CN1551886A - Specific human antibodies for selective cancer therapy - Google Patents

Abstract

The present invention relates 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 determined primarily by a first hypervariable region, and wherein the Fv is an scFv or a dsFv, and optionally has one or more tags. The enhanced binding is to a substantially exposed and/or overexpressed binding site located on or within the target, which target comprises a cell that facilitates binding relative to other cells on or within which the binding site is substantially absent and/or not expressed. The invention also relates to methods for isolating said peptides and polypeptides from phage display libraries, and to nucleic acid molecules encoding them. The invention provides pharmaceutical compositions comprising said peptides or polypeptides, as well as kits for the diagnosis and treatment of diseases, preferably cancer, most preferably acute myeloid leukemia.

Description

Specific human antibodies for selective cancer therapy

field of invention

The present invention relates to the field of tissue targeting and identification by means of phage display technology, and to peptides and polypeptides capable of specifically binding to target cells. The peptides and polypeptides are Fv molecules, constructs thereof, fragments of both or constructs of fragments. More specifically, the peptides and polypeptides may have anticancer activity, and/or be associated or conjugated to anticancer agents, particularly against blood-related cancers.

Background of the invention

Tissue-selective targeting of therapeutic agents is an emerging discipline in the pharmaceutical industry. New guidance-based approaches to cancer therapy have been designed to increase the specificity and efficacy of therapy while reducing toxicity, thereby enhancing overall efficacy. To target toxins, radionucleotides, and chemotherapeutic conjugates to tumors, mouse monoclonal antibodies (MAb's) directed against tumor-associated antigens have been employed. In addition, differentiation antigens of CD19, CD20, CD22 and CD25 have been used as cancer-specific targets in the treatment of hematopoietic malignancies. However, in-depth studies have shown that this method has several limitations. One limitation is the difficulty in isolating suitable monoclonal antibodies with selective binding capabilities. The second limitation is the need for high antibody immunogenicity as a prerequisite for successful antibody isolation. A third limitation is the induction of an immune response in patients to murine antibodies (human anti-mouse antibody-HAMA response), which often results in a short serum half-life and prevents repeated treatment, thus reducing the therapeutic value of said antibody . The latter limitation has given rise to interest in engineering chimeric or humanized monoclonal antibodies of murine origin, as well as in the discovery of human antibodies.

There are various factors that interfere with the therapeutic efficacy of monoclonal antibodies (Mabs) to treat cancer. Such factors include the specificity of antigen expression on tumor cells, the level of expression, antigen heterozygosity, and the accessibility of tumor material. Leukemias and lymphomas are generally more sensitive to treatment with antibodies than solid tumors such as carcinomas. MAbs can rapidly bind to leukemia cells and lymphoma cells in the bloodstream, and easily penetrate to malignant tumor cells in lymphoid tissues, thus making lymphoma the best candidate for MAb-based therapy. One ideal system involves identifying MAbs that produce markers that recognize stem cells on the cell surface of malignant tumor progeny cells.

To aid in the discovery/production of Mabs, phage libraries have been utilized to select for random single-chain Fvs (scFvs) that bind isolated, predetermined target proteins such as antibodies, hormones and receptors. In addition, the use of antibody display libraries in general, and phage scFv libraries in particular, facilitates additional approaches to the discovery of specific molecules with targeting specificity, not yet recognized and defined, cell surface components.

Leukemias, lymphomas, and myelomas are cancers that originate in the bone marrow and lymphoid tissue and involve the uncontrolled growth of cells. Acute lymphoblastic leukemia ("ALL") is a heterogeneous disease defined by specific clinical and immunological features. Like other forms of ALL, the cause of most B-cell ALL ("B-ALL") is unknown, although in many cases the disease results from acquired genetic changes in the DNA of a single cell, This change makes it abnormal and continuously amplifies.

AML is a heterozygous neoplasm whose progenitor cells normally give rise to terminally differentiated cells of the myeloid lineage (erythrocytes, granulocytes, monocytes, and platelets). Like other forms of neoplasm, AML is associated with acquired genetic changes that result in the replacement of normally differentiated myeloid cells by relatively undifferentiated blast cells exhibiting one or more types of early myeloid differentiation. AML is generally associated with the bone marrow and, to a lesser extent, with secondary hematopoietic organs. AML primarily affects adults, with a peak incidence between the ages of 15 and 40, although the disease is also known to affect children and the elderly. Nearly all AML patients require treatment immediately after diagnosis in order to achieve clinical remission in which there is no evidence of abnormal levels of circulating undifferentiated blast cells.

So far, various monoclonal antibodies capable of inducing cytolytic activity against tumor cells have been developed. A humanized form of the monoclonal antibody, MuMAb4D5, directed against the extracellular domain of the P185-growth factor receptor (HER2), has FDA approval and is being used to treat human breast cancer (US Patents 5,821,337 and 5,720,954). After binding, the antibody is capable of inhibiting tumor cell growth, which is dependent on the HER2 growth factor receptor. In addition, a chimeric antibody against CD20 that results in rapid clearance of peripheral B cells, including lymphoma-associated B cells, recently received FDA approval (US Patent 5,843,439). Binding of this antibody to target cells results in complement-dependent lysis. The product has recently been approved and is being used to treat low-grade B-cell non-Hodgkin's lymphoma.

Several other humanized and chimeric antibodies are in development or in clinical trials. In addition, a humanized Ig that can specifically react with the CD33 antigen expressed on normal myeloid cells and most types of myeloid leukemia cells was coupled to the anticancer drug calicheamicin, CMA-676 (Sievers et al. , Blood, 90 (10 Suppl. 1 Part 1), 504A (1997)). This conjugate, known as the drug Mylotarg, has recently been approved (Caron et al., Cancer Supplement, 73, 1049-1056 (1994)). Due to its cytolytic activity, another anti-CD33 antibody (HuM195) currently in clinical trials was conjugated to several cytotoxic agents, including gelonin toxin (McGraw et al., Cancer Immunol. Immunother, 39, 367-374 (1994 )) and radioactive isotopes ¹³¹ I (Caron et al., Blood 83, 1760-1768 (1994)), ⁹⁰ Y (Jurcic et al., Blood, 92, (10Suppl.Part 1-2), 613A (1998)) and ²¹³ Bi ( Sgouros et al., J. Nucl. Med., 38(5 Suppl.), 231P (1997)).

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

Although the above preliminary results appear promising, they suffer from the following limitations. Final products that include non-human sequences lead to problematic immune responses to non-human materials, such as HAMA. This HAMA response precludes repeated treatment and results in a short serum half-life of the product. In addition, the methods described above can only isolate one type of antibody, and only antibodies against known and purified antigens. In addition, the method is not selective in that the method can isolate antibodies against cell surface markers present on both normal and malignant cells.

Therefore, there is a need for a method that overcomes the aforementioned limitations. In addition, the method preferably enables the identification of target ligands or markers on cancer cells associated with, eg, mediating cancer cell metastasis. In addition, the method also enables the production of antibodies to the target. Phage display technology appears to have this capability.

The application of phage display technology has made it possible to isolate scFvs comprising complete human sequences. For example, a fully human antibody against the human TGFb2 receptor based on scFv clones from phage display technology has recently been developed. This scFv has strong anti-proliferative activity after conversion into fully human IgG4 capable of competing for binding to TGFb2 (Thompson et al., J. Immunol Methods, 227, 17-29 (1999)). This technique, known to those skilled in the art, is described in more detail in: Smith, Science, 228, 1315 (1985); Scott et al., Science, 249, 386-390 (1990); Cwirla et al., PNAS, 87, 6378-6382 (1990); Devlin et al., Science, 249, 404-406 (1990); Griffiths et al., EMBO J., 13(14), 3245-3260 (1994); Bass et al., Proteins, 8 , 309-314 (1990); McCafferty et al., Nature, 348, 552-554 (1990); Nissim et al., EMBO J., 13, 692-698 (1994); U.S. Pat.

Using this phage display technique, the inventors of the present invention have identified cellular markers present on cells in a diseased or malignant state. Accordingly, it is an object of the present invention to identify peptides and polypeptides that recognize cellular markers expressed on or within cells that are substantially exposed or overexpressed, especially in a diseased or malignant state.

Another object of the present invention is to use and extend phage display technology in order to identify said peptides and polypeptides.

Another object of the present invention is to identify said peptides and polypeptides by immunological cross-reactivity.

Another object of the present invention is that said peptides and polypeptides are entirely of human origin.

Another object of the invention is that said peptides and polypeptides are isolated with antigens which are not necessarily immunogenic.

Another object of the present invention is to provide peptides or polypeptides capable of preventing, delaying or curing cancer, especially blood-related cancers including leukemia or lymphoma.

Another object of the present invention is to provide local targeting of cancer cells using said peptides and polypeptides themselves, or associated or coupled with anticancer agents and/or diagnostic markers or markers.

Another object of the present invention is to provide a method for producing a directing agent to a desired ligand.

Yet another object of the present invention is to identify specific motifs that provide recognition of markers in cells overexpressed in malignant states and that can be used to construct targeting or diagnostic markers or markers for anticancer agents.

Yet another object of the present invention is to provide a composition comprising an effective amount of said peptide, polypeptide or motif bound or linked to an anticancer agent or diagnostic marker or marker.

SCFv has been shown to enter tissue and be cleared from the blood faster than full-sized antibodies because of its smaller size. Adams, G.P., et al., Br.J.Cancer77, 1405-1412(1988); Hudson, P.J., Curr.Opin.Immunol.11(5), 548-557(1999); Wu, A.M., et al., Tumor Targeting4, 47 (1999). Therefore, scFvs are often used in diagnostic methods such as tumor imaging that involve radiolabeling, which can be cleared from the body more quickly. Several cancer-targeting scFv multimers have recently been preclinically evaluated for in vivo stability and potency. Adams, G.P., et al., Br. J. Cancer 77, 1405-1412 (1988); Wu, A.M., et al., Tumor Targeting 4, 47 (1999).

Single-chain Fv (scFv) fragments consist of the variable heavy domain ( _VH ) and the variable domain of the light chain ( _VL ) of an antibody linked together by a polypeptide linker. The linker is long enough to allow the ( _VH ) and ( _VL ) domains to fold into a functional Fv domain, allowing the scFv to recognize and bind its target with similar or higher affinity than the parental antibody. Commonly used linkers include glycine and serine residues to provide flexibility and protease resistance.

Typically, scFv monomers are designed such that the C-terminus of the _VH domain is linked to the N-terminal residue of the _VL by a polypeptide linker. The reverse orientation is optionally used: the C-terminus of the _VL domain is linked to the N-terminal residue of the _VH via a polypeptide linker. Power, B., et al., J. Immun. Meth. 242, 193-204 (2000). The polypeptide linker is usually about 12 amino acids in length. When the length of the linker is reduced to about 3-12 amino acids, the scFvs can no longer fold into a functional Fv domain and instead associate with another scFv to form a diabody. Further reducing the length of the linker to less than 3 amino acids forces scFv to associate as trimers or tetramers, depending on the length, composition and orientation of the Fv domains of the linker. BE Powers, PJ Hudson, J. Immun. Meth. 242 (2000) 193-194.

Recently, it has been found that multivalent antibody fragments such as scFv dimers, trimers and tetramers generally have a higher apparent binding affinity for the target than the parental antibody. This higher affinity has several advantages, including desirable pharmacokinetic advantages for tumor-directed applications.

Thus, the greater binding affinity of the multivalent form is desirable for diagnostic and therapeutic regimens. For example, a scFv can be used as a blocking agent for binding to a target receptor and thus blocking the binding of the "native" ligand. In this case, a high association affinity between the scFv and the receptor is required in order to reduce the possibility of dissociation which could result in undesired binding of the natural ligand to the target. Additionally, this high affinity is particularly important when the target receptor is involved in adhesion or rolling or when the target receptor is present on cells such as platelets present at sites of high shear flow.

Therefore, the object of the present invention is the multivalent form of Y1 and Y17 scFv. Such multivalent forms include, but are not limited to, dimers, trimers, and tetramers, sometimes referred to herein as diabodies, triabodies, and tetrabodies, respectively.

Summary of the invention

The present invention provides peptides and polypeptides capable of selectively and/or specifically binding to target cells, particularly blood-related cancer cells, their construction, their use by themselves, or in association with one or more pharmaceutical agents. Combined or combined, coupled or fused uses.

One embodiment of the invention provides peptides or polypeptides comprising Fv molecules, constructs thereof, fragments of both or constructs of fragments, which have enhanced binding characteristics to facilitate selection relative to other cells Binds selectively and/or specifically to target cells, wherein the binding selectivity or specificity is primarily determined by the first hypervariable region, and wherein the Fv is a single chain ("scFv") or disulfide Fv ("dsFv"), and optionally with 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 both, or a construct of a fragment, which has enhanced binding characteristics to enable selective binds selectively and/or specifically to a substantially exposed and/or overexpressed binding site on or within a target cell, wherein said binding is substantially absent and/or expressed relative to thereon or in Other cells at the site are more conducive to binding to target cells, wherein the binding selectivity or specificity is mainly determined by the first hypervariable region, and wherein the Fv is scFv or dsFv, and optionally has one or Multiple tags.

In another embodiment of the present invention, peptides or polypeptides comprising Fv molecules, constructs thereof, fragments or constructs of fragments are provided which have enhanced binding characteristics so that they are more Facilitates selective and/or specific binding to target cells, wherein the Fv molecule comprises a first chain with first, second and third hypervariable regions, and has first, second and third hypervariable regions The second chain of the second chain, wherein one of the hypervariable regions of the first chain has a sequence selected from SEQ ID Nos: 8-24, and, wherein, one of the hypervariable regions of the second chain has a sequence selected from From the sequence of SEQ ID Nos: 1-6 and 125-202, and wherein the first, second and third hypervariable regions are CDR3, CDR2 and CDR1 regions, and wherein the Fv is scFv or dsFv, and either Selection has one or more tags.

In another embodiment of the invention,

(a) said first and second strands each comprise a first hypervariable region selected from SEQ ID NOs: 8-24,

(b) the first hypervariable region of the first strand and the second strand are identical and are selected from SEQ ID NOs: 8-24,

(c) the first hypervariable region of the first chain is selected from SEQ ID NOs: 8-24, and the first hypervariable region of the second chain is selected from SEQ ID Nos: 1-6 and 125-202 ,or

(d) the first hypervariable region of the first chain is selected from SEQ ID NOs: 1-6 and 125-202, and the first hypervariable region of the second chain is selected from SEQ ID NOs: 8-24 .

In another embodiment of the present invention there is provided a peptide or polypeptide comprising an Fv molecule, a construct thereof, a fragment or a construct of a fragment of both, which is capable of binding to a protein having a first state and a second state An unknown ligand on a first cell of wherein said binding is achieved in said second state by immunological cross-reactivity, binding specificity or selectivity with a ligand on a second cell, This is substantially not possible in the first state, and wherein the Fv is scFv or dsFv, optionally with one or more markers.

In another embodiment of the invention there is provided a method for identifying a targeting molecule that binds to an unknown immunologically cross-reactive binding site on a first and a second cell comprising

(a) one or more biopanning steps performed on the first target cells in a second state, other than the first state, in which they substantially expose or display substances including unknown the binding site of the ligand in order to generate the first population of recognition molecules;

(b) subsequent biopanning and/or selection steps, starting from the population of recognition molecules obtained in step (a), which include immunologically cross-reactive molecules with unknown ligands on said first cell Performed on a second cell with an unknown ligand binding site in order to produce a second population of recognition molecules;

(c) amplifying and purifying the second population of recognition molecules of step (b); and

(d) constructing a peptide or polypeptide comprising a targeting molecule having selectivity and/or specificity for the unknown ligand on the second cell from the recognition site of the purified recognition molecule of step (c) .

In another embodiment of the present invention, there is provided a binding motif comprising the amino acid sequence R ₁ -X PhePro-R ₂ , wherein R ₁ and R ₂ each comprise 0-15 amino acid residues, and wherein X is Arg, Gly or Lys.

In another embodiment of the present invention, there is provided a method for producing a directing agent, comprising the steps of:

a) Isolation and selection of one or more targeting molecules comprising a primary recognition site, including performing a biopanning procedure directly on the target cells or indirectly on the first in a second state instead of the first performing a biopanning procedure on a target cell and subsequently indirectly performing a biopanning procedure on a second target cell to produce one or more of said targeting molecules;

b) amplifying, purifying and identifying one or more targeting molecules; and

c) constructing a targeting agent from the one or more targeting molecules, alternatively, wherein the targeting agent may be a peptide, polypeptide, antibody or antibody fragment, or a multimer thereof.

Wherein, the targeting agent may be a peptide, a polypeptide, an antibody or an antibody fragment or a multimer thereof.

In another embodiment of the present invention, there is provided a peptide or polypeptide having the following molecular formula or structure:

A-X-B

Wherein, X is a hypervariable CDR3 region with a length of 3-30 amino acids; A and B can each be an amino acid chain with a length of 1-1000 amino acids, wherein A is an amino terminal and B is a carboxyl terminal.

Description of drawings

The present invention will be described in more detail below in conjunction with the accompanying drawings disclosed below only by way of example, rather than in a limiting manner, wherein:

Figure 1 shows phage clones bound to immobilized platelets as determined by EIA analysis. Data are presented as a function of absorbance at a wavelength of 405 nm.

Figures 2a, 2b and 2c show the binding of monocyte samples obtained from three AML patients to scFvs as determined by molecular FACS. The fluorescence intensity of cells bound by two FITC-labeled test samples (control scFv and scFv clone Y1 ) is shown.

Figure 3 shows the binding of Ficoll-purified Y-I to platelets (3a) and monocytes (3b) as determined by FACS analysis. The fluorescence intensity of cells bound by two FITC-labeled test samples (control scFv and scFv clone Y1 ) is shown.

Figure 4 shows the binding of FITC-labeled scFv clone Y1 to cord blood CD34+ stem cells. In the FLI-H channel, CD34+ gated cells in the FL3-H channel were analyzed for binding to FITC-labeled negative control scFv (Fig. 4a) or FITC-labeled scFv clone Y1 (Fig. 4b). Figure 4c shows the FSC and SSC dot plot analysis of the same FITC-labeled scFv clone Y1 samples as in Figure 4b. The circled portion in Figures 4b and 4c represents the subpopulation of CD34+ cells that binds scFv clone Y1.

Figure 5: FACS analysis of samples obtained from two patients with pre-B-ALL cells: one from a child (5a, 5c, 5e) and the other from an adult (5b, 5d, 5f). A double-staining method was employed using commercially available PE-labeled CD19 (marker of normal peripheral B-cells; Figure 5a, 5c) or PE-labeled CD34 (marker of stem cells, Figure 5d), while using FITC-labeled negative control scFv (5a, 5b) or FITC-labeled Y-I scFv (5c, 5d) were added. Figure 5b is a double negative control. Fluorescence intensity (x-axis) of cells bound by FITC-labeled sample (scFv clone Y1 ) in contrast to negative control staining pattern is presented (5e and 5f).

Figure 6: This figure presents the results of a comparative binding study performed with Jurkat cells. FACS analysis of binding of FITC-labeled Y-I scFv monomer, diabody and triabody, and negative control to Jurkat cells is provided.

Figure 7: This figure presents the results of a study comparing IgG-Y-I and scFv-Y1 binding. To compare the binding of the full-sized IgG-Y1 and the scFv-YI form, a double staining method was employed. 5 ng IgG-YI was used for FACS analysis of RAJI cells (YI negative cells; Figure 7a) and Jurkat cells (Y1 positive cells; Figure 7b). For detection, PE-labeled goat anti-human IgG was used. About 1 μg (200-fold) was used for scFv-YI-I binding, then stained with PE-labeled rabbit anti-scFv antibody, and subjected to FACS analysis (Fig. 7c).

Figure 8: This graph represents the binding comparison between YI dimer, Y1 scFv (CONY1) and Y1 IgG.

Figure 9: This graph represents the binding comparison between Y1 thioether dimer and Y1 scFv(CONY1).

Figure 10: This figure is the Superdex 75 curve of Y1cys-kak.

Figure 11: This graph represents the size of the dimer compared to the size of the monomer under reducing and non-reducing conditions.

Figure 12: This figure provides the results of the ELISA analysis.

Figure 13: This figure is a graph of the epitopes of anti-GPIbα antibodies.

Figure 14: This figure is the amino acid SEQ ID NO:.

Detailed description of the invention

Specificity is defined herein as the recognition of a target ligand by one or more domains in a peptide or polypeptide of the invention, and subsequent binding to it.

Selectivity is defined herein as the ability of a targeting molecule to select and bind to one type or cell state in a mixture of cell types or cell states, all of which may be specific for said targeting molecule.

Conservative amino acid substitutions are defined as changes in amino acid composition by altering one or two amino acids on a peptide, polypeptide or protein or a fragment thereof. The substitutions are made with amino acids of substantially similar properties (e.g., acidic, basic, aromatic, size, positively or negatively charged, polar or nonpolar) such that such substitutions do not significantly alter A characteristic (eg, charge, IEF, affinity, avidity, conformation, solubility) or activity of a peptide, polypeptide or protein. Typical substitutions that can be made in such conservative amino acid substitutions can include substitutions made in the following groups of amino acids:

(i) Glycine (G), Alanine (A), Valine (V), Leucine (L) and Isoleucine (I)

(ii) Aspartic acid (D) and glutamic acid (E)

(iii) Alanine (A), Serine (S) and Threonine (T)

(iv) Histidine (H), Lysine (K) and Arginine (R)

(v) Asparagine (N) and Glutamine (Q)

(vi) Phenylalanine (F), Tyrosine (Y) and Tryptophan (W)

Conservative amino acid substitutions can be made within and flanking hypervariable regions that primarily determine the selective and/or specific binding characteristics of the molecule, as well as in other parts of the molecule, e.g., the variable heavy chain cassette . Additionally or alternatively, full-sized antibodies, diabodies (dimers), triabodies (trimers), and/or tetrabodies (tetramers) may be formed by reconstituting the molecule or form small or microbodies. body modification.

In this specification and claims, Fv is defined as a molecule consisting of the heavy chain variable region of a human antibody and the light chain variable region of a human antibody, which may be the same or different, and wherein The heavy chain variable region is joined, linked, fused or covalently linked or associated with the light chain variable region.

A fragment of an Fv molecule is defined as any molecule that is smaller than the original Fv, yet retains the selective and/or specific binding characteristics of the original Fv. Examples of such fragments include, but are not limited to (1) minibodies, which include only a fragment of the Fv heavy chain, (2) minibodies, which include a small constituent unit of the antibody heavy chain variable region (PCT Application No. PCT/IL99/00581), (3) analogues comprising fragments of said light chain, and (4) analogues comprising functional units of the light chain variable region.

An anticancer agent is an agent having anticancer activity, ie any activity that inhibits the growth or differentiation of cancerous or immature precancerous cells, or any activity that inhibits metastasis of cancer cells. In the present invention, the anticancer agent is also an agent with anti-angiogenic activity, which can prevent, inhibit, delay or eliminate angiogenesis in tumor tissue, or an agent with anti-adhesion activity, which can inhibit, Delays or eliminates adhesion and metastatic invasion of cancer cells and precancerous cells.

Inhibition of cancer cell growth is defined herein as (i) inhibiting cancerous or metastatic growth, (ii) delaying cancerous or metastatic growth, (iii) completely inhibiting the growth process or metastatic process of cancer cells while maintaining all integrity and viability of said cells, or (iv) killing said cancer cells. More specifically, inhibition of cancerous growth may find particular application against blood-related cancers such as AML, multiple myeloma or chronic lymphocytic leukemia.

A phagemid is defined as a phage particle carrying a plasmid DNA, because with it, the phagemid particle no longer has enough space to accommodate the entire complement of the phage genome. Missing from the phage genome is the information necessary for packaging the phage particle. Therefore, in order to propagate the phage, the desired phage particle must be incubated with a helper phage strain that complements the missing packaging information.

As defined herein, a cassette for a polypeptide refers to a specific contiguous sequence of amino acids that functions as a framework and is considered and manipulated as a unit. Amino acids may be substituted, inserted, removed or linked at one or both ends thereof. Likewise, amino acid fragments may be substituted, inserted, deleted or joined at one or both ends thereof.

Herein, immunoglobulin (Ig) molecules are defined as any of five classes, IgG, IgA, IgD, IgE or IgM. The IgG class includes several subtypes including, but not limited to IgGl, IgG2, IgG3 and IgG4.

A pharmaceutical composition refers to a preparation, which includes the peptide or polypeptide of the present invention, and a pharmaceutically acceptable carrier, excipient or diluent thereof.

A pharmaceutical agent means an agent useful in the prophylactic treatment or diagnosis of a mammal including, but not limited to, a human, bovine, equine, porcine, murine, canine, feline, or any other warm-blooded animal. The pharmaceutical agent is selected from the group consisting of radioisotopes, toxins, oligonucleotides, recombinant proteins, antibody fragments, and anticancer agents. Examples of such pharmaceutical agents include, but are not limited to, antiviral agents, including acyclovir, ganciclovir, and zidovudine; antithrombotic/restenosis agents, including cilostazol, dalteparin sodium, radium Heparin, and aspirin; anti-inflammatory agents, including zaltoprofen, pranoprofen, droxicam, acetylsalicylic acid17, diclofenac, ibuprofen, dexibuprofen, sulindac, Naproxen, amtolmetin, celecoxib, indomethacin, rofecoxib, and nimesulide; anti-autoimmune agents, including leflunomide, denileukin diftitox, subreum, WinRho SDF, defibrotide, and cyclophosphamide; and anti-adhesion/anti-aggregation agents, including limaprost, clorcromene, and hyaluronic acid.

An anti-leukemic agent is an agent having anti-leukemic activity. For example, anti-leukemic agents include agents that inhibit or prevent the growth of leukemic cells or immature pre-leukemic cells, agents that kill leukemic or pre-leukemic cells, that enhance the susceptibility of leukemic or pre-leukemic cells to other anti-leukemic agents reagents, and reagents that inhibit the metastasis of leukemia cells. In the present invention, the anti-leukemia agent may also be an agent having anti-angiogenic activity, which prevents, inhibits, delays or eliminates 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 strength of the total non-covalent interaction between a single antigen-binding site on an antibody and an epitope is the affinity of the antibody for that epitope. Low-affinity antibodies bind antigen weakly and tend to dissociate easily, while high-affinity antibodies bind antigen more tightly and remain bound for a longer period of time. The term "avidity" is distinguished from affinity because the former reflects the valence of an antigen-antibody interaction.

Specificity of Antibody-Antigen Interactions: Although antigen-antibody reactions are specific, in some cases antibodies induced by one antigen can cross-react with another, unrelated antigen. This can occur if two different antigens share a homologous or similar epitope or its anchoring region, or if an antibody specific for an epitope binds an unrelated epitope with similar chemical properties. Cross reaction.

Blast cells are cells in the immature stage of cell development, which differ from resting cells in having a higher ratio of cytoplasm to nucleus.

Platelets are disc-shaped cytoplasmic fragments of megakaryocytes that are expelled from the bone marrow sinuses and subsequently circulate in the peripheral blood. Platelets have several physiological functions, including a major role in coagulation. Platelets consist of granules in the central part and hyaline protoplasm in the peripheral part, but without a well-defined nucleus.

The term "epitope" herein means an antigenic determinant or antigenic site capable of interacting with an antibody, antibody fragment, antibody complex or a complex including its binding fragment or T-cell receptor. Herein, the term epitope is used interchangeably with the terms ligand, domain, and binding domain.

A particular cell may express on its surface a protein that has a binding site (or epitope) for a particular antibody, but the binding site may be in a cryptic fashion (e.g., sterically obstructed or blocked, or lacking characteristics required for antibody binding) exist in the cells in a state that can be referred to as the first phase (phase I), for example, phase I can be a normal, healthy, non-diseased state. When the epitope is present in a cryptic form, it cannot be recognized by the specific antibody, ie there is no binding of the antibody to the epitope or to the specific cell in phase I. However, the epitope may become unmasked, for example, by modification that has occurred in itself, or by unblocking due to modification of adjacent or related molecules, or by a conformational change in one segment. Examples of modifications include changes in folding, changes in post-translational modifications, changes in phospholipidation, changes in sulfation, and changes in glycosylation, among others. The modification may occur when the cell enters a different state, which may be referred to as phase II (phase II), examples of a second state or phase include activation, proliferation, transformation, or malignancy state. When modified, the epitope is exposed and the antibody is able to bind it.

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

Polyclonal antibodies are the product of an immune response and are formed by a variety of different B-lymphocytes. Monoclonal antibodies originate from a single cell.

As used herein, the term agglutination refers to the process of causing suspended bacteria, cells, discs or other particles of similar size to stick together and form clumps. The process is similar to precipitation, but the particles are larger and exist in suspension rather than in solution.

The term "aggregation" denotes the in vitro and thrombin- and collagen-induced aggregation of platelets as part of a chain of mechanisms leading to the formation of a thrombus or hemostatic plug.

The expression pattern of a gene can be studied by analyzing the amount of the gene product produced under various conditions, at a specific time, in various tissues, and the like. A gene is considered "overexpressed" when the gene product is present in an amount higher than that present in normal controls, eg, non-diseased controls.

A promoter is a region on DNA to which RNA polymerase binds and initiates transcription.

Antibodies or immunoglobulins are protein molecules that bind antigens. They consist of four polypeptide chain (two heavy and two light chain) units linked together by disulfide bonds. Each chain has a constant region and a variable region. According to the heavy chain components of antibodies, antibodies can be divided into five types: IgG, IgM, IgA, IgD and IgE. Antibodies are produced by B lymphocytes, recognize specific foreign antigenic determinants, and facilitate clearance of the antigen.

Antibodies can be produced and used in a variety of forms, including antibody complexes. As used herein, the term "antibody complex" is used to denote a complex of one or more antibodies with another antibody or with one or more antibody fragments, or a complex of two or more antibody fragments body.

F(ab')2 fragments are divalent antigen-binding fragments of immunoglobulins obtained by pepsin digestion. It includes the light chain and part of the heavy chain.

The Fc fragment is the non-antigen binding portion of an immunoglobulin. It includes the carboxy-terminal portion of the heavy chain and the binding site for the Fc receptor.

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

A contaminating protein is a protein that is not specifically selected for use in a sample and may be present in that sample.

A peptidomimetic is a small molecule, peptide, polypeptide, lipid, polysaccharide, or conjugate thereof that has the same functional role or activity as another entity, such as an antibody.

Phagemids are plasmid vectors designed to include an origin of replication for filamentous bacteriophages such as M13 derived from Fd.

There are a variety of diseases that involve diseased, altered, or otherwise modified cells that express cell-specific and/or disease-specific ligands on their surface. By identifying, selecting, diagnosing, and treating each type of cell, the ligand can be used to achieve the identification, selection, diagnosis, and treatment of specific diseases. The invention provides peptides or polypeptides comprising Fv molecules, constructs thereof, fragments thereof, constructs of fragments thereof, or fragments of constructs, all of which have enhanced binding characteristics. This binding characteristic allows the peptide or polypeptide molecule to selectively and/or specifically bind to target cells more favorably than other cells. The binding specificity and/or selectivity is mainly determined by the first hypervariable region. The Fv may be scFv or dsFv.

The Fv molecules described above can be used to target diseased cells. For example, the diseased cells may be cancer cells. Examples of cancer types that can be specifically directed for diagnosis and/or treatment include, but are not limited to, carcinomas, sarcomas, leukemias, adenomas, lymphomas, myelomas, blastomas, seminomas, and melanomas. Leukemias, lymphomas, and myelomas are cancers that originate in the bone marrow and lymphoid tissue and are associated with uncontrolled cell growth.

In recent years, new methods have been developed for the diagnosis and treatment of diseases, especially cancer. These include tumor-targeting approaches that use targeting molecules that can be screened and produced in a variety of ways. One method for identifying potential targeting molecules is phage display. Phage display is a technique in which peptides, polypeptides, antibodies or proteins are produced and selected by expression and display on the surface of filamentous bacteriophage by fusion with a phage coat protein, the DNA encoding the displayed protein being located on the phage Inside the virus particle. scFv, produced by phage display technology, consists of the variable regions of each of the antibody heavy and light chains linked by a flexible amino acid polypeptide spacer (Nissim et al., EMBO J, 13, 692-698 (1994)).

A phage display library (also known as a phage peptide/antibody library, phage library, or peptide/antibody library) includes a large population of phages (usually 10 ⁸ -10 ⁹ ), and each phage particle has a different peptide or polypeptide sequence. The peptide or polypeptide fragments can be constructed in different lengths. The displayed peptides or polypeptides may be derived from, but are not necessarily limited to, human antibody heavy or light chains.

In the present invention, the scFv antibody library produced by phage display technology was used to obtain and produce targeting molecules. Flow cytometry, particularly fluorescence-activated cell sorting ("FACS"), is used to identify and isolate specific phage clones whose peptides or polypeptides recognize target cells. The scFv antibody fragment expressed by phage can be used for in vitro screening, enrichment and selection of high-affinity clones (US Patent 5,821,337; US Patent 5,720,954). Thus, this type of library provides an efficient means of producing new tools for research and clinical applications and has several advantages over conventional methods (Caron et al., Cancer Supplement, 73, 1049-1056 (1994)). Such libraries include the potential for a high diversity of antibody molecules (Nissim et al., EMBO J., 692-698 (1994)). In the present invention, stable human cDNA can be used as a continuous source of material for antibody production (US Patent 5,843,439). Molecular recognition and selection are not affected by the in vivo immunogenicity of candidate target proteins.

Although affinity selection of phage-displayed antibodies provides a useful method for enriching antigen-reactive scFvs from large libraries, it requires multiple steps to isolate individual clones, as well as characterize soluble scFvs. The scFvs themselves may be modified in order to increase their affinity and/or avidity by making conservative amino acid substitutions, or by producing fragments of the scFv, or constructs of such fragments.

The scFvs of the present invention specific to different human cells and tissues can be combined, combined, fused, or linked with various pharmaceutical agents and/or radioisotopes in a pharmaceutically effective amount, and optionally combined with a pharmaceutically effective carrier, so that Formation of drug-peptide compositions, fusions or conjugates having anti-disease and/or anti-cancer activity, and/or for diagnostic purposes of said diseases.

Phage clones are selected and identified by a multi-step method known as biopanning. Biopanning is performed by incubating phage-displayed protein ligand variants (phage display library) with one target, removing unbound phage by washing techniques, and specifically eluting bound phage. Optional amplification of eluted phage followed by additional rounds of binding and optional amplification to enrich the library for specific sequences favors phage cloning with antibody fragments exhibiting optimal binding ability to the target . After several rounds, single phage clones were characterized and the sequence of the peptide displayed by the clone was determined by sequencing the corresponding DNA of the phage virion.

The scFv obtained by this method is also called a lead compound. A lead compound is defined as a compound whose final form includes a core peptide or polypeptide. The lead compound can be modified and/or amplified, but it must retain the core peptide or polypeptide or some conservative modification thereof. For example, modification by amino acid substitution can be performed at the N-terminal, carboxy-terminal, or any of the CDR regions of Fv, or at upstream or downstream regions thereof. Modifications can also include, but are not limited to, fusion proteins, conjugation to drugs or toxins, construction of multimers, and expansion into complete antibody molecules. A preferred type of lead compound for use in the present invention is the scFv obtained as the end product of the biopanning process.

One embodiment of the invention provides at least one non-natural modification of a peptide or polypeptide of the invention. Such non-natural modifications may render the peptide or polypeptide more or less immunogenic. Non-natural modifications include, but are not limited to, peptoid modifications, semi-peptoid modifications, cyclic peptide modifications, N-terminal modifications, C-terminal modifications, peptide bond modifications, backbone modifications, and residue modifications.

The selection of antigen-specific phage antibodies largely depends on the biopanning of immobilized single antigens. Only limited selection was done with whole cells as targets. In the present invention, whole cells are used to select for specific antibodies that recognize determinants on the surface of leukemia cells, wherein the specific receptors are not previously known or characterized. This method does not allow easy adjustment of antigen concentration or removal of unwanted dominant antibody reactivity. In addition, the phage can be enriched for clones displaying multiple copies of scFv compared to clones with higher affinity. Nevertheless, the described advantages of this method make it a very valuable tool for isolating novel human antibody molecules.

One embodiment of the present invention provides peptides or polypeptides comprising Fv molecules, constructs thereof, fragments or constructs of fragments thereof, capable of binding to an unknown ligand on a first cell having a first and a second state. An entity, wherein said binding is achieved in a second state substantially incapable of being achieved in a first state, specifically or selectively binds to a ligand on a second cell by immunological cross-reactivity and wherein the Fv is scFvs or dsFv, and optionally has one or more markers.

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

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

Another embodiment provides the peptide or polypeptide of the present invention, wherein the first hypervariable region is a CDR3 region having an amino acid sequence selected from SEQ ID NOs: 8-24, and wherein the binding selectivity or specificity Sex is secondarily influenced by the second hypervariable region and/or the third hypervariable region and/or one or more upstream regions and/or one or more downstream regions flanking said first, second and third hypervariable regions respectively area impact.

Another embodiment provides a ligand for a second cell bound by a peptide or polypeptide of the invention. One such two-cell selection method is based on the principle that megakaryocytes are large multinucleated cells derived from hematopoietic stem cells in the bone marrow. Platelets can destroy the cytoplasm of megakaryocytes and enter the peripheral blood. In vitro, a variety of cytokines can directly affect stem cells. For example, thrombopoietin increases platelet count by directly increasing the differentiation of stem cells into giant cells. Thus, the cells are capable of expressing several cell surface markers that are also present in immature cells.

Malignant blood cells (leukemia and lymphoma) are characterized by immature cells that express cell surface proteins normally found in partially differentiated hematopoietic progenitor cells. Platelets are therefore an attractive source for the identification of immature cell surface markers expressed on diseased or malignant blood cells. In one method disclosed below, specific cells, such as, but not limited to, platelets with unknown ligands are used in an initial biopanning step. Subsequent clonal selection is performed with desired target cells whose surface markers are unknown, such as, but not limited to, AML cells. In this approach, phage clones obtained by biopanning on platelets can provide tools for recognizing and binding ligands located on diseased or malignant blood cells of interest.

The aforementioned target cells include cells obtained from isolated tissues. The isolated tissue may be diseased tissue, more specifically, cancerous tissue. Cancerous tissue may arise from any form of malignancy including, but not limited to, carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma, and melanoma.

In addition to the biopanning method described above, another method is based on the isolation of peptides or polypeptides capable of binding a ligand on a cell, which peptides or polypeptides are determined by panning directly on said ligand.

The invention provides peptides or polypeptides comprising Fv molecules, constructs thereof, fragments of both, or constructs of fragments. A construct can be a multimer (eg, diabody, triabody, tetrabody) or a full-sized Ig molecule. A fragment can be a body or a microbody. All derived constructs and fragments retain enhanced binding characteristics to favor selective and/or specific binding of target cells relative to other cells. The binding selectivity and/or specificity is mainly determined by the first hypervariable region, and the Fv therein is scFv or dsFv, and optionally has one or more markers.

In one embodiment of the invention, a marker is inserted or attached to the Fv peptide or polypeptide to facilitate the preparation and identification of said peptide or polypeptide and to aid in diagnosis. The label can subsequently 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.

A spacer segment of 0-20 amino acid residues in length may be used to link or bring together the two variable chains of the Fv molecule of the invention. The spacer segments may be branched or unbranched. The linker is preferably 0-15 amino acid residues, the most preferred linker being ( _Gly4Ser ) ₃ , in order to generate a single chain Fv ("scFv"). ScFv can be obtained from phage display libraries.

The Fv molecule itself consists of a first chain and a second chain, each chain including first, second and third hypervariable regions. The hypervariable loops located within the variable domains of the light and heavy chains are called complementarity determining regions (CDRs). Each of the heavy and light chains has CDR1, CDR2 and CDR3 regions. Such regions are believed to form antigen binding sites and may be specifically modified to produce enhanced binding activity. The essence of the majority of the variable region is the CDR3 region of the heavy chain. The CDR3 region is understood to be the most exposed region of the Ig molecule and, as demonstrated and provided herein, is the site that primarily determines the observed selective and/or specific binding characteristics.

One embodiment of the present invention provides peptides or polypeptides comprising Fv molecules, constructs thereof, fragments of both, or constructs of fragments, which have enhanced binding characteristics so as to be substantially free from said binding site. Cells at sites where expression is present and/or non-expressed are more favorable for selectively and/or specifically binding to substantially exposed and/or overexpressed sites on a target site comprising a cell, Wherein, the binding selectivity and specificity are mainly determined by the first hypervariable region, and the Fv therein is scFv or dsFv, and optionally has one or more markers.

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

Another embodiment of the present invention provides a peptide or polypeptide, wherein the first hypervariable region is a CDR3 region having an amino acid sequence selected from SEQ ID NOs: 8-24. And, wherein the binding selectivity or specificity is secondarily influenced by the second hypervariable region and/or the third hypervariable region and/or one or more upstream regions flanking said first, second and third hypervariable regions respectively region and/or one or more downstream regions, wherein the second and third hypervariable regions are CDR2 and CDR1 regions, respectively.

One embodiment of the invention provides peptides or polypeptides that bind to target cells that are activated, excited, modified, altered, dysregulated or diseased cells. Another embodiment of the present invention provides target cells that are cancer cells. The target cells may be selected from, but are not limited to, carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma, and melanoma. In a preferred embodiment, the cancer cells are leukemia cells, and in a most preferred embodiment, the leukemia cells are AML cells.

The peptide or polypeptide of the present invention may also be any construct or modified construct of Fv which retains one or more hypervariable regions of said heavy and/or light chain and which has selective and/or specific binding characteristics. Constructs or modified constructs include, but are not limited to, scFv, dsFv, multimers of scFv, such as dimers, trimers and tetramers, etc. (also known as diabodies, triabodies, tetrabodies ), and intact antibodies, and any other multimers thereof that can be constructed and that incorporate one or more hypervariable domains of said antibodies. A peptide or polypeptide of the invention is also a fragment of any construct or modified construct that possesses some or all of the binding characteristics of the original construct.

A peptide or polypeptide of the invention is also a construct that is a fragment of some or all of the selective and/or specific binding characteristics of the original construct. The Fvs disclosed herein are capable of selectively and/or specifically binding to target cells, and may be associated with, or conjugated to, an anticancer or antidisease agent.

The peptides, polypeptides, fragments thereof, constructs thereof and fragments of constructs of Fv molecules of the present invention can be produced in prokaryotic or eukaryotic expression systems. In one embodiment of the invention, said eukaryotic expression system is a mammalian expression system, and the peptide or polypeptide produced in said mammalian expression system is substantially free of mammalian contaminants after purification. A eukaryotic cell system as defined herein refers to an expression system for producing peptides or polypeptides by genetic engineering methods, wherein the host cells are eukaryotic cells. In another embodiment of the present invention, the prokaryotic system for producing the peptides or polypeptides of the present invention uses E. coli as the host for the expression vector. The peptide or polypeptide produced in the E. coli system is substantially free of E. coli contaminating proteins after purification. Use of prokaryotic expression systems may result in the addition of a methionine residue to the N-terminus of some or all of the sequences provided herein. Following peptide or polypeptide production, removal of the N-terminal methionine enables full expression of the peptide or polypeptide by methods generally known in the art, such as, but not limited to, the use of Aeromonas aminopeptidase under suitable conditions (US Patent 5,763,215).

The invention provides for the production of scFv based on the Fv peptides of the invention. Promoters incorporated into vectors for cloning and amplifying scFv in prokaryotic cells can be selected from a variety of candidate promoters. A promoter is a DNA sequence located upstream of a structural gene and capable of controlling gene expression. Promoters exist in chromosomes in the natural state of organisms, and can also be integrated into prokaryotic or eukaryotic expression vectors by engineering methods. By engineering a promoter at a specific locus of a DNA fragment of interest, fine-tuned and precisely controlled expression of a gene of interest is possible. In the present invention, several promoters were used on the construct including the gene encoding the selected Fv. Promoters include, but are not limited to the following promoters: deo, P1-P2, osmB, _λPL , β-lac-U5, SRα5 and CMV early promoters. Deo is a double-stranded DNA plasmid that, after introduction into a suitable E. coli host, enables the host to express the desired naturally occurring DNA of polypeptides or polypeptide analogs. A more complete description is provided in US Patent 5,795,776 (Fischer, August 18, 1998) and US Patent 5,945,304 (Fischer, August 31, 1999).

Expression from the E. coli osmB promoter is osmotically regulated. Vectors with this promoter can be used to produce high levels of a variety of recombinant eukaryotic and prokaryotic polypeptides under the control of the osmB promoter in E. coli hosts. A more complete description is provided in US Patent 5,795,776 (Fischer, August 18, 1998) and US Patent 5,945,304 (Fischer, August 31, 1999).

_λPL is a lambda phage promoter regulated by the thermolabile repressor cI ⁸⁵⁷ . A more comprehensive discussion can be found in Hendrix et al. Lambda II, Cold Spring Harbor Laboratory (1983).

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

SR _α 5 is a mammalian cDNA expression system consisting of the Simian virus 40 (SV40) early promoter and the long terminal repeat R-U5 fragment of human T-cell leukemia virus type 1. This expression system is 1 or 2 orders of magnitude more active than the SV40 early promoter in various cell types (Takebe et al., Molecular and Cellular Biology, 8, 466-472 (1988)).

Most preferably the human cytomegalovirus promoter known as the CMV mid/early enhancer/promoter is used in the present invention in order to facilitate constitutive expression of the cloned DNA insert in mammalian cells. CMV promoters are disclosed in Schmidt, E.V. et al., (1990) Mol. Cell. Biol., 10, 4406, and US Patents 5,168,062 and 5,385,839.

In a preferred embodiment of the present invention, the promoter for inducing the phagemid system in prokaryotic cells is selected from deo, osmB, _λPL , β-lac-U5, and CMV promoters. In a more preferred embodiment of the present invention, the β-lac-U promoter is used to induce the phagemid system in E. coli. In the most preferred embodiment, the CMV promoter is used.

In one embodiment of the invention, the peptide or polypeptide of the invention comprises: (a) a leader sequence present only in the coding sequence but absent in the mature protein; (b) a heavy chain of 135-145 amino acids in size The variable region of the variable region, including the first hypervariable region of 4-12 amino acids to be modified; (c) a spacer of ≤ 20 amino acids, which can be shortened or eliminated; (d) the same as described below The variable region of the specially modified light chain; (e) immediately followed by a marker sequence, which sequence is optionally not present in the final injectable preparation. The spacer segment in a ScFv is typically approximately 15 amino acid residues in length, enabling the two variable region chains (heavy and light) to fold into a functional Fv domain. Functional Fv domains retain binding activity with enhanced selectivity and/or specificity.

In another embodiment, (d) above is followed by a marker sequence or marker, which can be used for conjugation, diagnostic and/or identification purposes. In this embodiment, the label is designed to link the peptide or polypeptide of the invention to an agent for therapy or diagnosis of the target cell.

The spacer region of a ScFv can be linear or branched and usually consists of multiple glycine and serine residues of the formula (Gly ₄ Ser) _n and is usually 0-20 amino acids in total, preferably 0-15 amino acids in length , and is linear. Various multimers can be obtained by varying the spacer length as desired. In an embodiment of the present invention, the length of the spacer is 0-5 amino acids. In another embodiment, the spacer is less than 3 amino acids in length (see below for details).

The following are examples of amino acid sequences of scFv molecules of the invention:

ATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCGGCCCAGCCGGCC

61 ATGGCCGAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGGGGGTCCCTG

twenty one E V Q L V E S G G G V V R P G G S L

121 AGACTTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGGCATGAGCTGGGTCCGC

41 R L S C A A S G F T F D D Y G M S W V R

181 CAAGCTCCAGGGAAGGGGCTGGAGTGGGTCTCTGGTATTAATTGGAATGGTGGTAGCACA

61 Q A P G K G L E W V S G I N W N G G S T

241 GGTTATGCAGACTCTGTGAGGGGCCGATTCACCATTCTCCAGAGACAACGCCAAGAACTCC

81 G Y A D S V K G R F T I S R D N A K N S

301 CTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACCGGCCGGTGTATTACGTGGCAAGA

101 L Y L Q M N S L R A E D T A V Y Y C A R

361 ATGAGGGCTCCTGTGATTTGGGCCCAAGTAACCCTGGTCACCGTGTCGAGAGTGGGAGGC

121 W G Q G T L V T V S R G G G

421 GGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGTCTGAGCTGACACAGGACCCTGCT

141 G S G G G G G S G G G G S S E L T Q D P A

481 GTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGACAGCCTCAGAAGC

161 V S V A L G Q T V R I T C Q G D S L R S

541 TATTATGCAAGCTGGTACCAGCAGAAGCAGGACCAGGCCCCTTGTCTTGTCATCATGGGT

181 Y Y A S W Y Q Q K K P G Q A P V L V I Y G

601 AAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACA

201 K N N R P S G I P D R F S G S S S G N T

661 GCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTAACTCC

221 A S L T I T G A Q A E D E A D Y Y C N S

721 CGGGACAGCAGTGGTAACCATGTGGTATTCGGCGGAGGGACCAAAGCTGACCGTCCTAGGT

241 R D S S G N H V V F G G G T K L T V L G

781 GCGGCCGCAGAACAAAAACTCATCTCCAGAAGAGGATCTGAATGGGGCCGCATAG

261 A A A

Add a dotted line below the leading sequence. The _VH regions are encoded by the amino acid sequences in bold. This particular clone was derived from germline _VH3 -DP32; however, the germline of each clone depends on its specific origin (see below). The boxed amino acid sequence encodes the _VH -CDR3 from which all cloned hypervariable regions were derived. The spacer connecting the _VH and _VL regions is a flexible polypeptide encoded by the amino acids indicated by italics. Finally, the _VL region is shown. The fused _VL segments in all clones were derived from a single unmutated V gene of the germline IGLV3SI, here followed by the c-myc marker, with a wavy line below it. Its complete amino acid sequence is identical to SEQ ID NO:25.

First, the repertoire of _VH fragments (from 49 germlines) was prepared by PCR using the rearranged V-genes of unimmunized human peripheral blood lymphocytes (referred to as "naive") by the provider of the library. Composition"). The origin (germline) of a _VH -sequence can be identified by homology determination (Blast search) using one of the following websites:

The binding characteristics of an antibody can be optimized in one of several ways. Approaches to optimize antibodies for higher binding affinity compared to the original lead compound are based on substituting amino acid residues in the lead compound in order to introduce higher variability, or to extend the sequence. For example, an entire native _VL region can be replaced with a _VL region from a different antibody subtype.

Another approach to optimize binding affinity is to construct phagemid display mutagenesis libraries. In the phagemid display mutagenesis library, oligonucleotides are synthesized such that each amino acid sequence of the core sequences within the _VH and _VL CDR3 respectively is substituted with any other amino acid, preferably with conservation known in the art way instead. The present invention provides a set of special antibody scFv displayed on phage, wherein the displayed antibody fragments and soluble antibody fragments can be extracted from phage virus particles with the same biological activity.

Phage display libraries used in the present invention were constructed using naive human peripheral blood lymphocytes, and the Fv peptides were selected with previously uncharacterized and unpurified antigens on the surface of target cells. As used herein, a previously uncharacterized and unpurified antigen refers to a ligand present on the surface of cells that has not been identified, characterized, isolated, or purified by biochemical or molecular methods prior to the conduct of this study, and which was discovered or predicted in this study by the observed selective and/or specific binding to isolated antibody fragments.

The scFvs of the present invention exhibit enhanced binding to target cells. The enhanced binding is to a specific surface marker. Specific surface markers are molecules that are sequestered in the cell membrane and are accessible to circulating recognition molecules. The presence of surface markers enables the development of phage display technology through the biopanning technique disclosed herein. In the present invention, specific surface markers are used to characterize and differentiate various cell types and serve as binding sites for various forms of Fvs. Various hematopoietic cell types can be distinguished based on their characteristic surface markers. Similarly, diseased or cancer cells possess surface markers that are specific to that cell type and stage.

Screening of scFv clones can be achieved by two different biopanning methods:

1. Direct screening using diseased cells or cancer cells as target cells, and

2. Using steps such as a first, such as normal cell, in a second state, e.g., activated, excited, modified, altered, or dysregulated, for stepwise selection of the first in the second state The binding site of the species cell includes a substantially exposed or displayed unknown ligand. Through immunological cross-reactivity, the resulting clones can selectively and/or specifically bind new and unknown ligands on the second cell after a subsequent panning step or screening step. After further optional amplification and subsequent purification, a targeting molecule can be constructed from the recognition site of the purified recognition molecule, which is selective and/or specific for the unknown ligand on the second cell .

In one embodiment of the present invention, the first cell may be a normal cell, the first state is an inactive state, and the second state is an activated, excited, modified, altered, or disordered state. The second cell in said stepwise selection may be a human cell. In a preferred embodiment of the invention, the second type of cell in said stepwise selection is a diseased cell. In a more preferred embodiment of the present invention, the second cell in said stepwise selection is a cancer cell such as, but not limited to, carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blast tumor, seminoma, and melanoma. In a more preferred embodiment, said second cell is a leukemia cell. In a most preferred embodiment, said second cell is an AML cell.

A more preferred embodiment of the present invention provides a peptide or polypeptide, wherein the selective and/or specific binding of the peptide or polypeptide to the ligand on the second cell is mainly caused by the first high determined by the variable region. In another more preferred embodiment, the first hypervariable region is CDR3 having an amino acid sequence selected from SEQ ID NOs: 8-24.

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

The enhanced binding to cancer cells is most likely due to overexpression of the ligand in cancer cells and/or exposure of the binding site relative to expression in normal cells. As used herein, the term ligand overexpression is defined as the expression of a gene or its product, or the increased expression of a gene, that is normally silent in a particular cell type and/or at a particular stage of the cell cycle , the gene is expressed at basal levels under normal, non-malignant conditions in a particular cell type.

In a more preferred embodiment of the invention, the target cells of said biopanning method are contained in a cell suspension. Hematopoietic cells were obtained in suspension and subjected to biopanning involving mixing the phage library with the blood cell suspension followed by washing with several buffers. Phage are extracted from the human cells, amplified, and sequenced to display antibody fragments.

In another more preferred embodiment of the invention, said suspension of blood cells comprises leukemia cells. In a most preferred embodiment, said suspension of blood cells comprises AML cells. In another embodiment of the invention, said target cells are derived from an isolated organ or part thereof.

In another embodiment of the invention said target cell or said second cell is derived from a cell line. Cell lines can be cultured and manipulated such that they facilitate the determination of the binding characteristics of the Fv clones. Additionally, cell lines can be used to develop diagnostic kits.

In a preferred embodiment, the cell line is a hematopoietic cell line, such as, but not limited to, the following cell lines: Jurkat, Molt-4, HS-602, U937, TF-1, THP-1, KG-1 , ML-2 and HUT-78 cell lines.

In a preferred embodiment of the invention, the CDR3 region is constructed, inserted, linked or fused in or on any one of the 84 cassettes (SEQ ID NOs: 30-113). In a more preferred embodiment, the CDR3 region is constructed, inserted, linked or fused in 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 or on any one of. In a most preferred embodiment, the CDR3 region is constructed, inserted, linked or fused at the C-terminus of the cassette of SEQ ID NOs: 61, or any one of the above sequences having at least 90% sequence similarity to it superior.

In one embodiment, the amino acid sequence of the cassette is apparently fixed, while the substituted, inserted, or attached sequences may be highly variable. The cassette may consist of several domains, each of which includes a function important to the final construct. A cassette according to a specific embodiment of the present invention comprises, starting from the N-terminus, framework region 1 (FRI), CDRI, framework region 2 (FR2), CDR2, and framework region 3 (FR3).

In one embodiment of the invention, different regions within the cassette may be substituted. For example, the CDR2 and CDR1 hypervariable regions within the cassette may be substituted or modified by non-conservative or preferably conservative amino acid substitutions. More specifically, the CDR2 and CDR1 regions selected from the contiguous amino acid cassettes of SEQ ID NOs: 30-113 or fragments thereof may be substituted with SEQ ID NOs: 115 and 114, respectively. More specifically, selected from 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 the CDR2 and CDR1 regions of the consecutive amino acid boxes of fragments thereof, may be substituted with SEQ DD NOs: 115 and 114, respectively.

In a preferred embodiment of the present invention, said peptide or polypeptide comprises a heavy chain and a light chain, and each chain comprises first, second and third hypervariable regions which are CDR3, CDR2 and CDR1 respectively district. The binding selectivity and specificity are determined in particular by the CDR3 region of one 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 of the light chain and CDR1 regions, and preferably determined by the CDR2 and CDR1 regions of the heavy chain. Said binding selectivity and specificity are also aided by upstream or downstream regions flanking said 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 SEQ ID NOs: 8-24.

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

In a most preferred embodiment, the CDR3 region has the same amino acid sequence as SEQ ID NOs: 8.

In addition to the heavy and light chains, Fv includes a flexible spacer segment of 0-20 amino acid residues. The spacer segment can be branched or linear. Two possible sequences of this spacer are identical to SEQ ID NOs: 123 and 124.

A preferred embodiment of the present invention is a scFv having the same CDR3 sequence as SEQ ID NO:8 and the complete scFv sequence as SEQ ID NO:25.

Another preferred embodiment of the present invention is a scFv having the same CDR3 sequence as SEQ ID NO: 20 and the same complete scFv sequence as SEQ ID NO: 203.

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

In one embodiment of the invention, the Fv peptide comprises the CDR1 and CDR2 regions of the variable heavy chain, which itself comprises a cassette having an amino acid sequence selected from SEQ ID NOs: 30-113; the CDR3 region, preferably variable The CDR3 region of the heavy chain, which has an amino acid sequence selected from SEQ ID NO: 8-24; the upstream region flanking the CDR3 region, which has the amino acid sequence of SEQ ID NO: 117; the downstream region flanking the CDR3 region, which has The amino acid sequence of SEQ ID NO: 116; the spacer segment with 0-20 amino acid residues of SEQ ID NO: 123 or 124; the sequence is the variable light chain region of SEQ ID NO: 7.

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

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

Another preferred embodiment of the present invention provides an Fv molecule comprising a first chain having first, second and third hypervariable regions, and a chain having first, second and third hypervariable regions The second chain, wherein one of the hypervariable regions of the first chain has an amino acid sequence selected from SEQ ID NO: 8-24, and wherein one of the hypervariable regions of the second chain has an amino acid sequence selected from SEQ ID NO: : the amino acid sequences of 1-6 and 125-202, and wherein the first, second and third hypervariable regions are CDR3, CDR2 and CDR1 regions respectively, and wherein the Fv is scFv or dsFv, and selectively Has one or more tags.

Another embodiment of the present invention provides a peptide or polypeptide, (i) wherein said first chain and second chain each comprise a first hypervariable region selected from SEQ ID NOs: 8-24; Or (ii) wherein, the first hypervariable region of the first chain and the second chain is selected from SEQ ID NOs: 8-24 and identical thereto; or (iii) wherein, the first chain of The first hypervariable region is selected from SEQ ID NOs: 8-24, and the first hypervariable region of the second chain is selected from SEQ ID NOs: 1-6 and 125-202; or (iv) wherein, the first The first hypervariable region of one chain is selected from SEQ ID NOs: 1-6 and 125-202, and the first hypervariable region of said second chain is selected from SEQ ID NOs: 8-24.

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

For all amino acid sequences (e.g., CDR regions, CDR flanking regions) of ≤ 25 amino acid residues disclosed and specified herein, it can be understood and regarded as another embodiment of the present invention that the Said amino acid sequence includes within its scope one or two amino acid substitutions, and said substitutions are preferably conservative amino acid substitutions. For all amino acid sequences > 25 amino acid residues disclosed and specified herein, it is to be understood and regarded as another embodiment of the present invention that said amino acid sequences include within their scope the same as the original Amino acid sequences having >90% sequence similarity (Altschul et al., Nucleic Acids Res., 25, 3389-3402 (1997)). Similar or homologous amino acids are defined as non-identical amino acids having similar properties, eg, acidic, basic, aromatic, size, positively or negatively charged, polar, non-polar amino acids.

Percent amino acid similarity or homology or sequence similarity is determined by comparing the amino acid sequences of two different peptides or polypeptides. The two sequences are aligned, usually by using one of a variety of computer programs designed for this purpose, and the amino acid residues at each position are compared. Amino acid identity or homology is then determined. An algorithm is then applied to determine percent amino acid similarity. In general, comparing amino acid sequences is preferred because this greatly increases the sensitivity for detecting close relationships between peptide, polypeptide or protein molecules. For protein comparisons, the presence of conservative amino acid substitutions is taken into account, whereas mismatches may result in positive scores if non-identical amino acids have 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 interchanged between the two chains, and the three hypervariable regions within and/or between said chains exchange points between each other.

Those of ordinary skill in the art can understand that to verify the specific and/or selective binding of the peptide or polypeptide of the present invention, an appropriate negative control must be used. A suitable negative control may be a peptide or polypeptide whose amino acid sequence is nearly identical to the peptide or polypeptide of the invention, the only difference being the hypervariable CDR3 region. Another suitable negative control may be a peptide or polypeptide having the same size and/or overall three-dimensional structure as the peptide or polypeptide of the invention, but having a completely unrelated amino acid sequence. Another suitable negative control may be a peptide or polypeptide having completely different physical and chemical characteristics compared to the peptide or polypeptide of the invention. The negative controls used to develop the present invention were named N14, which has the same CDR3 sequence as SEQ ID NO:28, and C181, which has the same CDR3 sequence as SEQ ID NO:29. However, other negative controls may also be suitable.

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

In a preferred embodiment of the invention, in order to optimize the selective binding of the Fv, the CDR3 sequence conferring the main binding selectivity and/or specificity of the Fv can be transferred into any other heavy chain germline. More specifically, it can be transferred to one of 84 possible heavy chain germlines. These 84 lines (SEQ ID NOs: 30-113) included (a) the line in which the alleged phage clone was originally isolated, (b) the 48 other lines provided in the phage display library, and (c) 35 other strains claimed in the present invention (Tomlinson et al., J. Mol. Biol., 227(3):776-798 (1992)). The local linear or three-dimensional environment of the CDR3 region is consistent with the CDR3 region itself and may play a role in guiding or promoting proper CDR3 binding. For example, the present invention also includes peptides having any of the CDR3 sequences described in SEQ ID NOs: 8-24, 125, and derived from any of the 49 germline sequences (SEQ ID NOs: 3032, 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).

Germline DP-32 is a cassette for several clones of the invention. The C-terminus of this germline has been replaced with a consensus sequence to facilitate phage display library preparation. The carboxy-terminal amino acid of SEQ ID NO:61 has been replaced by the 7 amino acid sequence of SEQ ID NO:122.

The CDR3 region of the Fvs of the present invention may include the core sequence ^Arg / _Gly/Lys Phe Pro that can specifically bind to AML cells. Eight examples of the CDR3 region are shown in Table 2. Although the motif coincides in each case with the three N-terminal amino acid residues of the CDR3 region, it can also be located elsewhere in the CDR3 region. In addition, the motif is an anchor or binding region used to build or construct a part of a larger binding or targeting or recognition molecule or a binding motif used alone as a targeting vector.

In another embodiment of the present invention, a binding motif is provided, which includes the amino acid sequence R ₁ -X Phe Pro-R ₂ , wherein each of R ₁ and R ₂ includes 0-15, preferably 1-9 amino acid residues, and, where X is Arg, Gly or Lys. Most preferably, CDR3 comprises the amino acid sequence R ₁ -X Phe Pro-R ₂ , wherein R ₁ and R ₂ 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, 1-1000 amino acids may be added at the C-terminal or N-terminal of said peptide, while said peptide maintains its biological activity. In a preferred embodiment of the present invention, 150-500 amino acids can be added at the C-terminal or N-terminal of said peptide or polypeptide, while said peptide retains its biological activity. In a preferred embodiment of the present invention, 800-1000 amino acids can be added to the C-terminal or N-terminal of the peptide or polypeptide, while the peptide or polypeptide retains its biological activity.

An example of extending the core amino acid sequence is by constructing a full-sized immunoglobulin Ig using a lead compound as the core of the Ig, e.g. Activation of cytolytic activity induces endogenous cytolytic activity (eg, IgG1, IgG2 or IgG3). The full size Ig is likely to be of the class of immunoglobulins with strong binding antibodies such as IgG4. When bound, the full-sized Ig can act in one or more of a variety of ways, for example, by initiating an immune response as a marker of the body's defense mechanisms, transducing intracellular cell signaling, or causing damage to target cells. destroy.

A preferred embodiment of the invention provides Ig molecules expressed as recombinant polypeptides and produced in eukaryotic cell systems. In a preferred embodiment of the invention said Ig polypeptide is an IgG polypeptide and it is produced in a mammalian cell system. In a more preferred embodiment, said mammalian cell system includes a CMV promoter.

In a preferred embodiment of the present invention, said IgG molecule comprises CDR3, CDR2 and CDR1 hypervariable regions located on the light chain and the heavy chain. In a more preferred embodiment of the present invention, said Fv molecule comprises CDR3, CDR2 and CDR1 regions having SEQ ID NOs: 8, 115 and 114, respectively. The CDR3, CDR2 and CDR1 regions may be of the heavy chain and of the light chain.

Another preferred embodiment of the present invention provides a light chain having the same sequence as SEQ ID NO: 27 and a heavy chain having the same sequence as SEQ ID NO: 26, or a heavy chain having at least 90% sequence similarity thereto. chain and light chain IgG molecules. In a most preferred embodiment of the invention, the two heavy chains of IgG are identical and the two light chains of IgG are identical.

In another embodiment, the peptides of the invention are constructed to fold into a multivalent Fv format.

The invention provides Y1 or Y17 peptides or polypeptides comprising scFv molecules. Herein, a scFv is defined as a molecule consisting of the heavy chain variable region of a human antibody and the light chain variable region of a human antibody, which may be the same or different, and wherein the variable region of the heavy chain is identical to the variable region of the light chain The variable regions of the combination, linkage, fusion or covalent attachment or association.

The Y1 and Y17 scFv constructs may be multimers (eg, dimers, trimers, tetramers, etc.) of scFv molecules that incorporate one or more hypervariable domains of the Y1 or Y17 antibody. All scFv-derived constructs and fragments retain enhanced binding characteristics to favor selective and/or specific binding of target cells relative to other cells. The binding selectivity and/or specificity is mainly determined by the hypervariable regions.

The hypervariable loops within the light and heavy chain variable domains are defined as complementarity determining regions (CDRs). There are CDR1, CDR2 and CDR3 regions within the heavy and light chains, respectively. Among them, the CDR3 region of the heavy chain was the most varied. The CDR3 region is considered to be the most exposed region of the Ig molecule and, as disclosed herein, it is the site that primarily determines the observed selective and/or specific binding characteristics.

The Y1 and Y17 peptides of the invention can be constructed so as to fold into a multivalent Fv format. Multimeric forms of Y1 and Y17 were constructed to improve binding affinity and specificity, and to increase half-life in blood.

Other researchers have produced multivalent forms of scFv. One approach is to link the two scFvs with a linker. Another approach involves the use of disulfide linkages between the two scFvs. The simplest methods for producing dimeric or trimeric Fv are disclosed in: Holliger 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 dimers by adding the FOS sequence and the JUN protein region to form a leucine zipper between the C-terminals of the scFv. Kostelny SA et al., J Immunol.1992 Mar 1;148(5):1547-53; De Kruif J et al., J Biol Chem.1996 Mar 29;271(13):7630-4. Another method was devised to make tetramers by adding a streptavidin coding sequence at the C-terminus of the scFv. Streptavidin is composed of four subunits, so when the scFv-streptavidin is folded, the four subunits accommodate itself so as to form a tetramer. Kipriyanov SM et al., Hum Antibodies Hybridomas, 1995;6(3):93-101. In another approach, a free cysteine is introduced into the protein of interest for the preparation of dimers, trimers and tetramers. The protein of interest and the free cysteine were cross-linked using peptide-based cross-linkers with varying numbers (2-4) of maleimide groups. Cochran JR et al., Immunity, 2000 Mar;12(3):241-50.

In this system, phage libraries (as described above) are designed to display scFvs, which fold into a monovalent form of the Fv region of an antibody. Additionally, the constructs are suitable for bacterial expression, also as described above. The scFvs produced by genetic engineering methods include heavy and light chain variable regions connected by a continuously encoded 15 amino acid flexible peptide spacer. A preferred spacer is (Gly ₄ Ser) ₃ . The length of the spacer and its amino acid composition provide a modest spacer which allows the _VH and _VL domains to fold into a functional Fv domain which provides efficient binding to its target.

The present invention relates to Y1 and Y17 multimers prepared by any method known in the art. A preferred method of producing multimers, especially dimers, utilizes cysteine residues to form disulfide bonds between two monomers. In this embodiment, dimers are formed by adding a cysteine to the carboxyl terminus of the scFvs (referred to as Y1-cys scFv or Y1 dimer) in order to favor dimer formation. Following preparation of the DNA constructs (see Examples 2D and 6D) and use for transfection, Y1 dimers were expressed in production vectors and refolded in vitro. The proteins were analyzed by SDS-PAGE, HPLC, and FACS. Two liter batch fermentations of the antibodies were performed. Refolding in arginine following expression of Y1-cys in E. coli strain BL21. After refolding, dialysis and purification were performed by Q-sepharose and gel filtration (sephadex 75). Two peaks were detected by SDS-PAGE (non-reducing) and gel filtration. The two peaks were collected separately and analyzed by FACS. Monomer and dimer binding to Jurkat cells was examined by FACS. Binding of the dimer required only 1/100 the amount of monomeric antibody for the same level of staining, indicating the higher avidity of the dimer. Conditions for dimer refolding were determined and after dialysis, chromatography, and gel filtration steps produced material containing >90% dimer (mg amount). Purified dimers were characterized by gel filtration and SDS-PAGE analysis under oxidative conditions. The binding capacity of the dimer was confirmed by radioreceptor analysis, ELISA and FACS analysis.

The CONY1 scF antibody fragment is derived from Y1 scFv. The DNA sequence encoding the myc tag of the Y1 scFv was removed and replaced with a synthetic oligonucleotide DNA sequence encoding lysine-alanine-lysine (KAK).

To compare the binding of scFv monomer (also known as CONY1) to Y1 dimer, a binding competition experiment was performed in vitro with KG-1 cells. Additionally, the experiments compared the binding of intact Y1 IgG to scFv Y1 monomer. For this study, Y1 IgG was labeled with biotin. This study found that Y1 IgG can compete with IgG Y1-biotin. Irrelevant human IgG cannot compete with labeled Y1 IgG. Y1 scFvs (5 μg and 10 μg) could partially compete with Y1 IgG-biotin (50 ng). It was also demonstrated that lng of IgGY1-FITC bound to KG-1 cells (serum-free) to the same extent as 1 μg of scFv-FITC, however, most Y1 IgG binding was "blocked" in the presence of serum. This study also demonstrated that the binding capacity of Y1 dimers was at least 20 times higher than that of scFV monomers, a result confirmed by radioreceptor assays, ELISA or FACS analysis.

In another embodiment, in addition to cysteine, lysine-alanine-lysine (referred to as YI-cys-kak scFv) is added at the carboxyl terminus. 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 QAPVLVlYGK 180

181 NNRPSGIPDR FSGSSSGNTA SLTITGAQAE DEADYYCNSR

DSSGNHVVFG GGTKLTVLGG 240

241 GGCKAK

181 NNRPSGIPDR FSGSSSGNTA SLTITGAQAE DEADYYCNSR

DSSGNHVVFG GGTKLTVLGG 240

241 GGCKAK

Y1-cys-kak is produced in bacteria in the λ-pL vector. Expression in the lambda-pL vector was induced by raising the temperature to 42°C. Inclusion bodies were obtained from induced cultures and semi-purified by aqueous solution to remove undesired soluble proteins. By DTT reduction, the inclusion bodies were solubilized in guanidine and refolded in vitro in an arginine/OX-glutathione based solution. After refolding, the protein was dialyzed and concentrated into a buffer containing urea/phosphate buffer by tangential flow filtration. The protein was purified and concentrated by ion chromatography in SP-columns.

In order to obtain a higher level of expression of CONY1 scFv and Y1-cys-kak scFv in E. coli, we introduced an amino acid encoding an alanine residue at position 2 of the N-terminus of the scFv construct. With this newly modified construct we achieved a 4-fold level of expression.

ELISA analysis was performed to demonstrate the difference in binding of monomeric (CONY1 scFv - also known as Y1-kak) and dimeric Y1-kak (cysteine dimer) to the platelet-derived antigen GPIb (glycocalicin) . Polyclonal anti-single chain antibodies and/or novel anti- _VL (from rabbit) and anti-rabbit HRP were used to detect binding to GPIb. Dimers are approximately 20-100 times more active than monomers. For example, to achieve an OD unit of 0.8, 12.8 mg/ml of monomer was used in comparison to only 0.1 mg/ml of dimer. See Figure 12.

The dimers were characterized by SDS-page electrophoresis, gel filtration chromatography, ELISA, radioreceptor binding and FACS. Dimers have a higher apparent affinity than monomers due to avidity. This effect was confirmed on glycocalicin by ELISA, on KG-1 cells by FAGS, and by competition in a radioreceptor assay.

HPLC was performed after refolding and purification through a Superdex 75 gel filtration column to characterize the dimer. In Figure 10, Yl-cys-kak (dimer) is the first peak on the left (approximately 10.8 minutes), while the subsequent peaks are monomers (approximately 12 minutes). Based on protein size markers processed on the same column, the dimer is approximately 52 kDa and the monomer is 26 kDa. By changing the refolding conditions (concentration of oxidant and concentration of protein in refolding buffer), the equilibrium between dimers and monomers can be altered. The dimer and 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, monomer is observed due to reduction between the two monomers, and in the non-reduced form, two populations are observed (as seen in gel filtration experiments), the monomer fraction is approximately is 30kDa, and the dimer fraction is approximately 60kDa.

Additionally, FACS binding analysis performed on KG-1 cells confirmed that dimers are more sensitive than monomers when performing two- or three-step binding assays. Dimers labeled directly by FITC performed slightly better than monomers (using 10-fold less material). When the dimer was used as a competitor, radioreceptor assays on KG-1 cells showed that the dimer was 30-fold more efficient than the monomer.

Changing the length of the spacer segment is another preferred method for producing dimers, trimers, and tetramers (commonly referred to in the art as diabodies, triabodies, and tetrabodies, respectively). Dimers are formed under conditions where the spacer segment connecting the two variable chains of the scFv is shortened overall. This shortened spacer segment inhibits the folding of the two variable chains from the same molecule into a functional Fv domain. Instead, the domain is forced to pair with a complementary domain of another molecule so as to form two binding domains. In a preferred approach, a spacer fragment of only 5 amino acids (Gly ₄ Ser) is used for diabody construction. Such dimers may be formed from two identical scFvs, or may consist of populations of different scFvs, and retain the selective and/or specificity-enhanced activity of the parental scFv(s), and/or have increased binding strength or affinity.

In a similar format, triabodies are formed when the spacer segment linking the two variable chains of the scFv is shortened overall to less than 5 amino acid residues, avoiding the folding of the two variable chains from the same molecule into Functional Fv domains. Instead, three separate scFvs associate to form trimers. In a preferred method, the triabody is obtained by completely removing the flexible spacer. The triabody may consist of three identical scFvs, or of two or three different populations of scFvs, and retains the selective and/or specificity-enhanced activity of the parental scFv(s), and/or have increased binding strength or affinity.

Tetrabodies are formed in a similar manner under conditions in which the spacer fragment linking the two variable chains of the scFv is overall shortened to less than 5 amino acid residues, avoiding the folding of the two variable chains from the same molecule into a functional The Fv domain. Instead, four individual Fv molecules associate to form tetramers. Tetrabodies can be formed from four identical scFvs, or from 1-4 independent units from different scFv populations, and should retain the selective and/or specificity-enhanced binding activity of the parental scFv(s), and/or have increased binding strength or affinity.

Whether triabodies or tetrabodies are formed under conditions where the spacer fragments are generally less than 5 amino acid residues in length depends on the amino acid sequence of the particular scFv(s) in the mixture and the reaction conditions.

In a preferred method, tetramers are formed by biotin/streptavidin association. A novel fermentation construct (referred to herein as Y1-biomarker or Y1-B) that can be enzymatically labeled with biotin was prepared. A sequence that is a substrate for the BirA enzyme was added at the Y1 C-terminus. The BirA adds biotin on lysine residues within this sequence. Cloning and expression of the Y1-biomarker in E. coli. The inclusion body material is separated and refolded. The folded protein was >95% pure and >100 mg was obtained from 1 liter culture (small scale, non-optimized conditions). The molecular weight of this form was found to be similar to that of scFv according to HPLC, SDS-PAGE and mass spectrometry. The Y1-biomarker was found to be the most stable reagent for FACS analysis. However, when examining the binding of the Y1-biomarker to KG-1 cells in the presence of serum, higher concentrations (10-fold higher) were required to obtain comparable binding to that in the absence of serum. However, this construct has the advantage of specific biotinylation, wherein the binding site of the molecule remains intact. Additionally, each molecule is labeled with only one type of biotin - each molecule receives one biotin at its carboxyl terminus.

For limited labeling of one biotin/molecule at desired sites, tetramers with streptavidin can be produced. The tetramer was formed by incubating Y1-B with streptavidin-PE.

FACS analysis showed that the tetramer prepared by Y1-biomarker and streptavidin-PE was 100-1000 times more sensitive than Y1 scFv monomer in the absence of serum. Y1-biomarker tetramers with streptavidin-PE showed specific binding to one of the Y1-responsive cell lines (KG-1). The response is highly differential from background binding and has high sensitivity to detect small amounts of receptor. FACS assessment of normal whole blood with Y1-SAV tetramers revealed no hyperresponsive populations. Monocytes and granulocytes are positive to a lesser extent. In cell lines with positive results, such as KG-1 cells, the reactivity of the tetramer was at least 100-fold higher.

The tetramers are then incubated with cell samples. A low dose of Y1 tetramer (5 ng) bound the cell line (KG-1) well, producing a 10-20 fold higher response than previously observed with other Y1 antibody formats. Weak responses were observed when negative cell lines were examined with various doses of tetramer.

One embodiment of the invention provides a method for identifying targeting molecules that bind to unknown immunologically cross-reactive binding sites on first and second cells, the method comprising (a) on the first target cell One or more biopanning steps performed with the target cells in a second state other than the first state substantially exposing or displaying a binding site comprising an unknown ligand in order to produce a first population of recognition molecules (b) a subsequent biopanning and/or selection step starting from the pool of recognition molecules obtained in step (a), said step being carried out on a second cell displaying a binding sites of an unknown ligand immunologically cross-reactive with the unknown ligand of said first cell, in order to produce a second population of recognition molecules; (c) amplifying and purifying the second recognition of step (b) population of molecules; and (d) constructing from the recognition sites of the purified recognition molecules of step (c) peptides or polypeptides comprising targeting molecules that are selective and specific for the unknown ligand on the second cell / or specificity.

A preferred embodiment provides that the first cell is a normal cell, said first state is an inactive state, and said second state is an activated, excited, modified, altered or dysregulated state. In a more preferred embodiment, said second cell is a diseased cell. In a more preferred embodiment, said diseased cells are cancer cells. Cancer cells can be, but are not limited to, carcinomas, sarcomas, leukemias, adenomas, lymphomas, myelomas, blastomas, seminomas, and melanomas. In a more preferred embodiment, the cancer cells are leukemia cells. In a most preferred embodiment, said leukemia cells are AML cells.

One embodiment of the invention provides the use of a peptide or polypeptide optionally associated or attached, coupled, combined, linked or fused to a pharmaceutical agent for the manufacture of a medicament. In a preferred embodiment, the drug has activity against diseased cells. In a more preferred embodiment, the activity is against cancer cells. The cancer cells are, but are not limited to, carcinomas, sarcomas, leukemias, adenomas, lymphomas, myelomas, blastomas, seminomas, and melanomas. In a more preferred embodiment, the cancer cells are leukemia cells. In a most preferred embodiment, said leukemia cells are AML cells.

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

Another embodiment provides the use of a peptide or polypeptide of the invention associated or attached, coupled, combined, linked or fused to a pharmaceutical agent for the manufacture of a medicament. The drug may have the activity of treating diseased cells, more preferably specifically treating cancer cells. The cancer cells may be, but are not limited to, carcinomas, sarcomas, leukemias, adenomas, lymphomas, myelomas, blastomas, seminomas, and melanomas. In a more preferred embodiment, the medicament has anti-leukemic cell activity. In a most preferred embodiment, the medicament has activity in the treatment of AML cells. The activity of the drug against the cells may result in a delay of cancerous growth, complete inhibition of all growth, or killing of cancer cells.

In one embodiment of the invention, the activity of the medicament or pharmaceutical composition is cytostatic.

The peptides or polypeptides of the invention may be used for the preparation of compositions, preferably pharmaceutical compositions, for inhibiting the growth of cancer cells, preferably leukemia cells, most preferably AML cells. In a preferred embodiment of the present invention, the peptide or polypeptide of the present invention can be used to prepare a composition for inhibiting the growth of cancer cells, said composition comprising at least one drug ligand selective for said cancer cells and/or specific compounds.

The peptide or polypeptide of the present invention can be administered to patients alone, or as a drug or pharmaceutical composition in combination with a pharmaceutically effective amount of a pharmaceutical agent, a pharmaceutically effective carrier, and optionally associated, coupled, or linked with an adjuvant , or combined use. The pharmaceutical composition may include proteins, diluents, preservatives and antioxidants (see Osol et al. (eds.), Remington's Pharmaceutical Sciences (16th ed), Mack Publishing Company, (1980)).

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

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

In a preferred embodiment, the radioisotopes used include gamma emitters, positron emitters and x-ray emitters for localization and/or therapy, and beta emitters and alpha rays for therapy emitter.

In a specific embodiment of the ^present invention, ^the therapeutic isotope is selected from ¹¹¹ indium, ¹¹³ indium, ^99m rhenium, ¹⁰⁵ rhenium, ¹⁰¹ rhenium, 99m technetium, ^121m tellurium, 122m tellurium, ^125m tellurium, ¹⁶⁵ thulium, ¹⁶⁷ thulium, ¹⁶⁸ thulium ^{, 123} iodine, ¹²⁶ iodine, ¹³¹ iodine, ¹³³ iodine, ^81m krypton, ³³ xenon, ⁹⁰ yttrium, ²¹³ bismuth, ⁷⁷ bromine, ¹⁸ fluorine, ⁹⁵ ruthenium, ^{97 ruthenium, 103} ruthenium, ¹⁰⁵ ruthenium, ¹⁰⁷ mercury, ²⁰³ mercury , ^67Ga and ^68Ga , etc.

In another embodiment of the present invention, the anticancer agent is selected from doxorubicin, adriamycin, cisplatin, paclitaxel, calicheamicin, vincristine, cytarabine (Ara-C), cyclophosphamide, prednisone, daunorubicin, idarubicin, fludarabine, chlorambucil, interferon alfa, hydroxyurea, temozolomide, thalidomide, and blepharil Mycin, and its derivatives.

One embodiment of the invention provides a method of inhibiting the growth of cancer cells comprising contacting said cancer cells with an amount of a peptide or polypeptide of the invention. In a preferred embodiment, the cancer cells may be, but are not limited to, carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma, and melanoma. In a more preferred embodiment, the cancer cells are leukemia cells. In a most preferred embodiment, said leukemia cells are AML cells. One embodiment of the invention allows for in vivo and ex vivo treatment of said patient. A more preferred embodiment of the present invention may perform ex vivo purification of autologous bone marrow in order to remove abnormal stem cells.

In a more preferred embodiment of the invention, the blood of a leukemia patient can be circulated ex vivo through a system comprising a peptide or polypeptide of the invention coupled to an anticancer agent. After removal of bound cells and unbound anticancer agent, the blood cells can be reintroduced into the patient. Alternatively, the blood of a leukemia patient can be circulated ex vivo by a system comprising the peptide or polypeptide of the invention attached to a solid phase. Cells that have passed through the system and are not bound to the peptide or polypeptide of the invention attached to the solid phase can be reintroduced into the patient.

In another preferred embodiment of the invention, said peptide or polypeptide is used ex vivo in an autologous bone marrow suspension in order to remove abnormal stem cells prior to transplantation. Purification of abnormal stem cells can be carried out by passing the suspension through a solid support (such as, but not limited to, magnetic beads and affinity columns) on which the peptide or polypeptide of the present invention is bound (i.e. targeting molecule), construct, fragment, fragment of a construct or construct of fragments. The ex vivo bone marrow thus purified can then be used for autologous bone marrow transplantation. This preferred embodiment is based on the identification in the present invention of a phagemid clone (Y1) capable of binding stem cells released from the bone marrow of leukemia patients but not from the bone marrow of healthy donors. Similarly, Y1 phagemid clones were able to bind blast cells determined to be abnormal by FACS analysis, and to leukemic cells.

Blast cells are defined herein as primary cells, which are the precursors of all circulating cells in mammals. Due to their progenitor character, blast cells are not present in the circulatory system of adult organisms in significant numbers. The presence of circulating blast cells in the absence of exogenous stimuli may indicate malignancy. For example, a malignancy of the hematopoietic system, and its subsequent disappearance, may indicate a reduction in malignancy disease.

In another embodiment of the invention, said pharmaceutical composition is used for prophylactic purposes.

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

Herein, a mixture is defined as two or more molecules or particles of different types contained within one formulation. The different types of molecules form covalent or non-covalent chemical bonds.

In one embodiment of the invention, a peptide or polypeptide of the invention is linked, fused or conjugated to a pharmaceutical agent.

In another embodiment of the invention, the link between the peptide and the pharmaceutical agent is a direct link. In this context, a direct link between two or two molecules is obtained by a chemical bond between elements or groups of elements of said molecules. For example, the chemical bond can be an ionic bond, a covalent bond, a hydrophobic bond, a hydrophilic bond, an electrostatic bond, or a hydrogen bond. The linkage may be selected from, but not limited to, amine, carboxyl, amide, hydroxyl, peptide and disulfide linkages. The direct link may preferably be a protease resistant bond.

In another embodiment, the linkage between the peptide and the pharmaceutical agent is effected by a linker compound. In this specification and claims, a linker compound is defined as a compound that joins two or more moieties together. The linker can be linear or branched. Branched linker compounds may consist of bi-, tri-, or tetra- or more-branched compounds. The linker compound can be, but is not limited to, dicarboxylic acids, maleimide hydrazides, PDPH, carboxylic acid hydrazides and small peptides. Examples of other linker compounds include: dicarboxylic acids, such as succinic acid, glutaric acid, and adipic acid; maleimide hydrazides, such as N-[ε-maleimide caproic acid] hydrazide, 4-[ N-maleimidomethyl]cyclohexane-1-carboxyhydrazide and N-[κ-maleimidoundecanoic acid]hydrazide; PDPH linkers, such as those conjugated to sulfhydryl-responsive proteins (3-[2-pyridyldithio]propionohydrazide). selected from carboxylic acid hydrazides of 2-5 carbon atoms; and direct ligation using a small peptide linker between a free sugar such as the anticancer drug doxorubicin and the 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 Tag.

The peptides and polypeptides of the invention may be administered by any known method, such as intravenous, intramuscular, subcutaneous, topical, intratracheal, intrathecal, intraperitoneal, intralymphatic, intranasal, sublingual, oral, rectal, vaginal, respiratory , buccal, intradermal, percutaneous or intrapleural.

For intravenous administration, the formulations are preferably prepared so that the dosage administered to the patient is from about 0.1 mg to about 1000 mg of an effective amount of the desired composition. More preferably, the dose administered is in the range of about 1 mg to about 500 mg of the desired compound. The compositions of the present invention are effective over a wide dosage range and depend on various factors such as the specific disease to be treated, the half-life of the peptide or polypeptide-based pharmaceutical composition in the patient, the pharmaceutical The physical and chemical characteristics of the reagents and pharmaceutical compositions, the mode of administration of said pharmaceutical compositions, the particular conditions of the patient to be treated or diagnosed, and other factors deemed important by the treating physician.

Pharmaceutical compositions for oral administration may be in the form of tablets, liquids, emulsions, suspensions, syrups, pills, caplets or capsules. The pharmaceutical composition can also be administered with a device.

Pharmaceutical compositions for topical administration may be in the form of creams, ointments, lotions, patches, solutions, suspensions or gels.

Additionally, the pharmaceutical composition may be prepared as a solid, liquid, or sustained release formulation.

Compositions containing antibody fragments produced according to the present invention may include common pharmaceutically acceptable diluents or carriers. Tablets, pills, caplets, and capsules may include common excipients such as lactose, starch, and magnesium stearate. Suppositories may include excipients such as waxes and glycerin. Injectable solutions include sterile pyrogen-free vehicles, such as saline, and may include buffers, stabilizers or preservatives. Common enteric coatings can also be used.

The invention also includes methods of producing antibody fragments by synthetic methods known in the art.

One embodiment of the invention comprises a pharmaceutical composition comprising at least one peptide or polypeptide of the invention attached, coupled, combined, linked or fused to a imaging agent for diagnostic localization and /or visualize.

Another embodiment of the present invention provides a diagnostic kit for in vitro analysis of therapeutic effects before, during or after treatment comprising an imaging formulation comprising a peptide of the present invention linked to an indicator marker molecule. The present invention also provides a method for using the imaging agent for cancer, more specifically for diagnostic localization and/or imaging of tumors, comprising the following steps:

a) contacting said cells with said composition,

b) determining the radioactivity bound to said cells, and

c) Visualizing the tumor.

In a preferred embodiment of the present invention, the imaging agent of the kit is a fluorescent dye, and the kit provides detection of cancer, more specifically blood-related cancers, such as leukemia, lymphoma and analysis of therapeutic effects in myeloma. FACS analysis is used to determine the percentage of cells stained by the imaging agent, and the intensity of staining, at various stages of disease, eg, at diagnosis, during treatment, during recovery, and during relapse.

The present invention also provides a composition comprising an effective amount of an imaging agent, the peptide of the present invention and a pharmaceutically acceptable carrier.

In a preferred embodiment, the indicator molecule is any known marker known in the art, including, but not limited to, radioactive isotopes, X-ray-impermeable elements, paramagnetic ions, or fluorescent molecules, etc. .

In a specific embodiment of the present invention, the indicative radioactive isotope can be, but not limited to, ¹¹¹ indium, ¹¹³ indium, ^99m rhenium, ¹⁰⁵ rhenium, ¹⁰¹ rhenium, ^99m technetium, ^121m tellurium, ^122m tellurium, ^125m tellurium , ¹⁶⁵ thulium, ¹⁶⁷ thulium, ¹⁶⁸ thulium, ¹²³ iodine, ¹²⁶ iodine, ¹³¹ iodine, ¹³³ iodine, ^81m krypton, ³³ xenon, ⁹⁰ yttrium, ²¹³ bismuth, ⁷⁷ bromine, ¹⁸ fluorine, ⁹⁵ ruthenium, ⁹⁷ ruthenium, ¹⁰³ ruthenium, ¹⁰⁵ ruthenium, ¹⁰⁷ mercury, ²⁰³ mercury, ⁶⁷ gallium and ⁶⁸ gallium.

According to another preferred embodiment, said indicator marker molecule is a fluorescent marker molecule. According to a more preferred embodiment, the fluorescent marker molecule is fluorescein, phycoerythrin or rhodamine, or a modified or conjugated product thereof.

The present invention also relates to compositions comprising an effective amount of the imaging agent of the present invention, a pharmaceutical agent linked thereto, and a physiologically acceptable carrier.

The present invention also provides a method for imaging an organ or cell, comprising exposing the organ or cell to be imaged to the imaging agent of the present invention under conditions such that an imaging agent can bind to the organ or cell. agent, to visualize the bound imaging agent, and thus to visualize the organ or cell.

The invention also provides a method of treating an organ in the body comprising contacting the organ to be treated with a composition of the invention under conditions such that the composition binds the organ, so as to treat the organ.

In a preferred embodiment of the present invention, said peptide or polypeptide may be used to treat target malignant tumor cells, more particularly leukemia cells in whole blood, comprising monitoring said cells and imaging said cells, As analyzed by FACS. Tumor cell samples that achieve a higher (eg, 4-fold higher) score relative to normal cells are used for treatment.

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

In a most preferred embodiment, the peptide or polypeptide of the present invention is capable of specifically or selectively binding to AML cells. The present invention provides ligands that are present on AML cells that bind the peptides or polypeptides of the present invention, and also provide peptides or polypeptides that bind said ligands.

The novel antibody fragments of the invention, or their corresponding peptidomimetics, are used in the manufacture of compositions and medicaments for the treatment of various diseases and conditions.

The invention provides a method for producing targeting preparations, comprising the following steps:

a) directly on the target cells by the biopanning procedure, or indirectly on the first target cells in the second state instead of the first state by the biopanning procedure, and subsequently directly by the biopanning procedure isolating and selecting one or more targeting molecules comprising a primary recognition site on a second target cell for production of one or more said targeting molecules;

b) amplifying, purifying and identifying the one or more targeting molecules; and

c) constructing a targeting agent from the one or more targeting molecules or the recognition sites of the molecules, wherein the targeting agent can be a peptide, a polypeptide, an antibody or an antibody fragment or a multimer thereof.

The targeting agent can also be additionally configured to be coupled, attached, combined, linked or fused or associated with a pharmaceutical agent.

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

In another preferred embodiment of the present invention, the pharmaceutical agent is selected from radioisotopes, toxins, oligonucleotides, recombinant proteins, antibody fragments, and anticancer agents. The isotopes may be selected from ¹¹¹ indium, ¹¹³ indium, ^99m rhenium ^{, 105} rhenium, ¹⁰¹ rhenium, 99m technetium, ^121m tellurium, ^122m tellurium, ^{125m tellurium, 165 thulium, 167 thulium , 168} thulium, 123 iodine, ¹²⁶ iodine, ^{131 iodine} , ¹³³ iodine, ^81m krypton, ³³ xenon, ⁹⁰ yttrium, ²¹³ bismuth, ⁷⁷ bromine, ¹⁸ fluorine, ⁹⁵ ruthenium, ⁹⁷ ruthenium, ¹⁰³ ruthenium, ¹⁰⁵ ruthenium, ¹⁰⁷ mercury, ²⁰³ mercury, ⁶⁷ gallium and ⁶⁸ gallium.

In another embodiment, the toxin is selected from gelonin, Pseudomonas exotoxin (PE), PE40, PE38, diphtheria toxin, ricin, or modifications or derivatives thereof.

In another embodiment of the present invention, the anticancer agent is selected from the group consisting of doxorubicin, morpholino doxorubicin (MDOX), adriamycin, cisplatin, paclitaxel, and Chamomile californica Vincristine, cytarabine (Ara-C), cyclophosphamide, prednisone, daunorubicin, idarubicin, fludarabine, chlorambucil, interferon alpha, hydroxyl Urea, Temozolomide, Thalidomide, and Bleomycin, and their derivatives.

The present invention provides a method for identifying antibody fragments comprising: (a) biopanning comprising incubating a phage display library with blood-derived cells; (b) washing to remove unbound phage; (c) eluting the bound phage from the blood cells; (d) amplifying the resulting bound phage; and (e) determining the sequence of the displayed peptide of the bound phage to identify the peptide.

The present invention provides peptides or polypeptides having the following formula or structure:

A-X-B

Wherein, X is a hypervariable CDR3 region with 3-30 amino acids; and A and B can be amino acid chains with a length of 1-1000 amino acids, respectively, wherein A is the amino terminal and B is the carboxyl terminal.

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 has 5-13 amino acid residues.

In another embodiment, X in the above structural formula is an amino acid sequence selected from SEQ ID NOs: 8-24.

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

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

In a preferred embodiment of the present invention, X in the dimer molecule is an amino acid sequence selected from SEQ ID NOs: 8-24.

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

The invention provides the use of said peptide or polypeptide optionally associated or attached, coupled, combined, linked or fused to a pharmaceutical agent for the manufacture of a medicament.

The present invention also provides the use of the peptide or polypeptide for the production of a medicament with activity against diseased cells, more specifically against cancer cells. The cancer cells may be selected from carcinoma, sarcoma, leukemia, adenoma, lymphoma, myeloma, blastoma, seminoma, and melanoma. More specifically, the cancer cells may be leukemia cells, most specifically, the leukemia cells are AML cells.

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

Example

The following examples are provided for the understanding of the present invention, but are not intended to, and should not be construed as limiting the scope of the present invention in any way. Although specific reagents and reaction conditions are disclosed, modifications can be made and such modifications are considered to be within the scope of this invention. Accordingly, the following examples are provided to further illustrate the invention.

Example 1:

1. Preparation and protein purification of cells, bacterial strains, scFv phage display libraries, cell membranes for biopanning methods.

1.1 Preparation of leukemia cells. Blood samples were obtained from leukemia patients. Monocytes (primary cells) were separated from other blood cells on Ficoll mats (Iso-prep, Robbins Scientific Corp., Sunnyvale, CA, USA). Centrifuge at 110 x g for 25 minutes. Cells at the interface were collected and washed twice with PBS. Cells were then suspended in RPMI+10% fetal calf serum (FCS) and counted. For long-term storage, 10% FCS and 10% DMSO were added to the lymphocytes and then frozen at -70°C.

1.2 Preparation of fixed platelets. Platelet concentrates obtained from blood banks were incubated for 1 hour at 37°C. An equal volume of 2.0% paraformaldehyde was added and platelets were fixed at 40°C for 18 hours. Platelets were washed twice with cold saline (centrifuged at 2500 xg for 10 minutes). Resuspended in saline containing 0.01% HEPES and counted microscopically.

Validation of platelet sensitivity to plasma von Willebrand factor and ristocetin. Plasma von Willebrand (vWF; 18, ug/ml) and ristocetin (0.6 mg/ml) were added to fixed platelets to induce platelet aggregation and monitored by chronolog lumi-aggregator.

1.3 Bacterial Strains - TG-1 and HB2151: Prepare fresh bacterial cultures for infection by growing cells to an _A600 of 0.5-0.9 (exponentially growing cells). E. coli TG-1 cells were used for phage preparation, while E. coli HB2151 cells were used for scFv protein production.

1.4 Source of scFv display phage library. The ScFv library (Nissim et al., EMBO J., 13, 692-698 (1994)) was provided by Dr. A. Nissim with permission from MRC. The library was originally constructed as a phagemid library displaying ScFv fragments in which the _VH and _VL domains are linked by a flexible polypeptide. The ScFvs displayed in this phagemid library were fused to the N-terminus of the minor coat protein pill of the phage, which was then subcloned into the pHEN1 vector (Nissim et al., EMBO J., 13, 692-698 (1994)). First, a repertoire of antibody fragments (referred to as "native repertoire") is prepared by PCR from rearranged V-genes of immunized human peripheral blood lymphocytes. To diversify the repertoire, random nucleotide sequences encoding heavy chain CDR3s of 4-12 residues in length were introduced into a library of 49 cloned human _VH gene segments. The fused _VL segments in all clones were derived from a single unmutated V gene of the germline IGLV3S1, resulting in a single pot library of approximately ¹⁰⁸ clones.

1.5 Membrane preparations from AML cells. 1 ml of cold lysis solution (0.3 M sucrose, 5 mM EDTA, 1 mM PMSF) was added to the pellet containing 10 ⁸ washed cells, followed by centrifugation at 11,000 x g for 20 min at 4°C. The supernatant was discarded and the pellet resuspended in TE (10 mM Tris, 1 mM EDTA, 1 mM PMSF) and centrifuged as above. The final pellet was resuspended in 6 ml PBS at an _A280 of 0.4 and used to coat three Maxisorb immunotubes (NUNC) for 2 hours at 37°C. After coating, the tubes were rinsed 3 times with PBS and then blocked with MPBS (2% skim milk in PBS) for 2 hours at room temperature. The tubes were rinsed 3 more times with PBS prior to biopanning.

Example 2:

2. Manipulation of Phagemid Particles: Biopanning Method

2.1 Phagemid screening and amplification: through a four-step biopanning method, the library is screened for phagemids expressing epitopes of particular interest:

a) binding phagemid particles to a target site, more specifically, binding said phagemid particles to washed target cells or cell membranes

b) Removal of unbound phagemid particles, more specifically, removal of unbound phagemid particles by extensive washing

c) Elution of bound phagemid particles

d) Propagation and amplification of eluted phagemid particles, more specifically, propagation and amplification in E. coli

2.2 Cloning identification: The biopanning method of the four steps was generally repeated 3-5 times. Selected phagemid clones were propagated individually and further characterized by:

a) DNA sequencing

b) Ex vivo comparison of phage binding to several cell types

c) Infection of E. coli HB2151 for production of soluble scFv

2.3 Sequence analysis: Using upstream primer #203743 (5'-GAAATACCTATTGCCTACGG) and downstream primer #181390 (5'-TGAATTTTCTGTATGAGG), the encoded scFv DNA of about 800 bp in the phagemid particle was amplified by PCR. Using the ABI PRISM Big Dye Termination Cycle Sequencing Kit and the above primers, the DNA fragments were comprehensively identified from both ends by an automatic ABI PRISM DNA sequencer (310 Genetic Analyzer, Perkin Elmer). Two other primers located in the flexible polypeptide binding region between the heavy and light chains, primer #191181 (5'-CGATCCGCCACCGCCAGAG) and its complementary primer #191344 (5'-CTCTGGCGGTGGCGGATCG) were used for sequencing.

Example 3:

3. Biopanning method

3.1 Basic biopanning method: The biopanning method is an integral part of the above-mentioned phage display technology. Three biopanning methods were developed and employed in this study:

a) Method AM (AML membrane panning/bacterial elution followed by intact AML cell panning/trypsin elution)

b) Method YPR (Fixed Human Platelet Panning/Acid Elution)

c) Method YPNR (Fixed Human Platelet Panning/Acid Elution)

The above method is disclosed in detail below

3.1.1 Method AM

3.1.1.1 Prewash: Quickly thaw at 37°C a 1ml aliquot containing 2 x ¹⁰⁷ frozen AML cells from a patient stored at -70°C and dilute immediately into 10ml of cold 2% PBS-milk (MPBS). Cells were centrifuged at 120 xg for 5 minutes at room temperature (RT), resuspended in MPBS, and counted with a hemocytometer. Prepare cell membranes as described in section 1.5.

3.1.1.2 Selection was performed on immobilized AML cell membranes by adding 2 ml of MPBS containing 10 ¹² phagemids from the original Nissim library. The tubes were agitated gently for 30 minutes and then incubated without agitation for an additional 90 minutes, both at room temperature. After 3 rounds of panning on AML cell membranes, 1 round of panning was performed on intact AML cells.

3.1.1.3 Washing: In order to remove excess unbound phagemids, pour out the contents of the test tube, and wash the test tube 10 times with PBS, 0.1% Tween, and then wash 10 times with PBS only.

3.1.1.4 Elution: Exponentially growing E. coli TG-1 cells (2ml) were added directly to the tube and incubated at 37°C for 30 minutes with slow agitation. One sample was plated for titration and the remaining volume was plated for amplification as described above.

3.1.1.5 Amplification: Colonies from the large plates were scraped and pooled. A sample (approximately 10 ⁷ ) of ampicillin-resistant E. coli TG-1 cells was grown in liquid culture to an _A600 of approximately 0.5 and then infected with a helper phage (VSC-M13, Stratagene) to produce large amplified cells. Increased phagemid stocks. Phagemid (18a) was recovered by PEG precipitation method. The above amplified T16MI stock (approximately 10 ¹¹ phagemids/ml) was used for subsequent rounds of panning. The screening method was repeated for two more rounds using ¹⁰¹¹ phagemids from the previously amplified stocks. The amplified stock for the third panning method performed on immobilized membranes was named T16M3.

3.1.1.6 Re-panning on intact cells: The third membrane-panned amplified stock T16M3 was used to pan on intact AML cells. Selection was performed in a final volume of 0.5 ml MPBS containing 2 × ¹⁰⁷ cells and ¹⁰¹⁰ colony-forming units (CFU) of phagemids (Nissim library), and ¹⁰¹³ wild-type phage M13, slowly at 4 °C. Stir for 2 hours. Bound phagemids were eluted with 50 μl of trypsin:EDTA (0.25%:0.05%) and then neutralized by adding 50 μl of FCS. For titration and amplification, 1 ml of E. coli TG-I culture (A ₆₀₀ =0.5) was used. The expanded, final stock was designated T16M3.1.

3.1.2 Method YPR

3.1.2.1 Selection: Clonal selection was done by panning 10 ⁸ fixed human platelets with 10 ¹¹ phagemids (Nissim library) in 1 ml PBS/HEPES/1% BSA buffer. The binding was allowed to proceed for 1 hour at room temperature while mixing the samples by swirling.

3.1.2.2 Cell washing: Wash platelets 5 times by low-speed centrifugation (3500×g), and resuspend as above.

3.1.2.3 Elution: The first round of bound phagemids are eluted from the immobilized platelets by acid elution technique:

The platelets were incubated with 200 μl 0.1 M glycine (pH 2.2) for 10 minutes at room temperature. After neutralization with 0.5M Tris-HCl pH 8.0 and centrifugation, remaining platelet-bound phages were eluted by adding 200 μl trypsin-EDTA (0.25%/0.05) and neutralized by adding 50 μl FCS. The cells were removed by centrifugation, and the supernatants of eluted phage from the acid elution method and the trypsin elution method were collected and named YPR(a)-1 and YPR(t)-1 stocks, respectively. The stocks were then expanded by adding 1 ml of exponentially growing TG-1 cells for 30 minutes at 37°C. An aliquot of the sample was plated for titration and the remaining infected E. coli cells were plated on 2×TV/AMP 15 cm plates. Plates were incubated overnight at 30°C. Yield after each round of panning was determined by counting the number of colonies on the plate.

3.1.2.4 Amplification: Amplify the clone according to the method described in Section 3.1.1.5. Amplified stocks of approximately 10 ¹² phagemids/ml, designated R1(a) and R1(t) stocks from the acid-elution method and the trypsin-elution method, respectively, were pooled and used for subsequent rounds of panning.

3.1.2.5 The second and third rounds of panning were carried out according to the first round of panning method of the YPR method, with the following improvements: (i) For the second round of panning, use the same method as 10 ¹² R1( t) Combined 10 ¹² R1 (a), and (ii) elution was performed with glycine (pH 2.2) only. The amplified eluate from the second round of panning was designated as R2. (iii) For the third round of biopanning, 10 ¹² R2 was used, and the elution was performed as in the second round of panning. The stock for the third round of amplification was named R3.

3.1.3 YPNR method

3.1.3.1 Biopanning and washing are generally carried out as described in the YPR method. However, in the present invention, (i) elution is performed with glycine (pH 2.2) after each of three rounds of panning, and (ii) after the first round of panning and amplification, followed by two Round panning without amplification. The first, second and third rounds were named YPNRI, YPNR2 and YPNR3, respectively.

3.2 Screening of negative control scFv clones

3.2.1 N14 CDR3 sequence: For all binding experiments, one clone was picked from the native library (before selection). A phage stock and a soluble scFv designated N14 were prepared from this clone. The sequences indicated that it belongs to the V _H 4-DP65 gene family. The sequence of the 11-mer _VH -CDR3 encoded by this clone is designated as N14 CDR3, and its sequence is as follows (SEQ ID NO: 28):

Phe Leu Thr Tyr Asn Ser Tyr Glu Val Pro Thr

3.2.2 C181 CDR3 sequence: another negative clone C181 was used for binding analysis experiments. Clone C181 (reactive to recombinant hepatitis B virus [HBV] particles) belongs to the _VH3 -DP35 family, and the sequence of the 9-mer _VH- CDR3 encoded by this clone is named C181CDR3, and its sequence 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: The vector pHEN1 used for constructing the original phagemid library was designed with an amber stop codon encoded at the junction of scFv gene and pIII gene. Thus, when a specific cloned vector was introduced into E. coli HB2151 (which is a non-suppressor strain) by phagemid infection, this system was able to produce soluble scFv, which was secreted into the periplasm of the bacteria (Harrison et al., Methods in Enzymology, 267, 83 -109 (1996)). The scFv can then be conveniently recovered from the culture medium. Soluble scFvs were produced under the control of the lacZ promoter (Gilbert and Muller-Hill, PNAS (US), 58, 2415 (1967)), which was induced with IPTG.

Upstream of the amber mutation in the vector, a sequence encoding a c-myc tag (10 amino acids - Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu; SEQ ID NO: 123) was included. The C-terminus of the expressed scFv should have a c-myc tag detectable with a mouse anti-myc tag antibody (from European Collection of Cell Cultures (ECACC) 9E10-hybridoma).

4.2 Purification of scFv on a protein-A bead affinity column: the scFvs of the selected clone and the scFvs of the control clone C181 belong to the V _H 3 family and can be purified on a protein-A affinity column. Periplasmic fractions (100-250 ml) of induced cultures from each clone were prepared and incubated with Protein-A Sepharose beads. Bound scFvs were recovered from the column by acid elution (0.1M glycine, pH 3.0), and the eluate was neutralized with Tris, pH 8.0. The concentration of recovered protein was determined by _A280 assay followed by PBS buffer exchange by dialysis or on a G-25 sepharose column.

4.3 Purification of N14-scFv on Sephacryl S-200 column: The scFv of negative clone N14 belongs to the _VH4 gene family and therefore could not be purified on a protein-A affinity column. For scFv-N14 purification, the total protein in the periplasmic fraction of 200 ml induced cultures was precipitated by 60% ammonium sulfate. The pellet was resuspended in 2 ml 0.1 x PBS, 5 mM EDTA, 5 mMPMSF and loaded onto a Sephaeryl S-200 column (1.5 x 90 cm) pre-equilibrated with flow-through buffer (0.1 x PBS, 5 mM EDTA). Protein was isolated and fractions containing N14-scFv (detected by SDS-PAGE and Western analysis) were pooled, lyophilized, and resuspended in 1/10 volume of water. N14-scFv (unlabeled and FITC-labeled) were then used as negative controls in FACS analysis experiments.

4.4. Labeling of purified scFvs with FITC: Approximately 1 mg of purified FITC from each preparation was resuspended in PBS and conjugated using the Fluoro-Tag FITC commercial kit (Sigma cat. #FITC-1) , coupled to FITC according to the manufacturer's protocol.

4.5 Quality analysis of purified and labeled scFv

4.5.1 After purification and FITC labeling, each preparation (labeled and unlabeled) was analyzed by SDS-PAGE, Western blot, HPLC (A280 and A495) using a Superdex-75 column and fluorometry. feature. This analysis showed that the N14 scFv was 80% pure and the _VH3 clone was 90% pure, with approximately two FITC molecules coupled to each scFv molecule (F/P ratio 2:1).

4.5.2 Binding activity was assessed after FITC labeling in order to confirm retention of scFv specificity (see Example 5).

4.6 Biochemical characterization of phagemid clones: The constructs were evaluated with several types of assays, and the purity of various scFv preparations (see Example 8), including SDS-PAGE, mass spectrometry (applicable only in Y1 and Y17 scFvs) and HPLC. scFvs were identified by Western analysis and EIA; and scFv binding was characterized by FACS.

Example 5:

5. Binding assay

Binding of selected clones to cells was assessed at both the phagemid and soluble scFv levels.

5.1 Binding at the phagemid level

For this, phagemid stocks are prepared separately from each specific clone

5.1.1 Colony test: In one experiment, a mixture of ¹⁰⁹ specific phagemids from the biopanning method that were able to infect ampicillin-resistant E. coli and that were not ampicillin-resistant and were used as " 10 ¹¹ wild-type M13 phages of "blocker" were incubated with 10 ⁵ cells selected from a panel of cell types. After incubation and washing, bound phage were eluted with trypsin and an aliquot was used to infect E. coli TG-I. The E. coli were then plated on 2×TY/AMP and incubated overnight at 30°C. Count and compare the number of colonies obtained for each clone. The results provide an indication of the binding affinity and specificity of the phagemid.

5.1.2 White/blue colony test: In this test, each experiment includes an internal control, the specific phagemid is compared with the named Another control phagemid (Promega Corp., Madison, Wisconsin, USA) of pGEM7 was mixed. The pGEM7 phagemid is resistant to ampicillin. However, it cannot express any recombinant polypeptide at the N-terminus of its pill gene. Colonies were counted after TG-1 infection and incubation on plates containing 1 mM X-gal ampicillin. The colonies obtained containing pGEM7 were blue, while the colonies obtained from the specific phagemid were white. The enrichment factor from the input/output ratio of the white/blue colonies (grown on the same plate) was then calculated for each tube.

5.1.3 EIA of phagemids

5.1.3.1 Phagemids bound to selected cells: About 5×10 ⁵ selected cells were immobilized on the surface of a 24-well plate with acetone:methanol (1:1). The binding assay requires ¹⁰⁹ phagemids. Binding was performed 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 resulting color intensity is read at A ₄₀₅ by an ELISA plate reader and is directly proportional to the amount of bound phagemid.

5.1.3.2 Phagemids bound to immobilized platelets: Polystyrene microtiter plates were coated with 10 ⁸ immobilized platelets and incubated overnight at 4°C. Approximately ¹⁰¹⁰ phagemids were used to assess binding. Washing and incubation of plates and determination of binding levels were performed as described in Section 5.1.3.1 above.

5.1.4 Binding assays were performed on specific proteins selected from the group consisting of human growth hormone (hGH), fibrinogen, fibronectin, BSA, SM (skim milk) and glycocalicin (proteolytic fragment of GPIb). Binding was analyzed as follows. Wells of polystyrene microtiter plates were coated with 2 μg/well of one of the proteins to be tested. Coating was performed by incubation overnight at 4°C. Add approximately 10 ¹⁰ phagemids to test for binding. After extensive washing with PBS, the plates were incubated with rabbit anti-M13, anti-rabbit BRP and substrate. The level of binding was determined by the intensity of the color produced. Optical density was determined at _A405 . Repeat determinations were performed for each sample and the average value calculated.

5.2 Binding assay at scFv level: Binding of scFvs produced in the periplasm of HB2151 was compared in several cell types by two different assays, EIA analysis and FACS analysis.

5.2.1 EIA of soluble scFv: Approximately 5 x 105 ^AML cells were incubated with 5-10 μg total protein. Binding was performed for 1 h at 4 °C, followed by EIA analysis with mouse anti-myc antibody, anti-mouse HRP and substrate. After each step, excess unbound antibody was removed by washing the cells 3 times with PBS. The intensity of the color produced was read by ELISA plate reader ( _OD405 ). As noted above, color intensity is directly proportional to the level of binding.

5.2.2 FACS analysis of cells

5.2.2.1 Analysis of cells stained by the "three-step staining" method

Methods: FACS analysis was performed in order to test and confirm the specificity of the selected clones. First, a "three-step staining" method was established using crude extracts or purified unlabeled scFv, followed by a mouse anti-myc antibody, and finally a FITC or PE-conjugated anti-mouse antibody.

FACS analysis requires 5-8 x ¹⁰⁵ cells that have been purified by Ficoll and resuspended in PBS + 1% BSA, binding is performed at 4°C for 1 hour. After each step, cells were washed and resuspended in PBS+1% BSA. After the final staining step, cells were fixed by resuspension in PBS, 1% BSA, 2% formaldehyde and read by FACS (Becton-Dickinson).

5.2.2.2 Stain cells with FITC-labeled scFv by one staining step: FITC-labeled scFv was incubated with 5-8 x ¹⁰⁵ Ficoll purified cells in PBS + 1% BSA. Binding was performed at 4°C for 1 hour. Cells were then washed, fixed as described in 5.2.2.1 above, and read by FACS.

Example 6: panning and sequencing results

6.1 Results of the AM method

6.1.1 Panning results for the AM method: In the table below (Table 1) the estimated number of phagemids used for panning (input) and the estimated number of bound phagemids eluted in the AM method are summarized Quantity (output):

Table 1. Panning results from method AM Import stock cell source Elution output amplified stock 2×10 ¹¹ membrane of AML Bacteria TG-1 3×10 ⁴ T16M1 T16MI-10 ¹¹ membrane of AML Bacteria TG-1 6.4×10 ⁵ T16M2 T16M2-10 ¹¹ membrane of AML Bacteria TG-1 10 ⁶ T16M3 T16M3-10 ¹⁰ AML cells Trypsin 2×10 ⁶ T16M3.1

An enrichment in the yield (yield) obtained by each successful panning was found. In addition, there was no reduction in yield when T16M3 was used to pan AML intact cells, suggesting that the bound phagemids may be specific for components on the extracellular surface of the cells, or that the specific system may contain larger amounts of Binds non-specifically to phagemids.

6.1.2 Cloning sequence results of the AM method: Although clones were selected and sequenced from T16M1, T16M2 and 16M3 output stocks, the results provided below are mainly the results of clones from T16M3.1 output stocks (AML intact Cell panning). Clones AM10, AM11 and AM12 were identified in the T16M3 stock but not in subsequent output.

The amino acid sequences displayed in the _VH -CDR3 and their frequencies in the tested clones are summarized in Table 2 below.

        Table 2. Grams selected from T16M3 and T16M3.1 outputs according to the AM biopanning method

Long. clone number V _H -CDR3 size _VH -CDR3 sequence germline Frequency in T16M3 output Frequency in T16M3.1 outputs AM1 8 Pro Trp Asp Asp Val ThrPro Pro1 2 3 45 6 7 8 V _H 3-DP47 5/31 8/51 AM2 12 Gly Phe Pro Arg lleThr Pro Pro Ser AlaGlu Ile1 2 3 4 56 7 8 9 1011 12 V _H 3-DP46 11/31 20/51 AM3 5 Gly Phe Pro Met Pro1 2 3 4 5 V _H 3-DP46 1/31 2/51 AM6 10 Gly Phe Pro H is SerSer Ser Val SerArg1 2 3 4 56 7 8 9 10 V _H 3-DP46 4/31 6/51 AM7 11 A rg Phe Pro M etArg His Glu Lys ThrAsn Tyr1 2 3 4 56 7 8 9 1011 V _H 3-DP46 3/31 4/51 AM8 8 Arg Phe Pro Pro ThrAla Thr Ile1 2 3 4 56 7 8 V _H 3-DP46 6/31 8/51 AM9 7 Thr Gln Arg ArgAsp Leu Gly1 2 3 4 56 7 V _H 3-DP87 0/31 2/51 AM10 11 Lys Phe Pro GlyGly Thr Val ArgGly Leu Lys1 2 3 4 56 7 8 9 1011 V _H 3-DP46 0/31 1/31 AM11 12 Gly Phe Pro Val IleVal Glu Gln ArgGln Ser Thr1 2 3 4 56 7 8 9 1011 12 V _H 3-DP49 0/31 1/31 AM12 10 Arg Phe Pro Gln Arg Val Asp Asn Arg Val 1 2 3 4 56 7 8 9 10 V _H 3-DP46 0/31 1/31

The amino acid sequence ^Arg / _GlyPhePro was present in 7 of the 10 isolated clones presented in Table 2 and represented one of the motifs. Additionally, it should be noted that in each case the identified motifs represent the N-terminal 3 amino acids of the CDR 3 region. Thus, the motif may be an effective anchor or binding site, either alone or in combination with other amino acid residues extending to one or both ends of the CDR 3 region or as a larger peptide or polypeptide or Fv molecule a part of.

Other CDR3 regions with high binding force to AML cells can be constructed according to the core sequence ^Arg / _GlyPhePro . The CDR3 region can be constructed by adding, deleting or mutating any of the above 5-12-mers while maintaining the ^Arg / _Gly PhePro core sequence.

The CDR3 region of the present invention has the amino acid sequence R1- ^Arg / _GlyPhePro -R2, wherein R1 includes 0-15 amino acids, preferably 0-9, most preferably 0-1 amino acids, and R2 includes 1-15 amino acids , most preferably an amino acid sequence of 1-9 amino acids. R1 and R2 are amino acid sequences that do not negatively affect the specific binding of ^Arg / _Gly PhePro sequences to AML cells.

The CDR3 regions of the light chains cloned above are identical and are shown in SEQ ID NO:125.

6.2 Results of YPR and YPNR methods

6.2.1 Panning results for YPR and YPNR methods: The estimated number of phagemids used for panning (input), and the estimated number of eluted bound phagemids are summarized in the following tables (Tables 3, 4) Quantity (output).

Table 3. Panning results from the YPR method Import stock Elution output amplified stock Nissim Library Y10 ¹¹ acid trypsin 10 ⁷ 4×10 ⁷ R1(a)R1(t) Combined "R1(a)-10 ¹² , +R1(t)-10 ¹² ) acid 5×10 ⁵ R2 R2-10 ¹² acid 3×10 ⁸ R3

Table 3 shows that the trypsin elution yielded a 4-fold increase compared to the first round of acid elution.

Repanning without an amplification step following the YPNR method reduces the likelihood of preferentially amplifying phagemid infection or bacterial infection. The yields obtained are shown in Table 4.

Table 4. Panning results from the YPNR method Import stock Elution output amplified stock Nissim Library Y10 ¹¹ acid 3×10 ⁷ YPNR1 YPNR1-3×10 ⁷ acid 4×10 ⁵ YPNR2 YPNR2-4×10 ⁵ acid 10 ³ YPNR3

As expected, the results in Table 4 show a decrease in phage yield after each round of panning. This method is used to prevent bias due to the amplification of non-specific phage.

6.2.2 Cloned sequence results of YPR and YPNR methods

Several clones from the third panning of both methods were selected for sequencing. The amino acid sequence shown in Table 5 is the sequence of the heavy chain CDR3 region ( _VH -CDR3). Also shown in this table is the associated line and the frequency of occurrence of this sequence in the R3 output.

Table 5: Y-series clones selected with R3 output according to the YPR biopanning method

Clones isolated by the YPNR method were also predominantly Y1.

The light chain CDR3 regions of the above clones are identical and are shown in SEQ ID NO:125.

Embodiment 7:

7. Combining evaluation results

7.1 Binding of selected phagemid clones to AML cells (AM clone sequence): AM clones were used to perform binding assays for assessing phagemid binding to cells, such as the white/blue colony assay described in Example 5. With the exception of clone AM7, no dominant binding was detected to the test cells. Significant, but non-selective binding of clone AM7 to all target cells was observed as phagemid or purified scFv. The results confirmed that there was no enrichment of AM clonal series.

7.2 Combination of γ-cloning series

7.2.1 Phagemid binding - EIA using fixed platelets: Phage clones were tested for binding to fixed platelets by EIA after three rounds of panning with two different methods. Phagemid stocks were prepared with each selected clone and the clones were tested with two sets of EIA. Each sample was analyzed in duplicate and the average value was calculated. The results are summarized in Figure 1 and show that 6 out of 9 Y-series clones showed positive EIA reactions. The greatest degree of binding was associated with clones Y1, Y16, Y17 and Y-27. Phage stocks M13 (wild type phage) and E6 (selected on CLL leukemia cells) were used as negative controls. The dominant clone phage Y1 showed the strongest binding to immobilized platelets, which together with Y17 showed a significantly stronger binding ability than the M13 or E6 phage clones.

Embodiment 8:

8. Detailed characterization and cloning of scFvs

8.1 Structure and identification of scFv: The native construct of Y-I was evaluated by HPLC analysis with a Superdex 75 column and by mass spectrometry. The results of the former method indicated the presence of monomers, dimers and tetramers in this preparation. The sensitivity of mass spectrometry was sufficient to identify the expected molecular weight of 26.5 kD, and when the c-myc tag was cleaved, a molecular weight of 24 kD was obtained.

However, the results of SDS-PAGE indicated that the intact, unfragmented molecule had an apparent molecular weight of 30 kD, despite an inferred molecular weight of 26.5 kD based on the nucleic acid sequence and mass spectrometry results described above. Western analysis with a c-myc-specific antibody confirmed the 30 kD result of SDS-PAGE and supported the notion that the c-myc tag is present at the end of the intact molecule. The difference between the results of the two methods is due to the degree of precision of the method and the conditions under which the SDS-PAGE was run, factors that can alter the apparent molecular weight of the test protein.

8.2 Binding of platelet-selected clones to leukemic cells: As noted in the introduction, platelet cell surface markers can be expressed on immature hematopoietic cells. Binding of platelet-selected clones was tested by FACS analysis. FACS analysis was performed after staining of whole blood followed by RBC lysis or on isolated-prepared-(Ficoll cushion) purified monocytes. ScFvs were prepared from each clone, purified on protein-A, and FITC-labeled (as described in Sections 4.1-4.4). To enable production of intact scFv in non-repressor E. coli strain HB2151, the amber codon (TAG) present in the _VH -CDR3 of clone Y-27 was mutated to encode glutamic acid (GAG) by DNA site-directed mutagenesis . The target cells used in the study were isolated from fresh blood samples of various patients with leukemia. The samples were obtained from three medical centers in Israel.

Clones Y1 and Y17 exhibited preferential binding to the leukemic cells tested. All other Y-series clones produced only background levels of binding. Table 6 provides the binding of FITC-labeled Y-1 and Y-17 to various leukemia cells.

    Table 6.Y-I binding specificity to leukemia cells

^* not determined

Results expressed as fractions in Table 6 represent the proportion of patients whose cells were determined to react positively with each of the tested antibodies by FACS analysis. The numerator represents the number of positive patients and the denominator represents the total number of patients tested for a particular scFv/cell type combination. Y-17 was able to tightly bind to all tested cells. Therefore, this binding is considered to be non-cell selective. However, Y1 binding was found to be highly selective for several samples of leukemia cells, especially those in the acute phase. Y1-scFv binding was further analyzed as follows.

Representative results of YI binding to three AML samples are shown in FIG. 3 . In each case, the majority of the cell population emitted fluorescence intensities significantly higher than the background fluorescence intensity obtained by staining with the negative control scFv. The above results indicate that, for each patient, Y1 binds to a different fraction of the total cell population. The Y-1 peak on the right side of each graph is considered to represent the lowest number of Y1-bound cells in the population, and the proportion of total cells below this peak may represent the minimum proportion of Y1-bound cells in each sample .

8.3 Binding of Y-1 to normal blood cells: Analyze the binding of Y1 to Ficoll-purified normal blood cells according to different blood cell types. Although no binding to normal lymphocytes was detected, Y1 was able to bind purified Ficoll-purified monocytes from 9/28 subjects, platelets from 5/8 subjects, and erythrocytes from 1/4 subjects. blood cells (RBCs). However, CD14-specific antibodies were able to bind cells in all monocyte preparations, and many neutrophil preparations. A summary of the analysis is provided in Table 7.

Table 7. FACS analysis of scFv binding to Ficoll purified normal blood cells.

The above combined results represent the proportion of normal blood samples determined by FACS analysis that reacted positively with each of the tested antibodies. It should be noted that despite selection on fixed platelets, FITC-Y1 scFv exhibited lower binding to platelets.

Figure 4 shows the binding of Y1 to Ficoll purified platelets (4a) and to monocyte-gated cells (4b). The variation in the monocyte population was greater than that observed in the platelets, and the calculated mean fluorescence intensity was 30-fold and 5-fold higher than the negative control, respectively. This result is most likely due to the characteristic of repeated platelet attachment to Ficoll-purified monocytes. Subsequent experiments showed that, when analyzed in whole blood samples, Y1 binding was observed in the normal monocytes, granulocytes, platelets and RBCs tested. Similarly, no binding of Y1 to platelets was observed when the platelets were derived from platelet-rich plasma (PRP). Under the same binding conditions (in whole blood followed by RBC lysis in FACS lysis buffer [Becton Dickenson]), Y1 bound to leukemia cells in a manner similar to that obtained after Ficoll purification. Therefore, we can conclude that the Y-I epitope on platelets or monocytes is cryptic under native conditions. During Ficoll purification, the epitope is exposed, enabling it to be recognized by Y1, whereas for leukemia cells, the epitope is exposed under both purified and non-purified conditions.

Y1 was tested for binding to hematopoietic stem cells (CD34+ cells) in cord blood, in addition to normal hematopoietic progenitors of the lymphoid and myeloid lineages. Figure 5 shows the binding results of FITC-labeled scFv clones to cord blood CD34+ stem cells; Figure 5a shows the binding results of CD34+ gated cells and FITC-labeled negative control scFv, while Figure 5b shows the binding results of CD34+ gated cells and FITC-labeled scFv The same analysis results for clone Y1. Figure 5c shows the FSC and SSC dot plot analysis of the same FITC-labeled scFv clone Y-I sample as in Figure 5b. The results of this analysis indicated that there were two subsets of CD34+ stem cells from cord blood that differed in forward scatter (FSC), which represents cell size. Y1 can bind to smaller cells in both populations. The circled portion in Figures 5b and 5c represents the subpopulation of CD34+ cells that bind clone Y1 scFv. Further analysis revealed that the smaller cells were the dead cells present in the cell population, and that Y1 binding might indicate the presence of intracellular ligands recognized by Y1.

The experiment was also performed with peripheral blood cells from healthy donors pretreated with GM-CSF (GM-CSF treatment releases stem cell translocation into the blood). Results similar to those shown in Figure 5 were obtained.

8.4 Binding specificity of Y1 scFv compared to various cell markers on AML cells: Y1 staining of Ficoll-purified peripheral and myeloid cells from AML patients compared to staining of said cells by a panel of other antibodies . In Table 8 the results of the FACS analysis performed on samples from 14 patients are summarized. It should be noted that for all markers tested, including Y1, there was significant variation in the frequency of stained cells in preparations from individual individuals. The lack of correlation between the results for the various markers and the binding of Y1 suggests that Y1 cannot bind any of the ligands bound by the other markers tested, and that the Y-1 ligand does not constitute any of the cell surface markers tested.

Table 8. Comparison of binding of Y1 scFv and antibodies to various cell markers

^** BM/PB-bone marrow/peripheral blood

The above results are expressed as the percentage of cells in the Ficoll-purified samples of a particular patient, as determined by FACS analysis, which reacted positively with each independent antibody.

In terms of the concentration of Y1 required for binding detection (approximately 1 μg/5×10 ⁵ ), the above results indicate that Y1 scFv has a high binding ability to specific antibodies on AML cells.

In addition to the results shown in Table 8 showing the binding of Y1 to AML cells, we have demonstrated above (Table 6) that Y1 can also bind to most of the other types of leukemia cells tested, including B-ALL cells, however, The sample size of the other leukemia samples was limited. Figure 6 shows FACS analysis of Y1 scFv binding to pre-B-ALL cells obtained from two patients. A double staining method was used using commercially available PE-labeled CD 19 (marker of normal peripheral B cells, Figure 6a, 6c) or PE-labeled CD 34 (marker of stem cells, Figure 6d), while using FITC-labeled negative control scFv or FITC-labeled Y1 scFv. Figure 6b is a double negative control. Fluorescence intensity (X-axis) of cells bound by FITC-labeled samples (scFv clone Y1 ) is presented, compared to negative control staining patterns (6e and 6f). The results in Figure 6 indicate that the majority of the leukemic, pre-B-ALL cells in each of the two samples stained positive for Yl cells due to Y-I binding.

8.5 Binding of YI-scFv to cell lines: Several cell lines from malignant hematopoietic cell lines were screened for their ability to be recognized by Y1. FACS analysis indicated that Y1 was able to bind many of the cells tested (Table 9). It should be noted that only one human B-cell line and one mouse myeloid cell line were tested. Importantly, this binding was restricted to exponentially growing cells. Cells in quiescent phase were essentially unable to bind Y1, suggesting that Y1 ligand expression is regulated during the life cycle of the cells. In addition, the binding strength differs between the responding cells. This finding implies that there are differences in expression levels or affinities of the ligands in different cells.

Table 9. Binding of Y1 to hematopoietic cell lines type hyperresponsiveness moderate reactivity hyporesponsiveness human bone marrow KG-1; THP-1; U937; Tf-1; MEG HL-60; HEL; K-562; MC1010 NB-4 human B-cell Namalwa; Daudi; UMUC3, RAJI human T-cell Jurkat; Hs-602 CCRF-CEM; Molt-4; Hut-78; mouse bone marrow M1; P388D1; PU5-1.8; WEHI-274.1

8.6 Binding of purified Y1 in the presence of DTT: Once the Y1 clone has been selected, methods for scFv production are further developed. The results of the FTLC analysis of the Y1 batch indicated that the protein could multimerize, mainly forming monomers and tetramers, the ratio between these two forms differing from one preparation to another. To obtain consistent material, 5 mM DTT was added during affinity purification on a Protein-A Sepharose column and then removed by PBS buffer exchange. In fact, after DTT treatment, most (>90%) species appeared in the monomer fraction. There were no significant differences between the binding of the monomeric form of Y1 (purified in the presence of DTT and analyzed on HPLC) and the binding of the mixture of Y1 forms.

8.7 Y1 is a clone specific for leukemia cells: The Y1 cassette belongs to the _VH -DP32 germline. Several other clones from the same germline were isolated and described in Example 6. The clones included Y17, Y27 and Y-44, all of which differ in their primary sequence (germline cassette) only in its CDR3 region. However, Y1 exhibited selectivity for leukemia cells. The CD3 sequences of these clones are summarized in Table 10 and the binding characteristics of the clones are summarized in Table 11.

Table 10: CDR3 sequences of _VH3 -DP32 isolated clones.

Table 11: Binding characteristics of _VH3 -DP32 isolated clones clone number binding specificity Y1 Can bind to many leukemia cells Y17 Binds to all tested hematopoietic cells including normal lymphocytes Y-27 Does not bind to any hematopoietic cells tested Y-44 Does not bind to any hematopoietic cells tested

Tables 10 and 11 show that although the main sequence is identical between the four clones except for the _VH -CDR3 region, the binding characteristics are significantly different between the clones. This finding strengthens the idea that the sequence of the _VH -CDR3 region plays an important role in the specificity of the binding site for the antigen. It should be noted that neither the length of the CDR3 sequence nor the specific germline cassette in which this sequence is present appears to be a major determinant of binding specificity. Like Y1, Y17 and Y-27 each included a hexameric CDR3, and the heavy chains of all three clones were from the same germline. For Y17 and Y-27, no selective binding to hematopoietic cells was demonstrated.

Example 9

9.1 Construction of the triple antibody: the VL region and _{the VH} region of the vector pHEN-Y1 encoding the original Y1 were amplified by PCR. For the V _L PCR reaction, the sense oligonucleotide 5'-AACTCGAGTGAGCTGACACAGGACCCT, and the antisense oligonucleotide 5'-TTTGTCGACTCATTTCTTTTTTGCGGCCGCACC were used. A cDNA product of the expected size of approximately 350 bp was purified, sequenced, and digested with XhoI and NotI restriction enzymes.

The _VH region was amplified in the same way (using the sense oligonucleotide 5'-ATGAAATACCTATTGCCTACGG and the antisense oligonucleotide 5'-AACTCGAGACGGTGACCAGGGTACC). The _VH PCR product was digested with NcoI and XhoI restriction enzymes. The triple ligation method was used on the pHEN vector predigested with NcoI-NotI. The final vector was named pTria-Y1.

After E. coli transformation, several clones were picked for further analysis including DNA sequencing, protein expression, and extraction from the periplasmic space of the bacteria. SDS-PAGE analysis and Western blot analysis were performed under reducing conditions to confirm the size of the Y1 triabody.

9.2 Construction of double antibody

The above pTria-Y1 vector was linearized with XhoI restriction enzyme, and the synthetic complementary double-stranded oligonucleotides (5'-TCGAGAGGTGGAGGCGGT and 5'TCGAACCGCCTCCACCTC) were pre-annealed and ligated into the Y1 heavy chain and Y1 light chain on the XhoI site. This new vector was named pDia-Y1. The DNA sequence and protein expression were verified as described for the triple antibody.

9.3 Expression and purification of triabody and diabody

Expression in E. coli was generally as described above in connection with scFv-Y1. However, the method for purifying Y1 diabody and triabody from the periplasm of transformed E. coli cells is different. The scFv Y1 monomeric form can be purified on an affinity column of Protein-A Sepharose beads. However, this method does not efficiently purify Y1 in multimeric form. Therefore, periplasmic proteins extracted from the bacteria were precipitated overnight with 60% ammonium sulfate, resuspended in water, and loaded onto a Sephacryl-200 (Pharmacia) size exclusion column pre-equilibrated with 0.1×PBS . Fractions were collected and analyzed by HPLC and separate fractions containing dimer or trimer forms were collected for FITC labeling and FACS analysis.

9.4 Binding of Y1 double antibody and triple antibody to cells

FACS analysis of Jurkat cells was performed using the "three-step staining method". First, crude extracts or purified unlabeled scFv were stained, then with a mouse anti-myc antibody, and finally with a FITC- or PE-conjugated anti-mouse antibody. FACS analysis requires 5-8 x ¹⁰⁵ cells that are Ficoll purified and resuspended in PBS + 1% BSA. Binding was performed at 4°C for 1 hour. After each step, cells were washed and resuspended in PBS+1% BSA. After the final staining step, cells were fixed by resuspension in PBS, 1% BSA, 2% formaldehyde and read by FACS (Becton-Dickinson).

The binding of Y1-scFv was compared to that of diabodies and terabodies. In this analysis (Figure 7), the binding characteristics of all three forms were very similar, suggesting that the above-mentioned modifications on the molecule did not alter, eliminate or disrupt the apparent binding of Yl to its ligand.

9.5 Production of Y1-cys-kak (cysteine dimer)

Induce 1 liter of λpL-y1-cys-kak bacterial culture at 42°C for 2-3 hours. The culture was centrifuged at 5000 RPM for 30 minutes. Resuspend the pellet in 180 ml TE (50 mM Tris-HCl pH 7.4, 20 mM EDTA). 8ml of lysozyme (from 5mg/ml stock solution) was added and incubated for 1 hour. 20ml of 5M NaCl and 25ml of 25% Triton were added and incubated for another 1 hour. The mixture was centrifuged at 13000 RPM for 60 minutes at 4°C, and the supernatant was discarded. The pellet was resuspended in TE with the aid of a tissue blender (or homogenizer). This process was repeated 3-4 times until the inclusion bodies (precipitate) turned gray/light brown. The inclusion bodies were dissolved in 6M guanidine hydrochloride, 0.1M Tris pH 7.4, 2mM EDTA (1.5 g of inclusion bodies dissolved in 10 ml of lysis buffer provides approximately 10 mg/ml of dissolved protein). The solution was incubated for at least 4 hours. Protein concentration was determined and adjusted to 10 mg/ml. DTE was added to a final concentration of 65 mM and incubated overnight at room temperature. Refolding was initiated by diluting (dropwise) 10ml of protein into a solution containing 0.5M Arginine, 0.1M Tris pH 8, 2mM EDTA, 0.9mM GSSG. The refolded solution was incubated at approximately 10°C for 48 hours. The refolded solution containing the protein was dialyzed against a buffer containing 25 mM phosphate buffer pH 6, 100 mM urea and concentrated to 500 ml. The concentrated/dialyzed solution was bound to a SP-Sepharose column, and the protein was eluted by gradient NaCl (up to 1M).

9.6 Study of affinity of S-S Y1-dimer by radioreceptor binding assay (RRA) using KG-1 cells and comparison with CONY1 and Y1-IgG

The assay system includes the use of radioactive ligands prepared by iodination with ¹²⁵ I, Chloramine T on Y1-IgG constructs, or Bolton-Hunter reagent on CONY1 (Y1 scFV ) on the construct. Assay tubes contained 5 x ¹⁰⁶ KG-1 cells per 0.2 ml solution, and labeled tracer with varying amounts of unlabeled competitor, dissolved in PBS + 0.1% BSA, pH 7.4. After incubation with agitation for 1 hour at 4°C, the cells were washed extensively with cold buffer and used for radioactivity counting.

In RRA studies using labeled Y1-IgG, 2 ng/tube ^of125I -Y1-IgG was used and competed with each of the three molecules described above. The result is shown in Figure 8. The results presented in Figure 8 show that the SS Y1 dimer has a 30-fold higher affinity than CONY1. The approximate affinity of Y1-IgG estimated in this experiment was 2 x 10 ^-8 M. Accordingly, the corresponding affinity of the dimer is 4 x 10 ^-8 M.

In a second RRA study using labeled CONY1, ¹²⁵ I-Y1-IgG at 100 ng/tube was used and competed with each of the three molecules described above. The result is shown in Figure 9. The figure shows that the SS Y1 dimer has a 20-fold higher affinity than CONY1. The roughly estimated affinity of CONY1 in this experiment was 10 ⁻⁶ M. Accordingly, the corresponding affinity of the dimer is 5 x 10 ^-8 M.

9.7 ELISA analysis of GC (glycocalicin)

At 4°C, incubate 100 μl of purified glycocalicin on a 96-well flat-bottomed maxisorp plate overnight, wash the plate 3 times with PBST (PBS+0.05% tween), and then add 200 ml of PBST-milk (PBST+2% non-fat milk ) and incubated for 1 hour at room temperature. The plates were washed with PBST, and the monomers or dimers (100 μl) were added to PBST-emulsion at different concentrations and incubated for 1 hour at room temperature. The plates were then washed and anti- _VL polyclonal antibody (from immunized rabbit, _VL from Y1) (diluted 1:100 in PBST-milk) was added and incubated for 1 hour. The plates were washed and anti-rabbit HRP was added and incubated for an additional 1 hour. The plate was washed 5 times and 100 μl of TMB substrate was added, incubated for approximately 15 minutes, and then 100 μl of 0.5 H ₂ SO ₄ was added to stop the reaction. The optical density of the plate was measured at a wavelength of 450 nm in an ELISA reader.

9.8 Reactivity of Y1 with recombinant glycocalicin (GC) expressed in prokaryotic (Escherichia coli) system

A DNA fragment encoding the N-terminal soluble portion (GC, amino acids 1-493) of human platelet GP1b-glycocalicin was cloned into an IPTG-inducible prokaryotic vector cassette. E. coli (BL21 DE3) cells with newly constructed plasmids were grown to an O.D. of 0.7-0.8 at 37°C for 3 hours at 37°C for induction in the presence of IPTG. SDS-polyacrylamide gels loaded with native semi-purified human platelets from GC or E. coli cell lysates (total protein content) from induced and non-induced cells were analyzed. With scFv Y1-biotinylated, polyclonal rabbit anti-human GC antibody, commercially obtained human anti-mouse CD42 monoclonal antibody (SZ2 Immunotech, PM640 Serotec, HIP1 Pharmigen, AN51 DAKO) and anti-gp1ba N-terminal Polyclonal antibody (Sc-7071, Santa Cruz) was used for Western blot analysis. The above two polyclonal antibodies can recognize GC derived from recombinant bacteria and GC derived from natural human platelets. The scFv Y1 and commercially available antibodies can only recognize natural human-derived GCs, but cannot recognize the bacterial-derived recombinant platelet GCs.

Post-translational modifications such as glycosylation and sulfation are required for GC-binding scFv and commercially available antibodies. The prokaryotic (E. coli) system lacks post-translational modification mechanisms such as glycosylation and sulfation.

9.9 Preparation of tetramers of Y1

A construct was designed in which the following sequence LNDIFEAQKIEWHE was added at the C-terminus of Y1 by PCR and cloned into the IPGC inducible expression system. This clone was named Y1-biomarker. This sequence is a substrate for the enzyme BirA, which is capable of covalently attaching biotin to lysine (K) residues in the presence of free biotin (phenotype analysis of antigen-specific T lymphocytes. Science.1996 Oct 4;274(5284):94-6, Altman JD et al). The construct was produced as inclusion bodies in BL21 bacterial cells. Refold as above. The inclusion bodies were dissolved in guanidine-DTE. Refolding was performed by dilution in buffer containing arginine-tris-EDTA. After dialysis and concentration, HiTrapQ ion exchange purification was performed.

Purified Y1-biomarker scFv was incubated with BirA enzyme (purchased from Avidity) and biotin as recommended by the supplier. The biotinylated Y1-biomarker was analyzed by HABA assay (the analysis estimated the amount of biotin per molecule) and indicated the presence of approximately >0.8 biotin residues/molecule.

The biotinylated Y1-biomarker was incubated with streptavidin-PE (phycoerythrin) to form a complex and used for FACS experiments with KG-1 cells (Y1 positive). Streptavidin can bind up to 4 biotinylated Y-1-biomarker molecules. Due to the increased avidity, the sensitivity of the binding is increased by at least 100-fold.

The sequence of the Y1-biomarker is as follows:

1 MEVQLVESGG GVVRPGGSLR LSCAASGFTF DDYGMSWVRQ

41 APGKGLEWVS GINWNGGSTG YADSVKGRFT ISRDNAKNSL

81 YLQMNSLRAE DTAVYYCARM RAPVTWGQGT LVTVSRGGGG

121 SGGGGSGGGG SSELTQDPAV SVALGQTVRI TCQGDSLRSY

161 YASWYQQKPG QAPVLVIYGK NNRPSGIPDR FSGSSSGNTA

201 SLTITGAQAE DEADYYCNSR DSSGNNVVFG GGTKLTVLGG

241 GGLNDIFEAQ KIEWHE

Example 10: Construction of full size Y1-IgG1

Intact IgG molecules have several advantages over the Fv form, including a longer half-life in vivo and the potential to induce cellular responses in vivo, as induced by ADCC or CDC (complement-dependent cytotoxicity; Tomlinson, Current Opinions of Immunology, 5 , 83-89 (1993)) mediated response. By the molecular cloning method disclosed below, we have converted the Y1 Fv region into a full-sized Y1-IgG1 molecule. The construction of the Y1-IgG1 construct was accomplished by ligating the cDNA fragments to each other in the following order.

10.1 Leader sequences compatible with mammalian expression systems: Design an interchangeable system to allow easy insertion of the elerAents required for complete IgG molecules. The following complementary double-stranded oligonucleotides encoding the putative leader sequence were synthesized, annealed, and ligated into the Xhol site of a mammalian expression vector (under the control of the SRα5 promoter).

5′-

TCGACCTCATCACCATGGCCTGGGCTCTGCTGCTCCTCACCCTCCTCACTC

AGGACACAGGGTCCTGGGCCGAT

and

5′-

GATCGATTGCACCAGCTGGATATCGGCCCAGGACCCTGTGTCCTGAGTGA

GGAGGGTGAGGAGCAGCAGCCCAGGCCATGGTGATGAGG.

Two Kozak elements were included upstream of the initiation ATG codon. In addition, an internal EcoRV site was introduced at the putative cleavage site and XhoI site of the leader sequence to allow subcloning of its variable region. This modified vector was named pBJ-3.

10.2 Insert the _VL coding sequence from the Y1 scFv cDNA sequence between the leader sequence and the light region coding sequence of the construct. Similarly, the _VH coding sequence from the Y1 scFv cDNA sequence was inserted between the leader sequence and the heavy region coding sequence of the construct. This objective was achieved by PCR amplifying the vector pHEN-Y1 encoding the starting Y1 in order to obtain the _VL and _VH regions, respectively.

10.3 The oligonucleotides 5'-TTTGATATCCAGCTGGTGGAGTCTGGGGGA (sense) and 5'-GCTGACCTAGGACGGTCAGCTTGGT (antisense) were used in the V _L PCR reaction. A cDNA product with an expected size of approximately 350 bp was purified, sequenced, and digested with EcoRV and AvrII restriction enzymes. The _VH cDNA region was amplified and purified using the same method, using sense and antisense oligonucleotides 5'-GGGATATCCAGCTG(C/G)(A/T)GGAGTCGGGC and 5'-GGACTCGAGACGGTGACCAGGGTACCTTG, respectively.

10.4 Constant region: Synthesize the constant λ3 (CL-λ3) region and the constant heavy region CH1-CH3 for IgG1 cDNA according to the following methods.

10.4.1 For the constant CL-λ3 region, RT-PCR was performed on mRNA extracted from a pool of normal peripheral B cells (CD 19+ cells) using both sense 5′-CCGTCCTAGGTCAGCCCAAGGCTGC and antisense 5′-TTTGCGGCCGCTCATGAACATTCTGTAGGGGCCACTGT oligos Nucleotides. A PCR product of the expected size (approximately 400 bp) was purified, sequenced, and digested with Avrll and Notl restriction enzymes.

10.4.2 For the constant IgGl region (γ chain), a human B cell clone (CMV-clone #40) immortalized in BTG was selected for PCR amplification. This clone was shown to secrete IgGl against human CMV and was also shown to induce ADCC responses in in vitro assays. For CH1-CH3 cDNA, oligonucleotides 5′-CCGCTCGAGTGC(T/C)TCCACCAAGGGCCCATC(G/C)GTCTTC (sense) and 5′TTTGCGGCCGCTCATTTACCC(A/G)GAGACAGGGAGAGGCT (antisense) were synthesized and used in PCR Amplify. PCR products of the expected size (approximately 1500 bp) were purified, sequenced, and digested with Avrll and Notl restriction enzymes as described for the CL cDNA coding sequence.

10.5 For the final expression vector, perform a triple ligation approach with EcoRV-NotI predigested vector, EcoRV-AvrII variable cDNAs and AvrII-NotI constant regions. The final vectors for heavy and light chain expression were named Y-I-HC and Y1-LC, respectively.

10.6 Construct another vector pBJ-Y1-LP based on Y1-LC to enable double selection based on the puromycin resistance gene (PAC). In this vector, the neomycin resistance gene of the Y1-LC plasmid was replaced by an approximately 1600 bp fragment (from the pMCC-ZP vector) encoding the PAC gene.

10.7 The open reading frames (ORL) and their encoded amino acid sequences of Y-1-IgG-HC and Y1-IgG-LC are provided below:

10.7.1 ORF of Y1-IgG-HC (V _{H CH} 1 _CH 2 _CH 3)

1 ATGGCCTGGGCTCTGCTGCTCCTOACCCTCTCACTCAGGACACAGGGTCCTGGGCCGAT

1 M A W A L L L L T L L T Q D T G S W A D

61 ATCCAGCTGGTGGAGTCTGGGGGAGOTGTGGTACGGCCTGGGGOGTCCCTGAGACTCTCC

21 I Q L V E S G G G G V V R P G G S L R L S

121 TGTGCAGCCTCTGGATTCACCTTTGATGATTATGGCATGAGCTGGGTCCGCCAAGCTCCA

41 C A A A S G F T F D D Y G M S W V R Q A P

181 GGGAAGGGGCTGGAGTGGGTCTCTGGTATTAATTGGAATGGTGGTAGCACAGGTTATGCA

61 G K G L R W V S G I N W N G G S T G Y A

241 GACTCTGTGAAGGGCCGATTCACCATTCTCTAGAGACAACGCCAAGAACTCCCTGTATCTG

81 D S V K G R F T I S R D N A K N S L Y L

301 CAAATGAACAGTCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCAAGAATGAGGGCT

101 Q M N S L R A E D T A V Y Y C A R M R A

361 CCTGTGATTTGGGGCCAAGGTACCCTGGTCACCGTCTCGAGTGCTTCCACCAAGGGCCCA

121 P V I W G Q G T L V T V S S A S T K G P

421 TCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGC

141 S V F P L A P S S K S T S G G T A A L G

481 TGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTG

161 C L V K D Y F P E P V T V S W N S G A L

541 ACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGC

181 T S G V H T F P A V L Q S S G L Y S L S

601 AGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAAT

201 S V V T V P S S S L G T Q T Y I C N V N

661 CACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACT

221 H K P S N T K V D K R V E P K S C D K T

721 CACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACTGTCAGTCTTCOTCTTC

241 H T C P P C P A P E L L G G P S V F L F

781 CCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTG

261 P P P K P K D T L M I S R T P E V T C V V

841 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 K V

1021 TCCAACAAAGCCCTCCCAGCCCCCCATCGAGAAAACCATTCTCAAAGCCAAAGGGCAGCCC

341 S N K A L P A P I E K T I S K A K G Q P

1081 OGAGAACCACAGGTGTACACCCTGCCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTC

361 R E P Q V Y T L P P S R E E M T K N Q V

1141 AGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGC

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 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 K S L S L

1381 TCTCTGGGTAAATGA

461 S L G K *

10.7.2 ORF(V _L C _L ) of Y1-IgG-LC

1 ATGGCCTGGGCTCTGCTGCTCCTCACCCTCCTCACTCAGGACACAGGGTCCTGGGCCGAT

1 M A W A L 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 G Q T V R I T

1212 TGCCAAGGAGACAGCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAG

41 C Q G D S L R S Y Y A S W Y Q Q K P G Q

181 GCCCCTGTACTTGTCATCTATGGTAAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTC

161 A P V L V I Y G K N N R P S G I P D R F

241 TCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGAT

81 S G S S S S G N T A S L T I T G A Q A E D

301 GAGGCTGACTATTACTGTAACTCCCGGGACAGCAGTGGTAACCATGTGGTATTCGGCGGA

101 E A D Y Y C N S R D S S G N H V V F 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

421 CCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTC

141 P S S E E E L Q A N K A T L V C L I S D F

481 TACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTG

161 Y P G A V T V A W K A D S S P V K A G V

541 GAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGC

181 E T T T T P S K Q S N N K Y A A S S Y L S

601 CTGACGCCTGAGCAGTGGAAGTCCCACAAAAAGCTACAGCTGCCAGGTCACGCATGAAGGG

201 L T P E Q W K S H K S 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 *

Underline the leading sequence. The _VH and _VL regions are encoded by the bold amino acid sequence, respectively, followed by the IgGl (for heavy chain) or λ3 (for light chain) constant region sequence.

10.8 Expression of Y1 heavy chain and light chain in CHO cells

Vectors Y1-HC and Y1-LC were used for transfection and selection of stable cells expressing the heavy or light chain, respectively. After selection and cell growth on G418, the secreted protein in the supernatant was analyzed by capture EIA analysis and Western blot as described below to confirm IgGI expression.

10.8.1 Capture EIA analysis: the wells of a 96-well plate were pre-coated with mouse anti-human IgG Fc (Sigma), the supernatant obtained from the above method was added to the wells, and biotinylated Goat anti-gamma chain-specific antibody (Sigma), streptavidin-HRP and substrate detect the presence of heavy chain IgGl. Color development was monitored with an ELISA plate reader at A ₄₀₅ wavelength.

10.8.2 Western blot analysis: put the supernatant of the above cells on 12.5% SDS-PAGE for electrophoresis. Heavy chain expression was detected with (a) goat anti-human IgG-HRP (H+L; Sigma cat. no. A8667) and (b) biotinylated goat anti-human λ3 chain (Southern Biotechnology Association, cat. no. 2070-08) with For detection of light chain expression.

Expression of both chains was confirmed by the above analysis, and co-transfection was performed in order to obtain Y1-IgG1 at full size.

10.9 Expression and Purification of Y1-IgG

10.9.1 Cell culture and transfection: Culture CHO cells in F-12 medium containing 10% fetal bovine serum and 40 μg/ml gentamicin at 37°C in 5% CO ₂ air. 1 day before transfection, 0.8 × ¹⁰⁶ cells were seeded onto a 90 mm Petri dish. The cultures were co-transfected with 10 μg of light and heavy chain DNA by FuGene (Roche) transfection reagent technology. After 2 days of growth in non-selective medium, the cells were cultured for 10-12 days in F-12 medium containing 550 μg/ml neomycin and 3 μg/ml puromycin. The cells were trypsinized and cloned by limiting dilution at 0.5 cells/well in Costar 96-well plates. Single colonies were picked, grown on 6-well dishes, and transferred to flasks.

10.9.2 Determination of heavy and light chain secretion: The concentration of antibody secreted into the supernatant of transfected CHO cells was determined using a sandwich ELISA assay. To determine the concentration of the antibody, the following reagents were used: monoclonal anti-human IgG1 (Fc) (Sigma) as coating antibody, goat anti-human IgG (γ chain specific) biotin conjugate as detection reagent (Sigma ), and pure human IgG1, λ (Sigma) was used as a standard. Yield rates fluctuated between 3-4 μg/ml as confirmed by this ELISA analysis.

10.9.3 Mab Production and Purification from the Cells: Cells were grown in roller bottles to 1-2 Final concentration of ^x108 cells. For production, cells were cultured for an additional 2 days in the same medium containing 2% fetal bovine serum. Secreted antibodies were purified on protein G-sepharose columns (Pharmacia). Binding was performed in 20 mM sodium phosphate buffer, pH 7.0; elution was performed with 0.1 M glycine, pH 2.5-3-0. Quantity of purified antibody was determined by UV absorbance; purity was analyzed by SDS PAGE. Under non-denaturing conditions, intact IgG antibodies have their expected molecular weight of 160 kD. In denaturing gels, the heavy and light chains had expected molecular weights of 55 and 28 kD, respectively.

10.9.4 Binding of full size Y1-IgG molecules: Binding experiments were performed in order to determine the level of binding of Y1-IgG molecules and compare with the binding levels of scFv-Y1 molecules. A two-step staining method was employed in which 5 ng of Y1-IgG was reacted with RAJI cells (negative control, Figure 7a) and Jurkat cells (Y1 positive cells, Figure 7b). For detection, PE-labeled goat anti-human IgG was used. Similarly, 1 μg of scFv-Y1 was reacted with Jurkat cells (Fig. 7c), and P1-labeled rabbit anti-scFv was used for detection. The results showed that both Y1-IgG and scFv-Y1 could bind Jurkat cells, and approximately ¹⁰³ -fold higher scFv-Y1 molecules were required to obtain detection levels similar to Y1-IgG.

Brief description of the form

Table 1: Panning results from method AM. The estimated number of phagemids used for panning (input), and the estimated number of eluted bound phagemids (output) of the AM biopanning method for four consecutive steps were summarized. The cell source and elution medium for each yielding result are listed, along with the terms used to distinguish the individual stocks for each virion.

Table 2: Clones selected by AM biopanning method. The number of amino acids in the CDR3 region ( _VH -CDR3 size), and the CDR3 amino acid sequences of different types of clones isolated were summarized. In addition, the frequency of each type of clone in the two AM biopanning output T16M3 and T16M3.1 output is provided.

Table 3: Panning results from the YPR method. The estimated number of phagemids used for panning (input), and the estimated number of eluted bound phagemids (output) were summarized. The elution medium for each yielding result is listed, along with the terminology used to distinguish each individual stock.

Table 4: Panning results from the YPNR method. The estimated number of phagemids used for panning (input) and the estimated number of eluted bound phagemids (output) of the YPNR biopanning method for three consecutive steps were summarized. The elution medium for each yielding result is listed, along with the terminology used to distinguish each of the different stocks.

Table 5: Y-series clones selected by the YPR biopanning method with R3 output. Several different clones were identified in the R3 producing stock. The number of amino acid residues and the amino acid sequence contained in the _VH -CDR3 region of the identified clones are provided, along with the germline designation.

Table 6: Binding specificity of Y1 to leukemia cells. The results of binding experiments of three different scFv clones are presented, each capable of reacting with cell mixtures containing primarily each of seven different types of leukemia cells, as demonstrated by FACS analysis. The results represent the proportion of patients whose cells were confirmed to react positively with each antibody tested by FACS analysis. The numerator represents the number of positive patients, while the denominator represents the total number of patients tested for a particular scFv/leukemia cell type combination.

Table 7: FACS analysis of scFv binding to Ficoll purified normal blood cells. Three scFv clones were analyzed separately for binding to five different Ficoll-purified normal blood cell types. The binding results represent the proportion of normal blood samples that were confirmed to react positively with each of the tested antibodies by FACS analysis.

Table 8: Comparison of Y1 scFv and antibody binding to various cell markers. Results of scFv analysis stained by Y1 and a panel of other antibodies are presented. Ficoll-purified peripheral and bone marrow cells from ANE patients were prepared, and the binding specificity of Y1 scFv and that of various cell markers were studied comparatively on AML cells. Results are expressed as the percentage of cells in Ficoll-purified samples from a particular patient that were confirmed to react positively with each Fv by FACS analysis. Comparisons were made with four other antibodies: (1) CD13-marker of granulocytes and monocytes; (2) CD14-marker of monocytes and neutrophils; (3) CD33-marker of normal and leukemic myeloid cells and (4) CD34-stem cell marker.

Table 9: Binding of Y1 to hematopoietic cell lines. FACS analysis was performed to determine the binding of Y1 scFv to different types of human leukemia cell lines and to a murine cell line. Cell lines that positively bind Y1 (reactive) or fail to bind (anergic) are listed.

Table 10: CDR3 sequences of _VH3 -DP32 isolated clones.

Several clones based on the DP32 germline were isolated following different biopanning and selection methods. Clones Y1, Y17, Y-27 and Y-44 were identified during selection with platelet biopanning (YPR and YPNR methods). The _VH -CDR3 region sequence of each clone is provided.

Table 11: Binding characteristics of _VH3 -DP32 isolated clones. The binding specificity of DP32-derived clones to several hematopoietic cell types was tested by FACS analysis.

The invention has been described with reference to specific embodiments, materials and data. Alternatives for using or making the various aspects of the invention are possible, as will be appreciated by those skilled in the art. Such alternatives are considered to be within the intended and contemplated scope of the present invention as defined by the following claims.

sequence listing

<110> Biotech General Corporation

<210>1

<211>10

<212>PRT

<213> Human (Homo sapiens)

<400>1

Ser Ser Tyr Thr Ser Ser Ser Ser Thr Leu Val

1 5 10

<210>2

<211>10

<212>PRT

<213> people

<400>2

Ser Ser Tyr Thr Ser Ser Ser Ser Thr Leu Gly

1 5 10

<210>3

<211>9

<212>PRT

<213> people

<400>3

Gln Ser Tyr Asp Ser Asn Leu Arg Val

1 5

<210>4

<211>8

<212>PRT

<213> people

<400>4

Gln Gln Leu Asn Ser Tyr Pro Thr

1 5

<210>5

<211>11

<212>PRT

<213> people

<400>5

Asn Ser Arg Asp Ser Ser Gly Phe Gln Leu Val

1 5 10

<210>6

<211>9

<212>PRT

<213> people

<400>6

Gln Gln Ala Asn Ser Phe Pro Ile Thr

1 5

<210>7

<211>111

<212>PRT

<213> people

<400>7

Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln Thr

1 5 10 15

Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala Ser

20 25 30

Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr Gly

35 40 45

Lys Asn Asn Arg Pro Ser Gly rle Pro Asp Arg Phe Ser Gly Ser Ser

50 55 60

Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp

65 70 75 80

Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Asn His Val

85 90 95

Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Ala Ala Ala

100 105 110

<210>8

<211>6

<212>PRT

<213> people

<400>8

Met Arg Ala Pro Val Ile

1 5

<210>9

<211>8

<212>PRT

<213> people

<400>9

Pro Trp Asp Asp Val Thr Pro Pro

1 5

<210>10

<211>12

<212>PRT

<213> people

<400>10

Gly Phe Pro Arg Ile Thr Pro Pro Ser Ala Glu Ile

1 5 10

<210>11

<211>5

<212>PRT

<213> people

<400>11

Gly Phe Pro Met Pro

1 5

<210>12

<211>10

<212>PRT

<213> people

<400>12

Gly Phe Pro His Ser Ser Ser Ser Val Ser Arg

1 5 10

<210>13

<211>11

<212>PRT

<213> people

<400>13

Arg Phe Pro Met Arg His Glu Lys Thr Asn Tyr

1 5 5 10

<210>14

<211>8

<212>PRT

<213> people

<400>14

Arg Phe Pro Pro Thr Ala Thr Ile

1 5

<210>15

<211>7

<212>PRT

<213> people

<400>15

Thr Gln Arg Arg Asp Leu Gly

1 5

<210>16

<211>11

<212>PRT

<213> people

<400>16

Lys Phe Pro Gly Gly Thr Val Arg Gly Leu Lys

1 5 5 10

<210>17

<211>12

<212>PRT

<213> people

<400>17

Gly Phe Pro Val Ile Val Glu Glu Arg Gln Ser Thr

1 5 10

<210>18

<211>10

<212>PRT

<213> people

<400>18

Arg Phe Pro Gln Arg Val Asp Asn Arg Val

1 5 5 10

<210>19

<211>8

<212>PRT

<213> people

<400>19

Thr Gly Gln Ser Ile Lys Arg Ser

1 5

<210>20

<211>6

<212>PRT

<213> people

<400>20

Leu Thr His Pro Tyr Phe

1 5

<210>21

<211>6

<212>PRT

<213> people

<400>21

Leu Arg Pro Pro Gln Ser

1 5

<210>22

<211>11

<212>PRT

<213> people

<400>22

Thr Ser Lys Asn Thr Ser Ser Ser Lys Arg His

1 5 10

<210>23

<211>12

<212>PRT

<213> people

<400>23

Arg Tyr Tyr Cys Arg Ser Ser Asp Cys Thr Val Ser

1 5 5 10

<210>24

<211>10

<212>PRT

<213> people

<400>24

Phe Arg Arg Met Glu Thr Val Pro Ala Pro

1 5 5 10

<210>25

<211>277

<212>PRT

<213> people

<400>25

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala

1 5 10 15

Ala Gln Pro Ala Met Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly

20 25 30

Val Val Arg Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

35 40 45

Phe Thr Phe Asp Asp Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly

50 55 60

Lys Gly Leu Glu Trp Val Ser Gly Ile Asn Trp Asn Gly Gly Ser Thr

65 70 75 80

Gly Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

85 90 95

Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

100 105 110

Thr Ala Val Tyr Tyr Cys Ala Arg Met Arg Ala Pro Val Ile Trp Gly

115 120 125

Gln Gly Thr Leu Val Thr Val Ser Arg Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Ser Glu Leu Thr Gln Asp Pro Ala

145 150 155 160

Val Ser Val Ala Leu Gly Gln Thr Val Arg Ile Thr Cys Gln Gly Asp

165 170 175

Ser Leu Arg Ser Tyr Tyr Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln

180 185 190

Ala Pro Val Leu Val Ile Tyr Gly Lys Asn Asn Arg Pro Ser Gly Ile

195 200 205

Pro Asp Arg Phe Ser Gly Ser Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr

210 215 220

Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser

225 230 235 240

Arg Asp Ser Ser Gly Asn His Val Val Phe Gly Gly Gly Thr Lys Leu

245 250 255

Thr Val Leu Gly Ala Ala Ala Glu Gln Lys Leu Ile Ser Glu Glu Asp

260 265 270

Leu Asn Gly Ala Ala

275

<210>26

<211>464

<212>PRT

<213> people

<400>26

Met Ala Trp Ala Leu Leu Leu Leu Thr Leu Leu Thr Gln Asp Thr Gly

1 5 10 15

Ser Trp Ala Asp Ile Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Asp Asp Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ser Gly Ile Asn Trp Asn Gly Gly Ser Thr Gly Tyr Ala

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn

85 90 95

Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Met Arg Ala Pro Val Ile Trp Gly Gln Gly Thr

115 120 125

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

130 135 140

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

145 150 155 160

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

165 170 175

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

180 185 190

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val Val Thr Val Pro Ser Ser

195 200 205

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

210 215 220

Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr

225 230 235 240

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

245 250 255

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

260 265 270

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

275 280 285

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

290 295 300

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

305 310 315 320

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

325 330 335

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

340 345 350

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

355 360 365

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

370 375 380

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

385 390 395 400

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Ser Pro Val Leu Asp

405 410 415

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

420 425 430

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

435 440 445

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

450 455 460

<210>27

<211>233

<212>PRT

<213> people

<400>27

Met Ala Trp Ala Leu Leu Leu Leu Thr Leu Leu Thr Gln Asp Thr Gly

1 5 10 15

Ser Trp Ala Asp Ala Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala

20 25 30

Leu Gly Gln Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser

35 40 45

Tyr Tyr Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu

50 55 60

Val Ile Tyr Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe

65 70 75 80

Ser Gly Ser Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala

85 90 95

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser

100 105 110

Gly Asn His Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

115 120 125

Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu

130 135 140

Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe

145 150 155 160

Tyr Pro Gly Ala Val Thr Val Ala Tro Lys Ala Asp Ser Ser Pro Val

165 170 175

Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys

180 185 190

Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser

195 200 205

His Lys Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu

210 215 220

Lys Thr Val Ala Pro Thr Glu Cys Ser

225 230

<210>28

<211>11

<212>PRT

<213> people

<400>28

Phe Leu Thr Tyr Asn Ser Tyr Glu Val Pro Thr

1 5 5 10

<210>29

<211>9

<212>PRT

<213> people

<400>29

Thr Asn Trp Tyr Leu Arg Pro Leu Asn

1 5

<210>30

<211>98

<212>PRT

<213> people

<400>30

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Met His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Leu Val Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Glu Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr

<210>31

<211>98

<212>PRT

<213> people

<400>31

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Asp Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro G1y Gln Glu Leu Gly Trp Met

35 40 45

Gly Arg Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Thr Glu Leu Ser Ser Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg

<210>32

<211>98

<212>PRT

<213> people

<400>32

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Leu Thr Ghu Leu

20 25 30

Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Gly Phe Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr

<210>33

<211>98

<212>PRT

<213> people

<400>33

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ser Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Val Val Tyr Tyr Cys

85 90 95

Ala Arg

<210>34

<211>98

<212>PRT

<213> people

<400>34

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210>35

<211>98

<212>PRT

<213> people

<400>35

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Trp Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210>36

<211>98

<212>PRT

<213> people

<220>

<221>X

<222>(1)..(98)

<223>Xaa

<400>36

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Leu Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Xaa Gln Ala Pro Gly Gly Gly Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210>37

<211>98

<212>PRT

<213> people

<400>37

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Cys Met His Trp Val Arg Gln Val His Ala Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Val Cys Pro Ser Asp Gly Ser Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Ala Arg Val Thr Ile Thr Arg Asp Thr Ser Met Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Val Arg

<210>38

<211>98

<212>PRT

<213> people

<400>38

Gln Met Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Thr

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Ser Ser

20 25 30

Ala Val Gln Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Trp Ile Val Val Gly Ser Gly Asn Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Glu Arg Val Thr Ile Thr Arg Asp Met Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala

<210>39

<211>98

<212>PRT

<213> people

<400>39

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210>40

<211>98

<212>PRT

<213> people

<400>40

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210>41

<211>98

<212>PRT

<213> people

<400>41

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210>42

<211>98

<212>PRT

<213> people

<400>42

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ser Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Glu Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Met Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210>43

<211>98

<212>PRT

<213> people

<400>43

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Asn Thr Gly Asn Pro Thr Tyr Ala Gln Gly Phe

50 55 60

Thr Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Cys Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<2l0>44

<211>98

<212>PRT

<213> people

<400>44

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Asn Thr Gly Asn Pro Thr Tyr Ala Gln Gly Phe

50 55 60

Thr Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210>45

<211>98

<212>PRT

<213> people

<400>45

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asp Ile Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asn Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210>46

<211>98

<212>PRT

<213> people

<400>46

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210>47

<211>92

<212>PRT

<213> people

<400>47

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala

85 90

<210>48

<211>98

<212>PRT

<213> people

<400>48

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210>49

<211>98

<212>PRT

<213> people

<400>49

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Asn Ser Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210>50

<211>98

<212>PRT

<213> people

<400>50

Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Thr Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Tyr Arg

20 25 30

Tyr Leu His Trp Val Arg Gln Ala Pro Gly Gln Ala Leu Glu Trp Met

35 40 45

Gly Trp Ile Thr Pro Phe Asn Gly Asn Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Asp Arg Val Thr Ile Thr Arg Asp Arg Ser Met Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg

<210>51

<211>98

<212>PRT

<213> people

<400>51

Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Thr Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Tyr Arg

20 25 30

Tyr Leu His Trp Val Arg Gln Ala Pro Gly Gln Ala Leu Glu Trp Met

35 40 45

Gly Trp Ile Thr Pro Phe Asn Gly Asn Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Asp Arg Val Thr Ile Thr Arg Asp Arg Ser Met Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg

<210>52

<211>96

<212>PRT

<213> people

<400>52

Gln Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asn Ala

20 25 30

Arg Met Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu

35 40 45

Trp Leu Ala His Ile Phe Ser Asn Asp Glu Lys Ser Tyr Ser Thr Ser

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

<210>53

<211>99

<212>PRT

<213> people

<400>53

Gln Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

Glu Trp Cys Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp

35 40 45

Leu Ala Leu Ile Tyr Trp Asn Asp Asp Lys Arg Tyr Ser Pro Ser Leu

50 55 60

Lys Ser Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln Val Val

65 70 75 80

Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala His Arg

<210>54

<211>96

<212>PRT

<213> people

<400>54

Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

Gly Met Cys Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu

35 40 45

Trp Leu Ala Leu Ile Asp Trp Asp Asp Asp Lys Tyr Tyr Ser Thr Ser

50 55 60

Leu Lys Thr Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

<210>55

<211>96

<212>PRT

<213> people

<400>55

Gln Val Thr Leu Lys Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

Gly Met Arg Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu

35 40 45

Trp Leu Ala Arg Ile Asp Trp Asp Asp Asp Lys Phe Tyr Ser Thr Ser

50 55 60

Leu Lys Thr Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

<210>56

<211>100

<212>PRT

<213> people

<400>56

Gln Ile Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser

20 25 30

Gly Val Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu

35 40 45

Trp Leu Ala Leu Ile Tyr Trp Asn Asp Asp Lys Arg Tyr Ser Pro Ser

50 55 60

Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala His Arg

100

<210>57

<211>100

<212>PRT

<213> people

<400>57

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp His

20 25 30

Tyr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Thr Arg Asn Lys Ala Asn Ser Tyr Thr Thr Glu Tyr Ala Val

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg

100

<210>58

<211>100

<212>PRT

<213> people

<400>58

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp His

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Gln Gly Lys Gly Leu Glu Leu Val

35 40 45

Gly Leu Ile Arg Asn Lys Ala Asn Ser Tyr Thr Thr Glu Tyr Ala Ala

50 55 60

Ser Val Lys Gly Arg Leu Thr Ile Ser Arg Glu Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Ser Ser Leu Lys Thr Glu Asp Leu Ala Val Tyr

85 90 95

Tyr Cys Ala Arg

100

<210>59

<211>100

<212>PRT

<213> people

<400>59

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp His

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Gln Gly Lys Gly Leu Glu Leu Val

35 40 45

Gly Leu Ile Arg Asn Lys Ala Asn Ser Tyr Thr Thr Glu Tyr Ala Ala

50 55 60

Ser Val Lys Gly Arg Leu Thr Ile Ser Arg Glu Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Ser Ser Leu Lys Thr Glu Asp Leu Ala Val Tyr

85 90 95

Tyr Cys Ala Arg

100

<210>60

<211>98

<212>PRT

<213> people

<400>60

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Lys

<210>61

<211>98

<212>PRT

<213> people

<400>61

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro G1y Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Asn Trp Asn Gly Gly Ser Thr Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr His Cys

85 90 95

Ala Arg

<210>62

<211>98

<212>PRT

<213> people

<400>62

Glu Val Gln Leu Val Glu Ser Gly Gly Val Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Leu Ile Ser Trp Asp Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Lys

<210>63

<211>98

<212>PRT

<213> people

<400>63

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210>64

<211>100

<212>PRT

<213> people

<400>64

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala

50 55 60

Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Thr

100

<210>65

<211>98

<212>PRT

<213> people

<400>65

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Pro Ala Ser Gly Phe Thr Phe Ser Asn His

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Gly Asp Ser Gly Tyr Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Asn Asn Ser Pro Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Lys

<210>66

<211>98

<212>PRT

<213> people

<400>66

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn His

20 25 30

Tyr Thr Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ser Ser Ser Gly Asn Ser Gly Tyr Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Lys

<210>67

<211>98

<212>PRT

<213> people

<400>67

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ser

20 25 30

Asp Met Asn Trp Val His Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Val Ser Trp Asn Gly Ser Arg Thr His Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ser Arg Asn Thr Leu Tyr

65 70 75 80

Leu Gln Thr Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Arg

<210>68

<211>97

<212>PRT

<213> people

<400>68

Glu Val Gln Leu Val Glu Thr Gly Gly Gly Leu Ile Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg

<210>69

<211>97

<212>PRT

<213> people

<400>69

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg

<210>70

<211>97

<212>PRT

<213> people

<400>70

Glu Val Gln Leu Val His Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Gly Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys Ala

85 90 95

Arg

<210>71

<211>97

<212>PRT

<213> people

<400>71

Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Gly Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys Ala

85 90 95

Arg

<210>72

<211>98

<212>PRT

<213> people

<400>72

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val

35 40 45

Ser Ala Ile Ser Ser Asn Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Val Gln Met Ser Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Arg

<210>73

<211>35

<212>PRT

<213> people

<400>73

Thr Phe Ser Ser Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys

1 5 10 15

Gly Leu Glu Tyr Val Ser Ala Ile Ser Ser Asn Gly Gly Ser Thr Tyr

20 25 30

Tyr Ala Asp

35

<210>74

<211>98

<212>PRT

<213> people

<400>74

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210>75

<211>98

<212>PRT

<213> people

<400>75

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210>76

<211>98

<212>PRT

<213> people

<400>76

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210>77

<211>98

<212>PRT

<213> people

<400>77

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys

<210>78

<211>97

<212>PRT

<213> people

<400>78

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met His Trp Val Arg Gln Ala Thr Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Gly Thr Ala Gly Asp Thr Tyr Tyr Pro Gly Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg

<210>79

<211>98

<212>PRT

<213> people

<400>79

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Glu Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210>80

<211>98

<212>PRT

<213> people

<400>80

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys

<210>81

<211>98

<212>PRT

<213> people

<400>81

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

l 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210>82

<211>98

<212>PRT

<213> people

<400>82

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210>83

<211>97

<212>PRT

<213> people

<400>83

Glu Asp Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

l 5 10 15

Ser Leu Arg Pro Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr

20 25 30

Val Leu His Trp Val Arg Arg Ala Pro Gly Lys Gly Pro Glu Trp Val

35 40 45

Ser Ala Ile Gly Thr Gly Gly Asp Thr Tyr Tyr Ala Asp Ser Val Met

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Ile Ala Glu Asp Met Ala Val Tyr Tyr Cys Ala

85 90 95

Arg

<210>84

<211>98

<212>PRT

<213> people

<400>84

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Val Trp Val

35 40 45

Ser Arg Ile Asn Ser Asp Gly Ser Ser Thr Thr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210>85

<211>98

<212>PRT

<213> people

<400>85

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210>86

<211>97

<212>PRT

<213> people

<400>86

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Ile His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg

<210>87

<211>97

<212>PRT

<213> people

<400>87

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg

<210>88

<211>97

<212>PRT

<213> people

<400>88

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Val Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Asn Asn Pro Ser Leu Lys

50 55 60

Ser Arg Ala Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Asn Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Cys Cys Ala

85 90 95

Arg

<210>89

<211>99

<212>PRT

<213> people

<400>89

Gln Leu Gln Leu Gln Glu Ser Gly Ser Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly Tyr Ser Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Arg Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg

<210>90

<211>99

<212>PRT

<213> people

<400>90

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg

<210>91

<211>99

<212>PRT

<213> people

<400>91

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Gly

20 25 30

Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg

<210>92

<211>98

<212>PRT

<213> people

<400>92

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser Gly

20 25 30

Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210>93

<211>98

<212>PRT

<213> people

<400>93

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

l 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Ser Ser Gly

20 25 30

Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Ser Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210>94

<211>98

<212>PRT

<213> people

<400>94

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Asp

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser Ser Ser

20 25 30

Asn Trp Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Val Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210>95

<211>98

<212>PRT

<213> people

<400>95

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser Ser Ser

20 25 30

Asn Trp Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Ile Tyr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Val Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210>96

<211>98

<212>PRT

<213> people

<400>96

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Val Val Ser Gly Gly Ser Ile Ser Ser Ser Ser

20 25 30

Asn Trp Trp Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Glu Ile Tyr His Ser Gly Asn Pro Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Ile Ser Ile Asp Lys Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210>97

<211>98

<212>PRT

<213> people

<400>97

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Val Val Ser Gly Gly Ser Ile Ser Ser Ser Ser

20 25 30

Asn Trp Trp Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Glu Ile Tyr His Ser Gly Ser Pro Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210>98

<211>98

<212>PRT

<213> people

<400>98

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Pro Gly

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Gly Ser Ile Ser Ser Ser Ser

20 25 30

Asn Trp Trp Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Glu Ile Tyr His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val TyT Cys Cys

85 90 95

Ala Arg

<210>99

<211>98

<212>PRT

<213> people

<400>99

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gly

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Gly Ser Ile Ser Ser Ser Ser

20 25 30

Asn Trp Trp Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Glu Ile Tyr His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Ile Ser Val Asp Lys Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210>100

<211>99

<212>PRT

<213> people

<400>100

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser Ser

20 25 30

Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg

<210>101

<211>99

<212>PRT

<213> people

<400>101

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser Ser

20 25 30

Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn His Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg

<210>102

<211>97

<212>PRT

<213> people

<400>102

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Ala Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Tyr Thr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Asn Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg

<210>103

<211>97

<212>PRT

<213> people

<400>103

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg

<210>104

<211>97

<212>PRT

<213> people

<400>104

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Met Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg

<210>105

<211>97

<212>PRT

<213> people

<400>105

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Asp

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Tyr lle Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg

<210>106

<211>98

<212>PRT

<213> people

<400>106

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg

<210>107

<211>98

<212>PRT

<213> people

<400>107

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Pro Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg

<210>108

<211>98

<212>PRT

<213> people

<400>108

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg

<210>109

<211>98

<212>PRT

<213> people

<400>109

Glu Val Gln Leu Leu Gln Ser Ala Ala Glu Val Lys Arg Pro Gly Glu

1 5 10 15

Ser Leu Arg Ile Ser Cys Lys Thr Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Ile His Trp Val Arg Gln Met Pro Gly Lys Glu Leu Glu Trp Met

35 40 45

Gly Ser Ile Tyr Pro Gly Asn Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly His Val Thr Ile Ser Ala Asp Ser Ser Ser Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Ala Ala Met Tyr Tyr Cys

85 90 95

Val Arg

<210>110

<211>98

<212>PRT

<213> people

<400>110

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Ile Ser Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Ser Pro Ser Phe

50 55 60

Gln Gly His Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg

<210>111

<211>98

<212>PRT

<213> people

<400>111

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Ile Ser Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Ser Pro Ser Phe

50 55 60

Gln Gly His Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg

<210>112

<211>101

<212>PRT

<213> people

<400>112

Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala

50 55 60

Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg

100

<210>113

<211>87

<212>PRT

<213> people

<400>113

Arg Lys Leu Gly Ala Ser Val Lys Val Ser Arg Lys Ala Ser Ser Tyr

1 5 10 15

Thr Phe Thr Ser Tyr Asp Ile His Cys Val Arg Gln Ala Pro Gly Lys

20 25 30

Gly Leu Lys Gly Trp Met Gly Gly Ile Tyr Ser Gly Asn Gly Lys Thr

35 40 45

Gly Tyr Ala Gln Lys Phe Gln Arg Val Thr Met Thr Arg Asp Met Ser

50 55 60

Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Gln Arg Ser Glu Asp Ile

65 70 75 80

Asp Val Tyr Tyr Cys Ala Arg

85

<210>114

<211>5

<212>PRT

<213> people

<400>114

Asp Tyr Gly Met Ser

1 5

<210>115

<211>17

<212>PRT

<213> people

<400>115

Gly Ile Asn Trp Asn Gly Gly Ser Thr Gly Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210>116

<211>11

<212>PRT

<213> people

<400>116

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Arg

1 5 5 10

<210>117

<211>11

<212>PRT

<213> people

<400>117

Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

1 5 5 10

<210>118

<211>11

<212>PRT

<213> people

<400>118

Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn

1 5 5 10

<210>119

<211>8

<212>PRT

<213> people

<400>119

Gly Lys Gly Leu Glu Trp Val Ser

1 5

<210>120

<211>6

<212>PRT

<213> people

<400>120

Trp Val Arg Gln Ala Pro

1 5

<210>121

<211>11

<212>PRT

<213> people

<400>121

Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp

1 5 5 10

<210>122

<211>7

<212>PRT

<213> people

<400>122

Ala Val Tyr Tyr Cys Ala Arg

1 5

<210>123

<211>20

<212>PRT

<213> people

<400>123

Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210>124

<211>15

<212>PRT

<213> people

<400>124

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210>125

<211>9

<212>PRT

<213> people

<400>125

Asn Ser Arg Asp Ser Ser Gly Asn His

1 5

<210>126

<211>8

<212>PRT

<213> people

<400>126

Ala Ala Trp Asp Asp Ser Leu Val

1 5

<210>127

<211>8

<212>PRT

<213> people

<400>127

Met Gln Ser Ile Gln Leu Pro Thr

1 5

<210>128

<211>9

<212>PRT

<213> people

<400>128

Met Gln Ser Ile Gln Leu Pro Ala Thr

1 5

<210>129

<211>10

<212>PRT

<213> people

<400>129

Ala Ala Trp Asp Asp Gly Leu Ser Leu Val

1 5 5 10

<210>130

<211>10

<212>PRT

<213> people

<400>130

Ala Ala Trp Asp Asp Ser Leu Ser Gly Val

1 5 5 10

<210>131

<211>11

<212>PRT

<213> people

<400>131

Asn Ser Arg Asp Ser Ser Gly Ser Val Arg Val

1 5 5 10

<210>132

<211>9

<212>PRT

<213> people

<400>132

Leu Leu Tyr Tyr Gly Gly Ala Tyr Val

1 5

<210>133

<211>11

<212>PRT

<213> people

<400>133

Asn Ser Arg Asp Ser Ser Gly Val Ser Arg Val

1 5 5 10

<210>134

<211>10

<212>PRT

<213> people

<400>134

Ala Ala Trp Asp Asp Ser Leu Pro Tyr Val

1 5 5 10

<210>135

<211>12

<212>PRT

<213> people

<400>135

Ala Ala Trp Asp Asp Ser Leu Cys Pro Glu Phe Val

1 5 5 10

<210>136

<211>11

<212>PRT

<213> people

<400>136

Ala Ala Trp Asp Asp Ser Leu Ala Trp Phe Val

1 5 5 10

<210>137

<211>10

<212>PRT

<213> people

<400>137

Leu Ala Trp Asp Thr Ser Pro Arg Trp Val

1 5 5 10

<210>138

<211>10

<212>PRT

<213> people

<400>138

Thr Ala Trp Asp Asp Ser Leu Ala Val Val

1 5 5 10

<210>139

<211>11

<212>PRT

<213> people

<400>139

Asn Ser Arg Asp Ser Ser Gly Asn His Arg Val

1 5 5 10

<210>140

<211>9

<212>PRT

<213> people

<400>140

Gln Gln Tyr Gly Ser Ser Gln Arg Thr

1 5

<210>141

<211>10

<212>PRT

<213> people

<400>141

Ala Ala Trp Asp Asp Ser Leu Arg Leu Val

1 5 5 10

<210>142

<211>9

<212>PRT

<213> people

<400>142

Met Gln Gly Thr His Trp Arg Pro Thr

1 5

<210>143

<211>9

<212>PRT

<213> people

<400>143

Met Gln Gly Lys His Trp Pro Leu Thr

1 5

<210>144

<211>9

<212>PRT

<213> people

<400>144

Ala Ala Trp Asp Asp Ser Leu Gly Phe

1 5

<210>145

<211>9

<212>PRT

<213> people

<400>145

Met Gln Gly Thr His Arg Arg Ala Thr

1 5

<210>146

<211>9

<212>PRT

<213> people

<400>146

Met Gln Ala Leu Gln Thr Pro Leu Thr

1 5

<210>147

<211>9

<212>PRT

<213> people

<400>147

Met Arg Gly Thr His Arg Arg Ala Thr

1 5

<210>148

<211>9

<212>PRT

<213> people

<400>148

Met Gln Gly Thr His Trp His Pro Thr

1 5

<210>149

<211>8

<212>PRT

<213> people

<400>149

Met Gln Ala Leu Gln Ser Pro Thr

1 5

<210>150

<211>10

<212>PRT

<213> people

<400>150

Ala Ala Trp Asp Asp Ser Leu Ala Phe Val

1 5 5 10

<210>151

<211>8

<212>PRT

<213> people

<400>151

Met Gln Ala Leu Gln Thr Pro Thr

1 5

<210>152

<211>8

<212>PRT

<213> people

<400>152

Gln Gln Ser Tyr Ser Thr Arg Thr

1 5

<210>153

<211>9

<212>PRT

<213> people

<400>153

Met Gln Gly Thr His Trp Pro Phe Thr

1 5

<210>154

<211>9

<212>PRT

<213> people

<400>154

Met Gln Gly Thr His Trp Pro Ala Thr

1 5

<210>155

<211>10

<212>PRT

<213> people

<400>155

Ala Ala Trp Asp Asp Ser Leu Arg Ser Val

1 5 5 10

<210>156

<211>9

<212>PRT

<213> people

<400>156

Ala Ala Trp Asp Asp Ser Leu Leu Val

1 5

<210>157

<211>11

<212>PRT

<213> people

<400>157

Asp Ser Trp Asp Asn Ser Leu Val Ser Pro Val

1 5 5 10

<210>158

<211>9

<212>PRT

<213> people

<400>158

Met Gln Ala Leu Gln Ser Pro Ala Thr

1 5

<210>159

<211>9

<212>PRT

<213> people

<400>159

Met Gln Ala Leu Gln Thr Pro Val Thr

1 5

<210>160

<211>11

<212>PRT

<213> people

<400>160

Ala Ala Trp Asp Asp Ser Leu Ser Ala Tyr Val

1 5 5 10

<210>161

<211>11

<212>PRT

<213> people

<400>161

Asn Ser Arg Asp Ser Ser Ser Gly Arg Val Asn Val

1 5 5 10

<210>162

<211>8

<212>PRT

<213> people

<400>162

Met Gln Ala Leu Arg Thr Arg Thr

1 5

<210>163

<211>11

<212>PRT

<213> people

<400>163

Ala Ala Trp Asp Asp Ser Leu Phe Tyr Pro Val

1 5 5 10

<210>164

<211>9

<212>PRT

<213> people

<400>164

Met Gln Gly Thr His Trp Pro Val Thr

1 5

<210>165

<211>8

<212>PRT

<213> people

<400>165

Met Gln Gly Thr His Trp Arg Thr

1 5

<210>166

<211>10

<212>PRT

<213> people

<400>166

Ala Ala Trp Asp Asp Ser Leu Phe Tyr Val

1 5 5 10

<210>167

<211>9

<212>PRT

<213> people

<400>167

Met Gln Ser Ile Gln Leu Pro Leu Thr

1 5

<210>168

<211>11

<212>PRT

<213> people

<400>168

Ala Ala Trp Asp Asp Ser Leu Leu Gly Ser Val

1 5 5 10

<210>169

<211>9

<212>PRT

<213> people

<400>169

Cys Ser Tyr Ala Gly Ser Ser Tyr Val

1 5

<210>170

<211>8

<212>PRT

<213> people

<400>170

Gln Gln Asp Tyr Asn Leu Leu Thr

1 5

<210>171

<211>10

<212>PRT

<213> people

<400>171

Val Leu Tyr Met Gly Ser Gly Ser Ala Val

1 5 5 10

<210>172

<211>9

<212>PRT

<213> people

<400>172

Met Gln Arg Ile Glu Phe Pro Asn Thr

1 5

<210>173

<211>11

<212>PRT

<213> people

<400>173

Ala Ala Trp Asp Asp Ser Leu Ala Cys Ala Val

1 5 5 10

<210>174

<211>8

<212>PRT

<213> people

<400>174

Gln Gln Ala Asn Ser Phe Arg Thr

1 5

<210>175

<211>11

<212>PRT

<213> people

<400>175

Ala Ala Trp Asp Asp Ser Leu Ser Arg Pro Val

1 5 5 10

<210>176

<211>10

<212>PRT

<213> people

<400>176

Ala Ala Trp Asp Asp Ser Leu Tyr Asn Val

1 5 5 10

<210>177

<21>11

<212>PRT

<213> people

<400>177

Ala Ala Trp Asp Asp Ser Leu Asn Arg Asn Val

1 5 5 10

<210>178

<211>8

<212>PRT

<213> people

<400>178

Met Gln Val Leu Gln Thr Arg Thr

1 5

<210>179

<211>8

<212>PRT

<213> people

<400>179

Met Gln Ala Leu Gln Thr Arg Thr

1 5

<210>180

<211>8

<212>PRT

<213> people

<400>180

Gln Gln Ser Tyr Ser Thr Arg Met

1 5

<210>181

<211>8

<212>PRT

<213> people

<400>181

Met Gln Ala Leu Gln Thr Leu Thr

1 5

<210>182

<211>8

<212>PRT

<213> people

<400>182

Met Arg Ala Leu Gln Thr Pro Thr

1 5

<210>183

<211>11

<212>PRT

<213> people

<400>183

Ala Ala Trp Asp Asp Ser Leu Pro Gly Tyr Val

1 5 5 10

<210>184

<211>10

<212>PRT

<213> people

<400>184

Ala Ala Trp Asp Asp Ser Leu Gly Phe Val

1 5 5 10

<210>185

<211>10

<212>PRT

<213> people

<400>185

Ala Ala Trp Asp Asp Ser Leu Phe Leu Val

1 5 5 10

<210>186

<211>8

<212>PRT

<213> people

<400>186

Met Gln Ser Ile Gln Leu Arg Thr

1 5

<210>187

<211>10

<212>PRT

<213> people

<400>187

Ala Ala Trp Asp Asp Ser Leu Ser Ile Val

1 5 5 10

<210>188

<211>8

<212>PRT

<213> people

<400>188

Met Gln Gly Thr His Trp Pro Thr

1 5

<210>189

<211>8

<212>PRT

<213> people

<400>189

Met Gln Ala Leu His Thr Arg Thr

1 5

<210>190

<211>9

<212>PRT

<213> people

<400>190

Asn Ser Arg Asp Ser Ser Gly Ser Val

1 5

<210>191

<211>9

<212>PRT

<213> people

<400>191

Gln Gln Tyr Gly Ser Ser Pro Tyr Thr

1 5

<210>192

<211>8

<212>PRT

<213> people

<400>192

Gln Gln Ser Tyr Ser Thr Arg Thr

1 5

<210>193

<211>9

<212>PRT

<213> people

<400>193

Gln Gln Ala Asn Ser Phe Ala Ala Thr

1 5

<210>194

<211>9

<212>PRT

<213> people

<400>194

Gln Gln Ala Asn Ser Phe Pro Ala Thr

1 5

<210>195

<211>10

<212>PRT

<213> people

<400>195

Val Leu Tyr Met Gly Ser Gly Val Tyr Val

1 5 5 10

<210>196

<211>11

<212>PRT

<213> people

<400>196

Ala Ala Trp Asp Asp Ser Leu Trp Ser Ala Val

1 5 5 10

<210>197

<211>12

<212>PRT

<213> people

<400>197

Ala Ala Trp Asp Asp Ser Leu Pro Arg Arg Leu Val

1 5 5 10

<210>198

<211>11

<212>PRT

<213> people

<400>198

Ala Ala Trp Asp Asp Ser Leu Pro Ser Gly Val

1 5 5 10

<210>199

<211>8

<212>PRT

<213> people

<400>199

Met Gln Ala Leu Gln Thr Leu Thr

1 5

<210>200

<211>10

<212>PRT

<213> people

<400>200

Ala Ala Trp Asp Asp Gly Leu Leu Arg Val

1 5 5 10

<210>201

<211>10

<212>PRT

<213> people

<400>201

Ala Ala Trp Asp Asp Ser Leu Ala Leu Val

1 5 5 10

<210>202

<211>11

<212>PRT

<213> people

<400>202

Asn Ser Arg Asp Ser Ser Gly Phe Gln Leu Val

1 5 5 10

<210>203

<211>277

<212>PRT

<213> people

<400>203

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala

1 5 10 15

Ala Gln Pro Ala Met Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly

20 25 30

Val Val Arg Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

35 40 45

Phe Thr Phe Asp Asp Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly

50 55 60

Lys Gly Leu Glu Trp Val Ser Gly Ile Asn Trp Asn Gly Gly Ser Thr

65 70 75 80

Gly Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

85 90 95

Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

100 105 110

Thr Ala Val Tyr Tyr Cys Ala Arg Leu Thr His Pro Tyr Phe Trp Gly

115 120 125

Gln Gly Thr Leu Val Thr Val Ser Arg Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Ser Glu Leu Thr Gln Asp Pro Ala

145 150 155 160

Val Ser Val Ala Leu Gly Gln Thr Val Arg Ile Thr Cys Gln Gly Asp

165 170 175

Ser Leu Arg Ser Tyr Tyr Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln

180 185 190

Ala Pro Val Leu Val Ile Tyr Gly Lys Asn Asn Arg Pro Ser Gly Ile

195 200 205

Pro Asp Arg Phe Ser Gly Ser Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr

210 215 220

Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser

225 230 235 240

Arg Asp Ser Ser Gly Asn His Val Val Phe Gly Gly Gly Thr Lys Leu

245 250 255

Thr Val Leu Gly Ala Ala Ala Glu Gln Lys Leu Ile Ser Glu Glu Asp

260 265 270

Leu Asn Gly Ala Ala

275

Claims (259)

1. one kind comprises Fv molecule, its construct, both fragments or the peptide or the polypeptide of segmental construct, it have strengthened in conjunction with feature, so that compare with other cells, help optionally and/or specifically in conjunction with target cell, wherein, described in conjunction with selectivity or specificity mainly by the decision of first hypervariable region, and Fv wherein is scFv or dsFv, and optionally has one or more marks.

2. peptide as claimed in claim 1 or polypeptide, wherein, described first hypervariable region is the CDR3 district with the aminoacid sequence that is selected from SEQ ID NOs:8-24.

3. peptide as claimed in claim 1 or polypeptide, wherein, described first hypervariable region is the CDR3 district with the aminoacid sequence that is selected from SEQ ID NOs:8-24, and, secondly being subjected to second hypervariable region, the 3rd hypervariable region and/or being positioned at described first, second and/or the one or more upstreams of the 3rd hypervariable region flank or the influence of catchment wherein in conjunction with selectivity or specificity.

4. peptide as claimed in claim 2 or polypeptide, wherein, described peptide or polypeptide are the scFv with SEQ IDNO:25, first hypervariable region wherein is the CDR3 district identical with SEQ ID NO:8.

5. peptide as claimed in claim 1 or polypeptide, wherein, described scFv molecule comprises having 20 or the straight or branched stuffer fragment of amino-acid residue still less.

6. peptide as claimed in claim 5 or polypeptide, wherein, described stuffer fragment comprises SEQ ID NO:123 or SEQ ID NO:124.

7. peptide as claimed in claim 1 or polypeptide, wherein, described target cell is activated, excitement, that modify, that change, imbalance, cell unusual or pathology.

8. peptide as claimed in claim 7 or polypeptide, wherein, described sick cell is a cancer cells.

9. peptide as claimed in claim 7 or polypeptide, wherein, described cell is selected from cancer, sarcoma, leukemia, adenoma, lymphoma, myelomatosis, blastoma, spermocytoma and melanoma cells.

10. peptide as claimed in claim 9 or polypeptide, wherein, described cell is leukemia or myeloma cell.

11. peptide as claimed in claim 9 or polypeptide, wherein, described leukemia or myeloma cell are the B-cell malignancies.

12. as the peptide or the polypeptide of claim 10, wherein, described leukemia cell is acute myelocytic leukemia cell or B-cell malignancies.

13. peptide as claimed in claim 2 or polypeptide, the box that also comprises a continuous amino acid, this box has the aminoacid sequence that is selected from SEQ ID NOs:30-113, or has the aminoacid sequence of at least 90% amino acid similarity with described aminoacid sequence or its fragment, wherein, described box or fragment provide a kind of like this framework, wherein, and structure, insertion, connection, coupling, combination or merged CDR3 district with the aminoacid sequence that is selected from SEQ ID NOs:8-24.

14. as the peptide or the polypeptide of claim 13, wherein, described box has the SEQ of being selected from 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 aminoacid sequence, or have the aminoacid sequence of at least 90% amino acid similarity with described sequence.

15. as the peptide or the polypeptide of claim 13, wherein, described box has the aminoacid sequence of SEQ ID NO:61, or has at least 90% amino acid similarity with this sequence.

16. as the peptide or the polypeptide of claim 15, wherein, described box has the aminoacid sequence of SEQ ID NO:61, or has at least 90% amino acid similarity with this sequence.

17. as the peptide or the polypeptide of claim 15, wherein, 7 C-terminal amino-acid residues of SEQ ID NO:61 are replaced by 7 amino-acid residues of SEQ ID NO:122.

18. peptide as claimed in claim 3 or polypeptide, wherein, the described second and the 3rd hypervariable region is respectively CDR2 and CDR1 hypervariable region.

19. peptide as claimed in claim 2 or polypeptide, wherein, described CDR3 district has the aminoacid sequence of SEQ ID NO:8.

20. as the peptide or the polypeptide of claim 18, wherein, described CDR2 and CDR1 district have the aminoacid sequence of SEQ ID NO:115 and SEQ ID NO:114 respectively.

21. peptide as claimed in claim 3 or polypeptide, wherein, the described second and the 3rd hypervariable region is respectively CDR2 and CDR1 hypervariable region, and CDR3 wherein, and CDR2 and CDR1 district have SEQ ID NOs:8,115 and 114 aminoacid sequence respectively.

22. peptide as claimed in claim 3 or polypeptide, wherein, the upstream of CDR3 district flank has the aminoacid sequence of SEQ ID NO:117, and wherein the catchment of CDR3 district flank has the aminoacid sequence of SEQ ID NO:116.

23. peptide as claimed in claim 3 or polypeptide, wherein, described second hypervariable region is the CDR2 hypervariable region, and, wherein the upstream of CDR2 district flank has the aminoacid sequence of SEQ ID NO:119, and wherein the catchment of CDR2 district flank has the aminoacid sequence of SEQ ID NO:118.

24. peptide as claimed in claim 3 or polypeptide, wherein, described the 3rd hypervariable region is the CDR1 hypervariable region, and, wherein the upstream of CDR1 district flank has the aminoacid sequence of SEQ ID NO:120, and wherein the catchment of CDR1 district flank has the aminoacid sequence of SEQ ID NO:121.

25. as the peptide or the polypeptide of claim 18, wherein, described CDR2 and the CDR1 district that is selected from the box of 30-113 or its segmental continuous amino acid replaced by SEQ ID NOs:115 and 114 respectively.

26. as the peptide or the polypeptide of claim 18, wherein, described SEQ ID NOs:30-32,35, the 37-39 of being selected from, 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 the CDR2 and the CDR1 of the box of its segmental continuous amino acid replaced by SEQ ID NOs:115 and 114 respectively.

27. peptide as claimed in claim 3 or polypeptide, wherein,

(a) the described second and the 3rd hypervariable region is respectively CDR2 and CDR1 hypervariable region,

(b) described CDR3 aminoacid sequence is SEQ ID NO:8,

(c) described CDR2 aminoacid sequence is SEQ ID NO:115,

(d) described CDR1 aminoacid sequence is SEQ ID NO:114,

(e) upstream of CDR3 district flank has the aminoacid sequence of SEQ ID NO:117,

(f) catchment of CDR3 district flank has the aminoacid sequence of SEQ ID NO:116,

(g) upstream of CDR2 district flank has the aminoacid sequence of SEQ ID NO:119,

(h) catchment of CDR2 district flank has the aminoacid sequence of SEQ ID NO:118,

(i) upstream of CDR1 district flank have SEQ ID NO:121 aminoacid sequence and

(j) catchment of CDR1 district flank has the aminoacid sequence of SEQ ID NO:120.

28. peptide as claimed in claim 1 or polypeptide, wherein, described Fv is can be from the scFv of phage display library acquisition.

29. peptide or polypeptide as claim 28, wherein, described phage display library is that the people's that crosses with immunity peripheral blood lymphocyte makes up, and scFv peptide wherein is the antigen selection crossed with target cell lip-deep that do not characterize before in advance and purifying not.

30. a method that is used to select or identify the peptide or the polypeptide of claim 28 comprises biological elutriation, wherein, described biological elutriation comprises phage is incorporated into target, remove unconjugated phage, the phage of the phage of elution of bound and propagation and amplification wash-out.

31. one kind comprises the Fv molecule, its construct, both fragments, or the peptide of segmental construct or polypeptide, it have strengthened in conjunction with feature, so that can be optionally and/or specifically in conjunction with that expose and/or overexpression the basically binding site on the target cell or in the target cell, wherein, relatively thereon or inner other cells that do not have and/or express described binding site basically, more help in conjunction with target cell, wherein, describedly mainly determine by first hypervariable region in conjunction with selectivity or specificity, wherein, Fv is scFv or dsFv, and Fv wherein chooses wantonly has one or more marks.

32. as the peptide or the polypeptide of claim 31, wherein, described first hypervariable region is the CDR3 district with the aminoacid sequence that is selected from SEQ ID NOs:8-24.

33. peptide or polypeptide as claim 31, wherein, described first hypervariable region is the CDR3 district with the aminoacid sequence that is selected from SEQ ID NOs:8-24, and, secondly being subjected to second hypervariable region, the 3rd hypervariable region and/or being positioned at described first, second and/or the one or more upstreams of the 3rd hypervariable region flank or the influence of catchment wherein in conjunction with selectivity or specificity, and wherein, the described second and the 3rd hypervariable region is respectively CDR2 and CDR1 district.

34. one kind comprises the Fv molecule, its construct, the peptide of both fragments or segmental construct or polypeptide, it have strengthened in conjunction with feature, more help optionally and/or specifically in conjunction with target cell so that compare with other cells, wherein, described Fv molecule comprises having first, article one chain of the second and the 3rd hypervariable region, with have first, the second chain of the second and the 3rd hypervariable region, wherein, one of hypervariable region of described article one chain has the sequence that is selected from SEQ ID NOs:8-24, and, wherein one of hypervariable region of second chain has the sequence that is selected from SEQ ID NOs:1-6 and 125-202, and, wherein said first, the second and the 3rd hypervariable region is respectively CDR3, CDR2 and CDR1 district, wherein, Fv is scFv or dsFv, and wherein said Fv is optional has one or more marks.

35. as the peptide or the polypeptide of claim 34, wherein,

(a) described article one chain and second chain comprise first hypervariable region that is selected from SEQ ID NOs:8-24 separately; Or

(b) first hypervariable region of first hypervariable region of described article one chain and described second chain is identical, and is selected from SEQ ID NOs:8-24; Or

(c) first hypervariable region of described article one chain is selected from SEQ ID NOs:8-24, and first hypervariable region of described second chain is selected from SEQ ID NOs:1-6 and 125-202; Or

(d) first hypervariable region of described article one chain is selected from SEQ ID NOs:1-6 and 125-202, and first hypervariable region of described second chain is selected from SEQ ID NOs:8-24.

36. as the peptide or the polypeptide of claim 34, wherein, the second and the 3rd hypervariable region of described article one chain is respectively SEQ ID NOs:114 and 115.

37. one kind comprises Fv molecule, its construct, both fragments or the peptide or the polypeptide of segmental construct,

(a) its can be in conjunction with the unknown part on first kind of cell with first kind and second kind state, and wherein, described combination realizes under second kind of state, and can not under first kind of state, realize basically and

(b) by immune cross-reactivity, with ligand specificity on second kind of cell or optionally combine, and Fv wherein is scFv or dsFv, and Fv wherein is optional to have one or more marks.

38. as the peptide or the polypeptide of claim 37, wherein, described first kind of cell is normal cell.

39. as the peptide or the polypeptide of claim 37, wherein, described first kind of state is nonactivated state, described second kind of state be activated, excitement, the state of that modify, that change or imbalance.

40. as the peptide or the polypeptide of claim 37, wherein, described second kind of cell is sick cell.

41. as the peptide or the polypeptide of claim 40, wherein, described sick cell is a cancer cells.

42. as the peptide or the polypeptide of claim 40, wherein, described sick cell is selected from cancer, sarcoma, leukemia, adenoma, lymphoma, myelomatosis, blastoma, spermocytoma and melanoma cells.

43. as the peptide or the polypeptide of claim 42, wherein, described sick cell is the leukemia cell.

44. as the peptide or the polypeptide of claim 43, wherein, described leukemia cell is the acute myelocytic leukemia cell.

45. as the peptide or the polypeptide of claim 37, wherein, the selectivity of described peptide or polypeptide and the part of second kind of cell and/or specificity combine mainly and are determined by first hypervariable region.

46. as the peptide or the polypeptide of claim 45, wherein, described first hypervariable region is the CDR3 district with the aminoacid sequence that is selected from SEQ ID NOs:8-24.

47. peptide or polypeptide as claim 46, wherein, described first hypervariable region is the CDR3 district with the aminoacid sequence that is selected from SEQ ID NOs:8-24, and, secondly being subjected to second hypervariable region, the 3rd hypervariable region and/or laying respectively at described first, second and/or the one or more upstreams of the 3rd hypervariable region flank or the influence of catchment wherein in conjunction with selectivity or specificity.

48. a part, it is by described second kind of cell expressing, and it can combine with the peptide or the polypeptide of claim 37.

49. can discern and in conjunction with the molecule of the part of claim 48 for one kind.

50. a nucleic acid molecule, its claim 1 of encoding, any one peptide or polypeptide in 31,34 or 37.

51. as the nucleic acid molecule of claim 50, wherein, described nucleic acid is DNA.

52. as the peptide or the polypeptide of claim 37, wherein, first and second kinds of states of described first kind of cell are identical, and wherein, described first kind of cell is from a kind of clone.

53. as the peptide or the polypeptide of claim 52, wherein, described clone is selected from Jurkat, Molt-4, HS-602, U937, TF-1, THP-1, KG-1, ML-2 and HUT-78.

54. one kind is used to identify and can comprises in conjunction with the method for the guide molecule of the immune cross-reactivity binding site of the unknown on first kind and the second kind of cell

(a) carry out the biological elutriation of one or many on first kind of target cell, described target cell is in second kind of state, rather than first kind of state, and it exposes basically or shows the binding site that comprises at least a unknown part, so that produce first identification molecular group;

(b) first identification molecular group from step (a) comes into effect biological elutriation subsequently and/or selects step, this step is carried out on second kind of cell, described cell display comprises the binding site of at least a unknown part, the unknown part of this unknown part and described first kind of cell has immune cross-reactivity, so that produce second identification molecular group;

(c) second of amplification and purification step (b) identification molecular group; With

(d) recognition site by the identification molecule of the purifying of step (c) makes up peptide or polypeptide, and described peptide or polypeptide comprise that the unknown part on described second kind of cell is had selectivity and/or specific guide molecule.

55. as the method for claim 54, wherein, described first kind of cell is normal cell, and wherein said first kind of state is unactivated state, second kind of state is the state of activated, excitement, that modify, that change or imbalance.

56. as the method for claim 54, wherein, described second kind of cell is sick cell.

57. as the method for claim 56, wherein, described sick cell is a cancer cells.

58. as the method for claim 56, wherein, described cell is selected from cancer, sarcoma, leukemia, adenoma, lymphoma, myelomatosis, blastoma, spermocytoma and melanoma cells.

59. as the method for claim 58, wherein, described cell is the leukemia cell.

60. as the method for claim 59, wherein, described leukemia cell is the acute myelocytic leukemia cell.

61. will choose wantonly and associate with a kind of medicinal reagent or adhere to, coupling, associating, be connected or claim 1 or the peptide of claim 37 or the purposes that polypeptide is used to make medicine of fusion.

62. as the purposes of claim 61, wherein, described medicine has disease-resistant change cell activity.

63. as the purposes of claim 62, wherein, described sick cell is a cancer cells.

64. as the purposes of claim 62, wherein, described cell is selected from cancer, sarcoma, leukemia, adenoma, lymphoma, myelomatosis, blastoma, spermocytoma and melanoma cells.

65. as the purposes of claim 64, wherein, described cell is the leukemia cell.

66. as the purposes of claim 65, wherein, described leukemia cell is the acute myelocytic leukemia cell.

67. as optional and a kind of medicinal reagent association of medicine or adhere to, coupling, associating, be connected or the claim 1 of fusion or the peptide or the polypeptide of claim 37.

68. as the peptide or the polypeptide of claim 67, wherein, described medicine has disease-resistant change cell activity.

69. as the peptide or the polypeptide of claim 68, wherein, described sick cell is a cancer cells.

70. as the peptide or the polypeptide of claim 68, wherein, described cell is selected from cancer, sarcoma, leukemia, adenoma, lymphoma, myelomatosis, blastoma, spermocytoma and melanoma cells.

71. as the peptide or the polypeptide of claim 70, wherein, described cell is the leukemia cell.

72. as the peptide or the polypeptide of claim 71, wherein, described leukemia cell is the acute myelocytic leukemia cell.

73. the peptide of claim 1 or claim 37 or polypeptide are used to prepare the purposes of the composition that is used for suppressing sick cell or growth of cancer cells.

74. as the purposes of the peptide or the polypeptide of claim 73, wherein, described cell is the leukemia cell.

75. as the purposes of the peptide or the polypeptide of claim 74, wherein, described leukemia cell is the acute myelocytic leukemia cell.

76. the peptide of claim 1 or claim 37 or polypeptide be used to prepare be used for the purposes of composition of anticancer growth, described composition comprises at least a compound, and this compound has the selective and/or specific medicinal part of described cancer cells.

77. a composition, what comprise medicinal significant quantity associates with a kind of medicinal reagent or adheres to, coupling, associating, is connected or at least a peptide as claim 1 or claim 37 of fusion, and optionally comprises a kind of medicinal effective carrier.

78. as the composition of claim 77, wherein, described peptide or polypeptide are connected by linker compounds with described medicinal reagent.

79. as the composition of claim 78, wherein, described linker compounds is selected from dicarboxylic acid, maleimide hydrazides, PDPH, carboxylic acid hydrazides and little peptide.

80. as the composition of claim 79, wherein, described little peptide is selected from AU1, AU5, BTag, c-myc, FLAG, Glu-Glu, HA, His6, HSV, HTTPHH, IRS, KT3, PROTEIN C, STag , T7, V5, VSV-G and KAK-Tag.

81. as claim 1, any one peptide or polypeptide in 31,34 and 37, wherein, described mark is selected from AU1, AU5, BTag, c-myc, FLAG, Glu-Glu, HA, His6, HSV, HTTPHH, IRS, KT3, PROTEIN C, STag , T7, V5, VSV-G and KAK Tag.

82. as the composition of claim 77, wherein, described medicinal reagent is selected from radio isotope, toxin, oligonucleotide, recombinant protein, antibody fragment, and carcinostatic agent.

83. as the composition of claim 82, wherein, described isotropic substance is selected from indium, ¹¹¹Indium, ¹¹³Indium, ^99mRhenium, ¹⁰⁵Rhenium, ¹⁰¹Rhenium, ^99mTechnetium, ^121mTellurium, ^122mTellurium, ^125mTellurium, ¹⁶⁵Thulium, ¹⁶⁷Thulium, ¹⁶⁸Thulium, ¹²³Iodine, ¹²⁶Iodine, ¹³¹Iodine, ¹³³Iodine, ^81mKrypton, ³³Xenon, ⁹⁰Yttrium, ²¹³Bismuth, ⁷⁷Bromine, ¹⁸Fluorine, ⁹⁵Ruthenium, ⁹⁷Ruthenium, ¹⁰³Ruthenium, ¹⁰⁵Ruthenium, ¹⁰⁷Mercury, ²⁰³Mercury, ⁶⁷Gallium and ⁶⁸Gallium.

84. as the composition of claim 82, wherein, described toxin is selected from gelonin, Pseudomonas exotoxin (PE), PE40, PE38, diphtheria toxin, ricin, and modifier and derivative.

85. as the composition of claim 82, wherein, described carcinostatic agent is selected from Zorubicin, morpholino Zorubicin (MDOX), the A Deliya mycin, cis-platinum, taxol, calicheamicin, vincristine(VCR), cytosine arabinoside (Ara-C), endoxan, prednisone, daunorubicin, idarubicin, NSC-118218, Chlorambucil, interferon alpha, hydroxyurea, Temozolomide, Thalidomide, bleomycin, and derivative.

86. a cytostatic method, it comprises allows described cell contact with the peptide or the polypeptide of a certain amount of claim 1 or claim 37.

87. as the method for claim 86, wherein, described cell is selected from cancer, sarcoma, leukemia, adenoma, lymphoma, myelomatosis, blastoma, spermocytoma and melanoma.

88. as the method for claim 87, wherein, described cell is the leukemia cell.

89. as the method for claim 88, wherein, described leukemia cell is the acute myelocytic leukemia cell.

90. one kind is used for the diagnosis location of tumour and the pharmaceutical composition of video picture, it comprises with a kind of developer and adhering to, coupling, combination, be connected or at least a peptide as claim 1 or claim 37 of fusion.

91. a treatment suffers from the patient's of disease or cancer method, this method comprises peptide or the polypeptide of using the claim 1 or the claim 37 of the amount that can effectively treat described disease or cancer to described patient.

92. as the method for claim 91, wherein, described disease or cancer are selected from cancer, sarcoma, leukemia, adenoma, lymphoma, myelomatosis, blastoma, spermocytoma and melanoma.

93. as the method for claim 92, wherein, described disease is a leukemia.

94. as the method for claim 93, wherein, described leukemia is an acute myelocytic leukemia.

95. as the peptide or the polypeptide of claim 1 or claim 37, wherein, described Fv can be specifically or optionally in conjunction with acute myelocytic leukemia (AML) cell.

96. one kind be present in can be by the peptide of claim 95 or polypeptide bonded part on the AML cell.

97. an energy is in conjunction with the peptide or the polypeptide of the part of claim 96.

98. one kind be used for before the treatment, during or analyze the diagnostic kit of result of treatment afterwards, comprise with a kind of cue mark molecule attached, coupling, combination, be connected or the peptide or the polypeptide as claim 1 or claim 37 of fusion.

99. as the test kit of claim 98, wherein, described cue mark molecule is a fluorescent mark.

100. as the test kit of claim 99, wherein, described fluorescent mark is selected from fluorescein, rhodamine, phycoerythrin, and modifier and conjugate.

101. as the test kit of claim 98, wherein, described test kit is used to diagnose the illness or cancer.

102. as the peptide or the polypeptide of claim 1 or claim 37, wherein, described construct is the Ig polypeptide.

103. a method of producing the peptide or the polypeptide of claim 102, wherein, described Ig polypeptide is expressed as recombinant polypeptide, and produces in eukaryotic cell system.

104. as the method for claim 103, wherein, described eukaryotic system is the mammal cell line system.

105. as the peptide or the polypeptide of claim 102, wherein, described Ig polypeptide is the IgG polypeptide.

106. as the peptide or the polypeptide of claim 105, wherein, described IgG polypeptide comprises having SEQ ID NOs:8,115 and 114 CDR3, CDR2 and CDR1 district respectively.

107. as the IgG polypeptide of claim 106, wherein, described CDR3, CDR2 and CDR1 district are heavy chains.

108. as the IgG polypeptide of claim 106, wherein, described CDR3, CDR2 and CDR1 district are light chains.

109. as the IgG polypeptide of claim 102, wherein, described IgG has the heavy chain that comprises SEQ IDNO:26 and comprises the light chain of SEQ ID NO:27 or have the chain of at least 90% amino acid similarity with described chain.

110. a method that is used for the peptide or the polypeptide of production claim 1 or claim 37, wherein, described peptide or polypeptide produce in prokaryotic cell prokaryocyte system or eukaryotic cell system.

111. as the method for claim 110, wherein, described prokaryotic system comprises intestinal bacteria, described intestinal bacteria comprise a kind of expression vector, and described eukaryotic system is the mammal cell line system.

112. as the method for claim 111, wherein, the expression vector of described prokaryotic system comprises and is selected from osmB, deo, β-lac-U5, λ P _L, the promotor of SR α 5 and CMV.

113. comprise a kind of peptide or polypeptide of binding motif, described motif comprises aminoacid sequence R ₁-X Phe Pro-R ₂, wherein, R ₁And R ₂Comprise 0-15 amino-acid residue separately, and X wherein is Arg, Gly or Lys.

114. as peptide any in the claim 2,34 or 46 or polypeptide, described CDR3 comprises aminoacid sequence R ₁-X Phe Pro-R ₂, wherein, R ₁And R ₂Comprise 0-15 amino-acid residue separately, and X wherein is Arg, Gly or Lys.

115. as the peptide or the polypeptide of claim 1 or claim 37, wherein, described peptide or polypeptide comprise the modification that at least a non-natural exists.

116. as the peptide or the polypeptide of claim 115, wherein, the modification that described non-natural exists makes described peptide or polypeptide have higher immunogenicity or more stable.

117. as the peptide or the polypeptide of claim 116, wherein, described at least a modification is selected from the class peptide and modifies, half class peptide is modified, and cyclic peptide is modified, and N-is end modified, and C-is end modified, and peptide bond is modified, and backbone modifications and residue are modified.

118. as claim 1, any one peptide or polypeptide in 31,34,37 or 67 is used for the ex-vivo purging autologous bone marrow, so that remove abnormal cells.

119. a method of producing directed agents may further comprise the steps:

A) separation and selection comprise one or more guide molecules of a main recognition site, comprise directly and to carry out biological elutriation program on the target cell or to be connected on the first kind of target cell that is in second kind of state rather than first kind of state carrying out biological elutriation program, and be connected on subsequently and carry out biological elutriation program on second kind of target cell, so that produce one or more described guide molecules;

B) one or more guide molecules of amplification, purifying and evaluation; With

C) make up a kind of directed agents by described one or more guide molecules, perhaps, wherein, described directed agents can be peptide, polypeptide, antibody or antibody fragment, or its polymer.

120. as the method for claim 119, wherein, described directed agents with a kind of medicinal reagent coupling, adhere to, unite, be connected, merge or associate.

121. as the method for claim 119 and 120, wherein, described directed agents is disease-resistant or carcinostatic agent.

122. as the method for claim 120, wherein, described medicinal reagent is selected from radio isotope, toxin, oligonucleotide, recombinant protein, antibody fragment, and carcinostatic agent.

123. as the method for claim 122, wherein, described radio isotope is selected from ¹¹¹Indium, ¹¹³Indium, ^99mRhenium, ¹⁰⁵Rhenium, ¹⁰¹Rhenium, ^99mTechnetium, ^121mTellurium, ^122mTellurium, ^125mTellurium, ¹⁶⁵Thulium, ¹⁶⁷Thulium, ¹⁶⁸Thulium, ¹²³Iodine, ¹²⁶Iodine, ¹³¹Iodine, ¹³³Iodine, ^81mKrypton, ³³Xenon, ⁹⁰Yttrium, ²¹³Bismuth, ⁷⁷Bromine, ¹⁸Fluorine, ⁹⁵Ruthenium, ⁹⁷Ruthenium, ¹⁰³Ruthenium, ¹⁰⁵Ruthenium, ¹⁰⁷Mercury, ²⁰³Mercury, ⁶⁷Gallium and ⁶⁸Gallium.

124. as the method for claim 122, wherein, described toxin is selected from gelonin, Rhodopseudomonas extracellular toxin (PE), PE40, PE38, diphtheria toxin, ricin, and modifier and derivative.

125. as the method for claim 122, wherein, described carcinostatic agent is selected from vincristine(VCR), cytosine arabinoside (Ara-C), endoxan, prednisone, daunorubicin, idarubicin, NSC-118218, Chlorambucil, interferon alpha, hydroxyurea, Temozolomide, Thalidomide, bleomycin, and derivative.

126. have the peptide or the polypeptide of following general formula or structure:

A-X-B

Wherein, X has a 3-30 amino acid whose CDR3 district, A and B can each naturally length be the individual amino acid whose amino acid chain of 1-1000, wherein, A is a N-terminal, and B is a C-terminal.

127. as the peptide of claim 126, wherein, A is a 150-250 amino-acid residue, and B wherein is a 350-500 amino-acid residue.

128. as the peptide of claim 126, wherein, described CDR3 district is a 5-13 amino-acid residue.

129. as the peptide of claim 126, wherein, X is the aminoacid sequence that is selected from SEQ ID NOs:8-24.

130. as the peptide or the polypeptide of claim 127, it is bigger or a complete antibody or a polymeric part.

131. a dimer molecule, it comprises two kinds of peptides or polypeptide, and one of them is the peptide or the polypeptide of claim 126.

132. one kind comprises the two kinds of peptides of claim 126 or the dimer molecule of polypeptide, these two kinds of peptides or polypeptide are identical.

133. as the dimer molecule of claim 131 or 132, wherein, X is the aminoacid sequence that is selected from SEQ IDNOs:8-24.

134. a nucleic acid molecule, the dimer molecule of peptide of its coding claim 126 or polypeptide or coding claim 130.

135. as the method for claim 104, wherein, described mammal cell line turnkey is drawn together SR α 5 promotors.

136. as the method for claim 104, wherein, described mammal cell line turnkey is drawn together the CMV promotor.

137. peptide as described herein substantially or polypeptide.

138. one kind comprises Fv molecule, its construct, both fragments or the peptide or the polypeptide of segmental construct, it has enhanced in conjunction with feature, so that other cells more help optionally and/or specifically in conjunction with target cell relatively, wherein, describedly mainly determine by first hypervariable region in conjunction with selectivity or specificity, wherein, described first hypervariable region is the CDR3 district that comprises the aminoacid sequence that is selected from SEQ ID NOs:8 or 20, and, Fv wherein is scFv or dsFv, and optional has one or more marks.

139. as the peptide or the polypeptide of claim 138, wherein, describedly secondly be subjected to second hypervariable region in conjunction with selectivity or specificity, the 3rd hypervariable region, and/or be positioned at described first, second and/or the one or more upstreams of the 3rd hypervariable region flank or the influence of catchment.

140. as the peptide or the polypeptide of claim 138, wherein, described peptide or polypeptide are the scFv with SEQID NO:25, wherein, described first hypervariable region is the CDR3 district identical with SEQ ID NO:8.

141. as the peptide or the polypeptide of claim 138, wherein, described peptide or polypeptide are the scFv with SEQID NO:203, wherein, described first hypervariable region is the CDR3 district identical with SEQ ID NO:20.

142. as the peptide or the polypeptide of claim 138, wherein, described scFv molecule has 20 or the straight or branched stuffer fragment of amino-acid residue still less.

143. as the peptide or the polypeptide of claim 142, wherein, described stuffer fragment comprises SEQ IDNO:123 or SEQ ID NO:124.

144. as the peptide or the polypeptide of claim 138, wherein, described target cell is activated, excitement, that modify, that change, imbalance, cell unusual or pathology.

145. as the peptide or the polypeptide of claim 144, wherein, described sick cell is a cancer cells.

146. as the peptide or the polypeptide of claim 144, wherein, described cell is selected from cancer, sarcoma, leukemia, adenoma, lymphoma, myelomatosis, blastoma, spermocytoma and melanoma cells.

147. as the peptide or the polypeptide of claim 146, wherein, described cell is leukemia cell or myeloma cell.

148. as the peptide or the polypeptide of claim 146, wherein, described leukemia or myeloma cell are the B-cell malignancies.

149. as the peptide or the polypeptide of claim 147, wherein, described leukemia cell is acute myelocytic leukemia cell or B-malignant tumour.

150. peptide or polypeptide as claim 138, the box that also comprises a continuous amino acid, this box has the aminoacid sequence that is selected from SEQ ID NOs:30-113, or has the aminoacid sequence of at least 90% amino acid similarity with described aminoacid sequence or its fragment, wherein, described box or fragment provide a kind of like this framework, wherein, and structure, insertion, connection, coupling, combination or merged CDR3 district with the aminoacid sequence that is selected from SEQ ID NOs:8-24.

151. as the peptide or the polypeptide of claim 150, wherein, described box has the SEQ of being selected from IDNOs: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 aminoacid sequence, or have the aminoacid sequence of at least 90% amino acid similarity with described sequence.

152. as the peptide or the polypeptide of claim 150, wherein, described box has the aminoacid sequence of SEQ ID NO:61, or has at least 90% amino acid similarity with described sequence.

153. as the peptide or the polypeptide of claim 152, wherein, described box has the aminoacid sequence of SEQ ID NO:61, or has at least 90% amino acid similarity with described sequence.

154. as the peptide or the polypeptide of claim 152, wherein, seven C-terminal amino-acid residues of SEQ ID NO:61 are replaced by the seven amino acid residue of SEQ ID NO:122.

155. as the peptide or the polypeptide of claim 139, wherein, the described second and the 3rd hypervariable region is respectively CDR2 and CDR1 hypervariable region.

156. as the peptide or the polypeptide of claim 138, wherein, described CDR3 district has the aminoacid sequence of SEQ IDNO:8.

157. as the peptide or the polypeptide of claim 138, wherein, described CDR3 district has the aminoacid sequence of SEQ IDNO:20.

158. as the peptide or the polypeptide of claim 155, wherein, described CDR2 and CDR1 district have the aminoacid sequence of SEQ ID NO:115 and SEQ ID NO:114 respectively.

159. as the peptide or the polypeptide of claim 140, wherein, the described second and the 3rd hypervariable region is respectively CDR2 and CDR1 hypervariable region, and, CDR3 wherein, CDR2 and CDR1 district have SEQ ID NOs:8,115 and 114 aminoacid sequence respectively.

160. as the peptide or the polypeptide of claim 140, wherein, the described second and the 3rd hypervariable region is respectively CDR2 and CDR1 hypervariable region, and, CDR3 wherein, CDR2 and CDR1 district have SEQ ID NOs:20,115 and 114 aminoacid sequence respectively.

161. as the peptide or the polypeptide of claim 139, wherein, the upstream of CDR3 district flank has the aminoacid sequence of SEQ ID NO:117, and wherein the catchment of CDR3 district flank has the aminoacid sequence of SEQ ID NO:116.

162. as the peptide or the polypeptide of claim 139, wherein, described second hypervariable region is the CDR2 hypervariable region, and, wherein the upstream of CDR2 district flank has the aminoacid sequence of SEQ ID NO:119, and wherein the catchment of CDR2 district flank has the aminoacid sequence of SEQ ID NO:118.

163. as the peptide or the polypeptide of claim 139, wherein, described the 3rd hypervariable region is the CDR1 hypervariable region, and, wherein the upstream of CDR1 district flank has the aminoacid sequence of SEQ ID NO:121, and wherein the catchment of CDR1 district flank has the aminoacid sequence of SEQ ID NO:120.

164. as the peptide or the polypeptide of claim 155, wherein, described CDR2 and the CDR1 district that is selected from the box of SEQ ID NOs:30-113 or its segmental continuous amino acid replaced by SEQ IDNOs:115 and 114 respectively.

165. as the peptide or the polypeptide of claim 155, wherein, described SEQ ID NOs:30-32,35, the 37-39 of being selected from, 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 the CDR2 and the CDR1 district of the box of its segmental continuous amino acid replaced by SEQ ID NOs:115 and 114 respectively.

166. as the peptide or the polypeptide of claim 139, wherein

(a) the described second and the 3rd hypervariable region is respectively CDR2 and CDR1 hypervariable region,

(b) described CDR3 aminoacid sequence is SEQ ID NO:8,

(c) described CDR2 aminoacid sequence is SEQ ID NO:115,

(d) described CDR1 aminoacid sequence is SEQ ID NO:114,

(e) upstream of CDR3 district flank has the aminoacid sequence of SEQ ID NO:117,

(f) catchment of CDR3 district flank has the aminoacid sequence of SEQ ID NO:116,

(g) upstream of CDR2 district flank has the aminoacid sequence of SEQ ID NO:119,

(h) catchment of CDR2 district flank has the aminoacid sequence of SEQ ID NO:118,

(i) upstream of CDR1 district flank have SEQ ID NO:121 aminoacid sequence and

(j) catchment of CDR1 district flank has the aminoacid sequence of SEQ ID NO:120.

167. as the peptide or the polypeptide of claim 139, wherein

(a) the described second and the 3rd hypervariable region is respectively CDR2 and CDR1 hypervariable region,

(b) described CDR3 aminoacid sequence is SEQ ID NO:20,

(c) described CDR2 aminoacid sequence is SEQ ID NO:115,

(d) described CDR1 aminoacid sequence is SEQ ID NO:114,

(e) upstream of CDR3 district flank has the aminoacid sequence of SEQ ID NO:117,

(f) catchment of CDR3 district flank has the aminoacid sequence of SEQ ID NO:116,

(g) upstream of CDR2 district flank has the aminoacid sequence of SEQ ID NO:119,

(h) catchment of CDR2 district flank has the aminoacid sequence of SEQ ID NO:118,

(i) upstream of CDR1 district flank have SEQ ID NO:121 aminoacid sequence and

(j) catchment of CDR1 district flank has the aminoacid sequence of SEQ ID NO:120.

168. as the peptide or the polypeptide of claim 138, wherein, described Fv is the scFv that can obtain from phage display library.

169. peptide or polypeptide as claim 165, wherein, described phage display library is that the people's of immunity mistake of usefulness peripheral blood lymphocyte makes up, and, wherein, described scFv peptide be on the target cell surface with in the past not sign with unpurified antigen selection.

170. a method that is used to select or identify the peptide or the polypeptide of claim 165 comprises biological elutriation, wherein, described biological elutriation comprises phage is incorporated into target, remove unconjugated phage, the phage of the phage of elution of bound and propagation and amplification wash-out.

171. one kind comprises the Fv molecule, its construct, both fragments, or the peptide of segmental construct or polypeptide, it have strengthened in conjunction with feature, so that can be optionally and/or specifically in conjunction with that expose and/or overexpression the basically binding site on the target cell or in the target cell, wherein, relatively thereon or inner other cells that do not have and/or express described binding site basically, more help in conjunction with target cell, wherein, describedly mainly determine by first hypervariable region in conjunction with selectivity or specificity, wherein, described in conjunction with selectivity or specificity mainly by the decision of first hypervariable region, wherein, the CDR3 district that described first hypervariable region is made up of SEQ ID Nos:8 or 20, wherein, described Fv is scFv or dsFv, and Fv wherein chooses wantonly has one or more marks.

172. peptide or polypeptide as claim 171, wherein, describedly secondly be subjected to second hypervariable region, the 3rd hypervariable region and/or be positioned at described first, second and/or the one or more upstreams of the 3rd hypervariable region flank or the influence of catchment in conjunction with selectivity or specificity, and, wherein, the described second and the 3rd hypervariable region is respectively CDR2 and CDR1 district.

173. one kind comprises the Fv molecule, its construct, the peptide of both fragments or segmental construct or polypeptide, it have strengthened in conjunction with feature, more help optionally and/or specifically in conjunction with target cell so that compare with other cells, wherein, described Fv molecule comprises having first, article one chain of the second and the 3rd hypervariable region, with have first, the second chain of the second and the 3rd hypervariable region, wherein, one of hypervariable region of described article one chain comprises the sequence that is selected from SEQ ID NOs:8 or 20, and, one of hypervariable region of wherein said second chain has the sequence that is selected from SEQ IDNOs:1-6 and 125-202, and, wherein said first, the second and the 3rd hypervariable region is respectively CDR3, CDR2 and CDR1 district, wherein, Fv is scFv or dsFv, and wherein said Fv is optional has one or more marks.

174. as the peptide or the polypeptide of claim 173, wherein,

(a) first hypervariable region of first hypervariable region of described article one chain and described second chain is identical, and is selected from SEQ ID NO:8 or 20; Or

(b) first hypervariable region of described article one chain is selected from SEQ ID NOs:8 or 20, and first hypervariable region of described second chain is selected from SEQ ID NOs:1-6 and 125-202; Or

(d) first hypervariable region of described article one chain is selected from SEQ ID NOs:1-6 and 125-202, and first hypervariable region of described second chain is selected from SEQ ID NOs:8 or 20.

175. as the peptide or the polypeptide of claim 173, wherein, the second and the 3rd hypervariable region of described article one chain is respectively SEQ ID NOs:114 and 115.

176. one kind comprises Fv molecule, its construct, both fragments or the peptide or the polypeptide of segmental construct,

(a) its can be in conjunction with the unknown part on first kind of cell with first kind and second kind state, and wherein, described combination realizes under second kind of state, and can not under first kind of state, realize basically and

(b) pass through immune cross-reactivity, with ligand specificity on second kind of cell or optionally combine, and, Fv wherein is scFv or dsFv, and, Fv wherein selects to have one or more marks, and first hypervariable region wherein is the CDR3 with the aminoacid sequence that is selected from SEQ ID NOs8 or 20.

177. as the peptide or the polypeptide of claim 176, wherein, described first kind of cell is normal cell.

178. as the peptide or the polypeptide of claim 176, wherein, described first kind of state is nonactivated state, described second kind of state be activated, excitement, the state of that modify, that change or imbalance.

179. as the peptide or the polypeptide of claim 176, wherein, described second kind of cell is sick cell.

180. as the peptide or the polypeptide of claim 179, wherein, described sick cell is a cancer cells.

181. as the peptide or the polypeptide of claim 179, wherein, described sick cell is selected from cancer, sarcoma, leukemia, adenoma, lymphoma, myelomatosis, blastoma, spermocytoma and melanoma cells.

182. as the peptide or the polypeptide of claim 181, wherein, described sick cell is the leukemia cell.

183. as the peptide or the polypeptide of claim 182, wherein, described leukemia cell is the acute myelocytic leukemia cell.

184. as the peptide or the polypeptide of claim 176, wherein, the selectivity of described peptide or polypeptide and the part of second kind of cell and/or specificity combine mainly and are determined by first hypervariable region.

185. peptide or polypeptide as claim 176, wherein, secondly be subjected to second hypervariable region, the 3rd hypervariable region in conjunction with selectivity or specificity and/or lay respectively at described first, second and/or the one or more upstreams of the 3rd hypervariable region flank or the influence of catchment.

186. a part, it is by described second kind of cell expressing, and it can combine with the peptide or the polypeptide of claim 196.

187. can discern and in conjunction with the molecule of the part of claim 186 for one kind.

188. a nucleic acid molecule, its claim 138 of encoding, any one peptide or polypeptide in 171,173 or 176.

189. as the nucleic acid molecule of claim 188, wherein, described nucleic acid is DNA.

190. as the peptide or the polypeptide of claim 176, wherein, first and second kinds of states of described first kind of cell are identical, and wherein, described first kind of cell is from a kind of clone.

191. as the peptide or the polypeptide of claim 190, wherein, described clone is selected from Jurkat, Molt-4, HS-602, U937, TF-1, THP-1, KG-1, ML-2 and HUT-78.

192. will choose wantonly and associate with a kind of medicinal reagent or adhere to, coupling, associating, be connected or claim 138 or the peptide of claim 176 or the purposes that polypeptide is used to produce medicine of fusion.

193. as the purposes of claim 192, wherein, described medicine has disease-resistant change cell activity.

194. as the purposes of claim 193, wherein, described sick cell is a cancer cells.

195. as the purposes of claim 193, wherein, described cell is selected from cancer, sarcoma, leukemia, adenoma, lymphoma, myelomatosis, blastoma, spermocytoma and melanoma.

196. as the purposes of claim 195, wherein, described cell is the leukemia cell.

197. as the purposes of claim 196, wherein, described leukemia cell is the acute myelocytic leukemia cell.

198. as optional and a kind of medicinal reagent association of medicine or adhere to, coupling, associating, be connected or the claim 138 of fusion or the peptide or the polypeptide of claim 176.

199. as the peptide or the polypeptide of claim 198, wherein, described medicine has disease-resistant change cell activity.

200. as the peptide or the polypeptide of claim 199, wherein, described sick cell is a cancer cells.

201. as the peptide or the polypeptide of claim 199, wherein, described cell is selected from cancer, sarcoma, leukemia, adenoma, lymphoma, myelomatosis, blastoma, spermocytoma and melanoma cells.

202. as the peptide or the polypeptide of claim 201, wherein, described cell is the leukemia cell.

203. as the peptide or the polypeptide of claim 202, wherein, described leukemia cell is the acute myelocytic leukemia cell.

204. the peptide of claim 138 or claim 176 or polypeptide are used to prepare the purposes of the composition that is used for suppressing the sick cell growth.

205. as the purposes of the peptide or the polypeptide of claim 204, wherein, described cell is the leukemia cell.

206. as the purposes of the peptide or the polypeptide of claim 205, wherein, described leukemia cell is the acute myelocytic leukemia cell.

207. the peptide of claim 138 or claim 176 or polypeptide be used to prepare be used for the purposes of composition of anticancer growth, described composition comprises at least a compound, and this compound has the selective and/or specific medicinal part of described cancer cells.

208. a composition, what comprise medicinal significant quantity associates with a kind of medicinal reagent or adheres to, coupling, associating, is connected or at least a peptide as claim 138 or claim 176 of fusion, and the optional effectively carrier that comprises on a kind of pharmacology.

209. as the composition of claim 208, wherein, described peptide or polypeptide are connected by a kind of linker compounds with described medicinal reagent.

210. as the composition of claim 209, wherein, described linker compounds is selected from dicarboxylic acid, maleimide hydrazides, PDPH, carboxylic acid hydrazides and little peptide.

211. as the composition of claim 210, wherein, described little peptide is selected from AU1, AU5, BTag, c-myc, FLAG, Glu-Glu, HA, His6, HSV, HTTPHH, IRS, KT3, PROTEIN C, STag , T7, V5 and VSV-G.

212. as claim 138, any one peptide or polypeptide in 171,173 and 176, wherein, described mark is selected from AU1, AU5, BTag, c-myc, FLAG, Glu-Glu, HA, His6, HSV, HTTPHH, IRS, KT3, PROTEIN C, STag , T7, V5 and VSV-G.

213. as the composition of claim 208, wherein, described medicinal reagent is selected from radio isotope, toxin, oligonucleotide, recombinant protein, antibody fragment, and carcinostatic agent.

214. as the composition of claim 213, wherein, described radio isotope is selected from indium, ¹¹¹Indium, ¹¹³Indium, ^99mRhenium, ¹⁰⁵Rhenium, ¹⁰¹Rhenium, ^99mTechnetium, ^121mTellurium, ^122mTellurium, 1 ^25mTellurium, ¹⁶⁵Thulium, ¹⁶⁷Thulium, ¹⁶⁸Thulium, ¹²³Iodine, ¹²⁶Iodine, ¹³¹Iodine, ¹³³Iodine, ^81mKrypton, ³³Xenon, ⁹⁰Yttrium, ²¹³Bismuth, ⁷⁷Bromine, ¹⁸Fluorine, ⁹⁵Ruthenium, ⁹⁷Ruthenium, ¹⁰³Ruthenium, ¹⁰⁵Ruthenium, ¹⁰Mercury, ²⁰³Mercury, ⁶⁷Gallium and ⁶⁸Gallium.

215. as the composition of claim 213, wherein, described toxin is selected from gelonin, Pseudomonas exotoxin (PE), PE40, PE38, ricin, and modifier and derivative.

216. as the composition of claim 213, wherein, described carcinostatic agent is selected from Zorubicin, the A Deliya mycin, cis-platinum, taxol, calicheamicin, vincristine(VCR), cytosine arabinoside (Ara-C), endoxan, prednisone, daunorubicin, idarubicin, NSC-118218, Chlorambucil, interferon alpha, hydroxyurea, Temozolomide, Thalidomide and bleomycin, and derivative.

217. a cytostatic method, it comprises allows described cell contact with the peptide or the polypeptide of a certain amount of claim 138 or claim 176.

218. as the method for claim 217, wherein, described cell is selected from cancer, sarcoma, leukemia, adenoma, lymphoma, myelomatosis, blastoma, spermocytoma and melanoma.

219. as the method for claim 218, wherein, described cell is the leukemia cell.

220. as the method for claim 219, wherein, described leukemia cell is the acute myelocytic leukemia cell.

221. one kind is used for the diagnosis location of tumour and the pharmaceutical composition of video picture, it comprises with a kind of developer and adhering to, coupling, combination, be connected or at least a peptide as claim 138 or claim 176 of fusion.

222. a treatment suffers from the patient's of disease method, this method comprises peptide or the polypeptide of using the claim 138 or the claim 176 of the amount that can effectively treat described disease to described patient.

223. as the method for claim 222, wherein, described disease is selected from cancer, sarcoma, leukemia, adenoma, lymphoma, myelomatosis, blastoma, spermocytoma and melanoma.

224. as the method for claim 223, wherein, described disease is a leukemia.

225. as the method for claim 224, wherein, described leukemia is an acute myelocytic leukemia.

226. as the peptide or the polypeptide of claim 138 or claim 176, wherein, described Fv can be specifically or optionally in conjunction with acute myelocytic leukemia (AML) cell.

227. one kind is present on the AML cell, can be by the peptide of claim 226 or polypeptide bonded part.

228. an energy is in conjunction with the peptide or the polypeptide of the part of claim 227.

229. one kind be used for before the treatment, during or the diagnostic kit of analyzed in vitro result of treatment afterwards, comprise with a kind of cue mark molecule attached, coupling, combination, be connected or the peptide or the polypeptide as claim 138 or claim 176 of fusion.

230. as the test kit of claim 229, wherein, described cue mark molecule is a fluorescent mark.

231. as the test kit of claim 230, wherein, described fluorescent mark is selected from fluorescein, rhodamine, phycoerythrin, and modifier and conjugate.

232. as the test kit of claim 229, wherein, described test kit is used to diagnosing cancer.

233. as the peptide or the polypeptide of claim 139 or claim 176, wherein, described construct is the Ig polypeptide.

234. a method of producing the peptide or the polypeptide of claim 233, wherein, described Ig polypeptide is expressed as recombinant polypeptide, and produces in eukaryotic cell system.

235. as the method for claim 234, wherein, described eukaryotic system is the mammal cell line system.

236. as the peptide or the polypeptide of claim 233, wherein, described Ig polypeptide is the IgG polypeptide.

237. as the peptide or the polypeptide of claim 236, wherein, described IgG polypeptide comprises having SEQ ID NOs:8,115 and 114 CDR3, CDR2 and CDR1 district respectively.

238. as the IgG polypeptide of claim 236, wherein, described IgG polypeptide comprises having SEQ ID NOs:20,115 and 114 CDR3, CDR2 and CDR1 district respectively.

239. as the IgG polypeptide of claim 237, wherein, described CDR3, CDR2 and CDR1 district are heavy chains.

240. as the IgG polypeptide of claim 238, wherein, described CDR3, CDR2 and CDR1 district are heavy chains.

241. as the IgG polypeptide of claim 237, wherein, described CDR3, CDR2 and CDR1 district are light chains.

242. as the IgG polypeptide of claim 238, wherein, described CDR3, CDR2 and CDR1 district are light chains.

243. as the IgG polypeptide of claim 233, wherein, described IgG has the heavy chain that comprises SEQ IDNO:26 and comprises the light chain of SEQ ID NO:27 or have the chain of at least 90% amino acid similarity with described chain.

244. a method that is used for the peptide or the polypeptide of production claim 139 or claim 176, wherein, described peptide or polypeptide produce in prokaryotic cell prokaryocyte system or eukaryotic cell system.

245. as the method for claim 244, wherein, described prokaryotic system comprises intestinal bacteria, described intestinal bacteria comprise a kind of expression vector, and described eukaryotic system is the mammal cell line system.

246. as the method for claim 245, wherein, the expression vector of described prokaryotic system comprises and is selected from osmB, deo, β-lac-U5, λ P _LPromotor with CMV.

247. as peptide any in claim 138 or 176 or polypeptide, wherein, described CDR3 comprises aminoacid sequence R ₁-X Phe Pro-R ₂, wherein, R ₁And R ₂Comprise 0-15 amino-acid residue separately, and X wherein is Arg, Gly or Lys.

248. as the peptide or the polypeptide of claim 138 or claim 176, wherein, described peptide or polypeptide comprise the modification that at least a non-natural exists.

249. as the peptide or the polypeptide of claim 248, wherein, the modification that described non-natural exists makes described peptide or polypeptide have higher immunogenicity or more stable.

250. as the peptide or the polypeptide of claim 249, wherein, described at least a modification is selected from the class peptide and modifies, half class peptide is modified, and cyclic peptide is modified, and N-is end modified, and C-is end modified, and peptide bond is modified, and backbone modifications and residue are modified.

251. as claim 138, any one peptide or polypeptide in 171,173,176 or 198 is used for the ex-vivo purging autologous bone marrow, so that remove abnormal cells.

A 252. peptide or polypeptide with following general formula or structure:

A-X-B

Wherein, X is the hypermutation CDR3 district that comprises SEQ ID NOs:8 or 20; A and B can each naturally length be 1-1000 amino acid whose amino acid chain, wherein, A is a N-terminal, and B is a C-terminal.

253. as the peptide of claim 252, wherein, A is a 150-250 amino-acid residue, and B wherein is a 350-500 amino-acid residue.

254. as the peptide or the polypeptide of claim 253, it is bigger or a complete antibody or a polymeric part.

255. a dimer molecule, it comprises two kinds of peptides or polypeptide, and one of them is the peptide or the polypeptide of claim 252.

256. one kind comprises the two kinds of peptides of claim 252 or the dimer molecule of polypeptide, these two kinds of peptides or polypeptide are identical.

257. a nucleic acid molecule, the dimer molecule of peptide of its coding claim 252 or polypeptide or coding claim 254.

258. as the method for claim 235, wherein, described mammal cell line turnkey is drawn together SR α 5 promotors.

259. as the method for claim 235, wherein, described mammal cell line turnkey is drawn together the CMV promotor.

Images