MXPA03005944A - Specific human antibodies for selective cancer therapy. - Google Patents

Abstract

The present invention is directed to a peptide or polypeptide comprising an Fv molecule, a construct thereof, a fragment of either, or a construct of a fragment having enhanced binding characteristics so as to bind selectively and/or specifically to a target cell in favor of other cells, wherein the binding selectivity or specificity is primarily determined by a first hypervariable region, and wherein the Fv is a scFv or a dsFv, and optionally having one or more tags. The enhanced binding is directed to a substantially exposed and/or over-expressed binding site on or in a target comprising a cell in favor of other cells on or in which the binding site is not substantially available and/or expressed. The invention is further directed to a method for isolating such peptides and polypeptides from a phage display library and to the nucleic acid molecules encoding them. The invention provides for a pharmaceutical composition comprising the peptide or polypeptide and kits for diagnosis and treatment of disease, specifically cancer, most specifically acute myeloid leukemia.

Description

SPECIFIC HUMAN ANTIBODIES FOR SELECTIVE CANCER THERAPY FIELD OF THE INVENTION The present invention relates to the field of marking and identification of white tissue, with the aid of phage display technology, of peptides and polypeptides that specifically bind to target cells. Such peptides and polypeptides are Fv molecules, constructions thereof, fragments of some of them or constructions of a fragment. More particularly, the peptides and polypeptides may have anticancer activity, and / or be associated with, conjugated to, anti-cancer agents, especially blood-related cancers.

BACKGROUND OF THE INVENTION Selective target tissue labeling of therapeutic agents is a emerging discipline in the pharmaceutical industry. New cancer treatments based on white labeling have been designed to increase the specificity and potency of the treatment, simultaneously reducing toxicity, thereby improving overall efficacy. Monoclonal antibodies from mice (Mab) of antigens associated with tumors have been employed in an attempt to target conjugates of toxins, radionucleotides and chemotherapeutics to tumors. In addition, differentiation antigens, such as CD19, CD20, CD22 and CD25, have been exploited as cancer-specific targets in the treatment of hematopoietic diseases. Although widely studied, this approach has several limitations. One limitation is the difficulty of isolating appropriate monoclonal antibodies that show selective binding. A second limitation is the need for high immunogenicity to the antibody as a prerequisite for successful isolation of the antibody. A third limitation is the obtaining in the patient of an immune response against antibodies of mice (response to HAMA, human anti-mouse antibody) that often results in a shorter serum half-life, and prevents repeated treatments, thus decreasing the therapeutic value of the antibody. This latter limitation has stimulated interest in producing chimeric or humanized monoclonal antibodies of mouse origin, and in discovering human antibodies.

There are many factors that influence the therapeutic efficacy of monoclonal antibodies (Mab) to treat cancer. These factors include the specificity of antigen expression in tumor cells, the level of expression, antigenic heterogeneity, and the accessibility of tumor cells. Leukemia and lymphoma have been generally more sensitive to antibody treatment than solid tumors, such as carcinomas. The Mab quickly bind to leukemia and lymphoma cells in the bloodstream and easily penetrate the malignant cells in the "lymphatic ejido, making then the lymphoid tumors excellent candidates for Mab-based therapy. An ideal system would involve identifying a Mab. that recognizes a marker on the cell surface of stem cells that produce malignant progeny cells.

To assist in the discovery / production of Mab, phage libraries have been used to select single chain randomized Fv (scFv) that bind predetermined, isolated, white proteins, such as antibodies, hormones and receptors. In addition, the use of antibody-presenting libraries in general, and of phage-scFv libraries in particular, provides an alternative means to discover unique molecules to target cell-surface groups not yet recognized and not determined, specific.

Leukemia, lymphoma, and myeloma are cancers that originate in the bone marrow and lymphatic tissues and are involved 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 cases of ALL 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 causes it to become abnormal and multiply continuously.

AML is a heterogeneous group of neoplasms with a progenitor cell that, under normal conditions, gives rise to cells of differentiated terms of the myeloid series (erythrocytes, granulocytes, monocytes and platelets). As in the other forms of neoplasia, AML is associated with acquired genetic alterations that result in the replacement of myeloid cells differentiated normally 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 hemotopoietic organs. AML mainly affects adults, with peaks of incidence between 15 and 40 years, but it is also known to affect both children and older adults. Almost all patients with AML require treatment immediately after diagnosis to achieve clinical remission, in which there is no evidence of abnormal levels of undifferentiated blasts cells in circulation.

To date, a variety of monoclonal antibodies have been developed that induce cytolytic activity against tumor cells. A humanized version of the monoclonal antibody MuMAb4D5, targeted to the extracellular domain of P185, growth factor receptor (HER2), was approved by the FDA [Food and Drug Administration] and is being used to treat human breast cancer (US Patent). Nos. 5,821,337 and 5,720,954). After binding, the antibody is capable of inhibiting the growth of tumor cells that depends on the growth factor receptor HER2. In addition, a chimeric antibody to cD20, which causes rapid depletion of peripheral B cells, including those associated with lymphoma, was recently approved by the FDA (US Patent No. 5,843,439). The binding of this antibody to target cells results in complement-dependent lysis. This product has recently been approved and is currently being used in clinical medicine to treat non-Hodgkin's low-level B-cell lymphoma.

Various other humanized and chimeric antibodies are in development or in clinical trials. In addition, a humanized Ig that reacts specifically with the CD33 antigen, expressed in normal myeloid cells as well as in most types of myeloid leukemia cells, was conjugated to anti-cancer drug calicheamicin, CMA-676 (Sievers et al, Supplement Blood, 308 504a (1997)). This conjugate, called the drug MYLOTARG®, has recently received approval from the FDA (Caron et al, Cancer Supplement, 73, 1049-1056 (1994)). In light of its cytolytic activity, an additional anti-CD33 antibody (HumM195), currently in clinical trials, was conjugated to several cytotoxic agents, including the gelonin toxin (McGraw et al, Cancer Immunol. Immunother, 39, 367-374 ( 1994)) and radioisotopes 131I (Caron et al, Blood 83, 1760-1768 (1994)), 90Y (Jurcic et al, Supplement Blood, 92, 613a (1998)) and 213Bi (Humm et al, Supplement Blood, 38: 231P (1997)).

A chimeric antibody against the leukocyte antigen CD-45 (cHuLym3) is in the preclinical phase for the treatment of human leukemia and lymphoma (Sun et al, Cancer Immunol. Immunother., 48, 595-602 (2000)) as a conditioning for bone marrow transplant. In in vitro assays, lysis of specific cells was observed in ADCC assays (cytotoxicity mediated by antibody-dependent cells) (Henkart, Immunity, 1, 343-346 (1994); Squier and Cohen, Current Opin. Immunol., 6 , 447-452 (1994)).

Although these preliminary results seem promising, they have the following limitations. The final product comprises non-human sequences, which results in a problematic immune response to the non-human material, such as HAMA. This response to HAMA prevents repeated treatments and results in a shorter serum half-life for the product. In addition, the foregoing methods allow the isolation of a single species of antibody and only allow the isolation of antibodies against known and purified antigens. In addition, these methods are not selective as long as they allow the isolation of antibodies against cell surface markers that are present in normal cells as well as in malignant cells.

Therefore, a method that solves these limitations considered above would be desirable. In addition, such a method would ideally allow the identification of ligands or white markers in cancer cells or cells involved in the mediation of cancer cell metastases, for example. In addition, such a method would also allow the production of antibodies against such targets. Phage display technology seems to offer such capabilities.

The use of phage display technology has made it possible to isolate scFv comprising completely human sequences. For example, the fully human antibody against the human TGFβ2 receptor based on the scFv clone obtained from the phage display technology was recently developed. This scFv, converted to a fully human IgG4 that is capable of competing with the binding of TGFβ2 (Thompson et al, J. Immunol Methods, 227, 17-29 (1999)), has strong antiproliferative activity. This technology, known to an expert in the art, is described more specifically in the following publications: Smith, Science, 228, 1315 (1985); Scott et al, Science, 249, 386-390 (1990); C irla et al, PNAS, 87, 6378-6382 (1990); Devlin et al, Science, 249, 404-406 (1990); Griffiths et al, EMBO J., 13 (14), 3245-3260 (1994); Bass et al, Proteins, 8, 309-314 (1990); McCafferty et al, Nature, 348, 552-554 (1990); Nissim et al, EMBO J., 13, 692-698 (1994); U.S. Patent Nos. 5,427,908, 5,432,018, 5,223,409, and 5,403,484, lib.

Using this phage display technology, the inventors of the present invention have identified cell markers present in cells in sick or malignant state. Accordingly, an objective of the present invention is to identify peptides and polypeptides that recognize markers of cells that are substantially exposed or overexpressed, particularly in cells in sick or malignant state.

Another objective of the present invention is to use and expand the phage display technology as an aid to identify such peptides and polypeptides.

Another objective of the present invention is to identify such peptides and polypeptides by cross immunoreactivity.

Another objective of the present invention is that such peptides and polypeptides are of completely human origin.

Another objective of the present invention is that such peptides and polypeptides are isolated against antigens that may be necessarily immunogenic.

Another objective of the present invention is to provide peptides or polypeptides that prevent, retard or cure cancer, particularly blood-related cancers including leukemia and lymphoma.

Another objective of the present invention is to provide local labeling of cancer cells with such peptides and polypeptides alone, or associated with, or coupled to, an anticancer agent and / or a diagnostic marker or marker.

Another objective of the present invention is to provide a method for producing a labeling agent against desired ligands.

Another objective of the present invention is to identify specific motifs that provide for the recognition of markers of cells that are overexpressed in the malignant state and that can be used in the construction of a label or label for diagnosis or diagnosis for an anticancer agent.

Another objective of the present invention is to provide a composition comprising an effective amount of such peptides, polypeptides or motifs associated with, or coupled to, an anticancer agent or a diagnostic marker or marker.

It has been established that scFv penetrate the tissues and clear the blood more quickly than a full-size antibody because its size is smaller. Adams, G.P. et al, Br. J. Cancer 77, 1405-1412 (1988); Hudson, P.J., Cuur. Opin. Immunol. 11 (5), 548-557 (1999); Wu, A.M. et al, White Tumor Marking, 4, 47 (1999). Therefore, scFv are often employed in diagnostics consisting of radioactive labels such as tumor images to allow faster clearance of the body's radioactive label. Numerous scFv multimers indicating white cancers have recently been subjected to preclinical evaluation of stability and efficacy in vivo. Adams, G.P. et al, Br. J. Cancer 77, 1405-1412 (1988); Wu, A.M. et al, White Tumor Marking 4, 47 (1999).

Fragments of single chain Fv (scFv) are composed of the variable domains of the heavy (VH) and light (VL) chains of an antibody bound by a polypeptide linker. The linker is long enough to allow the (VH) and (VL) domains to fold into a functional Fv domain that allows the scFv to recognize and bind to its target with the similar or increased affinity of the parent antibody. A commonly used connector comprises glycine and serine residues to provide flexibility and resistance to the protease.

Generally, the scFv monomers are designed with the terminus of the C terminus of the VH domain linked by a polypeptide linker to the residue of the N terminus of the VL. Optionally an inverse orientation is employed: the terminus of the C terminus of the VL domain is linked to the N-terminus residue of VH through a polypeptide linker. Power, B. et al, J. Immunol. Meth. 242, 293-204 (2000). The polypeptide linker is about twelve amino acids in length. When the linker is reduced to three to twelve amino acids, the scFvs can not be folded into a functional Fv domain and instead associate with a second scFv to form a diabody. A further reduction in the length of the linker to less than three amino acids forces the association of scFv in trimers or tetramers, depending on the length of the linker, the composition and the orientations of the Fv domains. B.E. Powers, P.J. Hudson, J. Immun. Meth. 242 (2000 193-204.

Recently, it has been discovered that multivalent antibody fragments such as scFv dimers, trimer and tetramers often provide a higher apparent affinity compared to the binding of the parent antibody to the target. The higher affinity offers many advantages including the ideal pharmacokinetics for target tumor target applications.

The greater affinity of the binding of these multivalent forms is therefore desirable in diagnosis and therapeutic regimens. For example, a scFv can be used as a blocking agent to bind a target receptor thereby blocking the binding of the "native" ligand. In such cases, it is desirable to have a high affinity association between the scFv and the receptor to reduce the chances of dissociation, which may allow an undesirable binding of the natural ligand to the target. In addition, this high affinity is especially critical when white receptors are involved in adhesion and winding or when white receptors are in cells present in areas of high slip flow, such as platelets.

Accordingly, one object of the invention is the multivalent forms of sceFv of Yl and Y17. These multivalent forms include, but are not limited to, dimers, trimers, and tetramers, sometimes referred to as diabodies, triabodies, and tetrabodies, respectively.

EXTRACT OF THE INVENTION The present invention provides for the identification of peptides and polypeptides that selectively and / or specifically bind to target cells especially against blood-related cancer cells, their construction, their use alone or in association with, or combined, conjugated or fused with one or more pharmaceutical agents.

An embodiment of the present invention provides a peptide or polypeptide comprising an Fv molecule, a construction thereof, a fragment of some of them, or a construction of a fragment having improved binding characteristics so as to selectively bind and / or or specifically to a target cell in favor of other cells, wherein the selectivity or binding specificity is determined primarily by a first hypervariable region, and wherein the Fv is a single chain Fv ("scFv") or an Fv of disulfide ("dsFv"), and that optionally has one or more markers.

In another embodiment of the present invention, a peptide or polypeptide comprising an Fv molecule, a construction thereof, a fragment of some of them is provided., or a fragment construct having improved binding characteristics so that it binds selectively and / or specifically to a binding site substantially exposed and / or overexpressed in a target comprising a cell in favor of other cells, or where the binding site is not substantially available and / or exposed, wherein the selectivity or specificity of the binding is determined primarily by a first hypervariable region, and wherein the Fv is a scFv or a dsFv, and optionally has one or more markers .

In another embodiment of the present invention there is provided a peptide or polypeptide comprising an Fv molecule, a construct thereof, a fragment of some of them, or a construction of a fragment having improved binding characteristics so as to bind selectively and selectively. / or specifically to a white cell in favor of other cells, wherein the Fv molecule comprises a first chain having a first, second and third hyprvariable regions and a second chain having a first, second and third hypervariable regions, wherein one of the hypervariable regions of the first chain has a sequence selected from the group consisting of SEQ ID N °: 8-24 and wherein one of the hypervariable regions of the second chain has a sequence selected from the group comprising SEQ ID N °: 1-6 and 125-202, and wherein the first, second and third hypervariable regions are a region CDR3, CDR2 and CDR1, respectively, and wherein the Fv is a scFv or a dsFv, and optionally has one or more markers.

In another embodiment of the invention, (a) the first and second strands comprise each first hypervariable region selected from the group comprising SEQ ID NO: 8-24, (b) the first hypervariable regions of the first and second strands are identical and are selected from the group comprising SEQ ID NOS: 8-24, (c) the first hypervariable region of the first strand is selected from the group comprising SEQ ID NOS: 8-24 and the first hypervariable region of the second chain is selected from the group comprising SEQ ID N °: 1-6 and 125-202, (d) the first hypervariable region of the first chain is selected from the group comprising SEQ ID N °: 1-6 and 125-202, and the first hypervariable region of the second chain is selected from the group comprising SEQ ID N °: 8-24.

In yet another embodiment of the present invention there is provided a peptide or polypeptide comprising an Fv molecule, a construct thereof, a fragment of some of them, or a construction of a fragment that binds to an unknown ligand in a first cell having a first and second state, wherein the binding is effective in the second state but not substantially in the first state, and, by virtue of the cross-immunoreactivity, binds specifically or selectively to a ligand in a second cell and in where the Fv is a scFv or a dsFv and optionally has one or more markers.

In still another embodiment of the present invention there is provided a method for identifying a target molecule that binds to the unknown cross-linked immunoreactive binding site in first and second cells comprising (a) one or more steps of biowashing performed in a first cell blank which, in a second state but not in a first state, substantially exposes or shows the binding site comprising an unknown ligand, so as to produce a first population of recognition molecules; (b) subsequent steps of biowashing and / or selection beginning with the resultant existence of recognition molecules of step (a) that are performed in a second cell that has a binding site comprising an unknown ligand that has cross-linked immunoreactivity unknown ligand of the first cell so as to produce a second population of recognition molecules; (c) the amplification and purification steps of the second population of recognition molecules of step (b); and (e) construction from the recognition sites of the purified recognition molecules of peptides or polypeptides of step (c) comprising target molecules that are selective and / or specific for unknown ligands in the second cell.

In still another embodiment of the present invention there is provided a binding motif comprising the amino acid sequence of Ri-X Phe Pro-R2 wherein Ri and R2 each comprise from 0 to 15 amino acid residues, and wherein X is Arg. , Gly or Lys.

In still another embodiment of the present invention there is provided a method of producing a labeling agent comprising the following steps: (a) isolating and selecting one or more target molecules comprising a main recognition site by a process of biowashing directly on a white cell or by a process of biowashing indirectly on a first target cell in a second but not in a first state, and subsequently by a process of biowashing directly on a target cell to produce one or more of said target molecules; (b) amplification, purification and identification of one or more target molecules; (c) construction of a labeling agent from one or more target molecules or recognition sites thereof; wherein the labeling agent can be a peptide, polypeptide, antibody or antibody fragment or a multimer thereof.

In another embodiment of the present invention there is provided a peptide or polypeptide having the formula or structure: A-X-B Where X is a hypervariable CDR3 region of 3 to 30 amino acids; A and B are each amino acid chains of 1 to 1,000 amino acids in length, wherein A is the amino terminus and B is the carboxy terminus.

BRIEF DESCRIPTION OF THE DRAWINGS The invention is described herein in more detail, by way of example only, and as no form of limitation, with reference to the accompanying drawings described below, wherein: Figure 1 presents the binding of phage clones to fixed platelets, determined by the EIA assay. The data are presented according to an absorbance at 405 nm.

Figures 2a, 2b and 2c show the union of mononuclear cell samples obtained from three patients with individual AML to scFv, determined by FACS analysis. The intensity of the fluorescence of the bound cells is presented by the two assayed samples labeled by FITC (scFv control and Yl of scFv clone).

Figure 3 shows the binding of Yl to platelets (3a) and monocytes (3b) that have been purified with Ficoll, as determined by the FACS analysis. The intensity of the fluorescence of bound cells is presented by the two tested samples labeled with FITC (scFv control and Yl clone of scFv).

Figure 4 shows the binding of Yl of scFv clone labeled by FITC to blood CD34 + stem cells. Regulated CD34 + cells, in the FL3-H channel, were analyzed in the FLI-H channel for their binding to the negative control scFv labeled with FITC (Figure 4a) or YL of scFv labeled with FITC (Figure 4b) . Figure 4c presents a plot of FSC and SSC points of the same Yl sample of scFv clone labeled with FITC as in Figure 4b. The areas enclosed in circles in Figures 4b and 4c delineate the subpopulation of CD34 + cells that bind to the Yl of scFv clone.

Figure 5: FACS analyzes of samples obtained from two patients with pre-B-ALL cells are presented: one from a child (5a, 5c, 5e) and the other from an adult (5b, 5d, 5f). A double-staining procedure was used, using a CD19 labeled with available PE (a marker for normal peripheral B cells; Figure 5a, 5c) or a CD34 labeled with PE (a marker for stem cells; Figure 5d), together with a scFv negative control labeled with FITC (5a, 5b) or scFv of Yl labeled with FITC (5c, 5d). Figure 5b is a double negative control. The fluorescence intensity (X axis) of cells bound by the FITC-labeled sample (Yl of scFv clone) is presented, in relation to the staining pattern of the negative control (5e and 5f).

Figure 6: This figure provides results of a comparative study of junctions performed using Jurkat cells. The FACS analysis of Jurkat cell binding of FITC-labeled Yl scFv monomers, diabodies and triabodies is presented, together with a negative control.

Figure 7: This figure provides results of a study comparing the binding of IgG-Yl and scFv-Yl. Double-staining procedure was used to compare the binding of full-length IgG-Yl with that of the scFv-Yl form. Five YgG-Yl nanograms were used for FACS analysis in RAJI cells (Yl negative cells, Figure 7a) and in Jurkat cells (Yl positive cells, Figure 7b). For detection, goat anti-human IgG labeled with PE was used. For the binding of scFv-Yl-I 1 g (200 times) was used, followed by staining with anti-rabbit scFv antibodies labeled with PE and FACS analysis (Figure 7c).

Figure 8: This figure shows a comparison of junctions between a dimer of Y1, the scFv of Y1 (CONY1) and IgG of Y1.

Figure 9: This figure shows a comparison of junctions between a disulfide bridge dimer of Y1 and Y1 scFv (CONY1).

Figure 10: This figure is a graph of the Yl-cys-kak of the Superdex 75 profile.

Figure 11: This figure reveals the size of the dimers compared to the monomer under conditions of reduction and not reduction.

Figure 12: This figure provides results of an ELISA test.

Figure 13: This figure is a table of epitopes of anti-GPIba antibodies.

Figure 14: This figure is SEQ ID N °: of amino acids.

DETAILED DESCRIPTION OF THE INVENTION Specificity is defined herein as the recognition, by one or more domains of the peptide or polypeptide of the invention, of a target ligand and subsequent binding thereto.

Selectivity is defined here as the ability of a target molecule to choose and bind to a cell type or cell state from a mixture of cell types or cell states, all cell types or cell states can be specific to the cell. white molecule.

The substitution of conservative amino acids is defined as a change in the composition of amino acids 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, polar or non-polar) so that the substitution does not fundamentally alter the characteristics (ex. , isoelectric point, affinity, avidity, conformation, solubility) or activity of the peptide, polypeptide or protein. Typical substitutions that can be made for such substitution of conservative amino acids can be between the following amino acid groups: (i) glycine (G), alanine (A), valine (V), leucine (L), and isoleucine (I) ( ii) aspartic acid (D) and glutamic acid (E) (iii) alanine (A), serine (S) and threonine (T) (iv) histidine (H), lysine (K) and arginine (R) (v) asparagine (N) and glutamine (Q) (vi) phenylalanine (F), tyrosine (Y) and tryptophan () Conservative amino acid substitutions can be made inside, as well as on the sides of hypervariable regions primarily responsible for the selective and / or specific binding characteristics of the molecule, as well as other parts of the molecule, eg. variable heavy chain cassette. In addition or alternatively, the modifications may be accompanied by the reconstruction of the molecules to form full size antibodies, diabodies (dimers), triabodies (trimers) and / or tetrabodies (tetramers) or to form minibodies or microbodies.

As used in the specification and 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 selectively binds or associates with the variable region of the light chain.

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 (1) a minibody, comprising a fragment of the heavy chain only of Fv, (2) a microbody comprising a small fraction unit of the variable region of the heavy chain of the antibody (PCT Application No. PCT / IL99 / 00581), (3) similar bodies comprising a fragment of the light chain, and (4) similar bodies comprising a functional unit of a variable region of the light chain.

An anti-cancer agent is an agent with anti-cancer activity, i.e. any activity that inhibits the growth or differentiation of immature cancer or precancerous cells, or any activity that inhibits the metastasis of cancer cells. In the present invention, an anti-cancer agent is also an agent with anti-angiogenic activity that prevents, inhibits, retards or slows angiogenesis of tumor tissue or is also an agent with anti-adhesion activities that inhibits, prevents, retards or slows adhesion and Metastatic invasion of cancer and precancerous cells.

The inhibition of the growth of a cancer cell is defined here as (i) the prevention of cancerous or metastatic growth, (ii) slowing down the cancerous or metastatic growth, (iii) the total prevention of the growth process of the cancer cell or the metastatic process, leaving the cell intact and alive; or (iv) the elimination of the cancer cell. More specifically, the inhibition of cancerous growth can be applied especially against blood-related cells, e.g. AML, multiple myeloma, or chronic lymphatic leukemia.

A phagemid is defined as a phage particle that carries the plasmid DNA. 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. In order to propagate the phage, consequently, it is necessary to cultivate the desired phage particles together with a collaborating phage strain that complements the missing packaging information. applied to polypeptides and defined herein refers to a given sequence of 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 joined at one or both ends. Similarly, stretches of amino acids can be replaced, inserted, removed or attached to one or both ends.

As used herein, an immunoglobulin (Ig) molecule is defined as any of five classes, ie, IgG, IgA, IgD, IgE, or IgM. The IgG class includes several subclasses that include, but are not limited to, IgG1, IgG2, IgG3 and IgG4.

A "pharmaceutical composition" refers to a formulation comprising a peptide or polypeptide of the invention and a pharmaceutically acceptable carrier, excipient or diluent.

A "pharmaceutical agent" refers to an agent useful in the prophylactic treatment or diagnosis of a mammal that includes, but is not limited to, a human, bovine, equine, porcine, mouse, canine, feline, or any other warm-blooded animal. . The pharmaceutical agent is selected from the group comprising radioisotope, toxin, oligonucleotide, recombinant protein, antibody fragment, and anticancer agent. Examples of such pharmaceutical 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 asprin; anti-inflammatory agents which include zaltoprofen, pranoprofen, droxicam, acetyl salicylic 17, diclofenac, ibuprofen, dexibuprofen, sulindac, naproxen, antolmetin, celecoxib, indomethacin, rofecoxib, and nimesulid; anti-autoimmune agents which include leflunomide, denileucine diftitox, subreum, inRho SDF, defibrotide, and cyclophosphamide; and anti-adhesion / anti-aggregation agents which include limaprost, chlorchromen and hyaluronic acid.

The anti-leukemia agent is an agent with activity against leukemia. For example, antileukemia agents include agents that inhibit or slow 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 slows the vascularization of tumors.

The term "affinity" as used herein is a measure of the binding strength (association constant) between a receptor (eg, a binding site on an antibody) and a ligand (eg, an antigenic determinant). The resistance of the sum total of monovalent interactions between a single antigen-binding site in an antibody and a single epitope is the affinity of the antibody to that epitope. Antibodies with low affinity bind weakly to the antigen and tend to dissociate easily, while antibodies with high affinity bind to the antigen more closely 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 reaction of the antibody to the antigen is specific, in some cases the antibody produced by an antigen can cross-react with another unrelated antigen. Such cross-reactions occur if two different antigens share a homologous or similar epitope or an anchor region thereof or if antibodies specific for an epitope bind to an unrelated epitope that possesses similar chemical properties.

Blasto cells are cells in an immature stage of cell development distinguished by a ratio of the cytoplasm to the nucleus higher than a resting cell.

A platelet is a cytoplasmic fragment similar to a disk of a megakaryocyte spread in the medullary canal and then circulates in the bloodstream. Platelets have several physiological functions that include a fundamental role in coagulation. A platelet contains granules in the central part and peripherally, transparent protoplasm, but no defined nucleus.

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

A given cell can express on its surface a protein that has a binding site (or epitope) for a given antibody, but that binding site can exist in a cryptic form (eg, being blocked or spherically blocked, or lacking the characteristics necessary to be bound by an antibody) in the cell in a state, which may be referred to as a first step (step I). Stage I may be, for example, a normal, healthy, non-diseased 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 stage I. However, the epitope can be exposed by, eg. to undergo modifications itself, or to be unblocked because nearby or associated molecules are modified 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. Such modifications may occur when the cell enters a different state, which may be termed a second stage (stage II). Examples of second states, or steps, include activation, proliferation, transformation, or in a malignant state. When modified, the epitope can be exposed, and the antibody can bind.

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

Polyclonal antibodies are the product of an immune response and are formed by a number of different B lymphocytes. Raonoclonal antibodies are derived from a single cell.

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

The term aggregation means the formation of platelet clumps induced in vitro, and thrombin and collagen, as part of sequential mechanisms lead to the formation of a thrombus or hemostatic plug.

The expression pattern of a gene can be studied by analyzing the amount of the genetic product produced under different conditions, at specific times, in different tissues, etc. A gene is considered "overexpressed" when the amount of genetic product is higher than that found in a normal control, eg. a control not sick.

A promoter is a region in the DNA in which the RNA polymerase is initiated and transcription initiated.

Antibodies, or immunoglobulins, are protein molecules that bind to the antigen. They are composed of units of four polypeptide chains (2 heavy and 2 light) connected together by disulfide bonds. Each of the chains has a constant and variable region. They can be divided into five classes, IgG, IgM, IgA, IgD, and IgE, based on their heavy chain component. They are produced by B lymphocytes and recognize a particular foreign antigenic determinant and facilitate the clearance of that antigen.

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

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

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

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

The contaminating proteins are those proteins that are not specifically selected and that may be present in a sample.

The peptide mimetics are small molecules, peptides, polypeptides, lipids, polysaccharides, or conjugates thereof that have the same effect or functional activity of another entity such as an antibody.

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

There is a broad spectrum of diseases that involve diseased, altered or otherwise modified cells that express 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 molecule of the peptide or polypeptide 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 mainly by a first hypervariable region. The Fv can be a scFv or a dsFv. The Fv molecule described above can be used to target the diseased cell. The diseased cell can be, for example, a cancer cell. Examples of cancer types that may be amenable to diagnosis and / or treatment by a specific target direction include, but are not limited to, carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, bastoma, semmoma, 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.

New approaches have been developed to diagnose and treat diseases, particularly cancer, in recent years. Among them, is the approach of targeting the tumor, using white molecules that can be selected and produced in a variety of ways. One approach to identify possible target molecules is the presentation of phages. Phage display is a technique in which peptides, polypeptides, antibodies or proteins are generated and selected by their expression and presentation on the surface of a filamentous bacteriophage by fusion to a protein from the phage coat, with the DNA encoding the phage. protein presented that resides within the phage virion. The scFv produced by the phage display technique is composed of the variable domains of each of the heavy and light chains of the antibody, joined by a flexible amino acid polypeptide spacer (Nissim et al, EMBO J., 13, 692- 698 (1994)).

A phage display library (also referred to as a peptide / phage antibody library, phage library, or peptide / antibody library), comprises a large phage population (generally 108-109), each phage particle having a sequence of peptide or different polypeptide. These peptide or polypeptide fragments can be constructed to be of variable length. The presented peptide or polypeptide can be obtained from, but not limited to, heavy or light chains of human antibodies.

In the present invention, an scFv antibody library produced by the phage library technique was used to obtain and produce target molecules. Flow cytometry, particularly fluorescence activated cell sorting ("FACS"), was used to identify and isolate specific phage clones, whose peptide or polypeptide recognizes target cells. The fragments of scFv antibodies expressed by phages are docile to the evaluation, enrichment and in vitro selection of high affinity clones (US Patent 5,821,337, US Patent 5,720,954). Therefore, a library of this type offers a powerful means to generate new tools for research and clinical applications, and has numerous advantages over the conventional approach (Canon et al, Cancer Supplement, 73, 1049-1056 (1994)) . The library contains the potential for a high diversity of antibody molecules (Nissim et al, EMBO J, 692-698 (1994)). In the present case, stable human cDNA can be used as a continuous source of material for the production of antibodies (U.S. Patent 5,843,439). The recognition and selection of molecules is not influenced by the in vivo immunogenicity of the candidate target proteins.

Although the affinity selection of antibodies presented in phage provides a useful method for enriching scFv reactive to the antigen from large libraries, it requires several steps to isolate a single clone and characterize soluble scFv. The scFvs themselves can be modified to improve their affinities and / or avidity by making substitutions of conservative amino acids or by producing fragments of the scFv, or constructions of said fragments.

The scFvs of the present invention, specific for different human cells and tissues, can be associated, combined, fused, or connected to different pharmaceutical agents and / or radioactive isotopes in a pharmaceutically effective amount with, optionally a pharmaceutically effective carrier, to form compositions, fusions or drug-peptide conjugates, which have anti-disease and / or anti-cancer activity, and / or for diagnostic purposes thereof.

The phage clones are selected and identified through a multi-step procedure called biowashing. The biowashing is carried out by incubating variants of phage display protein ligands (a phage display library) with a blank, removing unbound phage by a washing technique, and specifically eluting the bound phage. The optionally eluted phage is amplified before being taken through additional cycles of binding and optional amplification that enriches the deposit of specific sequences in favor of those phage clones that carry fragments of antibodies that present the best binding to the target. After several rounds, the individual phage clones are characterized, and the sequences of the peptides presented by the clones are determined by the formation of corresponding DNA sequences of the phage virion.

The scFv obtained in this manner is also called the terminal compound. The terminal compound is defined as a compound, which final format comprises a central peptide or polypeptide. The terminal compound can be modified and / or expanded, but it must maintain the central peptide or polypeptide or some conservatively modified form thereof. Modifications by amino acid substitution can be made at the N-terminus, at the carboxy terminus, or at any of the CDR regions of an Fv or in the ascending or descending regions thereof, for example. The modifications may also include, but are not limited to, fused proteins, coupling to drugs or toxins, construction of multimers, and expansion of whole antibody molecules. A preferred category of terminal compound, provided in the present invention, is a scFv obtained as the final product of the biowashing process.

An embodiment of the invention provides at least one unnatural modification of the peptide or polypeptide of the invention. Unnatural modification can give the peptide or polypeptide more immunogenic or more stable. Unnatural modifications include, but are not limited to, peptoid modification, semi-peptide modifications, modification of cyclic peptide, modification of the N term, and modification of residues.

The selection of antigen-specific phage antibodies has depended to a large extent on the biolaunder against a single immobilized antigen. There has been limited selection using those cells as targets. In the present invention, whole cells were used to select specific antibodies that recognize surface determinants of leukemia cells, where the specific receptor was previously unknown or not characterized. This method does not allow easy adjustment of antigen concentration or removal of unwanted dominant antibody reactivities. In addition, phage can be enriched for those who have multiple copies of scFv, unlike those with higher affinity clones. In any case, the advantages of this approach make it an invaluable tool for isolating novel human antibody molecules.

An embodiment of the invention provides a peptide or polypeptide comprising an Fv molecule, a construction thereof, a fragment of some of them or a construction of a fragment that binds to an unknown ligand in a first cell having a primer and a second state, wherein the binding is effective in the second state but not substantially in the first condition and, by virtue of the cross-immunoreactivity, binds specifically or selectively to a ligand in a second cell, and wherein the Fv is a scFv or a dsFv, and optionally has one or more markers.

Another embodiment provides the peptide or polypeptide of the invention, wherein the selective and / or specific binding of the peptide or polypeptide to the ligand of the second cell is determined primarily by a first hypervariable region.

Yet another embodiment provides the peptide or polypeptide of the invention, wherein the first hypervariable region is a CDR3 region having an amino acid sequence selected from the group consisting of SEQ ID NOS: 8-24.

Yet another embodiment provides the peptide or polypeptide of the invention, wherein the first hypervariable region is a CDR3 region having an amino acid sequence selected from the group formed by SEQ ID NOS: 8-24 and wherein the selectivity or specificity of the junction is secondarily influenced by a second hypervariable region and / or by a third hypervariable region and / or by one or more of the ascending regions and / or by one or more of the descending regions flanking the first, second and third regions. hypervariable regions, respectively.

Another embodiment provides the ligand of the second cell bound by the peptide or polypeptide of the invention. · One such two-cell selection protocol was based on the following: Megakaryocytes are large multinucleated cells derived from hematopoietic stem cells in the bone marrow. Platelets break the cytoplasm of the megakaryocyte and enter the peripheral blood. In vitro, a wide range of cytokines directly affects stem cells. For example, thrombopoietin increases the platelet count directly increasing the differentiation of stem cells into megakaryocytes. Therefore, these cells express several cell surface markers that are also found in premature cells.

Malignant blood cells (leukemia and lymphoma) are characterized as immature cells that express cell surface proteins normally found in partially differentiated hemotopoietic progenitors. Therefore, platelets are an attractive source for the identification of surface markers of immature cells expressed in diseased or malignant blood cells. In a protocol considered below, specific cells such as, but not limited to, platelets, which transport unknown ligands, were used for the initial steps of the biowashing. The selection of subsequent clones was performed with a desired target cell, whose white cell surface markers are unknown, such as in non-exhaustive form AML cells. In this method, phage clones obtained by the platelet biowashing can provide tools to recognize and bind ligands in diseased or malignant blood cells of interest.

The target described above includes cells derived from an isolated tissue. The isolated tissue can be a diseased tissue and, more specifically, a cancerous tissue. The cancerous tissue can be obtained from any form of malignant cell that includes, but is not limited to, carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma and melanoma.

In addition to the initiation method described above, another approach is based on the isolation of a peptide or polypeptide that binds a ligand in a cell as determined by direct washing of the ligand.

The present invention provides a peptide or polypeptide comprising an Fv molecule, a construction thereof, a fragment of some of them, or a construction of a fragment. A construct can be a multimer (for example a diabody, triabody, tetrabody) or a full size Ig molecule; A fragment can be a minibody or a microbody. All the constructions and fragments obtained maintain the binding characteristics so that they bind selectively and / or specifically to a target cell in favor of other cells. The selectivity and / or specificity of the binding is determined primarily by a first hypervariable region, and wherein Fv is a scFv or a dsFv, and optionally has one or more markers.

In one embodiment of the invention, a label is inserted or attached to the Fv peptide or polypeptide to aid in the preparation and identification thereof, and in diagnosis. The marker can later be removed from the molecule. The marker can be, but not limited to, the following markers: AU1, AU5, BTag, c-myc, FLAG, Glu-Glu, HA, His6, HSV, HTTPHH, IRS, KT3, Protein C, S-TAG®, T7, V5, VSV-G, (Jarvik and Telmer, Ann. Rev. Gen., 32, 601-618 (1998)) and KAK (lysine-alanine-lysine). The marker is preferably c-myc or KAK.

The two variable chains of the Fv molecule of the present invention can be connected or joined together with a spacer of 0-20 amino acid residues in length. The separator can be branched or unbranched. Preferably, the linker is 0-15 amino acid residues, and more preferably the linker is (Gly <] Ser) 3 to give a single chain Fv ("scFv"). The scFv can be obtained from a phage display library.

The Fv molecule itself consists of a first chain and a second chain, each chain comprising first, second and third hypervariable regions. The hypervariable loops within the variable domains of the light and heavy chains are called Complementary Determining Regions (CDR). There are CDR1, CDR2 and CDR3 regions in 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 stated here is the site primarily responsible for the selective and / or specific binding characteristics observed.

An embodiment of the invention provides a peptide or polypeptide comprising an Fv molecule, a construct thereof, a fragment of some of them, or a construction of a fragment having improved binding characteristics so as to bind selectively and / or specifically to a binding site substantially exposed and / or overexpressed in a blank comprising a cell in favor of other cells in which the binding site is not substantially available and / or expressed, wherein the selectivity and / or specificity of the binding is primarily determined by a first hypervariable region and wherein Fv is a scFv or a dsFv, and optionally has one or more markers.

Another embodiment of the invention provides a peptide or polypeptide wherein the first hypervariable region is a CDR3 region having an amino acid sequence selected from the group consisting of SEQ ID NOS: 8-24.

Still another embodiment provides the peptide or polypeptide of the invention, wherein the first hypervariable region is a CDR3 region having an amino acid sequence selected from the group formed by SEQ ID NOS: 8-24 and wherein the selectivity and / or The specificity of the junction is secondarily influenced by a second hypervariable region and / or by a third hypervariable region and / or by one or more of the ascending regions and / or by one or more of the descending regions flanking the first, second and third hypervariable regions, respectively, wherein the second and third hypervariable regions are a CDR2 and a CDR1 region, respectively.

An embodiment of the invention provides a peptide or polypeptide that binds to a target cell that is an activated, excited, modified, changed, disrupted or diseased cell. Another embodiment of the invention provides the target cell which is a cancer cell. The target cell can be selected from the group consisting of, but not limited to, carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma and melanoma. In a preferred embodiment, the cancer cell is a leukemia cell. In the most preferred embodiment, the leukemia cell is an AML cell.

The peptide or polypeptide of the present invention is also any modified construction or construction of the Fv that maintains one or more of the hypervariable regions of the heavy and / or light chains and has selective and / or specific binding characteristics. The modified construction or construction includes, but is not limited to, scFv, dsFv, scFv multimers such as dimers, trimers, tetramers, and the like (also called diabody, triabody, tetrabody) and complete antibody, and any other multimer that can be constructed with them. , and that incorporates one or more of the hypervariable regions of the antibody. The peptide or polypeptide of the present invention is also a fragment of any modified construction or construction that has some or all of the binding characteristics of the original construct.

The peptide or polypeptide of the present invention is also a construction of a fragment that has some or all of the selective and / or specific binding characteristics of the original construct. The Fv described herein bind selectively and / or specifically to target cells and can be associated with, or conjugated to, anti-cancer agents, or anti-disease agents.

Peptides, polypeptides, fragments thereof, constructions thereof and fragments of their constructions of Fv molecules of the invention can be prepared in prokaryotic or eukaryotic expression systems. In one embodiment of the invention, the eukaryotic expression system is a mammalian system, and the peptide or polypeptide produced in the mammalian expression system, after purification, is substantially free of mammalian contaminants. A system of eukaryotic cells, defined in the present invention, refers to an expression system for producing peptides or polypeptides by genetic engineering methods, wherein the host cell is a eukaryote. In another embodiment of the invention, a 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 E. coli contaminating proteins. The use of a eukaryotic 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 by methods commonly known in the art, such as, but not limited to, the use of aeromonas aminopeptidase under suitable conditions (U.S. Patent No. 5,763,215).

The present invention provides the production of a scFv based on the Fv peptide of the invention. The promoters incorporated in the vectors used for the cloning and amplification of the scFv in the prokaryotic cells can be chosen from a wide selection. A promoter is a DNA sequence that is located on top of structural genes and is able to control the expression of genes. The promoters are in the natural state in the chromosome (s) of the organism and can also be manufactured in prokaryotic or eukaryotic expression vectors. The '* ¾í .. ^. · ....

Promoters manufactured at specific sites on the peptide of the desired DNA fragment provide finely tuned and precisely controlled expression of the gene of interest. In the present invention, several promoters were used in constructs including the gene encoding the chosen Fv. Promoters include, but are not limited to, the following: deo, P1-P2, osmB,? PL, P ~ lac-U5, SRa 5, and the early CMV promoter. Deo is a two-stranded DNA plasmid which, when introduced into a suitable host E.coli, makes the host capable of effecting the expression of the DNA encoding a desired natural polypeptide or a polypeptide analogous to it under the control of the derived deoxyribonucleotide promoter. of constitutive E. coli, deo P1-P2. A more complete description is provided in U.S. Patent No. 5,795,776 (Fischer, August 18, 1998) and in U.S. Patent No. 5,945,304 (Fischer, August 31, 1999).

The expression of the osmB promoter of E. coli is regulated by the osmotic pressure. The vectors carrying this promoter can be used to produce high levels of a wide variety of recombinant eukaryotic and prokaryotic polypeptides under control of the osmB promoter in a host E. coli. A more complete description is provided in U.S. Patent No. 5,795,776 (Fischer, August 18, 1998) and in U.S. Patent No. 5,945,304 (Fischer, August 31, 1999).

PL is a thermoinducible bacteriophage 8 promoter regulated by the thermolabile repressor 857. For a more complete discussion, see Hendrix et al. Lambda II, Cold Spring Harbor Laboratory (1983).

P-lac-U5 s a lacZ promoter (Gilbert and Muller-Hill, PNAS (US), 58, 2415 (1967).

SRa5 is a mammalian cDNA expression system composed of the early promoter of simian virus 40 (SV40) and the R-U5 segment of the long-term repeat of human type I T-cell leukemia virus. This expression system is 1 or 2 orders of magnitude more active than the SV40 early promoter in a wide variety of cell types (Takebe et al, Molecular and Cellular Biology, 8, 466-472 (1988).

The human cytomegalovirus promoter, termed the intermediate / early enhancer / CMV promoter is most preferably used in the present invention to promote the constitutive expression of DNA insertions of clones in mammalian cells. The CMV promoter is described in Schmidt, E.V. et al (1990) Mol. Cell. Biol., 10, 4406, and in U.S. Patent Nos. 5,168,062 and 5,385,839.

In a preferred embodiment of the invention, the promoter for the induction of the phagemid system in prokaryotes is selected from a group comprising the deo, osmB,? ^? -lac-U5, and CMV promoters. In a preferred embodiment of the invention, the p-lac-U5 promoter was used for the induction of the phagemid system in E. coli. In the most preferred embodiment, the CMV promoter is used.

In one embodiment of the invention, a peptide or polypeptide of the present invention comprises: (a) an initial sequence that is present only in the encoded sequence but is missing from the mature protein; (b) a variable region of a heavy chain in the order of 135-145 amino acids, including a first hypervariable region of 4-12 amino acids which is subject to modifications; (c) a spacer region of < 20 amino acids that can be shortened or eliminated; (d) variable region of a light chain that is also subject to specific modifications described here followed by; (e) a marker sequence for monitoring, which optionally is not present in the final injectable product. The separator, which generally has 15 amino acid residues in length in the scFv, allows the two variable regions (heavy and light) to bend in the functional Fv domain. The functional Fv domain maintains selective and / or specific enhanced binding activity.embodiment, (d) above is followed by a tag sequence or label that can be used for conjugation, diagnostic and / or identification purposes. In this embodiment, the marker is designed for the connection between the peptide or polypeptide of the invention and an agent for the treatment or diagnosis of the target cell.

The scFv separating region can be linear or branched, and is generally composed of glycine and serine residues, in multiples of the formula (Gly Ser > 3 and generally has a total of 0-20 amino acids in length, preferably 0- 15 amino acids in length and is linear By changing the length of the separator as appropriate, a variety of multimers can be obtained In one embodiment of the invention, the separator is 0-5 amino acids in length In another embodiment, the separator has < 3 amino acids in length (as detailed below).

An example of an amino acid sequence of a scFv molecule of the present invention is the following: ATGAAATACCTATTGCCTAC5GCAGCCGCTGGA? STTATTACTCGCG¾CCCR5CCGSCC 1 _? L _? _ "" J- ... "JL _ X. _ A. TO . A _ Je _. t .. & _ .4·. _ k. _ A ~ _ A_. £ L .. JL _ # 63 A GGCCGftC-GTGCAGCTGGTGGAGTCTGG GGAGGTGTGG ACGGCCTGGGGGGTCC TG 21"M__A E V Q L V S G G G V V R P G G S L 121 AGACTCTCCTGTGCAGCCTCTGGATtCACCTTTGATGATTAIGGCATGAGCTGGGTCCGC 41 R L S C A A S G F T IT D D Y G M S M V R 181 CAAGCTCCAGGGAAGGGGC GGAGTGGG CTC GGTATTAAT'ÍGGAATGGTGGTAGCACA 61 C A? GXGLSWVS 3 IX 'KGGST February 1 GGTTA7GCAGAGTCTGTGAG3GGCCGATTCACCAXCTCCAGASACAACGCCAAGAACTCC 81 GYADSGRFTI 3 RD fó AKNS 301 CTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACCGGCCGGTGTATTACGTGGCAAGA 101 LYLQMNSLRAEDTAVYYCAR 361 ATGAGGGCTCCTGTGA TTGGGCCCAAGTAACCCTGGTCACCGTGTCGAGAGTGGGAGGC 121 j MRAPV IT WGQGTLVTVSRGGG 421 GGTTCAGGCGGAGGTGGCrcrGGCGGTGGCGGATCGTCHGAGCTGhC ^ CA QliCCC GCR 141 SSGGGGSGGGGSSELTQDPA 481 GTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGACAGCCTCAGAAGC 161 VSVALGQTVRIXCQGD 5 LRS 541 TATTATGCAAGCTGGTACCAGCAGAAGCAGGACCAGGCCCCTTGTCTTGTCATCATGGGT 181 YYASWYQQKPGQAPVLVIYG 601 AAAAAC ACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTC AGGAAACACA 201 KNNRPSGIPDRFSGSSSGNT 661 GCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTAACTCC 221 ASLTITGAQAEDEADYYCNS 721 CGGGACAGCAGTGGTAACCATGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGT 241 RDSSG N H V V F G G G T K L T V L G 781 GCGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGGGCCGCATAG_261_A A E Q L I S E E D L N G._A A * The initial sequence is underlined with a dotted line. The VH region is encoded by the bold amino acid sequence. This specific clone is derived from the germline VH3-DP32; however, the germline of each clone depends on its particular origin (see below). The amino acid sequence enclosed in a box encodes the VH-CDR3 sequence, the hypervariable region among all the clones obtained from this library. The spacer region joining the VH and VL regions is a flexible polypeptide, encoded by amino acids shown in italics. Finally, the VL region is presented. The VL fragment fused in all the clones is obtained from a single non-mutated V gene of the IGLV3SI germ line, and is here followed by the c-myc marker, underlined with a full line. The complete amino acid sequence is identical to SEQ ID NO: 25.

The repertoires of VH fragments (49 l germ lines) were first generated by PCR from rearranged V genes of peripheral blood lymphocytes from unimmunised humans (termed "naive repertoire") by the library provider. The origin (germ line) of the VH sequence can be identified by a homology test (blasto search), using one of the following websites: The binding characteristics of an antibody can be optimized in one of several ways. One way to optimize an antibody to obtain a higher binding affinity in relation to the original terminal compound is based on the replacement of the amino acid residues of the terminal compound, to introduce greater variability, or to extend the sequence. For example, the entire original VL region can be replaced with a VL region of a different antibody subtype.

Another way to optimize the binding affinity is to build a phagemid display mutagenesis library. In a phagemid display mutagenesis library, the oligonucleotides are synthesized such that each amino acid of the central sequence within the VH and VL CDR3 is independently replaced by any other amino acid, preferably in a conservative form known in the art. The present invention provides a set of specific antibody scFvs displayed on the phage, wherein the fragments of antibodies presented and the fragments of soluble antibodies that can be extracted from the phage virions have the same biological activity.

The phage display library used here was constructed from peripheral blood lymphocytes of non-immunized human, and the Fv peptide was screened against previously uncharacterized and unpurified antigens on the surface of a target cell. As used herein, previously uncharacterized and unpurified antigens refer to ligands presented on the surface of cells that have not been identified, characterized, isolated or purified by biochemical or molecular means prior to the present work, and which are observed or predicted in the present work by virtue of the selective and / or specific binding to fragments of isolated antibodies observed.

The scFv of the present invention exhibits an improved binding to a target cell. The improved binding is directed to specific surface markers. Specific surface markers are molecules that are sequestered in the cell membrane and accessible to circulating recognition molecules. The presence of surface markers allowed the development of phage display technology through the biolavado technology described here. In the present invention, specific surface markers are used to characterize and differentiate between different types of cells, as well as to serve as the binding site for Fv in its different forms. A variety of hematopoietic cell types can be differentiated according to their characteristic surface markers and, similarly, diseased or cancerous cells have surface markers that are unique to their type and stage.

The selection of the scFv clone may be accompanied by two different biowashing strategies: 1. selection directly, using a diseased or cancerous cell as the target cell, and 2. selection in steps, using a first normal cell in a second activated state, excited, modified, changed or disturbed, whereby a binding site of the first cell in the second state comprises an unknown ligand that is substantially exposed or presented. By virtue of the cross immunoreactivity, the resulting clone can be joined, after the subsequent steps of biolaving and selection, selectively and / or specifies a new or unknown ligand in a second cell. After a further optional amplification and subsequent purification, the target molecules can be constructed from the recognition sites of the purified recognition molecules in a selective and / or specific manner for an unknown ligand in a second cell.

In one embodiment of the invention, the first cell can be a normal cell, the first one a non-activated state and the second one an activated, excited, modified, changed or disturbed state. The second cell in step selection can be a human cell. In another embodiment of the invention, the second cell in step selection is a diseased cell.

In a more preferred embodiment, the second cell in the stepped selection is a cancer cell such as, in form or restrictive, carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma, and melanoma. In a more preferred embodiment, the second cell is a leukemia cell. In the most preferred embodiment, the second cell is an AML cell.

A more preferred embodiment of the invention provides a peptide or polypeptide wherein the selective and / or specific binding of the peptide or polypeptide to the ligand of the second cell is determined primarily by a first variable region. In a still more preferred embodiment, the first hypervariable region is a CDR3 region having an amino acid sequence selected from the group consisting of SEQ ID N °: 8-24.

In another embodiment of the present invention, the ligand of the second cell bound by the peptide or polypeptide of the invention is provided. Another embodiment provides any molecule that recognizes and binds the ligand bound by the peptide or polypeptide of the invention.

Improved binding to a cancer cell is most likely due to overexpression of the ligand and / or exposure of the binding site in the cancer cell relative to expression in the normal cell. As used herein, the term "overexpression of the ligand" is defined as the expression of a gene or its normally silent product in the particular cell type and / or at a particular stage of the cell cycle, or the increased expression of a gene that it is expressed at basal levels under normal conditions, not malignant for that particular cell type.

In a more preferred embodiment of the invention, the target cell of the biowashing process is contained in a cell suspension. The hematopoietic cells are obtained in suspension, and the biowashing can be carried out by mixing a phage library with a suspension of blood cells, then washing with several buffers. The phages are extracted from the human cells, amplified, and the sequence of the presented antibody fragment is determined.

In a still more preferred embodiment of the invention, the blood cell suspension comprises leukemic cells. In the most preferred embodiment, the blood cell suspension comprises AML cells. In another embodiment of the present invention, the target cell is derived from an isolated organ or part thereof.

In another embodiment of the present invention, the target cell or the second cell is derived from a cell line. The cell lines can be cultured and manipulated in such a way that they can help in the determination of the binding characteristics of the Fv clones. In addition, cell lines can be useful in the development of diagnostic kits.

In a preferred embodiment, the cell line is a line of hematopoietic cells, such as non-exhaustively, the following lines: Jurkat cell lines, MOLT-4, HS-602, U937, TF-1, THP-1, KG-1, ML-2, and HUT-78.

In a preferred embodiment of the invention, the CDR3 region is constructed, inserted, coupled or fused to or on one of 84 cassettes (SEQ ID NO: 30-112). In a more preferred embodiment, the CDR3 region is constructed, inserted, coupled or fused in or on one of 49 cassettes (SEQ ID NOS: 30-32, 35, 37-39, 41, 43, 45, 46, 48, 51, 54, 57, 59-68, 70, 71, 76-85, 87, 89-92, 94, 97, 99, 103, 106, 112 and 113). In the most preferred embodiment, the CDR3 region is constructed, inserted, coupled or fused to or on the C-terminus of the cassette of SEQ ID NO: 61, or any of the preceding sequences having at least 90% similarity to the cassette. sequence to it.

In one embodiment, the amino acid sequence of the cassette is ostensibly fixed, whereas the sequence replaced, inserted or joined can be highly variable. The cassette can be composed of several domains, each of which covers a crucial function for the final construction. The cassette of a particular embodiment of the present invention comprises, from the N term, the frame region 1 (FR1), CDR1, frame region 2 (FR2), CDR2 and frame region 3 (FR3).

In an embodiment of the invention, it is possible to replace distinguishable regions within the cassette. For example, the CDR2 and CDR1 hypervariable regions of the cassette can be replaced or modified by non-conservative amino acid substitutions, or preferably, conservative. More specifically, the CDR2 and CDR1 regions of a cassette of consecutive amino acids selected from the group consisting of SEQ ID NO:. 30-113 or a fragment thereof may be replaced by SEQ ID NOS: 115 and 114, respectively. Even more specifically, the CDR2 and CDR1 regions of a consecutive amino acid cassette selected from the group comprising SEQ ID NOS: 30-32, 35, 37-39, 41, 43, 45, 46, 48, 51, 54, 57, 59-68, 70, 71, 76-85, 87, 89.-92, 94, 97, 99, 103, 106, 112, and 113 or fragments thereof can be replaced by SEQ ID NOS: 115 and 114, respectively.

In a preferred embodiment of the invention, the peptide or polypeptide comprises a heavy and a light chain, and each chain comprises a first, second and third hypervariable region which are the CDR3, CDR2 and CDR1 regions, respectively. The selectivity and specificity of the binding are determined in particular by the CDR3 region of a chain, possibly by the CDR3 region of the light chain and preferably by the CDR3 region of the heavy chain, and secondarily by the CDR2 and CDR1 regions of the chain light and preferably, heavy chain. The selectivity and specificity of the binding can also be secondarily influenced by ascending or descending regions flanking the first, second and / or third hypervariable regions.

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

In a more preferred embodiment, the CDR3 region of the heavy chain has an amino acid sequence selected from the group consisting of SEQ ID NOS: 8-24, CDR2 has an amino acid sequence identical to SEQ ID No. 115 and the The CDR1 region has an amino acid sequence identical to SEQ ID No. 114.

In the most preferred embodiment of the invention, the CDR3 region has an amino acid sequence identical to SEQ ID No. 8.

In addition to the heavy and light chain, Fv comprises a flexible separator of 0-20 amino acid residues. The separator can be a branched chain or a straight chain. Two possible sequences of the separator are identical to SEQ ID No. 124 ..

A preferred embodiment of the invention is a scFv with a sequence of CDR3 identical to SEQ ID No. 8 and a complete scFv sequence identical to SEQ ID No. 25.

Another preferred embodiment of the invention is a scFv with a CDR3 sequence identical to SEQ ID No. 20 and a complete scFv sequence identical to SEQ ID No. 203.

In the most preferred embodiment of the invention, the CDR3, CDR2 and CDR1 regions have the amino acid sequences SEQ ID No. 8, 115, and 114, respectively.

In one embodiment of the invention, the Fv peptide comprises a CDR1 and CDR2 region of the variable heavy chain, which alone comprises a cassette with an amino acid sequence selected from the group consisting of SEQ ID NOS: 30-113; a CDR3 region, preferably of the variable heavy chain, having an amino acid sequence selected from the group comprising SEQ ID NOS: 8-24; an ascending region flanking the CDR3 region having the amino acid sequence of SEQ ID NO: 117; a descending region flanking the CDR3 region having the amino acid sequence of SEQ ID No. 116; a 0-20 amino acid residue separator of SEQ ID No. 123 or 124; a variable light chain region whose sequence is SEQ ID N ° 7.

Similarly, in another embodiment the ascending region flanking the CDR2 region has the amino acid sequence of SEQ ID No. 119, the descending region flanking the CDR2 region has the amino acid sequence of SEQ ID No. 118, the ascending region flanking the CDR1 region has the amino acid sequence of SEQ ID No. 121, and the descending region flanking the CDR1 region has the amino acid sequence of SEQ ID 120.

A preferred embodiment of the invention provides a peptide or polypeptide wherein the second and third hypervariable regions are a hypervariable region CDR2 and CDR1, respectively, and wherein the amino acid sequence of CDR3 is SEQ ID NO: 8, wherein the Amino acid sequence of CDR2 is SEQ ID No. 115, wherein the amino acid sequence of CDR1 is SEQ ID No. 114, wherein the ascending region flanking the CDR3 region has the amino acid sequence of SEQ ID N 117, wherein the descending region flanking the CDR3 region has the amino acid sequence of SEQ ID No. 116, wherein the ascending region flanking the CDR2 region has the amino acid sequence of SEQ ID No. 119 , wherein the descending region flanking the CDR2 has the amino acid sequence of SEQ ID No. 118, wherein the ascending region flanking the CDR1 region has the amino acid sequence of SEQ ID No. 121 and wherein the region descending flanking the CDR1 region has the amino acid sequence of SEQ ID No. 120.

Another preferred embodiment of the invention provides an Fv molecule comprising a first strand having a first, second and third hypervariable regions and a second strand having a first, second and third hypervariable regions, wherein one of the hypervariable regions of the first chain has a sequence selected from the group consisting of SEQ ID N °: 8-24, and wherein one of the hypervariable regions of the second chain has a sequence selected from the group consisting of SEQ ID N ° 1-6 and 125- 202, and wherein the first, second and third hypervariable regions are a region CDR3, CDR2 and CDR1, respectively and wherein Fv is a scFv or a dsFv and optionally has one or more markers.

Another embodiment of the invention provides a peptide or polypeptide (i) wherein the first strand and the second strand each comprise the first hypervariable region selected from the group consisting of SEQ ID No. 8-24; or (ii) wherein the first hypervariable region of the first and second chains are identical and are selected from the group consisting of SEQ ID No. 8-24; or (iii) wherein the first hypervariable region of the first chain is selected from the group consisting of SEQ ID No. 8-24, and the first hypervariable region of the second chain is selected from the group consisting of SEQ ID No.:. 1-6 and 125-202; or (iv) wherein the first hypervariable region of the first chain is selected from the group consisting of SEQ ID No. 1-6 and 125-202 and the first hypervariable region of the second chain is selected from the group consisting of SEQ ID No. 8-24.

Another embodiment provides the peptide or polypeptide of the invention wherein a second and third hypervariable regions of the first strand are SEQ ID Nos. 114 and 115, respectively.

For all amino acid sequences of < 25 amino acid residues described and detailed herein (eg, CDR regions, regions flanking CDRs), it should be understood and considered as another embodiment of the invention that these amino acid sequences include within their scope one or two substitution (s) of amino acids and that preferably the substitutions are substitutions of conservative amino acids. For all amino acid sequences of > 25 amino acid residues described and detailed herein, it should be understood and considered as an embodiment of the invention that these amino acid sequences include within their scope an amino acid sequence with > 90% similarity of the sequence to the original sequence (Altschul et al, Nucleic Acids Res., 25, 3389-3402 (1997)). Similar amino acids or homologs are defined as non-identical amino acids that have similar properties, eg. acids, basic, aromatic, size, with positive or negative charge, polar or non-polar.

~: V- 'The percentage of amino acid similarity or homology or similarity of the sequence is determined by comparing the amino acid sequences of two different peptides or polypeptides. The two sequences are aligned, generally using one of a variety of computer programs for this purpose, and the amino acid residues are compared at each position. Then the identity or homology of the amino acids is determined. Then an algorithm can be applied to determine the percentage of similarity of the amino acids. It is generally preferable to compare amino acid sequences, due to the greatly increased sensitivity to the detection of subtle relationships between the molecules of the peptides, polypeptides or proteins. The comparison of proteins can take into account the presence of amino acid substitutions, so that a disparity can still give a positive rating if the non-identical amino acid has similar physical and / or chemical properties (Altschul et al, Nucleic Acids Res., 25 , 3389-3402 (1997)).

In one embodiment of the invention the three hypervariable regions of each of the light and heavy chains can be exchanged between the two chains and between the three hypervariable sites within and / or between chains.

One skilled in the art will recognize that demonstration of the specific and / or selective binding of the peptide or polypeptide of the invention necessitates the use of an appropriate negative control. A suitable negative control can be a peptide or polypeptide, whose amino acid sequence is almost identical to the peptide or polypeptide of the invention, with the only difference being that it is in the hypervariable CDR3 region. Another suitable negative control may be a peptide or polypeptide that is the same size and / or general three-dimensional structure as the peptide or polypeptide of the invention but has a totally unrelated amino acid sequence. Another suitable negative control may be a peptide or polypeptide with completely different physical and chemical characteristics, when compared to the peptide or polypeptide of the invention. The negative controls used in the development of the present invention are designated N14, which has the sequence of CD 3 identical to SEQ ID No. 28 and C181, which has a sequence of CDR3 identical to SEQ ID No. 29. Other controls Negatives, however, can be equally suitable.

Another embodiment provides a nucleic acid molecule, preferably a DNA molecule, which encodes the Fv peptide or polypeptide of the invention.

In one embodiment of the present invention and to optimize the selective binding of Fv, the sequences of the CDR3 that confer the selectivity and / or specificity of main binding to the Fv can be moved to any other germ line of the heavy chain. More particularly, they can move to one of 84 possible germ lines of the heavy chain. These 84 germ lines (SEQ ID N °: 30-113) comprise (a) the germline in which the claimed phage clone was originally isolated, (b) 48 additional germ lines available in the phage display library and ( c) 35 alternative germ lines claimed here (Tomlinson et al, J. Med. Biol., 227 (3): 776-798 (1992)). The linear or three-dimensional local means of the CDR3 region, in concert with the CDR3 region itself, can potentially play a role in guiding or encouraging the correct binding of CDR3. For example, peptides having any of the CDR3 sequences mentioned herein as SEQ ID N ° 8-24, 125, and derived from any of the 49 germline sequences (SEQ ID NOS: 30-32, 35, 37 -39, 41, 43, 45, 46, 48, 51, 54, 57-68, 70, 71, 76-85, 87, 89-92, 94, 97, 99, 103, 106, 112 and 113) also they are comprised by the present invention.

The DP-32 germline is the cassette for several clones of the present invention. The C-terminus of this germline has been replaced with a sequence by consensus to aid in the preparation of the phage display library. The seven amino acids with carboxy terminus of SEQ ID No. 61 have been replaced by the 7 amino acid sequence of SEQ ID No. 122.

The Fv CDR3 regions of the present invention may contain the Arg / Giy / LysPhe Pro central sequence that specifically binds to AML cells. Eight examples of such CDR3 regions are presented in Table 2. Although the motif coincides with the three amino acid residues of the N terminus of the CDR3 region in each case, it may also be located elsewhere in the CDR3 region. Alternatively, the motif is a binding motif that is used to construct an anchor or binding region of part of a binding or target molecule or recognition or is used only as a target vehicle.

In another embodiment of the present invention, a binding motif comprising the amino acid sequence of Ri-X Phe Pro-R2 is provided wherein Ri and 2 each comprise 0-15, preferably 1-9, amino acid residues and in where X is Arg, Gly, or Lys. More preferably, the CDR3 comprises the amino acid sequence of Ri -X Phe Pro-R2, wherein Ri and R2 each comprise 0-15 amino acid residues and wherein X is Arg, Gly or Lys.

In another preferred embodiment of the peptide or polypeptide of the present invention, 1-1,000 amino acids can be added to the C-terminus or the N-terminus of the peptide, while the peptide maintains its biological activity. In a preferred embodiment of the invention, 150-500 amino acids can be added to the C-terminus or the N-terminus of the peptide or polypeptide, while the peptide maintains its biological activity. In another preferred embodiment of the invention, 800-1,000 amino acids may be added to the C-terminus or N-terminus of the peptide or polypeptide, while the peptide or polypeptide retains its biological activity.

An example for extending the central amino acid sequence is by building a full length Ig immunoglobulin, using a terminal compound as the center of the Ig. The full-length Ig may, for example, belong to the class of immunoglobulin which can induce endogenous cytolytic activity through complement or activation of cellular cytolytic activity (eg IgG1, IgG2, or IgG3). The full-length Ig may belong to the immunoglobulin class of resistant binding antibodies (eg IgG4). By joining, the full-length Ig can act in one or more of many ways, for example acting as a flag for the body's defense mechanism to initiate an immune response, transducing the cell's intracellular signaling or causing damage to a target cell .

A preferred embodiment of the present invention provides an Ig molecule expressed as a recombinant polypeptide and produced in a system of eukaryotic cells. In a preferred embodiment of the invention, the Ig polypeptide is an IgG polypeptide and is produced in a mammalian cell system. In a preferred embodiment the mammalian cell system comprises the CMV promoter.

In a preferred embodiment of the invention, the IgG molecule comprises a hypervariable region CDR3, CDR2 and CDR1, both in the light chains and in the heavy chains. In a preferred embodiment of the invention, the Fv molecule comprises a CDR3, CDR2 and CDR1 region having SEQ ID Nos. 8, 115 and 114, respectively. The CDR3, CDR2 and CDR1 regions can be of the heavy chain or the light chain.

Another preferred embodiment of the invention provides an IgG molecule having a light chain with a sequence identical to SEQ ID No. 27 and a heavy chain with a sequence identical to SEQ ID No. 26, or a heavy chain and a chain light that they have at least 90% similarity of sequence with it. In a preferred embodiment of the invention the two heavy chains of the IgG are identical and the two light chains of the IgG are identical.

In another embodiment, the peptide of the present invention is constructed to fold in multivalent Fv forms.

The present invention provides a Yl or Y17 peptide or polypeptide comprising a scFv molecule. As used herein a scFv 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 may be the same or different, and wherein the The variable region of the heavy chain is connected, linked, fused or covalently linked, or associated with the variable region of the ivian chain.

A scFv construct of Y1 and Y17 can be a multimer (eg, a dimer, trimer, tetramer, and the like) of the scFv molecules that incorporate one or more of the hypervariable domains of the Y1 or Y17 antibody. All constructs and fragments derived from scFv retain improved binding characteristics so as to bind selectively and / or specifically to a target cell in favor of other cells. The selectivity and / or specificity is determined mainly by hypervariable regions.

The hypervariable loops within the variable domains of the light and heavy chains are called Complementary Determining Regions (CDR). There are CDR1, CDR2 and CDR3 regions in each of the heavy and light chains. 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 provided herein, is the site primarily responsible for the selective and / or specific binding characteristics observed.

The peptide of Yll and Y17 of the present invention can be constructed to be doubled in multivalent Fv forms. The multimeric forms of Yl and Y17 were constructed to improve the affinity and specificity and the increased half-life in the blood.

The multivalent forms of scFv have been produced by third parties. One approach has been to join two scFvs with connectors. Another approach is to use disulfide bonds between two scFvs for ligation. The simplest approach for the production of dimeric or trimeric Fv was reported by Hollinger et al., PNAS 90, 6444-6448 (1993) and A.Kortt et al, Protein Eng. , 10, 423-433 (1997). One such method was designed to make scFv dimers by adding a sequence of the FOS and JUN protein region to form a leucine lock between them at the scFv c-terminus. Kostelny SA et al, J. Immunol. March 1, 1992; 148 (5): 1547-53; De Kruif J et al, J Biol. Chem. March 29, 1996; 271 (13): 7630-. Another method was designed to manufacture tetramers by adding a sequence encoding streptavidin in the C term of scFv. Streptavidin is composed of 4 subunits so that when streptavidin is doubled, 4 subunits are accommodated to form a tetramer. Kipriyanov SM et al, Hum Antibodies Hybridomas, 1995; 6 (3): 93-101. In yet another method, to make dimers, trimers and tetramers, a free cysteine is introduced into the protein of interest. A peptide-based cross-linking with variable numbers (2 to 4) of maleimide groups was used to cross-link the protein of interest with the free cysteines. Cochran JR et al, Immunity, March 2000; 12 (3): .241-50.

In this system, the phage library (described hereinabove) was designed to present scFv, which can be doubled in the monovalent form of the Fv region of an antibody. In addition, also discussed above, the construction is suitable for bacterial expression. The genetically engineered scFvs comprise variable regions of heavy chains and light chains linked by a contiguously encoded 15 amino acid flexible peptide separator. The preferred separator is (Gly¾Ser) 3. The length of this separator, together with its amino acid constituents, provides an antibody scavenger, which allows the VH and VL regions to fold into a functional Fv domain that provides an efficient binding to this target.

The present invention relates to Yl and Y17 multimers prepared by any method known in the art. A preferred method for forming multimers, and especially dimers, employs the use of cysteine residues to form disulfide bonds between two monomers. In this embodiment, dimers are formed by adding a cysteine on the carboxyl terminus of the scFvs (termed the Yl-cys dimer scFv or Yl dimer) in order to facilitate the formation of the dimer. After the DNA construct was made (See Example 2D and 6D) and used for transfection, the Y1 dimers were expressed in a production vector and doubled again in vitro. The protein was analyzed by SDS-PAGE, HPLC and FACS. Two-liter fermentation batches of the antibodies were circulated. After expressing Yl-cys in strain BL21 of E. coli, the new bent was made in arginma. After folding again, the protein was dialysed and purified by Q-Sepharose and gel filtration (Sephadex 75). Two peaks were detected by SDS-PAGE (not reduced) and by gel filtration. The peaks were collected separately and analyzed by FACS. The binding of monomers and dimers to Jurkat cells was verified by FACS. The binding by dimers required only 1/100 of the amount of the monomeric antibody for the same level of coloration, which indicates that the dimer has greater avidity. The conditions for the new doubling of the dimer were determined, and the material comprising > 90¾ of dimers (mg amounts) occurred after the subsequent steps of dialysis, chromatography and gel filtration. The purified dimer was characterized by gel filtration and by SDS-PAGE analysis under oxidation conditions. The binding capacity of the dimer was confirmed by radio-receptor assay, ELISA and FACS analysis.

The scF antibody fragment CONY1 is derived from Yl scFv. The DNA sequence encoding the myc marker of Yl scFv was removed and replaced by the synthetic oligonucleotide DNA sequence encoding the amino acids lysine, alanine lysine (KAK).

To compare the binding of the monomer of scFv (also called CONY1) with the dimer of Yl, in vitro binding competition experiments were performed on KG-1 cells. In addition, these experiments also compared the binding of the complete Yl IgG with the scFv Yl monomers. To perform this study, Yl IgG was labeled with biotin. This study revealed that YG IgG competed with IgG Yl-biotin. Non-relevant human IgG did not compete with marked Yl IgG. Yl scFv (5 μg and 10 μg) partially competed with Yl IgG-Biotin (50 ng). The studies also showed that 1 ng of IgGY1-FITC bound to KG-1 cells (without serum) in the same magnitude as 1 μg of scFv-FITC, but in the presence of serum, most of the IgG binding of Y1 it's blocked". These samples also showed that the binding of the Yl dimer is at least 20 times greater than that of the scFv monomer analyzed by the radio-receptor, ELISA or FACS assay.

In yet another embodiment, a lysine-alanine-lysine was added in addition to the cysteine at the carboxyl terminus (termed scFv of Yl-cys-KAK). The amino acid sequence of this scFv construct is reproduced below. 1 MEVQLVESGG GVVRPGGSLR LSCAASGFTF DDYGMSWVRQ APGKGLEWVS GINWNGGSTG 60 61 YADSVKGRFT ISRDNAKNSL YLQMNSLRAE DTAVYYCARM RAPVIWGQGT LVTVSRGGGG 120,121 SGGGGSGGGG SSELTQDPAV SVALGQTVRI TCQGDSLRSY YASWYQQKPG QAPVLVIYGK 180,181 NNRPSGIPDR FSGSSSGNTA SLTITGAQAE DEADYYCNSR DSSGNHVVFG GGTKLTVLGG 240 241 Yl-cys GGCKAK The KAK-occurred in a vector? -PL in bacteria . Expression in the? -pL vector was induced by raising the temperature to 42 ° C. Induced culture inclusion bodies were obtained and semipurified with aqueous solutions, to remove undesirable soluble proteins. The inclusion bodies were solubilized in guanidine, reduced with DTT and folded back in vitro in a solution based on arginine / oxidized glutathione. After folding again, the protein was dialysed and concentrated by tangential flow filtration to a buffer containing urea / phosphate buffer. The protein was repurified and concentrated by ion chromatography on a SP column.

To obtain higher expression levels in E. coli of the CONY1 scFv, as well as in the Yl-cys-KAK scFv, we introduced the amino acid coding for the alanine residue in position 2 of the N-terminal sequence of the scFv construct. . An expression level of four folds was obtained with the newly modified construction.

An ELISA was performed to determine the differences in the binding between the monomer (C0NY1 scFv, also called Yl-KAK) and the dimer Yl-cys-KAK (the cysteine dimer) for the GPIb (glycocalicin) antigen obtained from platelets. A polyclonal single chain antibody and / or a novel polyclonal anti-VL (obtained from rabbits) and a rabbit anti-HRP, were used to detect binding to GPIb. The dimer was approximately 20-100 times more active than the monomer. For example, to reach 0.8 OD units, 12.8 mg / ml of the monomer was used compared to only 0.1 mq / ml of the dimer. See Figure 12.

The dimer was characterized by SDS-page electrophoresis, gel filtration chromatography, ELISA, radio-receptor binding and FACS. The apparent affinity of the dimer was higher than that of the monomer due to the effect of avidity. This effect was confirmed by ELISA to glycocalycinin, FACS to KG-1 cells, and competition in a radio-receptor assay.

HPLC was performed to profile the dimer after re-bending and purification from a Superdex 75 gel filtration column. In Figure 10, the Yl-cys-kak (dimer) is the first peak on the left (10, 8 minutes) and the next peak is the monomer (12 minutes). The dimer is approximately 52kDa and the monomer 26kDa, according to the protein size markers passed on the same column. The balance between the dimer and the monomer can be changed by varying the conditions of the new bending (concentration of the oxidized agent and the concentration of the protein in the newly folded buffer). The dimer and the monomer were separated by chromatography on a superdex 75 column.

In Figure 11, a gel with a mixed population of dimers and monomers is shown. In the reduced form, the monomers are seen due to the reduction between the two monomers and in the non-reduced form, two populations are seen (as in the gel filtration experiment) a monomer fraction of 30 kDa and a dimer of 60 kDa . In addition, FACS analysis of KG-1 cell binding showed that the dimer is more sensitive than the monomer when a two- or three-step binding assay was performed. The dimers directly marked by FITC showed a slight advantage (use of 10 times less material) on the monomer. The radio receptor assay in KG-1 cells, where the dimer was used as a competitor, showed that the dimer is 30x times more efficient than the monomer.

Varying the length of the spacers is another preferred method of forming dimers, trimers and tetramers (generally referred to in the art as diabodies, triabodies, and tetrabodies, respectively). Dimers are formed under conditions in which the separator that binds the two variable chains of a scFv is generally shortened. This separator prevents the two variable chains of the same molecule from folding into a functional Fv domain. Instead, domains are forced to pair with complementary domains of another molecule to create two binding domains. In a preferred method, a separator of only 5 amino acids (Gly <; Being) was used for the construction of diabodies. This dimer can be formed from two identical scFvs, or from two different scFv populations and maintain the improved selective and / or specific binding activity of the parental scFv and / or show increased binding strength or affinity. Similarly, triabodies are formed under conditions in which the separator that binds the two variable chains of a scFv is shortened to generally less than 5 amino acid residues, preventing the two variable chains of the same molecule from folding into a domain of Functional Fv. In contrast, three separate scFv molecules associate to form a trimer. In a preferred method, triabodies were obtained by removing this flexible separator completely. The triabody can be formed from three identical scFvs, or from two or three different scFv populations and maintain the selective and / or specific binding activity of the parental scFv, and / or show increased binding strength or affinity.The tetrabodies are formed in a similar manner under conditions in which the separator that binds the two variable chains of a scFv shortens to generally less than 5 amino acid residues, preventing the two variable chains of the same molecule from bending in a domain of Functional Fv. In contrast, four separate scFv molecules associate to form a tetramer. The tetrabody can be formed from four identical scFvs, or from 1-4 individual units from different scFv populations and must maintain the enhanced selective and / or specific binding activity of the parental scFv and / or show increased resistance or binding affinity.

If triabodies or tetrabodies are formed under conditions in which the spacer is generally less than 5 amino acid residues in length, it depends on the amino acid sequence of the particular scFv under the conditions of mixing and reaction.

In a preferred method, the tetramers are formed through an association of biotin / streptavidin. A novel fermentation construct was created which is capable of being enzymatically labeled with biotin (referred to herein as Yl-biomarker or Yl-B). A sequence that is a substrate for the BirA enzyme was added in the C-terminus of Yl. The BirA enzyme adds a biotin to the lysine residue within the sequence. The Yl-biomarker was cloned and expressed in E. coli. The inclusion body material was isolated and bent again. The purity of the folded protein was again > 95% and were obtained > 100 mg of a 1 L culture (small scale, non-optimized conditions). It was found that the molecular weight of this form was similar to that of scFv according to HPLC, SDS-PAGE, and mass spectroscopy. It was found that Yl-biomarker is the most consistent reagent for FACS analysis. However, when the binding of Yl-biomarker to KG-1 cells was examined in the presence of serum, high concentrations (10 times more) were required for comparable binding in the absence of serum. However, this construction offered the advantage of a specific biotinylation in which the binding site of the molecule remains intact. In addition, each molecule is marked by only one biotin, each molecule receives a biotin at the carboxyl end.

Limiting the labeling to a biotin / molecule in a desired location allowed the production of tetramers with idina strepta. The tetramers were formed by incubating Yl-B with streptavidin-PE.

The FACS analysis indicated that the tetramers manufactured by Yl-biomarker and streptavidin-PE were 100 to 1,000 times more sensitive than the monomers of Yl scFv in the absence of serum. The tetramers of Yl-biomarker with streptatividin-PE appear to bind specifically to one of the cell lines reactive with Yl (KG-1). The differential of the reaction, from the binding antecedent, was very high, and offered high sensitivity to detect low amounts of receptor. The FACS evaluation of normal whole blood with Yl-SAV tetramers indicated that no highly reactive population is present. Monocytes and granulocytes were positive to a large extent. In cell lines where a positive result was present, such as KG-1 cells, the tetramers were at least 100 times more reactive. Therefore, the tetramers were incubated with the cell samples. A low dose of Yl tetramers (5 ng) binds well to the cell line (KG-1) that provides a response 10 to 20 times higher than those previously observed with other forms of Yl antibodies. A minor reaction was observed when a negative cell line was examined with varying doses of the tetramers.

One embodiment of the invention provides a method for identifying a target molecule that binds to cross-linked immunoreactive binding sites unknown in first and second cells comprising (a) one or more steps of biowashing performed in a first target cell which, in a second state but not in a first state, exhibits or substantially presents a binding site comprising an unknown ligand so as to produce a first population of recognition molecules; (b) subsequent steps of biowashing and / or selection, beginning with the resulting existence of recognition molecules from step (a), which is performed in a second cell that has a binding site comprising an unknown ligand that has cross immunoreactivity to the unknown ligand of the first cell in order to produce a second population of recognition molecules; (c) amplification and purification of the second population of recognition molecules of step (b); and (d) construction from the recognition sites of the purified recognition molecules of step (c), of peptides or polypeptides comprising target molecules that are selective and / or specific for unknown ligands in the second cell.

A preferred embodiment provides that the first cell is a normal cell, the first state is a non-activated state and the second state is an activated, excited, modified, changed or disturbed state. In a more preferred embodiment the second cell is a diseased cell. In an even more preferred embodiment the diseased cell is a cancer cell. The cancer cell may be, but not limited to, carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma, and melanoma. In an even more preferred embodiment, the cancer cell is a leukemia cell. In a more preferred embodiment the leukemia cell is an AML cell.

An embodiment of the present invention provides the use of the peptide or polypeptide optionally in association with or bound, coupled, combined, connected or fused with a pharmaceutical agent, in the manufacture of a medicament. In a preferred embodiment the medicament has activity against a diseased cell. In an even more preferred embodiment, the activity is against a cancer cell. The cancer cell can be in non-exhaustive form carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma, and melanoma. In a still more preferred embodiment the cancer cell is a leukemia cell. In the most preferred embodiment the leukemia cell is an AML cell.

One embodiment of the invention provides a pharmaceutical composition comprising mixtures of different monomeric scFvs, and / or mixtures of diabodies or triabodies or tetrabodies constructed from different scFvs.

Another embodiment provides for the use of the peptide or polypeptide of the invention, in association with, or bound, coupled, combined, connected or fused with a pharmaceutical agent, in the manufacture of a medicament. The drug may have activity against diseased cells, and more specifically against cancer cells. Cancer cells may be, but not limited to, carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma, and melanoma. In a more preferred embodiment, the medicament is active against leukemia cells. In the most preferred embodiment the medicament is active against AML cells. The activity of the drug against these cells can cause the cancerous growth retardation, the complete prevention of any growth, or the elimination of cancer cells. In one embodiment of the invention, the activity of the medicament or the pharmaceutical composition is inhibiting cell growth.

The peptide or polypeptide of the invention can be used to prepare a composition, preferably a pharmaceutical composition, for use in inhibiting the growth of a cancer cell, preferably a leukemia cell and more preferably an AML cell. In one embodiment of the invention, the peptide or polypeptide can be used to prepare a composition for use in inhibiting the growth of a cancer cell, said composition comprising at least one compound having a selective and / or cell-specific pharmaceutical ligand. cancerous A peptide or polypeptide of the present invention can be administered alone to a patient, or as one comprising a medicament or a pharmaceutical composition, in association with, conjugated, connected, or fused with a pharmaceutically effective amount of a pharmaceutical agent, a carrier pharmaceutically effective and optionally, an adjuvant. Such pharmaceutical compositions can include proteins, diluents, preservatives and antioxidants (see Osol et al (editors), Remington Pharmaceutical Science (16th edition), Mack Publishing Company, (1980)).

In another embodiment, the pharmaceutical agent is an antibody or a fragment thereof that is connected to a peptide or polypeptide of the invention by a peptide bond.

In a preferred embodiment, the toxin is, for example, gelonin, Pseudomonas exotoxin (PE), PE40, PE38, diphtheria toxin, ricin, or modifications or derivatives thereof.

In a preferred embodiment, the radioisotopes used include gamma radiation emitters, positron emitters, and X-ray emitters that can be used for localization and / or therapy, and beta radiation emitters and alpha radiation emitters that can be used for therapy.

In a specific embodiment of the present invention, the therapeutic radioisotope is selected from the group comprising mindium, indiumium, 99m, 10renium, 101renium, 99mtecnetium, 1ntelurium, 122mtelurium, 125mtelurium, 165thulium, 167thulium, 168thulium, 123yode, 126yode, 131yode, 133yode, 81mkrypton, 33xenone, 90yrium, 213 bismuth, bromine, fluorine, ruthenium, ruthenium, ruthenium, ruthenium, mercury, mercury, gallium, and the like In another specific embodiment of the present invention, the anticancer agent is selected from the group consisting of It includes doxorubicin (adriamycin), morpholinodoxorubicin, methoxymorpholinyldoxorubicin, cis-platinum, taxol, calicheamicin, vincristine, cytarabine (Ara-C), cyclophosphamide, prednisone, daunorubicin, morpholinodaunorubicin, methoxymorpholinyldaunorubicin, idarubicin, chlorambucil, interferon alfa, hydroxyurea, temozolomide, thalidomide and bleomycin, and derivatives and combinations thereof.

An embodiment of the invention provides a method for inhibiting the growth of a cancer cell comprising contacting the cancer cell with an amount of the peptide or polypeptide of the invention. In a preferred embodiment, the cancer cells can be non-exhaustive, carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma, and melanoma. In a preferred embodiment, the cancer cell is a leukemia cell. In the most preferred embodiment the leukemia cell is an AML cell. An embodiment of the invention allows in vivo and ex vivo treatment of the patient. A more specific embodiment of the invention allows the ex vivo purging of the autogenous bone marrow to remove abnormal stem cells.

In a more specific embodiment of the invention, the blood of a patient with leukemia can be circulated ex vivo through a system comprising a peptide or polypeptide of the invention conjugated to an anticancer agent. After the removal of the bound cells and the bound anticancer agent, the blood cells can be reintroduced into the body of a patient. Alternatively, the blood of a patient with leukemia can be circulated ex vivo through a system comprising a peptide or polypeptide of the invention bound to a solid phase. Cells that pass through the system and that do not bind to the peptide or polypeptide of the invention bound to a solid phase can be re-introduced into the body of a patient.

In another preferred embodiment of the invention, the peptide or polypeptide is used for autogenous bone marrow ex vivo in suspension to remove abnormal stem cells before implantation. The purging of the abnormal stem cells can be done by passing the suspension on a solid support (such as, but not limited to, magnetic beads and affinity columns) to which the peptide or polypeptide of the invention binds (ie, the target molecule). ), constructions, fragments, fragments of constructions, or constructions of fragments of them. The bone marrow thus purged ex vivo can then be used for the transplantation of autogenous bone marrow. This preferred embodiment is based on the identification in the present invention of a phagemid clone (Yl) that binds to stem cells released from the bone marrow of patients with leukemia, but does not bind to stem cells released from the bone marrow of healthy donors. Similarly, the phagemid clone of Yl binds to blasto cells that the FACS analysis determined to be abnormal, as well as to leukemia cells.

Blasto cells defined as primary cells that are precursors of all circulating cells in the mammalian organism. Due to their parent characteristics, the blasto cells are not circulating in significant amounts in the adult organism. The presence of circulating blasto cells without exogenous stimulation may be an indication of malignancy, eg. of the hematopoietic system, and its subsequent disappearance may indicate the remission of the malignant disease.

In another embodiment of the invention, the pharmaceutical composition is used for prophylaxis.

In a preferred embodiment, two or more peptides or polypeptides of the invention are combined to form a mixture.

As used herein, a mixture is defined as two or more molecules or particles of different species that are combined in a single preparation. The different species of molecules do not form chemical bonds neither covalent nor non-covalent.

In one embodiment of the present invention, the peptide or polypeptide of the present invention is connected, fused or conjugated with a pharmaceutical agent.

In another embodiment of the present invention, the connection between the peptide and the pharmaceutical agent is a direct connection. As used herein, a direct connection between two or more neighboring molecules is obtained through a chemical bond between elements or groups of elements of 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 linkages can be selected in non-exhaustive form from a group comprising amine, carboxy, amide, hydroxyl, peptide and disulfide. The direct connection may preferably be a resistant protease bond.

In yet another embodiment, the connection between the peptide and the pharmaceutical agent is affected by a linker compound. As used herein in the specification and claims, a linker compound is defined as a compound that joins two or more groups together. The connector can be straight or branched chain. The branched linker compound may be composed of a double branch, a triple branch, or a four or more branching compound. The connecting compound can be, but not limited to, a dicarboxylic acid, a malemido hydrazide, PDPH, a hydrazide of carboxylic acid, and a small peptide. Examples of other linking compounds include: dicarboxylic acids such as succinic acid, glutaric acid, and adipic acid; maleimide hydrazides such as N- [α -maleimidocaproic acid] hydrazide, 4- [N-maleimidomethyl] cyclohexane-1-carboxylhydrazide, and N- [6-maleimidoundecanoic acid] hydraydide PDPH-linker such as (3- [2-pyridyl thio] ] propionyl hydrazide) conjugated to reactive sulfurhydryl protein; carboxylic acid hydrazides selected from 2-5 carbon atoms; and direct coupling using small peptide linkers between the free sugar of, for example, the anti-cancer drug doxorubicin and a scFv. Small peptides include, but are not limited to, AU1, AU5, BTag, c-myc, FLAG, Glu-Glu, HA, His6, HSV, HTTPHH, IRS, KT3, Protein C, S-TAG®, T7, V5, VSV-G and KAK marker.

Any known method for the administration of a peptide or polypeptide of the present invention can be used, such as: intravenous, intramuscular, subcutaneous, topical, intratracheal, intrathecal, intraperitoneal, intralymphatic, nasal, sublingual, oral, rectal, vaginal, respiratory, buccal, intradermal, transdermal or intrapleural.

For intravenous administration, the formulation is preferably prepared such that the amount administered to the patient is an effective amount of 0.1 mg to 1.000 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 over a wide range of dosages, and depend on factors such as the details of the disease to be treated, the half-life of the pharmaceutical composition based on peptides or polypeptides in the patient's body, the physical characteristics and chemical agents of the pharmaceutical agent and the pharmaceutical composition, the form of administration of the pharmaceutical composition, details of the patient to be treated or diagnosed, as well as other parameters considered important by the attending physician.

The pharmaceutical composition for oral administration may be in the form of a tablet, liquid, emulsion, suspension, syrup, pill, capsule. The pharmaceutical composition can also be administered in an apparatus. The pharmaceutical composition for topical administration may be in the form of a cream, ointment, lotion, patch, solution, suspension or gel.

In addition, the pharmaceutical composition can be prepared for the solid, liquid or sustained release formulation.

The compositions comprising the antibody fragments produced in accordance with the invention may comprise pharmaceutically acceptable diluents or carriers. The tablets, pills, capsules may include conventional excipients such as lactose, starch and magnesium stearate. Suppositories can include excipients such as waxes and glycerol. Injectable solutions comprise sterile pyrogen-free media such as saline, and may include buffering agents, stabilizing agents or preservatives. Conventional enteric coatings can also be used.

The present invention also encompasses a method of producing the antibody fragment by synthetic means known in the art.

An embodiment of the invention comprises a pharmaceutical composition comprising at least one peptide or polypeptide of the invention, attached, coupled, combined, connected or fused with an imaging agent for use in the diagnosis and / or image location of a tumor .

Another embodiment of the invention provides a diagnostic kit for the in vitro analysis of treatment efficacy before, during or after treatment, comprising an imaging agent comprising a peptide of the invention connected to a marker molecule. The invention also provides a method of using the imaging agent for the diagnosis and / or imaging of a cancer, more specifically, a tumor, comprising the following steps: a) putting the cells in contact with the composition, b ) measure the radioactivity bound to the cells, and c) visualize the tumor.

In a preferred embodiment of the invention, the kit imaging agent is a fluorescent dye and the kit provides analysis of the treatment efficacy of cancers, more specifically blood-related cancers, e.g. 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, eg. when diagnosing it, during treatment, during remission and during relapse. The invention further provides a composition comprising an effective amount of an imaging agent, the peptide of the invention and a physiologically acceptable carrier.

In a preferred embodiment, the indicator marker molecule is any marker known in the art, including, but not limited to, a radioactive isotope, an element that is opaque to X-rays, a paramagnetic ion, or a fluorescent molecule, and Similar.

In a specific embodiment of the present invention, the radioactive indicator isotope can be, in non-exhaustive form, luindium, indium, 9-meroium, 105-rhenium, 101-rhenium, 99-methyl-ether, 121-methyl, 122, ntelurium, 12-naphthyl, 165-thulium, 167-thulium, 168-thulium, Iodine, Iodine, Iodine, Iodine, 81mkrypton, "xenon, 90ithium, bismuth, 77bromo, 18fluor, 95rutenium, 9rutenium, 103rutenium, * 3rutennium, 107mercury, 20mercury, 67galio, and 68galio.

According to another preferred embodiment, the indicator marker molecule is a fluorescent marker molecule. According to a more preferred embodiment the fluorescent marker molecule is fluorescein, phycoerythrin, or rhodamine, or modifications or conjugates thereof.

The present invention also provides a composition comprising an effective amount of an imaging agent of the invention, a pharmaceutical agent connected thereto, and a physiologically acceptable carrier.

The invention also provides a method for producing images of an organ or cells which consists in placing the organ or the cells in contact with an imaging agent of the invention under conditions such that the imaging agent binds to the organ and to the cells, producing images of the united image agent and, thus, produce images of the organ or cells.

The present invention further provides a method for treating an organ in vivo which consists in placing the organ to be treated in contact with a composition of the invention under conditions such that the composition binds to the organ, thereby treating the organ.

In a preferred embodiment of the invention, the peptide or polypeptide can be used to target malignant cells, more particularly, leukemia cells in whole blood, monitoring and imaging cells, e.g. by means of FACS analysis. Specimens that receive higher scores (eg four times higher) for tumor cells relative to normal cells are subject to treatment.

The invention provides the treatment of a patient suffering from a cancer, comprising administering to the patient an amount of the peptide or polypeptide of the invention effective to treat cancer. In a preferred embodiment, the cancer is selected from the group comprising carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma, and melanoma. In a preferred embodiment the cancer is a leukemia and in the most preferred embodiment the leukemia is AML.

In the most preferred embodiment the polypeptide peptide of the invention binds specifically or selectively to AML cells. The invention provides a ligand presented in AML cells linked to the peptide or polypeptide of the invention, and further provides a peptide or polypeptide that binds said ligand.

The novel antibody fragments of the present invention or their corresponding peptidomimetics are used in the manufacture of compositions or medicaments for treating different diseases and conditions.

The present invention provides a method for the production of a targeting agent comprising the following steps: a) isolating and selecting one or more target molecules comprising a main recognition site by a process of biowashing directly in a target cell or by a process of indirectly washing in a first target cell in a second but not in a first state and subsequently by a process of biofilling directly in a second target cell to produce one or more of said target molecules; b) amplification, purification and identification of one or more target molecules; and c) construction of an agent directed to a target from one or more target molecules or recognition sites thereof where the targeting agent can be a peptide, polypeptide, antibody or antibody fragment or a multimer thereof.

The targeting agent can also be constructed so that it is coupled, combined, linked or fused with or in association with a pharmaceutical agent.

In a preferred embodiment of the invention the targeting agent is an anti-disease or anti-cancer agent.

In another preferred embodiment of the invention the pharmaceutical agent is selected from the group comprising radioisotope, toxin, oligonucleotide, recombinant protein, antibody fragment and anti-cancer agent. The radioisotope can be selected from the group consisting of inindium, indium, 99mg, 10nrn, 101renium, "" technetium, 121mtelurium, "12turium, 125mtelurium, 165thulium, 167thulium, 168thulium, 123iodo, 126yodo, 1yodo, 133yodo, 81mkrypton, 33xenon, 90iterium, Bismuth, bromine, fluorine, ruthenium, ruthenium, ruthenium, ruthenium, mercury, mercury, gallium, and gallium.

In yet another embodiment the toxin can be selected from the group comprising gelonin, Pseudomonas exotoxin (PE), PE40, PE38, diphtheria toxin, ricin or modifications or derivatives thereof.

In yet another embodiment of the invention the anticancer agent is selected from the group comprising doxorubicin (adriamycin), morpholinodoxorubicin, methoxymorpholydindoxdoxubicin, cis-platinum, taxol, calicheamicin, vincristine, cytarabine (Ara-C), cyclophosphamide, prednisone, daunorubicin, morpholinodaunorubicin, methoximor folidinyldaunorubicin, idarubicin, fludarabine, chlorambucil, interferon alfa, hydroxyurea, temozolomide, thalidomide and bleomycin, and derivatives thereof.

The present invention provides a method for the identification of antibody fragments by: (a) biolavado which consists in incubating a phage display library with cells obtained from the blood; (b) washing to remove unbound phages; (c) eluting the bound phage of the blood cells; (d) amplification of the resulting bound phage; and (e) determining the sequence of the peptide presented from the bound phage so as to identify the peptide.

The present invention provides a peptide or polypeptide having the formula or structure: A - X - B Where X is a hypervariable CDR3 region of 3 to 30 amino acids; and A and B can each be amino acid chains of 1 to 1,000 amino acids in length, wherein A is the amino terminus and B is the carboxy terminus.

In a preferred embodiment of the invention A is 150-250 amino acid residues and B is 350-500 amino acid residues.

In another preferred embodiment the CDR3 region of the peptide is 5-13 amino acid residues.

In another preferred embodiment X in the foregoing formula is an amino acid sequence selected from the group consisting of SEQ ID N °: 8-2.

In another embodiment of the invention, the peptide or polypeptide is a part of a larger or complete antibody or a multimer.

In yet another embodiment a dimeric molecule comprises two peptides or polypeptides, one of which is the peptide or polypeptide of the invention. The dimeric molecule can comprise two identical peptides or polypeptides of the invention.

In a preferred embodiment of the invention X is an amino acid sequence selected from the group consisting of SEQ ID NOS: 8-24 in said dimeric molecule.

Another embodiment provides a nucleic acid molecule that encodes the peptide or polypeptide or the dimeric molecule of the invention.

The invention provides for the use of the peptide or polypeptide, optionally in association with or bound, coupled, combined, connected, fused with a pharmaceutical agent, in the manufacture of a medicament.

The invention further provides for the use of the peptide or polypeptide in the manufacture of a medicament having activity against a diseased cell, more specifically a cancer cell. The cancer cell can be selected from the group comprising carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma, and melanoma. More specifically, the cancer cell can be a leukemia cell and more specifically, the leukemia cell can be an AML cell.

An interchangeable system, defined in the present invention and discussed below in the examples, is a nucleic acid construct that is designed to allow the exchange or replacement of a redefined variable region within said construct, without the need for further manipulation or reconstruction of the molecule. Such a system allows rapid and convenient preparation of the desired nucleic acid molecule.

EXAMPLES The following examples are set forth to assist in understanding the invention but are not intended and should not be construed as limiting in any way their scope. Although specific reagents and reaction conditions are described, modifications can be made that are encompassed by the scope of the invention. The following examples, accordingly, are provided to further illustrate the invention.

EXAMPLE 1 : 1. Preparation of cells, bacterial strains, scFv phage display library, cell membranes and protein purification for the biowashing process: 1. 1 Preparation of leukemia cells. Blood samples were obtained from patients with leukemia. Mononuclear cells (primary cells) were separated from the other blood cells in a Ficoll cushion (Iso-prep, Robbins Scientific Corp., Sunnyvale, CA, United States). The centrifugation was performed at 110 x g for 25 minutes. The cells at the interface were harvested and washed twice in PBS. Cells were suspended in RPMI + 10% fetal calf serum (FCS) and enumerated. During prolonged storage, 10% FCS and 10% DMSO were added to the lymphocytes which were then frozen at -70 ° C. 1. 2 Preparation of fixed platelets. A platelet concentrate obtained from a blood bank was incubated for 1 hour at 37 ° C.

An equal volume of 2.0% paraformaldehyde was added and the platelets were fixed for 18 hours at 40 ° C. The platelets were washed twice with cold saline (centrifugation for 10 minutes, at 2500 x g), resuspended in 0.01% HEPES in saline, and counted using a mycroscope.

The sensitivity of platelets to the von Willebrand factor of plasma and ristocetin was verified. Plasma von Willebrand factor (vWF; 18 μg / ml) and ristocetin (0.6 mg / ml) were added to the fixed platelets, and platelet aggregation was induced and monitored by a chronological lumi-aggregometer. 1. TG-1 and HB2151 of Bacterial Strains: New bacterial cultures were prepared for the infection by growing the cells at Aeoo of 0.5-0.9 (cells growing exponentially). E.coli TG-1 cells were used for the propagation of phages and HB2151 cells of E. coli for the production of scFv protein. 1. 4 Source of the scFv presentation phage library. The scFv library (Nissim et al, EMBO J, 13, 692-698 (1994) was provided by Dr. A. Nissim with the agreement of the MRC.The library was originally constructed as a phagemid library that presents fragments of scFv in which the VH and VL domains were linked by a flexible polypeptide The scFv presented in the phagemid library were fused to the N terminus of the pIII pIII minor coat protein, which was then subcloned into the vector pHENl (Nissim et al, EMBO J, 13, 692-698 (1994).) The repertoires of antibody fragments were first generated by PCR from rearranged V genes from peripheral blood lymphocytes from unimmunised humans (referred to as "naive repertoires"). In the repertoire, random nucleotide sequences encoding heavy chain CDR3 lengths of 4-12 residues were introduced into a bank of 49 segments of cloned human VH genes.V fragment fused in all clones are derived from a single non-mutated V gene from the IGLV3S1 germline, creating a single container library of approximately 109 clones. 1. 5. Preparation of membrane from A L cells. To the pellet containing 108 washed cells, 1 ml of cold lysis solution (0.3 M sucrose, 5 mM EDTA, 1 mM PMSF) was added, then centrifuged for 20 minutes at 11,000 x g at 4 ° C. The supernatant fluid was discarded, and the pellet was suspended again in TE (10 mM Tris, 1 mM EDTA, 1 mM PMSF) and centrifuged as before. The final pellet was suspended again in 6 ml of PBS at an A28o of 0.4 and was used to coat 3 MaxiSorp immuno-tubes (NUNC), at 37 ° C, for 2 hours. After coating, the tubes were rinsed 3 times with PBS, then blocked with MPBS (2% skim milk in PBS), at room temperature, for 2 hours. Before the bio-washing, the tubes were rinsed three more times with PBS.

EXAMPLE 2: 2. Handling of phagomid particles: biowashing procedure 2. 1. Selection and amplification of phagemids: Phagemids expressing epitopes of specific interest were selected from the library by a four-step biolamping process: a) Binding of phagemid particles to a target, more particularly binding of phagemid particles to washed white cells or cell membranes b) Removal of phagomid particles that did not join, more particularly removal by extensive washing c) Elution of phagemid particles that were joined d) Propagation and manipulation of eluted phagemid particles, more particularly propagation and amplification in E. coli. 2. 2 Clone Identification: The four step biowashing procedure was repeated 3 to 5 times in general. The selected phagemid clones were propagated individually, and were further characterized by: a) DNA sequence b) Ex vivo comparison of phage binding to several cell types c) HB2151 infection of E. coli to produce soluble scFv. 2. 3 Sequence Analysis: 800 bp-encoded scFv DNA within the phagemid particles was amplified by PCR using an upstream primer No. 203743 (5 '-GAATACCTATTGCCTACGG) and a downstream primer No. 181390 (5' -TGAATTTTCTGTATGAGG). The DNA fragments were fully sequenced from both ends with an automatic ABI PRISM DNA sequencer (Genetic Analyzer 310, Perkin Elmer) using the ABI PRISM Grand Dye Finishing Cycle sequencing kit and the preceding primers. Two additional primers, initiator No. 1911811 (5 '-CGTCCGCCACCGCCAGAG) and its complementary primer No. 191344 (5'-CTCTGGCGGTGGCGATCG), which are located in the flexible polypeptide linkage region between the heavy and light chains, were used.

EXAMPLE 3: 3. Biowaste protocols 3. 1 Basic biowashing protocols: The biowashing process is an integral part of the phage display technology described above. Three biowashing protocols were developed and used in the present work:. a) AM protocol (membrane washing of AML cells / bacterial elution, followed by washing of whole AML cells / elution with trypsin). b) YPR protocol (clean human platelet wash / acid elution) c) YPNR protocol (washing of fixed human platelets / acid elution) These protocols are described in detail below: 3.1.1 AM Protocol 3. 1.1.1 Prewash: Aliquots of 1 ml containing 2x10 'AML cells frozen from patients, stored at -70 ° C, rapidly melted at 37 ° C and were immediately diluted in 10 ml of PBS-Milk (MPBS) at 2%. The cells were centrifuged 5 'at 1120 x g at room temperature (RT), washed twice, resuspended in MPBS and counted with a hemocytometer. The cell membranes were prepared as described in Section 1.5. 3. 1.1.2 The selection was carried out on membranes of immobilized AML cells by adding 2 ml of MPBS containing 10l2 phagemid from the original Nissim library. The tube was stirred slowly for 30 minutes, then incubated for an additional 90 minutes without agitation, both steps at room temperature. After three months of washing on the membranes of AML cells, a wash lap was carried out in whole AML cells. 3. 1.1.3 Washing: To remove excess phagomids that did not bind, the contents of the tube were decanted and the tube was washed 10 times with PBS, 0.1% Tween, followed by 10 washes with PBS only. 3. 1.1.4 Elution: E.coli TG-1 cells growing exponentially (2 ml) were added directly to the tube and incubated with slow agitation at 37 ° C for 30 minutes. As before, an aliquot was placed on a plate for titration and the remaining volume was placed on plates for amplification. 3. 1.1.5 Amplification: The colonies of the large plates were scraped and placed in tanks. An aliquot (107) of E.coli TG-1 cells resistant to ampicillin were grown in a? ß? of 0.5, then they were infected with helper phage (VSC-M13, Stratagene) to produce a large amplified phagemid material. The phagomids were rescued by a PEG precipitation process (18a). The amplified 6MI IT material (1011 phagemid / ml) was used for subsequent washings. The selection procedure was repeated for two additional rounds, using 10n phagomids of the previously amplified material. The amplified material of the third washing procedure in immobilized membranes was called T16M3. 3. 1.1.6 Whole Cell Relaxation: The amplified material from the third membrane wash, T16M3, was used to wash intact AML cells. The selection was carried out in a final volume of 0.5 ml of MPBS containing 2xl07 cells and 1010 Colony Forming Units (CFU) of phagemid (Nissim library) and 1013 wild type M13 bacteriophages., with slow stirring for 2 hours at 4 ° C. The bound phagomids were eluted from the pellet of cells washed with 5 μ? of Trypsin: EDTA (0.25%: 0.05%), then neutralized by adding 50 μ? of FCS. For titration and amplification, 1 ml of E.coli TG-1 culture (A6oo = 0.5) was used. The amplified and final material was named T16M3.1. 3. 1.2 YPR Protocol 3. 1.2.1 Selection: The selection of clones was accompanied by the washing of 108 fixed human platelets with 1011 phagemid (Nissim library) in 1 ml of PBS / HEPES / 1% BSA buffer. The union was allowed to advance for one hour at room temperature while mixing the mixture by rotation. 3. 1.2.2 Cell Washing: The platelets were washed five times by low speed centrifugation (3,500 x g) and suspended again as before. 3. 1.2.3 Elution: The phagemids joined in the first round were eluted from fixed platelets by the acid elution technique: The platelets were incubated for 10 minutes at room temperature with 200 μ? of 0.1 M glycine (pH 2.2). After neutralization with 0.5 M Tris-HCl, at pH 8.0 and centrifugation, the phage bound to the remaining platelets was eluted by adding 200 μ? of trypsin-EDTA (0.25% / 0.05%) and neutralization by adding 50 μ? of FCS. The cells were removed by centrifugation, and supernatant fluids containing eluted phage, from the acid elution and trypsin protocols, were collected and designated YPR (a) -l and YPR (T) -1 materials, respectively. These materials were then amplified by adding 1 ml of TG-1 cells that grow exponentially for 30 minutes at 37 ° C. An aliquot was placed on a plate for titration, and the remaining infected E. coli cells were plated on 2 x TV / 15 cm AMP. The plates were incubated overnight at 30 ° C. The result after each wash lap was determined by counting the colonies on a titration plate. 3. 1.2.4 Amplification: The clones were amplified as described in section 3.1.1.5. The 1012 phagemid / ml amplified materials of the acid elution and trypsin protocols, designated materials R1 (a) and R1 (t), respectively, were combined and used for the subsequent washings. 3. 1.2.5 The second and third wash laps were carried out as described for the first wash turn of the YPR procedure with the following modifications: (i) For the second wash, 1012 of Rl (a), combined with 1012 of Rl (t) and (ii) the elution was carried out with glycine (pH 2.2) only. The amplified eluate of the second round was designated R2. (iii) For the third round of biowashing, 1012 of R2 were used, and the elution was carried out as in the second round. The amplified material of round three was named R3. 3. 1.3 YPNR Protocol 3. 1.3.1 The biowashing and washing were carried out essentially as described in the YPR protocol. However, in this protocol, (i) the elution was carried out after each of the three laps of glycine washing (pH 2.2) and (ii) the first washing and amplification were followed by two laps of washing later without amplification. The first, second and third rounds were designated YPNR1, YPNR2 and YPNR3, respectively. 3. 2 Selection of negative control scFv clones 3. 2.1 Sequence of N14 CDR3: For all binding experiments, a single clone was collected from the naive library (before selection). A phage material and a soluble scFv, designated N14, were prepared from this clone. The analysis of the sequence indicates that it belongs to the VH4-DP65 gene family. The sequence of the VH-CDR3 limera encoded by this clone, designated N14 CDR3, is as follows (SEQ ID N °: 28: Phe Leu Thr Tyr Asn Ser Tyr Glu Val Pro Thr 3. 2.2 Sequence of C181 CDR3: An additional negative clone, C181, was used in the binding analysis experiments. Clone C181 (recombinant hepatitis B virus reagent [HBV]) belongs to the VH3-DP35 family, and the sequence of the 9mera VH-CDR3 encoded by this clone, designated C181 CDR3, is as follows (SEQ ID No. 29): Thr Asn Trp Tyr Leu Arg Pro Leu Asn EXAMPLE 4: 4. Production, purification, labeling and characterization of scFv clones 4. 1 Production of soluble scFv: pHENl, a vector used to construct the original phagemid library, was designed with an amber stop codon encoded in the joint of the scFv gene and the pIII gene. Consequently, when the vectors of selected clones are introduced by infection of the phagemid in HB2151 of E. coli, which is a non-suppressor strain, this system allows the production and secretion of soluble scFv in the bacterial periplasm (Harrison et al., Methods de Enzimologia, 267, 83-109 (1996)). The scFv is then easily recoverable from the culture broth. Low soluble scFvs are produced in control of the lacZ promoter (Gilbert and Muller-Hill, PNAS (United States), 58, 2415 (1967)), which is induced with IPTG.

A sequence encoding the c-myc marker (10 amino acids -Glu Gln Lys Leu lie Ser Glu Glu Asp Leu; SEQ ID No.123) is contained in the vector ascending to the amber mutation. The C-terminus of the expressed scFv must carry the c-myc marker, which can be detected using mouse anti-myc marker antibodies (obtained from hybridoma 9E10 of the European Collection of Cell Cultures (ECACC)). 4. 2 Purification of scFv in an affinity column of Protein A beads: The scFv of selected clones and the control clone C181 all belong to the VH3 family, which allows purification on an affinity column of Protein A. The peroplasmic fractions (100-250 ml) of induced cultures of each clone were prepared and incubated with Sepharose beads of Protein A. The scFv bound were recovered from the column by acid eluate (0.1 M glycine, pH 3.0), followed by neutralization of the eluate with Tris, at pH 8.0. The concentration of the recovered protein was determined by measuring? 2? > followed by the exchange of PBS buffer by dialysis or on a Sepharose G-25 column. 4. 3 Purification of N14-scFv on a Sephacryl S-200 column: The scFv of negative clone N14 belongs to the family of VH4 genes and can not, therefore, be purified in an affinity column of Protein A. For the purification of scFv-N14, total protein was precipitated in the periplasmic fraction of a culture induced 200 ml with 60% ammonium sulfate. The pellet was resuspended in 2 ml of 0, lxPBS, 5 mM EDTA, 5 mM PMSF and loaded onto a Sephacryl S-200 column (1.5 x 90 cm) pre-equilibrated with current buffer (0.1 x PBS) , 5 mM EDTA). The proteins were fractionated and the fractions containing the N14-scFv (detected by SDS-PAGE and immunoblot analysis) were deposited, lyophilized, and suspended in 1/10 volume of H20. The N14-scFv (unlabeled and labeled with FITC) was then used as the negative control in FACS analysis experiments. 4. 4 Marking of scFv purified with FITC: Approximately one milligram of scFv purified from each preparation was resuspended in PBS and coupled to FITC using a commercial Fluoro FITC Marker conjugation kit (Sigma category N ° FITC-1), according to the manufacturer's instructions. 4. 5 Quality Analysis of purified and labeled scFv 4. 5.1 After purification and labeling with FITC, the profile of each preparation (labeled and unlabeled) was analyzed by SDS-PAGE, immunoblot, HPLC using a Superdex-75 column (A280 and A495) and fluorometry. The analyzes indicated an 80% purity of the N14 scFv, and a 90% purity for the VH3 clones, with approximately 2 FITC molecules conjugated to each scFv molecule (F / P ratio of 2: 1). 4. 5.2 Union activity after the mark with FITC was evaluated to verify the maintenance of the specificity of scFv (see Example 5). 4. 6 Biochemical characterization of phagemid clones: Various types of analysis were used to evaluate the structure and estimate the purity of different scFv preparations (see Example 8) including SDS-PAGE, mass spectroscopy (for scFv of Yl and Y17 only) and HPLC. Immunoblot analysis and EIA were used to identify the scFv; and FACS was used to characterize the binding of scFv.

EXAMPLE 5: 5. Union tests The binding of the selected clones to the cells was evaluated at two levels, the phagemid level and the level of soluble scFv. 5. 1 Union at the fagomido level To this end, a phagomid material was prepared individually from each of the selected clones. 5. 1.1 Colony test: In a set of experiments, a mixture of 109 specific phagemid, obtained from the biowashing protocol, carrying infected E. coli resistant to ampicillin, and 1011 wild type M13 phages, which do not carry resistance to ampicillin and serve As a "blocker", it was incubated with 105 cells, chosen from a panel of cell types. After incubation and washing, the bound phages were eluted with trypsin, and an aliquot was used to infect TG-1 of E. coli. The E. coli were then placed in 2xTY / AMP plates and incubated overnight at 30 ° C. The number of colonies obtained for each clone was calculated and compared. The results give a magnitude of the affinity and specificity of the phagomids. 5. 1.2 White / Blue Colony Assay: In this assay, in which each experiment includes an internal control, the specific phagomid was mixed in the same relation as in Section 5.1.1 above ie 1/100, with another control phagemid named pGEM7 (Promega Corp., Madison, Wisconsin, United States). This phagemid pGEM7 transports resistance to ampicillin; however, it does not express any recombinant polypeptide at the N-terminus of this pIII gene. After infection of TG-1 and incubation in ampicillin plates containing 1 M X-gal, the colonies were enumerated. The obtained colonies containing pGEM7 are blue, while the colonies obtained from specific phagemid are white. The enrichment factor was then calculated, derived from the input / output ratio of the white / blue colonies (which grew on the same plate) for each test tube. 5. 1.3 EIA of phagomids 5. 1.3.1 Phagomid binding to selected cells: Approximately 5xl05 of the selected cells were fixed with acetone: methanol (1: 1) on the surface of 24-well plates. The binding assay required 109 phagemid. Binding was carried out at 37 ° C for 1 hour, followed by extensive washing with PBS / Tween (0.05%). After extensive washing with PBS, the plates were incubated with rabbit anti-M13, anti-rabbit IgG-HRP and substrate. The intensity of the color produced was read by the ELISA plate reader, at A405, and was proportional to the level of bound phagomids. 5. 1.3.2 Phagemid binding to fixed platelets: Polystyrene microtitre plates were coated with 108 fixed platelets and incubated overnight at 4 ° C. Approximately 101C phagomids were used to evaluate the binding. The washing and incubation of the plates and determination of the level of binding was carried out as described in 5.1.3.1 above. 5. 1.4 Specific protein binding assays were performed, selected from the group consisting of human growth hormone (hGH), fibrinogen, fibronectin, BSA, SM (skimmed milk), and glycocalycin (proteolytic fragment of GPIb). The union was tested in the following manner. The polystyrene microtiter plate receptacles were coated with one of the proteins to be assayed, at 2 μg / well. The coating was allowed to advance during the overnight incubation at 4 ° C. Approximately 101C phagomids were added to the binding under test. After extensive washing with PBS, the plates were incubated with anti-rabbit M13, BRP anti-rabbit, and substrate. The level of the union was measured by the intensity of the color produced. The optical density was measured at? 405. Each sample was tested in duplicate and the average was calculated. 5. 2 Binding assays at the scFv level: The binding of the scFv produced in the periplasm of HB2151 was compared in several cell types by two different assays, by EIA and by FACS analysis. 5. 2.1 EIA of soluble scFv: Approximately 5xl05 AML cells were incubated with 5-10 μg of total protein. The binding was carried out at 4 ° C for 1 hour, followed by EIA, using anti-mouse myc antibodies, anti-mouse HRP, and a substrate. Unbound antibodies were removed after each step by washing cells three times with PBS. The intensity of the color produced is read with an ELISA plate reader (O.D. 05) · As before, the intensity of the color is proportional to the level of binding. 5. 2.2 FACS analysis of the cells 5. 2.2.1 Analysis of stained cells by a "three step staining" procedure: The FACS analysis was performed to test and confirm the specificity of the selected clones. Initially, a "three step staining" procedure was established, using crude extracts or purified unlabeled scFv, followed by mouse anti-myc antibodies and, finally, anti-mouse antibodies conjugated with FITC or PE.

The FACS analysis requires 5-8x105 cells, which have been purified with Ficoll and suspended again in PBS + 1% BSA. The union was carried out for 1 hour at 4 ° C. After each step, the beads were washed and suspended again in PBS + 1% BSA. After the final staining step, the cells were fixed by suspending them again in PBS, 1% BSA, 2% formaldehyde, then they were read with FACS (Becton-Dickinson). 5. 2.2.2 Staining of cells with scFv labeled with FITC, in a single staining step: scFv labeled with FITC was incubated with 5-8x10 cells purified with Ficoll in PBS + 1% BSA. The union was carried out for 1 hour at 4 ° C. The cells were then washed and fixed as in section 5.2.2.1 above and read with FACS.

EXAMPLE 6: Results of Washing and Sequence Formation 6. 1 Results of the AM Protocol 6. 1.1 Results of the Washing for the AM Protocol: The estimated number of phagemids used for washing (entry), and the estimated number of bound phagemids eluted in the AM protocol (output) is summarized in the following table (Table 1):.

Table 1. Wash results obtained from the AM Protocol Note the enrichment in the yield (output) obtained with each successive wash. In addition, there is no drop in the exit when T16; 3 was used to wash whole AML cells, which suggests that phagemid bound aon may be specific for components on the outer cell surface or that this specific system may contain a relatively high number of non-specific united phagomids. 6. 1.2 Results of Clone Sequences for the AM Protocol: Although clones were collected and sequences were formed from T16M1, T16M2 and T16M3 output materials, the results presented below are mainly from those clones that derived from the T16M3.1 output material (washing of intact AML cells). Clones AM10, AM11 and AM12 were identified in the material T16M3, but not in the posterior output.

The amino acid sequences presented in the VH-CDR3 and their frequency at the exit of the clones tested are summarized in Table 2.

Table 2. Clones selected after the AM biowashing protocol, from the outputs T16M3 and T16M3.1 Clone Size Sequence of V "-CDR3 Line Frequency Frequency No. of VH- Germinal in output on CDR3 of T16M3 of T16M3.1 AM1 8 Pro Trp Asp Asp Val Thr VK3-DP47 5/31 8/51 Pro Pro 1 2 3 4 5 6 7 8 AM2 12 Gly Phe Pro Arg lie VH3-DP46 11/31 20/511 Thr Pro Pro Ser Wing Glu lie 1 2 3 4 5 6 7 8 9 10 11 12 AM3 5 Gly Phe Pro Met Pro VH3-DP46 1/31 2/51 1 2 3 4 5 AM6 10 Gly Phe Pro His Ser VH3-DP46 4/31 6/51 Ser Ser Val Ser Arg 1 2 3 4 5 6 7 8 9 10 AM7 11 Arg Phe Pro Met VH3-DP46 3/31 4/51 Arg His Glu Lys Thr Asn Tyr 1 2 3 4 5 6 7 8 9 10 11 AMS 8 Arg Phe Pro Pro Thr VH3-DP46 6/31 8/51 Wing Thr lie 1 2 3 4 5 6 7 8 AM9 7 Thr Gin A g Arg VH3-DP46 0/31 2/51 Asp Leu Gly 1 2 3 4 5 6 7 AM10 11 Lys Phe Pro Gly VH3-DP46 0/31 11/31 Gly Thr Val Arg Gly Leu Lys 1 2 3 4 5 6 7 8 9 10 11 AM11 12 Gly Phe Pro Val Lie VH3-DP46 0/31 1/31 Val Glu Gln Arg Gin Ser Thr 1 2 3 4 5 6 7 8 9 10 11 12 AM12 10 Arg Phe Pro Gin VH3-DP46 0/31 1/31 Arg Val Asp Asn Arg Val 1 2 3 4 5 6 7 8 9 10 The amino acid sequence of Arg / GiyPhePro is present in seven of ten isolated clones presented in Table 2, and represents a motif there. In addition, note that the identified motif represents N-terminus amino acids of the CDR3 region in each case. Accordingly, this motif can be an anchor or effective binding site alone or in combination with other amino acid residues that extend beyond one or both ends of the CDR3 region or as part of a larger peptide or polypeptide or Fv molecule. .

Other CDR3 regions with high affinity for binding to AML cells can be constructed based on the central sequence Arg / ciyPhePro. They can be constructed by varying any of the 5-12mers by additions, deletions or mutations, keeping the Arg / GiyPhePro central sequence.

The CDR3 regions of the invention have the amino acid sequence Rl-Arg / GiyPhePro-R2, wherein Rl comprises 0-15 amino acids, preferably 0-9, more preferably 0-1 amino acid and R2 comprises an amino acid sequence of 0-15 amino acids, more preferably 1-9 amino acids. R1 and R2 are amino acid sequences that do not adversely affect the specific binding of the Arg / GiyPhePro sequence to AML cells.

The CDR3 region of the light chain of the preceding clones is identical to that cited in SEQ ID No. 125. 6. 2 Results in the YPR and YPNR Protocols 6. 2.1 Washing Results for the YPR and YPNR Protocols: The estimated number of phagomids used to wash (inlet) and the estimated number of bound phagemids eluted (output) are summarized in the following tables (Tables 3, 4).

Table 3. Washing results obtained from the YPR protocol Table 3 demonstrates that elution with trypsin gives a 4 times higher output compared to acid elution in the first round.

The rewet according to the YPNR protocol without the amplification step minimized the possibility of preferential amplification of the phagemid infection or bacterial infection. The resulting output is illustrated in Table 4.

Table . Washing results obtained from the YPNR protocol As expected, the results presented in Table 4 show a reduction in phage yield after each wash lap. This protocol was used to prevent the deviation due to the amplification of non-specific phages. 6. 2.2 Results of Clone Sequences for the YPR and YPNR protocols: They were selected! several clones of the third wash of both protocols to form sequences. The amino acid sequences presented in Table 5 are those of the CDR3 regions of the heavy chain (VH-CDR3). The germline and the frequency with which the sequences appeared at the output of R3 are also indicated in this table.

Table 5. Clones of the Y series selected after the YPR biowashing protocol with the R3 output Clone Size Sequence of VH-CDR3 Line Frequency No. of VK- Germinal CDR3 Yl 6 Met Arg Ala Pro VM-DP32 14/30 Val lie 1 2 3 4 5 6 8 Y16 6 Thr Gly Gln Ser VH3-DP26 1/30 lie Lys Arg Ser 1 2 3 4 5 6 7 8 Y17 6 Leu Thr His Pro VH3-DP32 7/30 Tyr Phe 1 2 3 4 5 6 Y-27 6 Leu Arg Pro Pro VH3-DP32 2/30 Glu Ser 1 2 3 4 5 6 Y-44 111 Thr Ser Lys Asn Thr VH3-DP32 2/30 Ser Ser Ser Lys Arg His 1 2 3 4 5 6 7 8 9 10 11? -45 12 Arg Tyr Tyr Cys Arg VH3-DP49 11/30 Ser Ser Asp Cys Thr Val Ser 1 2 3 4 5 6 7 8 9 10 11 12? -52 10 Phe Arg Arg Met VH3-DP49 1/30 Gln Thr Val Pro Wing Pro 1 2 3 4 5 6 7 8 9 10 Most of the clones isolated from the YPNR protocol were also Yl.

The CDR3 region of the light chain of the preceding clones is identical to that cited in SEQ ID No. 1125.

EXAMPLE 7: 7. Results of the Union Evaluation 7. 1 Binding of selected phagemid clones to AML cells (series of AM clones): The binding assay to evaluate phagemid binding to cells, the White / Blue colony assay described in Example 5, were performed with the AM clones. . With the exception of clone AM7, no preferential binding was detected to the cells tested. Significant but non-selective binding of the AM7 clone to the target cells was observed, such as a phagemid or as a purified scFv. The results show no enrichment for the AM clone series. 7. 2 Union of Clone Series Y 7. 2.1 EIA of Phagemid Unions using Fixed Platelets: After three laps of washing using two different protocols, the phage clones were tested by EIA for binding to fixed platelets. The phagemid material was prepared from each of the selected clones, and these clones were tested in two EIA groups. Each sample was tested in duplicate, and the average was calculated. The results are summarized in Figure 1 and indicate that six of the nine series of Y clones show a positive reaction to EIA. The highest level of binding was associated with clones Yl, Y16, Y17, and Y27. The phage (wild type bacteriophage) and E6 (selected in CLL leukemia cells) materials were used as negative controls. The dominant clone, phage Yl, showed the highest binding to fixed platelets and, together with Y17, showed a significantly higher binding than the P13 or E6 phage clones.

EXAMPLE 8: 8. Detailed characterization of scFv and union of clones 8. 1 Structure and identification of scFv: The native structure of Y-l was evaluated by HPLC analysis with a Superdex 75 column and by mass spectroscopy. The results of the first method indicate the presence of monomers, dimers, and tetramers in the preparation. The mass spectroscopy was sufficiently sensitive to identify the expected molecular weight of 26.5 kD and, in cases where the c-myc marker broke, a molecular weight of 24 kD was obtained.

The results of SDS-PAGE, however, indicate that the unbroken, intact molecule has an apparent molecular weight of 30 kD, despite the expected molecular weight being 26.5 kD, in accordance with the acid sequence nucleic acids and with the results of the preceding mass spectroscopy. Immunoblot analysis using specific c-myc antibodies confirmed the results of 30 kD of SDS-PAGE and supported the deduction that the c-myc marker is present at the end of the intact molecule. The discrepancy between the results of the two procedures is due to the level of precision of the methods as well as to the conditions of performance of SDS-PAGE that can alter the apparent molecular weight of the protein tested. 8. 2 Binding of selected platelets-clones to leukemic cells: As indicated in the introduction, markers on the surface of platelet cells can be expressed in premature hematopoietic cells. The binding of selected platelets-clones was tested by FACS analysis. The FACS analysis was performed after spotting whole blood, followed by lysis, or in mononuclear cells purified with Iso-prep (Ficoll cushion). The scFvs were prepared from each clone, purified in Protein A, and labeled with FITC (as described in Sections 4.1-4.4). To allow the production of intact scFv in the non-suppressive E. coli strain HB2151, the amber codon (TAG) found in the V "-CDR3 of clone Y27 was ruted by site-directed mutagenesis of the DNA to encode glutaric acid (GAG ). The target cells for each study were cells isolated from new blood samples from different patients with leukemia. Samples were obtained from three Medical Centers in Israel.

Clones Yl and Y17 showed a preferential binding to the leukemia cells tested while all the Y-series clones gave binding at background levels only. Table 6 presents the binding of Yl and Y17 labeled with FITC to a variety of leukemic cells.

Table 6. Specificity of Yl binding for leukemia cells B Cell Lineage * Undetermined.

The results, presented as fractions in Table 6, represent the fraction of patients, whose cells the FACS analysis identified that reacted positively with each antibody tested. The numerator represents the number of positive patients, the denominator represents the total number of patients tested for a combination of scFv / cell type. Y-17 bound strongly to all the cells tested; this union was considered non-selective. However, the binding of Yl was considered highly selective for several specimens of leukemic cells, especially those in the acute phase. The binding of Yl-scFv was further analyzed as described below.

The representative results of the binding of Yl to three samples of AML are presented in Figure 3. In each case, a high proportion of the cell population is fluorescent at an intensity significantly higher than the background fluorescence obtained by staining with the scFv negative control. These results indicate that, for each patient, Yl binds to a different fraction of the total cell population. The peak of Yl on the right in each graph is thought to represent the maximum number of cells that bind Yl in the population, the proportion of total cells that fall below this peak most likely represents the minimum proportion of cells that join Yl in each sample. 8. 3 Y-l binding to normal blood cells: The binding of Yl to purified normal blood cells was analyzed according to different types of blood cells. Although no binding to normal lymphocytes was detected, Yl bound to purified monocytes by Ficoll of 9/28 subjects, to platelets of 5/8 subjects, and to red blood cells (RCB) of 1/4 subjects. However, the CD14-specific antibodies bound to cells in all monocyte preparations and in many of the neutrophil preparations. A summary of this analysis is presented in Table 7.

Table 7. FACS analysis of the binding of scFv to normal blood cells purified by Ficoll Antibody Lymphocytes Monocytes Neutrophile Platelets Red blood cells N14 0/18 0/4 0/3 0/4 Yl 0/28 9/28 0/4 5/8 1/4 CD14 0/15 14/14 8 / 14 0/5 0/4 These junction results represent the fraction of normal blood samples that the FACS analysis identified as having reacted positively with each antibody tested. Note that, although it was selected on fixed platelets, FITC-Y1 scFv shows relatively low affinity to platelets. Figure 4 demonstrates the binding of Yl to platelets purified by Ficoll (4a) and cells closed with monocytes (4b). The change in the population of monocyte cells is greater than that observed in platelets, with a mean fluorescence calculated 30 times and 5 times higher, respectively, than the negative control. This observation is most likely due to the characteristic of platelets of adhering in multiples to monocytes purified by Ficoll. Subsequent experiments showed that, when tested in the whole blood samples, no binding to Yl was observed in any of the normal monocytes, granulocytes, platelets or red blood cells tested. Similarly, no binding of Yl to platelets was observed when obtained from platelet-rich plasma (PR?). Under the same binding conditions (in whole blood, followed by lysis of red blood cells with FACS lysis solution [Becton Dickenson]), Yl bound to leukemia cells in a manner similar to that obtained after purification by Ficoll. . Consequently, we conclude that, under natural conditions, the Y1 epitope in platelets or monocytes is hidden. During the Ficoll purification procedure the epitope is exposed, making it accessible for recognition by Yl, while for the leukemic cells the epitope is expressed under purified and unpurified conditions.

In addition to the progenitors of normal hematopoietic cells of the lymphatic and myeloid lineages, the binding of Yl to hematopoietic stem cells (CD34 + cells) in marrow blood was tested. Figure 5 depicts the results of the binding of scFv clones labeled with FITC to cord blood stem CD34 + cells; Figure 5a shows the results of the binding of CD34 + closed cells to the negative control scFv labeled by FITC and Figure 5b presents the same analysis for the binding of closed CD34 + cells to the scFv clone labeled by FITC. Figure 5c presents a graph plot analysis of FSC and SSC points from the same sample of Yl scFv clone labeled with FITC as in 5b. The results of this analysis indicated the presence of two sub-populations of DC34 + stem cells derived from the marrow blood, the differences in forward diffusion (FSC) are an indication of the size of the cells. Yl binds to the smaller size cells of the two populations. The areas enclosed in circles in Figures 5b and 5c delineate the sub-population of CD34 + cells that bind to the scFv of Yl clone. Another analysis indicated that the smaller cells are dead cells that are present in the cell population, and the binding of Y1 may possibly indicate the presence of an intracellular ligand recognized by Y1.

The experiment was performed on peripheral blood cells of healthy donors pretreated with GM-CSF (the GM-CSF treatment also mobilizes the release of stem cells into the bloodstream). Results similar to those presented in Figure 5 were obtained. 8. 4 Specification of Yl scFv binding compared to different cell markers in AML cells: Staining of Yl from peripheral cells purified by Ficoll and bone marrow cells from patients with AML was compared to staining of those cells by a panel of other antibodies. The results of such FACS analyzes, for samples obtained from 14 patients, are summarized in Table 8. Note that there is significant variability in the frequency of stained cells in preparations of different individuals for all of the markers tested, including Yl. The lack of correlation between the binding of different markers and that of Yl suggests that Yl does not bind to any of the ligands that are bound by the other markers tested, and that the ligand of Yl does not constitute any of the cell surface markers tested.

Table 8. Comparison of Yl scFv binding to antibody binding to different cell markers ** BM / PB: bone marrow / peripheral blood Results are expressed as the percentage of cells in samples purified by Ficoll from a given patient, that the FACS analysis identified that they reacted positively with each individual antibody.

In light of the concentration of Yl (1 μ? / 5? 105) required for the binding dection, the results indicate that Yl scFv has a relatively high binding affinity to the specific ligand of AML cells.

In addition to the results presented in Taba 8, which show the binding of Yl to AML cells, we have shown previously (Table 6) that Yl can also bind to most of the other types of leukemia cells tested, including cells of B-ALL, although the sample size for these other leukemia specimens was limited. Figure 6 presents a FACS analysis of the binding of Yl scFv to pre-B-ALL cells obtained from two patients. A double spotting procedure was used, using CD 19 labeled with PE (a marker for normal peripheral B cells; Figure 6a, 6c) or a CD 34 labeled with PE (a marker for stem cells; Figure 6d), together with a scFv negative control labeled with FITC or a Yl scFv labeled with FITC. Figure 6b is a double negative control. The intensity of the fluorescence (X axis) of the cells bound by the FITC-labeled sample (Yl clone of scFv) is presented, in relation to the staining pattern of the negative control (6e and 6f). The results of Figure 6 demonstrate that most of the leukaemic pre-B-ALL cells within each of the two samples tested are positive for staining with Y1 cells due to the binding of Y-1. 8. 5 Union of Yl-scFv to Cell Lines: Several lines of cells derived from malignant hematopoietic lineages were evaluated for their ability to be recognized by Yl. In analysis FACS indicates that Yl binds to many of the cells tested (Table 9). Note that only one line of human B cells and one line of mouse myeloid cells were tested. Fundamentally, this union was restricted to cells that grow exponentially. Cells in the stationary phase generally did not bind Yl, indicating that the expression of the Yl ligand is regulated during the life cycle of the cells. In addition, resistance to binding differs between the cells that react. This observation implies that there are differences in the levels of expression or in the affinity of the ligand in different cells.

Table 9. Union of Yl to Lines of Hematopoietic Cells 8. 6 Union of Yl purified in DTT presence: Once the Yl clone was selected, the process for producing the scFv was further developed. The results of the FTLC analysis of the lots of Yl indicated that the protein can be multimerized mainly with the formation of monomers and tetramers, the relationship between the two forms differs from one preparation to the next. To obtain a homogeneous material, 5 mM DTT was added during affinity purification on the Protein A Sepharose column followed by removal by changing PBS buffer. In fact, after treatment with DTT, most (> 90%) of the material was found in the monomeric fraction. No significant difference was found between the binding of the monomeric form of Yl (purified in the presence of DTT and analyzed on HPLC) and the binding of the mixture of Yl forms. 8. 7 Yl is a specific clone for leukemia cells: The cassette of Yl belongs to the germline VH-DP32. Several other clones, originating from the same germline, were isolated and detailed in Example 6. These clones include Y17, Y-27 and Y-44. The primary sequences (ie the germline cassette) of all these clones differ in their CDR3 regions only. However, only Yll shows selectivity for leukemic cells. The CD3 sequences of these clones are summarized in Table 10, and the binding profiles of the clones are summarized in Table 11.

Table 10: The CDR3 sequence of the isolated clones of VH3-DP32 Clone N ° Sequence of VH-CDR3 Germinal Line Yl Met Arg Ali Pro Val lie V; .: 3-DP32 1 2 3 4 5 6 Y17 Leu Thr His Pro Tyr Phe VH3-DP32 1 2 3 4 5 6 Y-27 Leu Arg Pro Pro Glu Ser VH3-DP32 1 2 3 4 5 6? -44 Thr Ser Lys Asn Thr Ser VH3-DP32 Ser Ser Lys Arg His 1 2 3 4 5 6 7 8 9 10 11 Table 11: Binding profile of isolated clones of VH3-DP32 Clone No. Specificity of binding Yl It binds to many leukemia cells Y17 It binds to all the hematopoietic cells tested, including normal lymphocytes Y-27 Does not bind to any of the hemoatopoietic cells tested Y- 44 Does not bind to any of the Hematopoietic cells tested Tables 10 and 11 indicate that, although the primary sequences are identical among the four clones with the exception of the VK-CDR3 region, the binding profiles differ significantly from one clone to another. This observation reinforces the concept that the sequence of the VH-CDR3 region plays an important role in the specificity of the antigen binding site. Note that neither the length of the CDR3 sequence nor the gerrnfiry cassette in which it is placed appears to be the main determinant of the binding specificity. Y17 and Y-27 each comprise a CDR3 6mera, as well as Y1, and the heavy chains of the three clones are obtained from the identical germline. In the case of Y17 and Y-27, selective binding to hematopoietic cells has not been demonstrated.

EXAMPLE 9: 9. 1 Construction of triabodies: The vector pHEN-Yl, which encodes the original Yl, was amplified using PCR for the VL and VH regions, individually. The sense oligonucleotide 5'-AACTCGAGTGAGCTACACAGGACCCT, and the antisense oligonucleotide 5 '-TTTGTCGACTCATTTCTTTTTTGCGGCCGCACC were used for the VL PCR reaction. The cDNA product of the expected size of 350 bp was purified, sequenced and digested with the restriction enzymes Xhol and Notl.

The same procedure was used to amplify the VH region (using the 5 'sense oligonucleotide -ATGAAATACCTATTGCCTACGG and the antisense oligonucleotide 5' -AACTCGAGACGGTGACCAGGGTACC). The VH PCR product was digested with the restriction enzymes Ncol and Xhol. A triple ligation procedure was used in the vector pHEN, predigested with Ncol-Notl. The final vector was named pTria-Yl.

After transformation of E.coli, several clones were collected for other analyzes, which included DNA sequence, protein expression, and extraction of bacteria from the periplasmic space. SDS-PAGE under reduced conditions and immunoblot analysis were performed to confirm the size of Yl triabodies. 9. 2 Diabody construction The pTria-Yl vector above was made linear with the restriction enzyme Xhol, and synthetic complementary double-stranded oligonucleotides (5 '-TCGAAGGTGGAGGCGGT and 5' TCGAACCGCCTCCACCTC) were prefixed and ligated into the Xhol site, between Yl heavy chains and Yl light chains. This new vector was called pDia-Yl. As described for triabodies, the DNA sequence and protein expression was confirmed. 9. 3 Expression and purification of diabodies and triabodies The expression of E.coli was essentially as described above for scFv-Yl. However, the purification of diabodies and triabodies of Y1 from the periplasm of the transformed E. coli cells was different. The monomeric form of scFv Yl can be purified on an affinity column of Protein A Sepharose beads. The multimeric forms of Yl, however, they are inefficiently purified by this procedure. Consequently, the periplasmic proteins extracted from the bacteria were precipitated overnight with 60% ammonium sulfate, resuspended in H20, and loaded onto a size exclusion column of Sephacryl-200 (Pharmacia) pre-equilibrated with 0 , lxPBS. The fractions were collected and analyzed by HPLC, and the separate fractions containing the dimers or trimeric forms were collected for FITC labeling and FACS analysis. 9. .4 Union of diabodies and triabodies from Yl to cells The FACS analysis was performed on Jurkat cells using a "three step staining procedure". First, raw or screened unflared scFv extracts were stained, then mouse anti-myc antibodies, and finally anti-mouse antibodies conjugated to FITC and PE. The FACS analysis requires 5-8x105 cells, which have been purified with Ficoll and have been resuspended in PBS + 1% BSA. The union was carried out for 1 hour at 4 ° C. After each step, the cells were washed and resuspended in PBS + 1% BSA. After the final staining step, the cells were fixed by suspending them again in PBS, 1% BSA, 2% formaldehyde, and then read by FACS (Becton-Dickinson).

The binding of Yl-scFv was compared with that of the diabodies and triabodies. In this analysis (Figure 7), the binding profile of the three forms was very similar, which indicates that the previous modifications in the molecule did not alter, hide or destroy the apparent binding affinity of Yl to its ligand. 9. 5 Production of Yl-cys-KAK (cysteine dimer) One liter of bacterial culture of ???, - ?? - ?? ß - ??? it was incubated at 42 ° C for 2-3 hours. This culture was centrifuged at 5,000 RPM for 30 minutes. The pellet was suspended again in 180 ml of TE (50 mM Tris-HCl pH 7.4, 20 mM EDTA). 8 ml of lysozyme (from a material of 5 mg / ml) was added and incubated for 1 hour. 20 ml of NaCl were added to 5M and 25 ml of 25% Triton and incubated for another hour. This mixture was centrifuged at 13,000 RPM for 60 minutes at 4 ° C. The supernatant was discarded. The pellet was suspended again in TE with the help of a tissue (or homogenizer). This process was repeated 3-4 times until the inclusion bodies (pellet) were gray / light brown. The inclusion bodies were solubilized in 6M Guanidine-HCl, 0.1M Tris at pH 7.4-4 mM EDTA (1.5 grams of inclusion bodies in 10 ml of solubilization buffer gave 10 mg / ml soluble protein ). This was incubated for at least 4 hours. The concentration of the protein was measured and brought to a concentration of 10 mg / ml. DTT was added to a final concentration of 65 mM and incubated overnight at room temperature. The new bending was initiated by diluting 10 ml of protein (drop by drop) to a solution containing 0.5 M Arginine, 0.1 M Tris at pH 8, 2 mM EDTA, 0.9 mM GSSG. The newly bent solution was incubated at 10 ° C for 48 hours. The newly folded solution containing the protein was analyzed in a buffer containing 25 mM phosphate buffer at pH 6, 100 mM Urea and concentrated to 500 ml. The concentrated / dialyzed solution was bound to a SP-sepharose column, and the protein was eluted with a gradient of NaCl (up to 1M). 9. 6 An Affinity Study of the Y-Dimero S-S compared to CONY1 and Yl IgG, using a Radio Receptor Binding Assay (RRA) with KG-1 Cells The assay system consisted of the use of radioactive ligands that were prepared by iodination with 2jI using chloramine T in the Yl-IgG construct or the Bolton-Hunter reagent in the construction of CONY1 (the Yl scFv). The test tubes contained 5xl06 KG-1 cells per 0.2 ml and a tracer labeled with varying amounts of unlabeled competitor, in PBS + 0.1% BSA, at pH 7.4. After incubation for 1 hour with shaking at 4 ° C, the cells were completely washed with cold buffer and collected for radioactivity counting.

In the RRA study using labeled Yl-IgG, 2 ng / tube of 123I-Yl-IgG was used and competition was performed with each of the three molecules. The results are given in Figure 8. These results presented in this figure demonstrate that the affinity of the S-S dimer of Yl was 30 times higher than that of CONY1. A rough estimate of the affinity of Yl-IgG in this experiment is 2x 10 ~ 8 M. The corresponding affinity of the dimer is, accordingly, 4 x 10"8 M.

In a second RRA study using labeled CONY1, 100 ng / tube of 123I-Yl-IgG was used and the competition was performed with each of the three molecules. The results are given in Figure 9. This figure shows that the affinity of the dimer S-S was 20 times higher than that of CONY1. A rough estimate of the affinity of CONY1 in this experiment is 10 ~ 6M. The corresponding affinity of the dimer is, accordingly, 5x 10 ~ 8M. 9. 7 ELISA to GC (glycocalicin) 100 μ? of purified glycocalicin were incubated on a maxisorp plate of 96 recipes, overnight at 4 ° C. The plate was washed with PBST (PBS + 0.05% tween) 3 times, then with 200 ml of PBST-milk (PBST + 2% skim milk), for 1 hour at room temperature. The plate was washed with PBST, and the monomer or dimer (100 μ) was added in PBST-milk at different concentrations for 1 hour at room temperature. The plate was then washed and polyclonal anti-VL (obtained from rabbits immunized with V-_ obtained from Yl) (diluted 1: 100 in PBST-milk) was added for one hour. The plate was washed and anti-rabbit HRP was added for another hour. The plate was washed 5 times and 100 μ? of TMB substrate for approximately 15 minutes, then 100 μ? of 0.5 H2SO4 to stop the reaction. The optical density of the plate was measured at 450 nm in an ELISA reader. 9. 8 Manufacture of Yl tetramers A construct was designed where the following sequence, LNDI FEAQKIEWHE, was added in the C-terminus of the Yl by PCR and cloning in an IPTG-inducible expression vector cassette. The clone was called Yl-biomarker. This sequence is a substrate for the BirA enzyme, which in the presence of free biotin, the enzyme is able to covalently connect biotin to the lysine residue (K) (phenotypic analysis of antigen-specific T lymphocytes.) Science, October 4, 1996; 274 (528): 94-6, Altman JD et al). This construction was produced as inclusion bodies in BL21 bacterial cells. The new bending was performed as previously described. The inclusion bodies were solubilized in guanidine-DTT. The new fold was made by diluting it in a buffer containing arginine-Tris-EDTA. Dialysis and concentration were performed by ion exchange purification of HiTrapQ.

The purified scFv Yl-biomarker was incubated with the enzyme BirA (acquired from Avidity) and biotin according to the supplier's recommendations. The biotinylated Yl-biomarker was analyzed by the HABA assay (which estimates the amount of biotin per molecule) and showed that there was > 0.8 biotin residues / molecule.

The biotinylated Yl-biomarker was incubated with Streptavidin-PE (Phycoerythrin) to form complexes and was used in FACS experiments using KG-1 cells (positive for Yl). Streptavidin can bind up to 4 molecules of biotinylated Yl-biomarker. The sensitivity of the union increased at least 100 times due to the increase in avidity. The Yl-biomarker sequence is as follows: 1 MEVQLVESGG GVVRPGGSLR LSCAASGFTF DDYGMSWVRQ 41 APGKGLEWVS GIN WNGGSTG YADSVKGRFT ISRDNAKNSL 81 YLQMNSLRAE DTAVYYCARM RAPVIWGQGT LVTVSRGGGG 121 SGGGGSGGGG SSELTQDPAV SVALGQTVR1 TCQGDSLRS and 161 YASWYQQKPG QAPVLV1YGK NNRPSGIPDR FSGSSSGNTA 201 SLTITGAQAE DEADYYCNSR DSSGNNVVFG GGTKLTVLGG 241 GGLNDIFEAQ KIEWHE putative were synthesized, and ligated into the fixed site XhoI of the mammalian expression vector (under the SRcc.5 promoter). 5'- TCGACCTCATCACCATGGCCTGGGCTCTGCTGCTCCTCACCCTCCTCACTC AGGACACAGGGTCCTGGGCCGAT Y 5 - . 5-G ATCG ATTGCACCAGC I GGATATCGGCCCAGG ACCCTG TG I CCTG AGTGA G GAGGGTGAGGAGCAGCAGCCCAGGCCATGGTGATGAGG.

Upstream of the initiation ATG codon, two Kozak elements were included. In addition, an internal EcoRV site was introduced between the putative cleavage site of the leader sequence and the Xhol site to allow subcloning of the variable regions. This modified vector was designated pBJ-3. 10. The sequence encoding VL derived from the cDNA sequence of Yl scFv was inserted between the leader sequence and the sequence encoding the constant light region. Similarly, the sequence encoding V;; derived from the cDNA sequence of Yl scFv was inserted between the leader sequence and the sequence encoding the constant heavy region. This was achieved by PCR amplification of the vector pHEN-Yl, which encodes the original Y1, to obtain the VL and VH regions, individually. 163 EXAMPLE 10: Construction of full-size Yl-IgGl Whole IgG molecules have advantages over Fv forms, including a longer half-life in vivo and the potential to induce a cellular response in vivo, such as those mediated by ADCC or CDC (complement-dependent cytotoxicity; Tomlinson , Current Opinions on Immunology, 5, 83-89 (1993)). Using a molecular cloning approach described below, we have converted Yl Fv regions into full-sized IgGl molecules. The construction of Yl-IgGl was achieved by joining fragments of cDNAs with each other in the following order: 10. 1 A compatible leader sequence for a mammalian expression system: An interchangeable system was designed to allow convenient insertion of required elerAents for a complete IgG molecule. The following complementary double-stranded oligonucleotides encode a leader sequence 10. 3 The 5 'oligonucleotides -TTTGATATCCAGCTGGTGGAGTCTGGGGA (sense) and 5'-GCTGACCTAGGACGGTCAGCTTGGT (counter-sense) were used for the VL PCR reaction. The cDNA product of the expected size of 350 bp was purified, sequenced and digested with the restriction enzymes EcoRV and Avrll. The same procedure was used to amplify and purify the VH cDNA region, using sense and contradictory oligonucleotides 5'-GGGATATCCAGCTG (C / G) GGAGTCGGGC and 5'-GGACTCGAGACGGTGACCAGGGTACCTTG, respectively. 10. 4 Constant regions: The constant region? 3. { C-X3) and the CH1-CH3 constant heavy regions obtained for the IgGII cDNA were individually synthesized as follows: 10.4.1 For the constant CL-3 region, RT-PCR was performed on the mAR extracted from a reservoir of normal peripheral B cells (CD 19+ cells) in combination with the oligonucleotides 5'-CCGTCCTAGGTCAGCCCAAGGCTGC sense and 5'- TTTGCGGCCGCTCATGAACATTCTGTAGGGGCCACTGT contrasentido. The PCR product of the expected size (400 bp) was purified, sequenced and digested with the restriction enzymes Avrll and Notl. 10. 4.2 For Isa constant IgGl regions (? Chain), a human B cell clone (CMV clone No. 40), immortalized in BTG, was selected for PCR amplification. It was shown that this clone secretes IgGl against human CMV and was also shown to induce the ADCC response in in vitro assays. For the CH1-CH3 cDNA, the oligonucleotides 5'-CCGCTCGAGTGC (T / C) TCCACCAAGGGCCCATC (G / C) GTCTTC (sense) and 5'-TTTGCGGCCGCTCATTTACCC (A / G) GAGACAGGGAGAGGCT (contradictory) were synthesized and used for the PCR amplification. As described for the sequence encoding the CL cDNA, the PCR product of the expected size (1500 bp) was purified, sequenced, and digested with the restriction enzymes Avrll and Notl. 10. For the final expression vectors, a triple ligation procedure was carried out using the vector previously digested with EcoRV-Notl, the variable cDNAs of EcoRV-AvrlI and the constant regions of Avrll-Notl. The final vectors for heavy chain and light chain expression were designated Y-l-HC and Yl-LC, respectively. 10. 6 An additional vector, pBJ-Yl-LP, was constructed based on the Yl-LC to allow double screening based on the puromycin-resistant gene (PAC). In this vector, the puromycin-resistant gene of the Yl-LC plasmid was replaced with a 1600 bp fragment encoding the PAC gene (of the pMCC-ZP vector). 10. 7 The open reading frame (ORF) of both Y-l-IgG-HC and Yl-IgG-LC and their encoded amino acid sequences are presented below: l ORF of Yl-IgG-HC (V "CH1 CH2 CH3) ATGGCCTGGGCTCTGCTGCtCCTOACCCTCCTCACTCAGGACACAGGGTCCTGGGCCGAT M A W A L L L T L L T Q D T G S W A D ATCCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGGGGGTCCCTGAGACTCTCC I Q L V E 3 G G O V V R P G G S L L S TGTGCAGCCTCTGGATTCACCTTTGATGATTATGGCATGAGCTGGGTCCGCCAAGCTCCA C A A S G F T F D D Y G M S W V R Q A P GGGAAGGGGCTGGAGTGGGTCTCTGGTATTAATTGGAATGGTGGTAGCACAGGTTATGCA G K G L E W V S G I N N G G S T G Y A GACTCTGTGAAGGGCCGATTCACCATCTCTAGAGACAACGCCAAGAACTCCCTGTATCTG D 3 V K G R F T I 8 R D N A K H 8 L Y L CAAATGAACAGTCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCAAGAATGAGGGCT Q M N S L R A R D T A V Y Y C A R M R A CCTGTGATTTGGGGCCAAGGTACCCTGGTCACCGTCTCGAGTGCTTCCACCAAGGGCCCA P V I W G Q G T L V T V S S A S T K G P TCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGC S V F P L A P S S K S T S G G T A A L G TGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTG C L V K D Y F P E P V T V S W N S G A L ACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGC T S G V H T F P A V L Q S S G L Y S L S AGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAAT S V V T V P S S S L G T Q T Y I C N V N CACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACT H K P S N T V D K R E P K S C D K T CACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACTGTCAGTCTTCOTCTTC H T C P P C P A P E L L G G P S V F L F CCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTG 261 P P K P K D T L I S R T P E V T C V V 8 1 GTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAG 281 V D V S H E D P E V K F N W Y V D G V E 901 GTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTC 301 V H N A K T K P R E E Q Y N S T Y R V V 961 AGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTC 321 S V L T V L H Q D W L N G K E Y K C V 1021 TCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCC 341 S N K A L P A P I E K T I S K A K G Q P 1081 OGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTC 361 R E P Q V Y T L P S R E E M T K N Q V 1141 AGCCTGACCTGCCTGGTCAAAGGCTTCTATCCGAGCGACATCGCCGTGGAGTGGGAGAGC 381 S L T C L V K G F Y P S D I A V E W E S 1201 AATGGGCAGCCGGAGAACAACTACAAGACCACGTCTCCCGTGCTGGACTCCGACGGCTCC 401 N G Q P E N N Y K T T S P V L D S D G S 1261 TTCTTCCTCTATAGCAAGCTCACCGTGCACAAGAGCAGGTGGCAGCAGGGGAACGTCTTC 421 F F L Y S K L T V D K S R W Q Q G N V F 1321 TCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTG 441 S C S V M H E A L H N H Y T Q S L S L 1381 TGTCTGGGTAAATGA 461 S L G K * 0. 7.2 The ORF of Yl-IgG-LC (VL CL) 1 ATGGCCTGGGCTCTGCTGCXCCTCACCCTCCTCACTCAGGACACAGGGTCCTGGGCCGAT 1 M A W A L L L T L L T Q D T G S W A D 61 GCAGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACA 21 A E L T Q D P A V S V A L O Q T V R I T 1212 TGCCAAGGAGACAGCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAG 41 C Q 0 D 8 L S Y Y A S W Y Q Q P G Q 181 GCCCCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTC 161 A P V L V I Y G K N M P S C I P D R F 241 TCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGAT 81 8 G 3 S S G N T A 8 L T I T G A Q A E D 301 GAGGCTGACTATTACTGTAACTCCCGGGACAGCAGTGGTAACCATGTGGTATTCGGCGGA 101 K A D Y Y C N S R D S S G N H V V r G G 361 GGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCG 121 G T K L T V L G Q P K A A P S V T L F P 21 CCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTC 141 P S S E L L A N K A L V C L I S D F 481 TACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTG 161 Y P G A V T V A K A D S S P V K A G V 541 GAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGC 181 T T T P S K Q S N N K Y A S S Y L S 601 CTGACGCCTGAGCAGTGGAAGTCCCACAAAAGCTACAGCTGCCAGGTCACGCATGAAGGG 201 L T P E Q W K S H K Y Y S C Q V T H E G 661 AGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCATGA 221 S T V E K T V A P T E C S * The leader sequence is underlined. The VH and VL regions are each encoded by amino acid sequences that are in bold, followed by the sequences of the constant regions IgGl (for the heavy chain) or? 3 (for the light region). 10. 8 Expression of the heavy and light chain of Yl in CHO cells The Yl-HC and Yl-LC vectors were used individually for the transfection and selection of stable cells expressing the heavy or light chains. After selection in G418 and cell growth, the protein secreted in the supernatant was analyzed for the expression of IgGl by the capture EIA assay and by immunoblot analysis, described below. 10. 8.1 Capture EIA Assay: The receptacles of 96-well plates were precoated with mouse anti-human IgGl Fe (Sigma). The preceding supernatant was added to the receptacles, and the presence of heavy chain IgG1 was detected with anti-γ chain specific antibody. biotinylated (Sigma), streptavidin-HRP and substrate. An ELISA plate reader monitored the development of color at A0s. 10. 8.2. Immunoblot analysis: The supernatant for the preceding cells was passed in 12.5% SDS-PAGE. The expression of each chain was detected with (a) goat anti-human IgG-HRP (H + L, Sigma Category No. A8667) for the detection of heavy chains and (b) biotinylated goat anti-human chain? 3 ( Southern Biotechnology Association, Category N ° 2070-08) for the detection of light chains.

The expression of both chains was confirmed with the preceding assays, and the joint transfection was carried out to obtain full-length Yl-IgGl. 10. 9 Expression and Purification of Yl-IgG 10. 9.1 Cell Culture and Transfection: CHO cells were cultured in the F-12 medium with 10% fetal calf serum and 40 μg mi of gentamycin at 37 ° C in an atmosphere of 5% C02. One day before transfection, 0.8 x 106 cells were germinated in 90 mm dishes. The cultures were transfected together with 10 μg of light and heavy chain DNA by the FuGene transfection reagent technique (Roche). After 2 days of growth in the non-selective medium, the cells were cultured for 10-12 days in the F-12 medium containing 550 μg / ml neomycin and 3 μg / ml puromycin. The cells were trypsinized and cloned by limiting the dilution of 0.5 cells / well in plates of 96 Costar receptacles. Individual colonies were collected, grown in plates of six receptacles and transfected into flasks. 10. 9.2 Determination of heavy and light chain secretion: A sandwich ELISA was used to determine the concentration of the secreted antibody in the supernatant of transfected CHO cells. To determine the concentration of the antibody, the following reagents were used: monoclonal anti-human IgGl (Fc) (Sigma) as the coated antibody, goat anti-human IgG (specific for chain?) Conjugate and biotin as the detector (Sigma), and pure human IgGl, lambda (Sigma) as standard. Based on this ELISA assay, the production index varied between 3-4 μg ml. 10. 9.3 Production and Purification of Mab from cells: Cells were grown in roller bottles at a final concentration of l-2xl08 cells per bottle in F-12 medium with 10% fetal calf serum, with a neomycin supplement and puromycin. For production, cells were cultured in the same medium, but with 2% fetal calf serum for another two days. The secreted antibody was purified on a G-Sepharose column (Pharmacia). The binding was in 20 mM sodium phosphate buffer, pH 7.0; the elution was carried out in 0.1 M glycine buffer, pH 2.5-3.0. The amount of the purified antibody was determined by ultraviolet radiation absorbance; the purity was analyzed by SDS-PAGE. Under non-denaturing conditions the complete IgG antibody has its expected molecular weight of 160 kD. In denaturing gels both heavy and light chains have the expected molecular weight size of 55 and 28 kD, respectively. 10. 9.4 Union of the full-length Yl-IgG molecule: Binding experiments were performed to determine the level of binding of the Yl-IgG molecule compared to the level of binding of the scFv-Yl molecule. A two step staining procedure was used, where 5 ng of Yl-IgG was reacted with both RAJI cells (negative control, Figure 7a) and Jurkat cells (Yl positive cells, Figure 7b). For detection, goat anti-human IgG labeled with PE was used. Similarly, 1 g of scFv-Yl reacted with Jurkat cells (Figure 7c) and PE-labeled rabbit anti-scFv was used for detection. The results indicate that both Yl-IgG and scFv-Yl bind to Jurkat cells, with approximately 103 times more scFv-Yl molecules necessary to obtain a detection level similar to that of Yl-IgG.

Brief Description of the Tables Table 1: Wash results obtained from the AM protocol. The estimated number of phagemids used for washing (inlet), and the estimated number of bound phagemids eluted (output) are summarized for the four consecutive steps of the AM biowashing protocol. The source of cells and the elution medium are listed for each output result, as well as the term used to distinguish each separate material.

Table 2: Selected clones following the biowaste protocol A.M. The number of amino acid residues in the CDR3 region (size of VH-CDR3) and the CDR3 amino acid sequences for the different types of isolated clones are summarized. In addition, the frequency of each of the types of clones is presented in the two outputs of the AM biowashing protocol, outputs T16M3 and T16M3.1.

Table 3: Results of biowashing obtained from the YPR protocol. The estimated number of phagomids used for washing (inlet) and the estimated number of bound phagemids eluted (output) are summarized. The elution medium is listed for each output result, as well as the term used to distinguish each separate material.

Table 4: Wash results obtained from the YPNR protocol. The estimated number of phagemids for washing (inlet) and the estimated number of phagemids attached eluded (output) for the three consecutive steps of the YPNR biowashing protocol are summarized. The elution medium is listed for each output result, as well as the term used to distinguish each separate material.

Table 5: Clones of the Y series selected following the YPR protocol with the R3 output. Several different clones were identified in the output material R3. The number of amino acid residues they comprise, and the amino acid sequences of the VH-CDR3 regions of the identified clones, as well as the germline designations are detailed.

Table 6: Specificity of Yl binding for leukemia cells. The results of the binding experiments of three different scFv clones are presented, each of which reacted with mixtures of cells containing mainly each of seven different types of leukemic cells, determined by FACS analysis. The results represent the fraction of patients, whose cells the FACS analysis identified that reacted positively with each antibody tested. The numerator represents the number of positive patients, the denominator indicates the total number of patients tested for a combination of certain scFv / lucémic cell types.

Table 7: FACS analysis of the binding of scFv to normal blood cells purified with Ficoll. Three clones are analyzed individually for binding to five different types of normal blood cells purified with Ficoll. These binding results represent the fraction of normal blood samples that the FACS analysis identified that reacted positively with each antibody tested.

Table 8: Comparison of the binding of Yl scFv with the binding of antibodies to different cell markers. The results of the FACS analysis of spotting by Yl and by a panel of other antibodies are presented. Ficoll-purified peripheral and bone marrow cells from patients with ANE were prepared and the specificity of Yl scFv binding compared to different cell markers in AML cells was studied. The results are expressed as the percentage of cells in the samples purified with Ficoll from a given patient, which the FACS analysis identified that reacted positively with each Fv. Four other antibodies were compared: (1) CD13, a marker for granulocytes and monocytes; (2) CD14, a marker for monocytes and neutrophils; (3) CD33, a marker for normal myeloid cells and leukemic myeloid cells; and (4) CD34, a marker for stem cells.

Table 9: Union of Yl to lines of hematopoietic cells. The FACS analysis was performed to determine the binding of Yl scFv to three different categories of human leukemia cell lines, and to a mouse cell line. The cell lines to which Yl is positively linked (reactive) and to which it did not bind (non-reactive) are listed.

Table 10: The CDR3 sequence of isolated clones of VH3-DP32.

Following different biolaving and selection procedures, several clones based on the DP32 germline were isolated. Clones Yl, Y17, Y-27 and Y-44 were identified during the selection of platelet biowashing (YPR and YPNR protocols). The sequence of the VH-CDR3 region of each of these clones is presented.

Table 11: Binding profile of isolated clones of VH3-DP32. The specificity of the binding of clones derived from DP32 to several hematopoietic cells was tested by FACS analysis.

The invention has been described with reference to specific examples, materials and data. As one skilled in the art will appreciate, there are alternative means to use and prepare the different aspects of the invention. Such alternative means should be construed as being included within the scope and spirit of the present invention defined by the following claims.

Claims (259)

1. CLAIMS 1. A peptide or polypeptide comprising an Fv molecule, a construct thereof, a fragment of some of them, or a construction of a fragment having improved binding characteristics so that it binds selectively and / or specifically to a target cell in favor of other cells, wherein the selectivity or specificity of the binding is primarily determined by a first hypervariable region, and wherein the Fv is a scFv or a dsFv, and optionally has one or more markers. 2. A peptide or polypeptide according to claim 1, wherein the first hypervariable region is the CDR3 region having an amino acid sequence selected from the group formed by SEQ ID No. 8-23. A peptide or polypeptide according to claim 1, wherein the first hypervariable region is the CDR3 region having an amino acid sequence selected from the group formed by SEQ ID No. 8-24, and wherein the selectivity or specificity of the junction is secondarily influenced by a second hypervariable region, by a third hypervariable region, and / or by one or more ascending regions or debris flanking the first, second and / or third hypervariable regions. 4. The peptide or polypeptide according to claim 2, wherein the peptide or polypeptide is a scFv having the SEQ ID No. 25 in which the first hypervariable region is the CDR3 region that is identical to SEQ ID No. 8. 5. The peptide or polypeptide according to claim 1, wherein the scFv molecule comprises a straight or branched chain separator of 20 or less amino acid residues. 6. The peptide or polypeptide according to claim 5, wherein the separator comprises SEQ ID No. 123 or SEQ ID No. 124. 7. The peptide or polypeptide according to claim 1, wherein the target cell is an activated, excited, modified, changed, disrupted, abnormal or diseased cell. 8. The peptide or polypeptide according to claim 7, wherein the diseased cell is a cancer cell. 3 9. The peptide or polypeptide according to the rei indication 7, wherein the cell is selected from the group consisting of carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma, and melanoma cells. 10. The peptide or polypeptide according to claim 9, wherein the cell is a leukemia or myeloma cell. 11. The peptide or polypeptide according to claim 9, wherein the leukemia or myeloma cell is a malignant B cell. 12. The peptide or polypeptide according to claim 10, wherein the leukemia cell is an acute myeloid leukemia cell or a malignant B cell. 13. The peptide or polypeptide according to claim 2, further comprising a cassette of consecutive amino acids having an amino acid sequence selected from the group formed by SEQ ID NOS: 30-113, or having at least 90% similarity of amino acids therewith, or a fragment thereof, wherein the cassette or fragment provides a framework in which a CDR3 region having an amino acid sequence selected from the group consisting of the amino acid sequence is constructed, inserted, linked, coupled, combined or fused. SEQ ID N °: 8-24. 4 14. The peptide or polypeptide according to claim 13, wherein the cassette has an amino acid sequence selected from the group consisting of SEQ ID NOS: 30-32, 33, 37-39, 41, 43, 45, 46, 48, 51, 54, 57, 59-68, 70, 71, 76-85, 87, 89-92, 94, 97, 99, 103, 106, 112, and 113, or having at least 90% of similarity of amino acids with them. 15. The peptide or polypeptide according to claim 13, wherein the cassette has the amino acid sequence of the SEQ ID No. 61 or has at least 90% similarity of amino acids with it. 16. The peptide or polypeptide according to the rei indication 15, wherein the cassette has the amino acid sequence of the SEQ ID No. 61 or has at least 90% similarity of amino acids with it. 17. The peptide or polypeptide according to claim 15, wherein the amino acid residues of the carboxy terminus of the SEQ ID No. 61 are replaced by seven amino acid residues of SEQ ID No. 122. 5 18. The peptide or polypeptide according to claim 3, wherein the second and third hypervariable regions are a hypervariable region CDR2 and a CDR1, respectively. 19. The peptide or polypeptide according to claim 2, wherein the CDR3 region has the amino acid sequence SEQ ID No. 8 .. 20. The peptide or polypeptide according to the rei indication 18, wherein the CDR2 and CDR1 regions have the amino acid sequences SEQ ID No. 115 and SEQ ID No. 114, respectively. 21. The peptide or polypeptide according to claim 3, wherein the second and third hypervariable regions are a hypervariable region CDR2 and CDR1 respectively and wherein the CDR3, CDR2 and CDR1 regions have the amino acid sequences SEQ ID NO: 8, 115 and 114, respectively. 22. The peptide or polypeptide according to claim 3, wherein the ascending region flanking the CDR3 region has the amino acid sequence of SEQ ID No. 117 and wherein the descending region flanking the CDR3 region has the amino acid sequence of SEQ ID N ° 116. 6 23. The peptide or polypeptide according to claim 3, wherein the second hypervariable region is the hypervariable region CDR2 and wherein the ascending region flanking the CDR2 region has the amino acid sequence of SEQ ID No. 119, and wherein the The descending region flanking the CDR2 region has the amino acid sequence of SEQ ID No. 118. 24. The peptide or polypeptide according to claim 3, wherein the third hypervariable region is the hypervariable region CDR1 and wherein the ascending region flanking the CDR1 region has the amino acid sequence of SEQ ID No. 121, and wherein the The descending region flanking the CDR1 region has the amino acid sequence of SEQ ID No. 120. 25. The peptide or polypeptide according to claim 18, wherein the CDR2 and CDR1 regions of a consecutive amino acid cassette selected from the group consisting of SEQ ID NOS 30-113 or a fragment thereof are replaced by SEQ ID N ° : 115 and 114, respectively. 26. The peptide or polypeptide according to claim 18, wherein the CDR2 and CDR1 regions of a cassette of the consecutive amino acids selected from the group consisting of SEQ ID NOS: 30-32, 35, 37-39, 41, 43, 45, 46, 48, 51, 54, 57, 59-7 68, 70, 71, 76-85, 87, 89-92, 94, 97, 99, 103, 106, 112, 113 or a fragment thereof are replaced by SEQ ID Nos. 115 and 114, respectively. 27. The peptide or polypeptide according to claim 3, wherein: (a) the second and third hypervariable regions are the hypervariable regions CDR2 and CDR1, respectively, (b) the amino acid sequence of CDR3 is SEQ ID No.8, (c) the amino acid sequence of CDR2 is SEQ ID No. 115, (d) the amino acid sequence of CDR1 is SEQ ID No. 114, (e) the ascending region flanking the CDR3 region has the amino acid sequence of SEQ ID 117, (f) the descending region flanking the CDR3 region has the amino acid sequence of SEQ ID No. 116, (g) the ascending region flanking the CDR2 region has the amino acid sequence of SEQ ID No. 1119, (h) the descending region flanking the CDR2 region has the amino acid sequence of SEQ ID No. 118, (i) the ascending region which flanks the CDR1 region has the amino acid sequence of SEQ ID No. 121, and (j) the descending region flanking the CDR1 region has the amino acid sequence of SEQ ID No. 120. 8 28. The peptide or polypeptide according to claim 1, wherein Fv is a scFv that can be obtained from a phage display library. 29. The peptide or polypeptide according to claim 28, wherein the phage display library was constructed from peripheral blood lymphocytes of a non-immunized human, and wherein the scFv peptide is selected against antigens previously not characterized and not purified on the surface of a white cell. 30. A method for selecting or identifying the peptide or polypeptide according to claim 18, comprising biowashing, wherein the biowashing comprises joining the phage to a target, removing the unbound phage, eluting the bound phage, and propagating and amplifying the phage eluted. 31. A peptide or polypeptide comprising an Fv molecule, a construct thereof, a fragment of some of them, or a fragment construct, having improved binding characteristics so that it binds selectively and / or specifically to a binding site substantially exposed and / or overexpressed or in a target cell, where binding to the target cell occurs in favor of other cells or where the binding site is not substantially available and / or expressed, wherein the selectivity or specificity of the binding is primarily determined by a first hypervariable region, wherein the Fv is a scFv or a dsFv, and wherein the Fv optionally has one or more markers. 32. The peptide or polypeptide according to claim 31, wherein the first hypervariable region is a CDR3 region having an amino acid sequence selected from the group formed by SEQ ID No. 8-24. 33. The peptide or polypeptide according to claim 31, wherein the first hypervariable region is the CDR3 region having an amino acid sequence selected from the group formed by SEQ ID No. 8-24 and wherein the selectivity or specificity of the binding it is secondarily influenced by a second hypervariable region, by a third hypervariable region, and / or by one or more ascending or descending regions flanking the first, the second and / or the third hypervariable regions and where the second and third regions Hypervariables are a CDR2 and CDR11 region, respectively. 34. A peptide or polypeptide comprising an Fv molecule, a construction thereof, a fragment of some of them, or a construction of a fragment having improved binding characteristics such that it binds selectively and / or specifically to a target cell in favor of other cells, wherein the Fv molecule comprises a first strand having a first, second and third hypervariable regions and a second chain having a first, a second and a third hypervariable region, wherein one of the hypervariable regions of the first chain has a sequence selected from the group consisting of SEQ ID No. 8-24, and wherein one of the hypervariable regions of the second strand have a sequence selected from the group consisting of SEQ ID Nos. 1-6 and 125-202, and wherein the first, second and third hypervariable regions are a CDR3, CDR2 and CDR1 region, respectively, where Fv is a scFv or a dsFv, and where Fv optionally has one or more markers. 35. The peptide or polypeptide according to claim 34, wherein (a) the first strand and the second strand each comprise a first hypervariable region selected from the group consisting of SEQ ID N ° 8-24; or (b) the first hypervariable region of the first strand and the first hypervariable region of the second strand are identical and are selected from the group consisting of SEQ ID No. 8-24; or 11 (c) the first hypervariable region of the first chain is selected from the group consisting of SEQ ID No. 8-24, and the first hypervariable region of the second chain is selected from the group consisting of SEQ ID No. 1-6 and 125-202; or (d) the first hypervariable region of the first chain is selected from the group consisting of SEQ ID No. 1-6 and 125-202 and the first hypervariable region of the second chain is selected from the group consisting of SEQ ID No. 8-24. 36. The peptide or polypeptide according to claim 34, wherein the second and third hypervariable regions of the first strand are SEQ ID Nos. 114 and 115, respectively. 37. A peptide or polypeptide comprising an Fv molecule, a construct thereof, a fragment of any of them, or a construct of a fragment that (a) binds to an unknown ligand in a first cell having a first and second state, in where the binding is effective in the second state but is not substantially effective in the first state; and (b) by virtue of the cross immunoreactivity, binds specifically or selectively to a ligand in a second cell and wherein the Fv is a scFv or a dsFv, and wherein the Fv optionally has one or more markers. 12 38. The peptide or polypeptide according to claim 37, wherein the first cell is a normal cell. 39. The peptide or polypeptide according to claim 37, wherein the first state is a non-activated state and the second state is an activated, excited, modified, changed or disturbed state. 40. The peptide or polypeptide according to claim 37, wherein the second cell is a diseased cell. 41. The peptide or polypeptide according to claim 40, wherein the diseased cell is a cancer cell. 42. The peptide or polypeptide according to the indication 40, wherein the diseased cell is selected from the group consisting of carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma and melanoma cells. 43. The peptide or polypeptide according to claim 42, wherein the diseased cell is a leukemia cell. 13 44. The peptide or polypeptide according to claim 43, wherein the leukemia cell is an acute myeloid leukemia cell. . Five. The peptide or polypeptide according to the rei indication 37, wherein the selective and / or specific binding of the peptide or polypeptide to the ligand of the second cell is determined primarily by a first hypervariable region. 46. The peptide or polypeptide according to claim 45, wherein the first hypervariable region is a CDR3 region having an amino acid sequence selected from the group formed by SEQ ID No. 8-24. 47. The peptide or polypeptide according to claim 46, wherein the first hypervariable region is a CDR3 region having an amino acid sequence selected from the group formed by SEQ ID No. 8-24 and wherein the selectivity or specificity of the binding it is secondarily influenced by a second hypervariable region, by a third hypervariable region, and / or by one or more ascending or descending regions flanking the first, second and third hypervariable regions. 14 48. A ligand expressed by the second cell and capable of being linked by the peptide or polypeptide according to claim 37. 49. A molecule that recognizes and binds to the ligand according to claim 48. 50. A nucleic acid molecule encoding the peptide or polypeptide according to any of claims 1, 31, 34, or 37. 51. The nucleic acid molecule according to claim 50, wherein the nucleic acid is DNA. 52. The peptide or polypeptide according to claim 37, wherein the first and second states of the first cell are the same, and wherein the first cell is obtained from a cell line. 53. The peptide or polypeptide according to claim 52, wherein the cell line is selected from the group consisting of Jurkat, MOLT-4, HS-602, U937, TF-1, THP-1, KG-1, ML-2 and HUT-78. fifteen 54. A method for identifying a target molecule, which binds to cross-linked immunoreactive binding sites unknown in first and second cells, comprising (a) performing one or more biolayevates from a first target cell that, in a second state but not a first state, substantially exposes or presents a binding site comprising at least one unknown ligand, thereby producing a first population of recognition molecules; (b) carrying out subsequent biolamping and / or selection steps, starting with the first population of recognition molecules of step (a), which is carried out in a second cell that has a binding site comprising at least one unknown ligand that has cross-immunoreactivity to the unknown ligand of the first cell so as to produce a second population of re-recognition molecules; (c) amplification and purification of the second population of recognition molecules of step (b); and (d) construction from the recognition sites of the purified recognition molecules of the peptides or polypeptides of step (c) comprising target molecules that are selective and / or specific for unknown ligands in the second cell. 16 55. The method according to claim 54, wherein the first cell is a normal cell and wherein the first state is a non-activated state and the second state is an activated, excited, modified, changed or disturbed state. 56. The method according to claim 54, wherein the second cell is a diseased cell. 57. The method according to claim 56, wherein the diseased cell is a cancer cell. 58. The method according to claim 56, wherein the cell is selected from the group consisting of carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma, and melanoma cells. 59. The method according to claim 58, wherein the cell is a leukemia cell. 60. The method according to claim 59, wherein the leukemia cell is an acute myeloid leukemia cell. 61. The use of the peptide or polypeptide according to claim 1 or claim 37, optionally in 17 association with or united, coupled, combined, connected or fused with a pharmaceutical agent, in the manufacture of a medicament. 62. The use according to claim 61, wherein the medicament has activity against a diseased cell. 63. Use according to the indication 62, where the diseased cell is a cancer cell. 64. The use according to claim 62, wherein the cell is selected from the group consisting of carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma and melanoma. 65. The use according to claim 64, wherein the cell is a leukemia cell. 66. The use according to claim 65, wherein the leukemia cell is an acute myeloid leukemia cell. 67. The peptide or polypeptide according to claim 1 or rei indication 37, optionally in association with or linked, coupled, combined, connected or fused with a pharmaceutical agent, for use as a medicine. 68. The peptide or polypeptide according to claim 67, wherein the medicament has activity against a diseased cell. 69. The peptide or polypeptide according to claim 68, wherein the diseased cell is a cancer cell. 70. The peptide or polypeptide according to claim 68, wherein the cell is selected from the group consisting of carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma, and melanoma cells. 71. The peptide or polypeptide according to claim 70, wherein the cell is a leukemia cell. 72. The peptide or polypeptide according to claim 71, wherein the leukemia cell is an acute myeloid leukemia cell. 73. The use of the peptide or polypeptide according to claim 1 or claim 37, for preparing a composition for use in inhibiting the growth of a diseased or cancerous cell. 74. The use of the peptide or polypeptide according to the rei indication 73, wherein the cell is a leukemia cell. 75. The use of the peptide or polypeptide according to claim 74, wherein the leukemia cell is an acute myeloid leukemia cell. 76. The use of the peptide or polypeptide according to claim 1 or claim 37, for preparing a composition for use in the inhibition of the growth of a cancer cell, said composition comprises at least one compound having a selective pharmaceutical ligand and / or specific for the cancer cell. 77. A composition comprising at least one peptide of claim 1 or claim 37, in association with, or bound to, coupled, combined, connected, or fused to a pharmaceutical agent in a pharmaceutically effective amount and, optionally, a pharmaceutically effective carrier. twenty 78. The composition according to claim 77, wherein the peptide or polypeptide and the pharmaceutical agent are connected through a connecting compound. 79. The composition according to the rei indication 78, wherein the connecting compound is selected from the group consisting of a dicarboxylic acid, a maleimido hydrazide, PDPH, a carboxylic acid hydrazide, and a small peptide. 80. The composition according to claim 79, wherein the small peptide is selected from the group consisting of AU1, AU5, BTag, c-myc, FLAG, Glu-Glu, HA, His6, HSV, HTTPHH, IRS, KT3, Protein C , S-TAG®, T7, V5, VSV-G, and KAK-Marker. 81. The peptide or polypeptide according to any of claims 1, 31, 34 and 37, wherein the marker is selected from the group consisting of: AU1, AU5, BTag, c-myc, FLAG, Glu-Glu, HA, His6, HSV, HTTPHH, IRS, KT3, Protein C, S-TAG®, T7, V5, VSV-G, and KAK-Marker. 82. The composition according to claim 77, wherein the pharmaceutical agent is selected from the group consisting of radioisotope, toxin, oligonucleotide, recombinant protein, antibody fragment, and anti-cancer agent. twenty-one 83. The composition according to claim 82, wherein the radioisotope is selected from the group consisting of inindium, indium, 99mrenium, 105renium, 101renium, 99: ntecnetium, 121, ntelurium, 122mtelurium, 125: ntelurium, 165thulium, 167thulium,, 68thulium, IOne, Iodo, Iodo, Iododo, 81 Krypton, 33xenon, 90ithrio, 213 bismuth, "bromine, 18fluor, ruthenium, 9rutenium, ruthenium," ° Druteny, 107mercury, 20mercury, 67galio, and 6galgal. 84. The composition according to claim 82, wherein the toxin is selected from the group consisting of gelonin, Pseudomonas exotoxin (PE), PE40, PE38, diphtheria toxin, ricin, and modifications and derivatives thereof. 85. The composition according to claim 82, wherein the anticancer agent is selected from the group consisting of doxorubicin (adriamycin), morpholinodoxorubicin, methoxymorpholydinyldoxorubicin, cis-platinum, taxol, calicheamicin, vincristine, cytarabine (Ara-C), cyclophosphamide, prednisone, daunorubicin, morpholinodaunorubicin, methoximorfolidinyldaunorubicin, idarubicin, fludarabine, chlorambucil, interferon alpha, hydroxyurea, temozolomide, thalidomide and bleomycin, and derivatives thereof. \ 22 86. A method for inhibiting the growth of a cell comprising bringing the cell into contact with an amount of the peptide or polypeptide according to claim 1 or claim 37. 87. The method according to claim 86, wherein the cell is selected from the group consisting of carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma and melanoma. 88. The method according to claim 87, wherein the cell is a leukemia cell. 89. The method according to claim 88, wherein the leukemia cell is an acute myloid leukemia cell. 90. A pharmaceutical composition comprising at least one peptide according to claim 1 or claim 37, linked, coupled, combined, connected or fused with an imaging agent for use in the diagnostic localization and imaging of a tumor . 91. A method for treating a patient suffering from a disease or cancer, comprising administering to the patient an amount of The peptide or polypeptide of claim 1 or claim 37 effective to treat the disease or cancer. 92. The method according to claim 91, wherein the disease or cancer is selected from the group consisting of carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma and melanoma. 93. The method according to the rei indication 92, where the disease is a leukemia. 94. The method according to claim 93, wherein the leukemia is an acute myeloid leukemia. 95. The method according to claim 1 or claim 37, wherein Fv binds specifically or selectively to acute myeloid leukemia (AML) cells. 96. A ligand presented in the AML cells bound by the peptide or polypeptide according to claim 95. 97. A peptide or polypeptide that binds to the ligand according to the indication 96. 24 98. A diagnostic kit for in vitro analysis of treatment efficacy before, during or after treatment, comprising the peptide or polypeptide according to claim 37, linked, coupled, combined, connected or fused to an indicator marker molecule. 99. The kit of claim 98, wherein the indicator marker molecule is a fluorescent label. 100. The kit of claim 99, wherein the fluorescent label is selected from the group promoted by fluorescein, rhodamine, phycoerythrin, and modifications and conjugates thereof. 101. The kit of claim 98, wherein the kit is used for the diagnosis of a disease or cancer. 102. The peptide or polypeptide of claim 1 or claim 37, wherein the construct is a 1 g polypeptide. 103. A method for producing the peptide or polypeptide of claim 102, wherein the 1 g polypeptide is expressed as a recombinant polypeptide and is produced in a system of eukaryotic cells. 25 104. The method of claim 103, wherein the eukaryotic system is a mammalian cell system. 105. The peptide or polypeptide of claim 102, wherein the 1 g polypeptide is an IgG polypeptide. 106. The peptide or polypeptide of claim 105, wherein the IgG polypeptide comprises a CDR3, CDR2 and CDR1 region having SEQ ID Nos. 8, 115 and 114, respectively. 107. The IgG polypeptide of claim 105, wherein the CDR3, CDR2 and CDR1 regions are of the heavy chain. 108. The IgG polypeptide of claim 106, wherein the CDR3, CDR2 and CDR1 regions are of the light chain. 109. The polypeptide of claim 102, wherein the IgG has a heavy chain comprising SEQ ID No. 26 and a light chain comprising SEQ ID No. 27 or chains having at least 90% similarity thereto. 110. A method for producing the peptide or polypeptide of claim 1 or claim 37, wherein the peptide or polypeptide is produced in a system of prokaryotic cells or in a system of eukaryotic cells. 111. The method of claim 110, wherein the prokaryotic system comprises E.coli, said E.coli comprises an expression vector, and the eukaryotic system is a mammalian cell system. 112. The method of claim 111, wherein the expression vector of the prokaryotic system comprises a promoter selected from the group consisting of osmB, deo, P ~ lac-U5, PLr SRa5, and CMV. 113. A peptide or polypeptide comprising a binding motif comprising an amino acid sequence of Ri ~ X Phe Pro-R2, wherein Ri and R2, wherein each amino acid sequence comprises 0-15 amino acid residues and wherein X is Arg , Gly or Lys. 114. The peptide or polypeptide of any of claims 2, 34 or 46, wherein the CDR3 comprises the amino acid sequence of Ri-X Phe Pro-R2, wherein Ri and R2 each comprise 0-15 amino acid residues and wherein X is Arg, Gl yo Lys. 27 115. The peptide or polypeptide of claim 1 or claim 37, wherein said peptide or polypeptide includes at least one unnatural modification. 116. The peptide or polypeptide of claim 115, wherein said unnatural modification makes the peptide or polypeptide more immunogenic or more stable. 117. The peptide or polypeptide of claim 116, wherein at least one modification is selected from the group consisting of peptoid modification, semi-peptide modification, cyclic peptide modification, N-terminus modification, C-terminus modification, peptide bond modification, modification of the main chain, and modification of waste. 118. The peptide or polypeptide of any of claims 1, 31, 34, 37 or 67, for purging ex vivo from the autogenous bone marrow to remove abnormal cells. 119. A method of producing an agent targeting a target comprising the following steps: a) isolating and selecting one or more molecules comprising a recognition site by a biowashing process 28 directly in a white cell or by a process of indirectly washing in a first target cell in a second but not in a first state and subsequently by a process of directly bluing in a second target molecule to produce one or more target molecules; b) amplification, purification and identification of one or more target molecules; and c) construction of an agent targeting the target from one or more target molecules or wherein the targeting agent may be a peptide, polypeptide, antibody or antibody fragment, or a multimer thereof. 120. The method of claim 119, wherein the targeted agent targets, binds, combines, connects, fuses or is in association with a pharmaceutical agent. 121. The method of claims 119 and 120, wherein the targeting agent is an anti-disease or anti-cancer agent. 122. The method of claim 120, wherein the pharmaceutical agent is selected from the group consisting of radioisotope, toxin, oligonucleotide, recombinant protein, antibody fragment and anti-cancer agent. 29 123. The method of claim 122, wherein the radioisotope is selected from the group consisting of niindium, indium, rhenium, rhenium, 101renium, 99mtecnetium, 121mtelurium, i2n "'tellurium, 125mtelurium, 165thulium, 167thulium, 168thulium, 123yodo, Iodine, Iodine, Iodine, 81mkrypton, 33xenone, 0itium, 213 bismuth, bromine, fluorine, ruthenium, ruthenium, ruthenium, ruthenium, mercury, mercury, gallium, and gallium. 124. The method according to claim 122, wherein the toxin is selected from the group consisting of gelonin, Pseudomonas exotoxin (PE), PE40, PE38, diphtheria toxin, ricin, and modifications and derivatives thereof. 125. The method according to claim 122, wherein the anticancer agent is selected from the group consisting of doxorubicin (adriamycin), morpholinodoxorubicin, methoxymorpholydinyldoxorubicin, cis-platinum, taxol, calicheamicin, vincristine, cytarabine (Ara-C), cyclophosphamide, prednisone, daunorubicin, morpholinodaunorubicin, metoximorfolidini ldaunorubicin, idarubicin, fludarabine, chlorambucil, interferon alfa, hydroxyurea, temozolomide, thalidomide and bleomycin, and derivatives thereof. 30 126. A peptide or polypeptide having the formula or structure: A-X-B Where X is a hypervariable CDR3 region of 3 to 30 amino acids; and A and B each can be amino acid chains of 1 to 1,000 amino acids in length where A is the amino terminus and B is the carboxy terminus. 127. The peptide of claim 126, wherein A is 150-250 amino acid residues and wherein B is 350-500 amino acid residues. 128. The peptide of the rei indication 126, wherein the CDR3 region is 5-13 amino acid residues. 129. The peptide or polypeptide of claim 126, wherein X is an amino acid sequence selected from the group consisting of SEQ ID No. 8-24. 130. The peptide or polypeptide of claim 127, which is part of an antibody or a larger or complete multimer. 131. A dimeric molecule comprising two peptides or polypeptides one of which is the peptide or polypeptide of claim 126. 31 132. A dimeric molecule comprising two peptides or polypeptides of claim 126 that are identical. 133. The dimeric molecule of claim 131 or claim 132, wherein X is an amino acid sequence selected from the group consisting of SEQ ID No. 8-24. 134. A nucleic acid molecule encoding the peptide or polypeptide of claim 1126 or the dimeric molecule of claim 130. 135. The method of claim 104, wherein the mammalian cell system comprises the SRa5 promoter. 136. The method of claim 104, wherein the mammalian cell system comprises the CMV promoter. 137. A peptide or polypeptide substantially described herein. 138. A peptide or polypeptide comprising an Fv molecule, a construction thereof, a fragment of some of them, or a construction of a fragment having improved binding characteristics so that it binds selectively and / or specifically to a protein. a white cell in favor of other cells, wherein the selectivity and / or specificity of the binding is mainly determined by a first hypervariable region, wherein the first hypervariable region is a CDR3 region comprising an amino acid sequence selected from the group consisting of SEQ ID No. 8 or 20, and wherein Fv is a scFv or a dsFv, and optionally has one or more markers. 139. A peptide or polypeptide of claim 138, wherein the selectivity or specificity of the binding is secondarily influenced by a second hypervariable region, by a third hypervariable region, and / or by one or more of the ascending or descending regions flanking the first, second and / or third hypervariable regions. 140. The peptide or polypeptide of claim 138, wherein the peptide or polypeptide is a scFv having SEQ ID No. 25 wherein the first hypervariable region is a CDR3 region that is identical to SEQ ID No. 8. 141. The peptide or polypeptide of claim 138, wherein the peptide or polypeptide is a scFv having SEQ ID No. 203 wherein the first hypervariable region is a CDR3 region that is identical to SEQ ID No. 20. 33 142. The peptide or polypeptide of claim 138, wherein the scFv molecule comprises a straight or branched chain spacer of 20 or less amino acid residues. 143. The peptide or polypeptide of claim 142, wherein the separator comprises SEQ ID No. 123 or SEQ ID No. 124. 144. The peptide or polypeptide of claim 138, wherein the target cell is an activated, excited, modified, changed, disrupted, abnormal or diseased cell. 145. The peptide or polypeptide of claim 144, wherein the diseased cell is a cancer cell. 146. The peptide or polypeptide of claim 144, wherein the cell is selected from the group consisting of carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma, and melanoma cells. 147. The peptide or polypeptide of claim 146, wherein the cell is a leukemia or myeloma cell. 3. 4 148. The peptide or polypeptide of claim 146, wherein the leukemia cell or myeloma is a malignant B cell. 149. The peptide or polypeptide of rei indication 147, wherein the leukemia cell is an acute myeloid leukemia cell or a malignant B cell. 150. The peptide or polypeptide of claim 138, further comprising a cassette of consecutive amino acids having an amino acid sequence selected from the group formed by SEQ ID No. 30-113 or having at least 90% amino acid similarity thereto. , or a fragment thereof, wherein the cassette or fragment provides a framework in which a CDR3 region having an amino acid sequence selected from the group formed by SEQ ID No. is constructed, inserted, linked, coupled, combined or fused. 8 or 20 151. The peptide or polypeptide of the rei indication 150, wherein the cassette has an amino acid sequence selected from the group consisting of SEQ ID NOS: 30-32, 33, 37-39, 41, 43, 45, 46, 48, 51, 54, 57, 59-68, 70, 71, 76-85, 87, 89-92, 94, 97, 99, 103, 106, 112 and 113 or having at least 90% amino acid similarity with they. 35 152. The peptide or polypeptide of claim 150, wherein the cassette has the amino acid sequence of SEQ ID No. 61, or has at least 90% similarity of amino acids therewith. 153. The peptide or polypeptide of claim 152, wherein the cassette has the amino acid sequence of SEQ ID No. 61, or at least 90% similarity of amino acids therewith. 154. The peptide or polypeptide of claim 152, wherein the seven amino acid residues of the carboxy terminus of SEQ ID 61 are replaced by the seven amino acid residues of SEQ ID No. 122. 155. The peptide or polypeptide of claim 139, wherein the second and third hypervariable regions are a hypervariable region CDR2 and CDR1, respectively. 156. The peptide or polypeptide of claim 138, wherein the CDR3 region has the amino acid sequence SEQ ID No. 8. 157. The peptide or polypeptide of claim 138, wherein the CDR3 region has the amino acid sequence SEQ ID No. 20. 36 158. The peptide or polypeptide of claim 155, wherein the CDR2 and CDR1 regions have the amino acid sequences SEQ ID No. 115 and SEQ ID No. 114, respectively. 159. The peptide or polypeptide of claim 140, wherein the second and third hypervariable regions are a hypervariable region CDR2 and CDR1 respectively and wherein the CDR3, CDR2 and CDR1 regions have the amino acid sequences SEQ ID No. 8, 115 and 114, respectively. 160. The peptide or polypeptide of claim 140, wherein the second and third hypervariable regions are a hypervariable region CDR2 and CDR1 respectively and wherein the CDR3, CDR2 and CDR11 regions have the amino acid sequences SEQ ID No. 20, 115 and 114, respectively. 161. The peptide or polypeptide of claim 139, wherein the ascending region flanking the CDR3 region has the amino acid sequence of SEQ ID No. 117, and wherein the descending region flanking the CDR3 region has the amino acid sequence of SEQ ID N ° 116 162. The peptide or polypeptide of claim 139, wherein the second hypervariable region is a hypervariable region CDR2 37 and wherein the ascending region flanking the CDR2 region has the amino acid sequence of SEQ ID No. 119 and wherein the descending region flanking the CDR2 region has the amino acid sequence of SEQ ID No. 118. 163. The peptide or polypeptide of claim 139, wherein the hypervariable region is a hypervariable region CDR1 and wherein the region flanking the CDR1 region has the amino acid sequence of SEQ ID No. 121, and wherein the descending region flanking to the CDR1 region has the amino acid sequence of SEQ ID No. 120. 164. The peptide or polypeptide of claim 155, wherein the CDR2 and CDR1 regions of a consecutive amino acid cassette selected from the group consisting of SEQ ID No. 30-113 or a fragment thereof are replaced by SEQ ID No. 115 and 114, respectively. 165. The peptide or polypeptide of claim 155, wherein the CDR2 and CDR1 regions of a consecutive amino acid cassette selected from the group consisting of SEQ ID NOS: 30-32, 37-39, 41, 43, 45, 46, 48 , 51, 54, 57, 59, 68, 70, 71, 76-85, 87, 89-92, 94, 97, 99, 103, 106, 12, and 113 or a fragment of 38 they are replaced by SEQ ID No. 115 and 114, respectively. 166. The peptide or polypeptide of claim 139, wherein (a) the second and third hypervariable regions are a hypervariable region CDR2 and CDR1, respectively, (b) the amino acid sequence of CDR3 is SEQ ID No. 8, (c) the amino acid sequence of CDR2 is SEQ ID No. 115, (d) the amino acid sequence of CDR2 is SEQ ID No. 114, (e) the ascending region flanking the CDR3 region has the amino acid sequence of SEQ ID No. 117, (f) the descending region flanking the CDR3 region has the amino acid sequence of SEQ ID No. 116, (g) the ascending region flanking the CDR2 region has the amino acid sequence of SEQ ID No. 119, (h) the descending region flanking the region CDR2 has the amino acid sequence of SEQ ID No. 118, (i) the ascending region flanking the CDR11 region has the amino acid sequence of SEQ ID No. 121, and (j) the descending region flanking the CDR1 region. has the amino acid sequence of SEQ ID No. 120,. 39 167. The peptide or polypeptide of claim 139, wherein (a) the second and third hypervariable regions are the hypervariable regions CDR2 and CDR1, respectively, (b) the amino acid sequence of CDR3 is SEQ ID No. 20, (c) the amino acid sequence of CDR2 is SEQ ID N ° 115, (d) the amino acid sequence of CDR1 is SEQ ID No. 114, (e) the ascending region flanking the CDR3 region has the amino acid sequence of SEQ ID No. 117, (f) the descending region flanking the CDR3 region has the amino acid sequence of SEQ ID No. 116, (g) ) the ascending region flanking the CDR2 region has the amino acid sequence of SEQ ID No. 119, (h) the descending region flanking the CDR2 region has the amino acid sequence of SEQ ID No. 118, (i) the the ascending region flanking the CDR1 region has the amino acid sequence of SEQ ID No. 121, and (j) the descemdemte region flanking the CDR1 region has the amino acid sequence of SEQ ID No. 120. 168. The peptide or polypeptide of claim 138, wherein Fv is a scFv that can be obtained from a phage display library. 40 169. The peptide or polypeptide of claim 165, wherein the phage display library was constructed from peripheral blood lymphocytes of a non-immunized human, and wherein the scFv peptide is selected against antigens not characterized and not previously purified on the surface of a white cell. 170. A method for selecting or identifying the peptide or polypeptide of claim 165, comprising the biowashing, wherein the biowashing comprises binding the phage to a target, removing the unbound phage, eluting the bound phage, and propagating and amplifying the eluted phage. . 171. A peptide or polypeptide comprising an Fv molecule, a construct thereof, a fragment of some of them, or a fragment construct, having improved binding characteristics so that it binds selectively and / or specifically to a binding site substantially exposed and / or overexpressed in a white cell, wherein binding to the target cell occurs in favor of other cells where the binding site is not substantially available and / or expressed, where the selectivity or specificity of the binding is mainly determined by a first hypervariable region, where the first hypervariable region is a CDR3 region formed by the 41 SEQ ID No. 8 or 20, wherein Fv is a scFv or a dsFv, and wherein Fv optionally has one or more markers. 172. The peptide or polypeptide of claim 171, wherein the selectivity or specificity of the binding is secondarily influenced by a second hypervariable region, by a third hypervariable region and / or by one or more ascending or descending regions flanking the first, second , and / or third hypervariable regions, and wherein the second and third hypervariable regions are a CDR2 and CDR1 region, respectively. 173. A peptide or polypeptide comprising an Fv molecule, a construction thereof, a fragment of some of them, or a construction of a fragment having improved binding characteristics so that it binds selectively and / or specifically to a target cell in favor of other cells, wherein the Fv molecule comprises a first strand having a first, a second and a third hypervariable regions and a second strand having a first, a second and a third hypervariable regions, wherein the hypervariable regions of the first chain comprise a sequence of SEQ ID No. 8 or 20, and wherein one of the hypervariable regions of the second chain has a sequence selected from the group consisting of SEQ ID No. 1-6 and 125-202, and wherein the first , second, and third regions 42 hypervariables are a region CDR3, CDR2 and CDR1, respectively, where Fv is a csFv or a dsFv, and where Fv optionally has one or more markers. 174. The peptide or polypeptide of claim 173, wherein (a) the first hypervariable region of the first strand and the first hypervariable region of the second strand are identical and are selected from the group consisting of SEQ ID No.: 8 or 20; or (b) the first hypervariable region of the first strand is selected from the group consisting of SEQ ID NO: 8 or 20, and the first hypervariable region of the second strand is selected from the group consisting of SEQ ID No. 1-6 and 125-202; or (c) the first hypervariable region of the first chain is selected from the group consisting of SEQ ID No. 1-6 and 125-202 and the first hypervariable region of the second chain is selected from the group consisting of SEQ ID No. 8 or twenty. 175. The peptide or polypeptide of claim 173, wherein the second and third hypervariable regions of the first strand are SEQ ID Nos. 114 and 115, respectively. 176. A peptide or polypeptide comprising an Fv molecule, a construction thereof, a fragment of some of them, or a construction of a fragment that (a) binds to an unknown ligand in a first cell having a first and a second state, wherein the binding is effective in the second state but not substantially effective in the first state; and (b) by virtue of the cross immunoreactivity, binds specifically or selectively to a ligand in a second cell, and wherein Fv is a scFv or a dsFv, and wherein Fv optionally has one or more markers, and wherein the The first hypervariable region is a CDR3 region having an amino acid sequence selected from the group consisting of SEQ ID No. 8 or 20. 177. The peptide or polypeptide of claim 176, wherein the first cell is a normal cell. 178. The peptide or polypeptide of claim 176, wherein the first state is a non-activated state and the second state is an activated, excited, modified, changed or disturbed state. 179. The peptide or polypeptide of claim 1176, wherein the second cell is a diseased cell. 180. The peptide or polypeptide of claim 179, wherein the diseased cell is a cancer cell. 44 181. The peptide or polypeptide of claim 179, wherein the diseased cell is selected from the group consisting of carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma, and melanoma cells. 182. The peptide or polypeptide of claim 181, wherein the diseased cell is a leukemia cell. 183. The peptide or polypeptide of claim 182, wherein the leukemia cell is an acute myeloid leukemia cell. 184. The peptide or polypeptide of claim 176, wherein the selective and / or specific binding of the peptide or polypeptide to the ligand of the second cell is determined primarily by a first hypervariable region. 185. The peptide or polypeptide of claim 176, wherein the selectivity and / or specificity is secondarily influenced by a second hypervariable region, by a third hypervariable region and / or by one or more of the ascending or descending regions flanking the first , second and third hypervariable regions, respectively. Four. Five 186. A ligand that is expressed by the second cell and that is capable of being linked by the peptide or polypeptide of claim 176. 187. A molecule that recognizes and binds to the ligand of claim 186. 188. A nucleic acid molecule encoding the peptide or polypeptide of any of claims 138, 171, 173 or 176. 189. The nucleic acid molecule of claim 188, wherein the nucleic acid is DNA. 190. The peptide or polypeptide of claim 176, wherein the first and second states of the first cell are the same, and wherein the first cell is obtained from a cell line. 191. The peptide or polypeptide of claim 190, wherein the cell line is selected from the group consisting of Jurkat, MOLT-4, HS-602, U937, TF-1, TH? -1, KG-1, and HUT-78 . 192. The use of the peptide or polypeptide of claim 138 or claim 176, optionally in association with or united, coupled, combined, connected, or fused with a pharmaceutical agent, in the manufacture of a medicament. 193. The use of claim 192, wherein the medicament has activity against a diseased cell. 194. The use of claim 193, wherein the diseased cell is a cancer cell. 195. The use of claim 193, wherein the cell is selected from the group consisting of carcinoma, sercoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma, and melanoma. 196. The use of claim 195, wherein the cell is a leukemia cell. 197. The use of claim 196, wherein the leukemia cell is an acute myeloid leukemia cell. 198. The peptide or polypeptide of claim 138 or claim 176, optionally in association with or bound, coupled, combined, bound or fused with a pharmaceutical agent, for use as a medicament. 47 199. The peptide or polypeptide of claim 198, wherein the medicament has activity against a diseased cell. 200. The peptide or polypeptide of claim 199, wherein the diseased cell is a cancer cell. 201. The peptide or polypeptide of claim 199, wherein the cell is selected from the group consisting of carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma, and melanoma cells. 202. The peptide or polypeptide of claim 201, wherein the cell is a leukemia cell. 203. The peptide or polypeptide of claim 202, wherein the leukemia cell is an acute myeloid leukemia cell. 204. The use of the peptide or polypeptide of claim 138 or claim 176, for preparing a composition for use in inhibiting the growth of a diseased cell. 205. The use of the peptide or polypeptide of claim 204, wherein the cell is a leukemia cell. 48 206. The use of the peptide or polypeptide of claim 205, wherein the leukemia cell is an acute myeloid leukemia cell. 207. The use of the peptide or polypeptide of claim 138 or claim 176, for preparing a composition for use in inhibiting the growth of a cancer cell, said composition comprises at least one compound having a selective pharmaceutical ligand and / or specific for the cancer cell. 208. A composition comprising at least one peptide of claim 138 or claim 176, in association with, or bound, coupled, combined, connected, or fused to a pharmaceutical agent in a pharmaceutically effective amount and optionally a pharmaceutically effective carrier. 209. The composition of claim 208, wherein the peptide or polypeptide and the pharmaceutical agent are connected through a linker compound. 210. The composition of claim 209, wherein the linking compound is selected from the group consisting of an acid 49 dicarboxylic, a maleimide hydrazide, PDPH, a carboxylic acid hydrazide, and a small peptide. 211. The composition of claim 209, wherein the small peptide is selected from the group consisting of AU1, AU5, BTag, c-myc, FLAG, Glu-Glu, HA, His6, HSV, HTTPHH, IRS, KT3, Protein C, S -TAG®, T7, V5, and VSV-G. 212. The peptide or polypeptide according to any of claims 137, 1170, 172, and 175, wherein the marker is selected from the group consisting of: AU1, AU5, BTag, c-myc, FLAG, Glu-Glu, HA, His6 , HSV, HTTPHH, IRS, KT3, Protein C, S-TAG®, T7, V5, and VSV-G. 213. The composition of claim 208, wherein the pharmaceutical agent is selected from the group consisting of radioisotope, toxin, oligonucleotide, recombinant protein, antibody fragment, and anti-cancer agent. 214. The composition of claim 213, wherein the radioisotope is selected from the group consisting of indium, 113indium, 9furanium, 105renium, 101renium, 99mnetnetium, 121muthion, 122mtelurium, 12S'T'telurium, 165thulium, 161thulium, 158thulium, 123th, 126th, 131th, , 133yodo, 81mkrypton, 33xenon, 0itrio, 13bismuto, 50 7 bromine, 18 fluorine, ruthenium, ruthenium, ruthenium, ruthenium, mercury, mercury, gallium, and gallium. 215. The composition according to claim 213, wherein the toxin is selected from the group consisting of gelonin, Pseudomonas exotoxin (PE), PE40, PE38, diphtheria toxin, ricin, and modifications and derivatives thereof. 216. The composition according to claim 212, in Wherein the anticancer agent is selected from the group consisting of doxorubicin (adriamycin), morpholinodoxorubicin, methoxymorpholydinyldoxorubicin, cis-platinum, taxol, calicheamicin, vincristine, cytarabine (Ara-C), cyclophosphamide, prednisone, daunorubicin, morpholinodaunorubicin, Methoxymorpholidinyldaunorubicin, idarubicin, fludarabine, chlorambucil, interferon alfa, hydroxyurea, temozolomide, thalidomide and bleomycin, and derivatives thereof. 217. A method for inhibiting the growth of a cell comprising bringing the cell into contact with an amount of the peptide or polypeptide of claim 138 or claim 176. ,?,? *,? '·. 51 218. The method of claim 217, wherein the cell is selected from the group consisting of carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma and melanoma. 219. The method of claim 218, wherein the cell is a leukemia cell. 220. The method of claim 219, wherein the leukemia cell is an acute leukemia cell. 221. A pharmaceutical composition comprising at least one peptide or polypeptide of claim 138 or claim 176, attached, coupled, combined, connected or fused to an imaging agent for use in diagnostic localization and imaging. A tumor . 222. A method for treating a patient suffering from a disease, comprising administering to the patient an amount of the peptide or polypeptide of claim 138 or claim 176 effective to treat the disease. 52 223. The method of claim 222, wherein the disease is selected from the group consisting of carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma, and melanoma. 224. The method of claim 223, wherein the disease is a leukemia. 225. The method of claim 224, wherein the leukemia is an acute myeloid leukemia. 226. The peptide or polypeptide of claim 138 or claim 176, wherein Fv specifies or selectively binds to acute myeloid leukemia (AML) cells. 227. A ligand presented in AML cells linked to a peptide or polypeptide of claim 226. 228. A peptide or polypeptide that binds to the ligand of claim 227. 229. A diagnostic kit for the in vitro analysis of treatment efficacy, before, during or after treatment, comprising the peptide or polypeptide of claim 138 or of claim 176, attached, coupled, combined, connected or fused to an indicator marker molecule. 230. The kit of claim 229, wherein the indicator marker molecule is a fluorescent label. 231. The kit of claim 230, wherein the fluorescent label is selected from the group consisting of fluorescein, rhodamine, phycoerythrin, and modifications or conjugates thereof. 232. The kit of claim 229, wherein the kit is used for the diagnosis of cancer. + 233. The peptide or polypeptide of claim 139 or claim 176, wherein the construct is a polypeptide of 1 g. 2. 34. A method for producing the peptide or polypeptide of claim 233, wherein the 1 g polypeptide is expressed as a recombinant polypeptide and is produced in a eukaryotic cell system. 235. The method of claim 234, wherein the eukaryotic system is a mammalian cell system. 54 236. The peptide or polypeptide of claim 233, wherein the 1 g polypeptide is an IgG polypeptide. 237. The peptide or polypeptide of claim 236, wherein the IgG polypeptide comprises a CDR3, CDR2 and CDR1 region having SEQ ID No. 8, 115 and 114, respectively. 238. The peptide or polypeptide of claim 236, wherein the IgG polypeptide comprises a CDR3, CDR2 and CDR1 region having SEQ ID No. 20, 115 and 114, respectively. 239. The IgG polypeptide of claim 237, wherein the CDR3, CDR2 and CDR1 regions are of the heavy chain. 240. The IgG polypeptide of claim 238, wherein the CDR3, CDR2 and CDR1 regions are of the heavy chain. 241. The IgG polypeptide of claim 237, wherein the CDR3, CDR2 and CDR1 regions are of the heavy chain. 242. The IgG polypeptide of claim 238, wherein the CDR3, CDR2 and CDR1 regions are of the light chain. 243. The polypeptide of claim 233, wherein the IgG has a heavy chain comprising SEQ ID No. 26 and a light chain comprising SEQ ID No. 27 or chains having at least 90% amino acid similarity. with her. 244. A method for producing the peptide or polypeptide of claim 139 or claim 176, wherein the peptide or polypeptide is produced in a prokaryotic cell system or a eukaryotic cell system. 245. The method of claim 244, wherein the prokaryotic system comprises E.coli, said E.coli comprises an expression vector and the eukaryotic system is a system of mammalian cells. 246. The method of claim 245, wherein the expression vector of the prokaryotic system comprises a promoter selected from the group consisting of osmB, deo, p-lac-U5, ??? and CMV. 247. The peptide or polypeptide of any of claims 138 or 176, wherein the CDR3 comprises the amino acid sequence of Rj-X Phe Pro-R2, wherein Ri and R2 each 56 one comprises 0-15 amino acid residues, and wherein X is Arg, Gly, or Lys. 248. The peptide or polypeptide of claim 138 or claim 176, wherein said peptide or polypeptide includes at least one unnatural modification. 249. The peptide or polypeptide of claim 248, wherein the non-natural modification makes the peptide or polypeptide more immunogenic or more stable. 250. The peptide or polypeptide of claim 249, wherein at least one modification is selected from the group consisting of peptoid modification, semi-peptide modification, cyclic peptide modification, N-terminus modification, C-terminus modification, peptide bond modification, modification of the main chain, and modification of waste. 251. The peptide or polypeptide of any of claims 138, 171, 173, 176 or 198, for purging ex vivo from the autogenous bone marrow to remove abnormal cells. 252. A peptide or polypeptide having the formula or structure: A-X-B 57 Where X is a hypervariable CDR3 region comprising SEQ ID No. 8 or 20 / and A and B can each be amino acid chains of 1 to 1000 amino acids in length wherein A is the amino terminus and B is the terminus of carboxy. 253. The peptide of claim 252, wherein A is 150-250 amino acid residues and wherein B is 350-500 amino acid residues. 254. The peptide or polypeptide of claim 253, which is part of an antibody or a larger or complete multimer. 255. A dimeric molecule comprising two peptides or polypeptides, one of which is the peptide or polypeptide of claim 252. 256. A dimeric molecule comprising two peptides or polypeptides of claim 252 that are identical. 257. A nucleic acid molecule encoding the peptide or polypeptide of claim 252 or the dimeric molecule of claim 254. 58 258. The method of claim 235, wherein the mammalian cell system comprises the SRa5 promoter. 259. The method of claim 235, wherein the mammalian cell system comprises the CMV promoter.