a fragment thereof according to claim 75 and an imaging agent. 78. The diagnostic, prognostic, or phase determination kit according to claim 77, wherein the imaging agent is a radioisotope. 79. A method of inducing antibody dependent cell cytotoxicity (ADCC) comprising administering to a patient in need thereof the pharmaceutical composition according to claim 76. 80. A method of stimulating a natural killer cell (NK) or a T cell comprising administering to a patient in need thereof the pharmaceutical composition according to claim 76. 81. A method of inducing cell death comprising administering to a patient in need thereof an antibody or a fragment thereof according to claim 75, wherein the antibody or fragment thereof that forms a complex with the agent enters the cell and the antibody or fragment thereof is broken from the agent, thereby releasing the agent. 82. A method of treating HIV which comprises administering to a patient in need thereof a composition according to claim 76. 83. The method according to claim 82, wherein the administration prevents the entry of HIV. 84. A method of treating a disease comprising administering to a patient in need thereof a pharmaceutical composition according to claim 1 or 76. 85. The method according to claim 84, wherein the method treats the winding of cells in a patient who needs it.