KR20010112915A - Neutrokine-alpha and neutrokine-alpha splice variant - Google Patents

Abstract

The present invention relates to a novel cytokine designated Neutrokine-alpha. The present invention also provides nucleic acid molecules encoding a neurotoxin-alpha SV polypeptide, an obvious splice variant of neurotoxin-alpha and neurotoxin-alpha, vectors, host cells and recombinant methods for producing them.

Description

Neutrokine-alpha and neutrokine-alpha splice variants < RTI ID = 0.0 >

Human tumor necrosis factor (TNF-alpha) and (TNF-beta or lymphotoxin) are related members of a broad class of polypeptide mediators including interferons, interleukins and growth factors, collectively referred to as cytokines [Beutler, B and Cerami, A., Annu. Rev. Immunol. 7: 625-655 (1989)). Sequence analysis of cytokine receptors showed that the membrane proteins of several subfamilies, (1) the Ig superfamily, (2) the hematopoietin (cytokine receptor superfamily) and (3) the tumor necrosis factor TNF) / nerve growth factor (NGF) receptor superfamilies were identified (Gruss and Dower, Blood 85 (12): 3378-3404 (1985) and Aggarwal and Natarajan, Eur. Cytokine Netw., 7 2): 93-124 (1996)). The TNF / NGF receptor superfamily contains 10 or more different proteins [see Gruss and Dower, supra]. Ligands for these receptors have been identified and belong to two or more cytokine superfamilies (see Gruss and Dower, supra).

Tumor necrosis factor (a mixture of TNF-alpha and TNF-beta) was first discovered as a result of its anti-tumor activity, but nowadays it has been found that a number of biologic agents, including mediators of apoptosis, cell activation and proliferation, Activity, and are recognized as expression cytokines that play an important role in immunomodulation and inflammation.

To date, members of the known TNF-ligand superfamily include TNF-alpha, TNF-beta (lymphotoxin-alpha), LT-beta, OX40L, Fas ligand, CD30L, CD27L. CD40L and 4-IBBL. The ligand of the TNF ligand superfamily is an acidic TNF-like molecule with about 20% sequence homology (range: 12% to 36%) in the extracellular domain and is mainly present in a membrane-bound form wherein the biologically active form Trimeric / multimeric complex. To date, the soluble forms of the TNF ligand superfamily have been identified only for TNF, LT-beta and Fas ligands (see Gruss, H. and Dower, SK, Blood, 85 (12): 3378-3404 ), The entire contents of which are incorporated herein by reference). These proteins are involved in the regulation of cell proliferation, activation and differentiation, including the regulation of cell survival or death by apoptosis or cytotoxicity [Armitage, R. J., Curr. Opin. Immunol. 6: 407 (1994) and Smith, C. A., Cell 75: 959 (1994)].

Tumor necrosis factor-alpha (TNF-alpha; also referred to as kakectin, hereinafter referred to as " TNF ") is a monocyte and macrophage that responds to endotoxin or other stimuli, Lt; RTI ID = 0.0 > al., ≪ / RTI > J. Biol. Chem. 262: 6951-6954 (1987)]. The membrane-bound 26 kD precursor form of TNF is also described (Kriegler, M. et al., Cell 53: 45-53 (1988)).

According to many evidences, TNF is a regulatory cytokine with multispecific biological activity. These activities include activation of polymorphonuclear leukocytes (Klebanoff, S.J., et al., Beutler, B. et al., Nature 316-552 (1985)), inhibition of lipoprotein lipase synthesis (" et al., J. Immunol. 136-4220 (1986); Perussia, B., et al., J. Immunol. 138: 765 (1987)], cell growth inhibition or cell growth stimulation [Vilctor, J. et al., J. Exp. Med. 163: 632 (1986); Sugarman, B. J. et al., Science 230: 943 (1985); Lachman, L.B. et al., J. Immunol. 138: 2913 (1987)], cytotoxic action on certain transformed cell types [see Lachman, L., et al. et al .; Darzynkiewicz, Z. et al., Canc. Res. 44: 83 (1984)], antiviral activity [Kohase, M. et al., Cell 45: 659 (1986); Wong, G.H.W. et al., Nature 323: 819 (1986)], stimulation of bone resorption (Bertolini, D.R. et al., Nature 319: 516 (1986); Saklatvala, J., Nature 322: 547 (1986)], stimulation of collagenase and prostaglandin E2 production [Dayer, J.M. et al., J. Exp. Med. 162: 2163 (1985)], and T cells [Tokota, S. et al., J. Immunol. 140: 531 (1988)], B cells [Kehrl, J. H. et al. et al., J. Exp. Med. 166: 786 (1987)], mononuclear cells [Philip, R. et al., Nature 323: 86 (1986)], thymocytes [Ranges, G.E. et al., J. Exp. Med. 167: 1472 (1988)] and the stimulation of cell-surface expression of major histocompatibility complex (MHC) class I and class II molecules [Collins, T. et al., Proc. Natl. Acad. Sci. USA 83: 446 (1986); Pujol-Borrel, R. et al., Nature 326: 304 (1987)].

TNF is an inflammatory component that causes tissue injury, for example, induction of coagulation promoting activity on vascular endothelial cells (Pober, J.S., et al., J. Immunol. 136: 1680 (1986)], increased adsorption of neutrophils and lymphocytes (Pober, J. S., et al., J. Immunol. 138: 3319 (1987)) and stimulation of platelet activating factor secretion from macrophages, neutrophils and vascular endothelial cells (see Camussi, G. et al., J. Exp. Med. 166: 1390 (1987).

Recent evidence indicates the incidence of many infections [Caeami, A. et al., Immunol. (Oliff, A. et al., Cell 50: 555 (1987)) and autoimmune diseases, such as autoimmune diseases Pathology and host pathology for transplants [Piguet, PF, et al., J. Exp. Med. 166-1280 (1987). The association of TNF with cancer and infectious pathology is mainly related to the cingulate state of the host. A major problem in cancer patients is weight loss associated with appetite loss in general. The enormous debility that is caused is known as " cachexia " (Kern, K. A. et al. J. Parent. Enter. Nutr. 12: 286-298 (1988). Cachexia includes progressive weight loss, loss of appetite and persistent body odor in response to malignant growth. Thus, cachexia conditions are associated with effective morbidity and cause the majority of cancer mortality rates. Many studies have suggested that TNF is a critical mediator of cachexia in cancer, infection pathology, and other catabolic states.

TNF is considered to play a key role in the pathophysiological consequences of Gram-negative sepsis and endotoxic shock including fever, discomfort, anorexia and cachexia (Michie, H. R. et al., Br. J. Surg. 76: 670-671 (1989); Debets, J. M. H. et al., Second Vienna Shock Forum, p. 463-466 (1989); Simpson, S. Q. et al., Crit. Care Clin. 5: 27-47 (1989). The endotoxin may be TNF [see Kornbluth, S.K. et al., J. Immunol. 137: 2585-2591 (1986)] and other monocyte / macrophage activating agents that stimulate the production and secretion of another cytokine. Because TNF can mimic many biological effects of endotoxin, it is inferred to be a key mediator responsible for the clinical symptoms of endotoxin-related diseases. TNF and other mononuclear cell-derived cytokines mediate metabolic and endocrine responses to endotoxins [Michie, H. R. et al. et al., N. Eng. J. Med. 318: 1481-1486 (1988). Administration of endotoxins to human volunteers results in acute illnesses of cold-like symptoms, including fever, tachycardia, increased metabolic rate, and stress hormone release (Revhaug, A. et al. Arch. Surg. 123: 162-170 (1988)]. Elevated levels of circulating TNF have also been found in patients with Gram-negative sepsis (Waage, A. et al., Lancet 1: 355-357 (1987); Hammerle, A. F. et al., Second Vienna Shock Forum p. 715-718 (1989); Debets, J. M. H. et al., Crit. Care Med. 17: 489-497 (1989); Calandra, T. et al., J. Infec. Dis. 161: 982-987 (1990).

Passive immunotherapy, focused on neutralizing TNF, has shown beneficial effects in these pathologies, based on increased TNF production and increased TNF levels in pathological conditions of gram-negative sepsis and endotoxemia, as discussed above . Reference is made to Cerami et al. EPO Patent Publication No. 0,212,489, dated March 4, 1987, describes antibodies against a " modulator " substance identified as carcitine (later found to be identical to TNF). Such antibodies have been found to be useful in diagnostic immunoassays and in the treatment of shock in bacterial infections. See Rubin et al. EPO Patent Publication No. 0,218,868, dated April 22, 1987) discloses a monoclonal antibody against human TNF, a hybridoma that secretes such an antibody, a method of producing such an antibody, and a method of immunizing such antibody Purpose is described. See, e.g., Yone et al .; EPO Patent Publication No. 0,288,088, dated October 26, 1988, describes the use of these antibodies in the diagnosis of anti-TNF antibodies and pathology, particularly Kawasaki's pathology and immunologic analysis of bacterial infections, including mAbs . Kawasaki disease (infant acute febrile mucocutaneous lymphadenitis syndrome) [Reference: Kawasaki, T., Allergy 16: 178 (1967); Kawasaki, T., Shonica (Pediatrics) 26: 935 (1985)] is considered to contain elevated levels of TNF associated with disease progression (Yone et al., Supra) .

Other investigators have described mAbs specific for recombinant human TNF with neutralizing activity in vitro [Liang, CM, et al. et al. Biochem. Biophys. Res. Comm. 137: 847-854 (1986); Meager, A. et al., Hybridoma 6: 305-311 (1987); Fendly et al., Hybridoma 6: 359-369 (1987); Bringman, T S et al., Hybridoma 6: 489-507 (1987); Hirai, M. et al., J. Immunol. Meth. 96: 57-62 (1987); Some of these mAbs have been used to map epitopes of human TNF and to develop enzyme immunoassays (Fendly et al., Supra; Hirai et al., Supra; Moller et al., Supra], are used to assist in the purification of recombinant TNF [Bringman et al., Supra]. However, these studies have shown that immunogenicity, lack of specificity and / Do not provide a basis for generating TNF neutralizing antibodies that can be used for in vivo diagnostic or therapeutic use in humans.

Neutralizing antisera or mAbs to TNF have been shown to alleviate harmful physiological changes and prevent death after catastrophic endotoxemia and bacteremia in non-human mammals. This effect has been demonstrated, for example, in rodent lethal analysis and primate pathology model systems [Mathison, J.C. et al., J. Clin. Invest. 81: 1925-1937 (1988); Beutler, B. et al., Science 229: 869-871 (1985); Tracey, K. J. et al., Nature 330: 662-664 (1987); Shimamoto, Y. et al., Immunol. Lett. 17: 311-318 (1988); Silva, A. T. et al., J. Infect. Dis. 162: 421-427 (1990); Opal, S. M. et al., J. Infect. Dis. 161: 1148-1152 (1990); Hinshaw, L. B. et al., Circ. Shock 30: 279-292 (1990)].

So far, experiments using anti-TNF mAb therapy in humans have been limited, but have shown beneficial therapeutic results, for example in arthritis and sepsis [Elliott, M. J. et al., Baillieres Clin. Rheumatol. 9: 633-52 (1995); Feldmann M. et al., Ann. N. Y. Acad. Sci. USA 766: 272-8 (1995); Van der Poll, T. et al., Shock 3: 1-12 (1995); Wherry et al., Crit. Care. Med. 21: S436-40 (1993); Tracey K. J., et al., Crit. Care Med. 21: S415-22 (1993)].

The development of mammals depends on both proliferation and differentiation of cells as well as programmed cell death resulting from apoptosis (Walker, et al., Methods Achiev. Exp. Pathol. 13: 18 (1988). Apoptosis plays a critical role in the destruction of immune thymocytes that recognize autoantigens. Failure of the normal removal process may play a role in autoimmune disease (Gammon et al., Immunology Today 12: 193 (1991)).

Itoh et al., Cell 66: 233 (1991) describes Fas / CD95, a cell surface antigen that mediates apoptosis and is involved in clonal deletion of T-cells. Fas is expressed in the ovaries of neutrophils, thymus, liver, heart, lung, and mature mice in addition to activated T-cells, B-cells (Watanabe-Fukunaga et al., J. Immunol. 148: 1274 (1992)). In experiments in which monoclonal Ab is cross-linked to Fas, apoptosis is induced (Yonehara et al., J. Exp. Med. 169: 1747 (1989); Trauth et al., Science 245: 301 (1989)]. In addition, there is an example that the binding of monoclonal Ab to Fas is a stimulus for T-cells under certain conditions (see Alderson et al., J. Exp. Med. 178: 2231 (1993)].

Fas antigen is a cell surface protein with a relative MW of 45 Kd. Both human and mouse genes for Fas were cloned by Watanabe-Fukunaga et al. (J. Immunol. 148: 1274 (1992)) and Itoh et al., Cell 66: 233 (1991)]. The proteins encoded by these genes are composed of two TNF receptors, a low affinity nerve growth factor receptor and a transmembrane protein that is structurally homologous to the nerve growth factor / tumor necrosis factor receptor superfamily including CD40, CD27, CD30 and OX40 Both are.

Recently, Fas ligands have been described (Suda et al., Cell 75: 1169 (1993)). The amino acid sequence is a type II epidermal protein in which the Fas ligand belongs to the TNF family. Thus, Fas ligand polypeptides include three major domains: the short intracellular domain of the amino terminus and the longer extracellular domain of the carboxy terminus linked by the hydrophobic transmembrane domain. Fas ligand is expressed in splenocytes and thymocytes closely related to T-cell mediated cytotoxicity. The MW of the purified Fas ligand is 40 kD.

Recently, Fas / Fas ligand interaction has been demonstrated to be required for post-activation apoptosis of T-cells (Ju et al., Nature 373: 444 (1995); Brunner et al., Nature 373: 441 (1995)]. Activation of T-cells induces both protein on the cell surface. Subsequent interaction between the ligand and the receptor causes cell apoptosis. This supports a possible regulatory role for apoptosis induced by Fas / Fas ligand interaction during a normal immune response.

Thus, there is a need to provide cytokines similar to TNF that are associated with pathological symptoms. These novel cytokines can be used to produce novel antibodies or other antagonists that bind to the TNF-like cytokine in question for the diagnosis and treatment of TNF-like cytokine related disorders.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided a novel extracellular domain of a neurotoxin-alpha polypeptide, a novel extracellular domain of a neurotoxin-alpha SV polypeptide and a biologically active, diagnostic or therapeutically useful fragment thereof , Analogs and derivatives thereof.

In accordance with another aspect of the present invention there is provided a human neurokinin-alpha or neurotoxin-alpha gene, including mRNA, DNA, cDNA and genomic DNA as well as analogs and biologically active, diagnostic or therapeutically useful fragments and derivatives thereof A separate nucleic acid molecule encoding the SV is provided.

The present invention encompasses a polynucleotide that encodes a cytokine and its apparent splice variant that are structurally similar to TNF and related cytokines and exhibit similar biological effects and activities or alternatively comprise a separate nucleic acid comprising such a polynucleotide Lt; / RTI > The cytokine is designated as neurotoxin-alpha and the present invention is characterized by the amino acid sequence of SEQ ID NO: 2 of FIGS. 1A and 1B or the cDNA clone (HNEDU15) deposited on October 22, 1996 and assigned ATCC No. 97768 Alpha < / RTI > polypeptide having at least a portion of the amino acid sequence being encoded. The nucleotide sequence (SEQ ID NO: 1) determined by sequencing the deposited neurotoxin-alpha clone shown in Figures 1A and 1B contains the N-terminal methionine, an estimated intracellular domain of about 46 amino acid residues, about 26 amino acids And an open reading frame encoding an entire extracellular domain of 285 amino acid residues comprising an estimated extracellular domain of about 213 amino acids and an estimated molecular weight of the complete protein of about 31 kDa. In the case of another type II transmembrane protein, the soluble form of neurotoxin-alpha is a complete neurotoxin-alpha polypeptide lacking all or part of the extracellular domain cleaved from the transmembrane domain and the transmembrane domain, Lt; RTI ID = 0.0 > extracellular < / RTI > domain.

The apparent splice variant of neurotoxin-alpha is designated as neurotoxin-alpha SV, and the present invention is based on the amino acid sequence of Figures 5A and 5B (SEQ ID NO: 19), or deposited on Dec. 10, 1998 and assigned ATCC No. 203518 alpha SV polypeptides comprising or alternatively consisting of at least a portion of the amino acid sequence encoded by the cDNA clone HDPMC52. The nucleotide sequence (SEQ ID NO: 18) determined by sequencing of the deposited neurotoxin-alpha SV clone shown in Figures 5A and 5B contains N-terminal methionine, an estimated intracellular domain of about 46 amino acid residues, about 26 amino acids And an open reading frame encoding an entire polypeptide consisting of an estimated extracellular domain of about 194 amino acids and consisting of 266 amino acid residues with an estimated molecular mass of the complete protein of about 29 kDa. In the case of another type II transmembrane protein, the soluble form of the neurotoxin-alpha SV is a complete neurotoxin-alpha SV polypeptide lacking all or part of the extracellular domain cleaved from the transmembrane domain and the transmembrane domain, Lt; RTI ID = 0.0 > extracellular < / RTI > domain linked to my domain.

Accordingly, one aspect of the present invention is directed to a nucleic acid encoding a full-length neurotoxin-encoding nucleic acid comprising (a) a full-length neurotoxin-encoding sequence encoded by a cDNA clone having the complete amino acid sequence of Figure 1A and 1B (SEQ ID NO: 2) or contained in a deposit of ATCC Accession No. 97768, (B) a nucleotide sequence encoding nucleotides 73 to 285 of Figure 1A and 1B (SEQ ID NO: 2), or a nucleotide sequence encoding a nucleotide sequence encoding a neurotoxin encoded by a clone contained in a deposit of ATCC Accession No. 97768 (C) a nucleotide sequence encoding a fragment of the polypeptide of (b) having a neurotoxin-alpha functional activity (e. G., Biological activity), (d) a nucleotide sequence encoding the deduced extracellular domain of the kin-alpha polypeptide ) Neurokinin-alpha intracellular domain (presumed to consist of amino acid residues from about 1 to about 46 of Figures 1A and 1B (SEQ ID NO: 2)) or ATCC Accession No. 97768 A nucleotide sequence encoding a polypeptide encoded by a clone contained in the deposit of (e) a neurotoxin-alpha envelope domain (consisting of amino acid residues from about 47 to about 72 of Figures 1A and 1B ), Or a nucleotide sequence encoding a polypeptide encoded by a clone contained in a deposit of ATCC Accession No. 97768, (f) a nucleotide sequence having extracellular and intracellular domains but lacking a dermal domain (G) a nucleotide sequence complementary to a nucleotide sequence of any one of the above (a), (b), (c), (d), (e) or (f) Polynucleotides having a nucleotide sequence selected from the group consisting of SEQ ID NOs: Jethro provides an isolated nucleic acid molecule consisting of.

A further aspect of the present invention is a nucleic acid sequence encoding a polypeptide comprising 80%, 85% or 90% sequence identity with any one of the nucleotide sequences of (a), (b), (c), (d), (e), (f) (A), (b), (c) or (c) under stringent hybridization conditions with a polynucleotide having a nucleotide sequence of at least 95%, 96%, 97%, 98% ), (d), (e), (f) or (g), or alternatively comprises a separate nucleic acid molecule consisting of such polynucleotides. The hybridizing polynucleotide does not hybridize under stringent hybridization conditions with a polynucleotide consisting of only the A residue or only the nucleotide sequence consisting of the T residue.

Another aspect of the present invention is a kit comprising (a) a cDNA clone contained in the ATCC deposit of ATCC Accession No. 203518, having the complete amino acid sequence (SEQ ID NO: 19) of Figures 5A and 5B or deposited on December 10, 1998 (B) a nucleotide sequence encoding the full-length Nutrokine-alpha SV polypeptide encoded by the nucleotide sequence of amino acid residues 73 to 266 of Figures 1A and 1B (SEQ ID NO: 2) 5A and 5B (SEQ ID NO: 19), which encodes the putative extracellular domain of a neurotoxin-alpha SV polypeptide encoded by a cDNA clone contained in SEQ ID NO: 203518, (c) a nucleotide sequence encoding a neurotoxin- Which encodes a polypeptide encoded by a cDNA clone contained in ATCC No. 203518 deposited on December 10, 1998, or a nucleotide sequence encoding a nucleotide sequence encoding a polypeptide encoded by a cDNA clone contained in ATCC No. 203518 deposited on December 10, (D) a neurotoxin-alpha SV epidermal domain (presumed to consist of amino acid residues from about 47 to about 72 amino acids in Figures 5A and 5B (SEQ ID NO: 19)), A nucleotide sequence encoding a polypeptide encoded by a cDNA clone contained in ATCC No. 203518, (e) a nucleotide sequence encoding a soluble neurotoxin-alpha SV polypeptide having extracellular and intracellular domains but lacking a transmembrane domain And (f) a polynucleotide having a nucleotide sequence selected from the group consisting of a nucleotide sequence complementary to any one of the nucleotide sequences of (a), (b), (c), (d) And provides separated nucleic acid molecules consisting of such polynucleotides.

Another aspect of the present invention is a nucleic acid molecule which is 80%, 85% or 90% or more, more preferably 80% or 85% or 90% or more homologous to any one of the nucleotide sequences of any one of the above (a), (b), (c) (A), (b), (c), (d), (e), or (f) of the polynucleotide having a nucleotide sequence identical to or greater than 95%, 96%, 97%, 98%, or 99% Of the polynucleotide hybridizes under stringent hybridization conditions, or alternatively comprises a separate nucleic acid molecule consisting of such a polynucleotide. The hybridizing polynucleotide does not hybridize under stringent hybridization conditions with a polynucleotide consisting of only the A residue or only the nucleotide sequence consisting of the T residue.

In one embodiment, the amino acid sequence of Gly-142 to Leu-266 as shown in Figures 5A and 5B (SEQ ID NO: 19), or the amino acid sequence of Gly-142 to Leu-266 by the clone HDPMC52 deposited with the ATCC Accession No. 203518 deposited on Dec. 10, 1998 A distinct splice variant of neurotoxin-alpha consisting of, or alternatively consisting of, at least a portion of the amino acid sequence being encoded is provided.

In a further aspect, the nucleic acid molecule of the present invention is a nucleic acid molecule having the amino acid sequence of (a), (b), (c), (d), (e), (f) Or alternatively consists of, such polynucleotides that encode the amino acid sequence of the epitope-bearing portion of the K-alpha SV polypeptide. A further nucleic acid aspect of the invention is a nucleic acid sequence comprising an amino acid sequence comprising at least 1 to 50, preferably at least 40, more preferably at least 30, even more preferably at most 20 amino acid additions, substitutions and / Or alternatively comprising a polynucleotide encoding an amino acid sequence of a neurotoxin-alpha or neurotoxin-alpha SV polypeptide having a polynucleotide comprising a polynucleotide of the present invention. Of course, the order of increasing the degree of preference is 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 or less, or 1 to 100, 1 to 50, 1 to 25, Alpha, or neurotoxin-alpha SV polypeptide having an amino acid sequence that contains the addition, substitution, and / or deletion of 1 to 15, 1 to 10, or 1 to 5 amino acids in the amino acid sequence of SEQ ID NO: Polynucleotides are highly preferred. Preservative substitution is preferred.

The present invention also relates to recombinant vectors comprising the isolated nucleic acid molecules of the invention, host cells containing said recombinant vectors, as well as methods for producing such vectors and host cells, and using them for recombinant techniques to produce neurotoxin-alpha polypeptides And a method for producing the same.

In a further aspect of the invention, recombinant prokaryotic and / or eukaryotic host cells containing the neurotoxin-alpha or neurotoxin-alpha SV nucleic acid sequences of the invention are cultured under conditions that promote expression of the polypeptide, There is provided a method of producing said polypeptide by recombinant techniques, including recovering the peptide.

(A) a full-length neurotoxin-encoding gene having the complete amino acid sequence shown in Figures 1A and 1B (position 1 to 285 of SEQ ID NO: 2) or encoded by a cDNA plasmid contained in a deposit of ATCC Accession No. 97768, (B) an amino acid sequence of a full-length neurotoxin-alpha polypeptide having the complete amino acid sequence (positions 2 to 285 of SEQ ID NO: 2) described in SEQ ID NO: 2 except for N-terminal methionine, (c) (D) a fragment of the polypeptide of (b) having a neurotoxin-alpha functional activity (e. G., Biological activity); (d) a polypeptide having an amino acid sequence at positions 73 to 285 shown in Figures 1A and 1B Amino acid sequence of the extracellular domain of the neurotoxin-alpha polypeptide encoded by the cDNA plasmid contained in the deposit of EP-A-97768, (e) amino acid sequence at positions 134 to 285 shown in Figures 1A and 1B (F) a nucleotide sequence encoding a neurotoxin-alpha intracellular domain (presumably comprising amino acid residues from about 1 to 46 shown in Figures 1A and 1B (SEQ ID NO: 2)), , Or an amino acid sequence encoded by a cDNA plasmid contained in a deposit of ATCC Accession No. 97768, (g) a nucleotide sequence encoding a neurotoxin-alpha alpha domain Amino acid sequence encoded by a cDNA plasmid contained in a deposit of ATCC Accession No. 97768, (h) an extracellular and intracellular domain, but lacking a transmembrane domain (A), (b), (c), (d), (e), (e), f), (g) and Comprises an amino acid sequence selected from the group consisting of a fragment of the polypeptide of (h), or, alternatively, the neutroavidin Keene separation consisting of such amino acid sequence - provides alpha polypeptide. The polypeptides of the present invention can also be produced by using the polypeptides described in (a), (b), (c), (d), (e), (f), (g), (h) 80%, 85% or 90%, more preferably 95% to 99% identical to the above, as well as polypeptides having 80%, 85% or 90% And polypeptides having an amino acid sequence having 95% or more similarity. A further aspect of the present invention is a method for producing a protein having the amino acid sequence described in (a), (b), (c), (d), (e), (f), (g), (h) Or a polypeptide consisting essentially of, or alternatively consisting of, the amino acid sequence of the epitope-containing portion of the neurotoxin-alpha polypeptide having the amino acid sequence of SEQ ID NO. The polypeptide having the amino acid sequence of the epitope-containing portion of the neurotoxin-alpha polypeptide according to the present invention has 4 to 8 or more, preferably 9 to 15 or more, 20 or more, 25 or more, 30 or more, Comprising a portion of the polypeptide consisting of at least 40, at least 50, more preferably at least about 30 to 50 amino acids, and also including the entire amino acid sequence of the polypeptide of the invention described above, Gt; epitope-containing < / RTI > polypeptides are included in the present invention.

A highly preferred embodiment of the present invention is a polynucleotide encoding a neurotoxin-alpha polypeptide having an amino acid sequence at positions 134 to 285 of Figure 1A and 1B (SEQ ID NO: 2) of 80%, 85%, 90% Or alternatively consisting of, a polynucleotide having a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical. A preferred embodiment of the invention comprises a polynucleotide having at least 90% identical nucleotide sequence to a polynucleotide sequence encoding a neurotoxin-alpha polypeptide having an amino acid sequence at positions 134 to 285 of Figure 1A and 1B (SEQ ID NO: 2) , Alternatively, a nucleic acid molecule comprising such a polynucleotide. A more preferred embodiment of the invention comprises a polynucleotide having a nucleotide sequence at least 95% identical to a polynucleotide sequence encoding a neurotoxin-alpha polypeptide having an amino acid sequence at positions 134 to 285 of Figure 1A and 1B (SEQ ID NO: 2) Or, alternatively, to a nucleic acid molecule comprising such a polynucleotide. A more preferred embodiment of the invention comprises a polynucleotide having a nucleotide sequence that is at least 96% identical to a polynucleotide sequence encoding a neurotoxin-alpha polypeptide having an amino acid sequence at positions 134 to 285 in Figures 1A and 1B (SEQ ID NO: 2) Or, alternatively, to a nucleic acid molecule comprising such a polynucleotide. In addition, a more preferred embodiment of the present invention is a polynucleotide encoding a neurotoxin-alpha polypeptide having an amino acid sequence at positions 134 to 285 of Figure 1A and 1B (SEQ ID NO: 2), wherein the polynucleotide has 97% or more identical nucleotide sequence Or alternatively, a nucleic acid molecule comprising such a polynucleotide. Further, a more preferred embodiment of the present invention is a polynucleotide encoding a neurotoxin-alpha polypeptide having an amino acid sequence at positions 134 to 285 shown in Figures 1A and 1B (SEQ ID NO: 2) with a nucleotide sequence that is 98% Polynucleotides, or, alternatively, nucleic acid molecules comprised of such polynucleotides. Further, a more preferred embodiment of the present invention is a polynucleotide encoding a neurotoxin-alpha polypeptide having an amino acid sequence at positions 134-285 of Figures 1A and 1B (SEQ ID NO: 2) Or alternatively, a nucleic acid molecule comprising such a polynucleotide.

The invention also encompasses polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these polynucleotides and nucleic acid molecules are also encompassed by the present invention.

(A) a cDNA plasmid containing the complete amino acid sequence shown in Figures 5A and 5B (position 1 to 266 of SEQ ID NO: 19) or contained in ATCC Accession No. 203518 deposited on December 10, 1998 Alpha SV polypeptides having the complete amino acid sequence shown in SEQ ID NO: 19 (positions 2 to 266 of SEQ ID NO: 19), except for the N-terminal methionine; (b) the amino acid sequence of the full length neurotoxin- (C) by a cDNA clone contained in ATCC Accession No. 203518, which has the amino acid sequence of positions 73 to 266 shown in Figures 5A and 5B (SEQ ID NO: 19) or deposited on Dec. 10, 1998 (D) an amino acid sequence of about 1 to 46 amino acids of the neurotoxin-alpha SV intracellular domain (Figures 5A and 5B (SEQ ID NO: 19), the amino acid sequence of the deduced extracellular domain of the neurotoxin- By doing (E) about 47 to about 72 amino acids of the neurotoxin-alpha SV transmembrane domain (Figures 5A and 5B (SEQ ID NO: 19)), or an amino acid sequence encoded by a cDNA clone contained in ATCC Accession No. 203518 Amino acid sequence encoded by a cDNA clone contained in ATCC Accession No. 203518 deposited on December 10, 1998, (f) an extracellular and intracellular domain (A), (b), (c), (c), (c) and (c), wherein the amino acid sequence of the soluble neurotoxin-alpha SV polypeptide lacking the transmembrane domain is deleted alpha SV polypeptides comprising, alternatively consisting of, an amino acid sequence selected from the group consisting of fragments of the polypeptides of (a), (d), (e) or (f). The polypeptide of the present invention may also contain 80%, 85% or 90% of the amino acid sequence described in (a), (b), (c), (d), (e) , More preferably 95% to 99%, as well as polypeptides having an amino acid sequence having 80%, 85% or 90% similarity, more preferably 95% or more similarity . A further aspect of the present invention is a neurotoxin-alpha SV polypeptide having an amino acid sequence as described in (a), (b), (c), (d), (e), (f) Or a polypeptide consisting essentially of, or alternatively consisting of, the amino acid sequence of the epitope-containing portion of the polypeptide. The peptide or polypeptide having the amino acid sequence of the epitope-containing portion of the neurotoxin-alpha SV polypeptide according to the present invention has 4 to 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, 50 More preferably at least about 30 to about 50 amino acids, and further comprising an epitope-containing poly (amino acid sequence) of less than or equal to the entire amino acid sequence of the polypeptide of the invention described above, Peptides are included in the present invention.

Certain conventional embodiments of the present invention are directed to the amino acid sequences described in (a), (b), (c), (d), (e), (f), (g), (h) Alpha or < RTI ID = 0.0 > neurotoxin-alpha < / RTI > SV polypeptide having the amino acid sequence of the epitope-containing portion of the neurotoxin-alpha or neurotoxin-alpha SV polypeptide. In another aspect, the present invention provides a method for producing a protein comprising the amino acid sequence described in (a), (b), (c), (d), (e), (f), (g), (h) (Or specifically) binds to a neurotoxin-alpha or neurotoxin-alpha SV polypeptide having an amino acid sequence of SEQ ID NO.

The present invention also provides a method of separating antibodies specifically binding (i.e., specifically) to a neurotoxin-alpha or neurotoxin-alpha SV polypeptide having an amino acid sequence as described herein. Such antibodies are diagnostic or therapeutically useful as described below.

The invention also relates to the use of soluble neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides, in particular human neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides and / or anti- Anti-neurotoxin-alpha SV antibodies and are useful for the treatment of host and tumor metastases, infections of bacteria, viruses and other parasites, immune deficiency, inflammatory diseases, lymphadenopathy, autoimmune diseases, , Prophylactic, prognostic and / or diagnostic, to stimulate peripheral tolerance, to destroy some transformed cell lines, to mediate cell activation, survival and proliferation, to mediate immune modulation and inflammatory responses, or to enhance or suppress immune responses Lt; RTI ID = 0.0 > pharmaceutically < / RTI >

In certain embodiments, the soluble neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides or agonists of the present invention are useful in the treatment of immune deficiency (e.g., severe combined immunodeficiency (SCID) -relaxation, SCID-autosomal, adenosine de (ADA deficiency), anti-gammaglobulinemia (XLA), Bruton's disease, congenital non-gammaglobulinemia, recurrent infantile non-gammaglobulinemia, acquired non-gammaglobulinemia, adult onset gamma globulin Specific IgG, non-gammaglobulinemia, non-classifiable immune deficiency syndrome (CVID) (acquisitive), hyperglycemia, hypogammaglobulinemia, hypogammaglobulinemia, hypogammaglobulinemia, infantile transient non-gammaglobulinemia, (WAS), IgM hyperinsulinemia deficiency, IgM hyperalgesia, selective IgA deficiency, IgG obesity deficiency (associated or non-IgA deficiency syndrome) ), Normal or elevated Ig antibody deficiency, immunodeficiency associated with thymic carcinoma, Ig oligosaccharidosis, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD), selective IgM immunodeficiency, degenerative non-gammaglobulinemia ), Neurogenic neutropenia, severe congenital leukopenia, thymic lymphoid hypoplasia with immune deficiency - amorphous or dysplasia, ataxia - capillary dilatation, meridian dwarfism, XLP, Prophylactic, therapeutic and / or prophylactic treatment of a condition associated with a syndrome-associated Ig immunodeficiency syndrome, purine type nucleoside phosphorylase deficiency (PNP), MHC type II deficiency (bare lymphocyte syndrome) and severe combined immunodeficiency syndrome) / RTI >

In certain embodiments, the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides or polynucleotides or agonists of the invention are administered to treat, prevent, prognose and / or diagnose a variety of common immunodeficiency disorders.

In certain embodiments, the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides or polynucleotides or agonists thereof of the invention are administered for the treatment, prophylaxis, prognosis and / or diagnosis of reactive non-gamma globulinemia.

In another specific embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides or polynucleotides or agonists thereof of the invention are administered to treat, prevent, prognose and / or diagnose severe combined immunodeficiency syndrome (SCID) do.

In another specific embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides or polynucleotides or agonists thereof of the invention are useful for treating, preventing, and prognosing Wiskott-Aldrich syndrome And / or diagnosed.

In another particular embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides or polynucleotides or agonists of the invention are used for the treatment, prophylaxis, prognosis and / or diagnosis of a refractory Ig deficiency with excess IgM Lt; / RTI >

In another embodiment, a neurotoxin-alpha alpha antagonist and / or a neurotoxin-alpha SV antagonist (e.g., an anti-neurotoxin-alpha antibody) is administered to a patient in need of treatment for autoimmune diseases (e.g., rheumatoid arthritis, systemic lupus erythematosus, idiopathic thrombocytopenia (Such as IgA nephropathy), autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, autoimmune hemolytic anemia, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, recurrent polychondritis, rheumatic heart disease, , Schizophrenia, autoimmune pneumonitis, Guillain-Barr syndrome, insulin-dependent diabetes mellitus and autoimmune diseases (eg, autoimmune diseases), multiple sclerosis, neuritis, uveitis conjunctivitis, polycystic dysplasia, purpura Inflammatory diseases, autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's thyroiditis, Goodpasture's syndrome, pemphigus, receptor autoimmunity, (A) Grave's Disease, (b) Myasthemia Gravis, and (c) insulin resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, anti-collagen antibodies (Eg, primary glomerulonephritis and IgA nephropathy), pemphigus vulgaris, Sjogren's syndrome, diabetes and adrenergic-acting drug resistance (eg, glomerulonephritis), scleroderma, mixed connective tissue disease, multiple myositis / dermatomyositis, malignant anemia, idiopathic Edison's disease, (Including adrenergic agonistic drug resistance with asthma or cystic fibrosis), chronic active hepatitis, primary biliary cirrhosis, endocrine disorders, albumin, vasculitis, post-MI, cardiac incision syndrome, urticaria, atopic dermatitis, asthma, inflammatory Prophylactic and / or diagnostic of symptoms associated with inflammation, myalgia and other inflammatory, granulomatous, degenerative and atrophic diseases) or autoimmune disease-related conditions. In a preferred embodiment, the anti-neurotoxin-alpha antibodies and / or anti-neurotoxin-alpha SV antibodies and / or other antagonists of the invention are used to treat, prevent, prognum and / or diagnose rheumatoid arthritis. In certain preferred embodiments, the anti-neurotoxin-alpha antibodies and / or anti-neurotoxin-alpha SV antibodies and / or other antagonists of the invention are used to treat, prevent, prognose and / or diagnose systemic lupus erythematosus. In another preferred specific embodiment, the anti-neutrophil-alpha antibody and / or the anti-neutrophil-alpha SV antibody and / or other antagonist of the present invention are used to treat, prevent, prognose and / or prevent idiopathic thrombocytopenia Diagnose. In another preferred specific embodiment, the anti-neurotoxin-alpha antibodies and / or anti-neurotoxin-alpha SV antibodies and / or other antagonists of the invention are used for the treatment, prophylaxis and / or diagnosis of IgA nephropathy. In another preferred particular embodiment, the anti-neurotoxin-alpha antibodies and / or anti-neurotoxin-alpha SV antibodies and / or other antagonists of the invention are used to treat autoimmune diseases and diseases and / Prophylactic, and / or diagnosed with the symptoms associated with the disease.

The present invention also relates to the use of a neurotoxin-alpha or neurotoxin-alpha SV polynucleotide, a neurotoxin-alpha or neurotoxin-alpha SV polypeptide and a neurotrophin-alpha SV polynucleotide for the administration to an in vitro cell, an ex vivo cell, an in vivo cell or a multicellular organism, / Or an anti-neurotoxin-alpha antibody or an anti-neurotoxin-alpha SV antibody. In a preferred embodiment, the compositions of the invention comprise a neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotide for the expression of a neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide in a host organism for the treatment of disease . In a most preferred embodiment, the compositions of the present invention are useful for the treatment of immunodeficiency and / or immunodeficiency associated conditions in neurotoxin-alpha and / or neurotrophin-alpha SV polypeptides for the expression of neurotoxin-alpha and / or neurotoxin- Or neurotoxin-alpha SV polynucleotides. Particularly preferred in this regard is the expression in a human patient for the treatment of disorders associated with abnormal endogenous activity of a neurotoxin-alpha or neurotoxin-alpha SV gene (e. G., By increasing the number of B- Expression to enhance normal B-cell function).

The present invention also relates to a method of screening a cell for expressing neurotoxin-alpha and / or neurotoxin-alpha SV by contacting the cell with a candidate compound, analyzing the cell reaction, Alpha and / or neurotoxin-alpha SV, comprising comparing a cell with a neurotoxin-alpha-SV, wherein the increased, compared to a standard cell response, A cellular response indicates that the compound is an efficacious agent and a reduced cellular response to the standard cellular response indicates that the compound is an antagonist.

In another embodiment, assays are provided for identifying neurotoxin-alpha and / or neurotoxin-alpha SV receptors as well as methods for selecting agonists and antagonists using such receptors. This assay involves measuring the effect of the candidate compound on the neurotoxin-alpha and / or neurotoxin-alpha SV, where the candidate compound binds to the neurotoxin-alpha and / or neurotoxin-alpha SV receptors. In particular, the method comprises contacting a neurotoxin-alpha and / or neurotoxin-alpha SV receptor with a neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide and a candidate compound of the present invention and administering neurotoxin-alpha and / Alpha < / RTI > and / or neurotoxin-alpha SV polypeptides that bind to the kin-alpha SV receptor are increased or decreased due to the presence of the candidate compound. Antagonists may be used in the treatment of septic shock, inflammation, cerebral malaria, activation of HIV virus, host rejection response to graft, bone resorption, rheumatoid arthritis, cachexia (debilitating or malnutrition), immune system function, lymphoma and autoimmune diseases : Rheumatoid arthritis and systemic lupus erythematosis).

The present inventors have found that neurotoxin-alpha inhibits the proliferation and proliferation of neurons in the kidney, lung, peripheral leukocyte, bone marrow, T cell lymphoma, B cell lymphoma, activated T cells, gastric cancer, smooth muscle, macrophages and cord blood tissue as well as in monocytic lineage cells Lt; / RTI > In addition, the present inventors have found that neurotoxin-alpha SV is highly expressed only in primary dendritic cells. The activation of many of these tissues and cells, such as tumor and tumor metastasis, infection of bacteria, viruses and other parasites, immunodeficiency (e.g. chronic immunodeficiency syndrome), septic shock, inflammation, cerebral malaria, HIV virus , Patients suffering from such diseases (for example, in relation to host rejection response to grafts, bone resorption, rheumatoid arthritis, autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus) and cachexia Standard " neurotoxin-alpha and / or neurotoxin-alpha SV gene expression levels, i. E., In the body fluids obtained from a subject, such as serum, plasma, urine, synovial fluid or spinal fluid or certain tissues Alpha < / RTI > and / or neurotoxin-alpha SV expression levels in body fluids or tissues from individuals who are not (A) assaying the level of neurotoxin-alpha and / or neurotoxin-alpha SV gene expression in an individual's cells or body fluids, and (b) There is provided a diagnostic method useful during diagnosis of a disease, including comparing neurotoxin-alpha and / or neurotoxin-alpha SV gene expression levels with standard neurotoxin-alpha and / or neurotoxin-alpha SV gene expression levels, Wherein an increase or decrease in the level of neurotoxin-alpha and / or neurotoxin-alpha SV gene expression assayed compared to standard expression levels indicates the presence or absence of disease.

A further aspect of the invention is a method of treating a neurodegenerative disorder in a body, comprising administering to the individual a therapeutically effective amount of a composition comprising an isolated neurotoxin-alpha and / or neurotoxin- alpha SV polypeptide or an agonist thereof according to the invention, Alpha and / or neurotoxin-alpha SV activity in a subject in need thereof.

Another further aspect of the invention is a method of treating a neurotoxin-alpha < RTI ID = 0.0 > (alpha) -alpha < / RTI > in a human body comprising administering to the individual a composition comprising a therapeutically effective amount of a separate neurotoxin- And / or a reduced level of neurotoxin-alpha SV activity. Preferred antagonists for use in the present invention are neurotoxin-alpha-specific and / or neurotoxin-alpha SV-specific antibodies.

The present invention relates to a novel cytokine named neurotoxin-alpha (" neurotoxin-alpha "). In addition, an apparent splice variant of neurotoxin-alpha was identified and named neurotoxin-alpha SV. In certain embodiments, the invention provides nucleic acid molecules encoding neurotoxin-alpha and neurotoxin-alpha SV polypeptides. In further embodiments, neurotoxin-alpha and neurotoxin-alpha SV polypeptides and vectors, host cells and recombinant methods for producing them are also provided.

The following drawings illustrate aspects of the present invention and are not intended to limit the scope of the invention as set forth in the claims.

Figures 1A and 1B show the nucleotides of Neutrokine-alpha (SEQ ID NO: 1) and the putative amino acid (SEQ ID NO: 2) sequences. Amino acids 1 to 46 represent the predicted intracellular domain, amino acids 47 to 72 represent the predicted transmembrane domain (double underlined sequence), and amino acids 73 to 285 represent the extracellular domain (remaining sequence). Potential asparagine-linked glycosylation sites are shown in Figures 1A and 1B and are asparagine-rich symbol N in the neurotoxin-alpha amino acid sequence, the first nucleotide encoding the asparagine residue in the neurotoxin-alpha nucleotide sequence, Indicated by a mark (#). Potential N-linked glycosylation sequences are found at the following positions in the neurotoxin-alpha amino acid sequence: N-124 to Q-127 (N-124, S-125, S- 242 to C-245 (N-242, N-243, S-244, C-245).

High identity regions between neurotoxin-alpha, neurotoxin-alpha SV, TNF-alpha, TNF-beta, LT-beta and closely related Fas ligands (these sequences are shown in FIG. 2) 1B. These regions are not limited, and the conserved domains (CD) -I, CD-II, CD-III, CD-IV, CD-V, CD-VI, CD- , CD-IX, CD-X and CD-XI.

Figures 2A and 2B are graphs showing neurotoxin-alpha (SEQ ID NO: 2) and neurotoxin-alpha SV (SEQ ID NO: 19), TNF-alpha (Figure 2A) measured by a "MegAlign" routine that is part of a computer program entitled "DNA * STAR" Beta] (" TNF beta " in Figures 2A and 2B; Genebank Z15026; SEQ ID NO: 4), lymphotoxin-beta And the FAS ligand (" FASL ", GenBank U11821, SEQ ID NO: 6) in the amino acid sequence shown in SEQ ID NO: 2 and 2B. Residues that match the conserved region are indicated by shading.

Figure 3 shows the results of analysis of the neurotoxin-alpha amino acid sequence. Beta, turn and coil regions by estimating the amino acid sequence of SEQ ID NO: 2 using the default parameters of the computer program described above; Hydrophilic and hydrophobic; Amphibolite area; Variable area; Antigen index and surface probability. In the "Antigenic Index-Jameson-Wolf" graph, the position of the region where the highly antigenic region of neurotoxin-alpha, that is, the epitope-containing peptide of the present invention, can be obtained is shown. The antigenic polypeptide comprises an amino acid residue of about Phe-115 to about Leu-147, about Ile-150 to about Tyr-163, about Ser-171 to about Phe-194, about Glu-223 to about Tyr-246, About Ser-271 to about Phe-278.

The data presented in Figure 3 is also presented in Table 1. The columns are denoted by the heading " Res ", " position " and Roman numerals I through XIV. The column heading characterizes the amino acid sequence described in Figure 3 and Table 1 as follows: " Res ": the amino acid residues of SEQ ID NO: 2 and 1A and 1B; &Quot; position ": the position of the corresponding residue within SEQ ID NO: 2 and Figs. 1A and 1B; I: Alpha, region-Garnier-Robson; II: Beta, area - Chou-Fasman; III: Beta, region-gamer-Robson; IV: Beta, area - Chow-Pasman; V: Turn, Area - Gannier - Robson; VI: Turn, Area - Chow-Parsman; VII: Coils, area - Gannier - Robson; VIII: hydrophilic float-kite-dolittle (Kyte Doolittle); IX: Hydrophobicity, also known as Hopp-Woods; X: alpha, amphibolite area - Eisenberg; XI: beta, amphipathic region - Eisenberg; XII: variable region - Karplus-Schlz; XIII: antigenic index - Jameson-Wolf; And XIV: Surface Probability Float - Emini.

Figures 4A, 4B and 4C illustrate the relationship between the neurotoxin-alpha nucleotide sequence measured from the human cDNA deposited at ATCC No. 97768 and the related persons of the invention designated HSOAD55 (SEQ ID NO: 7), HSLAH84 (SEQ ID NO: 8) and HLTBM08 The sequence of the cDNA clone is shown.

Figures 5A and 5B show nucleotide (SEQ ID NO: 18) and deduced amino acid (SEQ ID NO: 19) sequences of the neurotoxin-alpha SV protein. Amino acids 1 to 46 represent the predicted intracellular domain, amino acids 47 to 72 represent the predicted transmembrane domain (double underlined sequence), and amino acids 73 to 266 represent the extracellular domain (remaining sequence). The potential asparagine-linked glycosylation sites are shown in FIGS. 5A and 5B, with asparagine-rich N in the Nutrokine-alpha SV amino acid sequence and the first Nucleotide encoding the Asparagine residue in the Neutrokine-alpha SV Nucleotide sequence Indicated by a pound sign (#). The potential N-linked glycosylation sequences are found at the following positions in the neurotoxin-alpha SV amino acid sequence: N-124 to Q-127 (N-124, S-125, S-126, Q- -223 to C-226 (N-223, N-224, S-225, C-226). The antigenic polypeptide comprises an amino acid sequence of about Pro-32 to about Leu-47, about Glu-116 to about Ser-143, about Phe-153 to about Tyr-173, about Pro-218 to about Tyr-227 About Ala-232 to about Gln-241, about Ile-244 to about Ala-249, and about Ser-252 to about Val-257.

Highly identical regions of identity between neurotoxin-alpha, neurotoxin-alpha SV, TNF-alpha, TNF-beta, LT-beta and closely related Fas ligands (sequences of these sequences are shown in FIG. 2) With an underline. These conserved regions (of neurotoxin-alpha and neurotoxin-alpha SV) are conserved domains (CD) -I, CD-II, CD-III, CD-V, CD- CD-VII, CD-IX, CD-X and CD-XI. Neutrokine-alpha SV does not contain the sequence of CD-IV described in the legend of Figures 1A and 1B.

Additional sequences of APRIL, TNF alpha and LT alpha with the neurotoxin-alpha polypeptide sequence (SEQ ID NO: 2) are shown in FIG. 7A. In Figure 7A, the beta sheet area is marked as shown in Figure 7A legend.

Figure 6 shows an analysis of the neurotoxin-alpha SV amino acid sequence. Beta, turn and coil regions by estimating the amino acid sequence of SEQ ID NO: 19 using the default parameters of the computer program described above; Hydrophilic and hydrophobic; Amphibolite area; Variable area; Antigen index and surface probability. In the " antigen-index-Jameson-Wolf " graph, the position of the highly antigenic region of the neurotoxin-alpha protein (i.e., the region where the epitope-containing peptide of the present invention can be obtained) is displayed. Antigenic polypeptides include, but are not limited to, the amino acid residues from about SEQ ID NO: 19 about Pro-32 to about Leu-47, about Glu-116 to about Ser-143, about Phe-153 to about Tyr-173, about Pro- A polypeptide comprising about Tyr-227, about Ser-252 to about Thr-258, about Ala-232 to about Gln-241, about Ile-244 to about Ala-249 and about Ser-252 to Val- .

The data shown in FIG. 6 can be easily displayed in the form of a table similar to the data shown in Table 1. The exact form of the data described in Figure 6 can be created using the MegAlign component of the DNA * STAR computer sequencing package set as the default parameter. This is the same program used to create Figures 3 and 6 of the present application.

7A. Alpha, and LT-alpha (specifically, the human APRIL polypeptide (SEQ ID NO: 20; amino acid residues 115-250 of ATCC Accession No. AF046888) and the putative ligand- , Amino acid residues 88 to 233 of TNF alpha (SEQ ID NO: 3), and LT alpha (also amino acid residues 62 to 205 of SEQ ID NO: 4 of TNF-beta) of Zinbank Accession No. Z15026) ). It shows the estimated transmembrane domain of neurotoxin-alpha and shows the cleavage site of neurotoxin-alpha as an arrow. The sequence represented by the lines A to H represents the estimated beta-fleet sheet region.

7B. Expression of neurotoxin-alpha mRNA. Northern hybridization is performed using neurotoxin-alpha orf as a probe for screening of blots of poly (A) + RNA (Clontech) from the human tissue type spectrum and for screening of cancer cell lines. The 2.6kb neurotoxin-alpha mRNA is detected at high levels in the placenta, heart, lung, fetal liver, thymus and pancreas. In addition, 2.6kb of neurotoxin-alpha mRNA was detected in HL-60 and K562 cell lines.

8A and 8B. Neutrokine-alpha expression is increased after activation of human mononuclear cells by IFN-gamma. 8A. Flow cytometric analysis of neurokinin-alpha protein expression in mononuclear cells cultured in vitro. Purified mononuclear cells are cultured for 3 days in the presence or absence of IFN-gamma (100 U / ml). Cells were then stained with a neurotoxin-alpha-specific mAb (2E5) (solid line) or an isotype-matched control (IgG1) (dashed line). The comparative results are obtained using purified mononuclear cells from three different donors in three independent experiments. 8B. Neutrokine-alpha-specific TaqMan primers were prepared and used to assess the relative levels of neurotoxin-alpha mRNA expression in unstimulated mononuclear cells and mononuclear cells treated with IFN-gamma (100 U / mL) The nucleotide sequence is as follows: (a) Probe: 5'-CCA CCA GCT CCA GGA GAA GGC AAC TC-3 '(SEQ ID NO: 24); (b) 5 'amplification primer: 5'-ACC GCG GGA CTG AAA ATC T-3' (SEQ ID NO: 25); And (c) 3 'amplification primer: 5'-CAC GCT TAT TTC TGC TGT TCT GA-3' (SEQ ID NO: 26).

9A and 9B. Neutrokine-alpha is an efficacious B lymphocyte stimulating factor. 9A. The biological activity of neurotoxin-alpha is assessed in standard B-lymphocyte co-stimulatory assays using Staphylococcus aureus cowan 1 SAC as an antigenic priming agent. The SAC itself provides a base factor of 1427 +/- 316. Values are recorded as the mean +/- standard deviation of three wells. Similar results are obtained using purified CHO transfectants and recombinant neurotoxin-alpha purified from transiently transfected HEK 293T cells. 9B. Proliferation of tonsil B cells by neurotoxin-alpha and simultaneous stimulation with anti-IgM. Biological assays are performed as described for SAC, except that each well is precoated with a goat anti-human IgM antibody (10 micrograms / mL) in PBS.

10A and 10B. Neutrokine-alpha receptor expression in normal peripheral blood mononuclear cells and tumor cells. 10A. Human peripheral blood nucleated cells are obtained from normal volunteers and separated by density gradient centrifugation. Cells are stained with biotinylated neurotoxin-alpha and then stained with PE-conjugated streptavidin and FITC or PerPC conjugated CD3, CD20, CD14, CD56 and CD66b specific mAbs. Cells are analyzed on a Becton Dicknson FACScan using CellQuest software. The data represents one of four independent experiments. 10B. Neutrokine-alpha, which binds to the histioclast cell line U-937 and melanoma strain IM-9.

11A, 11B and 11C. In vivo efficacy of neurotoxin-alpha administration in BALB / cAnNCR mice. Formalin-fixed spleens are impregnated with paraffin and 5 micrometer sections are stained with hematoxylin and eosin (top panel). The lower panels were obtained from the same animals stained with anti-CD45R (B220) mAbs and incubated with horseradish peroxidase-coupled rabbit anti-rat Ig (mouse adsorbed) and substrate diaminobenzidine tetrahydrochloride (DAB) It is a developed intercept. The slide is stained with Meyer hematoxylin. CD45R (B220) expressing cells appear brown. 11B. Flow cell count analysis of normal (left panel) and neurotoxin-alpha treated (right panel) mice stained with PE-CD45R (B220) and FITC-ThB (Ly6D). 11C. Serum IgM, IgG and IgA levels in normal and neurotoxin-alpha treated mice.

The invention provides isolated nucleic acid molecules comprising a polynucleotide measured by encoding a neurotoxin-alpha polypeptide having the amino acid sequence shown in Figures 1A and 1B (SEQ ID NO: 2) and sequencing the cDNA clone. The nucleotide sequence shown in Figures 1A and 1B (SEQ ID NO: 1) was deposited at the American Type Culture Collection, 10801, Universuty Boulevard, Manassas, Va. 20110-2201 on October 22, 1996 and assigned ATCC Accession No. 97768 Clones of the received HNEDU15 are obtained by sequencing. The deposited clones are contained in the pBluescript SK (-) plasmid [manufacturer: Stratagene, La Jolla, CA].

The invention also provides isolated nucleic acid molecules comprising a polynucleotide determined by encoding a neurotoxin-alpha SV polypeptide having the amino acid sequence shown in Figures 5A and 5B (SEQ ID NO: 19) and sequencing the cDNA clone. The nucleotide sequences described in Figures 5A and 5B (SEQ ID NO: 18) were obtained by sequencing HDPMC52 clones deposited with the American Type Culture Collection on Dec. 10, 1996 and assigned ATCC Accession No. 203518. The deposited clones are contained in the pBluescript SK (-) plasmid [manufacturer: Stratagene, La Jolla, CA].

The neurotoxin-alpha and neurotoxin-alpha SV polypeptides of the invention share sequence homology with the decoy products of human mRNA for TNF-alpha, TNF-beta, LT beta, Fas ligand, APRIL and LT alpha : Figs. 2A, 2B and 7A). As noted above, TNF-alpha plays a role in the regulation of cytotoxicity, necrosis, apoptosis, co-stimulation, proliferation, lymph node formation, immunoglobulin class switching, differentiation, antiviral activity and adsorption molecules and other cytokines and growth factors Is considered to be an important cytokine.

Nucleic acid molecule

Unless otherwise specified, all nucleotide sequences determined by sequencing DNA molecules were determined using an automated DNA sequencer (e.g., Model 373, Applied Biosystems, Ind., Foster City, CA) All amino acid sequences of the polypeptides encoded by the DNA molecules are deduced by decoding the DNA sequences measured as above. Thus, as is known in the art for DNA sequences determined by such automated methods, all nucleotide sequences measured herein may contain some errors. The nucleotide sequence determined by automation is typically at least 90%, more usually at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more accurately determined by other methods including passive DNA sequence analysis well known in the art. Also, as is known in the art, one insertion or deletion in the nucleotide sequence measured relative to the actual sequence results in a frame shift in the decoding of the nucleotide sequence, and thus the predicted amino acid sequence encoded by the measured nucleotide sequence Will be completely different from the amino acid sequence that is actually encoded by the DNA molecule sequenced from the point of insertion or deletion.

A " nucleotide sequence " of a nucleic acid molecule or polynucleotide includes a DNA molecule or a polynucleotide (sequence of a deoxyribonucleotide), and a nucleotide sequence in which the respective thymidine deoxyribonucleotide (T) of the deoxyribonucleotide sequence described above is a ribonucleotide uridine Refers to an RNA molecule or polynucleotide corresponding to a ribonucleotide (A, G, C and U) sequence that has been replaced with a nucleotide (U).

When the information provided herein, for example, the nucleotide sequence of Figures 1A and 1B, is used, standard cloning and screening methods can be used, such as, for example, the method of cloning cDNA using mRNA as a starting material, The nucleic acid molecule of the present invention encoding the polypeptide can be obtained. As an example of the present invention, the nucleic acid molecules described in Figures 1A and 1B (SEQ ID NO: 1) are found in cDNA libraries derived from neutrophils. Expressed sequence tags corresponding to a portion of the neurotoxin-alpha cDNA were also found in kidney, lung, peripheral leukocyte, bone marrow, T cell lymphoma, B cell lymphoma, activated T cells, stomach cancer, smooth muscle, macrophages and spinal cord blood tissue do. It is also possible to use the nucleotide information provided in FIGS. 5A and 5B and to perform a standard cloning and screening method, such as the method of the present invention for encoding a neurotoxin-alpha SV polypeptide, according to a method of cloning cDNA using mRNA as a starting material Nucleic acid molecules can be obtained. As an example of the present invention, the nucleic acid molecules described in Figures 5A and 5B (SEQ ID NO: 18) are found in cDNA libraries derived from primary dendritic cells.

The neurotoxin-alpha plasmid HNEDU 15 deposited as ATCC Accession No. 97768 contains about 285 amino acid residues of the protein, an estimated intracellular domain of about 46 amino acids (Figures 1 to 46 of Figures 1A and 1B (SEQ ID NO: 2) (The amino acid residues from about 47 to about 72 amino acids underlined in Figures 1A and 1B (SEQ ID NO: 2)) of about 26 amino acids and an estimated extracellular domain of about 213 amino acids 1A and 1B (SEQ ID NO: 2)) and contains an open reading frame with an estimated molecular weight of about 31 kDa. The neurotoxin-alpha polypeptide shown in Figures 1A and 1B (SEQ ID NO: 2) is about 20% similar and about 10% identical to the human TNF-alpha available as accession number 339764 from Gene Bank.

The neurotoxin-alpha SV plasmid HDPMC52 deposited as ATCC Accession No. 203518 contains a protein of about 266 amino acid residues, an estimated intracellular domain of about 46 amino acids (Figures 1 to 46 in Figures 5A and 5B (The amino acid residues from about 47 to about 72 amino acids underlined in Figures 5A and 5B (SEQ ID NO: 19)) of about 26 amino acids and the estimated extracellular domain of about 194 amino acids 5A and 5B (SEQ ID NO: 19)) and contains an open reading frame with an estimated molecular weight of about 29 kDa. The neurotoxin-alpha SV polypeptides shown in Figures 5A and 5B (SEQ ID NO: 19) are about 33.9% similar and about 22.0% identical to the human TNF-alpha available from Gene Bank, Accession No. 339764.

As will be appreciated by those skilled in the art, due to the possibility of the sequence analysis errors discussed above, virtually complete neurotoxin-alpha and / or neurotoxin encoded by the deposited cDNA comprising about 285 and 266 amino acids, respectively - alpha SV polypeptides may be slightly shorter. In particular, the measured neurotoxin-alpha and neurotoxin-alpha SV coding sequences are located at nucleotide positions 210-212 of Figures 1A and 1B (SEQ ID NO: 1) and nucleotide positions 64-66 of Figures 5A and 5B Lt; RTI ID = 0.0 > methionine < / RTI > codon that can serve as another start codon for decoding the open reading frame. More typically, the actual open reading frame will contain ± 20 amino acids from the deduced from the first or second methionine codon from the N-terminus shown in Figures 1A and 1B (SEQ ID NO: 1) and Figures 5A and 5B , With a higher probability that there can be anywhere in the range of +/- 10 amino acids. Because the polypeptide domains described herein have been estimated by computer analysis, the extracellular, intracellular, and epidermal domains of the neurotoxin-alpha and neurotoxin-alpha SV polypeptides, according to the analytical criteria used to identify the various action domains It will also be appreciated that the exact " address " For example, the exact positions of the neurotoxin-alpha and neurotoxin-alpha SV extracellular domains in Figures 1A and 1B (SEQ ID NO: 2) and Figures 5A and 5B (SEQ ID NO: 19) (E.g., the address can range from about 1 to about 20 residues, with a higher probability of about 1 to 5 residues). In this case, the start and end points of the transmembrane domain and the extracellular domain were estimated based on the identification of the hydrophobic amino acid sequence at the positions described above, as shown in Figs. 3 and 6 and Table 1. In any case, as further discussed below, the present invention also encompasses the use of the N-terminus of the extracellular domain described herein constituting a soluble form of the extracellular domain of the neurotoxin-alpha and neurotoxin-alpha SV polypeptides A polypeptide having one or more amino acids deleted from the N-terminus and / or the C-terminus of the complete polypeptide.

As indicated, the nucleic acid molecules and polynucleotides of the present invention may be in the form of DNA, including cDNA and genomic DNA obtained, for example, by cloning or produced by synthesis, or in the form of RNA, such as mRNA. The DNA may be double-stranded or double-stranded. The stranded DNA or RNA may be a cipher chain, also known as a sense strand, or a non-cipher strand, also referred to as an antisense strand.

An "isolated" nucleic acid molecule refers to a nucleic acid molecule (DNA or RNA) that has been removed from its natural environment. For example, recombinant DNA molecules contained within a vector are considered isolated for purposes of the present invention. Another example of a separate DNA molecule includes a purified (partially or substantially) DNA molecule in a recombinant DNA molecule or solution maintained in a heterologous host cell. The isolated RNA molecules include RNA transcripts in vivo or in vitro of the DNA molecules according to the invention. However, clones that are members of a library (e.g., a genomic or cDNA library) that have not been separated from another member of the library (e.g., in the form of a homogeneous solution containing the clone and another member of the library) or cells Or a nucleic acid contained in a removed chromosome (e.g., a "dispersed chromosome" such as in a karyotype) is not isolated for the purposes of the present invention. As further discussed herein, isolated nucleic acid molecules according to the present invention can be prepared naturally, recombinantly or synthetically.

The isolated nucleic acid molecule of the present invention may comprise an open reading frame (ORF) with an initiation codon at positions 147-149 in the nucleotide sequence shown in Figures 1A and 1B (SEQ ID NO: 1), alternatively this open reading frame And DNA molecules constituted. In addition, the isolated nucleic acid molecule of the present invention may comprise or alternatively comprise a sequence that is substantially different from the sequence described above, but may comprise DNA molecules encoding the neurotoxin-alpha protein due to the degeneracy of the genetic code . Of course, genetic codes are well known in the art. Thus, it is conventional for those skilled in the art to prepare dextrose variants as described above. In another embodiment, the invention comprises a sequence encoding a neurotoxin-alpha polypeptide having an amino acid sequence encoded by the cDNA contained in the plasmid of ATCC Accession No. 97768, or alternatively comprises a sequence encoding such a sequence ≪ / RTI > Preferably, the nucleic acid molecule comprises a sequence encoding an extracellular domain or a mature or soluble polypeptide sequence of the polypeptide encoded by the cDNA contained in the plasmid of ATCC Accession No. 97768, or alternatively, .

The isolated nucleic acid molecule of the present invention also comprises a DNA molecule comprising an open reading frame (ORF) with an initiation codon at positions 1-3 of the nucleotide sequence shown in Figures 5A and 5B (SEQ ID NO: 18). Also, the isolated nucleic acid molecule of the present invention may comprise, or alternatively consist of, sequences that are substantially different from the sequences described above, or that encode a neurotoxin-alpha SV polypeptide because of the degradation of the genetic code Lt; / RTI > Of course, genetic codes are well known in the art. Thus, it is conventional for those skilled in the art to produce dextrose variants as described above. In another embodiment, the invention includes, or alternatively comprises, a sequence encoding a neurotoxin-alpha SV polypeptide having an amino acid sequence encoded by the cDNA contained in the plasmid of ATCC Accession No. 203518 Thereby providing a separated nucleic acid molecule. Preferably, the nucleic acid molecule comprises a sequence encoding an extracellular domain or a mature or soluble polypeptide sequence of the polypeptide encoded by the cDNA contained in the plasmid of ATCC Accession No. 203518, or alternatively, .

The present invention also provides a nucleic acid molecule having a nucleotide sequence complementary to the nucleotide sequence shown in Figures 1A and 1B (SEQ ID NO: 1) or the nucleotide sequence of the neurotoxin-alpha cDNA contained in the plasmid of ATCC Accession No. 97768 Or, alternatively, provides a separate nucleic acid molecule consisting of such molecules. The present invention also relates to a nucleotide sequence shown in Figures 5A and 5B (SEQ ID NO: 18) or a nucleotide sequence of a neurotoxin-alpha SV cDNA contained in the plasmid of ATCC Accession No. 203518, or a nucleic acid having a sequence complementary to one of these sequences Or alternatively, a separate nucleic acid molecule comprised of such a molecule. Such isolated molecules, especially DNA molecules, include, but are not limited to, probes for genetic mapping by in situ hybridization with chromosomes and neurotoxin-alpha and < RTI ID = 0.0 > And has utility as a probe for detecting the expression of neurotoxin-alpha SV.

In one embodiment, a polynucleotide of the invention comprises, or alternatively consists of, the sequence set forth in SEQ ID NO: 22. The sequence provided as SEQ ID NO: 22 is constructed from several duplicate mouse EST sequences (AI182472, AA422749, AA254047 and AI122485) obtained from Gene Bank. EST sequence to generate a neurotoxin-alpha-like polynucleotide sequence provided as SEQ ID NO: 22. The amino acid sequence obtained from decoding SEQ ID NO: 22 is provided as SEQ ID NO: Fragments, variants and derivatives of the sequences provided as SEQ ID NO: 22 and SEQ ID NO: 23 are also included in the present invention.

In another embodiment, the polynucleotide of the invention comprises or alternatively consists of a sequence encoding the amino acid sequence shown in SEQ ID NO: 27 and / or SEQ ID NO: 28, fragments, variants and derivatives thereof, Nucleotides are also included in the present invention. For example, certain embodiments of the invention relate to polypeptides that are 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acids 68-219 of SEQ ID NO: Or alternatively comprises a polynucleotide consisting of such a sequence. The amino acid sequence obtained from the decoding of SEQ ID NO: 27 is provided as SEQ ID NO: 28. Polypeptides comprising, or alternatively consisting of, the amino acid sequence of SEQ ID NO: 28 and fragments, variants and derivatives of the sequences provided as SEQ ID NO: 28 are also encompassed by the invention. For example, certain embodiments of the invention relate to polypeptides that are 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acids 68-219 of SEQ ID NO: Or alternatively consisting of, such sequences. Nucleic acid molecules having the sequence provided as SEQ ID NO: 27 are obtained using RT-PCR from PBMCs of cyanomologous monkeys (i.e., macaques) using two degenerate primers. In summary, total RNA is prepared from cyanomolacus monkey PBMC using Trizol (vendor: Life Technologies, Inc., Rockville, Md.) According to the manufacturer's protocol. Subsequently, a single-stranded cDNA was synthesized from a cyano-morphological monkey PBMC preparation according to a standard method using oligo-dT primer. Neutrokine-alpha-specific primers are designed based on conserved regions between mouse and human neurokinin-alpha molecules (SEQ ID NOS: 22 and 1, respectively). Subsequently, PCR is performed using the cDNA template in combination with the following two degenerate oligonucleotide primers to prepare a cyanohomolagos monkey neurotoxin-alpha nucleic acid molecule. 5 'primer: 5'-TAC CAG ITG GCI GCC ITG CAA G-3' (SEQ ID NO: 35) and 3 'primer: 5'-GTI ACA GCA GTT TIA IIG CAC C-3' (SEQ ID NO: 36). In the sequence of decoction primers (SEQ ID NOS: 35 and 36), "I" refers to deoxyinosine or dideoxyinosine.

In another embodiment, the polynucleotide of the invention comprises, or alternatively consists of, the sequence shown in SEQ ID NO: 29 and / or the sequence encoding the amino acid sequence set forth in SEQ ID NO: 30, fragments, variants and derivatives thereof . These polynucleotides are also included in the present invention. For example, certain embodiments of the invention relate to polypeptides that are 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acids 68-219 of SEQ ID NO: Or alternatively comprises a polynucleotide consisting of such a sequence. The amino acid sequence obtained from the decoding of SEQ ID NO: 29 is provided as SEQ ID NO: 30. Polypeptides comprising, alternatively consisting of, the amino acid sequence of SEQ ID NO: 30 and fragments, variants and derivatives of the sequences provided as SEQ ID NO: 29 and SEQ ID NO: 30 are also encompassed by the present invention. For example, certain embodiments of the invention relate to polypeptides that are 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acids 68-219 of SEQ ID NO: Or alternatively consisting of, such sequences. The nucleic acid molecule having the sequence provided as SEQ ID NO: 29 is obtained using RT-PCR from rhesus monkey PBMC using two degenerative primers. In summary, total RNA is prepared from Rhesus monkey PBMC using trizol (Life Technologies, Inc., Rockville, Md.) According to the manufacturer's protocol. Subsequently, a single-stranded cDNA is synthesized from a Rhesus monkey PBMC preparation according to a standard method using oligo-dT primer. Neutrokine-alpha-specific primers are designed based on conserved regions between mouse and human neurokinin-alpha molecules (SEQ ID NOS: 22 and 1, respectively). Then, PCR is carried out using the cDNA template in combination with the following two degenerate oligonucleotide primers to prepare a Rhesus monkey neurotoxin-alpha nucleic acid molecule. 5 'primer: 5'-TAC CAG ITG GCI GCC ITG CAA G-3' (SEQ ID NO: 35) and 3 'primer: 5'-GTI ACA GCA GTT TIA IIG CAC C-3' (SEQ ID NO: 36). In the sequence of decoction primers (SEQ ID NOS: 35 and 36), "I" refers to deoxyinosine or dideoxyinosine.

The present invention also provides a nucleic acid molecule having a nucleotide sequence associated with a broad region of SEQ ID NO: 1 and SEQ ID NO: 18, measured from the following related cDNA clones: HSOAD55 (SEQ ID NO: 7), HSLAH84 (SEQ ID NO: 8) and HLTBM08 .

The invention also relates to a nucleic acid molecule encoding a portion of the nucleotide sequence described herein and to a fragment of the isolated nucleic acid molecule described herein. In one embodiment, the invention provides a polynucleotide having a nucleotide sequence representing a portion of SEQ ID NO: 1 consisting of nucleotides 1 to 1001 in position 1 of SEQ ID NO: 1. In another aspect, the invention provides a polynucleotide having a nucleotide sequence representing a portion of SEQ ID NO: 18 consisting of positions 1 to 798 of SEQ ID NO: 18.

The invention also relates to fragments of nucleic acid molecules (i. E., Polynucleotides) as described herein. For example, the nucleotide sequence of the cDNA contained in the plasmid of ATCC Accession No. 97768, the nucleotide sequence encoding the polypeptide encoded by the cDNA contained in the plasmid of ATCC Accession No. 97768, the nucleotide sequence of SEQ ID NO: 1, the nucleotide sequence of SEQ ID NO: 2 The nucleotide sequence encoding the polypeptide encoded by the cDNA contained in the plasmid of the ATCC Accession No. 203518, the nucleotide sequence encoding the polypeptide encoded by the nucleotide sequence of SEQ ID NO: 18, the nucleotide sequence encoding the polypeptide encoded by the cDNA contained in the plasmid of the ATCC Accession No. 203518, A nucleotide sequence encoding a polypeptide sequence of SEQ ID NO: 20 or a fragment of a nucleic acid molecule having a complementary strand thereof is at least 15 nt in length, preferably at least 20 nt or at least 25 nt in length, more preferably at least 30 nt in length, Preferably 40, 50 and means 100, 150, 200, 250, 300, 325, 350, 375, 400, 450 or at least 500 nt fragment. These fragments have numerous uses including, but not limited to, diagnostic probes and primers as discussed herein. Of course, the larger fragment, e. G., A fragment having a length of 501 to 1500 nt, may contain the nucleotide sequence of the cDNA contained in the plasmid of ATCC Accession No. 97768, the nucleotide sequence of SEQ ID NO: 1, the cDNA contained in the plasmid of ATCC Accession No. 203518 And the nucleotide sequence of SEQ ID NO: 18, are useful in accordance with the present invention, as well as the corresponding fragments in all cases. Preferred nucleic acid fragments of the present invention are the nucleic acid fragments of the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides identified in Figures 1A and 1B (SEQ ID NO: 2) and Figures 5A and 5B (SEQ ID NO: 19, respectively) Or a nucleic acid molecule encoding an epitope-containing portion, or, alternatively, a polypeptide consisting of such portions. Polypeptides encoded by these polynucleotide fragments are also included in the present invention.

A nucleotide sequence of SEQ ID NO: 27, a nucleotide sequence of SEQ ID NO: 29, a nucleotide sequence encoding the polypeptide sequence of SEQ ID NO: 23, a nucleotide sequence encoding the polypeptide sequence of SEQ ID NO: 28, a nucleotide sequence of SEQ ID NO: A nucleotide sequence encoding a polypeptide sequence of SEQ ID NO: 30 or a fragment of a nucleic acid molecule having a complementary strand thereof is at least 15 nt in length, preferably at least 20 nt in length, more preferably at least 30 nt in length, Means a fragment of at least 40, 50, 100, 150, 200, 250, 300, 325, 350, 375, 400, 450 or 500 nt. These fragments have numerous uses including, but not limited to, many uses including diagnostic probes and primers as discussed herein. Of course, a larger fragment, e. G., A fragment having a length of 501 to 1500 nt, may also comprise a nucleotide sequence of SEQ ID NO: 21, a nucleotide sequence of SEQ ID NO: 22, a nucleotide sequence of SEQ ID NO: 27, a nucleotide sequence of SEQ ID NO: Encoding nucleotide sequence, a nucleotide sequence encoding the polypeptide sequence of SEQ ID NO: 28, a nucleotide sequence encoding the polypeptide sequence of SEQ ID NO: 30, or a complementary strand thereof, but in most cases corresponding to the corresponding fragments Do. Polypeptides encoded by these polynucleotide fragments are also included in the present invention.

Representative examples of the neurotoxin-alpha polynucleotide fragments of the present invention include nucleotides 1 to 50, 51 to 100, 101 to 146, 147 to 200, 201 to 250, 251 300 to 300, 301 to 350, 351 to 400, 401 to 450, 451 to 500, 501 to 550, 551 to 600, 600 to 650, 651 to 700, 701 to 750, 751 to 800, 800 to 850, 851 to 900, 901 to 950, 951 to 1000, 1001 to 1050 and / 1082, a complementary strand thereof, or a cDNA contained in the plasmid of ATCC Accession No. 97768, or alternatively, a fragment consisting of such a sequence. In the context of the present invention, " about " includes the range specifically mentioned and includes ranges as large as or smaller than nucleotides of several (5, 4, 3, 2, 1) at either or both ends.

Representative examples of the neurotoxin-alpha SV polynucleotide fragments of the present invention include nucleotides about 1 to 50 nucleotides, 51 to 100 nucleotides, 101 to 150 nucleotides, 151 to 200 nucleotides, 201 to 250 nucleotides, 251 to 300, 301 to 350, 351 to 400, 401 to 450, 451 to 500, 501 to 550, 551 to 600, 600 to 650, 651 Or a cDNA contained in the plasmid of SEQ ID NO: 701 to 750, 751 to 800, 800 to 850 and / or 851 to 900, the complementary strand thereof, or the ATCC Accession No. 203518, Alternatively, fragments comprising such sequences are included. In the context of the present invention, " about " includes the range specifically mentioned and includes ranges as large as or smaller than nucleotides of several (5, 4, 3, 2, 1) at either or both ends.

In certain preferred embodiments, polynucleotides of the invention comprise nucleotide residues 571 to 627, 580 to 627, 590 to 627, 600 to 627, 610 to 627, 571 to 620 580 to 620, 590 to 620, 600 to 620, 571 to 610, 580 to 610, 590 to 610, 571 to 600, 580 to 600 and / Or 571 to 590, or alternatively consists of such residues.

In certain other preferred embodiments, the polynucleotide of the invention comprises a nucleotide residue of SEQ ID NO: 18

Or alternatively consists of such residues.

In certain further preferred embodiments, the polynucleotide of the invention comprises a nucleotide residue of SEQ ID NO: 1

Or alternatively consists of such residues.

In a further preferred embodiment, the polynucleotide of the invention comprises a nucleotide residue of SEQ ID NO: 1

Or alternatively consists of such residues.

Preferably, the polynucleotide fragment of the invention encodes a polypeptide that demonstrates neurotoxin-alpha and / or neurotoxin-alpha SV functional activity. A polypeptide demonstrating " functional activity " means a polypeptide capable of exhibiting at least one known functional activity associated with a full-length and / or secreted neurotoxin-alpha polypeptide and / or a neurotoxin-alpha SV polypeptide. Such functional activities include, but are not limited to, biological activity (eg, ability to stimulate B cell proliferation, survival, differentiation and / or activation), antigenic [anti-neurotoxin-alpha and / or anti- Ability to bind (or compete with) the alpha-LV antibody (or compete with the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides for binding), immunogenicity (ability to bind neurotoxin-alpha and / or neurotoxin- alpha SV polypeptides The ability to form multimers with the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides of the present invention and the ability to form multimers with respect to the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides ≪ / RTI > receptor or ligand.

In a further specific embodiment, the polynucleotide fragment of the invention comprises an amino acid sequence of amino acid 1 to 46 of SEQ ID NO: 2, an estimated transmembrane domain (amino acids 47 to 72 of SEQ ID NO: 2) (Amino acids 73-285 of SEQ ID NO: 2) or putative TNF conserved domains (amino acids 191-284 of SEQ ID NO: 2), or alternatively encodes a polypeptide consisting of these domains. In a further embodiment, the polynucleotide fragment of the invention encodes a polypeptide comprising, or alternatively consisting of, any combination of 1, 2, 3 or 4 of the domains described above. Polypeptides encoded by these polynucleotides are also encompassed by the present invention.

In a further particular embodiment, the polynucleotide fragment of the present invention comprises the putative intracellular domain (amino acids 1 to 46 of SEQ ID NO: 19), the putative transmembrane domain (amino acids 47 to 72 of SEQ ID NO: 19) of neurotoxin alpha SV, (Amino acids 73 to 266 of SEQ ID NO: 19) or the putative TNF conserved domain (amino acids 172 to 265 of SEQ ID NO: 19), or alternatively encodes a polypeptide consisting of these domains. In a further embodiment, the polynucleotide fragment of the invention encodes a polypeptide comprising, or alternatively consisting of, any combination of 1, 2, 3 or 4 of the domains described above. Polypeptides encoded by these polynucleotides are also included in the present invention.

In another embodiment, the polynucleotide fragment of the present invention comprises residues Met-1 to Lys-113, Leu-114 to Thr-141, Ile-142 to Lys-160, Gly-161 to Gln- 199 to Ala-248 and Gly-250 to Leu-285, or alternatively consists of such polynucleotides. In addition, polynucleotides encoding two, three, four, five, or more combinations of these amino acid sequences are also included in the present invention. Polypeptides encoded by these polynucleotides are also included in the present invention.

In another embodiment, the polynucleotide fragment of the present invention comprises residues Met-1 to Lys-113, Leu-114 to Thr-141, Gly-142 to Gln-179, Val-180 to Ala- 230 to Leu-266, or alternatively consists of such polynucleotides. In addition, polynucleotides encoding two, three, four, five, or more combinations of these amino acid sequences are also included in the present invention. Polypeptides encoded by these polynucleotides are also included in the present invention.

In another embodiment, the polynucleotide fragment of the present invention comprises at least one of the residues Met-1 to Lys-106, Leu-107 to Thr-134, Glu-135 to Asn-165, Ile-167 to Lys- Or alternatively consists of such a polynucleotide encoding a polynucleotide encoding an amino acid sequence selected from the group consisting of Gln-185 to Gln-224, Val-225 to Ala-272 and Gly-273 to Leu-309. In addition, polynucleotides encoding two, three, four, five, or more combinations of these amino acid sequences are also included in the present invention. Polypeptides encoded by these polynucleotides are also included in the present invention.

In another embodiment, the polynucleotide fragment of the present invention comprises residues Tyr-1 to Lys-47, Leu-48 to Thr-75, Ile-76 to Lys-94, Gly-95 to Gln- 133 to Ala-182 and Gly-183 to Ala-219, or alternatively consists of such polynucleotides. In addition, polynucleotides encoding two, three, four, five, or more combinations of these amino acid sequences are also included in the present invention. Polypeptides encoded by these polynucleotides are also included in the present invention.

In another embodiment, the polynucleotide fragment of the present invention comprises residues Tyr-1 to Lys-47, Leu-48 to Thr-75, Ile-76 to Lys-94, Gly-95 to Gln- 133 to Ala-182 and Gly-183 to Ala-219, or alternatively consists of such polynucleotides. In addition, polynucleotides encoding two, three, four, five, or more combinations of these amino acid sequences are also included in the present invention. Polypeptides encoded by these polynucleotides are also included in the present invention.

In another embodiment, the polynucleotide of the invention comprises, or alternatively consists of, the sequence set forth in SEQ ID NO: 21. The sequence described as SEQ ID NO: 21 encodes a polypeptide consisting of the initiation methionine residues linked to residues Ala-134 to Leu-285 of the neurotoxin-alpha polypeptide sequence described as SEQ ID NO: 2. Polypeptides encoded by these polynucleotides are also included in the present invention.

In a further particular preferred embodiment, the polynucleotide of the invention comprises a nucleotide residue of SEQ ID NO: 21

Or alternatively consists of such residues. Polypeptides encoded by these polynucleotides are also included in the present invention.

Thus, certain embodiments of the present invention encompass amino acid sequences of beta-betit sheet regions A, A ', B, B', C, D, E, F, G, or H described in Figures 7A and 6, And more particularly to a polynucleotide encoding a polypeptide consisting of such a sequence. A further aspect of the present invention is that a combination of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the beta-pleated sheet areas AH described in Figures 7A and 6, And in particular to polynucleotides encoding such neurotoxin-alpha polypeptide. A further preferred aspect of the invention is the use of the neurotoxin-alpha amino acid sequence of the beta-fit sheet regions A, A ', B, B', C, D, E, F, G or H described in Figures 7A and 6 Or alternatively consists of such sequences. A further aspect of the invention is that the beta, beta, beta, beta, alpha, alpha, alpha, beta, , Alternatively, a neu- trocin-alpha polypeptide of this combination.

In certain other preferred embodiments, polynucleotides of the invention comprise nucleotide residues 34-57, 118-123, 133-141, 151-159, 175-216, 232-255, 280-315, 328-357, 370 -393 and / or 430-456, or alternatively consists of such residues. Polypeptides encoded by these polynucleotides are also included in the present invention. These polynucleotide and polypeptide fragments correspond to the putative beta-fleeting sheet region described in Figure 7A. In certain embodiments, a polynucleotide of the invention comprises a polynucleotide sequence that encodes 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the beta-fitsheet domains described above, %, 96%, 97%, 98% or 99% identical or more of the same polynucleotide sequence. The present invention also encompasses polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these polynucleotide sequences are also encompassed by the present invention. In another aspect, the invention includes polynucleotides that hybridize to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the beta-fitsheet regions described above under stringent hybridization conditions Lt; RTI ID = 0.0 > nucleic acid < / RTI > The meaning of the term " stringent conditions " as used herein is set forth below.

In a further preferred embodiment, the polynucleotides of the invention comprise nucleotide residues 576-599, 660-665, 675-683, 693-701, 717-758, 774-803, 822-857, 870-899, 912 -935 and / or 972-998, or alternatively consists of such residues. Polypeptides encoded by these polynucleotide fragments are also included in the present invention. These polynucleotide and polypeptide fragments correspond to the putative beta-fleeting sheet region described in Figure 7A.

In another further preferred embodiment, the polynucleotides of the invention comprise nucleotide residues 457-462, 472-480, 490-498, 514-555, 571-600, 619-654, 667-696, 699-732, / Or 769-795, or alternatively consists of such residues. Polypeptides encoded by these polynucleotide fragments are also included in the present invention. These polynucleotide and polypeptide fragments correspond to the putative beta-fleeting sheet region described in Figure 7A.

In yet another preferred embodiment, polynucleotides of the invention comprise nucleotide residues 124-129, 139-147, 157-165, 181-222, 238-267, 286-321, 334-363, 376-399 And / or 436-462, or alternatively consists of such residues. Polypeptides encoded by these polynucleotide fragments are also included in the present invention. These polynucleotide and polypeptide fragments correspond to the putative beta-fleeting sheet region described in Figure 7A. Polypeptides comprising, or alternatively consisting of, these amino acid sequences in combination with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of these regions are encompassed by the present invention.

The relative positions of several intron / exon boundaries for mouse neurotoxin-alpha (SEQ ID NO: 22 and SEQ ID NO: 23) were determined based on sequencing of mouse genomic DNA. The apparent second exon (sometimes referred to as "exon 2") from the 5 'end of the murine neurotoxin-alpha genomic clone consists of Tyr-187 to Gln-222 of the sequence described in SEQ ID NO: 23. The apparent third exon (sometimes referred to as "exon 3") from the 5 'end of the mouse neurotoxin-alpha genomic clone includes Val-223 to Gly-273 of the sequence described in SEQ ID NO: 23.

Thus, in one embodiment, the invention provides a polynucleotide encoding, or alternatively comprising, the polypeptide consisting of the amino acid sequence of residues Tyr-187 to Gln-222 in SEQ ID NO: 23. The present invention also provides a polynucleotide sequence that is 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a polynucleotide sequence encoding the murine neurotoxin-alpha polypeptide described above Or, alternatively, to a nucleic acid molecule comprising such a sequence. The present invention also encompasses polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and / or polynucleotide sequences are also encompassed by the present invention.

In another embodiment, the invention provides polynucleotides encoding, or alternatively encoding, a polypeptide consisting of the amino acid sequence of residues Val-223 to Gly-273 in SEQ ID NO: 23. The present invention also provides a polynucleotide sequence that is 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a polynucleotide sequence encoding the murine neurotoxin-alpha polypeptide described above Or, alternatively, to a nucleic acid molecule comprising such a sequence. The present invention also encompasses polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and / or polynucleotide sequences are also encompassed by the present invention.

In addition, the relative positions of the intron / exon boundaries corresponding to human neurokinin-alpha (SEQ ID NO: 1 and SEQ ID NO: 2) were determined based on the sequence of mouse and human neurokinin-alpha polypeptide sequences. The apparent second exon (sometimes referred to as "exon 2") from the 5 'end of human neurokinin-alpha consists of Tyr-163 to Gln-198 of the sequence described in SEQ ID NO: 2. The apparent third exon (sometimes referred to as "exon 3") from the 5 'end of the human neurokinin-alpha consists of Val-199 to Gly-249 of the sequence described in SEQ ID NO: 2.

Thus, in one embodiment, the invention provides polynucleotides encoding, or alternatively encoding, a polypeptide consisting of the amino acid sequence of residues Tyr-163 to Gln-198 in SEQ ID NO: 2. The invention also encompasses polynucleotide sequences that are 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a polynucleotide sequence encoding the neurotoxin-alpha polypeptide described above Or, alternatively, consists of such sequences. The present invention also encompasses polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and / or polynucleotide sequences are also encompassed by the present invention.

In another embodiment, the invention provides polynucleotides encoding, or alternatively encoding, a polypeptide consisting of the amino acid sequence of residues Val-199 to Gly-249 in SEQ ID NO: 2. The invention also encompasses polynucleotide sequences that are 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a polynucleotide sequence encoding the neurotoxin-alpha polypeptide described above Or, alternatively, consists of such sequences. The present invention also encompasses polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and / or polynucleotide sequences are also encompassed by the present invention.

The functional activity of the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides and fragments, variant derivatives and analogs thereof can be assayed by a variety of methods as described herein and well-known in the art.

(S) and / or neurotoxin-alpha SV receptor (s) on B cells, or a combination of the neurotoxin-alpha receptor (s) and / or neurotoxin-alpha SV receptor In one embodiment that tests for the ability to bind or compete with the full-length neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides for binding, there may be mentioned, but not limited to, radioimmunoassays, ELISAs ), "Sandwich" immunoassay, immunoassay assay, gel diffusion settling, immunodiffusion assay, in situ immunoassay (using colloidal gold, enzyme or radioactive isotope labels), western blot, precipitation reaction, Including competitive and non-competitive assay systems using techniques such as gel electrophoresis (eg, gel agglutination assays, erythrocyte agglutination assays), complement fixation assays, immunofluorescence assays, protein A assays and immuno electrophoresis assays, There can be a number of immunoassay known. In one embodiment, antibody binding is detected by detecting the label in the first antibody. In another embodiment, the first antibody is detected by detecting binding of the second antibody or reagent to the first antibody. In a further embodiment, the second antibody is labeled. Many means for detecting binding in immunoassays are known in the art and are within the scope of the present invention.

In another embodiment, when identifying a neurotoxin-alpha and / or neurotoxin-alpha SV ligand and evaluating the ability of the polypeptide fragments, variants or derivatives of the invention to massimize the oligonucleotides, reduction and non-reducing gel chromatography, Binding can be assayed by means known in the art, such as affinity chromatography and affinity blotting (see generally Phizicky, E. et al., 1995, Microbiol. Rev. 59: 94-123). In another embodiment, the physiological correlations of the binding (signal transduction) of neurotoxin-alpha and / or neurotoxin-alpha SV to its substrate can be assayed.

Also, assays described herein (see Examples 6 and 7) and those described in the art are routinely applied to detect and quantify neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides and fragments, variants, It is believed that the analog stimulates B cell proliferation, differentiation and / or activation in the case of neurotoxin-alpha and / or neurotoxin-alpha SV-associated biological activities (e.g., neurotoxin-alpha and / or neurotoxin- ≪ / RTI > inhibiting and / or prolonging B cell survival in vitro or in vivo).

Other methods are well known to those skilled in the art and these methods are within the scope of the present invention.

In a further embodiment, the polynucleotides of the present invention comprise, or alternatively encode, a polypeptide consisting of such properties, the neurotoxin-alpha and neurotoxin-alpha SV. In this regard, preferred embodiments of the present invention are directed to alpha-helical and alpha-helical forming regions (" alpha-region "), beta-sheet and beta-sheet forming regions (&Quot; beta-region "), a turn and turn-forming region (" turn- region "), a coil and coil- Surface-forming regions, and high antigen-index regions, or alternatively comprises fragments of such regions.

It is believed that one or more of the beta-fleet sheet regions of neurotoxin-alpha described in Figure 7 is important for the interaction between dimerization and neurotoxin-alpha and its ligands.

Specific preferred areas in this regard are described in FIG. 3 (Table 1). The data described in Figure 3 and Table 1 are simply different in form and results from those obtained when analyzing the amino acid sequence of SEQ ID NO: 2 using the default parameters of the DNA * STAR computer algorithm and simply provide the same results.

The preferred regions described above in Figure 3 and Table 1 include, but are not limited to, regions of the above type identified by analysis of the amino acid sequences described in Figures 1A and 1B. Such preferred regions, as described in FIG. 3 and Table 1, include the Ganner-Robson alpha-region, the beta-region, the turn-region and the coil-region, the Chow-Parsmann alpha-region, Eosinberg alpha-and beta-amphipathic regions, kaplus-Schultz flexible regions, eminic surface-forming regions, and the Jamesson-Wolf region of high antigenic index. A highly preferred polynucleotide in this regard includes a region of neurotoxin-alpha and / or neurotoxin-alpha SV that combines several structural features, such as several (e.g., 1, 2, 3 or 4) Or alternatively, a polynucleotide encoding a polypeptide consisting of such regions. Polypeptides encoded by these polynucleotides are also included in the present invention.

In addition, the data set forth in columns VIII, IX, XIII and XIV of Table 1 can be routinely used to determine the area of neurotoxin-alpha that exhibits a high degree of latency for antigenicity (column VIII in Table 1 Column IX of Table 1 represents the hydrophobicity according to the Hope-Woods; Column XIII of Table 1 represents the antigenic index of Jameson-Wolf; Column XIV of Table 1 represents the hydrophilic surface according to the eminyi Probability). IX, XIII, and / or IV by selecting a value that indicates the region of the polypeptide that is likely to be exposed to the surface of the polypeptide in an environment where antigen recognition may occur in the initiation of an immune response Measure from the provided data. The data provided in Figure 6 can also be conventionally provided in a similar table format by simply examining the amino acid sequences described in Figure 6 (SEQ ID NO: 19) using modules and algorithms of DNA * STAR that are fixed with default parameters. As noted above, the amino acid sequence provided in Figure 6 can also be used to determine the region of the neurotoxin-alpha that is indicative of a high degree of antigenicity for the provided antigenicity (as in Figure 6) or as a table (as in Table 1) .

Additional preferred nucleic acid fragments according to the present invention comprise, or alternatively comprise, nucleic acid molecules consisting of, or alternatively consisting of, sequences encoding at least one epitope-containing portion of the neurotoxin-alpha. In particular, such nucleic acid fragments of the invention comprise an amino acid sequence of about Phe-115 to about Leu-147, about Ile-150 to about Tyr-163, about Ser-171 to about Phe-194, about Glu- Tyr-246 and about Ser-271 to about Phe-278, or alternatively comprises a nucleic acid molecule consisting of such sequence. As used herein, " about " means a range that is as large as or slightly larger than the range specifically mentioned and amino or carboxy terminus, or 5, 4, 3, 2 or 1. Polypeptides encoded by these nucleic acid molecules are also included in the present invention. A polypeptide fragment containing an antigenic epitope of neurotoxin-alpha can be readily determined by one of ordinary skill in the art using the assay described above for the Jamesson-Wolf antigen index as described in FIG. Methods for measuring such other epitope-containing portions of neurotoxin-alpha are described in detail below.

Additional preferred nucleic acid fragments according to the present invention comprise or alternatively comprise a nucleic acid molecule comprising a sequence encoding at least one epitope-containing portion of a neurotoxin-alpha SV. In particular, the nucleic acid fragment of the present invention comprises about amino acid sequence about Pro-32 to about Leu-47, about Glu-116 to about Ser-143, about Phe-153 to about Tyr-173, about Pro-218 to about Encoding a polypeptide selected from the group consisting of Tyr-227, about Ser-252 to about Thr-258, about Ala-232 to about Gln-241, about Ile-244 to about Ala-249 and about Ser-252 to about Vla-257 Or, alternatively, comprises nucleic acid molecules comprised of such sequences. As used herein, " about " means a range that is as large as or slightly larger than the range specifically mentioned and amino or carboxy terminus, or 5, 4, 3, 2 or 1. Polypeptides encoded by these nucleic acid molecules are also included in the present invention. Polypeptide fragments containing an antigenic epitope of neurotoxin-alpha SV can be readily determined by those skilled in the art using the assay described above for the Jamesson-Wolf antigen index as described in FIG. Methods for measuring such other epitope-containing portions of the neurotoxin-alpha SV are described in detail below.

In certain embodiments, the polynucleotides of the present invention have lengths of 100,000 kb, 50,000 kb, 10,000 kb, 1,000 kb, 500 kb, 400 kb, 350 kb, 300 kb, 250 kb, 200 kb, 175 kb, 150 kb, 125 kb , 100 kb, 75 kb, 50 kb, 40 kb, 30 kb, 25 kb, 20 kb, 15 kb, 10 kb, 7.5 kb or 5 kb.

In a further embodiment, the polynucleotide of the invention comprises at least 15, at least 30, at least 50, at least 100, at least 250, at least 500 or at least 1000 consecutive nucleotides of the neurotoxin-alpha coding sequence 500 kb, 250 kb, 200 kb, 150 kb, 100 kb of genomic DNA flanking the 5 'or 3' coded nucleotides described in Figures IA and IB (SEQ ID NO: 1) or Figures 5A and 5B kb, 75 kb, 50 kb, 30 kb, 25 kb, 20 kb, 15 kb, 10 kb or 5 kb or the like. In a further embodiment, the polynucleotide of the invention comprises at least 15, at least 30, at least 50, at least 100, at least 250, at least 500 or at least 1000 consecutive nucleotides of the neurotoxin-alpha coding sequence It does not include all or part of the neurotoxin-alpha intron. In another embodiment, the nucleic acid comprising a neurotoxin-alpha coding sequence does not contain a sequence of a genomic flanking gene (i.e., 5 'or 3' of the neurotoxin-alpha gene in the genome). In another embodiment, polynucleotides of the invention contain a coding sequence of 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2 or more genomic flanking genes Do not.

In another embodiment, the invention provides a nucleic acid molecule of the invention as described above, for example a sequence complementary to the coding and / or non-coding sequence described in Figures IA and IB (SEQ ID NO: 1), a deposit of ATCC Accession No. 97768 The sequence of the cDNA clone contained in water, the sequence complementary to the coding sequence and / or non-coding sequence deposited in Figures 5A and 5B (SEQ ID NO: 18), the sequence of the cDNA clone contained in the deposit of ATCC Accession No. 203518, A sequence complementary to the coding sequence and / or non-coding sequence described in SEQ ID NO: 21 and / or non-coding sequence (i.e., transcribed, A sequence complementary to the coding sequence and / or the non-coding sequence, a coding sequence shown in SEQ ID NO: 29 and / or a sequence complementary to the non-coding sequence, or fragments thereof (such as an open reading frame or fragment thereof) Of the polynucleotide It provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions minutes. &Quot; Strict hybridization conditions " include 50% formamide, 5 x SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate and 20 micrograms / ml < / RTI > denatured and truncated salmon sperm DNA at 42 < 0 > C overnight, followed by washing the filter at about 65 [deg.] C in 0.1x SSC.

A polynucleotide that hybridizes to a " portion " of a polynucleotide is at least about 15 nucleotides (nt) in the reference polynucleotide, preferably at least about 20 nt, more preferably at least about 30 nt, even more preferably at least 30- Means a polynucleotide (DNA or RNA) that hybridizes to 70 or 80-150 nucleotides. These include, but are not limited to, diagnostic probes and primers as described above and described in more detail below. By way of example, a portion of a polynucleotide that is "about 20 or more in length in length" includes a reference polynucleotide (eg, a sequence of one or both of the deposited cDNAs, a complementary strand of the nucleotide sequence described in Figures IA and IB (SEQ ID NO: 1) The complementary strand of the nucleotide sequence described in Figures 5A and 5B (SEQ ID NO: 18), the complementary strand of the nucleotide sequence described in SEQ ID NO: 21, the complementary strand of the nucleotide sequence described in SEQ ID NO: 22, the complementary strand of the nucleotide sequence described in SEQ ID NO: (I. E., 5, 4, 3, 2, 1 or 0) at one or both ends of the nucleotide sequence of the nucleotide sequence of the nucleotide sequence shown in SEQ ID NO: 29) do. Of course, the 3 'terminal Poly (A) tracks of the polyA sequences (e.g., the neurotoxin-alpha cDNA described in Figures 1A and 1B (SEQ ID NO: 1), the neurotoxin- alpha SVs described in Figures 5A and 5B terminus poly (A) of the cDNA or the 3 'terminal poly (A) track of the neurotoxin-alpha SV cDNA described in SEQ ID NO: 22) or a complementary sequence of the T (or U) Are not included in the polynucleotides of the present invention used to hybridize to a portion of the nucleic acid of the present invention. Since such polynucleotides hybridize to nucleic acid molecules containing poly (A) contiguous sequences or their complementary sequences (e. G., Double-stranded cDNA clones generated using oligo dT as a primer).

As mentioned, the nucleic acid molecules of the invention encoding a neurotoxin-alpha polypeptide or a neurotoxin-alpha SV polypeptide may include, but are not limited to, polynucleotides encoding the amino acid sequences of the individual extracellular domains of the polypeptide Additional sequences such as the coding sequence for the extracellular domain of the individual polypeptide and the intracellular and transmembrane domain sequences or the coding sequence for the pre-, pro- or prepro-protein sequence, Or may contain the coding sequence of the individual extracellular domain of the nonexistent polypeptide.

Also encoded by the nucleic acid of the present invention may include but are not limited to additional non-coded sequences including introns and non-cryptic 5 ' and 3 ' sequences (e.g., splicing such as ribosome binding and stability of mRNA, A sequence that is transcribed and compared that plays a role in mRNA processing and transcription, including a < RTI ID = 0.0 > dynylated < / RTI > signal) and additional coding sequences that encode additional amino acids, such as providing additional functions.

Thus, a sequence encoding a polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of the fused polypeptide. In a particularly preferred embodiment of this aspect of the invention, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in the pQE vector (QIAGEN, Inc., 9259, 9459 Chatteward, Ithone Avenue, CA) For example, Gentz et al., Proc. Natl. Acad. Sci. USA 86: 821-824, hexa-histidine provides convenient purification of the fusion protein. The "HA" tag corresponds to an epitope derived from the influenza hemagglutinin protein described in Wilson et al., Cell 37: 767 (1984), another peptide useful for purification. As discussed below, other such fusion proteins include neurotoxin-alpha or neurotoxin-alpha SV polypeptide polypeptides fused to Fc at the N- or C-terminus.

The invention also relates to variants of the nucleic acid molecules of the invention that encode a portion, derivative or analog of the Neutrokine-alpha or Neutrokine-alpha SV polypeptide of SEQ ID NO: 2. Variants can occur naturally, such as natural allelic variants. &Quot; Allelic variants " are one of several different forms of genes occupying a given locus in an organism's chromosome (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)). Non-native variants include, but are not limited to, oligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis, site directed mutagenesis (see for example Carter et al., Nucl. Acids Res. 13: 4331 (1986); (See, for example, Wells et al., Gene 34: 315 (1985)), cassette mutagenesis (see e.g., Zoller et al., Nucl. Acids Res. 10: 6487 al., Phiols. Trans. R. Soc. London SerA 317: 415 (1986)).

Such variants include those produced by nucleotide substitution, deletion or addition. Substitution, deletion or addition may involve one or more nucleotides. Variations can be made in the cryptographic domain, in the non-cryptographic domains, or both. Variations in the coding region can lead to conserved or unstored amino acid substitutions, deletions or additions. Particularly preferred among these are silent substitutions, additions and deletions that do not alter the properties and activity of the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides or portions thereof. Also particularly preferred in this regard are conservative substitutions.

Additional embodiments of the present invention may include one or more, no more than 50, more preferably no more than 40, even more preferably no more than 30, even more preferably no more than 20, 10-20, 5-10, Alpha and / or neurotoxin-alpha SV polypeptides having an amino acid sequence containing 1-5, 3-5, or 1-3 conserved amino acid substitutions (e.g., the neurotoxin-alpha and / / RTI > and / or a neurotoxin-alpha SV polypeptide fragment). Of course, in order of increasing preference, neurotoxin-alpha and / or neurotoxin-binding proteins having amino acid sequences containing 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conserved amino acid substitutions, A polynucleotide encoding the amino acid sequence of the alpha SV polypeptide is highly preferred.

Additional aspects include (a) a nucleotide sequence encoding a neurotoxin-alpha polypeptide having the complete amino acid sequence set forth in Figures IA and IB (i.e., positions 1 to 285 of SEQ ID NO: 2), (b) a N-terminal methionine A nucleotide sequence encoding a neurotoxin-alpha polypeptide having the complete amino acid sequence of SEQ ID NO: 2 (i.e., position 2 to 285 of SEQ ID NO: 2), (c) a nucleotide sequence encoding a neurotoxin-alpha functional activity (e.g., an antigen or a biological activity) (D) a nucleotide sequence encoding the putative extracellular domain of a neurotoxin-alpha polypeptide having an amino acid sequence at position 73 to 285 in Figures 1A and 1B (SEQ ID NO: 2), a fragment of the polypeptide of (b) (e) a nucleotide sequence encoding a neurotoxin-alpha polypeptide having an amino acid sequence at positions 134 to 285 in Figures 1A and 1B (SEQ ID NO: 2), (f) A nucleotide sequence encoding a neurotoxin-alpha polypeptide having the complete amino acid sequence encoded by the contained cDNA clone, (g) a nucleotide sequence encoding a neurotransmitter having an amino acid sequence encoded by a cDNA clone contained in a deposit of ATCC Accession No. 97768 (A), (b), (c), (d), (e), (f) or (g) of the nucleotide sequence coding for the extracellular domain of the kin-alpha polypeptide 85% or 90% identical, more preferably 95%, 96%, 97%, 98% or 99% identical polynucleotides to the polynucleotide selected from the group consisting of complementary nucleotide sequences Or alternatively comprises separate nucleic acid molecules of such polynucleotides. The invention also encompasses polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these polynucleotides and nucleic acid molecules are also included in the present invention.

A highly preferred embodiment of the present invention is a polynucleotide encoding a neurotoxin-alpha polypeptide having an amino acid sequence at positions 134 to 285 in Figures 1A and 1B (SEQ ID NO: 2), wherein the nucleotide sequence is 80%, 85% or 90% , More preferably 95%, 96%, 97%, 98%, 99% or 100% identical polynucleotides, or alternatively comprises separate nucleic acid molecules consisting of such polynucleotides. A preferred embodiment of the present invention comprises a polynucleotide encoding a neurotoxin-alpha polypeptide having an amino acid sequence at positions 134 to 285 in Figures 1A and 1B (SEQ ID NO: 2), or a polynucleotide comprising a nucleotide sequence at least 90% And more particularly, to nucleic acid molecules comprising such polynucleotides. A more preferred embodiment of the present invention comprises a polynucleotide encoding a neurotoxin-alpha polypeptide having an amino acid sequence at positions 134 to 285 in Figures 1A and 1B (SEQ ID NO: 2), or a polynucleotide having at least 95% Or alternatively a nucleic acid molecule comprising such a polynucleotide. A still more preferred embodiment of the invention comprises a polynucleotide encoding a neurotoxin-alpha polypeptide having an amino acid sequence at positions 134 to 285 in Figure 1A and 1B (SEQ ID NO: 2) and a polynucleotide having a nucleotide sequence at least 96% identical , Alternatively, a nucleic acid molecule comprising such a polynucleotide.

Further, a more preferred embodiment of the present invention is a polynucleotide encoding a neurotoxin-alpha polypeptide having an amino acid sequence at positions 134 to 285 in Figure 1A and 1B (SEQ ID NO: 2) and a polynucleotide having a nucleotide sequence at least 97% identical Or, alternatively, to a nucleic acid molecule comprising such a polynucleotide. Further, a more preferred embodiment of the present invention relates to a polynucleotide encoding a neurotoxin-alpha polypeptide having an amino acid sequence at positions 134 to 285 in Figures 1A and 1B (SEQ ID NO: 2) and a polynucleotide with a nucleotide sequence at least 98% Or, alternatively, to a nucleic acid molecule comprising such a polynucleotide. Further, a more preferred embodiment of the present invention is a polynucleotide encoding a neurotoxin-alpha polypeptide having an amino acid sequence at positions 134 to 285 in Figures 1A and 1B (SEQ ID NO: 2) and a polynucleotide having a nucleotide sequence at least 99% Or, alternatively, to a nucleic acid molecule comprising such a polynucleotide.

A further aspect of the invention is the use of a polypeptide having an amino acid sequence containing more than one, not more than 50, more preferably not more than 40, even more preferably not more than 30, even more preferably not more than 20 conservative amino acid substitutions The present invention relates to a separate nucleic acid molecule comprising a polynucleotide encoding an amino acid sequence of a neurotoxin-alpha SV polypeptide (e.g., the neurotoxin-alpha SV polypeptide fragment described herein). Of course, in order of increasing preference, the order of preference is 7-10, 5-10, 3-7, 3-5, 2-5, 1-5, 1-3, 10, 9, 8, 7, 6, A polynucleotide encoding the amino acid sequence of a neurotoxin-alpha SV polypeptide having an amino acid sequence containing 3, 2 or 1 conservative amino acid substitutions is highly preferred.

Additional aspects include (a) a nucleotide sequence encoding a neurotoxin-alpha SV polypeptide having the complete amino acid sequence shown in Figures 5A and 5B (i.e., positions 1 to 266 of SEQ ID NO: 19), (b) a N-terminal methionine Alpha SV polypeptide having the complete amino acid sequence of SEQ ID NO: 19 (i.e., position 2 to 266 of SEQ ID NO: 2), (c) a nucleotide sequence that encodes a nucleotide sequence at positions 73 to 285 in SEQ ID NOs: 5A and 5B Alpha SV polypeptide having the amino acid sequence of SEQ ID NO: 2, (d) a nucleotide sequence encoding the neurotoxin-alpha SV polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in the deposit of ATCC Accession No. 203518 (E) an amino acid sequence encoded by a cDNA clone contained in a deposit of ATCC Accession No. 203518; (F) a nucleotide sequence encoding a nucleotide sequence complementary to the nucleotide sequence of (a), (b), (c), (d) , Polynucleotides selected from the group consisting of polynucleotides comprising at least 80%, 85% or 90% identical, more preferably at least 95%, 96%, 97%, 98% or 99% And encompasses isolated nucleic acid molecules consisting of such polynucleotides.

The present invention also encompasses nucleotide 1 and nucleotide 1 except for the nucleotide sequence of nucleotides 797 to 1082, 810 to 1082 and 346 to 542 as measured from the four cDNA clones described above, preferably in Figures 1A and 1B (SEQ ID NO: 1) At least about 10 consecutive nucleotides, at least about 20 consecutive nucleotides, at least about 25 consecutive nucleotides, or at least about 30 consecutive nucleotides, preferably at least about 40 consecutive nucleotides, or at least about 50 consecutive nucleotides of the sequence of nucleotides 1082 Or a polynucleotide comprising, alternatively consisting of, such sequences, at least 90% or at least 95% identical to any portion. In addition, the present invention also encompasses about 10 or more consecutive nucleotides, about 20 or more consecutive nucleotides, about 25 consecutive nucleotides, about 25 consecutive nucleotides, about 25 consecutive nucleotides, about 25 consecutive nucleotides, Or at least 90 consecutive nucleotides or at least about 30 consecutive nucleotides, preferably at least about 40 consecutive nucleotides or at least about 90 consecutive nucleotides, Lt; RTI ID = 0.0 > polynucleotides < / RTI > In addition, the present invention also encompasses a nucleotide sequence having at least about 10 consecutive nucleotides, at least about 20 consecutive nucleotides, at least about 25 consecutive nucleotides, or about 30 consecutive nucleotides, preferably at least about 30 consecutive nucleotides, Or comprises, alternatively, a polynucleotide consisting of, or alternatively consisting of, at least 90% or at least 95% identical sequences to any portion of at least about 40 consecutive nucleotides, or at least about 40 consecutive nucleotides, or at least about 50 consecutive nucleotides, do. In addition, the present invention also encompasses a nucleotide sequence having at least about 10 consecutive nucleotides, at least about 20 consecutive nucleotides, at least about 25 consecutive nucleotides, or about 30 consecutive nucleotides, preferably at least about 30 consecutive nucleotides, Or comprises, alternatively, a polynucleotide consisting of, or alternatively consisting of, at least 90% or at least 95% identical sequences to any portion of at least about 40 consecutive nucleotides, or at least about 40 consecutive nucleotides, or at least about 50 consecutive nucleotides, do. In addition, the present invention also encompasses a nucleotide sequence having at least about 10 consecutive nucleotides, at least about 20 consecutive nucleotides, at least about 25 consecutive nucleotides, or at least about 30 consecutive nucleotides, preferably at least about 30 consecutive nucleotides, Or comprises, alternatively, a polynucleotide consisting of, or alternatively consisting of, at least 90% or at least 95% identical sequences to any portion of at least about 40 consecutive nucleotides, or at least about 40 consecutive nucleotides, or at least about 50 consecutive nucleotides, do. The invention also encompasses a nucleic acid molecule comprising at least about 10 consecutive nucleotides, at least about 20 consecutive nucleotides, at least about 25 consecutive nucleotides, or at least about 30 consecutive nucleotides, preferably at least about 30 consecutive nucleotides, Or comprises, alternatively, a polynucleotide consisting of, or alternatively consisting of, at least 90% or at least 95% identical sequences to any portion of at least about 40 consecutive nucleotides, or at least about 40 consecutive nucleotides, or at least about 50 consecutive nucleotides, do. As used herein, " about " includes those that are greater or less than the number of amino acids at one or both ends (i.e., 5, 4, 3, 2 or 1) from a specifically specified range.

A polynucleotide having, for example, a " same " nucleotide sequence that is at least 95% identical to a reference nucleotide sequence encoding a neurotoxin-alpha and / or neurotoxin- alpha SV polypeptide, may be constructed from a nucleotide sequence of a polynucleotide, Means that the reference sequence is identical to the reference sequence except that it may contain no more than five mismatches per 100 nucleotides of the reference nucleotide sequence encoding the neurotoxin-alpha SV polypeptide. In other words, 5% or less of the nucleotides of the reference sequence may be deleted or substituted with other nucleotides to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to the reference nucleotide sequence, or the number of nucleotides to 5% of the total nucleotides of the reference sequence May be inserted into the reference sequence. These mutations of the reference sequence may be located at the 5 ' or 3 ' end of the reference nucleotide sequence, at any position between their terminal positions, individually interspersed among the nucleotides in the reference sequence, or interspersed within one or more contiguous groups within the reference sequence Lt; / RTI > The reference sequence is the complete nucleotide sequence encoding the neurotoxin-alpha or neurotoxin-alpha SV described in Figures 1A and 1B (SEQ ID NO: 1) and Figures 5A and 5B (SEQ ID NO: 18, respectively) Alpha, or all neurotoxin-alpha or neurotoxin-alpha SV polynucleotide fragments described herein, such as the neurotoxin-alpha polynucleotide described in SEQ ID NOS: 22, 27,

Indeed, it is contemplated that the particular nucleic acid molecule may be modified by, for example, the nucleotide sequence described in Figures 1A and 1B, the nucleotide sequence shown in Figures 5A and 5B, the nucleotide sequence of the deposited cDNA clone, or the neurotoxin- 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to all neurotoxin-alpha polynucleotides such as the alpha polynucleotide, or fragments thereof, Science Drive 575 University Research Park, Genetics < / RTI > Computer Group, version 8 for Unix). Best-fit uses Smith and Waterman's local homology algorithm to find the optimal homology fragment between two sequences (Advances in Applied Mathematics 2: 482-489 (1981)). When using a best-fit or other sequence alignment program to determine if a particular sequence is 95% identical to the reference sequence of the present invention, of course, the percentage of identity is calculated for the full length of the reference nucleotide sequence and the percentage of the nucleotide in the reference sequence A variable is set such that a gap of homology is allowed to be less than 5% of the total number.

In a particular embodiment, the identity (also referred to as the full sequence alignment) between the reference (query) sequence (the present invention sequence) and the corresponding sequence is determined by the algorithm of Brutagh and colleagues (Comp. App. Biosic. 237-245) using the FASTDB computer program. In a sequence, both the query and the corresponding sequence are DNA sequences. RNA sequences can be compared by converting U to T. The result of the entire sequence alignment appears as% identity. The preferred variables used in the FASTDB sequence of the DNA sequence to calculate percent identity are: Matrix = Unitary, k-tuple = 4, mismatch penalty point = 1, binding penalty point = 30, random group length = 0, 1, gap penalty point = 5, gap size penalty point = 0.05, window size = 500 or the length of the corresponding nucleotide sequence, whichever is shorter. According to this embodiment, the result should be manually corrected if the sequence is shorter than the query sequence due to a 5 'or 3' deletion rather than an internal deletion. This is because the FASTDB program does not calculate the 5 'and 3' truncations of the sequence when calculating percent identity. For the corresponding sequence truncated at the 5 'or 3' end of the query sequence, the percent identity is calculated as the percentage of the total base in the query sequence and the number of bases in the query sequence 5 'and 3' of the corresponding sequence that are not matched / . Whether the nucleotides are matched / ordered is determined as a result of the FASTDB sequence alignment. This percentage is then subtracted from the% identity calculated by the FASTDB program using a particular variable to obtain the final% identity score. This corrected score is used for the purposes of this embodiment. For the purpose of manually adjusting the percent identity score, only bases other than the 5 'and 3' bases of the corresponding sequence indicated by the FASTDB sequence that are not matched / aligned with the query sequence are counted. For example, 90 nucleotide sequences are aligned with 100 nucleotide query sequences to determine% identity. Deletions occur at the 5 'end of the sequence, so that the FASTDB sequence does not show match / alignment in the first 10 bases at the 5' end. The ten unpaired bases represent 10% of the sequence (the number of bases at the 5 'and 3' ends that are unmatched / the total number of bases in the query sequence) from the% identity score calculated by the FASTDB program 10% is deducted. If the remaining 90 bases are perfectly matched, the final% identity will be 90%. As another example, 90 bases corresponding sequences were compared to 100 bases query sequences. At this point, the deletion is internal deletion, so there is no base at the 5 'or 3' of the corresponding sequence that is not matched / aligned with the query. In this case, the percent identity calculated by FASTDB is not manually corrected. Again, only 5 ' and 3 ' bases of the corresponding sequence that are not matched / aligned with the query sequence are manually corrected. No other manual correction is made for the purposes of the present invention.

The invention encompasses nucleic acid sequences (i. E., Polynucleotides) (e. G., Polynucleotides) as described herein, whether or not encoding polypeptides having neurotoxin-alpha and / or neurotoxin- alpha SV functional activity The nucleotide sequence of the cDNA described in Figures 1A and 1B (SEQ ID NO: 1), or the nucleic acid sequence of the deposited cDNA), which is 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% Nucleic acid molecule. The present invention also relates to a nucleic acid sequence as described in Figures 5A and 5B (SEQ ID NO: 18), or a nucleic acid sequence having 80%, 85% 90%, 92%, 95%, 96%, 97%, 98% or 99% identical. The invention also relates to a nucleic acid sequence encoding a polypeptide having 80%, 85%, 90%, 92%, 95% or more identity with the nucleic acid sequence described in SEQ ID NO: 21, 22, 27 or 28, , 96%, 97%, 98% or 99% identical to the nucleic acid molecule. This means that even if a particular nucleic acid molecule does not encode a polypeptide having neurotoxin-alpha and / or neurotoxin-alpha SV activity, the skilled artisan will use the nucleic acid molecule as, for example, a hybridization probe or a polymerase chain reaction (PCR) primer I know how to do it. Uses of the nucleic acid molecules of the invention that do not encode a polypeptide having neurotoxin-alpha and / or neurotoxin-alpha SV activity include, among others, (1) the use of a neurotoxin-alpha and / or neurotoxin- alpha SV gene And / or neurotoxins as described in (2) Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York - In situ hybridization to metaphase chromosomal variants to provide the correct chromosomal location of alpha SV genes and normal blot analysis to detect neurotoxin-alpha and / or neurotoxin-alpha SV mRNA in specific tissues.

However, preferred is a nucleic acid sequence described herein that actually encodes a polypeptide having a neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide polypeptide activity (e. G., Biological activity) ), A nucleic acid molecule having the same nucleotide sequence as the nucleotide sequence of SEQ ID NO: 1 or a fragment of the nucleotide sequence of the deposited cDNA or a fragment thereof) of 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% to be. Also preferred is a nucleic acid sequence as described in Figures 5A and 5B (SEQ ID NO: 18) that actually encodes a polypeptide having a neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide functional activity (e.g., biological activity) is a nucleic acid molecule that is 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of the cDNA. Also preferred are nucleic acid sequences and sequences described in SEQ ID NOS: 21, 22, 27, or 28 that actually encode polypeptides having neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide functional activity (e.g., biological activity) 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical.

"Polypeptides having neurotoxin-alpha polypeptide functional activity" (eg, biological activity) and "polypeptides having neurotoxin-alpha SV polypeptide functional activity" (eg, biological activity) Refers to a polypeptide that does not necessarily correspond to the activity of the extracellular domain or full-length neurotoxin-alpha or neurotoxin-alpha SV polypeptide of the invention, but exhibits similar activity, For example, the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide functional activity may be determined by comparing the polypeptide sequences described herein with complete neurotoxin-alpha and / or neurotoxin-alpha SV or neurotoxin-alpha and / or The ability to form multimer (e. G., Homodimers and homotrimers) with the extracellular domain of neurotoxin-alpha SV and its ability to bind neurotoxin-alpha and / or neurotoxin-alpha SV ligands have. In addition, the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide functional activity may be used to determine the ability of the polypeptides of the invention to induce lymphocyte (e.g., B cell) proliferation, differentiation, or activation and / Measurement can be performed. These activity assays are described herein (see, e.g., Example 6) and can be routinely performed using techniques known in the art. In addition, the neurotoxin-alpha or neurotoxin-alpha SV polypeptides of the present invention modulate cell proliferation, cytotoxicity, cell survival and apoptosis. In vitro cell proliferation, cytotoxicity, cell survival and cell death assays for measuring the effect of a protein on a specific cell are performed using reagents that are well known and readily available in the art for detecting cell replication and / or death . For example, many such assays for TNF-related protein activity are described in various references cited herein. Briefly, an example of such an assay is to harvest human or animal (e.g., mouse) cells and harvest (1) a transfected host cell-supernatant containing a neurotoxin-alpha protein (or candidate polypeptide) or (2) Mixing the cells with an infected host cell-supernatant control, and measuring the effect on cell number or survival after incubation for a specified period of time. Cell proliferation and / or survival modulating activity that can be measured in this type of assay is useful for treating tumors, tumor metastases, infections, autoimmune diseases, inflammation and other immune-related diseases.

Neutrokine-alpha modulates cell proliferation and differentiation in a dose-dependent manner in the assay. Thus, it is preferred that a " polypeptide having a neurotoxin-alpha polypeptide functional activity " (e. G., Biological activity) comprises polypeptides that exhibit the same cell regulation (especially immunomodulatory) activity in a dose-dependent manner in the assay . Although the degree of dose-dependent activity need not be the same as that of the neurotoxin-alpha polypeptide, preferably the " polypeptide having the neurotoxin-alpha polypeptide functional activity " (I. E., The candidate polypeptide exhibits greater activity as compared to the reference neurotoxin-alpha polypeptide, or less than about 25-fold, preferably less than about 10-fold less activity than the reference neurotoxin-alpha polypeptide ).

In certain preferred embodiments, "polypeptides having a neurotoxin-alpha polypeptide functional activity" (eg, biological activity) and "polypeptides having a neurotoxin-alpha SV polypeptide functional activity" (eg, biological activity) 8A, 8B, 9A, 9B, 10, 11, 12A and 12B, and polypeptides also exhibiting the same B cell (or other cell type) modulation (particularly immunomodulatory) activity as described in Example 6.

Like other members of the TNF family, neurotoxin-alpha is active against leukocytes including, for example, mononuclear cells, lymphocytes (e.g., B cells) and neutrophils. For this reason, neurotoxin-alpha is active in inducing proliferation, differentiation and migration of these cell types. Such activity is useful for immune enhancement or inhibition, bone marrow protection, hepatocyte mobilization, acute and chronic inflammation control, and leukemia. Assays for measuring such activity are known in the art (see, e.g., Peters et al., Immun. Today 17: 273 (1996); Young et al., J. Exp. Med. 182: 1111 (1995); Caux et al., Nature 390: 258 (1992); And Santiago-Schwarz et al., Adv. Exp. Med. Biol. 378: 7 (1995)).

Of course, due to the degeneracy of the genetic code, those skilled in the art will appreciate that the nucleic acid sequence contained in the cDNA clone deposited in ATCC Accession No. 97768 or the nucleic acid sequence described in Figures 1A and 1B (SEQ ID NO: 1) , Nucleic acid molecules that are 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical or more are "polypeptides having a" neurotoxin-alpha polypeptide functional activity " You will immediately recognize that you are encrypting. Those skilled in the art will also recognize that the nucleic acid sequences contained in the cDNA clones deposited in ATCC Accession No. 203518 or the nucleic acid sequences set forth in Figures 5A and 5B (SEQ ID NO: 18) and sequences having 80%, 85%, 90%, 92% , 96%, 97%, 98% or 99% or more of the nucleic acid molecules encode a polypeptide having "neurotoxin-alpha SV polypeptide functional activity" (eg, biological activity). In fact, this would be obvious to those skilled in the art, without performing the above-described comparison assays, since all of the degenerate variants of their nucleotide sequences encode the same polypeptide. It will also be recognized in the art that suitable numbers will also encode neurotoxin-alpha and / or neurotoxin-alpha SV activity, even in the case of nucleic acid molecules that are not degenerate variants. This is because those skilled in the art are fully aware of amino acid substitutions that are less likely or unlikely to significantly affect the protein action (e.g., substituting aliphatic amino acids with secondary aliphatic amino acids), as more fully described below.

Similarly, polynucleotides encoding polypeptides containing all or a portion of regions V-142 to K-160 in SEQ ID NO: 2 may be used to detect and / or detect the expression of a neurotoxin-alpha or neurotoxin-alpha SV polynucleotide Gt; polynucleotides < / RTI & In addition, a polynucleotide linking the amino acid residues T-141 and G-142 of the neurotoxin-alpha SV polypeptide described in SEQ ID NO: 19 (in which the V-142 to K-160 amino acid sequences of the neurotoxin- ) Appears to be useful both diagnostically and therapeutically. Such a T-141 / G-142 spanning polynucleotide will exhibit a much higher probability of hybridization with the neurotoxin-alpha SV polynucleotide than with the neurotoxin-alpha polynucleotide. Some non-limiting, non-exclusive listing of the neurotoxin-alpha SV polypeptides encoded by the polynucleotides of the present invention include polypeptides comprising or consisting of amino acid sequences selected from among the following: G -121 to E-163; E-122 to E-163; G-123 to E-163; N-124 to E-163; S-125 to E-163; S-126 to E-163; Q-127 to E-163; N-128 E163; S-129 to E-163; R-130 to E-163; N131 to E-163; K-132 to E-163; R-133 to E-163; A-134 to E-163; V-135 to E-163; Q-136 to E-163; G-137 to E-163; P-138 to E-163; E-139 to E-163; E-140 to E-163; T-141 to E-163; G-142 to E-163; S-143 to E-163; Y-144 to E-163; T-145 to E-163; F-146 to E-163; V-147 to E-163; P-148 to E-163; W-149 to E-163; L-150 to E-163; L-151 to E-163; S-152 to E-163; F-153 to E-163; K-154 to E-163; R-155 to E-163; G-156 to E-163; S-157 to E-163; A-158 to E-163; L-159 to E-163; E-160 to E-163; E-161 to E-163; K-162 to E-163; G-121 to K-162; G-121 to E-161; G-121 to E-160; G-121 to L-159; G-121 to A-158; G-121 to S-157; G-121 to G-156; G-121 to R-155; G-121 to K-154; G-121 to F-153; G-121 to S-152; G-121 to L-151; G-121 to L-150; G-121 to W-149; G-121 to P-148; G-121 to V-147; G-121 to F-146; G-121 to T-145; G-121 to Y-144; G-121 to S-143; G-121 to G-142; G-121 to T-141; G-121 to E-140; G-121 to E-139; G-121 to P-138; G-121 to G-137; G-121 to Q-136; G-121 to V-135; G-121A-134; G-121 to R-133; G-121 to K-132; G-121 to N-131; G-121 to R-130; G-121 to S-129; G-121 to N-128; G-121 to Q-127; G-121 to S-126; G-121 to S-125; G-121 to N-124; G-121 to G-123; And G-121 to E-122. Polypeptides encoded by these polynucleotides are also included in the present invention.

Vector and host cell

The present invention also relates to a vector comprising a separate DNA molecule of the invention, a host cell genetically engineered with this recombinant vector or engineering engineered to produce a polypeptide of the invention, and a recombinant or recombinant vector, Alpha-and / or neurotoxin-alpha SV polypeptides or fragments thereof.

In one embodiment, a polynucleotide of the invention is linked to a vector (e. G., A cloning or expression vector). The vector may be, for example, a phage, a plasmid, a virus or a retroviral vector. The retroviral vector may be a replication competent or a replication defective. In the latter case, virus proliferation generally occurs only in a complementary host cell. The polynucleotide may be linked to a vector containing a selectable marker for propagation in the host. Introduction of a vector construct into a host cell may be accomplished by any method known in the art including but not limited to calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, , ≪ / RTI > and the like. Such methods are described in many standard laboratory manuals such as Davis et al., Basic Methods in Molecular Biology (1986).

Generally, the recombinant expression vector will contain a replication origin and a selectable marker (e.g., an ampicillin resistant gene and a Saccharomyces cerevisiae TRP1 gene of E. coli) that allows transformation of the host and a transcription of a downstream structural sequence And a promoter derived from a highly-expressed gene. Such a promoter may be derived from an operon encoding a corresponding process enzyme, such as 3-phosphoglycerate kinase (PGK), an a-factor, an acid phosphatase or a heat shock protein. The heterologous structural sequence is combined with a leader sequence that is capable of inducing the initiation and termination of the translation at an appropriate stage and, preferably, the secretion of the protein that is detoxified into the periplasmics space or extracellular medium. Optionally, the heterologous sequence may encode a fusion protein comprising an N-terminal identifying peptide that provides the desired characteristics, e. G., Stabilization or simple purification of the expressed recombinant product.

In one embodiment, the DNA of the present invention comprises a phage lambda PL promoter, (E. G., A promoter or enhancer) such as E. coli lac, trp, phoA and tac promoters, SV40 early and late promoters, and retroviral LTR promoters. Other suitable promoters are known to those skilled in the art.

As mentioned, the expression vector preferably comprises one or more selectable markers. Such markers include dihydrofolate reductase, G418 or neomycin resistance gene for eukaryotic cell culture, And tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of suitable hosts include, but are not limited to, Bacterial cells such as E. coli, Streptomyces and Salmonella typhimurium cells; Yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); Insect cells such as Drosophila S2 and Spodoptera Sf9 cells; Animal cells such as CHO, COS, 293 and bowel melanoma cells; And plant cells. Culture media and conditions suitable for the host cells described above are known in the art.

The host cell may be a lower eukaryotic cell, such as a higher eukaryotic cell or yeast cell, such as a mammalian cell (e. G., A human derived cell), or the host cell may be a prokaryotic cell, such as a bacterial cell. Host strains may be selected to modulate the expression of the inserted gene sequence or to modify and process the gene product in the specific manner desired. Expression from a particular promoter can be elevated in the presence of a particular inducer and thus the expression of the genetically engineered polypeptide can be regulated. In addition, other host cells have features and specific mechanisms for processing and transformation (e. G., Phosphorylation, cleavage) after detoxification and detoxification of the protein. Appropriate cell lines may be selected to ensure the desired transformation and processing of the expressed foreign protein. Selection of appropriate vectors and promoters for expression in host cells is a well known procedure and techniques essential for the construction of expression vectors, introduction of vectors into hosts and expression in hosts are conventional in the art.

An expression vector useful in bacteria is constructed by inserting a structural DNA sequence encoding the desired protein along with a suitable initiation and termination signal into the operative reading with the acting promoter. The vector comprises one or more phenotype selection markers and a replication origin to ensure maintenance within the host and to provide amplification if necessary. Suitable prokaryotic hosts for transformation, although they may be used as a matter of choice for others, E. coli, Bacillus subtilis, Salmonella typhimurium and Pseudomonas, Streptomyces and Staphylococcus species. Expression vectors suitable for use in bacteria may include, but are not limited to, selectable markers derived from commercially available plasmids containing the genetic elements of the well-known cloning vector pBR322 (ATCC 37017) and bacterial replication origin . Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotech, Madison, Wis.). These pBR322 " backbone " moieties are combined with the appropriate expression vector and the structural sequence to be expressed. Preferred vectors for use in bacteria include pHE4-5 (ATCC Accession No. 209311 and its variants), pQE70, pQE60 and pQE-9 available from QIAGEN, Inc; PBS vector, phage script vector, blue script vector, pNH8A, pNH16a, pNH18A, pNH46A available from Stratagene; And ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Preferred expression vectors for use in yeast systems include but are not limited to pYES2, pYD1, pTEE1 / Zeo, pYES2 / GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL- pPIC3.5K, pPIC9K, and PAO815, both available from Invitrogen, Carlsbad, CA. Preferred eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be apparent to those skilled in the art.

After transforming an appropriate host strain and growing it to a suitable cell density, the selected promoter is induced by appropriate means (e.g., temperature conversion or chemical induction) and the cells are cultured for an additional period of time. Cells are typically harvested by centrifugation and disrupted by physical or chemical means, and the resulting crude extract is retained for further purification.

The microbial cells used for expression of the protein can be disrupted by convenient methods including freeze-thaw cycles, sonication, mechanical disruption or the use of cytolytic agents, and such methods are well known to those skilled in the art.

In one embodiment, yeast Pichia pastoris is used to express the neurotoxin-alpha protein in the eukaryotic system. Pichia pastoris is a methanol constituent yeast that can metabolize methanol to a unique carbon source. The main step in the methanol metabolism pathway is the oxidation of methanol to formaldehyde using O ₂ . This reaction is catalyzed by an enzyme alcohol oxidase. In order to metabolize methanol as its sole carbon source Pichia pastoris is partly due to the relatively low affinity of alcohol oxidase for the O ₂ must generate high levels of alcohol oxidase. Eventually, in the growth medium dependent on methanol as the main carbon source, the promoter region of one of the two alcohol oxidase genes (AOX1) is highly active. In the presence of methanol, the alcohol oxidase produced from the AOX1 gene accounts for up to about 30% of the total soluble proteins of P. falciparum (Ellis, SB, et al., Mol. Cell. Biol. 5: 21 (1985); Koutz, PJ et al., Yeast 5: 167-77 (1989); Tschopp, JF, et al., Nucl. Acids Res. 15: 3859-76 (1987)). Thus, under the transcriptional control of all or part of the AOX1 regulatory sequences, heterologous coding sequences such as, for example, the neurotoxin-alpha or neurotoxin-alpha SV polynucleotides of the invention can be detected at very high levels in the P.kyota yeast grown in the presence of methanol Lt; / RTI >

As an example, see " Pichia Protocols: Methods in Molecular Biology, " Higgins and J. Cregg, eds. Plasmid vector pPIC9K was used to express DNA encoding the neurotoxin-alpha or neurotoxin-alpha SV polypeptide in the Pichia yeast system as described in The Humana Press, Totowa, NJ, 1998 and described herein do. This expression vector has a strong AOX1 promoter linked to a Pichia pastoris alkaline phosphatase (PHO) secretion signal peptide (i. E., A leader) located upstream of the multiple cloning site, resulting in the expression of the neurotoxin-alpha or neurotoxin- Expression and secretion.

As can be readily appreciated by those skilled in the art, many other yeast vectors can be used in place of pPIC9K, including pYES2, pYD1, pTEF1 / Zeo, pYES2 / GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2 , pHIL-S1, pPIC3.5K, and PAO815. However, the proposed expression construct should provide a properly positioned signal for transcription, translation, secretion (if necessary), etc., including the essential in-frame AUG.

In one embodiment, the high level expression of the heterologous coding sequence, such as the neurotoxin-alpha or neurotoxin-alpha SV polynucleotide of the present invention, clones the heterologous polynucleotide of the invention into an expression vector such as pGAPZ or pGAPZ alpha, Lt; RTI ID = 0.0 > yeast < / RTI > culture.

Transcription of DNA encoding the polypeptide of the present invention by higher eukaryotic cells is increased by inserting an enhancer sequence into the vector. Enhancers are usually cis-acting elements of about 10 to 300 bp DNA that act on the promoter to increase transcription. Examples thereof include the SV40 enhancer of bp 100 to 270 located at the latter half of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer located at the latter half of the replication origin, and the adenovirus enhancer.

A variety of mammalian cell culture systems can also be used to express recombinant proteins. Examples of mammalian expression systems include the COS-7 strain of monkey kidney fibroblasts described in Gluzman, Cell 23: 175 (1981) and other cell lines capable of expressing the fitness vector (eg, C127, 3T3, CHO, And BHK cell lines). Mammalian expression vectors include a replication origin, a suitable promoter and enhancer and other necessary ribosome binding sites, a polyadenylation site, a splice donor and acceptor site, a transcription termination sequence and a 5 'flanking non-promoter sequence. DNA sequences and polyadenylation sites derived from the SV40 splice can be used to provide the necessary non-specific gene elements.

In certain embodiments, constructs designed to express some of the extracellular domains of neurotoxin-alpha (e.g., amino acid residues Ala-134 to Leu-285) are preferred. One of ordinary skill in the art can design polynucleotide primers and generate expression constructs therefrom using the polynucleotide and polypeptide sequences provided in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, SEQ ID NO: 18 and SEQ ID NO: 19, respectively.

In another embodiment, constructs designed to express the fully-predicted extracellular domain of neurotoxin-alpha (i. E., Amino acid residues Gln-73 to Leu-285) are preferred. One of ordinary skill in the art can design polynucleotide primers and generate expression constructs therefrom using the polynucleotide and polypeptide sequences provided in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, SEQ ID NO: 18 and SEQ ID NO: 19, respectively.

In addition to including host cells containing the vector constructs discussed herein, the present invention also encompasses the use of a neurotoxin-alpha polynucleotide of the invention (e. G., A neurotoxin-alpha coding sequence) (Eg, a heterologous polynucleotide sequence) operatively linked to an endogenous neurotoxin-alpha polynucleotide and activating, mutating and / or amplifying an endogenous neurotoxin-alpha polynucleotide, Primary, secondary, and immortal host cells. For example, a heterologous regulatory region (e. G., A promoter and / or enhancer) and an endogenous neurotoxin-alpha polynucleotide sequence may be used for homologous recombination (see, e. G., The United States of America Patent No. 5,461,670, International Publication No. WO 96/29411, published September 26, 1996; International Publication No. WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. USA 86: 8932-8935 (1989); and Zijlstra et al., Nature 342-435-438 (1989)). The entire contents of each of the above documents are incorporated herein by reference.

The host cells described below can be used in a conventional manner to generate a gene product that is encoded by recombinant techniques. Alternatively, a cell-free detoxification system can be used to generate the polypeptide of the present invention from RNA derived from the DNA construct of the present invention.

The polypeptides of the present invention may be expressed or synthesized in a modified form such as a fusion protein (including polypeptides linked to a heterologous protein sequence (via a peptide bond) of different proteins) and may contain additional secretion domains as well as secretory signals . Such fusion proteins can be obtained by linking the polynucleotides of the present invention and the nucleic acid sequences encoding the desired amino acid sequence to each other by methods known in the art within a suitable reading frame and expressing the fusion protein product by methods known in the art have. Alternatively, such fusion proteins can be made by protein synthesis techniques, for example, by the use of peptide synthesizers. Thus, for example, additional amino acids, particularly regions of charged amino acids, can be added to the N-terminus of the polypeptide to enhance stability and persistence during, during, or subsequent processing and storage in the host cell. Peptide moieties may also be added to the polypeptide to facilitate purification. Such regions can be removed prior to the final preparation of the polypeptide. It is a familiar and common skill in the art to add peptide moieties to polypeptides to facilitate secretion or release, to improve stability, and to facilitate purification.

In one embodiment, a polynucleotide encoding the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide of the invention is fused to a pelB pectytialase signal sequence to express and purify such polypeptides in Gram- (See, for example, U.S. Patent Nos. 5,576,195 and 5,846,818). The entire contents of these patents are incorporated herein by reference.

Preferred fusion proteins include heterologous regions from immunoglobulins useful for stabilizing and purifying proteins. For example, EP-A-0 464 533 (Canadian patent number 2045869) describes a fusion protein comprising several parts of the constant region of an immunoglobulin molecule with another protein or a portion thereof. In many cases, the Fc portion of the fusion protein is very advantageous for use in therapy and diagnosis and thus provides, for example, improved pharmacokinetic properties (EP-A 032 262). On the other hand, in some applications it may be desirable to remove the Fc portion after the fusion protein has been expressed, detected and purified in an advantageous manner as described. This is the case where it has been proven to be a hindrance in using the Fc portion in therapy and diagnosis, for example when the fusion protein is used as an antigen for immunization. In drug development, human proteins such as, for example, hIL-5 have been fused with the Fc portion for the purpose of high-throughput screening assays to identify antagonists of hIL-5 (see D. Bennet et al., J Molecular Recognition 8: 52-58 (1995) and K. Johanson et al., J. Biol. Chem. 270: 9459-9471 (1995)).

The polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures and products produced by recombinant techniques from prokaryotic or eukaryotic hosts, including, for example, bacteria, yeast, higher plants, insects and mammalian cells . Depending on the host used in the recombinant production procedure, the polypeptides of the invention may be glycosylated or non-glycosylated. In addition, the polypeptides of the present invention may also comprise a first modified methionine residue in some cases as a result of a host-mediated process.

The polypeptides of the present invention can be chemically synthesized using techniques known in the art (see, for example, Creighton, 1983, Proteins: Structures and Molecular Principles, WH Freeman & et al., 1984, Nature 310: 105-111). For example, a peptide corresponding to a fragment of the complete neurotoxin-alpha or neurotoxin-alpha SV polypeptide of the present invention can be synthesized using a peptide synthesizer. Also, if desired, non-classical amino acids or chemical amino acid analogs can be introduced as substitutions or additions into the neurotoxin-alpha or neurotoxin-alpha SV polynucleotide sequences. Unconventional amino acids include, but are not limited to, the D-isomer of a common amino acid, 2,4-diaminobutyric acid, a-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2- aminobutyric acid, g-Abu, e Aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-aminobutyric acid, (Eg, b-methyl amino acid, Ca-methyl amino acid, Na-methyl amino acid) and common amino acid analogs, including but not limited to glycine, butyl glycine, t-butyl alanine, phenyl glycine, cyclohexyl alanine, b- alanine, fluoro- do. Also, the amino acid may be D (right turn) or L (left turn).

The present invention also provides a method for modifying an antibody molecule or other cell ligand that is modified by, for example, glycosylation, acetylation, phosphorylation, amidation, derivatization by a known protecting / blocking group, proteolytic cleavage, Alpha or < RTI ID = 0.0 > neurotoxin-alpha < / RTI > SV polypeptides that can be distinguished. It is not limited to, any of the numerous chemical modification also, but cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH _4, by acetylation, formylation, oxidation, reduction, two NIKA My metabolism synthesized in the presence of God Can be accomplished by well known techniques involving specific chemical cleavage.

Additional post-translational modifications included by the present invention include, for example, N-terminal or C-terminal processing, linking of chemical moieties to amino acid backbones, chemical modification of N-linked or O- linked carbohydrate chains, Including the addition or deletion of N-terminal methionine residues as a result of expression. Polypeptides can also be modified to detect labels such as enzymes, fluorescence, isotopes or affinity labels to enable detection and isolation of the protein. In addition, the polypeptide of the present invention can be modified by iodination.

In one embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides of the invention may also be labeled with biotin. In other related embodiments, the biotinylated neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides of the invention may be conjugated to, for example, imaging agents or one or more neurotoxin-alpha and / or neurotoxin- alpha SV receptor (s) Or as a means for identifying other co-receptor or co-ligand molecules.

The present invention also relates to chemically modified derivatives of neurotoxin-alpha or neurotoxin-alpha SV, which can provide additional advantages such as increased solubility, stability and in vivo or in vitro circulation time or reduced immunogenicity of the polypeptide (Cf. U.S. Patent No. 4,179,337). Chemical moieties for derivatization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol / propylene glycol copolymer, carboxymethyl cellulose, dextran, polyvinyl alcohol, and the like. The polypeptide may be modified at any position within the molecule and at a predetermined position within the molecule and may comprise one, two, three or more linked chemical moieties.

The polymer may be of any molecular weight and may be branched or unbranched. For polyethylene glycol, the preferred molecular weight is from about 1 kDa to about 100 kDa for convenient manipulation and preparation, and the term " about " refers to the molecular weight at the time of the preparation of polyethylene glycol and some molecules may weigh more or slightly less have. Different sizes may be used depending upon the desired therapeutic profile (eg, the duration of the desired sustained release, the effect on biological activity, the ease of handling, the degree or lack of antigenicity and other known effects of polyethylene glycol on the therapeutic protein or analog) have. For example, polyethylene glycols can be used at about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 50,000, 15,000, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000 or 100,000 kDa.

As mentioned above, polyethylene glycol may have a branched structure. Side-chain polyethylene glycols are described, for example, in U.S. Patent Nos. 5,643,575; Morpurgo, et al., Appl. Biochem. Biotechnol. 56: 59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18: 2745-2750 (1999); And Caliceti et al., Bioconjug. Chem. 10: 638-646 (1999). The contents of each of the above references are incorporated herein by reference.

The polyethylene glycol molecule (or other chemical moiety) must be linked to the protein in view of its effect on the functional or antigenic domain of the protein. There are many binding methods available to those skilled in the art (reference example: EP 0 401 384 (binding PEG to G-CSF) and Malik et al., Exp. Hematol. 20: 1028-1035 (1992) Phenylation of GM-CSF Using Silyl Chloride)]. For example, the polyethylene glycol can be covalently bonded to the amino acid residue through a reactive group such as a free amino or carboxyl group. The reaction group is a group to which an activated polyethylene glycol molecule can be bonded. Amino acid residues having a free amino group may include, for example, lysine residues and N-terminal amino acid residues; Amino acid residues having a free carboxyl group can include an aspartic acid residue, a glutamic acid residue, and a C-terminal amino acid residue. The sulfhydryl group can also be used as a reaction group to bond polyethylene glycol molecules. N-terminal or linkage to an amino group such as a bond to a lysine group is preferred for therapeutic purposes.

As suggested above, polyethylene glycol can be linked to proteins via linkage with any one of many amino acid residues. For example, polyethylene glycol can be linked to proteins via covalent linkage with lysine, histidine, aspartic acid, glutamic acid, or cysteine residues. One or more reactive chemistries may be used to convert the polyethylene glycol to one or more types of amino acid residues of a protein (e.g., lysine, histidine, aspartic acid, glutamic acid, Glutamic acid, cysteine, and combinations thereof).

Proteins chemically modified at the N-terminus may be specifically required. (By molecular weight, side chainization, etc.), the ratio of the polyethylene glycol molecule to the protein (or peptide) molecule in the reaction mixture, the type of pheization reaction to be performed, and the ratio of the selected N - the method of obtaining the terminal pheizing protein. The method of obtaining an N-terminal pheizing agent (i. E., Separating this moiety from other monopepanated moieties if desired) can be accomplished by purification of the N-terminal pheizing agent from the group of pheizing protein molecules. Certain proteins that are chemically modified at the N-terminus can be achieved by reductive alkylation, which exhibits distinct reactivity of other types of primary amino groups (lysine to N-terminal) being used for derivatization in certain proteins. Substantially specific derivatization of the protein at the N-terminus is achieved with the carbonyl group containing polymer under suitable reaction conditions.

As described above, the pervaporation of proteins according to the present invention can be accomplished by many means. For example, polyethylene glycol may be attached to the protein directly or via an intermediate linker. Linker-less systems for linking polyethylene glycols to proteins are described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9: 249-304 (1992); Francis et al., Intern. J. of Hematol. 68: 1-18 (1998); U.S. Patent No. 4,002,531; U.S. Patent No. 5,349,052; WO 95/06058; And WO 98/32466. Each of which is incorporated herein by reference.

One system for direct linking of polyethylene glycols to amino acid residues of proteins without intermediate linkers is to use trisylated MPEG generated by modifying monomethoxypolyethylene glycol (MPEG) using trexylyl chloride (ClSO ₂ CH ₂ CF ₃ ) use. When the protein is reacted with the tracerized MPEG, the polyethylene glycol is directly linked to the amino group of the protein. Accordingly, the present invention includes a protein-polyethylene glycol conjugate produced by reacting the protein of the present invention with a polyethylene glycol molecule having a 2,2,2-trifluoroethanesulfonyl group.

Polyethylene glycol can also be linked to proteins using many different intermediate linkers. For example, U. S. Patent No. 5,612, 460 describes urethane linkers for linking polyethylene glycols to proteins. The entire contents of this patent are incorporated herein by reference. The protein-polyethylene glycol conjugate in which polyethylene glycol is linked to the protein by a linker can also be coupled to MPEG-succinimidyl succinate, MPEG activated with 1,1'-carbonyldiimidazole, MPEG-2,4,5-trichloro Phenyl carbonate, MPEG-p-nitrophenol carbonate, and several MPEG-succinate derivatives. Many additional polyethylene glycol derivatives and reaction chemistries for linking polyethylene glycols to proteins are described in WO 98/32466. The entire contents of this patent are incorporated herein by reference. The papermilled protein products produced using the reaction chemistries described herein are included within the scope of the present invention.

The number of polyethylene glycol moieties linked to each protein of the present invention (i.e., degree of substitution) may also vary. For example, the pegylated protein of the invention can be linked to an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20 or more polyethylene glycol molecules have. Similarly, the average degree of substitution can be from 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12 , 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol residues. Methods for measuring the degree of substitution are described, for example, in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9: 249-304 (1992).

Neutrokine-alpha and / or neurotoxin-alpha SV polypeptides include, but are not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, And can be recovered and purified by known methods, including chemical chromatography, hydroxyapatite chromatography and lectin chromatography. The most preferred method for purification is high performance liquid chromatography (HPLC).

Neutrokine-alpha polypeptide

The neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides of the present invention may be monomers or multimers (i.e., dimers, trimers, tetramers, and higher oligomers). Accordingly, the invention relates to monomers and oligomers of the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides according to the invention, to a process for their preparation and to compositions (preferably pharmaceutical compositions) containing them . In certain embodiments, the polypeptides of the invention are monomers, dimers, trimers, or tetramers. In a further embodiment, the oligomer of the invention is dimer, trimer or tetramer.

The multimer of the present invention may be homopolymer or heteropolymer. The term homologous multimer used herein refers to the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides of the present invention (the neurotoxin-alpha and / or neurotoxin-alpha SV fragments, variants and fusion proteins described herein And the like). These homomultimeters may contain a neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide having the same or a different amino acid sequence. In certain embodiments, homomultimeters of the invention are multimers that contain only the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides having the same amino acid sequence. In another specific embodiment, the homomultimeters of the present invention are oligomers containing neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides having different amino acid sequences. In certain embodiments, the oligomers of the invention may be homodimers (e.g., containing a neurotoxin-alpha and / or neurotoxin-alpha SV having the same or different amino acid sequences) or homotrimers (e.g., the same or different amino acid sequences Alpha and / or neurotoxin-alpha SV with < / RTI > In a preferred embodiment, the oligomer of the present invention is a homotrimer. In a further embodiment, the homomeric multimer of the present invention is a homomeric or higher homomeric, more homomeric or more homomeric.

As used herein, the term heterodimer means a multimer containing a heterologous polypeptide (i.e., a polypeptide of a different protein) in addition to the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides of the invention. In certain embodiments, the oligomers of the invention are heterodimers, heterotrimers, or heterotrimers. In a further embodiment, the oligomer of the invention is at least a heterodimer, a heteromer or a heteromer. In further conventional embodiments, the heterodimers of the invention contain CD40 ligand polypeptide sequence (s) or biologically active fragment (s) or variant (s) thereof.

The oligomers of the present invention may result in hydrophobic, hydrophilic, ionic and / or covalent bonds and / or may be indirectly linked by, for example, liposomal formation. Thus, in one embodiment, a multimer of the invention, such as a homodimer or homotrimer, is formed when the polypeptide of the invention is contacted with one other polypeptide in solution. In another embodiment, a heterodimer of the invention, such as a heterodimer or heterotrimer, comprises a polypeptide of the invention in a solution in which the antibody to the polypeptide of the present invention (in a heterologous polypeptide sequence in the fusion protein of the invention) Lt; / RTI > antibody). In another embodiment, the oligomers of the present invention are formed by covalent bonds with the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides of the invention and / or covalent bonds therebetween. Such covalent bonds include one or more amino acid residues contained in the polypeptide sequence (e. G., A residue described in SEQ ID NO. 2 or SEQ ID NO. 19, or a residue contained in a polypeptide encoded by a clone deposited in connection with the present disclosure). As an example, covalent linkages are bridges between cysteine residues located within polypeptide polypeptides that interact in native polypeptides. As another example, covalent bonds are the result of chemical or recombinant manipulation. Alternatively, the covalent bond may involve one or more amino acid residues contained in the heterologous polypeptide sequence in the neurotoxin-alpha and / or neurotoxin-alpha SV fusion protein. As an example, covalent bonds are made between heterologous sequences contained in the fusion proteins of the present invention (see, e.g., U.S. Patent No. 5,478,925). As a specific example, the covalent bond is made between the heterologous sequences contained in the neurotoxin-alpha-Fc and / or neurotoxin-alpha SV-Fc fusion proteins of the invention described herein. As another specific example, the covalent attachment of a fusion protein according to the present invention occurs between heterologous polypeptide sequences from other TNF class ligand / receptor members capable of forming covalently linked multimers, such as osteoprotegerin : International Publication No. WO 98/49305). The entire contents of this patent are incorporated herein by reference. As another specific example, the covalent attachment of a fusion protein according to the present invention occurs between heterologous polypeptide sequences from CD40L or soluble fragments thereof. In another embodiment, two or more neurotoxin-alpha and / or neurotoxin-alpha polypeptides according to the invention are linked via a synthetic linker (e.g., a peptide, carbohydrate or soluble polymer linker). Examples include the peptide linkers described in U.S. Patent No. 5,073,627. The contents of this patent are incorporated herein by reference. Proteins comprising a plurality of neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides separated by a peptide linker can be generated by conventional recombinant DNA techniques.

The method for producing the multimeric neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides according to the present invention is characterized in that the neuquinone-alpha and / or neurotoxin-alpha fusion proteins fused to a leucine zipper or isoleucine zipper polypeptide sequence SV polypeptides. The lucine zipper or isoleucine zipper domains are polypeptides that promote the mass multiplication of the proteins in which they are found. Lewis zippers were first identified in several DNA-binding proteins (Landschulz et al., Science 240: 1759, (1988)) and have since been found in many other proteins. Among the known lucine zipper or isoleucine zipper, natural peptides and derivatives thereof are dimerized or trimerized. Examples of suitable leucine zipper domains for producing soluble multimeric neurotoxin-alpha and / or neurotoxin-alpha SV proteins include those described in PCT Patent Publication No. WO 94/10308. The contents of this patent are incorporated herein by reference. A recombinant fusion protein comprising soluble neurotoxin-alpha and / or neurotoxin-alpha SV fused to a dimer or trimerizing peptide in solution is expressed in a suitable host cell using techniques known in the art and isolated from the culture supernatant The resulting soluble multimeric neurotoxin-alpha and / or neurotoxin-alpha SV is recovered.

It is believed that certain members of the TNF family of proteins are present in a trimer form (Beutler and Huffel, Science 264: 667, 1994; Banner et al., Cell 73: 431, 1993). Thus, trimeric neurotoxin-alpha and / or neurotoxin-alpha SV can provide the advantage of increased biological activity. A preferred leucine zipper residue is one that preferentially forms trimers. One example is a leucine zipper derived from pulmonary supernatant protein D (SPD) as described in Hoppe et al., FEBS Letters 344: 191, (1994) and US Patent Application 08 / 446,922. The contents of these documents are incorporated herein by reference. Other peptides derived from natural trimeric proteins can be used to prepare trimeric neurotoxin-alpha and / or neurotoxin-alpha SVs.

As another example, the protein of the present invention is linked by the interaction between a flag polypeptide sequence contained in the flag-neurotoxin alpha or flag-neurotoxin-alpha SV fusion protein of the present invention. In a further embodiment, the protein of the invention is linked by the interaction between the anti-flag antibody and the heterologous polypeptide sequence contained in the flag-neurotoxin alpha or flag-neurotoxin-alpha SV fusion protein of the invention.

The oligomers of the present invention can be produced using chemical techniques known in the art. For example, polypeptide peptides that need to be included in the oligomers of the present invention can be chemically crosslinked using linker molecule length optimization techniques and linker molecules known in the art (see, e.g., U.S. Patent No. 5,478,925 ). The entire contents of this patent are incorporated herein by reference. In addition, the oligomers of the present invention can be produced using techniques known in the art to form one or more intermolecular bridges between cysteine residues located within the sequence of a polypeptide that need to be contained in the multimers E.g., U.S. Patent No. 5,478,925). The entire contents of this patent are incorporated herein by reference. In addition, application of techniques known in the art can be used to form liposomes containing polypeptide components that need to be included in the multimers of the present invention (see, e.g., U.S. Patent No. 5,478,925). The contents of this patent are incorporated herein by reference.

Alternatively, the oligomers of the present invention can be formed using genetic engineering techniques known in the art. In one embodiment, the polypeptides contained in the oligomers of the invention are recombinantly produced using fusion protein techniques as described herein or known in the art (see, e.g., U.S. Patent No. 5,478,925). The entire contents of this patent are incorporated herein by reference. In a particular embodiment, a polynucleotide encoding a homodimer of the invention comprises a polynucleotide sequence encoding a polypeptide of the invention linked to a sequence encoding a linker polypeptide, and then further comprises a nucleotide sequence encoding an N-terminal To a synthetic polynucleotide encoding the decryption product (lacking the leader sequence) of the polypeptide in the opposite direction (see, for example, U.S. Patent No. 5,478,925). The entire contents of this patent are incorporated herein by reference. In another embodiment, recombinant techniques according to the present invention described herein or known in the art are applied to produce recombinant polypeptides according to the present invention that contain a transmembrane domain and can be introduced into liposomes by membrane reconstitution techniques (see, e.g., U.S. Patent No. 5,478,925). The entire contents of this patent are incorporated herein by reference.

In one embodiment, the invention provides an isolated nucleic acid molecule comprising an amino acid sequence encoded by a cDNA clone contained in ATCC 97768 or a portion of a separate neurotoxin-alpha polypeptide or polypeptide thereof having the amino acid sequence shown in Figures IA and IB (SEQ ID NO: 2) (I. E., A fragment). ≪ / RTI > In yet another embodiment, the invention provides an isolated nucleic acid molecule comprising an amino acid sequence encoded by a cDNA clone contained in ATCC 203518 or a separate neurotoxin-alpha SV polypeptide or amino acid sequence thereof as shown in Figures 5A and 5B (SEQ ID NO: 19) (I. E., A fragment). ≪ / RTI >

The polypeptide fragment of the present invention is contained in SEQ ID NO: 2, or is encoded by the cDNA contained in the plasmid of ATCC Accession No. 97768, or the nucleotide sequence contained in the deposited clone or the nucleotide sequence contained in the nucleotide sequence shown in FIG. 1A-B (SEQ ID NO: 1) Or alternatively consisting of, an amino acid sequence that is encoded by a nucleic acid that hybridizes (e.g., under stringent hybridization conditions) with a complementary strand of the nucleotide sequence of interest.

In addition, the polypeptide fragment of the present invention may comprise a nucleotide sequence contained in SEQ ID NO: 19, or a nucleotide sequence encoded by the cDNA contained in the plasmid of ATCC Accession No. 203518 or contained in the deposited clone, , Or alternatively consists of, such sequences as are encoded by a nucleic acid that hybridizes (e.g., under stringent hybridization conditions) with the complementary strand of the nucleotide sequence described in SEQ ID NO: 1.

In addition, the polypeptide fragment of the present invention comprises an amino acid sequence encoded by a nucleic acid that hybridizes (e.g., under hybridization conditions described herein) to the complementary strand of the nucleotide sequence described in SEQ ID NO: 21, or alternatively Includes polypeptides consisting of such sequences.

The polypeptide fragment of the present invention also includes an amino acid sequence that is contained in SEQ ID NO: 23 or that is encoded by a nucleic acid that hybridizes with the complementary strand of the nucleotide sequence described in SEQ ID NO: 22 (eg, under the hybridization conditions described herein) Or, alternatively, a polypeptide consisting of such sequences.

The polypeptide fragment of the present invention also includes an amino acid sequence contained in SEQ ID NO: 28 or encoded by a nucleic acid that hybridizes with the complementary strand of the nucleotide sequence described in SEQ ID NO: 27 (for example, under the hybridization conditions described herein) Or, alternatively, a polypeptide consisting of such sequences.

The polypeptide fragment of the present invention also includes an amino acid sequence that is contained in SEQ ID NO: 30 or is encoded by a nucleic acid that hybridizes with the complementary strand of the nucleotide sequence shown in SEQ ID NO: 29 (eg, under hybridization conditions described herein) , Alternatively a polypeptide consisting of such sequences.

The polypeptide fragment of the present invention is contained in SEQ ID NO: 2, or is encoded by the cDNA contained in the deposited clone, the nucleotide sequence contained in the deposited clone or the nucleotide sequence described in FIGS. 1A and 1B (SEQ ID NO: 1) Or alternatively consists of, an amino acid sequence encoded by a nucleic acid that hybridizes with its complementary strand (e.g., under stringent hybridization conditions). Protein fragments may be present independently or in a larger polypeptide that contains some or one region most preferably as a single continuous region. Representative examples of polypeptide fragments according to the invention include about 1 to 50, 51 to 100, 101 to 150, 151 to 200, 201 to 250 and / or 251 to 285 of amino acid residues of SEQ ID NO: 2, alternatively And fragments consisting of such residues. In addition, the polypeptide fragment may be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175 or more than 200 amino acids in length.

In a particular embodiment, the polypeptide fragment of the invention comprises the amino acid residues 1-46, 31-44, 47-72, 73-285, 73-83, 94-102, 148-152 shown in Figures 1A and 1B , 166-181, 185-209, 210-221, 226-237, 244-249, 253-265, and / or 277-284, or alternatively consists of such residues.

The neurotoxin of the present invention comprising 19 amino acid residue insertions not found in the neurotoxin-alpha SV polypeptide sequence (i. E., The amino acid residues Val-142 to Lys-160 in the sequences described in Figures 1A and 1B and SEQ ID NO: 2) It will be appreciated by those skilled in the art that mutations targeting regions of the alpha-polypeptide may affect the observed biological activity of the neurotoxin-alpha polypeptides. More specifically, the partial, non-limiting, non-exclusive list of residues in the neurotoxin-alpha polypeptide sequence that may be the target of the mutation comprises the following amino acid residues in the neurotoxin-alpha polypeptide sequence of SEQ ID NO: 2 V-142; T-143; Q-144; D-145; C-146; L-147; Q-148, L-149; I-150; A-151; D-152; S-153; E-154; T-155; P-156; T-157; I-158; Q-159; And K-160. Polynucleotides encoding the neurotoxin-alpha polypeptide having one or more mutations in regions V-142 to K-160 of SEQ ID NO: 2 are contemplated. Polypeptides encoded by these polynucleotides are also included in the present invention.

Polypeptide fragments may be present independently or in the context of larger polypeptides in which the fragments occupy some or one region most preferably as a single continuous region. Representative examples of polypeptide fragments according to the present invention include amino acid residues of about 1 to 15, 16 to 30, 31 to 46, 47 to 55, 56 to 72, 73 to 104, 105 to 163, 163 to 188, 186 To 210 and 210 to 284, or alternatively comprises fragments consisting of such residues. Additional representative examples of polypeptide fragments according to the invention include amino acid residues of SEQ ID NO: 19 of about 1-143, 1-150, 47-143, 47-150, 73-143, 73-150, 100-150, 140-145, 142 to 148, 140 to 150, 140 to 200, 140 to 225 and 140 to 266, or alternatively, fragments consisting of such residues. In addition, the polypeptide fragment may be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175 or more than 200 amino acids in length. As used herein, " about " means a range that is larger or smaller by several in the amino acid and carboxy termini, i.e., 5, 4, 3, 2 or 1 amino acid residues in the specified range. Polynucleotides encoding these polypeptide fragments are also encompassed by the present invention.

Additional preferred embodiments include the putative intracellular domain of neurotoxin-alpha (amino acid residues 1 to 46 of SEQ ID NO: 2), the putative transmembrane domain of neurotoxin-alpha (amino acid residues 47 to 72 of SEQ ID NO: 2), neurotoxin- (Amino acid residues 73-285 of SEQ ID NO: 2), the deduced TNF conserved domain of neurotoxin-alpha (amino acids 191-284 of SEQ ID NO: 2), and the neurotoxin (Including amino acid residues 1 to 46 fused to amino acid residues 73 to 285 of SEQ ID NO: 2) fused to the putative extracellular domain of alpha. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

Further, a further preferred embodiment is the use of the putative intracellular domain of the neurotoxin-alpha SV (amino acid residues 1 to 46 of SEQ ID NO: 19), the putative transmembrane domain of neurotoxin-alpha SV (amino acid residues 47 to 72 of SEQ ID NO: 19) (The amino acid residues 73 to 266 of SEQ ID NO: 19), the putative TNF conserved domain of the neurotoxin-alpha SV (amino acids 172 to 265 of SEQ ID NO: 19) Polypeptide polypeptide and a polypeptide comprising or consisting of an estimated intracellular domain fused to the putative extracellular domain of neurotoxin-alpha SV (amino acid residues 1 to 46 fused to amino acid residues 73 to 266 of SEQ ID NO: 19) . Polynucleotides encoding these polypeptides are also encompassed by the present invention.

Certain further aspects of the invention include polypeptides fragments comprising or consisting of the putative beta-fitsite sheet regions described in Figure 7A. These polypeptides fragments of the present invention comprise the amino acid residues Gln-144 to Ala-151, Phe-172 to Lys-173, Ala-177 to Glu-179, Asn-183 to Ile-185, Gly-191 to Lys Leu-284, Leu-226 to Pro-237, Asn-242 to Ala-251, Gly-256 to Ile-263 and / or Val-276 to Leu-284, And the like. In another non-regulatory embodiment, these polypeptide fragments of the invention comprise the amino acid residues Phe-153 to Lys-154, Ala-158 to Glu-160, Asn-164 to Ile-166, Gly-172 to Lys -185, His-191 to Val-200, Leu-207 to Pro-218, Asn-223 to Ala-232, Gly-237 to Ile-244 and / or Vla-257 to Leu- Phe-42 to Lys-43, Ala-47 to Glu-49, Asn-53 to Ile-55, Gly-61 to Pro-74, His-80 to Val-89, Leu- 112 to Ala-121, Gly-126 to Ile-133 and / or Asp-146 to Leu-154. In a further non-limiting embodiment, the polypeptides fragments of the invention may also comprise amino acid residues Gln-78 to Ala-85, Phe-106 to Lys-107, Ala-111 to Glu-113, Asn- Ile-119, Gly-125 to Lys-138, His-144 to Val-153, Leu-160 to Pro-171, Asn-176 to Ala-185, Gly-190 to Ile-197 and / Leu-218 and the amino acid residues Gln-78 to Ala-85, Phe-106 to Lys-107, Ala-111 to Glu-113, Asn-117 to Ile-119, Gly-125 to Lys- -144 to Val-153, Leu-160 to Pro-171, Asn-176 to Ala-185, Gly-190 to Ile-197 and / or Val-210 to Leu-218. Polynucleotide fragments encoding these polypeptides are also provided by the present invention.

An example of a non-limiting example of a polypeptide according to the invention comprising, or alternatively consisting of, a combination of amino acid sequences according to the present invention is a polypeptide comprising a sequence of [Gly-250 to Leu-285] Leu-114 to Thr-141] fused to [Ile-142 to Lys-160] fused to [Gly-161 to Gln-198] fused to [Met- 1 to Lys-113]; Ile-142 to Lys-160 fused to [Gly-161 to Gln-198] fused to [Val-199 to Ala-248] fused to [Gly-250 to Leu-285] [Met-1 to Lys-113]; Or [Leu-114 to Thr-141] fused to [Ile-142 to Lys-160] fused to [Gly-161 to Gln-198] fused to [Gly-250 to Leu-285] Fused [Met-1 to Lys-113]. Other combinations may include polypeptide fragments that have been combined in other orders than those described above (e.g., a polypeptide fragment fused to [Gly-250 to Leu-285] fused to [Ile-142 to Lys-160] [Leu-114 to Thr-141] fused to [Val-199 to Ala-248]. Other combinations may also include heterologous polypeptide fragments as described herein and / or other polypeptides and polypeptide fragments of the invention (eg, [Gly-250 to Leu-285] of SEQ ID NO: 2 fused to a FLAG tag [Met-1 to Lys-113] fused to [Leu-114 to Thr-141] fused to [Ile-142 to Lys-160] fused to the fused [Gly-161 to Gln-198]. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

180-fused to [Gly-230 to Leu-266] of SEQ ID NO: 19 as a further non-limiting example of a polypeptide according to the invention comprising, or alternatively consisting of, [Met-1 to Lys-113] fused to [Leu-114 to Thr-141] fused to [Gly-142 to Gln-179] fused to Ala-229; [Met-1 to Lys-113] fused to [Gly-142 to Gln-179] fused to [Val-180 to Ala-229] fused to [Gly-230 to Leu-266] of SEQ ID NO: 19; Or [Met-1 to Lys-113] fused to [Leu-114 to Thr-141] fused to [Gly-142 to Gln-179] fused to [Gly-230 to Leu-266] . Other combinations of the polypeptide fragments combined in other orders than those described above can be included (e.g., a fusion of [Gly-230 to Leu-266] fused to [Gly-142 to Gln-179] [Leu-114 to Thr-141] fused to [Val-180 to Ala-229]. Other combinations may also include heterologous polypeptide fragments as described herein and / or other polypeptides and polypeptide fragments of the invention (e.g., [Gly-230 to Leu-266] of SEQ ID NO: 19 fused to a FLAG tag [Met-1 to Lys-113] fused to [Leu-114 to Thr-141] fused to the fused [Gly-142 to Gln-179]. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

225, which is fused to [Gly-273 to Leu-309] of SEQ ID NO: 23 as a further non-limiting example of a polypeptide according to the present invention comprising, or alternatively consists of, Leu-107 to Thr-134 fused to [Ile-167 to Lys-184] fused to [Gly-185 to Gln-224] fused to [Ala- 106]; Ile-167 to Lys-184] fused to [Gly-185 to Gln-224] fused to [Val-225 to Ala-272] fused to [Gly-273 to Leu-309] [Met-1 to Lys-106] fused to [Glu-135 to Asn-165]; Or [Glu-135 to Asn-165] fused to [Ile-167 to Lys-184] fused to [Gly-185 to Gln-224] fused to [Gly-273 to Leu-309] And [Met-1 to Lys-106] fused to fused [Leu-107 to Thr-134]. Other combinations may include polypeptide fragments that are combined in a sequence other than those described above (e.g., a polypeptide fragment fused to [Leu-107 to Thr-134] fused to [Gly-273 to Leu-309] [Met-1 to Lys-106] fused to [Gly-185 to Gln-224] fused to [Ile-167 to Lys-184] fused to [Val-225 to Ala-272]. Other combinations may also include heterologous polypeptide fragments as described herein and / or other polypeptides and polypeptide fragments of the invention (e.g., [Gly-273 to Leu-309] of SEQ ID NO: 23 fused to a FLAG tag Fused to [Glu-135 to Asn-165] fused to [Ile-167 to Lys-184] fused to [Gly-185 to Gln-224] fused to the fused [Val-225 to Ala-272] [Met-1 to Lys-106]). Polynucleotides encoding these polypeptides are also encompassed by the present invention.

133 to Ala-219] fused to [Gly-183 to Ala-219] of SEQ ID NO: 28 as a further non-limiting example of a polypeptide according to the invention comprising, or alternatively consisting of, Leu-48 to Thr-75] fused to [Ile-76 to Lys-94] fused to [Gly-95 to Gln-132] fused to [Leu- 47]; [Tyr-1 to Lys-47] fused to [Leu-48 to Thr-75] fused to [Ile-76 to Lys-94] fused to [Val-133 to Ala-182] of SEQ ID NO: 28; Or [Tyr-1 to Lys-47] fused to [Ile-76 to Lys-94] fused to [Val-133 to Ala-182] fused to [Gly-183 to Ala-219] . Other combinations of polypeptide fragments other than those described above may be included (e.g., a fusion of [Val-133 to Ala-182] fused to [Leu-48 to Thr-75] [Tyr-1 to Lys-47] fused to [Gly-183 to Ala-219]. Other combinations may also include heterologous polypeptide fragments and / or other polypeptides and polypeptide fragments of the invention described herein (e.g., [Val-133 to Ala-182] of SEQ ID NO: 28 fused to an Fc receptor tag, [Leu-48 to Thr-75] fused to [Ile-76 to Lys-94] fused to [Gly-95 to Gln-132] Polynucleotides encoding these polypeptides are also encompassed by the present invention.

133 to Ala-219] of SEQ ID NO: 30 as a further non-limiting example of a polypeptide according to the invention comprising, or alternatively consisting of, a combination of amino acid sequences of SEQ ID NOs: Leu-48 to Thr-75] fused to [Ile-76 to Lys-94] fused to [Gly-95 to Gln-132] fused to [Leu- 47]; [Tyr-1 to Lys-47] fused to [Leu-48 to Thr-75] fused to [Ile-76 to Lys-94] fused to [Val-133 to Ala-182] of SEQ ID NO: 30; Or [Tyr-1 to Lys-47] fused to [Ile-76 to Lys-94] fused to [Val-133 to Ala-182] fused to [Gly-183 to Ala-219] . Other combinations may include polypeptide fragments that are combined in a sequence other than those described above (e.g., fused to [Val-133 to Ala-182] fused to [Leu-48 to Thr-75 of SEQ ID NO: 30] [Tyr-1 to Lys-47] fused to [Gly-183 to Ala-219]. Other combinations may also include heterologous polypeptide fragments as described herein and / or other polypeptides and polypeptide fragments of the invention (e.g., [Val-133 to Ala-182] of SEQ ID NO: 30 fused to an Fc tag [Leu-48 to Thr-75] fused to [Ile-76 to Lys-94] fused to the fused [Gly-95 to Gln-132]. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

A further aspect of the present invention is the use of a Ganer-Robson alpha-region, a beta-region, a turn-region and a coil-region as described in Figures 3 and 6 and Table 1 and described herein, The present invention relates to the use of the Jamesson-Tropsch suppositories of the Eisenberg alpha-and beta-amphipathic regions, the Caplus-Schultz bend region, the eminite surface-forming region and the high antigenic index, Alpha < / RTI > and / or neurotoxin-alpha SV polypeptide fragments comprising, or alternatively consisting of, such regions as the active region of the polypeptide of the invention, such as the wild-type region. In a preferred embodiment, the polypeptide fragment of the invention is antigenic. The data described in columns VIII, IX, XIII and XIV of Table 1 can be routinely used to determine regions of neurotoxin-alpha that exhibit a high degree of antigenicity potential. Regions of high antigenicity can be detected in columns VIII, IX, XIII and / or IV by selecting a value that represents a region of the polypeptide that is likely to be exposed to the surface of the polypeptide in an environment where antigen recognition may occur in the initiation of the immune response Measure from the described data. A highly preferred fragment of the present invention includes a region of neurotoxin-alpha and / or neurotoxin-alpha SV that combines several structural features, such as several (e.g., 1, 2, 3 or 4) And the like. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

In another embodiment, the invention provides polypeptides comprising, or alternatively consisting of, epitope-containing portions of polypeptides according to the invention. Polynucleotides encoding these polypeptides are also encompassed by the present invention. The epitope of this polypeptide portion is an immunogenic or antigenic epitope of the polypeptide according to the invention. An " immunogenic epitope " is defined as a portion of a protein that triggers an antibody response when the complete protein is an immunogen. On the other hand, the region of a protein molecule to which an antibody can bind is defined as an " antigenic epitope ". The number of immunogenic epitopes of a protein is generally less than the number of antigenic epitopes (see, e.g., Geysen et al., Proc. Natl. Acad. Sci. USA 81: 3998-4002 (1983)).

With respect to the selection of polypeptides containing an antigenic epitope (i. E., Containing a region of a protein molecule capable of binding to an antibody), relatively short synthetic peptides that mimic a portion of the protein sequence are reacted with partially simulated proteins (See, for example, Sutcliffe, JG, Shinnick, TM, Green, N. and Learner, RA (1983) "Antibodies that react with selected sites on proteins", Science, 219 : 660-666). Peptides that can induce protein-reactive sera are commonly found in the primary sequence of a protein and can be characterized by a set of simple chemical rules and can be found in the immunodominant region of a complete protein (i. E., An immunogenic epitope) The terminal is not limited to anywhere. Accordingly, the antigenic epitope-containing peptides and polypeptides according to the present invention are useful for inducing antibodies (including monoclonal antibodies) that specifically bind to the polypeptides of the present invention (Wilson et al., Cell 37: 767-778 (1984) at 777).

The antigenic epitope-containing peptides and polypeptides of the present invention preferably contain at least 4, at least 6, at least 7 amino acid sequences contained in the amino acid sequence of the polypeptide of the present invention, At least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, Contain at least 50 amino acid sequences, and most preferably contain about 15 to about 30 amino acid sequences. Preferred polypeptides comprising an immunogenic or antigenic epitope are polypeptides of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 Or 100 amino acid residues. Additional conventional preferred antigenic epitopes include the antigenic epitopes described herein as well as portions thereof.

Non-limiting examples of antigenic polypeptides or peptides that can be used to generate neurotoxin-alpha-and / or neurotoxin-alpha SV-specific antibodies include the following: Figures 1A and 1B (SEQ ID NO: 2) A polypeptide comprising, or alternatively consisting of, the amino acid residues of about Phe-115 to about Leu-147 as shown; Polypeptides comprising, or alternatively consisting of, amino acid residues from about Ile-150 to about Tyr-163 shown in Figures 1A and 1B (SEQ ID NO: 2); A polypeptide comprising, or alternatively consisting of, the amino acid residues of about Ser-171 to about Phe-194 shown in Figures 1A and 1B (SEQ ID NO: 2); A polypeptide comprising, or alternatively consisting of, the amino acid residues of about Glu-223 to about Tyr-246 shown in Figures 1A and 1B (SEQ ID NO: 2); And a polypeptide comprising, or alternatively consisting of, the amino acid residues of about Ser-271 to about Phe-278 shown in Figures 1A and 1B (SEQ ID NO: 2). As used herein, " about " means a greater or lesser extent of amino acids in either or both amino- and carboxy-termini in the specified range, i.e., 5, 4, 3, 2 or 1 amino acid residues. These polypeptide fragments were determined to contain an antigenic epitope of the neurotoxin-alpha polypeptide by analysis of the Jamesson-Wolf antigen index, as described in FIG. 3 and Table 1.

Non-limiting examples of antigenic polypeptides or peptides that can be used to generate neurotoxin-alpha-and / or neurotoxin-alpha SV-specific antibodies include the following: Figures 5A and 5B (SEQ ID NO: 19) A polypeptide comprising, or alternatively consisting of, the amino acid residues of about Pro-32 to about Leu-47 as shown; Polypeptides comprising, or alternatively consisting of, amino acid residues from about Glu-116 to about Ser-143 shown in Figures 5A and 5B (SEQ ID NO: 19); Polypeptides comprising, or alternatively consisting of, the amino acid residues of about Phe-153 to about Tyr-173 shown in Figures 5A and 5B (SEQ ID NO: 19); Polypeptides comprising, or alternatively consisting of, the amino acid residues of about Pro-218 to about Tyr-227 shown in Figures 5A and 5B (SEQ ID NO: 19); Polypeptides comprising, or alternatively consisting of, the amino acid residues of about Ala-232 to about Gln-241 shown in Figures 5A and 5B (SEQ ID NO: 19); A polypeptide comprising, or alternatively consisting of, the amino acid residues of about Ile-244 to about Ala-249 shown in Figures 5A and 5B (SEQ ID NO: 19); And a polypeptide comprising, or alternatively consisting of, the amino acid residues of about Ser-252 to about Val-257 shown in Figures 5A and 5B (SEQ ID NO: 19). As used herein, " about " means a greater or lesser extent of amino acids in either or both amino- and carboxy-termini in the specified range, i.e., 5, 4, 3, 2 or 1 amino acid residues. Polynucleotides encoding these polypeptides, or the present invention. These polypeptide fragments were analyzed by the analysis of the Jameson-Wolf antigen index described in Figure 6 above and the antigens of the neurotoxin-alpha polypeptide by a table for the data in Figure 6 obtained by the proteome component of the DNA * STAR computer program Lt; RTI ID = 0.0 > epitope. ≪ / RTI >

The epitope-containing peptides and polypeptides of the present invention can be produced by conventional means (Houghten, RA (1985) General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids Proc. Natl. Acad. Sci. USA 82: 5131-5135). This " simultaneous multiple peptide synthesis (SMPS) " method is also described in U.S. Patent No. 4,631,211 (1986) to Houghten et al.

The epitope-containing peptides and polypeptides of the present invention are used to induce antibodies according to methods well known in the art, including but not limited to those described in Sutcliffe et al., Supra; Wilson et al .; Chow USA et al., Proc Natl Acad Sci USA 82: 910-914, and Bittle, FJ et al., J. Gen. Virol. 66: 2347-2354 (1985)). The immunogenic epitope-containing peptide of the present invention, i.e., a portion of the protein that causes the antibody reaction when the complete protein is an immunogen, is identified according to methods known in the art (see, for example, Geysen et al., Supra). Geysen U.S. Pat. No. 5,194,392 (1990) also discloses the sequence of a monomer that is a topological equivalent of an epitope (i. E., &Quot; mimotope ") complementary to a specific paratope (antigen binding site) ≪ / RTI > is detected or measured. More generally, US Patent 4,433,092 (1989) of Geysen describes a method for detecting or measuring the sequence of a monomer that is a topological equivalent of a ligand complementary to the ligand binding site of the particular receptor. Similarly, U.S. Patent No. 5,480,971 to Houghten, RA et al. (1996) for a mixture of alkylated oligopeptides includes linear C1-C7-alkyl and alkylated oligopeptides and sets and libraries of such peptides, as well as preferential binding with corresponding receptor molecules Lt; RTI ID = 0.0 > oligopeptide < / RTI > sets and libraries to determine the sequence of the alkylated oligopeptides. Thus, non-peptide analogs of the epitope-containing peptides of the invention can also be routinely produced by these methods.

The present invention encompasses an epitope of a polypeptide having the amino acid sequence of SEQ ID NO: 2 or a polynucleotide sequence contained in ATCC Accession No. 97768 or a complementary sequence of the sequence of SEQ ID NO: 1, or a cDNA sequence contained in ATCC Accession No. 97768 (E. G., Under hybridization conditions described herein) of a polypeptide encoded by a polynucleotide, or alternatively comprises a polypeptide consisting of such an epitope. The invention also encompasses a polynucleotide sequence comprising, or alternatively consisting of, a sequence encoding the epitope of the polypeptide sequence of the invention (e. G., The sequence described in SEQ ID NO: 1), a polynucleotide sequence encoding the epitope of the invention Polynucleotide sequences of the complementary strand of the polynucleotide sequence and polynucleotide sequences that hybridize with the complementary strand (e.g., under the hybridization conditions described herein).

In addition, the present invention encompasses an epitope of a polypeptide having the amino acid sequence of SEQ ID NO: 19, which is encoded by the cDNA sequence contained in ATCC Accession No. 203518 or a complementary sequence of the sequence of SEQ ID NO: 18 or a cDNA contained in ATCC Accession No. 203518 Includes, or alternatively comprises, an epitope of a polypeptide sequence that is encoded by a polynucleotide that hybridizes with the sequence (e. G., Under hybridization conditions described herein). Alternatively, the polypeptide comprises such an epitope. The invention also encompasses polynucleotide sequences comprising, or alternatively consisting of, a sequence encoding an epitope of the polypeptide sequence of the invention (e. G., The sequence described in SEQ ID NO: 18), a polynucleotide sequence encoding the epitope of the invention Polynucleotide sequences of the complementary strand of the polynucleotide sequence and polynucleotide sequences that hybridize with the complementary strand (e.g., under the hybridization conditions described herein).

The term " epitope " as used herein refers to a portion of a polypeptide having an antigenic or immunogenic activity in an animal, preferably a mammal, most preferably a human. In a preferred embodiment, the invention encompasses polypeptides containing epitopes as well as polynucleotides encoding the polypeptides. As used herein, an " immunogenic epitope " is defined as a portion of a protein that elicits an antibody response in an animal when measured by methods known in the art, for example, by methods of generating antibodies described in the following references : Geysen et al., Proc Natl Acad Sci USA 81: 3998-4002 (1983)). As used herein, " antigenic epitope " is intended to include a portion of a protein that is capable of immunologically binding to an antigen of the antibody when measured by immunoassays as described herein, eg, Is defined. Immunospecific binding excludes non-specific binding, but does not necessarily exclude cross-reactivity with other antigens. An antigenic epitope does not necessarily have to be immunogenic.

Fragments that act as epitopes can be prepared by conventional means (see for example Houghten, Proc. Natl. Acad. Sci. USA 82: 5131-5135 (1985) and U.S. Patent 4,631,211).

In the present invention, the antigenic epitope preferably has 4 or more, 5 or more, 6 or more, 7 or more, more preferably 8 or more, 9 or more, 10 or more, 11 or more, 12 or more , 13, 14, 15, 20, 25, 30, 40, 50, and most preferably about 15 to about 30 amino acids . Preferred polypeptides comprising an immunogenic or antigenic epitope are polypeptides of length 10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95 Or more than 100 amino acids. Additional conventional preferred antigenic epitopes include the antigenic epitopes described herein as well as portions thereof. An antigenic epitope is useful, for example, to elicit an antibody (including a monoclonal antibody) that specifically binds to an epitope. Preferred antigenic epitopes include the antigenic epitopes described herein as well as combinations of 2, 3, 4, 5 or more of these antigenic epitopes. Antigenic epitopes can be used as target molecules in immunoassays (see for example Wilson et al., Cell 37: 767-778 (1984); Sutcliffe et al., Science 219: 660-666 (1983)).

Similarly, immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art (see, for example, Sutcliffe et al., Supra; Wilson et al .; Chow et al., Proc. Natl. Acad Sci USA 82: 910-914; and Bittle et al., J. Gen. Virol. 66: 2347-2354 (1985)). Preferred antigenic epitopes comprise the immunogenic epitopes described herein as well as combinations of 2, 3, 4, 5 or more of these immunogenic epitopes. A polypeptide comprising one or more immunogenic epitopes may be provided to induce an antibody response with an animal system (e. G., Rabbit or mouse) with a carrier protein such as albumin, or if the polypeptide is of sufficient length ) The polypeptide may be provided without a carrier. However, it has been suggested that an immunogenic epitope comprising from 8 to 10 fewer amino acids is sufficient (e. G., In Western blotting) to induce antibodies that are capable of binding very little with a linear epitope in the modified polypeptide.

The epitope-containing polypeptides of the present invention can be used to induce antibodies according to methods well known in the art, including, but not limited to, in vivo immunization, in vitro immunization and phage display methods (see, e.g., Wilton et al .; and Bittle et al., J. Gen. Virol., 66: 2347-2345 (1985)). When in vivo immunization is used, the animal can be immunized with a free peptide. However, anti-peptide antibody titer can be elevated by linking peptides to macromolecular carriers such as clam hemaglissia (KLH) or tetanus toxin. For example, a peptide containing a cysteine residue can be linked to a carrier using a linker such as maleimido benzoyl-N-hydroxysuccinimide ester (MBS) while the other peptide is linked to a more common linker such as glutaraldehyde Can be used to connect to the carrier. Animals such as rabbits, rats and mice can be administered by intraperitoneal and / or intradermal injection of an emulsion containing, for example, about 100 micrograms of peptide or carrier protein and a Freund ' s adjuvant or other excipient known to stimulate the immune response Free or carrier-bound peptide. Several inoculation scans may be required, for example, at intervals of about two weeks, to provide useful potency of anti-peptide antibodies that can be assayed, for example, by ELISA assays using free peptide adsorbed on a solid surface. Reverse transcription of anti-peptide antibodies in sera from immunized animals can be increased by the selection of anti-peptide antibodies, for example by adsorption of the peptide to a solid support and elution of the selected antibodies according to methods well known in the art have.

As those skilled in the art are aware and discussed above, the polypeptides of the present invention, including immunogenic or antigenic epitopes, may be fused to other polypeptide sequences. For example, the polypeptides of the invention can be fused with the constant domain of an immunoglobulin (IgA, IgE, IgG, IgM) or a portion thereof (CH1, CH2, CH3 or a combination and a portion thereof) to generate a chimeric polypeptide . Such fusion proteins can promote purification and increase the half-life in vivo. This has been demonstrated for chimeric proteins consisting of the first two domains of human CD4-polypeptide and several domains of the constant region of the heavy or light chain of mammalian immunoglobulins (see for example EP394,827; Traunecker et al., Nature, 331: 84-86 (1988)). Increased delivery of antigen to the immune system along with epithelial barriers has been demonstrated for antigens conjugated to FcRn binding partners such as IgG or Fc fragments (see, e.g., PCT Publication Nos. WO 96/22024 and WO 99/04813). IgG fusion proteins having a disulfide-linked dimer structure due to IgG partial disulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than a single monomer polypeptide or fragment thereof (see, e.g., Fountoulakis et al., J The nucleic acid encoding the epitope can also be used in conjunction with an epitope tag (e.g., a hemagglutinin (" HA ") tag or a flag (e. G. The system described by Janknecht et al., For example, allows easy purification of unmodified fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. USA, 88: 9872-897). In this system, the gene is inserted into the amino-terminal tag consisting of 6 histidine residues Such as fusion dokjeok is cloned into a vaccinia recombination plasmid ah tag matrix for a fusion protein - the loaded agarose column - acts as a binding domain to extract acetic acid Ni ²⁺ nitrile from cells infected with a recombinant vaccinia virus And the histidine-tagged protein can be selectively eluted with the imidazole-containing buffer.

In another embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides and epitope-containing fragments thereof of the invention are fused with heterologous antigens (e.g., polypeptides, carbohydrates, phospholipids or nucleic acids). In certain embodiments, the heterologous antigen is an immunogen.

In a more specific embodiment, the heterologous antigen is the gp120 protein of HIV or a fragment thereof. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

In another embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides and epitope-containing fragments thereof of the invention are fused with a polypeptide sequence (or biologically active fragment or variant thereof) of another TNF ligand system member . In certain embodiments, the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides of the invention are fused to a CD40L polypeptide sequence. In a preferred embodiment, the CD40L polypeptide sequence is soluble.

Alpha and / or neurotoxin-alpha SVs using techniques of gene-shuffling, motif-shuffling, exon-shuffling and / or codon-shuffling (collectively referred to as "DNA shuffling" Activity and thus effectively produce agonists and antagonists of neurotoxin-alpha and / or neurotoxin-alpha SV (see, for example, U.S. Patent Nos. 5,605,793, 5,605,793, 5,811,238, 5,830,721 , 5,834,252 and 5,837,458 and Patten, PA, et al., Curr. Opinion Biotechnol. 8: 724-33 (1997); Harayama, S. Trends Biotechnol. 16 (2): 76-82 (1998); Lans et al., J. Mol. Biol. 287: 265-76 (1999), and Lorenzo, MM and Blasco, R. Biotechniques 24 (2): 308-13 (1998)). Each of the above patents and documents is incorporated herein by reference. In one embodiment, mutations of the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides and corresponding polypeptides can be achieved by DNA shuffling. DNA shuffling involves the insertion of two or more DNA fragments into a desired neurotoxin-alpha and / or neurotoxin-alpha SV molecule by homologous recombination or site-specific recombination. In another embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides and corresponding polypeptides can be mutated by application to random mutation induction by error-directed PCR, random nucleotide insertion or other methods prior to recombination have. In yet another embodiment, one or more components, motifs, regions, fragments, domains, fragments, etc. of a neurotoxin-alpha and / or neurotoxin-alpha SV are linked to one or more components, motifs, Fragments, and the like. In a preferred embodiment, the heterologous molecule is selected from the group consisting of TNF-alpha, lymphotoxin-alpha (also known as LT-alpha, also known as TNF-beta), LT-beta (found in complex heterotrimeric LT- , OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO 96/14328), AIM-I (International Publication No. WO 97/34911), APRIL OPG, OX40 and nerve growth factor (NGF), and Fas, CD30, CD27, CD40 and 4-IBB, as well as endocaine-alpha (International Publication No. WO 98/07880) Soluble form, TR2 (International Publication No. WO 96/34095), DR3 (International Publication No. WO 97/33904), DR4 (International Publication No. WO 98/32856), TR4 (International Publication No. WO 98/30693), TR6 (International Publication No. WO 98/30694), TR7 (International Publication No. WO 98/41629), TRANK, TR9 (International Publication No. WO 98/56892), TR10 (International Publication No. WO 98/54202), 312C2 / 06842), TR12, CAD and v-FLIP. In a further embodiment, the heterologous molecule includes all members of the TNF family.

In a preferred embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides (including biologically active fragments or variants thereof) of the invention are fused with soluble CD40L polypeptides or biologically active fragments or variants thereof.

Protein manipulation can be used to enhance or modify the characteristics of the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides. Recombinant DNA techniques known to those skilled in the art can be used to generate new mutant proteins or muteins, including one or more amino acid substitutions, deletions, additions, or fusion proteins. Such modified polypeptides may, for example, exhibit enhanced activity or increased stability. In addition, they can be purified at a higher yield and exhibit better solubility than the corresponding native polypeptide at least under certain purification and storage conditions. For example, it is known in the art that in the case of many proteins, including extracellular domains or mature forms of secreted proteins, one or more amino acids from the N-terminus or C-terminus can be deleted without substantial loss of biological function have. See, e.g., Ron et al., J. Biol. Chem., 268: 2984-2988 (1993)) describes a modified KGF protein that exhibits heparin binding activity although 3, 8 or 27 amino-terminal amino acid residues have been deleted.

In the present case, since the protein of the present invention is a member of the TNF polypeptide family, deletion of the N-terminal amino acid to the Gly (G) residue at position 191 in Figures 1A and 1B (SEQ ID NO: 2) , B cells) to stimulate proliferation, differentiation and / or activation and cytotoxicity to appropriate target cells. Polypeptides with additional N-terminal deletions, including the Gly (G) residues, will retain biological activity because this residue in the TNF-related polypeptide is known to be within the starting point of the conserved domain required for biological activity Not expected. However, even if one or more biological functions of the protein are modified or lost when one or more amino acids from the N-terminus of the protein are deleted, other functional activities can still be maintained. Thus, the ability of a shortened protein to induce and / or bind to an antibody that recognizes the complete or extracellular domain of a protein is maintained when the subunits of the complete or extracellular domain of the protein are removed from the N- Will be. Whether a particular polypeptide lacking the N-terminal residue of the complete protein retains such immunological activity can be readily determined by conventional methods as described herein and known in the art.

Thus, the present invention also provides a polypeptide having deletion of one or more residues from the amino terminus of the amino acid sequence of the neurotoxin-alpha shown in Figures 1A and 1B (SEQ ID NO: 2) to the glycine residue at position 191 (amino acid terminus to GIy191 residue) And polynucleotides encoding such polypeptides. In particular, the present invention encompasses a polypeptide comprising the amino acid sequence of residue n ¹ -285 in SEQ ID NO: 2, wherein n ¹ is an integer in the range of amino acid positions 2-190 of the amino acid sequence of SEQ ID NO: 2, Provides a polypeptide consisting of such sequences. Polynucleotides encoding these polypeptides are also encompassed by the present invention. More particularly, the present invention relates to a polypeptide comprising the amino acid sequence of residues 2-285 of SEQ ID NO: 2,

And 190-285, or alternatively comprises a polynucleotide encoding a polypeptide consisting of such a sequence. Polypeptides encoded by these polynucleotides are also included in the present invention. The present invention also provides a polynucleotide encoding a neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide as described above, which is 80%, 85%, 90%, 92%, 95%, 96% 98% or 99% identical to or more than the polynucleotide sequence of SEQ ID NO. The invention also encompasses polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and / or polynucleotide sequences are also encompassed by the present invention. In addition, the present invention encompasses amino acid sequences which comprise at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% Polypeptides composed of sequences and polynucleotides encoding such polypeptides are included in the present invention.

In addition, since the putative extracellular domain of neurotoxin-alpha according to the present invention can maintain its biological activity on its own, it is possible to predict from the putative extracellular domain of the polypeptide (positions Gln-73 to Leu-285 of SEQ ID NO: 2) Deletion of the N- and C- terminal amino acid residues may result in some biological activity such as, for example, ligand binding, stimulation of lymphocyte (e.g., B cell) proliferation, differentiation and / or activation and modulation of cell replication or control of target cell activity . However, even if one or more biological functions of the polypeptide are altered or lost when one or more amino acids are deleted from the N-terminus of the putative extracellular domain of the neurotoxin-alpha polypeptide, other functional activity is still retained . Thus, the ability of a shortened protein to induce and / or bind to an antibody that recognizes the complete or mature or extracellular domain of the polypeptide is determined by determining that less than a major portion of the residues of the complete or mature or extracellular domain of the polypeptide is N- Will be retained when removed from the terminus. Whether a particular polypeptide lacking the N-terminal residue of the complete polypeptide retains such immunological activity can be readily determined by conventional methods known in the art and known in the art.

Thus, the present invention also provides a polypeptide wherein one or more residues are deleted from the amino terminus of the amino acid sequence of neurotoxin-alpha described in SEQ ID NO: 2 to the glycine residue at position 280, and a polynucleotide encoding such a polypeptide. In particular, the present invention residues in SEQ ID NO: 2, n ² -285 (where, n ² is an integer in SEQ ID NO: 2 within the range of the amino acid position of amino acid residues 73-280, and 73 is a neutroavidin Keene described in SEQ ID NO: 2-alpha polypeptide Or the position of the first residue from the N-terminus of the putative extracellular domain), or alternatively comprises a polypeptide consisting of such sequence. Polynucleotides encoding these polypeptides are also encompassed by the present invention. More particularly, in certain embodiments, the present invention provides a compound having the structure of residues Q-73 to L-285 of SEQ ID NO: 2; G-74 to L-285; D-75 to L-285; L-76 to L-285; A-77 to L-285; S-78 to L-285; L-79 to L-285; R-80 to L-285; A-81 to L-285; E-82 to L-285; L-83 to L-285; Q-84 to L-285; G-85 to L-285; H-86 to L-285; H-87 to L-285; A-88 to L-285; E-89 to L-285; K-90 to L-285; L-91 to L-285; P-92 to L-285; A-93 to L-285; G-94 to L-285; A-95 to L-285; G-96 to L-285; A-97 to L-285; P-98 to L-285; A-99 to L-285; A-100 to L-285; G-101 to L-285; L-102 to L-285; E-103 to L-285; E-104 to L-285; A-105 to L-285; P-106 to L-285; A-107 to L-285; V-108 to L-285; T-109 to L-285; A-110 to L-285; G-111 to L-285; L-112 to L-285; K-113 to L-285; I-114 to L-285; F-115 to L-285; E-116 to L-285; P-117 to L-285; P-118 to L-285; A-119 to L-285; P-120 to L-285; G-121 to L-285; E-122 to L-285; G-123 to L-285; N-124 to L-285; S-125 to L-285; S-126 to L-285; Q-127 to L-285; N-128 to L-285; S-129 to L-285; R-130 to L-285; N-131 to L-285; K-132 to L-285; R-133 to L-285; A-134 to L-285; V-135 to L-285; Q-136 to L-285; G-137 to L-285; P-138 to L-285; L-139 to L-285; E-140 to L-285; T-141 to L-285; V-142 to L-285; T-143 to L-285; Q-144 to L-285; D-145 to L-285; C-146 to L-285; L-147 to L-285; Q-148 to L-285; L-149 to L-285; I-150 to L-285; A-151 to L-285; D-152 to L-285; S-153 to L-285; E-154 to L-285; T-155 to L-285; P-156 to L-285; T-157 to L-285; I-158 to L-285; Q-159 to L-285; K-160 to L-285; G-161 to L-285; S-162 to L-285; Y-163 to L-285; T-164 to L-285; F-165 to L-285; V-166 to L-285; P-167 to L-285; W-168 to L-285; L-169 to L-285; L-170 to L-285; S-171 to L-285; F-172 to L-285; K-173 to L-285; R-174 to L-285; G-175 to L-285; S-176 to L-285; A-177 to L-285; L-178 to L-285; E-179 to L-285; E-180 to L-285; K-181 to L-285; E-182 to L-285; N-183 to L-285; K-184 to L-285; I-185 to L-285; L-186 to L-285; V-187 to L-285; K-188 to L-285; E-189 to L-285; T-190 to L-285; G-191 to L-285; Y-192 to L-285; F-193 to L-285; F-194 to L-285; I-195 to L-285; Y-196 to L-285; G-197 to L-285; Q-198 to L-285; V-199 to L-285; L-200 to L-285; Y-201 to L-285; T-202 to L-285; D-203 to L-285; K-204 to L-285; T-205 to L-285; Y-206 to L-285; A-207 to L-285; M-208 to L-285; G-209 to L-285; H-210 to L-285; L-211 to L-285; I-212 to L-285; Q-213 to L-285; R-214 to L-285; K-215 to L-285; K-216 to L-285; V-217 to L-285; H-218 to L-285; V-219 to L-285; F-220 to L-285; G-221 to L-285; D-222 to L-285; E-223 to L-285; L-224 to L-285; S-225 to L-285; L-226 to L-285; V-227 to L-285; T-228 to L-285; L-229 to L-285; F-230 to L-285; R-231 to L-285; C-232 to L-285; I-233 to L-285; Q-234 to L-285; N-235 to L-285; N-236 to L-285; P-237 to L-285; E-238 to L-285; T-239 to L-285; L-240 to L-285; P-241 to L-285; N-242 to L-285; N-243 to L-285; S-244 to L-285; C-245 to L-285; Y-246 to L-285; S-247 to L-285; A-248 to L-285; G-249 to L-285; I-250 to L-285; A-251 to L-285; K-252 to L-285; L-253 to L-285; E-254 to L-285; E-255 to L-285; G-256 to L-285; D-257 to L-285; E-258 to L-285; L-259 to L-285; Q-260 to L-285; L-261 to L-285; A-262 to L-285; I-263 to L-285; P-264 to L-285; R-265 to L-285; E-266 to L-285; N-267 to L-285; A-268 to L-285; Q-269 to L-285; I-270 to L-285; S-271 to L-285; L-272 to L-285; D-273 to L-285; G-274 to L-285; D-275 to L-285; V-276 to L-285; T-277 to L-285; F-278 to L-285; F-279 to L-285 and G-280 to L-285, or alternatively, a polynucleotide encoding a polypeptide consisting of such a sequence. Polypeptides encoded by these polynucleotides are also included in the present invention. The present invention also provides a polynucleotide encoding a neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide as described above, which is 80%, 85%, 90%, 92%, 95%, 96% 98% or 99% identical to or more than the polynucleotide sequence of SEQ ID NO. The invention also encompasses polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and / or polynucleotide sequences are also encompassed by the present invention. In addition, the present invention encompasses amino acid sequences which comprise at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% Polypeptides composed of sequences and polynucleotides encoding such polypeptides are included in the present invention.

A highly preferred embodiment of the present invention is a polynucleotide encoding a neurotoxin-alpha polypeptide having an amino acid sequence at position 134-285 in Figures 1A and 1B (SEQ ID NO: 2) that is 80%, 85%, 90% Or alternatively consisting of, a polynucleotide having at least 95%, 96%, 97%, 98%, 99% or 100% identical nucleotide sequences. A preferred embodiment of the invention comprises a polynucleotide having at least 90% identical nucleotide sequence to a polynucleotide sequence encoding a neurotoxin-alpha polypeptide having an amino acid sequence at position 134-285 in Figures 1A and 1B (SEQ ID NO: 2) , Alternatively, a nucleic acid molecule comprising such a polynucleotide. A more preferred embodiment of the invention comprises a polynucleotide having a nucleotide sequence at least 95% identical to a polynucleotide sequence encoding a neurotoxin-alpha polypeptide having an amino acid sequence at position 134-285 in Figures 1A and 1B (SEQ ID NO: 2) Or, alternatively, to a nucleic acid molecule comprising such a polynucleotide. A more preferred embodiment of the invention comprises a polynucleotide having a nucleotide sequence at least 96% identical to a polynucleotide sequence encoding a neurotoxin-alpha polypeptide having an amino acid sequence at positions 134-285 in Figures 1A and 1B (SEQ ID NO: 2) Or, alternatively, to a nucleic acid molecule comprising such a polynucleotide. Further, a more preferred embodiment of the present invention is a polynucleotide encoding a neurotoxin-alpha polypeptide having an amino acid sequence at positions 134-285 in Figures 1A and 1B (SEQ ID NO: 2), wherein the polynucleotide sequence has at least 97% identical nucleotide sequence Or alternatively, a nucleic acid molecule comprising such a polynucleotide. In a further preferred embodiment of the present invention, a polynucleotide sequence encoding a neurotoxin-alpha polypeptide having an amino acid sequence at positions 134-285 in Figures 1A and 1B (SEQ ID NO: 2) Or alternatively, a nucleic acid molecule comprising such a polynucleotide. In a further preferred embodiment of the present invention, a polynucleotide sequence encoding a neurotoxin-alpha polypeptide having an amino acid sequence at position 134-285 in Figure 1A and 1B (SEQ ID NO: 2) Or alternatively, a nucleic acid molecule comprising such a polynucleotide.

In a particular embodiment, polypeptides comprising, or alternatively consisting of, one of the following N-terminal deleted polypeptide fragments, Neutrokine-alpha and / or the following are preferred: amino acid residues Ala-71 Leu-285, amino acid residues Ala-81 to Leu-285, amino acid residues Leu-112 to Leu-285, amino acid residues Ala-134 to Leu-285, amino acid residues Leu-147 to Leu- Leu-285. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

Similarly, many examples of biologically functional C-terminal deletion muteins are known. For example, interferon gamma shows up to 10-fold greater activity by deleting 8-10 amino acid residues from the carboxy terminus of this protein (Dobeli et al., J. Biotechnology 7: 199-2 (1998)). Because this protein is a member of the TNF polypeptide family, deletion of the C-terminal amino acid to the lysine residue at position 284 is not, but is not limited to, all biological activity, for example, ligand binding, lymphocyte (e.g., B cell) And / or the ability to stimulate activation and regulation of cell replication. Polypeptides having deletions of up to about 10 additional C-terminal residues (i.e., up to the glycine residue at position 274) may also be present in a conserved TNF domain wherein such polypeptide extends to about Leu-284 of SEQ ID NO: 2 Although one addition is deleted, some activity such as receptor binding can be maintained. However, even if deletion of one or more amino acids from the C-terminus of the protein induces modification or loss in one or more biological functions of the protein, other functional activity can still be maintained. Thus, the ability of a shortened protein to induce and / or bind an antibody that recognizes a complete or mature protein will be retained when less than a major portion of the complete or mature protein residue is removed from the C-terminus. Whether a particular polypeptide lacking the C-terminal residue of a complete protein retains such immunological activity can be readily determined by conventional methods as described herein and known in the art.

Thus, the present invention also provides polypeptides in which one or more residues are deleted from the carboxy terminus of the amino acid sequence of the neurotoxin-alpha polypeptide shown in Figures 1A and 1B (SEQ ID NO: 2) to the glycine residue at position 274, and polypeptides encoding such polypeptides Polynucleotide. In particular, the present invention encompasses a polynucleotide comprising or consisting of the amino acid sequence of residue 1-m ¹ in SEQ ID NO: 2, wherein m ¹ is an integer within the amino acid position of amino acid residue 274-284 in SEQ ID NO: 2, ≪ / RTI > Polynucleotides encoding these polypeptides are also encompassed by the present invention. More particularly, in certain embodiments, the present invention provides a compound of formula (I) wherein residues 1-274, 1-275, 1-276, 1-277, 1-278, 1-279, 1-280, 1-281, 1-282, 1-283 and 1-284, or alternatively comprises a polynucleotide encoding a polypeptide consisting of such a sequence. Polypeptides encoded by these polynucleotides are also included in the present invention. The present invention also provides a polynucleotide encoding a neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide as described above, which is 80%, 85%, 90%, 92%, 95%, 96% 98% or 99% identical to or more than the polynucleotide sequence of SEQ ID NO. The invention also encompasses polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and / or polynucleotide sequences are also encompassed by the present invention. In addition, the present invention encompasses amino acid sequences which comprise at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% Polypeptides composed of sequences and polynucleotides encoding such polypeptides are included in the present invention.

In addition, one or more amino acids deleted from both the amino and carboxyl ends, which may be generally described as having the moiety n ¹ -m ¹ of SEQ ID NO: 2, wherein n ¹ and m ¹ are integers as defined above Or a polypeptide consisting of such amino acid is provided. In addition, a portion of the complete neurotrophin-alpha amino acid sequence encoded by the deposited cDNA clone contained in ATCC Accession No. 97768, wherein a portion is derived from the amino terminus of the complete amino acid sequence encoded by the cDNA clone in the deposited plasmid Quot; refers to a polypeptide comprising, alternatively consisting of, a portion of such a polypeptide, excluding 1 to 190 amino acids or excluding 1 to 11 amino acids from the C-terminus or a combined deletion from the N-terminus and the C-terminus) Lt; RTI ID = 0.0 > nucleotide < / RTI > Polynucleotides encoding all of the deletion polypeptides are also encompassed by the present invention.

Similarly, deletion of the C-terminal amino acid residue of the putative extracellular domain of neurotoxin-alpha from SEQ ID NO: 2 to the leucine residue at position 79 can be achieved by, for example, ligand binding, lymphocyte (e.g., B cell) / RTI ID = 0.0 > and / or < / RTI > stimulation of activation and regulation of cell replication or regulation of target cell activation. Polypeptides with additional C-terminal deletions, including Leu-79 of SEQ ID NO: 2, are not expected to retain biological activity.

However, even if deletion of one or more amino acids from the C-terminus of the protein induces modification or loss in one or more biological functions of the protein, other functional activity can still be maintained. Thus, the ability of a shortened protein to induce and / or bind an antibody that recognizes a complete, mature, or extracellular form of the polypeptide may be complete or mature, or less than a major portion of the extracellular protein residues may be removed from the C-terminus Will be maintained. Whether a particular polypeptide lacking the C-terminal residue of a complete protein retains such immunological activity can be readily determined by conventional methods as described herein and known in the art.

Thus, the present invention also relates to polypeptides wherein one or more residues are deleted from the carboxy terminus of the amino acid sequence of the putative extracellular domain of the neurotoxin-alpha polypeptide described in SEQ ID NO: 2 to the glycine residue at position 2 to position 79, A polynucleotide encoding the peptide is provided. In particular, the present invention SEQ ID NO: 2, residues 73-m ² in (where, m is an integer from ² SEQ ID NO: 2 within the range of the amino acid position of amino acid residues 79-285 and residues 78 neutroavidin Kin-alpha polypeptide (described in SEQ ID NO: 2 Terminus of the predicted extracellular domain of the polypeptide), or, alternatively, provides a polypeptide consisting of such sequence. Polynucleotides encoding these polypeptides are also encompassed by the present invention. More particularly, in certain embodiments, the present invention provides a polypeptide comprising the amino acid sequence of residues Q-73 to Leu-285 of SEQ ID NO: 2; Q-73 to L-284; Q-73 to K-283; Q-73 to L-282; Q-73 to A-281; Q-73 to G-280; Q-73 to F-279; Q-73 to F-278; Q-73 to T-277; Q-73 to V-276; Q-73 to D-275; Q-73 to G-274; Q-73 to D-273; Q-73 to L-272; Q-73 to S-271; Q-73 to I-270; Q-73 to Q-269; Q-73 to A-268; Q-73 to N-267; Q-73 to E-266; Q-73 to R-265; Q-73 to P-264; Q-73 to I-263; Q-73 to A-262; Q-73 to L-261; Q-73 to Q-260; Q-73 to L-259; Q-73 to E-258; Q-73 to D-257; Q-73 to G-256; Q-73 to E-255; Q-73 to E-254; Q-73 to L-253; Q-73 to K-252; Q-73 to A-251; Q-73 to I-250; Q-73 to G-249; Q-73 to A-248; Q-73 to S-247; Q-73 to Y-246; Q-73 to K-245; Q-73 to S-244; Q-73 to N-243; Q-73 to N-242; Q-73 to P-241; Q-73 to L-240; Q-73 to T-239; Q-73 to E-238; Q-73 to P-237; Q-73 to M-236; Q-73 to N-235; Q-73 to Q-234; Q-73 to I-233; Q-73 to K-232; Q-73 to R-231; Q-73 to F-230; Q-73 to L-229; Q-73 to T-228; Q-73 to V-227; Q-73 to L-226; Q-73 to S-225; Q-73 to L-224; Q-73 to E-223; Q-73 to D-222; Q-73 to G-221; Q-73 to F-220; Q-73 to V-219; Q-73 to H-218; Q-73 to V-217; Q-73 to K-216; Q-73 to K-215; Q-73 to R-214; Q-73 to Q-213; Q-73 to I-212; Q-73 to L-211; Q-73 to H-210; Q-73 to G-209; Q-73 to M-208; Q-73 to A-207; Q-73 to Y-206; Q-73 to T-205; Q-73 to K-204; Q-73 to D-203; Q-73 to T-202; Q-73 to Y-201; Q-73 to L-200; Q-73 to V-199; Q-73 to Q-198; Q-73 to G-197; Q-73 to Y-196; Q-73 to I-195; Q-73 to F-194; Q-73 to F-193; Q-73 to Y-192; Q-73 to G-191; Q-73 to T-190; Q-73 to E-189; Q-73 to K-188; Q-73 to V-187; Q-73 to L-186; Q-73 to I-185; Q-73 to K-184; Q-73 to N-183; Q-73 to E-182; Q-73 to K-181; Q-73 to E-180; Q-73 to E-179; Q-73 to L-178; Q-73 to A-177; Q-73 to S-176; Q-73 to G-175; Q-73 to R-174; Q-73 to K-173; Q-73 to F-172; Q-73 to S-171; Q-73 to L-170; Q-73 to L-169; Q-73 to W-168; Q-73 to P-167; Q-73 to V-166; Q-73 to F-165; Q-73 to T-164; Q-73 to Y-163; Q-73 to S-162; Q-73 to G-161; Q-73 to K-160; Q-73 to Q-159; Q-73 to I-158; Q-73 to T-157; Q-73 to P-156; Q-73 to T-155; Q-73 to E-154; Q-73 to S-153; Q-73 to D-152; Q-73 to A-151; Q-73 to I-150; Q-73 to L-149; Q-73 to Q-148; Q-73 to L-147; Q-73 to C-146; Q-73 to D-145; Q-73 to Q-144; Q-73 to T-143; Q-73 to V-142; Q-73 to T-141; Q-73 to E-140; Q-73 to E-139; Q-73 to P-138; Q-73 to G-137; Q-73 to Q-136; Q-73 to V-135; Q-73 to A-134; Q-73 to R-133; Q-73 to K-132; Q-73 to N-131; Q-73 to R-130; Q-73 to S-129; Q-73 to N-128; Q-73 to Q-127; Q-73 to S-126; Q-73 to S-125; Q-73 to N-124; Q-73 to G-123; Q-73 to E-122; Q-73 to G-121; Q-73 to P-120; Q-73 to A-119; Q-73 to P-118; Q-73 to P-117; Q-73 to E-116; Q-73 to F-115; Q-73 to I-114; Q-73 to K-113; Q-73 to L-112; Q-73 to G-111; Q-73 to A-110; Q-73 to T-109; Q-73 to V-108; Q-73 to A-107; Q-73 to P-106; Q-73 to A-105; Q-73 to E-104; Q-73 to E-103; Q-73 to L-102; Q-73 to G-101; Q-73 to A-100; Q-73 to K-99; Q-73 to P-98; Q-73 to A-97; Q-73 to G-96; Q-73 to A-95; Q-73 to G-94; Q-73 to A-93; Q-73 to P-92; Q-73 to L-91; Q-73 to K-90; Q-73 to E-89; Q-73 to A-88; Q-73 to H-87; Q-73 to H-86; Q-73 to G-85; Q-73 to Q-84; Q-73 to L-83; Q-73 to E-82; Q-73 to A-81; Q-73 to R-80, and Q-73 to L-79, or alternatively, a polynucleotide encoding a polypeptide consisting of such sequence. Polypeptides encoded by these polynucleotides are also included in the present invention. The present invention also provides a polynucleotide encoding a neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide as described above, which is 80%, 85%, 90%, 92%, 95%, 96% 98% or 99% identical to or more than the polynucleotide sequence of SEQ ID NO. The invention also encompasses polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and / or polynucleotide sequences are also encompassed by the present invention. In addition, the present invention encompasses amino acid sequences which comprise at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% Polypeptides composed of sequences and polynucleotides encoding such polypeptides are included in the present invention.

The present invention also relates to the use of an amino acid sequence of the deduced extracellular domain of neurotoxin-alpha, which can be generally described as having the moiety n ¹ -m ¹ of SEQ ID NO: 2, wherein n ¹ and m ¹ are integers as defined above And at least one amino acid is deleted from both the carboxyl terminus.

In another embodiment, a portion of the extracellular domain of the neurotoxin-alpha amino acid sequence encoded by the cDNA plasmid contained in the deposit of ATCC Accession No. 97768, wherein a portion of the cDNA contained in the deposit of ATCC Accession No. 97768 The C-terminal portion of the extracellular domain of the amino acid sequence encoded by the cDNA plasmid contained in the deposit of ATCC Accession No. 97768, except for 1 to about 206 amino acids from the amino terminus of the extracellular domain of the amino acid sequence encoded by the plasmid, Refers to the combined deletion from the N-terminus and the C-terminus of the complete extracellular domain of the amino acid sequence encoded by the cDNA plasmid contained within the deposit of ATCC Accession No. 97768, excluding 1 to about 206 amino acids from the terminus ) Encoding a polypeptide consisting of a nucleotide Sequence.

As mentioned above, although deletion of one or more amino acids from the N-terminus of a polypeptide may result in modification or loss of one or more functional activity (e. G., Biological activity) of the polypeptide, other functional or biological activity Can be maintained. Thus, the ability of the abbreviated neurotoxin-alpha to induce and / or bind to an antibody that recognizes the full-length or mature form of the polypeptide or the extracellular domain will depend on the overall or mature form of the polypeptide or the residue of the extracellular domain Will be retained when less than the middle major portion is removed from the N-terminus. Whether a particular polypeptide lacking the N-terminal residue of the complete polypeptide retains such immunological activity can be readily determined by routine methods as described herein and known in the art. Neutrokine-alpha muteins with a large number of deleted N-terminal amino acid residues are unlikely to be able to maintain some functional (e.g., biological or immunogenic) activity. In fact, peptides composed of fewer than six neurotoxin-alpha amino acid residues can often cause an immune response.

Accordingly, the present invention also provides a polypeptide having deletion of one or more residues from the amino terminal of the putative full-length amino acid sequence of Neutrokine-alpha described in SEQ ID NO: 2 to the glycine residue at position 280 described in SEQ ID NO: 2, Gt; polynucleotide < / RTI > In particular, comprising the amino acid sequence of the invention residues n ³ -285 of the sequence described in SEQ ID NO: 2 (where, n ³ is an integer in the range of the amino acid residues 1 to 280 in the amino acid sequence of SEQ ID NO: 2, amino acid positions) Polypeptide.

More particularly, the invention relates to a polypeptide comprising residues D-2 to L-285 of SEQ ID NO: 2; D-3 to L-285; S-4 to L-285; T-5 to L-285; E-6 to L-285; R-7 to L-285; E-8 to L-285; Q-9 to L-285; S-10 to L-285; R-11 to L-285; L-12 to L-285; T-13 to L-285; S-14 to L-285; C-15 to L-285; L-16 to L-285; K-17 to L-285; K-18 to L-285; R-19 to L-285; E-20 to L-285; E-21 to L-285; M-22 to L-285; K-23 to L-285; L-24 to L-285; K-25 to L-285; E-26 to L-285; C-27 to L-285; V-28 to L-285; S-29 to L-285; I-30 to L-285; L-31 to L-285; P-32 to L-285; R-33 to L-285; K-34 to L-285; E-35 to L-285; S-36 to L-285; P-37 to L-285; S-38 to L-285; V-39 to L-285; R-40 to L-285; S-41 to L-285; S-42 to L-285; K-43 to L-285; D-44 to L-285; G-45 to L-285; K-46 to L-285; L-47 to L-285; L-48 to L-285; A-49 to L-285; A-50 to L-285; T-51 to L-285; L-52 to L-285; L-53 to L-285; L-54 to L-285; A-55 to L-285; L-56 to L-285; L-57 to L-285; S-58 to L-285; C-59 to L-285; C-60 to L-285; L-61 to L-285; T-62 to L-285; V-63 to L-285; V-64 to L-285; S-65 to L-285; F-66 to L-285; Y-67 to L-285; Q-68 to L-285; V-69 to L-285; A-70 to L-285; A-71 to L-285; L-72 to L-285; Q-73 to L-285; G-74 to L-285; D-75 to L-285; L-76 to L-285; A-77 to L-285; S-78 to L-285; L-79 to L-285; R-80 to L-285; A-81 to L-285; E-82 to L-285; L-83 to L-285; Q-84 to L-285; G-85 to L-285; H-86 to L-285; H-87 to L-285; A-88 to L-285; E-89 to L-285; K-90 to L-285; L-91 to L-285; P-92 to L-285; A-93 to L-285; G-94 to L-285; A-95 to L-285; G-96 to L-285; A-97 to L-285; P-98 to L-285; K-99 to L-285; A-100 to L-285; G-101 to L-285; L-102 to L-285; E-103 to L-285; E-104 to L-285; A-105 to L-285; P-106 to L-285; A-107 to L-285; V-108 to L-285; T-109 to L-285; A-110 to L-285; G-111 to L-285; L-112 to L-285; K-113 to L-285; I-114 to L-285; F-115 to L-285; E-116 to L-285; P-117 to L-285; P-118 to L-285; A-119 to L-285; P-120 to L-285; G-121 to L-285; E-122 to L-285; G-123 to L-285; N-124 to L-285; S-125 to L-285; S-126 to L-285; Q-127 to L-285; N-128 to L-285; S-129 to L-285; R-130 to L-285; N-131 to L-285; K-132 to L-285; R-133 to L-285; A-134 to L-285; V-135 to L-285; Q-136 to L-285; G-137 to L-285; P-138 to L-285; E-139 to L-285; E-140 to L-285; T-141 to L-285; V-142 to L-285; T-143 to L-285; Q-144 to L-285; D-145 to L-285; C-146 to L-285; L-147 to L-285; Q-148 to L-285; L-149 to L-285; I-150 to L-285; A-151 to L-285; D-152 to L-285; S-153 to L-285; E-154 to L-285; T-155 to L-285; P-156 to L-285; T-157 to L-285; I-158 to L-285; Q-159 to L-285; K-160 to L-285; G-161 to L-285; S-162 to L-285; Y-163 to L-285; T-164 to L-285; F-165 to L-285; V-166 to L-285; P-167 to L-285; W-168 to L-285; L-169 to L-285; L-170 to L-285; S-171 to L-285; F-172 to L-285; K-173 to L-285; R-174 to L-285; G-175 to L-285; S-176 to L-285; A-177 to L-285; L-178 to L-285; E-179 to L-285; E-180 to L-285; K-181 to L-285; E-182 to L-285; N-183 to L-285; K-184 to L-285; I-185 to L-285; L-186 to L-285; V-187 to L-285; K-188 to L-285; E-189 to L-285; T-190 to L-285; G-191 to L-285; Y-192 to L-285; F-193 to L-285; F-194 to L-285; I-195 to L-285; Y-196 to L-285; G-197 to L-285; Q-198 to L-285; V-199 to L-285; L-200 to L-285; Y-201 to L-285; T-202 to L-285; D-203 to L-285; K-204 to L-285; T-205 to L-285; Y-206 to L-285; A-207 to L-285; M-208 to L-285; G-209 to L-285; H-210 to L-285; L-211 to L-285; I-212 to L-285; Q-213 to L-285; R-214 to L-285; K-215 to L-285; K-216 to L-285; V-217 to L-285; H-218 to L-285; V-219 to L-285; F-220 to L-285; G-221 to L-285; D-222 to L-285; E-223 to L-285; L-224 to L-285; S-225 to L-285; L-226 to L-285; V-227 to L-285; T-228 to L-285; L-229 to L-285; F-230 to L-285; R-231 to L-285; C-232 to L-285; I-233 to L-285; Q-234 to L-285; N-235 to L-285; M-236 to L-285; P-237 to L-285; E-238 to L-285; T-239 to L-285; L-240 to L-285; P-241 to L-285; N-242 to L-285; N-243 to L-285; S-244 to L-285; C-245 to L-285; Y-246 to L-285; S-247 to L-285; A-248 to L-285; G-249 to L-285; I-250 to L-285; A-251 to L-285; K-252 to L-285; L-253 to L-285; E-254 to L-285; E-255 to L-285; G-256 to L-285; D-257 to L-285; E-258 to L-285; L-259 to L-285; Q-260 to L-285; L-261 to L-285; A-262 to L-285; I-263 to L-285; P-264 to L-285; R-265 to L-285; E-266 to L-285; N-267 to L-285; A-268 to L-285; Q-269 to L-285; I-270 to L-285; S-271 to L-285; L-272 to L-285; D-273 to L-285; G-274 to L-285; D-275 to L-285; V-276 to L-285; T-277 to L-285; F-278 to L-285; F-279 to L-285 and G-280 to L-285, or alternatively, a polynucleotide encoding a polypeptide consisting of such a sequence. The present application discloses a polynucleotide encoding a neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide as described above that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97% And more preferably 99% or more of the same polynucleotide sequence. The invention also encompasses polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and / or polynucleotide sequences are also encompassed by the present invention. In addition, the present invention encompasses amino acid sequences which comprise at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% Polypeptides composed of sequences and polynucleotides encoding such polypeptides are included in the present invention.

As noted above, although deletion of one or more amino acids from the C-terminus of a protein will induce modification or loss in one or more functional activities (e. G., Biological activity) of the protein, other functional or biological activities will still be maintained . Thus, the ability of abbreviated neurotoxin-alpha mutein to induce and / or bind to an antibody that recognizes a complete or mature form of the polypeptide or an extracellular domain is a complete or mature form of the polypeptide or an extracellular domain Will be retained when less than the major portion of the residues of the C-terminus is removed from the C-terminus. Whether a particular polypeptide lacking the C-terminal residue of the complete polypeptide retains such immunological activity can be readily determined by conventional methods as described herein and known in the art. Neutrokine-alpha muteins with a large number of deleted C-terminal amino acid residues are unlikely to be able to maintain some functional (e.g., biological or immunogenic) activity. In fact, peptides composed of fewer than six neurotoxin-alpha amino acid residues can often cause an immune response.

Thus, in another aspect, the invention also provides a polypeptide having one or more residues deleted from the carboxy terminus of the amino acid sequence of the neurotoxin-alpha described in SEQ ID NO: 2 to the glutamic acid residue at position 6 described in SEQ ID NO: 2, Lt; RTI ID = 0.0 > polynucleotides < / RTI > In particular, the present invention is a moiety 1-m ³ of the sequence described in SEQ ID NO: 2 comprising the amino acid sequence of (here, m ³ is an integer in the range of of SEQ ID NO: 2, amino acid residues in the amino acid sequence of 6 to 284 amino acid positions) Polypeptide.

More particularly, the invention relates to a polypeptide comprising residues M-1 to L-284 of SEQ ID NO: 2; M-1 to K-283; M-1 to L-282; M-1 to A-281; M-1 to G-280; M-1 to F-279; M-1 to F-278; M-1 to T-277; M-1 to V-276; M-1 to D-275; M-1 to G-274; M-1 to D-273; M-1 to L-272; M-1 to S-271; M-1 to I-270; M-1 to Q-269; M-1 to A-268; M-1 to N-267; M-1 to E-266; M-1 to R-265; M-1 to P-264; M-1 to I-263; M-1 to A-262; M-1 to L-261; M-1 to Q-260; M-1 to L-259; M-1 to E-258; M-1 to D-257; M-1 to G-256; M-1 to E-255; M-1 to E-254; M-1 to L-253; M-1 to K-252; M-1 to A-251; M-1 to I-250; M-1 to G-249; M-1 to A-248; M-1 to S-247; M-1 to Y-246; M-1 to K-245; M-1 to S-244; M-1 to N-243; M-1 to N-242; M-1 to P-241; M-1 to L-240; M-1 to T-239; M-1 to E-238; M-1 to P-237; M-1 to M-236; M-1 to N-235; M-1 to Q-234; M-1 to I-233; M-1 to C-232; M-1 to R-231; M-1 to F-230; M-1 to L-229; M-1 to T-228; M-1 to V-227; M-1 to L-226; M-1 to S-225; M-1 to L-224; M-1 to E-223; M-1 to D-222; M-1 to G-221; M-1 to F-220; M-1 to V-219; M-1 to H-218; M-1 to V-217; M-1 to K-216; M-1 to K-215; M-1 to R-214; M-1 to Q-213; M-1 to I-212; M-1 to L-211; M-1 to H-210; M-1 to G-209; M-1 to M-208; M-1 to A-207; M-1 to Y-206; M-1 to T-205; M-1 to K-204; M-1 to D-203; M-1 to T-202; M-1 to Y-201; M-1 to L-200; M-1 to V-199; M-1 to Q-198; M-1 to G-197; M-1 to Y-196; M-1 to I-195; M-1 to F-194; M-1 to F-193; M-1 to Y-192; M-1 to G-191; M-1 to T-190; M-1 to E-189; M-1 to K-188; M-1 to V-187; M-1 to L-186; M-1 to I-185; M-1 to K-184; M-1 to N-183; M-1 to E-182; M-1 to K-181; M-1 to E-180; M-1 to E-179; M-1 to L-178; M-1 to A-177; M-1 to S-176; M-1 to G-175; M-1 to R-174; M-1 to K-173; M-1 to F-172; M-1 to S-171; M-1 to L-170; M-1 to L-169; M-1 to W-168; M-1 to P-167; M-1 to V-166; M-1 to F-165; M-1 to T-164; M-1 to Y-163; M-1 to S-162; M-1 to G-161; M-1 to K-160; M-1 to Q-159; M-1 to I-158; M-1 to T-157; M-1 to P-156; M-1 to T-155; M-1 to E-154; M-1 to S-153; M-1 to D-152; M-1 to A-151; M-1 to I-150; M-1 to L-149; M-1 to Q-148; M-1 to L-147; M-1 to C-146; M-1 to D-145; M-1 to Q-144; M-1 to T-143; M-1 to V-142; M-1 to T-141; M-1 to E-140; M-1 to E-139; M-1 to P-138; M-1 to G-137; M-1 to Q-136; M-1 to V-135; M-1 to A-134; M-1 to R-133; M-1 to K-132; M-1 to N-131; M-1 to R-130; M-1 to S-129; M-1 to N-128; M-1 to Q-127; M-1 to S-126; M-1 to S-125; M-1 to N-124; M-1 to G-123; M-1 to E-122; M-1 to G-121; M-1 to P-120; M-1 to A-119; M-1 to P-118; M-1 to P-117; M-1 to E-116; M-1 to F-115; M-1 to I-114; M-1 to K-113; M-1 to L-112; M-1 to G-111; M-1 to A-110; M-1 to T-109; M-1 to V-108; M-1 to A-107; M-1 to P-106; M-1 to A-105; M-1 to E-104; M-1 to E-103; M-1 to L-102; M-1 to G-101; M-1 to A-100; M-1 to K-99; M-1 to P-98; M-1 to A-97; M-1 to G-96; M-1 to A-95; M-1 to G-94; M-1 to A-93; M-1 to P-92; M-1 to L-91; M-1 to K-90; M-1 to E-89; M-1 to A-88; M-1 to H-87; M-1 to H-86; M-1 to G-85; M-1 to Q-84; M-1 to L-83; M-1 to E-82; M-1 to A-81; M-1 to R-80; M-1 to L-79; M-1 to S-78; M-1 to A-77; M-1 to L-76; M-1 to D-75; M-1 to G-74; M-1 to Q-73; M-1 to L-72; M-1 to A-71; M-1 to A-70; M-1 to V-69; M-1 to Q-68; M-1 to Y-67; M-1 to F-66; M-1 to S-65; M-1 to V-64; M-1 to V-63; M-1 to T-62; M-1 to L-61; M-1 to C-60; M-1 to C-59; M-1 to S-58; M-1 to L-57; M-1 to L-56; M-1 to A-55; M-1 to L-54; M-1 to L-53; M-1 to L-52; M-1 to T-51; M-1 to A-50; M-1 to A-49 M-1 to L-48; M-1 to L-47; M-1 to K-46; M-1 to G-45; M-1 to D-44; M-1 to K-43; M-1 to S-42; M-1 to S-41; M-1 to R-40; M-1 to V-39; M-1 to S-38; M-1 to P-37; M-1 to S-36; M-1 to E-35; M-1 to K-34; M-1 to R-33; M-1 to P-32; M-1 to L-31; M-1 to I-30; M-1 to S-29; M-1 to V-28; M-1 to C-27; M-1 to E-26; M-1 to K-25; M-1 to L-24; M-1 to K-23; M-1 to M-22; M-1 to E-21; M-1 to E-20; M-1 to R-19; M-1 to K-18; M-1 to K-17; M-1 to L-16; M-1 to C-15; M-1 to S-14; M-1 to T-13; M-1 to L-12; M-1 to R-11; M-1 to S-10; M-1 to Q-9; M-1 to E-8; M-1 to R-7 and M-1 to E-6, or alternatively, a polynucleotide encoding a polypeptide consisting of such a sequence. The present application discloses a polynucleotide encoding a neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide as described above that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97% And more preferably 99% or more of the same polynucleotide sequence. The invention also encompasses polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and / or polynucleotide sequences are also encompassed by the present invention. In addition, the present invention encompasses amino acid sequences which comprise at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% Polypeptides composed of sequences and polynucleotides encoding such polypeptides are included in the present invention.

The present invention also relates to amino and carboxyl ends of a neurotoxin-alpha polypeptide, which may be generally described as having the moiety n ³ -m ³ of SEQ ID NO: 2, wherein n ³ and m ³ are integers as defined above Lt; RTI ID = 0.0 > amino < / RTI >

In addition, although the putative extracellular domain of the neurotoxin-alpha SV polypeptide according to the present invention may exhibit functional activity itself (e. G., Biological activity), it is preferred that the polypeptide of position Gln-73 to Leu- Deletion of the N- and C-terminal amino acid residues of the putative extracellular domain of the peptide can result in, for example, ligand binding, stimulation of lymphocyte (e.g., B cell) proliferation, differentiation and / Some biological activities such as regulation of activation can be maintained. However, although deletion of one or more amino acids from the N-terminus of the putative extracellular domain of the neurotoxin-alpha SV polypeptide induces modification or loss in one or more biological functions of the protein, other functional activities are still retained . Thus, the ability of a shortened protein to induce and / or bind an antibody that recognizes a complete, mature, or extracellular domain of a polypeptide means that less than a major portion of the residues of the complete, mature, or extracellular domain of the polypeptide is N- Will be retained when removed from the terminus. Whether a particular polypeptide lacking the N-terminal residue of the complete polypeptide retains such immunological activity can be readily determined by routine methods as described herein and known in the art.

Thus, the present invention also provides a polypeptide wherein one or more residues are deleted from the amino terminus of the amino acid sequence of the neurotoxin-alpha SV described in SEQ ID NO: 19 to the glycine residue at position 261, and a polynucleotide encoding such a polypeptide. In particular, the invention residues n ⁴ -266 in SEQ ID NO: 19 (where, n ⁴ is an integer in the range of from amino acid position of amino acid residues 73-261 in the amino acid sequence of SEQ ID NO: 19 and 261 are outside the estimated cell described in SEQ ID NO: 19 Terminal position of the first residue from the N-terminus of the domain neurotoxin-alpha SV polypeptide), or alternatively comprises a polypeptide consisting of such sequence.

More particularly, in certain embodiments, the present invention provides a compound having the structure of residues Q-73 to L-266 of SEQ ID NO: 19; G-74 to L-266; D-75 to L-266; L-76 to L-266; A-77 to L-266; S-78 to L-266; L-79 to L-266; R-80 to L-266; A-81 to L-266; E-82 to L-266; L-83 to L-266; Q-84 to L-266; G-85 to L-266; H-86 to L-266; H-87 to L-266; A-88 to L-266; E-89 to L-266; K-90 to L-266; L-91 to L-266; P-92 to L-266; A-93 to L-266; G-94 to L-266; A-95 to L-266; G-96 to L-266; A-97 to L-266; P-98 to L-266; K-99 to L-266; A-100 to L-266; G-101 to L-266; L-102 to L-266; E-103 to L-266; E-104 to L-266; A-105 to L-266; P-106 to L-266; A-107 to L-266; V-108 to L-266; T-109 to L-266; A-110 to L-266; G-111 to L-266; L-112 to L-266; K-113 to L-266; I-114 to L-266; F-115 to L-266; E-116 to L-266; P-117 to L-266; P-118 to L-266; A-119 to L-266; P-120 to L-266; G-121 to L-266; E-122 to L-266; G-123 to L-266; N-124 to L-266; S-125 to L-266; S-126 to L-266; Q-127 to L-266; N-128 to L-266; S-129 to L-266; R-130 to L-266; N-131 to L-266; K-132 to L-266; R-133 to L-266; A-134 to L-266; V-135 to L-266; Q-136 to L-266; G-137 to L-266; P-138 to L-266; E-139 to L-266; E-140 to L-266; T-141 to L-266; G-142 to L-266; S-143 to L-266; Y-144 to L-266; T-145 to L-266; F-146 to L-266; V-147 to L-266; P-148 to L-266; W-149 to L-266; L-150 to L-266; L-151 to L-266; S-152 to L-266; F-153 to L-266; K-154 to L-266; R-155 to L-266; G-156 to L-266; S-157 to L-266; A-158 to L-266; L-159 to L-266; E-160 to L-266; E-161 to L-266; K-162 to L-266; E-163 to L-266; N-164 to L-266; K-165 to L-266; I-166 to L-266; L-167 to L-266; V-168 to L-266; K-169 to L-266; E-170 to L-266; T-171 to L-266; G-172 to L-266; Y-173 to L-266; F-174 to L-266; F-175 to L-266; I-176 to L-266; Y-177 to L-266; G-178 to L-266; Q-179 to L-266; V-180 to L-266; L-181 to L-266; Y-182 to L-266; T-183 to L-266; D-184 to L-266; K-185 to L-266; T-186 to L-266; Y-187 to L-266; A-188 to L-266; M-189 to L-266; G-190 to L-266; H-191 to L-266; L-192 to L-266; I-193 to L-266; Q-194 to L-266; R-195 to L-266; K-196 to L-266; K-197 to L-266; V-198 to L-266; H-199 to L-266; V-200 to L-266; F-201 to L-266; G-202 to L-266; D-203 to L-266; E-204 to L-266; L-205 to L-266; S-206 to L-266; L-207 to L-266; V-208 to L-266; T-209 to L-266; L-210 to L-266; F-211 to L-266; R-212 to L-266; C-213 to L-266; I-214 to L-266; Q-215 to L-266; N-216 to L-266; M-217 to L-266; P-218 to L-266; E-219 to L-266; T-220 to L-266; L-221 to L-266; P-222 to L-266; N-223 to L-266; N-224 to L-266; S-225 to L-266; C-226 to L-266; Y-227 to L-266; S-228 to L-266; A-229 to L-266; G-230 to L-266; I-231 to L-266; A-232 to L-266; K-233 to L-266; L-234 to L-266; E-235 to L-266; E-236 to L-266; G-237 to L-266; D-238 to L-266; E-239 to L-266; L-240 to L-266; Q-241 to L-266; L-242 to L-266; A-243 to L-266; I-244 to L-266; P-245 to L-266; R-246 to L-266; E-247 to L-266; N-248 to L-266; A-249 to L-266; Q-250 to L-266; I-251 to L-266; S-252 to L-266; L-253 to L-266; D-254 to L-266; G-255 to L-266; D-256 to L-266; V-257 to L-266; T-258 to L-266; F-259 to L-266; F-260 to L-266 and G-261 to L-266, or alternatively, a polynucleotide encoding a polypeptide consisting of such a sequence. The present application discloses a polynucleotide encoding a neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide as described above that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97% And more preferably 99% or more of the same polynucleotide sequence. The invention also encompasses polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and / or polynucleotide sequences are also encompassed by the present invention. In addition, the present invention encompasses amino acid sequences which comprise at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% Polypeptides composed of sequences and polynucleotides encoding such polypeptides are included in the present invention.

Similarly, deletion of the C-terminal amino acid residue of the putative extracellular domain of the neurotoxin-alpha SV from the sequence 19 to the leucine residue at position 79 can be achieved by, for example, ligand binding, lymphocyte (e.g., B cell) And / or some functional activity such as the ability to stimulate activation, modulation of cell replication, target cell activity, and / or modulation of immunogenicity. Polypeptides with additional C-terminal deletions, including Leu-79 of SEQ ID NO: 19, are not expected to retain biological activity.

However, although deletion of one or more amino acids from the C-terminus of the polypeptide may result in modification or loss in one or more functional activities (e. G., Biological activity) of the polypeptide, other functional activities may still be retained. Thus, the ability of a shortened protein to induce and / or bind an antibody that recognizes a complete, mature, or extracellular domain of the polypeptide is less potent than that of the full, mature, or extracellular domain of the polypeptide, Will be retained when removed from the terminus. Whether a particular polypeptide lacking the C-terminal residue of the complete polypeptide retains such immunological activity can be readily determined by conventional methods as described herein and known in the art.

Thus, the present invention also provides polypeptides wherein one or more residues are deleted from the carboxy terminus of the amino acid sequence of the putative extracellular domain of the neurotoxin-alpha SV described in SEQ ID NO: 19 to the leucine residue at position 19 to position 79, A polynucleotide encoding the peptide is provided. In particular, the present invention relates to a polypeptide having the amino acid sequence of residue 73-m ⁴ of the amino acid sequence of SEQ ID NO: 19, wherein m ⁴ is an integer belonging to the amino acid position of the amino acid residue 79-266 in the amino acid sequence of SEQ ID NO: 19 Lt; / RTI >

More particularly, in certain embodiments, the invention encompasses residues Q-73 to L-265 of SEQ ID NO: 19; Q-73 to K-264; Q-73 to L-263; Q-73 to A-262; Q-73 to G-261; Q-73 to F-260; Q-73 to F-259; Q-73 to T-258; Q-73 to V-257; Q-73 to D-256; Q-73 to G-255; Q-73 to D-254; Q-73 to L-253; Q-73 to S-252; Q-73 to I-251; Q-73 to Q-250; Q-73 to A-249; Q-73 to N-248; Q-73 to E-247; Q-73 to R-246; Q-73 to P-245; Q-73 to I-244; Q-73 to A-243; Q-73 to L-242; Q-73 to Q-241; Q-73 to L-240; Q-73 to E-239; Q-73 to D-238; Q-73 to G-237; Q-73 to E-236; Q-73 to E-235; Q-73 to L-234; Q-73 to K-233; Q-73 to A-232; Q-73 to I-231; Q-73 to G-230; Q-73 to A-229; Q-73 to S-228; Q-73 to Y-227; Q-73 to C-226; Q-73 to S-225; Q-73 to N-224; Q-73 to N-223; Q-73 to P-222; Q-73 to L-221; Q-73 to T-220; Q-73 to E-219; Q-73 to P-218; Q-73 to M-217; Q-73 to N-216; Q-73 to Q-215; Q-73 to I-214; Q-73 to C-213; Q-73 to R-212; Q-73 to F-211; Q-73 to L-210; Q-73 to T-209; Q-73 to V-208; Q-73 to L-207; Q-73 to S-206; Q-73 to L-205; Q-73 to E-204; Q-73 to D-203; Q-73 to G-202; Q-73 to F-201; Q-73 to V-200; Q-73 to H-199; Q-73 to V-198; Q-73 to K-197; Q-73 to K-196; Q-73 to R-195; Q-73 to Q-194; Q-73 to I-193; Q-73 to L-192; Q-73 to H-191; Q-73 to G-190; Q-73 to Q-189; Q-73 to A-188; Q-73 to Y-187; Q-73 to T-186; Q-73 to K-185; Q-73 to D-184; Q-73 to T-183; Q-73 to Y-182; Q-73 to L-181; Q-73 to V-180; Q-73 to Q-179; Q-73 to G-178; Q-73 to Y-177; Q-73 to I-176; Q-73 to F-175; Q-73 to F-174; Q-73 to Y-173; Q-73 to G-172; Q-73 to T-171; Q-73 to E-170; Q-73 to K-169; Q-73 to V-168; Q-73 to L-167; Q-73 to I-166; Q-73 to K-165; Q-73 to N-164; Q-73 to E-163; Q-73 to K-162; Q-73 to E-161; Q-73 to E-160; Q-73 to L-159; Q-73 to A-158; Q-73 to S-157; Q-73 to G-156; Q-73 to R-155; Q-73 to K-154; Q-73 to F-153; Q-73 to S-152; Q-73 to L-151; Q-73 to L-150; Q-73 to W-149; Q-73 to P-148; Q-73 to V-147; Q-73 to F-146; Q-73 to T-145; Q-73 to Y-144; Q-73 to S-143; Q-73 to G-142; Q-73 to T-141; Q-73 to E-140; Q-73 to E-139; Q-73 to P-138; Q-73 to G-137; Q-73 to Q-136; Q-73 to V-135; Q-73 to A-134; Q-73 to R-133; Q-73 to K-132; Q-73 to N-131; Q-73 to R-130; Q-73 to S-129; Q-73 to N-128; Q-73 to Q-127; Q-73 to S-126; Q-73 to S-125; Q-73 to N-124; Q-73 to G-123; Q-73 to E-122; Q-73 to G-121; Q-73 to P-120; Q-73 to A-119; Q-73 to P-118; Q-73 to P-117; Q-73 to E-116; Q-73 to F-115; Q-73 to I-114; Q-73 to K-113; Q-73 to L-112; Q-73 to G-111; Q-73 to A-110; Q-73 to T-109; Q-73 to V-108; Q-73 to A-107; Q-73 to P-106; Q-73 to A-105; Q-73 to E-104; Q-73 to E-103; Q-73 to L-102; Q-73 to G-101; Q-73 to A-100; Q-73 to K-99; Q-73 to P-98; Q-73 to A-97; Q-73 to G-96; Q-73 to A-95; Q-73 to G-94; Q-73 to A-93; Q-73 to P-92; Q-73 to L-91; Q-73 to K-90; Q-73 to E-89; Q-73 to A-88; Q-73 to H-87; Q-73 to H-86; Q-73 to G-85; Q-73 to Q-84; Q-73 to L-83; Q-73 to E-82; Q-73 to A-81; Q-73 to R-80; Q-73 to L-79 and Q-73 to S-78, or alternatively, a polynucleotide encoding a polypeptide consisting of such sequence. Also provided herein is a polynucleotide sequence encoding a neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide as described above that is 80%, 85%, 90%, 92%, 95%, 96% Or 99% or more of the same polynucleotide sequence, or, alternatively, consists of such sequence. The invention also encompasses polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and / or polynucleotide sequences are also encompassed by the present invention. In addition, the present invention encompasses amino acid sequences which comprise at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% Polypeptides composed of sequences and polynucleotides encoding such polypeptides are included in the present invention.

In addition, the present invention relates to the use of the deduced extracellular domain of the neurotoxin-alpha SV, which can be generally described as having the residue n ⁴ -m ⁴ of SEQ ID NO: 19, wherein n ⁴ and m ⁴ are integers as defined above At least one amino acid is deleted from both the amino and carboxyl termini.

In another embodiment, a portion of the extracellular domain of the neurotoxin-alpha SV amino acid sequence encoded by the cDNA clone contained in the deposit of ATCC Accession No. 203518, wherein part is contained in a deposit of ATCC Accession No. 203518 Of the extracellular domain of the amino acid sequence encoded by the cDNA clone contained in the deposit of ATCC Accession No. 203518, or by excluding the 1 to about 260 amino acids from the amino terminus of the extracellular domain of the amino acid sequence encoded by the cloned cDNA clone Refers to the combined deletion from the amino terminus and the carboxy terminus of the complete extracellular domain of the amino acid sequence encoded by a cDNA clone contained within the deposit of ATCC Accession No. 203518, excluding 1 to about 187 amino acids from the carboxy terminus ) Encoding a polypeptide consisting of a nucleotide It includes sequences de.

As noted above, although deletion of one or more amino acids from the N-terminus of the polypeptide will induce modification or loss in one or more functional activities (e. G., Biological activity) of the polypeptide, other functional activities are still retained . Thus, the ability of a shortened neurotoxin-alpha SV to induce and / or bind to an antibody that recognizes the full-length or mature form of the polypeptide or an extracellular domain is the ability of the polypeptide to bind to the full-length or mature form of the polypeptide, It will be maintained when less than the major portion of the residue is removed from the N-terminus. Whether a particular polypeptide lacking the N-terminal residue of the complete polypeptide retains such immunological activity can be readily determined by conventional methods as described herein and known in the art. Neutrokine-alpha SV muteins with multiple deleted N-terminal amino acid residues are unlikely to be able to maintain some functional (e.g., immunogenic) activity. In fact, peptides composed of as few as six small numbers of neurotoxin-alpha SV amino acid residues can often cause an immune response.

Thus, the present invention also provides polypeptides wherein one or more residues are deleted from the amino terminal of the putative full-length amino acid sequence of the neurotoxin-alpha SV described in SEQ ID NO: 19 to the glycine residue at position 261 described in SEQ ID NO: 19, Encoding polynucleotides. In particular, the invention encompasses a polypeptide comprising the amino acid sequence of residue n ⁵ -266 of the sequence described in SEQ ID NO: 19, wherein n ⁵ is an integer in the range of amino acid positions 1 to 261 of the amino acid sequence of SEQ ID NO: 19 Polypeptide.

More particularly, the invention relates to a polypeptide comprising residues D-2 to L-266 of SEQ ID NO: 19; D-3 to L-266; S-4 to L-266; T-5 to L-266; E-6 to L-266; R-7 to L-266; E-8 to L-266; Q-9 to L-266; S-10 to L-266; R-11 to L-266; L-12 to L-266; T-13 to L-266; S-14 to L-266; C-15 to L-266; L-16 to L-266; K-17 to L-266; K-18 to L-266; R-19 to L-266; E-20 to L-266; E-21 to L-266; M-22 to L-266; K-23 to L-266; L-24 to L-266; K-25 to L-266; E-26 to L-266; C-27 to L-266; V-28 to L-266; S-29 to L-266; I-30 to L-266; L-31 to L-266; P-32 to L-266; R-33 to L-266; K-34 to L-266; E-35 to L-266; S-36 to L-266; P-37 to L-266; S-38 to L-266; V-39 to L-266; R-40 to L-266; S-41 to L-266; S-42 to L-266; K-43 to L-266; D-44 to L-266; G-45 to L-266; K-46 to L-266; L-47 to L-266; L-48 to L-266; A-49 to L-266; A-50 to L-266; T-51 to L-266; L-52 to L-266; L-53 to L-266; L-54 to L-266; A-55 to L-266; L-56 to L-266; L-57 to L-266; S-58 to L-266; C-59 to L-266; C-60 to L-266; L-61 to L-266; T-62 to L-266; V-63 to L-266; V-64 to L-266; S-65 to L-266; F-66 to L-266; Y-67 to L-266; Q-68 to L-266; V-69 to L-266; A-70 to L-266; A-71 to L-266; L-72 to L-266; Q-73 to L-266; G-74 to L-266; D-75 to L-266; L-76 to L-266; A-77 to L-266; S-78 to L-266; L-79 to L-266; R-80 to L-266; A-81 to L-266; E-82 to L-266; L-83 to L-266; Q-84 to L-266; G-85 to L-266; H-86 to L-266; H-87 to L-266; A-88 to L-266; E-89 to L-266; K-90 to L-266; L-91 to L-266; P-92 to L-266; A-93 to L-266; G-94 to L-266; A-95 to L-266; G-96 to L-266; A-97 to L-266; P-98 to L-266; K-99 to L-266; A-100 to L-266; G-101 to L-266; L-102 to L-266; E-103 to L-266; E-104 to L-266; A-105 to L-266; P-106 to L-266; A-107 to L-266; V-108 to L-266; T-109 to L-266; A-110 to L-266; G-111 to L-266; L-112 to L-266; K-113 to L-266; I-114 to L-266; F-115 to L-266; E-116 to L-266; P-117 to L-266; P-118 to L-266; A-119 to L-266; P-120 to L-266; G-121 to L-266; E-122 to L-266; G-123 to L-266; N-124 to L-266; S-125 to L-266; S-126 to L-266; Q-127 to L-266; N-128 to L-266; S-129 to L-266; R-130 to L-266; N-131 to L-266; K-132 to L-266; R-133 to L-266; A-134 to L-266; V-135 to L-266; Q-136 to L-266; G-137 to L-266; P-138 to L-266; E-139 to L-266; E-140 to L-266; T-141 to L-266; G-142 to L-266; S-143 to L-266; Y-144 to L-266; T-145 to L-266; F-146 to L-266; V-147 to L-266; P-148 to L-266; W-149 to L-266; L-150 to L-266; L-151 to L-266; S-152 to L-266; F-153 to L-266; K-154 to L-266; R-155 to L-266; G-156 to L-266; S-157 to L-266; A-158 to L-266; L-159 to L-266; E-160 to L-266; E-161 to L-266; K-162 to L-266; E-163 to L-266; N-164 to L-266; K-165 to L-266; I-166 to L-266; L-167 to L-266; V-168 to L-266; K-169 to L-266; E-170 to L-266; T-171 to L-266; G-172 to L-266; Y-173 to L-266; F-174 to L-266; F-175 to L-266; I-176 to L-266; Y-177 to L-266; G-178 to L-266; Q-179 to L-266; V-180 to L-266; L-181 to L-266; Y-182 to L-266; T-183 to L-266; D-184 to L-266; K-185 to L-266; T-186 to L-266; Y-187 to L-266; A-188 to L-266; M-189 to L-266; G-190 to L-266; H-191 to L-266; L-192 to L-266; I-193 to L-266; Q-194 to L-266; R-195 to L-266; K-196 to L-266; K-197 to L-266; V-198 to L-266; H-199 to L-266; V-200 to L-266; F-201 to L-266; G-202 to L-266; D-203 to L-266; E-204 to L-266; L-205 to L-266; S-206 to L-266; L-207 to L-266; V-208 to L-266; T-209 to L-266; L-210 to L-266; F-211 to L-266; R-212 to L-266; C-213 to L-266; I-214 to L-266; Q-215 to L-266; N-216 to L-266; M-217 to L-266; P-218 to L-266; E-219 to L-266; T-220 to L-266; L-221 to L-266; P-222 to L-266; N-223 to L-266; N-224 to L-266; S-225 to L-266; C-226 to L-266; Y-227 to L-266; S-228 to L-266; A-229 to L-266; G-230 to L-266; I-231 to L-266; A-232 to L-266; K-233 to L-266; L-234 to L-266; E-235 to L-266; E-236 to L-266; G-237 to L-266; D-238 to L-266; E-239 to L-266; L-240 to L-266; Q-241 to L-266; L-242 to L-266; A-243 to L-266; I-244 to L-266; P-245 to L-266; R-246 to L-266; E-247 to L-266; N-248 to L-266; A-249 to L-266; Q-250 to L-266; I-251 to L-266; S-252 to L-266; L-253 to L-266; D-254 to L-266; G-255 to L-266; D-256 to L-266; V-257 to L-266; T-258 to L-266; F-259 to L-266; F-260 to L-266 and G-261 to L-266, or alternatively, a polynucleotide encoding a polypeptide consisting of such a sequence. Also provided herein is a polynucleotide sequence encoding a neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide as described above that is 80%, 85%, 90%, 92%, 95%, 96% Or 99% or more of the same polynucleotide sequence, or, alternatively, consists of such sequence. The invention also encompasses polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and / or polynucleotide sequences are also encompassed by the present invention. In addition, the present invention encompasses amino acid sequences which comprise at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% Polypeptides composed of sequences and polynucleotides encoding such polypeptides are included in the present invention.

As noted above, although deletion of one or more amino acids from the C-terminus of a protein may result in alteration or loss of one or more functional activities (e. G., Biological activity) of the protein of interest, other functional activities may still be maintained have. Thus, the ability of the shortened neurotoxin-alpha SV mutein to induce and / or bind to an antibody that recognizes the complete or mature form of the polypeptide or the extracellular domain is the complete or mature form of the polypeptide, Terminal portion of the residues of the domain will be retained when removed from the C-terminus. Whether or not a particular polypeptide lacking the C-terminal residue of the complete polypeptide retains such immunological activity can be readily determined by routine methods as described herein and known in the art. Neutrokine-alpha muteins with multiple deleted C-terminal amino acid residues are unlikely to be able to maintain some functional (e.g., immunogenic) activity. In fact, peptides composed of as few as six of the neurotoxin-alpha SV amino acid residues can often cause an immune response.

Thus, the present invention also relates to polypeptides wherein one or more residues are deleted from the carboxy terminus of the amino acid sequence of the neurotoxin-alpha SV described in SEQ ID NO: 19 to the glutamic acid residue at position 6 described in SEQ ID NO: 19, and poly Nucleotides. In particular, the invention encompasses a polynucleotide comprising an amino acid sequence of residue 1-m ⁵ of the sequence described in SEQ ID NO: 19, wherein m ⁵ is an integer in the range of amino acid positions 6 to 265 in the amino acid sequence of SEQ ID NO: 19 Polypeptide.

More particularly, the invention relates to a polypeptide comprising residues M-1 to L-265 of SEQ ID NO: 19; M-1 to K-264; M-1 to L-263; M-1 to A-262; M-1 to G-261; M-1 to F-260; M-1 to F-259; M-1 to T-258; M-1 to V-257; M-1 to D-256; M-1 to G-255; M-1 to D-254; M-1 to L-253; M-1 to S-252; M-1 to I-251; M-1 to Q-250; M-1 to A-249; M-1 to N-248; M-1 to E-247; M-1 to R-246; M-1 to P-245; M-1 to I-244; M-1 to A-243; M-1 to L-242; M-1 to Q-241; M-1 to L-240; M-1 to E-239; M-1 to D-238; M-1 to G-237; M-1 to E-236; M-1 to E-235; M-1 to L-234; M-1 to K-233; M-1 to A-232; M-1 to I-231; M-1 to G-230; M-1 to A-229; M-1 to S-228; M-1 to Y-227; M-1 to C-226; M-1 to S-225; M-1 to N-224; M-1 to N-223; M-1 to P-222; M-1 to L-221; M-1 to T-220; M-1 to E-219; M-1 to P-218; M-1 to M-217; M-1 to N-216; M-1 to Q-215; M-1 to I-214; M-1 to C-213; M-1 to R-212; M-1 to F-211; M-1 to L-210; M-1 to T-209; M-1 to V-208; M-1 to L-207; M-1 to S-206; M-1 to L-205; M-1 to E-204; M-1 to D-203; M-1 to G-202; M-1 to F-201; M-1 to V-200; M-1 to H-199; M-1 to V-198; M-1 to K-197; M-1 to K-196; M-1 to R-195; M-1 to Q-194; M-1 to I-193; M-1 to L-192; M-1 to H-191; M-1 to G-190; M-1 to M-189; M-1 to A-188; M-1 to Y-187; M-1 to T-186; M-1 to K-185; M-1 to D-184; M-1 to T-183; M-1 to Y-182; M-1 to L-181; M-1 to V-180; M-1 to Q-179; M-1 to G-178; M-1 to Y-177; M-1 to I-176; M-1 to F-175; M-1 to F-174; M-1 to Y-173; M-1 to G-172; M-1 to T-171; M-1 to E-170; M-1 to K-169; M-1 to V-168; M-1 to L-167; M-1 to I-166; M-1 to K-165; M-1 to N-164; M-1 to E-163; M-1 to K-162; M-1 to E-161; M-1 to E-160; M-1 to L-159; M-1 to A-158; M-1 to S-157; M-1 to G-156; M-1 to R-155; M-1 to K-154; M-1 to F-153; M-1 to S-152; M-1 to L-151; M-1 to L-150; M-1 to W-149; M-1 to P-148; M-1 to V-147; M-1 to F-146; M-1 to T-145; M-1 to Y-144; M-1 to S-143; M-1 to G-142; M-1 to T-141; M-1 to E-140; M-1 to E-139; M-1 to P-138; M-1 to G-137; M-1 to Q-136; M-1 to V-135; M-1 to A-134; M-1 to R-133; M-1 to K-132; M-1 to N-131; M-1 to R-130; M-1 to S-129; M-1 to N-128; M-1 to Q-127; M-1 to S-126; M-1 to S-125; M-1 to N-124; M-1 to G-123; M-1 to E-122; M-1 to G-121; M-1 to P-120; M-1 to A-119; M-1 to P-118; M-1 to P-117; M-1 to E-116; M-1 to F-115; M-1 to I-114; M-1 to K-113; M-1 to L-112; M-1 to G-111; M-1 to A-110; M-1 to T-109; M-1 to V-108; M-1 to A-107; M-1 to P-106; M-1 to A-105; M-1 to E-104; M-1 to E-103; M-1 to L-102; M-1 to G-101; M-1 to A-100; M-1 to K-99; M-1 to P-98; M-1 to A-97; M-1 to G-96; M-1 to A-95; M-1 to G-94; M-1 to A-93; M-1 to P-92; M-1 to L-91; M-1 to K-90; M-1 to E-89; M-1 to A-88; M-1 to H-87; M-1 to H-86; M-1 to G-85; M-1 to Q-84; M-1 to L-83; M-1 to E-82; M-1 to A-81; M-1 to R-80; M-1 to L-79; M-1 to S-78; M-1 to A-77; M-1 to L-76; M-1 to D-75; M-1 to G-74; M-1 to Q-73; M-1 to L-72; M-1 to A-71; M-1 to A-70; M-1 to V-69; M-1 to Q-68; M-1 to Y-67; M-1 to F-66; M-1 to S-65; M-1 to V-64; M-1 to V-63; M-1 to T-62; M-1 to L-61; M-1 to C-60; M-1 to C-59; M-1 to S-58; M-1 to L-57; M-1 to L-56; M-1 to A-55; M-1 to L-54; M-1 to L-53; M-1 to L-52; M-1 to T-51; M-1 to A-50; M-1 to A-49; M-1 to L-48; M-1 to L-47; M-1 to K-46; M-1 to G-45; M-1 to D-44; M-1 to K-43; M-1 to S-42; M-1 to S-41; M-1 to R-40; M-1 to V-39; M-1 to S-38; M-1 to P-37; M-1 to S-36; M-1 to E-35; M-1 to K-34; M-1 to R-33; M-1 to P-32; M-1 to L-31; M-1 to I-30; M-1 to S-29; M-1 to V-28; M-1 to C-27; M-1 to E-26; M-1 to K-25; M-1 to L-24; M-1 to K-23; M-1 to M-22; M-1 to E-21; M-1 to E-20; M-1 to R-19; M-1 to K-18; M-1 to K-17; M-1 to L-16; M-1 to C-15; M-1 to S-14; M-1 to T-13; M-1 to L-12; M-1 to R-11; M-1 to S-10; M-1 to Q-9; M-1 to E-8; M-1 to R-7 and M-1 to E-6, or alternatively, a polynucleotide encoding a polypeptide consisting of such a sequence. The present application discloses a polynucleotide encoding a neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide as described above that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97% And more preferably 99% or more of the same polynucleotide sequence. The invention also encompasses polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and / or polynucleotide sequences are also encompassed by the present invention. In addition, the present invention encompasses amino acid sequences which comprise at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% Polypeptides composed of sequences and polynucleotides encoding such polypeptides are included in the present invention.

The invention also encompasses both the amino and carboxyl termini of the neurotoxin-alpha polypeptide, which may be generally described as having the moiety n ⁵ -m ⁵ of SEQ ID NO: 19, wherein n ⁵ and m ⁵ are integers as defined above Lt; RTI ID = 0.0 > 1, < / RTI >

In a further aspect, the invention provides a polypeptide comprising the amino acid sequence of residue 134-m ⁶ in SEQ ID NO: 2, wherein m ⁶ is an integer from 140 to 285, corresponding to the position of the amino acid residue in SEQ ID NO: 2 . For example, the invention encompasses residues A-134 to Leu-285 of SEQ ID NO: 2; A-134 to L-284; A-134 to K-283; A-134 to L-282; A-134 to A-281; A-134 to G-280; A-134 to F-279; A-134 to F-278; A-134 to T-277; A-134 to V-276; A-134 to D-275; A-134 to G-274; A-134 to D-273; A-134 to L-272; A-134 to S-271; A-134 to I-270; A-134 to Q-269; A-134 to A-268; A-134 to N-267; A-134 to E-266; A-134 to R-265; A-134 to P-264; A-134 to I-263; A-134 to A-262; A-134 to L-261; A-134 to Q-260; A-134 to L-259; A-134 to E-258; A-134 to D-257; A-134 to G-256; A-134 to E-255; A-134 to E-254; A-134 to L-253; A-134 to K-252; A-134 to A-251; A-134 to I-250; A-134 to G-249; A-134 to A-248; A-134 to S-247; A-134 to Y-246; A-134 to C-245; A-134 to S-244; A-134 to N-243; A-134 to N-242; A-134 to P-241; A-134 to L-240; A-134 to T-239; A-134 to E-238; A-134 to P-237; A-134 to M-236; A-134 to N-235; A-134 to Q-234; A-134 to I-233; A-134 to C-232; A-134 to R-231; A-134 to F-230; A-134 to L-229; A-134 to T-228; A-134 to V-227; A-134 to L-226; A-134 to S-225; A-134 to L-224; A-134 to E-223; A-134 to D-222; A-134 to G-221; A-134 to F-220; A-134 to V-219; A-134 to H-218; A-134 to V-217; A-134 to K-216; A-134 to K-215; A-134 to R-214; A-134 to Q-213; A-134 to I-212; A-134 to L-211; A-134 to H-210; A-134 to G-209; A-134 to M-208; A-134 to A-207; A-134 to Y-206; A-134 to T-205; A-134 to K-204; A-134 to D-203; A-134 to T-202; A-134 to Y-201; A-134 to L-200; A-134 to V-199; A-134 to Q-198; A-134 to G-197; A-134 to Y-196; A-134 to I-195; A-134 to F-194; A-134 to F-193; A-134 to Y-192; A-134 to G-191; A-134 to T-190; A-134 to E-189; A-134 to K-188; A-134 to V-187; A-134 to L-186; A-134 to I-185; A-134 to K-184; A-134 to N-183; A-134 to E-182; A-134 to K-181; A-134 to E-180; A-134 to E-179; A-134 to L-178; A-134 to A-177; A-134 to S-176; A-134 to G-175; A-134 to R-174; A-134 to K-173; A-134 to F-172; A-134 to S-171; A-134 to L-170; A-134 to L-169; A-134 to W-168; A-134 to P-167; A-134 to V-166; A-134 to F-165; A-134 to T-164; A-134 to Y-163; A-134 to S-162; A-134 to G-161; A-134 to K-160; A-134 to Q-159; A-134 to I-158; A-134 to T-157; A-134 to P-156; A-134 to T-155; A-134 to E-154; A-134 to S-153; A-134 to D-152; A-134 to A-151; A-134 to I-150; A-134 to L-149; A-134 to Q-148; A-134 to L-147; A-134 to C-146; A-134 to D-145; A-134 to Q-144; A-134 to T-143; A polynucleotide encoding, or alternatively consisting of, an amino acid sequence selected from the group consisting of A-134 to V-142 and A-134 to T-141. Also provided herein is a polynucleotide sequence encoding a neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide as described above that is 80%, 85%, 90%, 92%, 95%, 96% Or 99% or more of the same polynucleotide sequence, or, alternatively, consists of such sequence. The invention also encompasses polynucleotide sequences fused to heterologous polynucleotide sequences. Polypeptides encoded by these nucleic acid and / or polynucleotide sequences are also encompassed by the present invention. In addition, the present invention encompasses amino acid sequences which comprise at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% Polypeptides composed of sequences and polynucleotides encoding such polypeptides are included in the present invention.

Additional preferred polypeptide fragments of the invention comprise residues M-1 to C-15 of SEQ ID NO: 2; D-2 to L-16; D-3 to K-17; S-4 to K-18; T-5 to R-19; E-6 to E-20; R-7 to E-21; E-8 to M-22; Q-9 to K-23; S-10 to L-24; R-11 to K-25; L-12 to E-26; T-13 to C-27; S-14 to V-28; K-15 to S-29; L-16 to I-30; K-17 to L-31; K-18 to P-32; R-19 to R-33; E-20 to K-34; E-21 to E-35; M-22 to S-36; K-23 to P-37; L-24 to S-38; K-25 to V-39; E-26 to R-40; C-27 to S-41; V-28 to S-42; S-29 to K-43; I-30 to D-44; L-31 to G-45; P-32 to K-46; R-33 to L-47; K-34 to L-48; E-35 to A-49; S-36 to A-50; P-37 to T-51; S-38 to L-52; V-39 to L-53; R-40 to L-54; S-41 to A-55; A-42 to L-56; K-43 to L-57; D-44 to S-58; G-45 to C-59; K-46 to C-60; L-47 to L-61; L-48 to T-62; L-49 to V-63; A-50 to V-64; T-51 to S-65; L-52 to F-66; L-53 to Y-67; L-54 to Q-68; A-55 to V-69; L-56 to A-70; L-57 to A-71; S-58 to L-72; C-59 to Q-73; C-60 to G-74; L-61 to D-75; T-62 to L-76; V-63 to A-77; V-64 to S-78; S-65 to L-79; F-66 to R-80; Y-67 to A-81; Q-68 to E-82; V-69 to L-83; A-70 to Q-84; A-71 to G-85; L-72 to H-86; Q-73 to H-87; G-74 to A-88; D-75 to E-89; L-76 to K-90; A-77 to L-91; S-78 to P-92; L-79 to A-93; R-80 to G-94; A-81 to A-95; E-82 to G-96; L-83 to A-97; Q-84 to P-98; G-85 to K-99; H-86 to A-100; H-87 to G-101; A-88 to L-102; E-89 to E-103; K-90 to E-104; L-91 to A-105; P-92 to P-106; A-93 to A-107; G-94 to V-108; A-95 to T-109; G-96 to A-110; A-97 to G-111; P-98 to L-112; K-99 to K-113; A-100 to I-114; G-101 to F-115; L-102 to E-116; E-103 to P-117; E-104 to P-118; A-105 to A-119; P-106 to P-120; A-107 to G-121; V-108 to E-122; T-109 to G-123; A-110 to N-124; G-111 to S-125; L-112 to S-126; K-113 to Q-127; I-114 to N-128; F-115 to S-129; E-116 to R-130; P-117 to N-131; P-118 to K-132; A-119 to R-133; P-120 to A-134; G-121 to V-135; E-122 to Q-136; G-123 to G-137; N-124 to P-138; S-125 to E-139; S-126 to E-140; Q-127 to T-141; N-128 to V-142; S-129 to T-143; R-130 to Q-144; N-131 to D-145; K-132 to C-146; R-133 to L-147; A-134 to Q-148; V-135 to L-149; Q-136 to I-150; G-137 to A-151; P-138 to D-152; E-139 to S-153; E-140 to E-154; T-141 to T-155; V-142 to P-156; T-143 to T-157; Q-144 to I-158; D-145 to Q-159; C-146 to K-160; L-147 to G-161; Q-148 to S-162; L-149 to Y-163; I-150 to T-164; A-151 to F-165; D-152 to V-166; S-153 to P-167; E-154 to W-168; T-155 to L-169; P-156 to L-170; T-157 to S-171; I-158 to F-172; Q-159 to K-173; K-160 to R-174; G-161 to G-175; S-162 to S-176; Y-163 to A-177; T-164 to L-178; F-165 to E-179; V-166 to E-180; P-167 to K-181; W-168 to E-182; L-169 to N-183; L-170 to K-184; S-171 to I-185; F-172 to L-186; K-173 to V-187; R-174 to K-188; G-175 to E-189; S-176 to T-190; A-177 to G-191; L-178 to Y-192; E-179 to F-193; E-180 to F-194; K-181 to I-195; E-182 to Y-196; N-183 to G-197; K-184 to Q-198; I-185 to V-199; L-186 to L-200; V-187 to Y-201; K-188 to T-202; E-189 to D-203; T-190 to K-204; G-191 to T-205; Y-192 to Y-206; F-193 to A-207; F-194 to M-208; I-195 to G-209; Y-196 to H-210; G-197 to L-211; Q-198 to I-212; V-199 to Q-213; L-200 to R-214; Y-201 to K-215; T-202 to K-216; D-203 to V-217; K-204 to H-218; T-205 to V-219; Y-206 to F-220; A-207 to G-221; M-208 to D-222; G-209 to E-223; H-210 to L-224; L-211 to S-225; I-212 to L-226; Q-213 to V-227; R-214 to T-228; K-215 to L-229; K-216 to F-230; V-217 to R-231; H-218 to C-232; V-219 to I-233; F-220 to Q-234; G-221 to N-235; D-222 to M-236; E-223 to P-237; L-224 to E-238; S-225 to T-239; L-226 to L-240; V-227 to P-241; T-228 to N-242; L-229 to N-243; F-230 to S-244; R-231 to C-245; C-232 to Y-246; I-233 to S-247; Q-234 to A-248; N-235 to G-249; M-236 to I-250; P-237 to A-251; E-238 to K-252; T-239 to L-253; L-240 to E-254; P-241 to E-255; N-242 to G-256; N-243 to D-257; S-244 to E-258; C-245 to L-259; Y-246 to Q-260; S-247 to L-261; A-248 to A-262; G-249 to I-263; I-250 to P-264; A-251 to R-265; K-252 to E-266; L-253 to N-267; E-254 to A-268; E-255 to Q-269; G-256 to I-270; D-257 to S-271; E-258 to L-272; L-259 to D-273; Q-260 to G-274; L-261 to D-275; A-262 to V-276; I-263 to T-277; P-264 to F-278; R-265 to F-279; E-266 to G-280; N-267 to A-281; A-268 to L-282; Q-269 to K-283; I-270 to L-284 and S-271 to L-285, or alternatively consists of such residues. Preferably, these polypeptide fragments have at least one functional activity (e. G., Biological activity, antigenicity and immunogenicity) of the neurotoxin-alpha and / or neurotoxin- alpha SV polypeptides according to the invention, , ≪ / RTI > as described for example in < RTI ID = 0.0 > In addition, the present invention encompasses amino acid sequences which comprise at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% ≪ / RTI > The invention also encompasses the amino acid sequences fused to heterologous amino acid sequences as described herein. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

Additional preferred polypeptide fragments of the invention comprise residues M-1 to C-15 of SEQ ID NO: 19; D-2 to L-16; D-3 to K-17; S-4 to K-18; T-5 to R-19; E-6 to E-20; R-7 to E-21; E-8 to M-22; Q-9 to K-23; S-10 to L-24; R-11 to K-25; L-12 to E-26; T-13 to C-27; S-14 to V-28; C-15 to S-29; L-16 to I-30; K-17 to L-31; K-18 to P-32; R-19 to R-33; E-20 to K-34; E-21 to E-35; M-22 to S-36; K-23 to P-37; L-24 to S-38; K-25 to V-39; E-26 to R-40; C-27 to S-41; V-28 to S-42; S-29 to K-43; I-30 to D-44; L-31 to G-45; P-32 to K-46; R-33 to L-47; K-34 to L-48; E-35 to A-49; S-36 to A-50; P-37 to T-51; S-38 to L-52; V-39 to L-53; R-40 to L-54; S-41 to A-55; A-42 to L-56; K-43 to L-57; D-44 to S-58; G-45 to C-59; K-46 to C-60; L-47 to L-61; L-48 to T-62; L-49 to V-63; A-50 to V-64; T-51 to S-65; L-52 to F-66; L-53 to Y-67; L-54 to Q-68; A-55 to V-69; L-56 to A-70; L-57 to A-71; S-58 to L-72; C-59 to Q-73; C-60 to G-74; L-61 to D-75; T-62 to L-76; V-63 to A-77; V-64 to S-78; S-65 to L-79; F-66 to R-80; Y-67 to A-81; Q-68 to E-82; V-69 to L-83; A-70 to Q-84; A-71 to G-85; L-72 to H-86; Q-73 to H-87; G-74 to A-88; D-75 to E-89; L-76 to K-90; A-77 to L-91; S-78 to P-92; L-79 to A-93; R-80 to G-94; A-81 to A-95; E-82 to G-96; L-83 to A-97; Q-84 to P-98; G-85 to K-99; H-86 to A-100; H-87 to G-101; A-88 to L-102; E-89 to E-103; K-90 to E-104; L-91 to A-105; P-92 to P-106; A-93 to A-107; G-94 to V-108; A-95 to T-109; G-96 to A-110; A-97 to G-111; P-98 to L-112; K-99 to K-113; A-100 to I-114; G-101 to F-115; L-102 to E-116; E-103 to P-117; E-104 to P-118; A-105 to A-119; P-106 to P-120; A-107 to G-121; V-108 to E-122; T-109 to G-123; A-110 to N-124; G-111 to S-125; L-112 to S-126; K-113 to Q-127; I-114 to N-128; F-115 to S-129; E-116 to R-130; P-117 to N-131; P-118 to K-132; A-119 to R-133; P-120 to A-134; G-121 to V-135; E-122 to Q-136; G-123 to G-137; N-124 to P-138; S-125 to E-139; S-126 to E-140; Q-127 to T-141; N-128 to G-142; S-129 to S-143; R-130 to Y-144; N-131 to D-145; K-132 to F-146; R-133 to V-147; A-134 to P-148; V-135 to W-149; Q-136 to L-150; G-137 to L-151; P-138 to S-152; E-139 to F-153; E-140 to K-154; T-141 to R-155; G-142 to G-156; S-143 to S-157; Y-144 to A-158; T-145 to L-159; F-146 to E-160; V-147 to E-161; P-148 to K-162; W-149 to E-163; L-150 to N-164; L-151 to K-165; S-152 to I-166; F-153 to L-167; K-154 to V-168; R-155 to K-169; G-156 to E-170; S-157 to T-171; A-158 to G-172; L-159 to Y-173; E-160 to F-174; E-161 to F-175; K-162 to I-176; E-163 to Y-177; N-164 to G-178; K-165 to Q-179; I-166 to V-180; L-167 to L-181; V-168 to Y-182; K-169 to T-183; E-170 to D-184; T-171 to K-185; G-172 to T-186; Y-173 to Y-187; F-174 to A-188; F-175 to M-189; I-176 to G-190; Y-177 to H-191; G-178 to L-192; Q-179 to I-193; V-180 to Q-194; L-181 to R-195; Y-182 to K-196; T-183 to K-197; D-184 to V-198; K-185 to H-199; T-186 to V-200; Y-187 to F-201; A-188 to G-202; M-189 to D-203; G-190 to E-204; H-191 to L-205; L-192 to S-206; I-193 to L-207; Q-194 to V-208; R-195 to T-209; K-196 to L-210; K-197 to F-211; V-198 to R-212; H-199 to C-213; V-200 to I-214; F-201 to Q-215; G-202 to N-216; D-203 to M-217; E-204 to P-218; L-205 to E-219; S-206 to T-220; L-207 to L-221; V-208 to P-222; T-209 to N-223; L-210 to N-224; F-211 to S-225; R-212 to C-226; C-213 to Y-227; I-214 to S-228; Q-215 to A-229; N-216 to G-230; M-217 to I-231; P-218 to A-232; E-219 to K-233; T-220 to L-234; L-221 to E-235; P-222 to E-236; N-223 to G-237; N-224 to D-238; S-225 to E-239; C-226 to L-240; Y-227 to Q-241; S-228 to L-242; A-229 to A-243; G-230 to I-244; I-231 to P-245; A-232 to R-246; K-233 to E-247; L-234 to N-248; E-235 to A-249; E-236 to Q-250; G-237 to I-251; D-238 to S-252; E-239 to L-253; L-240 to D-254; Q-241 to G-255; L-242 to D-256; A-243 to V-257; I-244 to T-258; P-245 to F-259; R-246 to F-260; E-247 to G-261; N-248 to A-262; A-249 to L-263; Q-250 to K-264; I-251 to L-265 and S-252 to L-266, or alternatively consists of such residues. Preferably, these polypeptide fragments have at least one functional activity (e. G., Biological activity, antigenicity and immunogenicity) of the neurotoxin-alpha and / or neurotoxin- alpha SV polypeptides according to the invention, , ≪ / RTI > as described for example in < RTI ID = 0.0 > In addition, the present invention encompasses amino acid sequences which comprise at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% ≪ / RTI > The present invention also encompasses the above amino acid sequences fused to heterologous amino acid sequences as described herein. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

Additional preferred polypeptide fragments of the invention comprise residues M-1 to F-15 of SEQ ID NO: 38; D-2 to C-16; E-3 to S-17; S-4 to E-18; A-5 to K-19; K-6 to G-20; T-7 to E-21; L-8 to D-22; P-9 to M-23; P-10 to K-24; P-11 to V-25; C-12 to G-26; L-13 to Y-27; C-14 to D-28; F-15 to P-29; C-16 to I-30; S-17 to T-31; E-18 to P-32; K-19 to Q-33; G-20 to K-34; E-21 to E-35; D-22 to E-36; M-23 to G-37; K-24 to A-38; V-25 to W-39; G-26 to F-40; Y-27 to G-41; D-28 to I-42; P-29 to C-43; I-30 to R-44; T-31 to D-45; P-32 to G-46; Q-33 to R-47; K-34 to L-48; E-35 to L-49; E-36 to A-50; G-37 to A-51; A-38 to T-52; W-39 to L-53; F-40 to L-54; G-41 to L-55; I-42 to A-56; C-43 to L-57; R-44 to L-58; D-45 to S-59; G-46 to S-60; R-47 to S-61; L-48 to F-62; L-49 to T-63; A-50 to A-64; A-51 to M-65; T-52 to S-66; L-53 to L-67; L-54 to Y-68; L-55 to Q-69; A-56 to L-70; L-57 to A-71; L-58 to A-72; S-59 to L-73; S-60 to Q-74; S-61 to A-75; F-62 to D-76; T-63 to L-77; A-64 to M-78; M-65 to N-79; S-66 to L-80; L-67 to R-81; Y-68 to M-82; Q-69 to E-83; L-70 to L-84; A-71 to Q-85; A-72 to S-86; L-73 to Y-87; Q-74 to R-88; A-75 to G-89; D-76 to S-90; L-77 to A-91; M-78 to T-92; N-79 to P-93; L-80 to A-94; R-81 to A-95; M-82 to A-96; E-83 to G-97; L-84 to A-98; Q-85 to P-99; S-86 to E-100; Y-87 to L-101; R-88 to T-102; G-89 to A-103; S-90 to G-104; A-91 to V-105; T-92 to K-106; P-93 to L-107; A-94 to L-108; A-95 to T-109; A-96 to P-110; G-97 to A-111; A-98 to A-112; P-99 to P-113; E-100 to R-114; L-101 to P-115; T-102 to H-116; A-103 to N-117; G-104 to S-118; V-105 to S-119; K-106 to R-120; L-107 to G-121; L-108 to H-122; T-109 to R-123; P-110 to N-124; A-111 to R-125; A-112 to R-126; P-113 to A-127; R-114 to F-128; P-115 to Q-129; H-116 to G-130; N-117 to P-131; S-118 to E-132; S-119 to E-133; R-120 to T-134; G-121 to E-135; H-122 to Q-136; R-123 to D-137; N-124 to V-138; R-125 to D-139; R-126 to L-140; A-127 to S-141; F-128 to A-142; Q-129 to P-143; G-130 to P-144; P-131 to A-145; E-132 to P-146; E-133 to C-147; T-134 to L-148; E-135 to P-149; Q-136 to G-150; D-137 to C-151; V-138 to R-152; D-139 to H-153; L-140 to S-154; S-141 to Q-155; A-142 to H-156; P-143 to D-157; P-144 to D-158; A-145 to N-159; P-146 to G-160; C-147 to M-161; L-148 to N-162; P-149 to L-163; G-150 to R-164; C-151 to N-165; R-152 to I-166; H-153 to I-167; S-154 to Q-168; Q-155 to D-169; H-156 to C-170; D-157 to L-171; D-158 to Q-172; N-159 to L-173; G-160 to I-174; M-161 to A-175; N-162 to D-176; L-163 to S-177; R-164 to D-178; N-165 to T-179; I-166 to P-180; I-167 to A-181; Q-168 to L-182; D-169 to E-183; C-170 to E-184; L-171 to K-185; Q-172 to E-186; L-173 to N-187; I-174 to K-188; A-175 to I-189; D-176 to V-190; S-177 to V-191; D-178 to R-192; T-179 to Q-193; P-180 to T-194; A-181 to G-195; L-182 to Y-196; E-183 to F-197; E-184 to F-198; K-185 to I-199; E-186 to Y-200; N-187 to S-201; K-188 to Q-202; I-189 to V-203; V-190 to L-204; V-191 to Y-205; R-192 to T-206; Q-193 to D-207; T-194 to P-208; G-195 to I-209; Y-196 to F-210; F-197 to A-211; F-198 to M-212; I-199 to G-213; Y-200 to H-214; S-201 to V-215; Q-202 to I-216; V-203 to Q-217; L-204 to R-218; Y-205 to K-219; T-206 to K-220; D-207 to V-221; P-208 to H-222; I-209 to V-223; F-210 to F-224; A-211 to G-225; M-212 to D-226; G-213 to E-227; H-214 to L-228; V-215 to S-229; I-216 to L-230; Q-217 to V-231; R-218 to T-232; K-219 to L-233; K-220 to F-234; V-221 to R-235; H-222 to C-236; V-223 to I-237; F-224 to Q-238; G-225 to N-239; D-226 to M-240; E-227 to P-241; L-228 to K-242; S-229 to T-243; L-230 to L-244; V-231 to P-245; T-232 to N-246; L-233 to N-247; F-234 to S-248; R-235 to C-249; C-236 to Y-250; I-237 to S-251; Q-238 to A-252; N-239 to G-253; M-240 to I-254; P-241 to A-255; K-242 to R-256; T-243 to L-257; L-244 to E-258; P-245 to E-259; N-246 to G-260; N-247 to D-261; S-248 to E-262; C-249 to I-263; Y-250 to Q-264; S-251 to L-265; A-252 to A-266; G-253 to I-267; I-254 to P-268; A-255 to R-269; R-256 to E-270; L-257 to N-271; E-258 to A-272; E-259 to Q-273; G-260 to I-274; D-261 to S-275; E-262 to R-276; I-263 to N-277; Q-264 to G-278; L-265 to D-279; A-266 to D-280; I-267 to T-281; P-268 to F-282; R-269 to F-283; E-270 to G-284; N-271 to L-285 A-272; L-286; Q-273 to K-287; I-274 to L-288 and S-275 to L-289, or alternatively consists of such residues. Preferably, these polypeptide fragments have at least one functional activity (e. G., Biological activity, antigenicity and immunogenicity) of the neurotoxin-alpha and / or neurotoxin- alpha SV polypeptides according to the invention, , ≪ / RTI > as described for example in < RTI ID = 0.0 > In addition, the present invention encompasses amino acid sequences which comprise at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% ≪ / RTI > The invention also encompasses the amino acid sequences fused to heterologous amino acid sequences as described herein. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

Those skilled in the art will appreciate that some amino acid sequences of the neurotoxin-alpha and neurotoxin-alpha SV polypeptides may be varied without significant effect on the structure or function of the polypeptide. It should be kept in mind that when the difference in sequence is taken into account, an important region for measuring activity is present in the polypeptide.

Thus, the present invention contemplates a mutation of a neurotoxin-alpha polypeptide comprising a region of a neurotoxin-alpha polypeptide, such as a polypeptide fragment that exhibits a neurotoxin-alpha polypeptide functional activity (e. G., Biological activity) . In addition, the present invention relates to a neurotoxin-alpha SV polypeptide comprising a region of a neurotoxin-alpha SV polypeptide, such as a polypeptide fragment that exhibits a neurotoxin-alpha SV polypeptide functional activity (e. G., Biological activity) Includes variations of the peptides. Such variants include deletions, insertions, inversions, repeats, and type substitutions selected in accordance with general rules known in the art so as to have little effect on activity. For example, guidance on how to expressively produce amino acid substitutions of silence can be found in Bowie. J. U. et al., &Quot; Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions ", Science 247: 1306-1310 (1990). Here, the authors suggest that there are two main ways to study resistance to mutations in amino acid sequences. The first method relies on the process of evolution in which mutations are either allowed or rejected by natural selection. The second method uses screening or screening to identify sequences that maintain genetic engineering and functionality that introduce amino acid variations at specific locations in the cloned gene.

As the authors have stated, proteins from these studies have been found to be surprisingly intolerant of amino acid substitutions. The authors also indicate which amino acid variations can be accommodated at specific locations in the protein. For example, most buried amino acid residues require a non-polar side chain while the characteristics of the side chain are generally conserved in general. Other phenotypic silencing substitutions are described in this reference [Biwie, J. U. et al.] And references cited herein. Preservative substitutions typically include substitution of one of the aliphatic amino acids Ala, Val, Leu, and Ile for another, exchange of the acidic residues Asp and GIu, substitution between the amide residues Asn and Gln, exchange of the basic residues Lys and Arg, Substitution between residues Phe and Thr.

Thus, fragments, derivatives or analogs of those encoded by the polypeptides or deposited cDNA plasmids depicted in Figures 1A and 1B (SEQ ID NO: 2) are those that (i) are amino acid residues in which at least one of the amino acid residues is non- (Ii) one or more of the amino acid residues comprises a substituent group, (iii) the extracellular region of the polypeptide is replaced with an extracellular domain of the polypeptide, Domain is fused with another compound such as a compound that increases the half-life of the polypeptide (e. G., Polyethylene glycol) or (iv) the additional amino acid is fused to the extracellular domain of the polypeptide, e. G., An IgG Fc fusion region peptide or leader Secretory sequence or a sequence or proprotein sequence used to purify the extracellular domain of the polypeptide It may be connected. Such fragments, derivatives and analogs are within the scope of those skilled in the art from the teachings herein.

In addition, fragments, derivatives or analogs of those encoded by the polypeptides or deposited cDNA plasmids depicted in Figures 5A and 5B (SEQ ID NO: 19) can be used to detect (i) amino acid residues in which at least one of the amino acid residues is non- (Ii) one or more of the amino acid residues comprises a substituent group, (iii) the extracellular domain of the polypeptide, Domain is fused with another compound such as a compound that increases the half-life of the polypeptide (e. G., Polyethylene glycol) or (iv) the additional amino acid is fused to an extracellular domain of the polypeptide, e. G., Another TNF ligand family member CD40 ligand), a biologically active fragment of IgG Fc fusion region peptide or leader or secretory sequence or polypeptide May be linked to a sequence or a proprotein sequence used to purify the extracellular domain of the gene. Such fragments, derivatives and analogs are within the scope of those skilled in the art from the teachings herein.

Thus, the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides of the present invention may comprise one or more amino acid substitutions, deletions or additions by natural mutation or manipulation of humans. As noted, minor mutations are preferred, such as secondary amino acid substitutions that do not significantly affect folding or activity of the protein (see Table 2)

Conservative amino acid substitution Aromatic Phenylalanine tryptophan tyrosine Hydrophobicity Leucine isoleucine valine polarity Glutamine asparagine Basic Arginine lysine histidine acid Aspartic acid glutamic acid small type Alanine serine threonine methionine glycine

In one embodiment of the invention, the polypeptide is an amino acid that contains more than one, not more than 50, preferably not more than 40, more preferably not more than 30, even more preferably not more than 20 conservative amino acid substitutions Or alternatively consists of, the amino acid sequence of a neurotoxin-alpha or neurotoxin-alpha SV polypeptide having a sequence. Of course, those polypeptides comprising an amino acid sequence of a neurotoxin-alpha polypeptide containing one or more, 10, 9, 8, 7, 6, 5, 4, 3, 2 or fewer conservative amino acid substitutions in increasing order of preference Peptides or polypeptides having an amino acid sequence are highly preferred.

For example, site directed mutations at the amino acid level of neurotoxin-alpha can be made by substituting a conservative substitution for a particular amino acid. Preferred conservative substitution mutations of the neurotoxin-alpha amino acid sequence provided in SEQ ID NO: 2 include substitution of M1 with A, G, I, L, S, T or V; D2 is replaced by E; D3 is replaced by E; S4 is substituted with A, G, I, L, T, M or V; T5 is substituted with A, I, L, S, M or V; E6 is substituted by D; R7 is H or K; E8 is replaced by D; Q9 is substituted by N; S10 is substituted with A, G, I, L, T, M or V; R < 11 > is H or K; L12 is substituted with A, G, I, S, T, M or V; T13 is substituted by A; G, I, L, S, M or V; S14 is substituted with A, G, I, L, T, M or V; L16 is substituted with A, G, I, S, T, M or V; K17 is H or R; K18 is substituted by H or R; R19 is H or K; E20 is replaced by D; E21 is substituted by D; M22 is substituted with A, G, I, L, S, T or V; K23 is substituted by H or R; L24 is substituted with A, G, I, S, T, M or V; K25 is H or substituted by R; E26 is substituted by D; V28 is substituted with A, G, I, L, S, T or M; S29 is substituted with A, G, I, L, T, M or V; I30 is replaced by A, G, L, S, T, M or V; L31 is substituted with A, G, I, S, T, M or V; R33 is substituted with H or K; K34 is H or R; E35 is replaced by D; S36 is substituted with A, G, I, L, T, M or V; S38 is substituted with A, G, I, L, T, M or V; V39 is replaced by A, G, I, L, S, T or M; R40 is substituted with H or K; S41 is substituted with A, G, I, L, T, M or V; S42 is substituted with A, G, I, L, T, M or V; K43 is substituted by H or R; D44 is replaced by E; G45 is substituted with A, I, L, S, T, M or V; K46 is substituted by H or R; L47 is substituted with A, G, I, S, T, M or V; L48 is substituted with A, G, I, S, T, M or V; A49 is substituted with G, I, L, S, T, M or V; A50 is substituted with G, I, L, S, T, M or V; T51 is substituted with A, G, I, L, S, M or V; L52 is substituted with A, G, I, S, T, M or V; L53 is substituted with A, G, I, S, T, M or V; L54 is substituted with A, G, I, S, T, M or V; A55 is substituted with G, I, L, S, T, M or V; L56 is substituted with A, G, I, S, T, M or V; L57 is substituted with A, G, I, S, T, M or V; S58 is substituted with A, G, I, S, T, M or V; L61 is substituted with A, G, I, S, T, M or V; T62 is substituted with A, G, I, S, T, M or V; V63 is substituted with A, G, I, L, S, T or V; V64 is substituted with A, G, I, S, T, M or V; S65 is substituted with A, G, I, L, T, M or V; F66 is substituted with W or Y; Y67 is substituted by F or W; Q68 is replaced by N; V69 is substituted with A, G, I, L, S, T or M; A70 is substituted with G, I, L, S, T, M or V; A71 is substituted with G, I, L, S, T, M or V; L72 is substituted with A, G, I, S, T, M or V; Q73 is replaced by N; G74 is substituted with A, I, L, S, T, M or V; D75 is replaced by E; L76 is substituted with A, G, I, S, T, M or V; A77 is substituted with G, I, L, S, T, M or V; S78 is substituted with A, G, I, L, T, M or V; L79 is substituted with A, G, I, S, T, M or V; R80 is H or K; A81 is substituted with G, I, L, S, T, M or V; E82 is replaced by D; L83 is substituted with A, G, I, S, T, M or V; Q84 is substituted by N; G85 is substituted with A, I, L, S, T, M or V; H86 is substituted with K or R; H87 is substituted with K or R; A88 is substituted with G, I, L, S, T, M or V; E89 is replaced by D; K90 is replaced by H or R; L91 is substituted with A, G, I, S, T, M or V; A93 is substituted with G, I, L, S, T, M or V; G94 is substituted with A, I, L, S, T, M or V; A95 is substituted with G, I, L, S, T, M or V; G96 is substituted with A, I, L, S, T, M or V; A97 is substituted with G, I, L, S, T, M or V; K99 is H or substituted by R; A100 is substituted with G, I, L, S, T, M or V; G101 is substituted with A, I, L, S, T, M or V; L102 is substituted with A, G, I, S, T, M or V; E103 is substituted by D; E104 is replaced by D; A105 is substituted with G, I, L, S, T, M or V; A107 is substituted with G, I, L, S, T, M or V; V108 is substituted with A, G, I, S, T, M or V; T109 is substituted with A, G, I, S, T, M or V; A110 is substituted with G, I, L, S, T or M; G111 is substituted with A, I, L, S, T, M or V; L112 is substituted with A, G, I, S, T, M or V; K113 is substituted by H or R; I114 is substituted with A, G, L, S, T, M or V; F115 is substituted with W or Y; E116 is substituted by D; A119 is substituted with G, I, L, S, T, M or V; G121 is substituted with A, I, L, S, T, M or V; E122 is replaced by D; G123 is substituted with A, I, L, S, T, M or V; N124 is substituted by Q; S125 is substituted with A, G, I, L, T, M or V; S126 is substituted with A, G, I, L, T, M or V; Q127 is substituted by N; N128 is substituted by Q; S129 is substituted with A, G, I, L, T, M or V; R130 is replaced by H or K; N131 is substituted by Q; K132 is replaced by H or R; R133 is substituted with H or K; A134 is substituted with G, I, L, S, T, M or V; V135 is substituted with A, G, I, L, T or M; Q136 is substituted by N; G137 is substituted with A, I, L, S, T, M or V; E139 is replaced by D; E140 is replaced by D; T141 is substituted with A, G, I, L, S, M or V; V142 is substituted with A, G, I, L, S, T or M; T143 is substituted with A, G, I, L, S, M or V; Q144 is substituted by N; D145 is replaced by E; L147 is substituted with A, G, I, S, T, M or V; Q148 is substituted by N; L149 is substituted with A, G, I, S, T, M or V; I150 is replaced by A, G, L, S, T, M or V; A151 is substituted with G, I, L, S, T, M or V; D152 is replaced by E; S153 is substituted with A, G, I, L, T, M or V; E154 is replaced by D; T155 is substituted with A, G, I, L, S, M or V; T157 is substituted with A, G, I, L, S, M or V; I158 is substituted with A, G, L, S, T, M or V; Q159 is substituted by N; K160 is replaced by H or R; G161 is substituted with A, I, L, S, T, M or V; S162 is substituted with A, G, I, L, T, M or V; Y163 is substituted with F or W; T164 is substituted with A, G, I, L, S, M or V; F165 is replaced by W or Y; V166 is substituted with A, G, I, L, S, T or M; L169 is substituted with A, G, I, S, T, M or V; Substituting L170 with A, G, I, S, T, M or V; S171 is substituted with A, G, I, L, T, M or V; F172 is substituted with W or Y; K173 is substituted by H or R; R174 is substituted with H or K; G175 is substituted with A, I, L, S, T, M or V; S176 is substituted with A, G, I, L, T, M or V; A177 is substituted with G, I, L, S, T, M or V; L178 is substituted with A, G, I, S, T, M or V; E179 is substituted by D; E180 is replaced by D; K181 is substituted with H or R; E182 is substituted by D; N183 is substituted by Q; K184 is substituted by H or R; I185 is substituted with A, G, L, S, T, M or V; L186 is substituted with A, G, I, S, T, M or V; V187 is substituted with A, G, I, L, S, T or M; K188 is substituted with H or R; E189 is substituted by D; T190 is substituted with A, G, I, L, S, M or V; G191 is substituted with A, I, L, S, T, M or V; Y192 is substituted with F or W; F193 is substituted with W or Y; F194 is substituted with W or Y; I195 is substituted with A, G, L, S, T, M or V; Y196 is substituted with F or W; G197 is substituted with A, I, L, S, T, M or V; Q198 is substituted by N; V199 is replaced by A, G, I, L, S, T or M; L200 is substituted with A, G, I, S, T, M or V; Y201 is substituted by F or W; T202 is substituted with A, G, I, L, S, M or V; D203 is replaced by E; K204 is substituted by H or R; T205 is substituted with A, G, I, L, S, M or V; Y206 is substituted with F or W; A207 is substituted with G, I, L, S, T, M or V; M208 is substituted with A, G, I, L, S, T or V; G209 is substituted with A, I, L, S, T, M or V; H210 is replaced by K or R; L211 is substituted with A, G, I, S, T, M or V; I212 is substituted with A, G, L, S, T, M or V; Q213 is substituted by N; R214 is H or K; K215 is H or R; K216 is H or substituted by R; V217 is substituted with A, G, I, L, S, T or M; H218 is substituted with K or R; V219 is substituted with A, G, I, L, S, T or M; F220 is replaced by W or Y; G221 is substituted with A, I, L, S, T, M or V; D222 is replaced by E; E223 is substituted by D; L224 is substituted with A, G, I, S, T, M or V; S225 is substituted with A, G, I, L, T, M or V; L226 is substituted with A, G, I, S, T, M or V; V227 is substituted with A, G, I, L, S, T or M; T228 is substituted with A, G, I, L, S, M or V; L229 is substituted with A, G, I, S, T, M or V; F230 is replaced with W or Y; R231 is substituted with H or K; I233 is substituted with A, G, L, S, T, M or V; Q234 is substituted by N; N235 is substituted by Q; M236 substituted with A, G, I, L, S, T or V; E238 is substituted by D; T239 is substituted with A, G, I, L, S, M or V; E238 is substituted by D; T239 is substituted with A, G, I, L, S, M or V; L240 is replaced with A, G, I, S, T, M or V; N242 is substituted by Q; N243 is substituted by Q; S244 is substituted with A, G, I, L, T, M or V; Y246 is substituted by F or W; S247 is substituted with A, G, I, L, T, M or V; A248 is substituted with G, I, L, S, T, M or V; G249 is substituted with A, I, L, S, T, M or V; I250 is replaced by A, G, L, S, T, M or V; A251 is substituted with G, I, L, S, T, M or V; K252 is H or R; L253 is substituted with A, G, I, S, T, M or V; E254 is replaced by D; E255 is replaced by D; G256 is substituted with A, I, L, S, T, M or V; D257 is replaced by E; E258 is substituted by D; L259 is substituted with A, G, I, S, T, M or V; Q260 is replaced by N; L261 is substituted with A, G, I, S, T, M or V; A262 is substituted with G, I, L, S, T, M or V; I263 is substituted with A, G, L, S, T, M or V; R265 is replaced by H or K; E266 is substituted by D; N267 is substituted by Q; A268 is substituted with G, I, L, S, T, M or V; Q269 is substituted by N; I270 is replaced by A, G, L, S, T, M or V; S271 is substituted with A, G, I, L, T, M or V; L272 is substituted with A, G, I, S, T, M or V; D273 is replaced by E; G274 is substituted with A, I, L, S, T, M or V; D275 is replaced by E; V276 is substituted with A, G, I, L, S, T or M; T277 is substituted with A, G, I, L, S, M or V; F278 is replaced by W or Y; F279 is replaced by W or Y; G280 is substituted with A, I, L, S, T, M or V; A281 is substituted with G, I, L, S, T, M or V; L282 is substituted with A, G, I, S, T, M or V; K283 is substituted by H or R; L284 is substituted with A, G, I, S, T, M or V and / or L285 is substituted with A, G, I, S, T, M or V. Polynucleotides encoding these polypeptides are also encompassed by the present invention. The neurotoxin-alpha protein products of the present invention can be routinely screened for neurotoxin-alpha and / or neurotoxin-alpha SV functional activity and / or physical properties (e.g., increased or decreased stability and / or solubility) . Preferably, the resulting protein of the invention has increased and / or reduced neurotoxin-alpha and / or neurotoxin-alpha SV functional activity. More preferably, the neurotoxin-alpha and / or neurotoxin-alpha SV protein products of the present invention have one or more increased and / or reduced neurotoxin-alpha and / or neurotoxin-alpha SV functional activity and / Respectively.

In another embodiment, site directed mutations at the amino acid level of the neurotoxin-alpha SV can be made by substituting a particular amino acid with a conservative substitution. Preferred conservative substitution mutations of the neurotoxin-alpha SV amino acid sequence provided in SEQ ID NO: 19 include those wherein M1 is substituted with A, G, I, L, S, T or V; D2 is replaced by E; D3 is replaced by E; S4 is substituted with A, G, I, L, T, M or V; T5 is substituted with A, I, L, S, M or V; E6 is substituted by D; R7 is H or K; E8 is replaced by D; Q9 is substituted by N; S10 is substituted with A, G, I, L, T, M or V; R < 11 > is H or K; L12 is substituted with A, G, I, S, T, M or V; T13 is substituted with A, G, I, L, S, M or V; S14 is substituted with A, G, I, L, T, M or V; L16 is substituted with A, G, I, S, T, M or V; K17 is H or R; K18 is substituted by H or R; R19 is H or K; E20 is replaced by D; E21 is substituted by D; M22 is substituted with A, G, I, L, S, T or V; K23 is substituted by H or R; L24 is substituted with A, G, I, S, T, M or V; K25 is H or substituted by R; E26 is substituted by D; V28 is substituted with A, G, I, L, S, T or M; S29 is substituted with A, G, I, L, T, M or V; I30 is replaced by A, G, L, S, T, M or V; L31 is substituted with A, G, I, S, T, M or V; R33 is substituted with H or K; K34 is H or R; E35 is replaced by D; S36 is substituted with A, G, I, L, T, M or V; S38 is substituted with A, G, I, L, T, M or V; V39 is replaced by A, G, I, L, S, T or M; R40 is substituted with H or K; S41 is substituted with A, G, I, L, T, M or V; S42 is substituted with A, G, I, L, T, M or V; K43 is substituted by H or R; D44 is replaced by E; G45 is substituted with A, I, L, S, T, M or V; K46 is substituted by H or R; L47 is substituted with A, G, I, S, T, M or V; L48 is substituted with A, G, I, S, T, M or V; A49 is substituted with G, I, L, S, T, M or V; A50 is substituted with G, I, L, S, T, M or V; T51 is substituted with A, G, I, L, S, M or V; L52 is substituted with A, G, I, S, T, M or V; L53 is substituted with A, G, I, S, T, M or V; L54 is substituted with A, G, I, S, T, M or V; A55 is substituted with G, I, L, S, T, M or V; L56 is substituted with A, G, I, S, T, M or V; L57 is substituted with A, G, I, S, T, M or V; S58 is substituted with A, G, I, S, T, M or V; L61 is substituted with A, G, I, S, T, M or V; T62 is substituted with A, G, I, S, T, M or V; V63 is substituted with A, G, I, L, S, T or V; V64 is substituted with A, G, I, S, T, M or V; S65 is substituted with A, G, I, L, T, M or V; F66 is substituted with W or Y; Y67 is substituted by F or W; Q68 is replaced by N; V69 is substituted with A, G, I, L, S, T or M; A70 is substituted with G, I, L, S, T, M or V; A71 is substituted with G, I, L, S, T, M or V; L72 is substituted with A, G, I, S, T, M or V; Q73 is replaced by N; G74 is substituted with A, I, L, S, T, M or V; D75 is replaced by E; L76 is substituted with A, G, I, S, T, M or V; A77 is substituted with G, I, L, S, T, M or V; S78 is substituted with A, G, I, L, T, M or V; L79 is substituted with A, G, I, S, T, M or V; R80 is H or K; A81 is substituted with G, I, L, S, T, M or V; E82 is replaced by D; L83 is substituted with A, G, I, S, T, M or V; Q84 is substituted by N; G85 is substituted with A, I, L, S, T, M or V; H86 is substituted with K or R; H87 is substituted with K or R; A88 is substituted with G, I, L, S, T, M or V; E89 is replaced by D; K90 is replaced by H or R; L91 is substituted with A, G, I, S, T, M or V; A93 is substituted with G, I, L, S, T, M or V; G94 is substituted with A, I, L, S, T, M or V; A95 is substituted with G, I, L, S, T, M or V; G96 is substituted with A, I, L, S, T, M or V; A97 is substituted with G, I, L, S, T, M or V; K99 is H or substituted by R; A100 is substituted with G, I, L, S, T, M or V; G101 is substituted with A, I, L, S, T, M or V; L102 is substituted with A, G, I, S, T, M or V; E103 is substituted by D; E104 is replaced by D; A105 is substituted with G, I, L, S, T, M or V; A107 is substituted with G, I, L, S, T, M or V; V108 is replaced by A, G, I, L, S, T or M; T109 is substituted with A, G, I, S, T, M or V; A110 is substituted with G, I, L, S, T, M or V; G111 is substituted with A, I, L, S, T, M or V; L112 is substituted with A, G, I, S, T, M or V; K113 is substituted by H or R; I114 is substituted with A, G, L, S, T, M or V; F115 is substituted with W or Y; E116 is substituted by D; A119 is substituted with G, I, L, S, T, M or V; G121 is substituted with A, I, L, S, T, M or V; E122 is replaced by D; G123 is substituted with A, I, L, S, T, M or V; N124 is substituted by Q; S125 is substituted with A, G, I, L, T, M or V; S126 is substituted with A, G, I, L, T, M or V; Q127 is substituted by N; N128 is substituted by Q; S129 is substituted with A, G, I, L, T, M or V; R130 is replaced by H or K; N131 is substituted by Q; K132 is replaced by H or R; R133 is substituted with H or K; A134 is substituted with G, I, L, S, T, M or V; V135 is substituted with A, G, I, L, T, M or V; Q136 is substituted by N; G137 is substituted with A, I, L, S, T, M or V; E139 is replaced by D; E140 is replaced by D; T141 is substituted with A, G, I, L, S, M or V; G142 is substituted with A, I, L, S, T, M or V; S143 is substituted with A, G, I, L, T, M or V; Y144 is substituted with F or W; T145 is substituted with A, G, I, L, S, M or V; F146 is replaced by W or Y; V147 is substituted with A, G, I, L, S, T or M; W149 is substituted by F or Y; Substitution of L150 with A, G, I, S, T, M or V; L151 is substituted with A, G, I, S, T, M or V; S152 is substituted with A, G, I, L, T, M or V; F153 is substituted with W or Y; K154 is substituted by H or R; R155 is substituted with H or K; G156 is substituted with A, I, L, S, T, M or V; S157 is substituted with A, G, I, L, T, M or V; A158 is substituted with G, I, L, S, T, M or V; L159 is substituted with A, G, I, S, T, M or V; E160 is replaced by D; E161 is substituted by D; K162 is substituted by H or R; E163 is substituted by D; N164 is substituted by Q; K165 is substituted by H or R; I166 is substituted with A, G, L, S, T, M or V; L167 is substituted with A, G, I, S, T, M or V; V168 is substituted with A, G, I, L, S, T or M; K169 is substituted by H or R; E170 is replaced by D; T171 is substituted with A, G, I, L, S, M or V; G172 is substituted with A, I, L, S, T, M or V; Y173 is substituted with F or W; F174 is substituted with W or Y; F175 is replaced by W or Y; I176 is substituted with A, G, L, S, T, M or V; Y177 is substituted with F or W; G178 is substituted with A, I, L, S, T, M or V; Q179 is substituted by N; V180 is replaced by A, G, I, L, S, T or M; L181 is substituted with A, G, I, S, T, M or V; Y182 is substituted with F or W; T183 is substituted with A, G, I, L, S, M or V; D184 is replaced by E; K185 is H or R; T186 is substituted with A, G, I, L, S, M or V; Y187 is substituted by F or W; A188 is substituted with G, I, L, S, T, M or V; M189 is substituted with A, G, I, L, S, T or V; G190 is substituted with A, I, L, S, T, M or V; H191 is substituted with K or R; L192 is substituted with A, G, I, S, T, M or V; I193 is substituted with A, G, L, S, T, M or V; Q194 is substituted by N; R195 is substituted by H or K; K196 is substituted by H or R; K197 is H or substituted by R; V198 is substituted with A, G, I, L, S, T or M; H199 is substituted with K or R; V200 is replaced by A, G, I, L, S, T or M; F201 is substituted with W or Y; G202 is substituted with A, I, L, S, T, M or V; D203 is replaced by E; E204 is replaced by D; L205 is substituted with A, G, I, S, T, M or V; S206 is substituted with A, G, I, L, T, M or V; L207 is substituted with A, G, I, S, T, M or V; V208 is replaced with A, G, I, L, S, T or M; T209 is substituted with A, G, I, L, S, M or V; L210 is substituted with A, G, I, S, T, M or V; F211 is substituted with W or Y; R212 is replaced by H or K; I214 is substituted with A, G, L, S, T, M or V; Q215 is substituted by N; N216 is substituted by Q; M217 is substituted with A, G, I, L, S, T or V; E219 is substituted by D; T220 is substituted with A, G, I, L, S, M or V; L221 is substituted with A, G, I, S, T, M or V; N223 is substituted by Q; N224 is replaced by Q; S225 is substituted with A, G, I, L, T, M or V; Y227 is substituted by F or W; S228 is substituted with A, G, I, L, T, M or V; A229 is substituted with G, I, L, S, T, M or V; G230 is substituted with A, I, L, S, T, M or V; I231 is substituted with A, G, L, S, T, M or V; A232 is substituted with G, I, L, S, T, M or V; K233 is substituted by H or R; L234 is substituted with A, G, I, S, T, M or V; E235 is replaced by D; E236 is substituted by D; G237 is substituted with A, I, L, S, T, M or V; D238 is replaced by E; E239 is replaced by D; L240 is replaced with A, G, I, S, T, M or V; Q241 is substituted by N; L242 is substituted with A, G, I, S, T, M or V; A243 is substituted with G, I, L, S, T, M or V; I244 is substituted with A, G, L, S, T, M or V; R246 is substituted with H or K; E247 is substituted by D; N248 is substituted by Q; A249 is substituted with G, I, L, S, T, M or V; Q250 replaced by N; I251 is substituted with A, G, L, S, T, M or V; S252 is substituted with A, G, I, L, T, M or V; L253 is substituted with A, G, I, S, T, M or V; D254 is replaced by E; G255 is substituted with A, I, L, S, T, M or V; D256 is replaced by E; V257 is substituted with A, G, I, L, S, T or M; T258 is substituted with A, G, I, L, S, M or V; F259 is replaced by W or Y; F260 is replaced by W or Y; G261 is substituted with A, I, L, S, T, M or V; A262 is substituted with G, I, L, S, T, M or V; L263 is substituted with A, G, I, S, T, M or V; K264 is replaced by H or R; L265 is substituted by A, G, I, S, T, M or V and / or L266 is substituted by A, G, I, S, T, M or V. Polynucleotides encoding these polypeptides are also encompassed by the present invention. The neurotoxin-alpha protein products of the present invention can be routinely screened for neurotoxin-alpha and / or neurotoxin-alpha SV functional activity and / or physical properties (e.g., increased or decreased stability and / or solubility) . Preferably, the resulting protein of the invention has increased and / or reduced neurotoxin-alpha and / or neurotoxin-alpha SV functional activity. More preferably, the neurotoxin-alpha and / or neurotoxin-alpha SV protein products of the present invention have one or more increased and / or reduced neurotoxin-alpha and / or neurotoxin-alpha SV functional activity and / Respectively.

In another embodiment, site directed mutations at amino acid levels of neurotoxin-alpha can be made by substituting a particular amino acid with a conservative substitution. Preferred conservative substitution mutations of the neurotoxin-alpha amino acid sequence provided in SEQ ID NO: 23 include those wherein R1 is replaced by H or K; V2 is substituted with A, G, I, L, S, T or M; V3 is replaced by A, G, I, L, S, T or M; D4 is replaced by E; L5 is substituted with A, G, I, S, T, M or V; S6 is substituted with A, G, I, L, T, M or V; A7 is substituted with G, I, L, S, T, M or V; A10 is substituted with G, I, L, S, T, M or V; L13 is substituted with A, G, I, S, T, M or V; G15 is substituted with A, I, L, S, T, M or V; R17 is substituted with H or K; H18 is substituted with K or R; S19 is substituted with A, G, I, L, T, M or V; Q20 is replaced by N; H21 is substituted with K or R; D22 is replaced by E; D23 is substituted by E; N24 is substituted by Q; G25 is substituted with A, I, L, S, T, M or V; M26 is substituted with A, G, I, L, S, T or V; N27 is substituted by Q; L28 is substituted with A, G, I, S, T, M or V; R29 is substituted by H or K; N30 is substituted by Q; R31 is substituted with H or K; T32 is substituted with A, G, I, L, S, M or V; Y33 is substituted by F or W; T34 is substituted with A, G, I, L, S, M or V; F35 is substituted with W or Y; V36 is replaced by A, G, I, L, S, T or M; W38 is substituted by F or Y; L39 is substituted with A, G, I, S, T, M or V; L40 is substituted with A, G, I, S, T, M or V; S41 is substituted with A, G, I, L, T, M or V; F42 is substituted with W or Y; K43 is substituted by H or R; R44 is H or K; G45 is substituted with A, I, L, S, T, M or V; N46 is substituted by Q; A47 is substituted with G, I, L, S, T, M or V; L48 is substituted with A, G, I, S, T, M or V; E49 is replaced by D; E50 is replaced by D; K51 is substituted by H or R; E52 is replaced by D; N53 is substituted by Q; K54 is substituted by H or R; I55 is substituted with A, G, L, S, T, M or V; V56 is substituted with A, G, I, L, S, T or M; V57 is replaced by A, G, I, L, S, T or M; R58 is H or K; Q59 is substituted by N; T60 is substituted with A, G, I, L, S, M or V; G61 is substituted with A, G, I, L, S, M or V; Y62 is substituted by F or W; F63 is substituted with W or Y; F64 is replaced by W or Y; I65 substituted with A, G, L, S, T, M or V; Y66 is substituted by F or W; S67 is substituted with A, G, I, L, T, M or V; Q68 is replaced by N; V69 is substituted with A, G, I, L, S, T or M; L70 is substituted with A, G, I, S, T, M or V; Y71 is substituted by F or W; T72 is substituted with A, G, I, L, S, M or V; D73 is replaced by E; I75 is replaced by A, G, L, S, T, M or V; F76 is replaced by W or Y; A77 is substituted with G, I, L, S, T, M or V; M78 is substituted with A, G, I, L, S, T or V; G79 is substituted with A, I, L, S, T, M or V; H80 is substituted with K or R; V81 is substituted with A, G, I, L, S, T or M; I82 is substituted with A, G, L, S, T, M or V; Q83 is substituted by N; R84 is H or K; K85 is H or substituted by R; K86 is H or substituted by R; V87 is substituted with A, G, I, L, S, T or M; H88 is substituted with K or R; V89 is substituted with A, G, I, L, S, T or M; F90 is replaced with W or Y; G91 is substituted with A, I, L, S, T, M or V; D92 is replaced by E; E93 is replaced by D; L94 is substituted with A, G, I, S, T, M or V; S95 is substituted with A, G, I, L, T, M or V; L96 is substituted with A, G, I, S, T, M or V; V97 is substituted with A, G, I, L, S, T or M; T98 is substituted with A, G, I, L, S, M or V; L99 is substituted with A, G, I, S, T, M or V; F100 is replaced with W or Y; R101 is substituted with H or K; I103 is substituted with A, G, L, S, T, M or V; Q104 is substituted by N; N105 is substituted by Q; M106 is substituted with A, G, I, L, S, T or V; K108 is replaced by H or R; T109 is substituted with A, G, I, L, S, M or V; L110 is substituted with A, G, I, S, T, M or V; N112 is substituted by Q; N113 is substituted by Q; S114 is substituted with A, G, I, L, T, M or V; Y116 is substituted with F or W; S117 is substituted with A, G, I, L, T, M or V; A118 is substituted with G, I, L, S, T, M or V; G119 is substituted with A, I, L, S, T, M or V; I120 is substituted with A, G, L, S, T, M or V; A121 is substituted with G, I, L, S, T, M or V; R122 is substituted with H or K; L123 is substituted with A, G, I, S, T, M or V; E124 is replaced by D; E125 is replaced by D; G126 is substituted with A, I, L, S, T, M or V; D127 is replaced by E; E128 is replaced by D; I129 is substituted with A, G, L, S, T, M or V; Q130 is replaced by N; L131 is substituted with A, G, I, S, T, M or V; A132 is substituted with G, I, L, S, T, M or V; I133 is substituted with A, G, L, S, T, M or V; R135 is substituted with H or K; E136 is replaced by D; N137 is substituted by Q; A138 is substituted with G, I, L, S, T, M or V; Q139 is substituted by N; I140 is substituted with A, G, L, S, T, M or V; S141 is substituted with A, G, I, L, T, M or V; R142 is substituted with H or K; N143 is substituted by Q; G144 is substituted with A, I, L, S, T, M or V; D145 is replaced by E; D146 is substituted by E; T147 is substituted with A, G, I, L, S, M or V; F148 is replaced with W or Y; F149 is substituted with W or Y; G150 is substituted with A, I, L, S, T, M or V; A151 is substituted with G, I, L, S, T, M or V; L152 is substituted with A, G, I, S, T, M or V; K153 is substituted by H or R; L154 is substituted by A, G, I, S, T, M or V and / or L155 is substituted by A, G, I, S, T, M or V. Polynucleotides encoding these polypeptides are also encompassed by the present invention. The neurotoxin-alpha protein products of the present invention can be routinely screened for neurotoxin-alpha and / or neurotoxin-alpha SV functional activity and / or physical properties (e.g., increased or decreased stability and / or solubility) . Preferably, the resulting protein of the invention has increased and / or reduced neurotoxin-alpha and / or neurotoxin-alpha SV functional activity. More preferably, the neurotoxin-alpha and / or neurotoxin-alpha SV protein products of the present invention have one or more increased and / or reduced neurotoxin-alpha and / or neurotoxin-alpha SV functional activity and / Respectively.

In another embodiment, site directed mutations at the amino acid level of neurotoxin-alpha can be made by substituting a particular amino acid with a conservative substitution. Preferred conservative substitution mutations of the neurotoxin-alpha amino acid sequence provided in SEQ ID NO: 38 include those wherein M1 is substituted with A, G, I, L, S, T or V; D2 is replaced by E; E3 is replaced by D; S4 is substituted with A, G, I, L, T, M or V; A5 is substituted with G, I, L, S, T, M or V; K6 is H or substituted by R; T7 is substituted with A, G, I, L, S, M or V; L8 is substituted with A, G, I, S, T, M or V; L13 is substituted with A, G, I, S, T, M or V; F15 is substituted with W or Y; S17 is substituted with A, G, I, L, T, M or V; E18 is substituted by D; K19 is H or substituted by R; G20 is substituted with A, I, L, S, T, M or V; E21 is substituted by D; D22 is replaced by E; M23 is substituted with A, G, I, L, S, T or V; K24 is H or R; V25 is substituted with A, G, I, L, S, T or M; G26 is substituted with A, I, L, S, T, M or V; Y27 is substituted by F or W; D28 is substituted by E; I30 is replaced by A, G, L, S, T, M or V; T31 is substituted with A, G, I, L, S, M or V; Q33 is substituted by N; K34 is H or R; E35 is replaced by D; E36 is substituted by D; G37 is substituted with A, I, L, S, T, M or V; A38 is substituted with G, I, L, S, T, M or V; W39 is substituted by F or Y; F40 is replaced with W or Y; G41 is substituted with A, I, L, S, T, M or V; I42 is substituted with A, G, L, S, T, M or V; R44 is H or K; D45 is replaced by E; G46 is substituted with A, I, L, S, T, M or V; R47 is substituted by H or K; L48 is substituted with A, G, I, S, T, M or V; L49 is substituted with A, G, I, S, T, M or V; A50 is substituted with G, I, L, S, T, M or V; A51 is substituted with G, I, L, S, T, M or V; T52 is substituted with A, G, I, L, S, M or V; L53 is substituted with A, G, I, S, T, M or V; L54 is substituted with A, G, I, S, T, M or V; L55 is substituted with A, G, I, S, T, M or V; A56 is substituted with G, I, L, S, T, M or V; L57 is substituted with A, G, I, S, T, M or V; L58 is substituted with A, G, I, S, T, M or V; S59 is substituted with A, G, I, L, T, M or V; S60 is substituted with A, G, I, L, T, M or V; S61 is substituted with A, G, I, L, T, M or V; F62 is replaced with W or Y; T63 is substituted with A, G, I, L, S, M or V; A64 is substituted with G, I, L, S, T, M or V; M65 is substituted with A, G, I, L, S, T or V; S66 is substituted with A, G, I, L, T, M or V; L67 is substituted with A, G, I, S, T, M or V; Y68 is substituted by F or W; Q69 is substituted by N; L70 is substituted with A, G, I, S, T, M or V; A71 is substituted with G, I, L, S, T, M or V; A72 is substituted with G, I, L, S, T, M or V; L73 is substituted with A, G, I, S, T, M or V; Q74 is substituted by N; A75 is substituted with G, I, L, S, T, M or V; D76 is replaced by E; L77 is substituted with A, G, I, S, T, M or V; M78 is substituted with A, G, I, L, S, T or V; N79 is substituted by Q; L80 is substituted with A, G, I, S, T, M or V; R81 is substituted by H or K; M82 is substituted with A, G, I, L, S, T or V; E83 is replaced by D; L84 is substituted with A, G, I, S, T, M or V; Q85 is substituted by N; S86 is substituted with A, G, I, L, T, M or V; Y87 is substituted by F or W; R88 is H or K; G89 is substituted with A, I, L, S, T, M or V; S90 is substituted with A, G, I, L, T, M or V; A91 is substituted with G, I, L, S, T, M or V; T92 is substituted with A, G, I, L, S, M or V; A94 is substituted with G, I, L, S, T, M or V; A95 is substituted with G, I, L, S, T, M or V; A96 is substituted with G, I, L, S, T, M or V; G97 is substituted with A, I, L, S, T, M or V; A98 is substituted with G, I, L, S, T, M or V; E100 is replaced by D; L101 is substituted with A, G, I, S, T, M or V; T102 is substituted with A, G, I, L, S, M or V; A103 is substituted with G, I, L, S, T, M or V; G104 is substituted with A, I, L, S, T, M or V; V105 is substituted with A, G, I, L, S, T or M; K106 is replaced by H or R; L107 is substituted with A, G, I, S, T, M or V; L108 is substituted with A, G, I, S, T, M or V; T109 is substituted with A, G, I, L, S, M or V; A111 is substituted with G, I, L, S, T, M or V; A112 is substituted with G, I, L, S, T, M or V; R < 114 > is H or K; H116 is substituted with K or R; N117 is substituted by Q; S118 is substituted with A, G, I, L, T, M or V; S119 is substituted with A, G, I, L, T, M or V; R120 is replaced by H or K; G121 is substituted with A, I, L, S, T, M or V; H122 is substituted with K or R; R123 is substituted with H or K; N124 is substituted with Q; R125 is substituted with H or K; R126 is substituted with H or K; A127 is substituted with G, I, L, S, T, M or V; F128 is replaced with W or Y; Q129 is substituted by N; G130 is substituted with A, I, L, S, T, M or V; E132 is replaced by D; E133 is substituted by D; T134 is substituted with A, G, I, L, S, M or V; E135 is replaced by D; Q136 is substituted by N; D137 is replaced by E; V138 is substituted with A, G, I, L, S, T or M; D139 is replaced by E; L140 is substituted with A, G, I, S, T, M or V; S141 is substituted with A, G, I, L, T, M or V; A142 is substituted with G, I, L, S, T, M or V; A145 is substituted with G, I, L, S, T, M or V; L148 is substituted with A, G, I, S, T, M or V; G150 is substituted with A, I, L, S, T, M or V; R152 is substituted with H or K; H153 is substituted with K or R; S154 is substituted with A, G, I, L, T, M or V; Q155 is substituted by N; H156 is substituted with K or R; D157 is replaced by E; D158 is replaced by E; N159 is substituted by Q; G160 is substituted with A, I, L, S, T, M or V; M161 is substituted with A, G, I, L, S, T or V; N162 is substituted by Q; L163 is substituted with A, G, I, S, T, M or V; R164 is substituted with H or K; N165 is substituted by Q; I166 is substituted with A, G, L, S, T, M or V; I167 is substituted with A, G, L, S, T, M or V; Q168 is substituted by N; D169 is replaced by E; L171 is substituted with A, G, I, S, T, M or V; Q172 is substituted by N; L173 is substituted with A, G, I, S, T, M or V; I174 is substituted with A, G, L, S, T, M or V; A175 is substituted with G, I, L, S, T, M or V; D176 is replaced by E; S177 is substituted with A, G, I, L, T, M or V; D178 is replaced by E; T179 is substituted with A, G, I, L, S, M or V; A181 is substituted with G, I, L, S, T, M or V; L182 is substituted with A, G, I, S, T, M or V; E183 is substituted by D; E184 is substituted by D; K185 is H or R; E186 is substituted by D; N187 is substituted with Q; K188 is substituted with H or R; I188 is substituted with A, G, L, S, T, M or V; V190 is replaced by A, G, I, L, S, T or M; V191 is substituted with A, G, I, L, S, T or M; R192 is substituted with H or K; Q193 is substituted by N; T194 is substituted with A, G, I, L, S, M or V; G195 is substituted with A, I, L, S, T, M or V; Y196 is substituted with F or W; F197 is substituted with W or Y; F198 is replaced with W or Y; I199 is substituted with A, G, L, S, T, M or V; Y200 is substituted by F or W; S201 is substituted with A, G, I, L, T, M or V; Q202 is substituted by N; V203 is substituted with A, G, I, L, S, T or M; L204 is substituted with A, G, I, S, T, M or V; Y205 is substituted with F or W; T206 is substituted with A, G, I, L, S, M or V; D207 is replaced by E; I209 is substituted with A, G, L, S, T, M or V; F210 is replaced with W or Y; A211 is substituted with G, I, L, S, T, M or V; M212 is substituted with A, G, I, L, S, T or V; G213 is substituted with A, I, L, S, T, M or V; H214 is substituted with K or R; V215 is substituted with A, G, I, L, S, T or M; I216 is substituted with A, G, L, S, T, M or V; Q217 is substituted by N; R218 is substituted with H or K; K219 is H or R; K220 is substituted by H or R; V221 is substituted with A, G, I, L, S, T or M; H222 is substituted with K or R; V223 is substituted with A, G, I, L, S, T or M; F224 is replaced with W or Y; G225 is substituted with A, I, L, S, T, M or V; D226 is replaced by E; E227 is substituted by D; L228 is substituted with A, G, I, S, T, M or V; S229 is substituted with A, G, I, L, T, M or V; L230 is replaced by A, G, I, S, T, M or V; V231 is substituted with A, G, I, L, S, T or M; T232 is substituted with A, G, I, L, S, M or V; L233 is substituted with A, G, I, S, T, M or V; F234 is substituted with W or Y; R235 is replaced by H or K; I237 is substituted with A, G, L, S, T, M or V; Q238 is substituted by N; N239 is substituted by Q; M240 is replaced with A, G, I, L, S, T or V; K242 is H or substituted by R; T243 is substituted with A, G, I, L, S, M or V; L244 is substituted with A, G, I, S, T, M or V; N246 is substituted by Q; N247 is substituted by Q; S248 is substituted with A, G, I, L, T, M or V; Y250 is substituted with F or W; S251 is substituted with A, G, I, L, T, M or V; A252 is substituted with G, I, L, S, T, M or V; G253 is substituted with A, I, L, S, T, M or V; I254 is substituted with A, G, L, S, T, M or V; A255 is substituted with G, I, L, S, T, M or V; R256 is substituted with H or K; L257 is substituted with A, G, I, S, T, M or V; E258 is substituted by D; E259 is replaced by D; G260 is substituted with A, I, L, S, T, M or V; D261 is replaced by E; E262 is replaced by D; I263 is substituted with A, G, L, S, T, M or V; Q264 is replaced by N; L265 is replaced with A, G, I, S, T, M or V; A266 is substituted with G, I, L, S, T, M or V; I267 is substituted with A, G, L, S, T, M or V; R269 is substituted with H or K; E270 is replaced by D; N271 is substituted by Q; A272 is substituted with G, I, L, S, T, M or V; Q273 is replaced by N; I274 is substituted with A, G, L, S, T, M or V; S275 is substituted with A, G, I, L, T, M or V; R276 is replaced by H or K; N277 is substituted by Q; G278 is substituted with A, I, L, S, T, M or V; D279 is replaced by E; D280 is replaced by E; T281 is substituted with A, G, I, L, S, M or V; F282 is replaced by W or Y; F283 is substituted with W or Y; G284 is substituted with A, I, L, S, T, M or V; A285 is substituted with G, I, L, S, T, M or V; L286 is substituted with A, G, I, S, T, M or V; K287 is H or substituted by R; L288 is substituted by A, G, I, S, T, M or V and / or L289 is substituted by A, G, I, S, T, M or V. Polynucleotides encoding these polypeptides are also encompassed by the present invention. The neurotoxin-alpha protein products of the present invention can be routinely screened for neurotoxin-alpha and / or neurotoxin-alpha SV functional activity and / or physical properties (e.g., increased or decreased stability and / or solubility) . Preferably, the resulting protein of the invention has increased and / or reduced neurotoxin-alpha and / or neurotoxin-alpha SV functional activity. More preferably, the neurotoxin-alpha and / or neurotoxin-alpha SV protein products of the present invention have one or more increased and / or reduced neurotoxin-alpha and / or neurotoxin-alpha SV functional activity and / Respectively.

The amino acid of the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide according to the present invention essential for function can be identified by methods known in the art such as site directed mutagenesis or alanine-scanning mutagenesis [ (Cunningham and Wells, Science 244: 1081-1085 (1989)). The latter procedure introduces one alanine mutation at every residue in the molecule. The resulting mutant molecules are then tested for functional activity such as ligand binding and the ability to stimulate lymphocyte (e.g., B cell) proliferation, differentiation, and / or activation.

Particularly advantageous is the replacement of the charged amino acid with another charged or neutral amino acid, which can provide proteins with highly favorably improved properties such as decreased aggregation. Aggregation can cause problems as well as reduce activity when making pharmaceuticals, since aggregates can be immunogenic [Pinckard et al., Clin. Exp. Immunol. 2: 331-340 (1967); Robbins et al., Diabets 36: 838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10: 307-377 (1993)].

In another aspect, the invention provides a polypeptide having an amino acid sequence containing a non-conservative substitution of the amino acid sequence provided in SEQ ID NO: 2. For example, non-conservative substitutions of the neurotoxin-alpha protein sequence provided in SEQ ID NO: 2 include substitution of M1 with D, E, H, K, R, N, Q, F, W, Y, P or C; D2 is H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; D3 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; S4 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T5 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E6 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; R7 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E8 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; Q9 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; Wherein S10 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; R11 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L12 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T13 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S14 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; C15 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; L16 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K17 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K18 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; R19 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E20 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E21 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; M22 is substituted with D, E, H, K, R, F, W, Y, P or C; K23 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L24 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K25 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E26 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; C27 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; V28 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S29 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; I30 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L31 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P32 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; R33 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K34 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E35 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; S36 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P37 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; S38 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V39 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; R40 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; S41 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S42 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K43 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; D44 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G45 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K46 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L47 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L48 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A49 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A50 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T51 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L52 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L53 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L54 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A55 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L56 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L57 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S58 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; C59 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; C60 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; L61 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T62 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V63 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V64 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S65 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F66 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; Y67 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; Q68 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; V69 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A70 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A71 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L72 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q73 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; G74 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D75 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L76 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A77 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S78 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L79 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; R80 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; A81 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E82 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L83 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q84 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; G85 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; H86 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; H87 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; A88 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E89 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K90 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L91 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P92 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; A93 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G94 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A95 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G96 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A97 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P98 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; K99 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; A100 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G101 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L102 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E103 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E104 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; A105 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P106 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; A107 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V108 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T109 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A110 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G111 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L112 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K113 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; I114 is D, E, H, K, R, N, Q, F, W, Y, P or C; F115 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; E116 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; P117 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; P118 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; A119 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P120 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; G121 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E122 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G123 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; N124 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; S125 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S126 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q127 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; N128 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; S129 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; R130 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N131 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; K132 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; R133 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; A134 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V135 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q136 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; G137 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P138 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; E139 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E140 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T141 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V142 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T143 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q144 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; D145 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; C146 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; L147 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q148 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; L149 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; I150 is D, E, H, K, R, N, Q, F, W, Y, P or C; A151 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D152 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; S153 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E154 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T155 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P156 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; T157 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; I158 is D, E, H, K, R, N, Q, F, W, Y, P or C; Q159 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; K160 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G161 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S162 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y163 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; T164 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F165 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; V166 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P167 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; W168 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; L169 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L170 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S171 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F172 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; K173 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; R174 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G175 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S176 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A177 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L178 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E179 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E180 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K181 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E182 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N183 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; K184 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; I185 is D, E, H, K, R, N, Q, F, W, Y, P or C; L186 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V187 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K188 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E189 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T190 is replaced by D, E, H, K, R, N, Q, F, W, Y, P or C; G191 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y192 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; F193 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; F194 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; I195 is D, E, H, K, R, N, Q, F, W, Y, P or C; Y196 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; G197 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q198 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; V199 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L200 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y201 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; T202 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D203 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K204 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T205 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y206 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; A207 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; M208 is substituted with D, E, H, K, R, F, W, Y, P or C; G209 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; H210 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L211 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L212 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q213 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; R214 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K215 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K216 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; V217 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; H218 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; V219 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F220 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; G221 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D222 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E223 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L224 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S225 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L226 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V227 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T228 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L229 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F230 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; R231 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; C232 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; I233 is D, E, H, K, R, N, Q, F, W, Y, P or C; Q234 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; N235 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; M236 is substituted with D, E, H, K, R, F, W, Y, P or C; P237 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; E238 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T239 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L240 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P241 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; N242 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; N243 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; S244 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; C245 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; Y246 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; S247 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A248 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G249 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; I250 is D, E, H, K, R, N, Q, F, W, Y, P or C; A251 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K252 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L253 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E254 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E255 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G256 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D257 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E258 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L259 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q260 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; L261 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A262 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; I263 is D, E, H, K, R, N, Q, F, W, Y, P or C; P264 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; R265 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E266 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N267 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; A268 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q269 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; I270 is D, E, H, K, R, N, Q, F, W, Y, P or C; S271 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L272 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D273 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G274 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D275 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; V276 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T277 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F278 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; F279 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; G280 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A281 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L282 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K283 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L284 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C and / or L285 is D, E, H, K, Y, P, or C. Polynucleotides encoding these polypeptides are also encompassed by the present invention. The neurotoxin-alpha protein product of the present invention may be used to treat neurotoxin-alpha and / or neurotoxin-alpha SV functional activity and / or physical properties (e.g., increased or decreased stability and / or Solubility) can be routinely selected. Preferably, the resulting protein of the invention has increased and / or reduced neurotoxin-alpha and / or neurotoxin-alpha SV functional activity. More preferably, the neurotoxin-alpha and / or neurotoxin-alpha SV protein products of the present invention have one or more increased and / or reduced neurotoxin-alpha and / or neurotoxin-alpha SV functional activity and / Respectively.

In a further embodiment, the neurotoxin-alpha polypeptides of the invention comprise one or more amino acids substituted with a substituted amino acid (conserved or unconstrained) as described above (e.g., 2, 3, 4, 5, 6, 7 , 8, 9, 10, 15, 20, 30 and 50), or alternatively consists of such amino acids.

In another embodiment of the invention, the non-conservative substitutions of the neurotoxin-alpha SV protein sequences provided in SEQ ID NO: 19 include those wherein M1 is D, E, H, K, R, N, Q, F, W, ; D2 is H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; D3 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; S4 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T5 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E6 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; R7 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E8 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; Q9 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; Wherein S10 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; R11 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L12 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T13 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S14 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; C15 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; L16 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K17 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K18 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; R19 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E20 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E21 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; M22 is substituted with D, E, H, K, R, F, W, Y, P or C; K23 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L24 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K25 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E26 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; C27 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; V28 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S29 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; I30 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L31 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P32 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; R33 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K34 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E35 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; S36 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P37 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; S38 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V39 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; R40 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; S41 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S42 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K43 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; D44 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G45 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K46 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L47 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L48 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A49 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A50 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T51 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L52 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L53 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L54 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A55 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L56 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L57 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S58 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; C59 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; C60 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; L61 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T62 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V63 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V64 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S65 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F66 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; Y67 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; Q68 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; V69 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A70 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A71 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L72 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q73 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; G74 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D75 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L76 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A77 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S78 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L79 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; R80 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; A81 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E82 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L83 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q84 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; G85 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; H86 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; H87 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; A88 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E89 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K90 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L91 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P92 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; A93 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G94 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A95 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G96 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A97 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P98 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; K99 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; A100 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G101 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L102 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E103 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E104 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; A105 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P106 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; A107 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V108 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T109 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A110 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G111 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L112 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K113 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; I114 is D, E, H, K, R, N, Q, F, W, Y, P or C; F115 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; E116 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; P117 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; P118 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; A119 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P120 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; G121 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E122 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G123 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; N124 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; S125 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S126 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q127 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; N128 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; S129 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; R130 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N131 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; K132 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; R133 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; A134 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V135 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q136 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; G137 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P138 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; E139 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E140 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T141 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G142 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S143 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y144 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; T145 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F146 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; V147 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P148 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; W149 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; L150 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L151 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S152 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F153 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; K154 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; R155 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G156 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S157 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A158 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L159 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E160 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E161 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K162 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E163 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N164 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; K165 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; I166 is D, E, H, K, R, N, Q, F, W, Y, P or C; L167 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V168 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K169 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E170 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T171 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G172 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y173 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; F174 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; F175 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; I176 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y177 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; G178 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q179 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; V180 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L181 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y182 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; T183 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D184 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K185 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T186 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y187 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; A188 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; M189 is substituted with D, E, H, K, R, F, W, Y, P or C; G190 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; H191 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L192 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; I193 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q194 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; R195 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K196 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K197 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; V198 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; H199 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; V200 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F201 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; G202 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D203 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E204 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L205 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Wherein S206 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L207 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V208 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T209 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L210 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F211 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; R212 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; C213 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; I214 is D, E, H, K, R, N, Q, F, W, Y, P or C; Q215 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; N216 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; M217 is substituted with D, E, H, K, R, F, W, Y, P or C; P218 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; E219 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T220 is D, E, R, N, Q, F, W, Y, P or C; L221 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P222 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; N223 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; N224 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; S225 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; C226 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; Y227 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; S228 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A229 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G230 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; I231 is D, E, H, K, R, N, Q, F, W, Y, P or C; A232 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K233 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L234 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E235 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E236 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G237 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D238 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E239 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L240 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q241 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; L242 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A243 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; I244 is D, E, H, K, R, N, Q, F, W, Y, P or C; P245 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; R246 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E247 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N248 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; A249 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q250 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; I251 is D, E, H, K, R, N, Q, F, W, Y, P or C; S252 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L253 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D254 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G255 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D256 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; V257 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T258 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F259 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; F260 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; G261 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A262 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L263 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K264 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L265 is D, E, H, K, R, N, Q, F, W, Y, P or C and / Y, P, or C. Polynucleotides encoding these polypeptides are also encompassed by the present invention. The neurotoxin-alpha protein product of the present invention may be used to treat neurotoxin-alpha and / or neurotoxin-alpha SV functional activity and / or physical properties (e.g., increased or decreased stability and / or Solubility) can be routinely selected. Preferably, the resulting protein of the invention has increased and / or reduced neurotoxin-alpha and / or neurotoxin-alpha SV functional activity. More preferably, the neurotoxin-alpha and / or neurotoxin-alpha SV protein products of the present invention have one or more increased and / or reduced neurotoxin-alpha and / or neurotoxin-alpha SV functional activity and / Respectively.

In a further embodiment, the neurotoxin-alpha polypeptides of the invention comprise one or more amino acids substituted with a substituted amino acid (conserved or unconstrained) as described above (e.g., 2, 3, 4, 5, 6, 7 , 8, 9, 10, 15, 20, 30 and 50), or alternatively consists of such amino acids.

For example, preferred non-conservative substitutions of the neurotoxin-alpha protein sequence provided in SEQ ID NO: 23 include those in which R1 is D, E, A, G, I, L, S, T, M, V, , Y, P or C; V2 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V3 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D4 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L5 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S6 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A7 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P8 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; P9 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; A10 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P11 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; C12 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; L13 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P14 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; G15 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; C16 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; R17 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; H18 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; S19 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q20 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; H21 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; D22 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; D23 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N24 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; G25 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; M26 is substituted with D, E, H, K, R, F, W, Y, P or C; N27 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; L28 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; R29 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N30 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; R31 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T32 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y33 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; T34 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F35 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; V36 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P37 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; W38 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; L39 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L40 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S41 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F42 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; K43 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; R44 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G45 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; N46 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; A47 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L48 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E49 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E50 is replaced by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K51 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E52 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N53 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; K54 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; I55 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V56 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V57 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; R58 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; Q59 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; T60 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G61 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y62 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; F63 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; F64 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; I65 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y66 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; S67 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q68 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; V69 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L70 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y71 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; T72 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D73 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; P74 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; I75 is D, E, H, K, R, N, Q, F, W, Y, P or C; F76 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; A77 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; M78 is substituted with D, E, H, K, R, F, W, Y, P or C; G79 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; H80 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; V81 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; I82 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q83 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; R84 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K85 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, L, S, T, M, V, N, Q, F, W, Y, P or C; V87 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; H88 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; V89 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F90 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; G91 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D92 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E93 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L94 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S95 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L96 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V97 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T98 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L99 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F100 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; R101 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; C102 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; I103 is D, E, H, K, R, N, Q, F, W, Y, P or C; Q104 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; N105 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; M106 is substituted with D, E, H, K, R, F, W, Y, P or C; P107 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; K108 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T109 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L110 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P111 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; N112 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; N113 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; S114 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; C115 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; Y116 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; S117 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A118 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G119 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; I120 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A121 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; R122 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L123 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E124 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E125 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G126 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D127 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E128 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; I129 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q130 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; L131 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A132 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; I133 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P134 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; R135 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E136 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N137 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; A138 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q139 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; I140 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S141 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; R142 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N143 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; G144 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D145 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; D146 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T147 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F148 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; F149 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; G150 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A151 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L152 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K153 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L154 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C and / or L155 is substituted with D, E, H, K, Y, P, or C. Polynucleotides encoding these polypeptides are also encompassed by the present invention. The neurotoxin-alpha protein product of the present invention may be used to treat neurotoxin-alpha and / or neurotoxin-alpha SV functional activity and / or physical properties (e.g., increased or decreased stability and / or Solubility) can be routinely selected. Preferably, the resulting protein of the invention has increased and / or reduced neurotoxin-alpha and / or neurotoxin-alpha SV functional activity. More preferably, the neurotoxin-alpha and / or neurotoxin-alpha SV protein products of the present invention have one or more increased and / or reduced neurotoxin-alpha and / or neurotoxin-alpha SV functional activity and / Respectively.

In a further embodiment, the neurotoxin-alpha polypeptides of the invention comprise one or more amino acids substituted with a substituted amino acid (conserved or unconstrained) as described above (e.g., 2, 3, 4, 5, 6, 7 , 8, 9, 10, 15, 20, 30 and 50), or alternatively consists of such amino acids.

For example, preferred non-conservative substitutions of the neurotoxin-alpha protein sequence provided in SEQ ID NO: 38 include those wherein M1 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D2 is H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E3 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; S4 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A5 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K6 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T7 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L8 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P9 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; P10 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; P11 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; C12 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; L13 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; C14 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; F15 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; C16 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; S17 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E18 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K19 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G20 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E21 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; D22 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; M23 is substituted with D, E, H, K, R, F, W, Y, P or C; K24 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; V25 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G26 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y27 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; D28 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; P29 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; I30 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T31 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P32 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; Q33 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; K34 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E35 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E36 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G37 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A38 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; W39 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; F40 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; G41 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; I42 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; C43 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; R44 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; D45 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G46 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; R47 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L48 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L49 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A50 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A51 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T52 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L53 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L54 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L55 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A56 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L57 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L58 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S59 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S60 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S61 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F62 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; T63 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A64 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; M65 is substituted with D, E, H, K, R, F, W, Y, P or C; S66 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L67 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y68 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; Q69 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; L70 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A71 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A72 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L73 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q74 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; A75 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D76 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L77 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; M78 is substituted with D, E, H, K, R, F, W, Y, P or C; N79 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; L80 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; R81 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; M82 is substituted with D, E, H, K, R, F, W, Y, P or C; Substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L84 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q85 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; S86 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y87 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; R88 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G89 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S90 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A91 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T92 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P93 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; A94 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A95 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A96 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G97 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A98 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P99 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; E100 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L101 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T102 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A103 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G104 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V105 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K106 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L107 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L108 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T109 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P110 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; A111 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A112 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P113 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; R114 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; P115 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; H116 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N117 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; S118 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S119 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; R120 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G121 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; H122 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; R123 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N124 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; R125 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; R126 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; A127 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F128 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; Q129 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; G130 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P131 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; E132 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E133 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T134 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E135 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; Q136 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; D137 is H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; V138 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D139 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L140 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S141 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A142 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P143 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; P144 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; A145 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P146 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; C147 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; L148 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P149 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; G150 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; C151 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; R152 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; H153 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; S154 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q155 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; H156 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; D157 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; D158 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N159 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; G160 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; M161 is substituted with D, E, H, K, R, F, W, Y, P or C; N162 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; L163 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; R164 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N165 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; I166 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; I167 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q168 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; D169 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; C170 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; L171 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q172 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; L173 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; I174 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A175 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D176 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; S177 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D178 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T179 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P180 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; A181 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L182 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E183 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E184 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K185 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E186 is H, K, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N187 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; K188 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; I189 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V190 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V191 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; R192 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; Q193 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; T194 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G195 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y196 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; F197 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; F198 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; I199 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y200 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; Wherein S201 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q202 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; V203 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L204 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y205 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; T206 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D207 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; P208 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; I209 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F210 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; A211 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; M212 is substituted with D, E, H, K, R, F, W, Y, P or C; G213 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; H214 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; V215 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; I216 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q217 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; R218 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K219 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K220 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; V221 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; H222 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; V223 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F224 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; G225 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D226 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E227 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L228 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S229 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L230 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V231 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T232 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L233 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F234 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; R235 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; C236 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; I237 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q238 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; N239 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; M240 is substituted with D, E, H, K, R, F, W, Y, P or C; P241 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; K242 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T243 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L244 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P245 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; N246 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; N247 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; S248 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; C249 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; Y250 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; S251 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A252 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G253 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; I254 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A255 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; R256 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L257 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E258 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E259 is substituted by H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G260 is D, E, R, N, Q, F, W, Y, P or C; D261 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E262 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; I263 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q264 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; L265 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A266 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; I267 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P268 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; R269 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E270 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N271 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; A272 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q273 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; I274 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S275 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; R276 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N277 is substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; G278 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D279 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; D280 is substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T281 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F282 is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; F283 is substituted by D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; G284 is D, E, N, Q, F, W, Y, P or C; A285 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L286 is substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K287 is substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; And L289 is substituted with D, E, H, K, R, N, Q, F, W, Y, P, or C. Polynucleotides encoding these polypeptides are also encompassed by the present invention. The neurotoxin-alpha protein product of the present invention may be used to treat neurotoxin-alpha and / or neurotoxin-alpha SV functional activity and / or physical properties (e.g., increased or decreased stability and / or Solubility) can be routinely selected. Preferably, the resulting protein of the invention has increased and / or reduced neurotoxin-alpha and / or neurotoxin-alpha SV functional activity. More preferably, the neurotoxin-alpha and / or neurotoxin-alpha SV protein products of the present invention have one or more increased and / or reduced neurotoxin-alpha and / or neurotoxin-alpha SV functional activity and / Respectively.

In a further embodiment, the neurotoxin-alpha polypeptides of the invention comprise one or more amino acids substituted with a substituted amino acid (conserved or unconstrained) as described above (e.g., 2, 3, 4, 5, 6, 7 , 8, 9, 10, 15, 20, 30 and 50), or alternatively consists of such amino acids.

Substitution of amino acids can also alter the selectivity of ligand binding to cell surface receptors. For example, Ostade et al., Nature 361: 266-268 (1993) describe specific mutations that result in selective binding of TNF-alpha to only one of the two types of TNF receptors known. Because neurotoxin-alpha and neurotoxin-alpha SV are members of the TNF polypeptide family, mutations similar to those of TNF-alpha appear to have similar effects as in neurotoxin-alpha and / or neurotoxin-alpha SV.

Sites that are critical for ligand-receptor binding can also be measured by structural analysis such as metastasis, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol. Biol. 224: 899-904 de Vos et al. Science 255: 306-312 (1992)).

Because the neurotoxin-alpha is a member of the TNF-related protein family, in order to control rather than completely eliminate the functional activity (e.g., biological activity) of neurotoxin-alpha, the mutation is in the sequence encoding amino acids in the TNF- , TNF-alpha, TNF-beta, LT-beta and Fas ligands (see, for example, the Gly-191 to Leu-284 in Figures 1A and 1B 2A-B). ≪ / RTI > By producing specific mutations of the neurotoxin-alpha at positions where such conserved amino acids are typically found in the associated TNF, the neurotoxin-alpha mutein acts as an antagonist and thereby acts as a lymphocyte (e.g., B cell) proliferation, differentiation and / Or < / RTI > Thus, the polypeptides of the present invention include a neurotoxin-alpha mutant. Such neurotoxin-alpha mutants comprise, or alternatively consist of, full-length or preferably extracellular domain fragments, variants or derivatives of the neurotoxin-alpha amino acid sequence shown in Figures 1A and 1B (SEQ ID NO: 2) . Polynucleotides encoding the neurotoxin-alpha mutants are also encompassed by the present invention.

Because the neurotoxin-alpha SV is a member of the TNF-related protein family, in order to control rather than completely eliminate the functional activity (e. G., Biological activity) of the neurotoxin-alpha SV, the mutant encodes an amino acid in the TNF- (E.g., TNF-alpha, TNF-beta, LT-beta, and Fas ligand) (see, e.g., Gly-172 to Leu-265 in Figure 5A and 5B , Fig. 2A-B), which are not conserved in all, most or several members of this region. By producing specific mutations of the neurotoxin-alpha SV at positions where such conserved amino acids are typically found in the associated TNF, the neurotoxin-alpha SV mutein acts as an antagonist and thereby acts as a lymphocyte (e.g., B cell) Differentiation and / or activation. Thus, the polypeptides of the invention include a neurotoxin-alpha SV mutant. Such neurotoxin-alpha SV mutants comprise, or alternatively comprise, full-length or preferably extracellular domain fragments, variants or derivatives of the neurotoxin-alpha amino acid sequence shown in Figures 5A and 5B . Polynucleotides encoding the neurotoxin-alpha SV mutants are also encompassed by the present invention.

In addition, those skilled in the art will appreciate that incorporation of 19 amino acid residues (i.e., the amino acid residues Val-142 to Lys-160 of the sequences described in Figures 1A and 1B and SEQ ID NO: 2) that are not found in the neurotoxin-alpha SV polypeptide sequence It will be appreciated that a mutation targeted to the region of the neurotoxin-alpha polypeptide of the present invention may affect the observed functional activity (e. G., Biological activity) of the neurotoxin-alpha polypeptide. More specifically, a partial, non-limiting, non-exclusive list of residues of the neurotoxin-alpha polypeptide sequence that may be a target of mutation includes the following amino acid residues of the neurotoxin-alpha polypeptide sequence described in SEQ ID NO: 2 Lt; / RTI >:V-142;T-143;Q-144;D-145;C-146;L-147;Q-148;L-149;I-150;A-151;D-152;S-153;E-154;T-155;P-156;T-157;I-158; Q-159 and K-160.

Recombinant DNA techniques known in the art (see, for example, DNA shuffling above) can be used to generate new mutant proteins or muteins comprising one or more amino acid substitutions, deletions, additions or fusion proteins. Such modified polypeptides may, for example, exhibit enhanced activity or increased stability. In addition, they can be purified at higher yields and exhibit better solubility under at least certain purification and storage conditions, as compared to corresponding natural polypeptides.

Thus, the present invention also provides a method of producing a neurokinin-alpha and / or neurotoxin-alpha SV polypeptide, wherein one or more amino acid residues are deleted, added, or substituted so that expression of the neurotoxin-alpha and / Alpha < / RTI > and / or neurotoxin-alpha SV derivatives and analogs. For example, the cysteine residue may be deleted or substituted with another amino acid residue to remove the disulfide bridge; For example, the N-linked glycosylation site can be modified or eliminated to achieve expression of a homogeneous product that is more readily recovered and purified from a yeast host known to hyperglycosylate the N-linked site. Finally, multiple amino acid substitutions at one or both of the first or third amino acid positions in at least one of the glycosylation recognition sequences in the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides according to the present invention and / Amino acid deletions at the second position of any one or more of these recognition sequences will prevent glycosylation of the neurotoxin-alpha and / or neurotoxin-alpha SVs in the modified tripeptide sequence (see, e.g., Miyajimo et al., EMBO J 5 (6): 1193-1197).

Additionally, one or more of the amino acid residues of the polypeptide according to the invention (e.g., arginine and lysine residues) may be deleted or substituted with other amino acids to remove undesirable processing by, for example, purines or proteases such as hexyne . One possible outcome of such a mutation is that the neurotoxin-alpha polypeptide of the invention is not cleaved and released from the cell surface.

In certain embodiments, Lys-132 and / or Arg-133 of the neurotoxin-alpha sequence described in SEQ ID NO: 2 are mutated or deleted together with other amino acid residues to form a soluble form of neurokinin-alpha Is prevented or reduced. In a more particular embodiment, Lys-132 of the neurotoxin-alpha sequence described in SEQ ID NO: 2 is mutated to Ala-132. In another typical embodiment, Arg-133 of the neurotoxin-alpha sequence described in SEQ ID NO: 2 is mutated to Ala-133. These mutated proteins and / or polynucleotides encoding these proteins can be obtained by manipulating cells expressing the neurotoxin-alpha polypeptide to allow the polypeptide to be retained on the surface of the engineered cells, for example by in vitro or gene therapy .

In certain embodiments, the Cys-146 of the neurotoxin-alpha sequence described in SEQ ID NO: 2 is mutated or deleted together with other amino acid residues, for example when expressed in an expression system (essentially as described in Example 1) Lt; RTI ID = 0.0 > oligomerization < / RTI > of neurotoxin-alpha polypeptide. In certain embodiments, Cys-146 is substituted with a serine amino acid residue. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

In another specific embodiment, Cys-232 of the neurotoxin-alpha sequence described in SEQ ID NO: 2 is mutated or deleted together with other amino acid residues, for example when expressed in an expression system (essentially as described in Example 1) ) ≪ / RTI > mutagenic neurotoxin-alpha polypeptide. In certain embodiments, Cys-232 is substituted with a serine amino acid residue. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

In another specific embodiment, the Cys-245 of the neurotoxin-alpha sequence described in SEQ ID NO: 2 is mutated or deleted together with other amino acid residues, for example when expressed in an expression system (essentially as described in Example 1) ) ≪ / RTI > mutagenic neurotoxin-alpha polypeptide. In certain embodiments, Cys-245 is substituted with a serine amino acid residue. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

The polypeptides of the present invention are preferably provided in a separate form and are preferably substantially purified. The recombinantly produced forms of the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides can be substantially purified by the one-step method described in Smith and Johnson, Gene 67: 31-40 (1988) .

The polypeptide of the present invention is a complete polypeptide encoded by a deposited cDNA comprising the intracellular, epidermal and extracellular domains of the polypeptide encoded by the deposited cDNA (ATCC Accession No. 97768), the polypeptide encoded by the deposited cDNA (The amino acid residues 1-285 of SEQ ID NO: 2), the domain of the cells of FIGS. 1A and 1B (SEQ ID NO: 1), the full polypeptide of SEQ ID NO: (Amino acid residues 73-285 of SEQ ID NO: 2), except for the intracellular and transmembrane domains of FIGS. 1A and 1B, as well as 80%, 85%, 90% More preferably at least 95% similarity, even more preferably at least 96%, 97%, 98% or 99% similarity. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

The polypeptide of the present invention may be a complete polypeptide encoded by a deposited cDNA comprising the intracellular, epidermal and extracellular domains of the polypeptide encoded by the deposited cDNA (ATCC Accession No. 203518), the deposited cDNA 5A and 5B except for the intact and extracellular domain of the protein, the complete polypeptide of Figures 5A and 5B (amino acid residues 1-266 of SEQ ID NO: 19), the intracellular and transmembrane domains except for the intracellular and transmembrane domains of the protein, More preferably at least 95% homology, more preferably at least 96%, at least 97%, more preferably at least 95% homology to the above described extracellular domain (amino acid residues 73-266 of SEQ ID NO: 19) , ≪ / RTI >%, 98% or 99% similarity. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

In addition, the polypeptides of the present invention may encode polypeptides encoded by the deposited cDNAs (ATCC Accession No. 97768) or polypeptides of Figures 1A and 1B (SEQ ID NO: 2) at greater than or equal to 80%, or more preferably at least 85% , More preferably at least 90% or at least 95%, even more preferably at least 96%, 97%, 98% or 99% identical to the amino acid sequence of the polypeptide . Polynucleotides encoding these polypeptides are also encompassed by the present invention.

In addition, the polypeptide of the present invention may be a polypeptide encoded by the deposited cDNA (ATCC Accession No. 203518) or a polypeptide encoded by a polypeptide of Figures 5A and 5B (SEQ ID NO: 19) of 80% or more or 85% , More preferably at least 90% or at least 95%, even more preferably at least 96%, 97%, 98% or 99% identical to the amino acid sequence of the polypeptide . Polynucleotides encoding these polypeptides are also encompassed by the present invention.

The% similarity of the two polypeptides was determined using the best fit program (Wisconsin Sequence Analysis Package, version 8 for Unix by Genetics Computer Group, Inc., Madison Scientific Drive 575 University Research Park, Wis. 53711, ≪ / RTI > Best-fit uses Smith and Waterman's local homology algorithm (Advances in Applied Mathematics 2: 482-489) to find the optimal fragment of similarity between two sequences.

A polypeptide having an amino acid sequence that is " identical " for example at least 95% identical to a reference amino acid sequence of a neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide is a polypeptide having an amino acid sequence of Neutrokine-alpha and / Means that the reference sequence is identical to the reference sequence except that it may contain no more than 5 amino acid variations per 100 amino acids in the reference amino acid of the Kinet-alpha SV polypeptide. In other words, to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the reference amino acid sequence, 5% of the amino acid residues in the reference sequence may be deleted or replaced with another amino acid, or 5% of the total amino acid residues in the reference sequence Can be inserted into the reference sequence. These mutations of the reference sequence can occur anywhere between the amino or carboxy terminal positions of the reference amino acid sequence or between the terminal positions of the reference sequences individually or interspersed in one or more contiguous groups within the reference sequence.

As a practical matter, it is contemplated that a particular polypeptide may be, for example, an amino acid sequence as set forth in Figures 1A, 1B and 1C (SEQ ID NO: 2) or a deposited cDNA clone HNEDU15 (ATCC Accession No. 97768) or a fragment thereof, And 80%, 85%, 90%, 95%, 96%, 97%, 98% and 98% of the amino acid sequence encoded by the deposited cDNA clone HDPMC52 (ATCC Accession No. 203518) %, Or 99% or more identical can typically be measured using known computer programs such as the Best-Fit program (Wisconsin Sequence Analysis Package, version 8 for Unix, Genetics Computer Group, Madison Science Drive 575 University Research Park, Madison, Wisconsin) . When using a best-fit or other sequence alignment program to determine if a particular sequence is 95% identical to a reference sequence of the invention, of course, the percentage of identity is calculated for the full length of the reference amino acid sequence and the amino acid residue Of the total number of the non-homogeneous gaps is allowed to be set to 5% or less.

In certain embodiments, the identity between the reference (query) sequence (the present invention sequence) and the corresponding sequence is determined using the FASTDB computer program based on the Brutagh's algorithm (Comp. App. Biosic. 6: 237-245 . Preferred variables used in the FASTDB amino acid sequence are: Matrix = PAM 0, k-tuple = 2, mismatch penalty point = 1, binding penalty point = 20, random group length = 0, critical score = , The gap penalty point = 5, the gap size penalty point = 0.05, the window size = 500 or the shortest of the amino acid sequence. According to this embodiment, manual correction should be made to the result if the sequence is not due to an internal deletion but shorter than the query sequence due to N- or C-terminal deletions. This is because the FASTDB program does not calculate the N- and C-terminal truncations of the sequence when calculating the overall identity%. For the corresponding sequences truncated at the N- and C-termini of the query sequence, percent identity is calculated as the percentage of total bases in the query sequence, calculated as the number of bases in the query sequence, N- and C- . Whether the residues are aligned / aligned is measured as a result of the FASTDB sequence alignment. This percentage is then subtracted from the% identity calculated by the FASTDB program using a particular variable to obtain the final% identity score. This final percent identity score is used for the purposes of the present invention. Only the N- and C-terminal residues of the corresponding sequence that are not matched / aligned with the query sequence are considered for the purpose of manually adjusting the percent identity score. That is, only query residue positions other than the most N- and C-terminal residues of the sequence are considered. For example, 90% amino acid sequences are aligned with 100 residue query sequences to determine% identity. Deletions occur at the N-terminus of the corresponding sequence, so that the FASTDB sequence does not show the match / alignment of the first 10 residues at the N-terminus. Ten unpaired residues represent 10% of the sequence (the number of residues at the unconjugated N- and C-termini / the total number of residues in the query sequence), so the% identity of identity calculated by the FASTDB program 10% from the previous year. If the remaining 90 residues are perfectly matched, the final identity percent will be 90%. As another example, 90 residues corresponding sequences were compared to 100 residue query sequences. At this point, deletion is internal deletion, so there is no residue at the N- or C-terminus of the corresponding sequence that is not matched / aligned with the query. In this case, the percent identity calculated by FASTDB is not manually corrected. Once again, only the positions of residues other than the N- and C-termini of the sequence as shown in the FASTDB sequence that are not matched / aligned with the query sequence are manually corrected. No other manual correction is made for the purposes of the present invention.

The polypeptides of the present invention may be used as molecular weight markers on SDS-PAGE gels or molecular sieve gel filtration columns using, but not limited to, methods known to those skilled in the art. In addition, as described in detail below, the polypeptides of the present invention may be useful in assays to detect neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides, including but not limited to, neurotoxin-alpha and / Or to induce polyclonal and monoclonal antibodies useful as agonists and antagonists capable of enhancing or inhibiting neurotoxin-alpha SV function. In addition, the polypeptides of the present invention have therapeutic uses as described herein. Such polypeptides can also be used in a yeast pair-hybrid system to " capture " neurotoxin-alpha and / or neurotoxin-alpha SV, which is also a candidate agonist and antagonist according to the present invention. Yeast pair hybrid systems are described in Fields and Song, Nature 340: 245-246 (1989).

Transgenics and "knockout"

The polypeptides of the present invention may also be expressed in transgenic animals. Including but not limited to mice, rats, rabbits, hamsters, guinea pigs, pigs, pigs, goats, sheep, cows and non-human primates (eg, baboons, monkeys and chimpanzees) It can be used to generate genetic animals. In certain embodiments, the polypeptides of the invention are expressed in the human body as part of a gene therapy protocol using techniques described herein or known in the art.

Any technique known in the art can be used to introduce a transgene (i. E., A polynucleotide of the invention) into an animal to produce the original line of the transgenic animal. Such techniques include but are not limited to proton nuclear microinjection (Paterson et al., Appl. Microbiol. Biotechnol. 40: 691-698 (1994); Carver et al., Biotechnology (NY) 11: 1263-1270 (Wright et al., Biotechnology (NY) 9: 830-834 (1991) and Hoppe et al., USP 4,873,191 (1989)); Germ cells (Van der Putten et al., Proc. Natl. Acad. Sci., USA 82: 6148-6152 (1985)), retroviral mediated gene transfer into embryonic cells or intraparenchymal cells; Gene tracing in embryonic cells (Thompson et al., Cell 56: 313-321 (1989)); Electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol. 3: 1803-1814 (1983)); Introduction of polynucleotides of the present invention using a gene gun (Ulmer et al., Science 259: 1745 (1993)); Introduction of nucleic acid constructs into the pluripotent stem cells and delivery of the liver cells into embryonic cells; And sperm-mediated gene transfer (Lavitrano et al., Cell 57: 717-723 (1989)). The contents of which are incorporated herein by reference. The technique is described in Gordon, " Transgenic Animals, " Intl. Rev. Cytol. 115: 171-229 (1989). The entire contents of this document are incorporated herein by reference. See also: US Pat. No. 5,464,764 (Capecchi, et al., Positive-Negative Selection Methods and Vectors); U.S. Patent No. 5,631,153 (Capecchi, et al., Cells and Non-Human Organisms Containing Predetermined Genomic Modifications and Positive-Negative Selection Methods and Vectors for Making Same); U.S. Patent No. 4,736,866 (Leder et al., Transgenic Non-Human Animals); And U.S. Patent No. 4,873,191 (Wangner, et al., Genetic Transformation of Zygotes). The entire contents of each of these patents are incorporated herein by reference.

Any technique known in the art produces transgenic clones containing polynucleotides of the invention (e.g., nuclear transfer from cultured germ cells derived from dormant cells, fetal cells or mature cells to nucleated oocytes) (Campell et al., Nature 380: 64-66 (1996); Wilmut et al., Nature 385: 810-813 (1997)).

The present invention provides an animal (i. E., A mosaic animal or a chimeric animal) that contains a transgene in some cells, but not in all cells, as well as transgenic animals that contain the transgene in every cell. The transgene can be integrated as a single transgene or as multiple copies, such as in a cone catheter (e.g., a head-to-head colony or a head-to-tail colony). Transgenic genes are described, for example, in Lasko et al., Proc. Natl. Acad. Sci. USA 89: 6232-6236 (1992)). ≪ / RTI > The regulatory sequences required for such cell-type specific activation will depend on the particular cell type in question and will be apparent to those skilled in the art. If it is necessary for the polynucleotide transgene to integrate into the chromosomal region of the endogenous gene, tracing the gene is desirable. Briefly, when this technique is used, a vector containing some nucleotide sequences homologous to the endogenous gene is designed to integrate through homologous recombination with the chromosomal sequence and disrupt the function of the nucleotide sequence of the endogenous gene. Also, according to the teachings described in Gu et al., Science 265: 103-106 (1994), transgenes can be selectively introduced into specific types of cells to inactivate endogenous genes only in that type of cell have. The regulatory sequence required for such cell-type specific inactivation will depend on the particular cell type involved and will be apparent to those skilled in the art. In addition to expressing the polypeptide of the present invention in a sporadic or tissue-specific manner in a transgenic animal, those skilled in the art will readily appreciate that the expression of a polypeptide (e. G., Genetically or chemically regulated expression) The product can be formed.

Once the transgenic animal has been generated, the expression of the recombinant gene can be assayed using standard techniques. Initial screening can be accomplished by performing an analysis on animal tissues that demonstrates that integration of the transgene was accomplished by Southern blotting analysis or by PCR techniques. Also, by using techniques including, but not limited to, Northern blotting analysis of tissue samples obtained from animals, in situ hybridization assays and reverse transcriptase-PCR (rt-PCR) and TaqMan PCR, The level of mRNA expression of the transgene in the tissue can be assessed. Samples of transgenic gene-expressing tissue can also be assessed immunocytochemically or immunohistochemically using antibodies specific for the transgene product.

Once the aid animals have been generated, they can be breeded, allotted, crossbred or crossbreed to produce populations of specific animals. Examples of such sarcoma include, but are not limited to, crossbreeding of an assisted animal with one or more integration sites to establish a separate system, expression of the transgene at a higher level due to the effect of additional expression of each transgene Generation of homozygous animals for a given integration site by crossing a heterozygous transgenic animal to increase homologous propagation, expression of distinct lines to generate the transgenic group and eliminate the need for screening of the animal by DNA analysis. To produce a heterozygous or homozygous group and breeding to set the transgene on a distinct background suitable for that experimental model.

The transgenic and " knockout " animals of the present invention can be used to study the biological action of neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides, including but not limited to abnormal neurotrophin-alpha and / or neurotrophin- Is used as an animal model system useful for studying symptoms and / or diseases associated with SV expression, and for screening compounds effective in relieving such symptoms and / or diseases.

In a further aspect of the invention, a genetically engineered cell to express the polypeptide of the invention, or a genetically engineered cell (knockout), so as not to express the polypeptide of the invention, is administered to the patient in vivo. Such cells can be obtained from a patient (i. E., An animal including a human) or an MHC-matched donor, but not limited to fibroblasts, bone marrow cells, blood cells (i.e., lymphocytes), adipocytes, muscle cells, endothelial cells, . (Including, but not limited to, plasmid, cosmid, YAC, DNA, electroporation, liposomes, etc.) (using a vector that integrates a viral vector, preferably a transgene into a cellular genome) To genetically engineer these cells according to recombinant DNA technology to introduce the coding sequence of the polypeptide according to the present invention into the cell or disrupt the coding sequence and / or endogenous control sequence associated with the polypeptide of the present invention. The coding sequence of a polypeptide according to the present invention may be set under the control of a strong constitutive or inducible promoter or promoter / enhancer so that the expression and preferably secretion of the polypeptide according to the invention is effected. Engineered cells that express and preferably secrete the polypeptides of the present invention can be injected systemically into the patient (e. G., Circulating or intraperitoneally).

Alternatively, the cells can be introduced into the matrix or inserted into the body. For example, genetically engineered fibroblasts can be implanted as part of skin graft tissue and genetically engineered endothelial cells can be implanted as part of lymph or vascular graft tissue [ 5,399,349 and Mulligan & Wilson USP 5,460,959]. The entire contents of these documents are incorporated herein by reference.

If the cells to be administered are non-autologous or non-MHC compatible cells, these cells may be administered using well known techniques to inhibit the development of a host immune response to the introduced cells. For example, cells may be introduced into a capsule form, which allows the component to be exchanged for an adjacent extracellular environment, while the introduced cells are not recognized by the host immune system.

Antibody

The present invention also relates to the use of the polypeptides, polypeptide fragments or variants of SEQ ID NO: 2 and / or SEQ ID NO: 19 and / or the epitopes of the invention (determined by immunoassays well known in the art for the detection of specific antibody- And T-cell antigen receptor (TCR) that immunospecifically binds to T-cell antigen receptor (TCR). Antibodies of the invention may be derived from, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F (ab ' (Anti-Id) antibody (including an anti-id antibody to the antibody of the present invention) and an epitope-binding fragment of the antibody. As used herein, the term " antibody " refers to a molecule that contains an immunoglobulin molecule and an immunologically active portion of the immunoglobulin molecule, i. E., An antigen binding site that immunospecifically binds to the antigen. IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or immunogens, IgA (IgG1, IgG2, Lt; / RTI > IgA1 and IgA2) or a subclass. Immunoglobulins can have both heavy and light chains. The sequences of IgG, IgE, IgM, IgD, IgA and IgY heavy chains can be paired with light chains of the kappa or lambda form.

Most preferably, the antibody is a human antigen binding antibody fragment of the invention, including but not limited to Fab, Fab 'and F (ab') 2, Fd, single chain Fvs (scFv), single chain antibodies, disulfide- Linked Fvs (sdFv) and fragments comprising the VL or VH domains. An antigen-binding antibody fragment comprising a single chain antibody may comprise only a variable region or may comprise this variable region in combination with all or a portion of the hinge region, CHl, CH2 and CH3 domains. The invention also encompasses any combination of variable and hinge regions, CH1, CH2 and CH3 domains. The antibodies of the present invention may be derived from animals, including birds and mammals. Preferably, the antibody is derived from a human, mouse (mouse and rat), monkey, sheep, rabbit, goat, guinea pig, camel, horse or chicken. As used herein, " human " antibodies include antibodies having an amino acid sequence of a human immunoglobulin, and include antibodies isolated from human immunoglobulin libraries, or those described below and described, for example, in U.S. Patent No. 5,939,598 (Kucherlapatic et al. An antibody isolated from an animal transgenic for one or more human immunoglobulins that do not express endogenous immunoglobulin as described.

The antibodies of the present invention may be monospecific, bispecific, trispecific, or multispecific. Multispecific antibodies may be specific for the different epitopes of the polypeptides according to the invention or specific for the polypeptides of the invention and for heterologous epitopes (e. G., Heterologous polypeptides or solid support materials) WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol. 147: 60-69 (1991); USP 4,474,893, 4,714,681, 4,925,648, 5,573,920, 5,601,819,; Kostelny et al., J. Immunol. 148: 1547-1553 (1992)).

The antibodies of the present invention may be described or specified in terms of epitopes or portions of the polypeptides of the invention that they recognize or specifically bind to. The epitope (s) or portion of the polypeptide may be specified by the size of the contiguous amino acid residues or as listed in the tables and figures, for example, by N-terminal and C-terminal positions as described herein. Antibodies that specifically bind to any epitope or polypeptide of the invention may also be excluded. Accordingly, the present invention encompasses antibodies specifically binding to the polypeptides of the present invention and contemplates the exclusion of such antibodies.

In certain embodiments, an antibody of the invention comprises an amino acid sequence selected from the group consisting of Phe-115 to Leu-147, Ile-150 to Tyr-163, Ser-171 to Phe-194, Glu-223 to Tyr- To < RTI ID = 0.0 > Phe-278. ≪ / RTI > In another particular embodiment, an antibody of the invention comprises an amino acid sequence selected from the group consisting of Phe-115 to Leu-147, Ile-150 to Tyr-163, Ser-171 to Phe-194, Glu-223 to Tyr- 271 to < RTI ID = 0.0 > Phe-278 < / RTI > In a preferred embodiment, the antibody of the invention binds to a polypeptide comprising Glu-223 to Tyr-246 of SEQ ID NO: 2. In another preferred embodiment, the antibody of the invention binds to a polypeptide consisting of Glu-223 to Tyr-246 of SEQ ID NO: 2. In yet another preferred embodiment, the antibody of the invention binds to a polypeptide consisting of Phe-230 to Asn-242 of SEQ ID NO: 2. In further preferred and conventional embodiments, the antibodies of the invention inhibit one or more biological activities of the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides according to the invention through specific binding. In a more preferred embodiment, the antibodies of the invention inhibit neurotoxin-alpha and / or neurotoxin-alpha SV-mediated B cell proliferation.

The antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind to any other analogs, orthologs or homologs of the polypeptides according to the invention are also included. More than 95%, more than 90%, more than 85%, more than 80%, more than 75%, more than 70%, more than 65%, more than 60%, more than 55%, more than 50% identity to the polypeptide of the present invention As measured using methods known and described herein) are also encompassed by the present invention. In certain embodiments, the antibodies of the invention cross-react with mouse, rat and / or rabbit homologues of human proteins and their corresponding epitopes. Identity of less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50% with the polypeptide of the present invention Lt; RTI ID = 0.0 > and / or < / RTI > measured using methods described herein) are also included in the present invention. In certain embodiments, the cross-reactivity described above is used to identify any one particular antigenic or immunogenic polypeptide or a combination of 2, 3, 4, 5 or more of the specific antigenic and / or immunogenic polypeptides described herein Lt; / RTI > Also included are antibodies that bind to polypeptides encoded by polynucleotides that hybridize with the polynucleotides of the invention under stringent hybridization conditions as described herein. The antibodies of the present invention may also be described or specified in terms of their binding affinity for the polypeptides of the present invention. Preferred binding affinities include 5 x 10 ^-5 M, 10 ^-5 M, 5 x 10 ^-6 , 10 ^-6 , 5 x 10 ^-7 M, 10 ^-7 M, 5 x 10 ^-8 M, 10 ^{-8 M, 5 x 10 -9 M,} 10 -9 M, 5 x 10 -10 M, 10 -10 M, 5 x 10 -11 M, 10 -11 M, 5 x 10 -12 M, 10 -12 M, 5 x 10 ^-13 M, 10 ^-13 M, 5 x 10 ^-14 M, 10 ^-14 M, 5 x 10 ^-15 M and dissociation constants of less than 10 ^-15 M or Kd.

The present invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as measured by methods known in the art for measuring competitive binding, e. G., The immunoassays described herein. In a preferred embodiment, the antibody competitively inhibits binding to the epitope to greater than 95%, greater than 90%, greater than 85%, greater than 80%, greater than 75%, greater than 70%, greater than 60%, or greater than 50%.

The antibodies of the present invention may act as agonists or antagonists of the polypeptides according to the invention. For example, the invention includes antibodies that partially or completely block receptor / ligand interactions with the polypeptides of the invention. Preferably, an antibody of the invention binds to an antigenic epitope or a portion thereof described herein. The invention is characterized by both a receptor-specific antibody and a ligand-specific antibody. In addition, the invention features a receptor-specific antibody that does not inhibit ligand binding but inhibits receptor activation. Receptor activation (i. E. Signal transmission) may be measured by techniques described herein or by techniques known in the art. For example, receptor activation can be measured by detecting phosphorylation of the receptor or its substrate (e.g., tyrosine or serine / threonine) by immunoprecipitation followed by western blot analysis (e.g., as described above). In certain embodiments, the activity of inhibiting ligand activity or receptor activity to greater than 95%, 90%, 85%, 80%, 75%, 70%, 60%, or 50% Antibodies are provided.

The present invention also features antibodies recognizing receptor-ligand complexes as well as receptor-specific antibodies that inhibit both ligand binding and receptor activation, and preferably antibodies that do not specifically recognize unbound receptor or unbound ligand . Likewise, it includes neutralizing antibodies which bind to the ligand and inhibit the binding of the ligand to the receptor, as well as antigens that bind to the ligand to inhibit receptor activation but do not inhibit the ligand from binding to the receptor. Also included are antibodies that activate the receptor. These antibodies can act as receptor agonists, i. E., By enhancing or activating all or part of the biological activity induced by ligand-mediated receptor activation, for example, by inducing dimerization of the receptor. Antibodies can be specified as agonists, antagonists or inverse agonists for biological activity, including specific biological activities of the peptides of the invention described herein. Such antibody agonists can be prepared using methods known in the art (see PCT Publication WO 96/40281; USP 5,811,097; Deng et al. Blood 92 (6): 1981-1988 (1998); Chen et al. Cancer Res. 58 (16): 3668-3678 (1998); Harrop et al. J. Immunol. 161 (4): 1786-1794 (1998); Zhu et al. Cancer Res. 58 (15): 3209-3214 (1998); Yoon et al. J. Immunol. 160 (7): 3170-3179 (1998); Prat et al. J. Cell. Sci. 111 (Pt 2): 237-247 (1998); Pitard et al. J. Immunol. Methods 205 (2): 177-190 (1997); Liautard et al. Cytokine 9 (4): 233-241 (1997); Carlson et al. J. Biol. Chem. 272 (17): 11295-11301 (1997); Taryman et al. Neuron 14 (4): 755-762 (1995); Muller et al. Structure 6 (9): 1153-1167 (1998); Bartunek et al. Cytokine 8 (1): 14-20 (1996)]. The entire contents of which are incorporated herein by reference.

The antibodies of the present invention are used in methods known in the art for purifying, detecting and tracking the polypeptides of the present invention, including, but not limited to, in vitro and in vivo diagnostic and therapeutic methods. For example, antibodies are used in immunoassays to qualitatively and quantitatively measure the levels of polypeptides according to the invention in biological samples (Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988). The entire contents of this document are incorporated herein by reference.

The antibodies of the present invention may be used alone or in combination with other compositions. In addition, the antibody may be recombinantly fused at the N-or C-terminus of the heterologous polypeptide or may be chemically conjugated (including covalent and non-covalent bonds) to the polypeptide or other composition. For example, an antibody of the present invention may be recombinantly fused or conjugated to a molecule of interest as a label in a detection assay and to a dominant molecule such as a heterologous polypeptide, drug or toxin (see PCT publication WO 92/08495; WO 91/14438; WO 89/12624; USP 5,314,995 and EPO 396,387].

The antibodies of the present invention include modified derivatives wherein any type of molecule is covalently bound to the antibody and the antibody is not prevented from generating an anti-genotypic response by such covalent linkage. For example, but not by way of limitation, antibody derivatives may be modified by glycosylation, acetylation, phe- nylation, phosphorylation, amidation, derivatization by known protective / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins And the like. Many chemical transformations can be performed by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tocinamicin, and the like. In addition, the derivative may contain one or more unconventional amino acids.

The antibodies of the present invention can be produced by any suitable method known in the art. The polyclonal antibody to the antigen can be produced by a variety of methods well known in the art. For example, the polypeptides of the present invention may be administered to several host animals, including but not limited to rabbits, mice, rats, etc., to induce the production of sera containing polyclonal antibodies specific for the antigen have. A variety of adjuvants may be used to increase the immune response depending on the host species, including but not limited to Freund's solution (complete and incomplete), inorganic gel (e.g., aluminum hydroxide), surface active substances Tin), pluronic acid polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol and potentially useful adjuvants such as BCG (Bacillary Calmeter- Guerin) and Corynebacterium parvum ). Such adjuvants are also well known in the art.

Monoclonal antibodies can be produced using a variety of techniques known in the art, including the use of hybridomas, recombinant and phage display techniques, or a combination of these techniques. See, for example, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Monoclonal antibodies can be prepared using hybridoma technology, including those taught in Hammerling, et al., In: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981). The entire contents of which are incorporated herein by reference. The term " monoclonal antibody " as used herein is not limited to an antibody produced through hybridoma technology. The term " monoclonal antibody " refers to an antibody derived from a monoclonal, including eukaryotic, prokaryotic or phage clones, and is not a method of producing it.

A " monoclonal antibody " can comprise, or alternatively consist of, two proteins, a heavy chain and a light chain.

Methods for producing and screening for specific antibodies using hybridoma technology are conventional and are well known in the art and are discussed in detail in the Examples (e.g., Example 9). As a non-limiting example, mice can be immunized with the polypeptides of the invention or cells expressing such peptides. Once an immune response is detected, for example, an antibody specific for an antigen is detected in mouse serum, the mouse spleen is harvested and splenocytes are isolated. The splenocytes are then fused with suitable myeloma cells (e. G., Cells from cell line SP20 obtained from the ATCC) by techniques well known in the art. The hybridomas are selected and cloned by limiting dilution. The cells that secrete the antibody capable of binding the hybridoma clone to the polypeptide of the present invention are then assayed by methods known in the art. By immunizing a mouse with a positive hybridoma clone, it is possible to generate plural liquids containing generally high-level antibodies.

Therefore, the present invention is preferably a method of culturing a hybridoma cell which is produced by fusing spleen cells isolated from a mouse immunized with the antigen of the present invention with myeloma cells and secreting the antibody of the present invention, A method for producing a monoclonal antibody comprising selecting a hybridoma resulting from fusion against a hybridoma clone secreting an antibody capable of binding to a peptide, as well as an antibody produced by such a method.

Antibody fragments that recognize a particular epitope can be generated by known techniques. For example, the Fab and F (ab ') 2 fragments of the invention can be used for proteolysis of immunoglobulin molecules using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F (ab') 2 fragments) Lt; / RTI > The F (ab ') 2 fragment contains a variable region, a light chain constant region and a heavy chain CH1 domain.

For example, the antibodies of the present invention can be prepared using various phage display methods known in the art. In the phage display method, functional antibody domains are displayed on the surface of a phage particle having a polynucleotide sequence encoding them. In certain embodiments, such a phage can be used to display an antigen-binding domain expressed from a repertoire or a combined antibody library (e. G., Human or mouse). A phage that expresses an antigen-binding domain that binds to the antigen can be screened or identified, for example, using an antigen or labeled antigen bound or captured on a solid surface or bead. The phages used in these methods are typically scaffold phage comprising fd and M13 expressed from a phage with a disulfide stabilized Fv antibody domain, Fab or Fv recombinantly fused to a phage gene III or gene VIII protein. Examples of phage display methods that can be used to prepare antibodies of the invention are described in Brinkman et al. J. Immunol. Methods 182: 41-50 (1995); Ames et al. J. Immunol. Methods 184: 177-186 (1995); Kettleborough et al. Eur. J. Immunol. 24: 952-958 (1994); Persic et al. Gene 187 9-18 (1997); Burton et al. Advances in Immunology 57: 191-280 (1994); PCT / GB91 / 01134; PCT Publication WO 90/02809; WO91 / 10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; And USP 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108. The entire contents of which are incorporated herein by reference.

After screening the phage, the antibody coding region from the phage can be isolated and used to form a whole antibody or any other desired antigen-binding fragment, as described in that document, and can be used in mammalian cells, insect cells, plant cells, Can be expressed in any desired host, including yeast and bacteria. For example, techniques for recombinantly producing Fab, Fab 'and F (ab') 2 fragments are also described in PCT publication WO 92/22324; Mullinax et al. BioTechniques 12 (6): 864-869 (1992); Sawai et al. AJRI 34: 26-34 (1995); And Better et al. Science 240: 1041-1043 (1998)) can be used according to methods known in the art. The entire contents of which are incorporated herein by reference.

Examples of techniques that can be used to generate single chain Fvs and antibodies are described in USP 4,946,778 and 5,258,498; Huston et al. Methods in Enzymology 203: 46-88 (1991); Shu et al. PNAS 90: 7995-7999 (1993); And Skerra et al. Science 240: 1038-1040 (1998). For some uses, including the use of human in vivo antibodies and in vitro detection assays, it may be desirable to use chimeric, humanized or human antibodies. Chimeric antibodies include molecules in which different portions of the antibody are derived from other animal species, for example antibodies having a variable region derived from a monoclonal antibody of a mouse and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art (Morrison, Science 229: 1202 (1985); Oi et al., BioTechniques 4: 214 (1986); Gillies, et al. (1989) J. Immunol. Methods 125: 191-202; And USP 5,807,715, 4,816, 567 and 4,816, 397]. The entire contents of each of these documents are incorporated herein by reference. Humanized antibodies are antibody molecules from non-human species antibodies that bind to the desired antigen with one or more complementarity determining regions (CDRs) from non-human species and a framework region from human immunoglobulin molecules. Often, the framework residues in the human framework region are displaced with the corresponding residues from the CDR donor antibody to modify (preferably improve) the antigen binding. These skeleton substitutions are identified by methods well known in the art. For example, the interaction between a CDR and a skeletal residue is modeled to identify skeletal residues important for antigen binding, and sequences are compared to identify specific skeletal residues at specific positions (See, for example, Queen et al., U.S. Pat. No. 5,585,089 and Riechmann et al., Nature 332: 323 (1988)). The entire contents of each of these documents are incorporated herein by reference. Antibodies can be used in conjunction with CDR-grafting (EP 239,400; PCT Publication WO 91/09967; USP 5,225,539,5,530,101 and 5,585,089), surface coating or resurfacing (EP 592,106; EP O 519 596; Padlan Molecualr Immunology 28 (4/5) Roguska et al., PNAS 91: 969-973 (1994)) and chain shuffling (USP 5,565,332) And the like.

Complete human antibodies are particularly desirable for the treatment of human patients. Human antibodies can be prepared by a variety of methods known in the art, including the phage display method described above, using antibody libraries derived from human immunoglobulin sequences (see USP 4,444,887 and 4,716,111 and PCT Publication WO 98 / 46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741. The entire contents of each of the above documents are incorporated herein by reference.

Human antibodies can also be generated using transgenic mice that are unable to express functional endogenous immunoglobulin, but can express human immunoglobulin genes. For example, human heavy and light chain immunoglobulin gene complexes can be introduced into mouse embryonic cells by random or by homologous gene recombination. Alternatively, human variable regions, constant regions, and diversity regions can be introduced into mouse embryonic cells in addition to human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes can be made non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region inhibits endogenous antibody production. Modified embryonic cells are expanded and microinjected into undifferentiated embryonic cells to produce chimeric mice. Chimeric mice are then raised to produce homozygous descendants that express human antibodies. The transgenic mice are immunized in the normal manner with the selected antigen (e. G., Whole or part of the polypeptide according to the invention). Monoclonal antibodies to the antigens can be obtained from immunized transgenic mice using conventional hybridoma techniques. Human immunoglobulin transgenes retained by transgenic mice rearrange during B cell differentiation and then undergo class switching and somatic mutation. Thus, using such techniques, therapeutically useful IgG, IgA, IgM and IgE antibodies can be generated. Such techniques for generating human antibodies are described in Lonberg and Huszar, Int. Rev. Immunol. 13: 65-93 (1995). The above techniques for preparing human antibodies and human monoclonal antibodies and the proposal for preparing such antibodies are described in detail in the following references: PCT publication WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European Patent 0 598 877; U.S. Patent Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, 5,885,793, 5,916,771 and 5,939,598. The entire contents of each of these documents are incorporated herein by reference. In addition, techniques similar to those described above can be used to immunize human antibodies against a particular antigen from companies such as Abgenix, Inc. (Fremont, Calif.) And Gelpharm (Josse, CA) Can be obtained.

Whole human antibodies that recognize a particular epitope can be generated using a technique called " guided selection ". This method uses specific non-human monoclonal antibodies (e. G. Mouse antibodies) to guide selection of whole human antibodies that recognize the same epitope (Jespers et al., Bio / technology 12: 899-903 ).

In addition, antibodies to the polypeptides of the invention can be used in turn in accordance with techniques well known to those skilled in the art to produce anti-genotype antibodies that "mimic" the polypeptides of the invention (see Greenspan & Bona, FASEB J. 7 (5): 437-444 (1989) and Nissinoff, J. Immunol. 147 (8): 2429-2438 (1991)). For example, an antibody that binds to a polypeptide of the invention and competitively inhibits polypeptide multimerization and / or binding of the polypeptide to the ligand may be used to " mimic " the polypeptide multimerization and / Lt; RTI ID = 0.0 > and / or < / RTI > its ligand and neutralize it. Such neutralizing anti-genotypes or Fab fragments of such anti-genotypes can be used in therapeutic regimens to neutralize polypeptide ligands. For example, such anti-genotype antibodies can be used to bind the peptides of the invention and / or their ligands / receptors, thereby blocking their biological activity.

Polynucleotides encoding antibodies

The present invention provides polynucleotides and fragments thereof comprising a nucleotide encoding the antibody of the invention. The invention also encompasses antibodies that specifically bind to a polypeptide of the invention, preferably a polypeptide having an amino acid sequence of SEQ ID NO: 2, under stringent or less stringent conditions as defined below And a polynucleotide that hybridizes with a polynucleotide encoding the antibody. In another preferred embodiment, the antibody specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 19. In another preferred embodiment, the antibody specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 23. In another preferred embodiment, the antibody specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 28. In another preferred embodiment, the antibody specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 30.

The polynucleotide can be obtained by a method known in the art, and the nucleotide sequence of the polynucleotide can also be determined by methods known in the art. For example, where the nucleotide sequence of an antibody is known, the polynucleotide encoding the antibody may be prepared from a chemically synthesized oligonucleotide as described, for example, in Kutmeier et al., BioTechniques 17: 242 (1994) Can be assembled. In summary, this includes synthesis of overlapping oligonucleotides containing a portion of the sequence encoding the antibody, annealing and linking of these oligonucleotides, and amplification of linked oligonucleotides by PCR.

Alternatively, the polynucleotide encoding the antibody may be generated from a nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, the nucleic acid encoding the immunoglobulin can be chemically synthesized or synthesized using a suitable source such as an antibody cDNA library, Or hybridized at the 3 ' and 5 ' ends of the sequence from a cDNA library generated from a cell (e. G., A hybridoma cell selected to express an antibody of the invention) or a nucleic acid isolated therefrom, For example, by PCR amplification using a synthetic primer capable of hybridizing with a specific primer or by using an oligonucleotide probe specific for a specific gene sequence, for example, by identifying a cDNA clone from a cDNA library encoding the antibody. The amplified nucleic acid generated by PCR can then be cloned into a cloning vector that can be cloned using methods well known in the art.

Once the nucleotide sequence of the antibody and the corresponding amino acid sequence are determined, methods known in the art for the manipulation of nucleotide sequences, such as recombinant DNA technology, site directed mutagenesis, PCR, etc. (see, for example, Sambrook 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Ausubel et al. ) Can be used to manipulate the nucleotide sequences of the antibodies to generate antibodies with different amino acid sequences. For example, amino acid substitutions, deletions and / or insertions.

In certain embodiments, the amino acid sequences of the heavy and / or light chain variable domains can be examined by methods well known in the art to identify sequences of complementarity determining regions (CDRs). For example, regions of sequence and variability can be measured compared to known amino acid sequences of other heavy and light chain variable regions. Using conventional recombinant DNA techniques, as described above, one or more CDRs can be inserted into the framework region, such as a human bone region, to humanize non-human antibodies. The skeletal region may be naturally occurring or may be a consensus skeletal region and is preferably a human skeletal region (see, for example, Chothia et al., J Mol Biol. 278: 457-479 (1998) to be. Preferably, the polynucleotide produced by the combination of the framework region and the CDRs encodes an antibody that specifically binds to a polypeptide of the invention. Preferably, as discussed above, one or more amino acid substitutions can be made within the framework region, and preferably amino acid substitutions enhance binding of the antibody to the antigen. In addition, such methods can be used to participate in internal chain disulfide linkages to create amino acid substitutions or deletions of one or more variable region cysteine residues to produce antibody molecules lacking one or more internal chain disulfide bonds. Other variations of polynucleotides are included in the present invention and belong to the art.

Also, techniques developed to generate " chimeric antibodies " by splicing genes from mouse antibody molecules of appropriate antigen specificity with genes from human antibody molecules of appropriate biological activity (Morrison et al., Proc., Natl. Takeda et al., Nature 314: 452-454 (1985)) can be used. As described above, chimeric antibodies are molecules derived from animal species in which the different parts are different, such as antibodies (e.g., humanized antibodies) having a variable region derived from a mouse mAb and a human immunoglobulin constant region.

Alternatively, techniques for the production of single chain antibodies (U.S. Patent No. 4,946,778; Bird, Science 242: 423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 1988); and Ward et al., Nature 334: 544-54 (1989)) can be used to generate single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region through an amino acid bridge to yield a single chain polypeptide. Also, this. Techniques for assembly of functional Fv fragments within a cola can be used (Skerra et al., Science 242: 1038-1041 (1988)).

Method for producing antibody

The antibodies of the present invention may be prepared by methods known in the art for the synthesis of antibodies, in particular by chemical synthesis, preferably by recombinant expression techniques.

Recombinant expression of an antibody of the present invention or a fragment, derivative or analog thereof (heavy chain or light chain of an antibody of the present invention or single chain antibody of the present invention) requires construction of an expression vector containing a polynucleotide encoding the antibody . Once a polynucleotide encoding the heavy or light chain of the antibody molecule or antibody of the present invention, or a portion thereof (preferably containing a heavy chain or light chain variable domain) has been obtained, the vector for the production of the antibody molecule is known in the art Can be prepared by recombinant DNA techniques using known techniques. Thus, a method for producing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence is described herein. Methods well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcription and translation control signals. These methods include, for example, in vitro recombinant DNA technology, synthetic techniques and in vivo gene recombination. Thus, the invention provides a replicative vector in which a nucleotide sequence encoding the antibody molecule of the invention or its heavy or light or heavy or light chain variable domain is operably linked to a promoter. Such a vector may contain a nucleotide sequence encoding a constant region of an antibody molecule (see, e.g., PCT Publication No. WO 86/05807; WO 89/01036; and US Patent No. 5,122,464). To express the full heavy or light chain.

The expression vector is delivered to the host cell by conventional techniques, and the transfected cells are then cultured by conventional techniques to produce the antibody of the present invention. Accordingly, the present invention includes a host cell comprising a polynucleotide encoding an antibody of the invention or its heavy or light chain or single chain antibody of the invention operatively linked to a heterologous promoter. In a preferred embodiment for the expression of a double-chain antibody, a vector encoding both heavy and light chains can be simultaneously expressed in host cells to express a complete immunoglobulin molecule, as described in detail below.

A variety of host-expression vector systems can be used to express the antibody molecule of the invention. Such a host-expressing system can produce the corresponding coding sequence and then represent a vehicle that can be purified, but when transformed or transfected with the appropriate nucleotide coding sequence, the cell of the invention capable of expressing the antibody molecule at that position . These include, but are not limited to, recombinant bacteriophage DNA containing the antibody coding sequence, plasmids DNA or bacteria transformed with a cosmid DNA expression vector (e. G. E. coli, Bacillus subtilis) A yeast transformed with a recombinant yeast expression vector (e.g., Saccharomyces, Pichia), a recombinant virus expression vector containing an antibody coding sequence (e.g., a vaccinia virus), a recombinant virus expression vector containing an antibody coding sequence A plant cell system transformed with a recombinant plasmid expression vector (e.g., Ti plasmid) or a genome-derived promoter (e.g., a metallothionein promoter) infected with a maize virus CaMV virus mosaic virus TMV ) Or mammalian viruses (e. G., The adenovirus late promoter (Eg, COS, CHO, BHK, 293, and 3T3 cells) carrying a recombinant expression construct containing a recombinant expression vector (eg, Vaccinia virus 7.5K promoter). Preferably, bacterial cells (e.g., Escherichia coli) and more preferably eukaryotic cells are used for expression of recombinant antibody molecules, particularly for expression of complete recombinant antibody molecules. For example, mammalian cells such as cha nuclear hamster ovary cells (CHO) in combination with vectors such as the major intermediate early gene promoter elements from human cytomegalovirus are effective expression systems for antibodies (Foecking et al., Gene 45 : 101 (1986); Cockett et al., Bio / Technology 8: 2 (1990)).

In bacterial systems, a number of expression vectors can be advantageously selected depending on the use of the expressed antibody molecule. For example, a vector that induces a high level of expression of a fusion protein product that is readily purified when a large amount of the protein is to be produced to produce a pharmaceutical composition of an antibody molecule may be desirable. Such vectors include, but are not limited to, those in which the antibody coding sequences are individually linked within a frame of a vector having the lacZ coding region so that a fusion protein is produced. Coli expression vector pUR278 (Ruther et al., EMBO J. 2: 1791 (1983)); pIN vector (Inouye & Inouye, Nucleic Acids Res. 13: 3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24: 5503-5509 (1989)). In addition, the pGEX vector can be used to express an exogenous polypeptide as a fusion protein with glutathione S-transferase (GST). At one time, such fusion proteins are soluble and can easily be purified by elution in the presence of free glutathione followed by binding and adsorption from the dissolved cells to the matrix glutathione-agarose beads. The pGEX vector may contain a thrombin or factor Xa protease cleavage site to release the target gene product cloned from the GST residue.

For insect systems, autograph californica nuclear polyhedrosis virus (AcNPV) is used as a vector for expressing foreign genes. The virus grows in Spodoptera frugiperda cells. Antibody coding sequences may be individually cloned into non-essential regions of the virus (e.g., polyhedrin genes) and placed under the control of an AcNPV promoter (e.g., a polyhedrin promoter).

In the case of mammalian host cells, a number of viral-based expression systems may be used. When adenovirus is used as an expression vector, the antibody coding sequence may be linked to an adenovirus transcription / translation regulatory complex (e.g., a late promoter and a trimer leader sequence). This chimeric gene can then be inserted into the adenoviral genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e. G., Region E1 or E3) will produce a recombinant virus that is viable in the infected host and capable of expressing the antibody molecule (see, e.g., Logan & Shenk, Proc. Natl. Acad. Sci. USA 81: 355-359 (1984)). Certain initiation signals may also be required for efficient decoding of inserted antibody coding sequences. These signals include the ATG start codon and the adjacent sequence. In addition, the initiation codon must be located on the same top of the reading frame of the desired coding sequence to ensure complete detoxification of the insert. These exogenous detoxification regulatory signals and initiation codons can be of multiple origin, whether natural or synthetic. Expression efficiency can be enhanced by insertion of appropriate transcription enhancer elements, transcription terminators, etc. (Bittner et al., Methods in Enzymol, 153: 51-544 (1987)).

In addition, host cell strains may be selected that modulate the expression of the inserted sequences or modify and process the gene product in a desired specific manner. Such modifications (eg, glycosylation) and processing (eg, cleavage) of the protein product may be important for the function of the protein. Different host cells have characteristic and unique mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems may be selected to ensure correct transformation and processing of expressed foreign proteins. In this regard, eukaryotic host cells bearing cellular machinery for proper processing of the primary transcript, glycosylation of the gene product, and phosphorylation may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3 and WI38 and specifically include breast cancer cell lines such as BT483, Hs578T, HTB2, BT20 and T47D and CRL7030 and Hs578Bst Lt; RTI ID = 0.0 > cell line. ≪ / RTI >

Stable expression is desirable for long term high yield production of recombinant protein. For example, a cell line stably expressing an antibody molecule can be genetically engineered. Rather than using an expression vector containing the viral replication origin, the host cell can be transformed with DNA regulated by appropriate expression control elements (e.g., promoter, enhancer, sequence, transcription terminator, polyadenylation site, etc.) have. After introduction of the foreign DNA, the genetically engineered cells are grown in the proliferation medium for 1 to 2 days, and then transferred to the selection medium. Selection markers in recombinant plasmids provide resistance to selection and allow cells to stably integrate plasmids into their chromosomes and grow to form focus, which can then be cloned and expanded into cell lines. This method can be advantageously used to engineer a cell line expressing an antibody molecule. Such engineered cell lines may be particularly useful for screening and evaluating compounds that interact directly or indirectly with antibody molecules.

Many screening systems can be used, including but not limited to, simple herpesvirus thymidine kinase (Wigler et al., Cell 11: 223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Genes can be used in tk-, hgprt-, or aprt- cells, respectively, as shown in Fig. 1 (b) In addition, antimetabolite resistance can be used based on screening for the following genes: dhfr (Wigler et al., Natl. Acad. Sci. USA 77: 357 (198)), which provides resistance to methotrexate USA 78: 1527 (1981); gpt (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78: 2072 (1981); neo (Clinical Pharmacy 12: 488-505; Wu and Wu, Bioth) which provides resistance to aminoglycoside G-418 Rev. Pharmacol. 32: 573-596 (1993); Mulligan, Science 260: 926-932 (1993); and Morgan and Anderson, Ann. Rev. 3: 87-95 (1991); Hygro (Santerre et al., Gene 30: 147 (1984)), which provided resistance to hygromycin, Methods commonly known in the art of recombinant DNA technology can be routinely applied to screen for the desired recombinant clones, such as those described in Ausubel et al. (eds), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150: 1 (1981). The entire contents of each of the above documents are incorporated herein by reference.

The level of expression of the antibody molecule can be increased by vector amplification (see, e.g., Bebbington and Hentschel, The use of vectors based on gene amplification for expression of cloned genes in mammalian cells in DNA cloning, vol. , New York, 1987). When the marker is amplifiable in a vector system expressing the antibody, an increase in the level of inhibitor present in the culture of the host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, the production of the antibody will also increase (Crouse et al., Mol. Cell. Biol. 3: 257 (1983)).

The host cell may be co-transfected with the two expression vectors of the present invention (a first vector encoding a heavy chain-derived polypeptide and a second vector encoding a light chain-derived polypeptide). The two vectors may contain the same selectable marker allowing the same expression of the heavy and light chain polypeptides. Alternatively, a single vector capable of encoding and expressing both the heavy and light chain polypeptides can be used. In this case, the light chain should be placed in front of the heavy chain to avoid excess toxic glass heavy chain (Proudfoot, Nature 322: 52 (1986); Kohler, Proc. Natl. Acad Sci. USA 77: 2197 (1980)). The coding sequences of the heavy and light chains may comprise cDNA or genomic DNA.

Once the antibody molecule of the present invention has been produced by an animal, chemically synthesized, or recombinantly expressed, it may be purified by methods known in the art for purification of immunoglobulin molecules, for example, chromatography (e.g., ion exchange, Affinity, particularly by affinity for specific antigens according to protein A, and size-specific column chromatography), centrifugation, differential solubility or other standard techniques for purification of proteins. In addition, the antibodies or fragments thereof of the invention can be fused to heterologous polypeptide sequences as described herein or known in the art to facilitate purification.

The invention includes antibodies that are recombinantly fused to a polypeptide of the invention (or a portion thereof, preferably 10, 20, 30, 40, 50, 60, 70, 80, 90 or more than 100 amino acids of the polypeptide) And a fusion protein formed by chemically linking. Fusion does not necessarily have to be direct, but can be done through linker sequences. The antibody may be specific for an antigen other than the polypeptides of the invention (or a portion thereof, preferably 10, 20, 30, 40, 50, 60, 70, 80, 90 or more than 100 amino acids of the polypeptide) . For example, the polypeptides of the present invention can target specific cell types in vitro or in vivo by fusing or ligating the polypeptides of the present invention to antibodies specific for particular cell surface receptors. Antibodies fused or linked to the polypeptides of the present invention can also be used in in vitro immunoassay and purification methods according to methods known in the art (see Harbor et al .; WO 93/21232; EP 439,095; Gillies et al., PNAS 89: 1428-1432 (1992); Fell et al., J. Immunol. 146: < RTI ID = 0.0 > 2446-2452 (1991)). The entire contents of each of the above documents are incorporated herein by reference.

The invention also encompasses compositions comprising a polypeptide of the invention fused or linked to another antibody domain other than a variable region. For example, the polypeptides of the invention may be fused or linked to an antibody Fc region or a portion thereof. The antibody portion fused to the polypeptide of the present invention may comprise a constant region, a hinge region, a CH1 domain, a CH2 domain and a CH3 domain or a complete domain or any combination thereof. In addition, the polypeptide may be fused or bound to a portion of the antibody to form a multimer. For example, an Fc portion fused to a polypeptide of the invention can form a dimer through a disulfide bond between the Fc portions. By fusing the polypeptide to a portion of IgA and IgM, a higher oligomer form can be produced. Methods of fusing or linking the polypeptides of the invention to antibody portions are known in the art (see, for example, U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,112,946 ; Zheng et al., J. Immunol. 154 (1991); < RTI ID = 0.0 > : 5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA 89: 11337-11341 (1992)). The entire contents of each of the above documents are incorporated herein by reference.

As discussed above, polypeptides corresponding to the polypeptides, polypeptide fragments or variants of SEQ ID NO: 2 may be fused or linked to a portion of the antibody to increase the in vivo half-life of the polypeptide or use methods known in the art Can be used for immunoassay. In addition, the polypeptide corresponding to SEQ ID NO: 2 can be fused or linked to the antibody portion to facilitate purification. In addition, as discussed above, polypeptides corresponding to the polypeptides, polypeptide fragments or variants of SEQ ID NO: 19 can be fused or linked to a portion of the antibody to increase the in vivo half-life of the polypeptide, Can be used for immunoassay. In addition, the polypeptide corresponding to SEQ ID NO: 19 can be fused or linked to the antibody portion to facilitate purification. As reported examples, chimeric proteins consisting of the first two domains of human CD4-polypeptide and the various domains of the constant region of the heavy or light chain of mammalian immunoglobulins have been described (EP 394,827; Traunecker et al., Nature 331: 84-86 (1988)). The polypeptides of the invention fused or linked to an antibody having a disulfide-linked dimer structure (due to IgG) may also be more effective in binding and neutralizing molecules other than monomer-secreted proteins or fragments thereof (Fountoulakis et al. , J. Biochem., 270: 3958-3964 (1995)). In many cases, the Fc portion in the fusion protein can be beneficial for therapy and diagnosis and as a result can provide, for example, improved pharmacokinetics (EP A 232 262). Alternatively, it may be desirable to delete the Fc portion after the fusion protein has been expressed, deleted, and purified. For example, the Fc portion can interfere with therapy and diagnosis when the fusion protein is used as an antigen for immunization. In drug development, human proteins such as, for example, hIL-5 were fused with the Fc portion for the purpose of high throughput screening assays to identify antagonists of hIL-5 (see, for example, Bennett et al., J. Molecular Recognition 8: 52-58 (1995); Johanson et al., J. Biol. Chem. 270: 9459-9471 (1995)).

In addition, the antibody or fragment thereof of the present invention can be fused with a marker sequence such as a peptide that facilitates purification. In a preferred embodiment, the marker amino acid sequence is exemplified by a hexa-histidine peptide such as the tag provided by the pQE vector (QIAGEN, Inc., 9259, Sewardton Avenue, CA 91311) among many commercially available. See, e.g., Gentz et al., Proc. Natl. Acad. Sci. USA 86: 821-824 (1984), hexa-histidine provides convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the "HA" tag (Wilson et al., Cell 37: 767 (1984)), corresponding to an epitope derived from the influenza hemagglutinin protein, and the "flag" tag .

The invention further encompasses antibodies or fragments thereof linked to a diagnostic or therapeutic agent. Antibodies can be used diagnostically to monitor the development or progression of a tumor, for example, as part of a clinical trial procedure, for example, to measure the efficacy of a therapeutic regimen being performed. Antibodies can be linked to detectable material to facilitate detection. Examples of detectable substances include various enzymes, submerged constituents, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomography, and non-radioactive paramagnetic metal ions. The detectable material may be indirectly coupled to or coupled to the antibody (or fragment thereof) via an intermediate (e. G., A linker known in the art) using techniques known in the art. For example, U.S. Patent No. 4,741,900 can be referred to as a metal ion that can be bound to an antibody for use as a diagnostic agent according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase or acetylcholinesterase. Examples of suitable subcombinated complexes include streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dsyl chloride or picoerythrin. Examples of luminescent materials include luminol. Examples of bioluminescent materials include luciferase, luciferin and aequorin. Examples of suitable radioactive materials include ¹²⁵ I, ¹³¹ I, ¹¹¹ In, or ⁹⁹ Tc.

The antibody or fragment thereof may also be linked to therapeutic moieties such as cytotoxins (e. G., Cell proliferation inhibitors or cytotoxic agents), therapeutic agents, or radioactive metal ions (e. ^G. Alpha-emitters such as ²¹³ Bi). Cytotoxins or cytotoxic agents include all substances that are harmful to the cell. Examples include but are not limited to paclitaxel, cytosalacin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenofoside, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, Dexamethasone, glucocorticoid, proline, tetracaine, lidocaine, propranolol, and furomycin, and analogs or homologs thereof. Therapeutic agents include but are not limited to antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., (BCNU), cyclostaspicide, cyclopa mide, pilaster, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (e. G. (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly called daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, Include mitramycin and anthramycin (AMC) and anti-mitotic agents (e.g., vincristine and vinblastine).

The binding agents of the present invention may be used to alter a given biological response and the therapeutic agent or drug moiety should not be construed as being limited to conventional chemotherapeutic agents. For example, the drug moiety may be a protein or polypeptide having the desired biological activity. Examples of such proteins include toxins such as abrin, lysine A, pseudomonas exotoxin or diphtheria toxin; A TNF-alpha, an AIM (see WO 97/33899), a tumor necrosis factor, an alpha-interferon, a beta-interferon, a nerve growth factor, a platelet derived growth factor, a tissue plasminogen activator or an apoptotic agent ), AIM II (see WO 97/34911), Fas ligand (Takahashi et al., Int. Immunol. 6: 1567-1574 (1994)), VEGI (WO 99/23105), CD40 ligand, (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-1), or an anti-angiogenic agent (e.g., angiostatin or endostatin); or a biological response modifier such as lipocaine, 6), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF) or other growth factors.

Antibodies can also be linked to solid supports particularly useful for immunoassay or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

Techniques for linking such therapeutic moieties to antibodies are well known (see for example, Arnon et al., &Quot; Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy ", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. &Quot; Antibodies For DrugDelivery ", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (Eds.), Pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, " Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review ", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (Eds.), Pp. 475-506 (1985); " Analysis, Results, And Future Prospective Of The Therapeutic Use Of Raidolabeled Antibody In Cancer Therapy ", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. 1985), and Thorpe et al., &Quot; The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates ", Immunol. Rev. 62: 119-58 (1982)).

Alternatively, the antibody may be conjugated to a second antibody to form an antibody heterozygote, as described in Seal, U.S. Pat. No. 4,676,980. The entire contents of these patents are incorporated herein by reference.

Antibodies can be used as therapeutic agents either alone or in combination with cytotoxic factor (s) and / or cytokine (s) with or without such therapeutic moieties linked thereto.

Immunophenotype test

The antibodies of the invention can be used for immunophenotype testing of cell lines and biological samples. The detoxification products of the genes according to the present invention may be useful as cell-specific markers, and more particularly as cell markers that are differentially expressed at different stages of differentiation and / or maturation of a particular cell type. Monoclonal antibodies to a combination of a particular epitope or epitope enable selection of the cell population expressing the marker. A variety of techniques using monoclonal antibodies can be used to screen for cell populations expressing markers, including magnet separation using antibody-coated magnetic beads, " panning " with antibodies attached to solid matrices panning " and fluorocytometry (see, for example, U.S. Patent No. 5,985,660 and Morrison et al., Cell. 96: 737-49 (1999)).

These techniques are known as "non-autologous" implants in implants for the prevention of cells and graft-versus-host disease (GVHD) as evidenced by hematologic malignancy (ie minimal residual disease (MRD) in acute leukemia patients) Allowing selection of specific groups of cells. Alternatively, these techniques allow for the selection of hematopoietic cells and progenitor cells that can multiply and / or differentiate, as can be seen in human umbilical cells.

Assay of antibody binding

The antibodies of the invention can be assayed for immunospecific binding by methods known in the art. Immunoassays that may be used include, but are not limited to, Western blotting, radioimmunoassay, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitation reactions, Assay, red cell coagulation assay, complement-fix assay, immunoradiometric assay, fluorescence immunoassay, and protein A immunoassay. Such assays are conventional and well known in the art (see, for example, Ausubel et al., Eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York). The entire contents of which are incorporated herein by reference. The exemplified immunoassays are briefly described below, but they are not intended to limit the invention.

Immunoprecipitation assays generally involve suspending the cell population in RIPA buffer (1% NP-40 or Triton X-100, 1% sodium dithiothreitol) supplemented with protein phosphatase and / or protease inhibitors (eg EDTA, PMSF, aprotinin, sodium vanadate) (For example, 1 to 20 mM) at 4 占 폚, followed by incubation at 4 占 폚 for a predetermined period of time (for example, 1 to 30 minutes) 4 hours), protein A and / or protein G sepharose beads are added to the cell lysate and incubated at 4 캜 for about 1 hour or longer, the beads are washed in lysis buffer, and the beads are washed with SDS / Resuspension in sample buffer. The ability of the antibody to immunoprecipitate a particular antigen can be assessed, for example, by Western blot analysis. Those skilled in the art are well aware of the variables that can be modified to increase the binding of the antibody to the antigen and to reduce the background (e. G., Pre-removal of cell lysate with sepharose beads). For more information on immunoprecipitation origins, see Ausubel et al., Eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.

Western blotting analysis generally involves preparing protein samples, electrophoresis protein samples on polyacrylamide gels (e.g., 8% to 20% SDS-PAGE depending on the molecular weight of the antigen), and transferring samples from polyacrylamide gels to nitro The membrane is transferred to a membrane such as cellulose, PVDF or nylon and the membrane is blocked in blocking solution (e.g. PBS + 3% BSA or non-fat milk), the membrane is washed in washing buffer (e.g. PBS-Tween 20) The membranes are washed in wash buffer and the membranes are incubated with a diluted enzyme substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecules (e.g., ³² P or ¹²⁵ Blocking the membrane with a secondary antibody that recognizes the primary antibody (e.g., an anti-human antibody) bound to I), washing the membrane in washing buffer, and detecting the presence of the antigen. Those skilled in the art are familiar with variables that can be changed to increase the detected signal and reduce background noise. Further details regarding Western blotting can be found in Ausubel et al., Eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1].

ELISA is used to prepare the antigen, coat the wells of the 96 well microtiter plate with antigen, add the antibody conjugated to a detectable compound such as an enzyme substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well Incubating for a period of time, and detecting the presence of the antigen. In an ELISA, the antibody does not need to be bound to a detectable antibody. Alternatively, a secondary antibody (which recognizes the antibody) bound to a detectable compound can be added to the well. Alternatively, instead of coating the well with the antigen, the antibody can be coated on the well. In this case, the secondary antibody bound to the detectable compound can be added after addition of the corresponding antigen to the coated well. Those skilled in the art are well aware of the variables that can be changed to increase the detected signal as well as other variables of the ELISA known in the art. Additional information on ELISA can be found in Ausubel et al., Eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1].

The binding affinity of an antibody to an antigen and the dissociation rate of an antigen-antibody interaction can be measured by competitive binding assays. One example of a competitive binding assay is a radioactive immunoassay, which method comprises detecting the antibody bound to the cultured and labeled in the presence of an unlabelled antigen in the increase with the labeled antigen (e.g., ³ H or ¹²⁵ I) with the antibody-antigen to be. From this data, the affinity and binding dissociation rate of the antibody for a particular antigen can be measured by a Scatchard float assay. Competition with secondary antibodies can also be measured using radioimmunoassay. In this case, the antigen is incubated in the presence of the corresponding antibody conjugated to the labeled compound (e.g., ³ H or ¹²⁵ I) and increasing amounts of unlabeled secondary antibody.

Treatment purpose

The invention also relates to antibody-based therapies comprising administering an antibody of the invention to an animal, preferably a mammal, most preferably a human patient, for the treatment of one or more of the described diseases, disorders or conditions. Therapeutic compounds of the invention include, but are not limited to, an antibody of the invention (including fragments, analogs and derivatives thereof as described herein) and a nucleic acid encoding the antibody of the invention Fragments, analogs and derivatives, and anti-individual specific antibodies). An antibody of the invention can be used to treat, inhibit or prevent a disease, disorder, or condition associated with abnormal expression and / or activity of a polypeptide of the invention, including one or more of the diseases, disorders, or conditions described herein. Examples of autoimmune diseases, diseases or conditions associated with the disease or disorder include, but are not limited to, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia, autoimmune hemolytic anemia, hemolytic anemia, Neurodegenerative disorders such as dermatitis, allergic encephalitis, myocarditis, recurrent polychondritis, rheumatic heart disease, glomerulonephritis (e.g., IgA nephropathy), multiple sclerosis, neuritis, uveitis conjunctivitis, Autoimmune thyroiditis, hypothyroidism (i. E., Hashimoto's thyroiditis, systemic lupus erythematosus, dyslipidemia syndrome, < RTI ID = , Pemphigus, receptor autoimmunity (e.g., (a) Grave's disease, (b) myasthenia gravis, and (c) insulin (Eg, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, scleroderma with anti-collagen antibodies, mixed connective tissue disease, multiple myositis / dermatomyositis, malignant anemia, idiopathic Edison's disease, (Including glomerulonephritis, primary glomerulonephritis and IgA nephropathy), pemphigus vulgaris, Sjogren's syndrome, diabetes and adrenergic aggressive drug resistance (including adrenergic agonistic drug resistance with asthma or cystic fibrosis), chronic active hepatitis, primary biliary cirrhosis, Asthma, inflammatory muscular disease, and other inflammatory, granulomatous, degenerative, and atrophic diseases. The term " asthma "

In certain embodiments, the antibodies of the invention are used to treat, inhibit, prognose, diagnose or prevent rheumatoid arthritis.

In another specific embodiment, the antibodies of the invention are used to treat, inhibit, prognose, diagnose or prevent systemic lupus erythematosus.

The treatment and / or prevention of a disease, disorder or condition associated with abnormal expression and / or activity of the polypeptide of the present invention includes alleviating symptoms associated with the disease, disorder or condition, including but not limited to those. In addition, the antibodies of the invention can be used to target and kill cells that express neurotoxin-alpha at the surface and / or cells to which neurotoxin-alpha is bound to the surface. The antibodies of the present invention may be provided in a pharmaceutically acceptable composition as is known in the art or as described in the art.

As a summary of the ways in which the antibodies of the present invention can be used therapeutically, the polynucleotides or polypeptides of the invention may be conjugated locally or systemically to the body, for example, to complement (CDC) or effector cells (ADCC) RTI ID = 0.0 > cytotoxic < / RTI > Some of these methods are described in more detail below. One skilled in the art according to the teachings provided herein will be familiar with methods for using the antibodies of the present invention for diagnostic, monitoring, or therapeutic purposes without undue experimentation.

The antibodies of the present invention may be used in combination with other monoclonal or chimeric antibodies or with hematopoietic growth factors such as IL-2, IL-3 and IL-7, which serve to increase the number or activity of effector cells that interact with the antibody ) Or limpokine, and can be advantageously used.

The antibodies of the present invention may be administered alone or in combination with other therapeutic types (e.g., radiation therapy, chemotherapy, hormone therapy, immunotherapy, anti-tumor agents, antibiotics and immunoglobulins). In general, it is preferred to administer the same species of lineage or species reactivity (in the case of antibodies) as that of the patient. Thus, in a preferred embodiment, human antibodies, fragments, derivatives, analogs or nucleic acids are administered to a human patient for treatment or prevention.

For both the immunoassay of the polynucleotides or polypeptides of the present invention (including fragments thereof) and the treatment of diseases associated therewith, the polypeptides or polynucleotides of the present invention, their affinity and / Or an in vivo inhibiting and / or neutralizing antibody of efficacy. Such an antibody, fragment or region preferably has affinity for the polynucleotide or polypeptide of the present invention, including fragments thereof. Preferred binding affinities include 5 x 10 ^-5 M, 10 ^-5 M, 5 x 10 ^-6 , 10 ^-6 , 5 x 10 ^-7 M, 10 ^-7 M, 5 x 10 ^-8 M, 10 ^{-8 M, 5 x 10 -9 M,} 10 -9 M, 5 x 10 -10 M, 10 -10 M, 5 x 10 -11 M, 10 -11 M, 5 x 10 -12 M, 10 -12 M, 5 x 10 ^-13 M, 10 ^-13 M, 5 x 10 ^-14 M, 10 ^-14 M, 5 x 10 ^-15 M and dissociation constants of less than 10 ^-15 M or Kd.

Gene therapy

In certain embodiments, a nucleic acid comprising a sequence encoding an antibody or a functional derivative thereof is administered to treat, inhibit or prevent a disease or disorder associated with abnormal expression and / or activity of the polypeptide of the invention through gene therapy . Gene therapy refers to therapy performed by administering to a patient an expressed or expressible nucleic acid. In this aspect of the invention, the nucleic acids produce their encoded proteins mediating the therapeutic effect.

Any of the gene therapy methods used in the art may be used in accordance with the present invention. An exemplary method is described below.

Gene therapy is generally described in Goldspiel et al., Clinical Pharmacy 12: 488-505 (1993); Wu and Wu, Biotherapy 3: 87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32: 573-596 (1993); Mulligan, Science 260: 926-932 (1993); And Morgan and Anderson, Ann, Rev. Biochem. 62: 191-217 (1993); May, TIBTECH 11 (5): 155-215 (1993). Methods that can be used as is commonly known in the art of recombinant DNA technology are described in Ausubel et al., (Eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); And Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

In a preferred embodiment, the compound comprises a nucleic acid sequence encoding an antibody, and the nucleic acid sequence is part of an expression vector that expresses an antibody or fragment thereof or a chimeric protein or heavy or light chain in a suitable host. In particular, such nucleic acid sequences are operably linked to a promoter in the antibody coding region, and the promoter is inducible or constitutive and optionally tissue-specific. In another specific embodiment, nucleic acid molecules are used that are flanked by regions in which the antibody coding sequences and other desired sequences facilitate homologous recombination at desired locations in the genome, resulting in chromosomal expression of the antibody-encoding nucleic acid ( Natl. Acad. Sci. USA 86: 8932-8935 (1989); Zijlstra et al., Nature 342: 435-438 (1989)). In certain embodiments, the expressed antibody molecule is a single-chain antibody, and as another strategy, the nucleic acid sequence comprises a sequence encoding both the antibody's light chain and light chain or fragments thereof.

Transmission of the nucleic acid into the patient may be direct, in which case the patient is exposed directly to the nucleic acid or nucleic acid-containing vector. In addition, this transfer may be indirect, in which case the cell is first transformed into the nucleic acid in vitro and then transplanted into the patient. Both of these methods are known as in vivo or in vitro gene therapy, respectively.

In certain embodiments, the nucleic acid sequence is administered directly in vivo, and the nucleic acid is expressed in vivo to produce a product that is encoded. This can be accomplished by a number of methods known in the art, for example by constructing the nucleic acid sequence as part of a suitable nucleic acid expression vector and expressing the vector by deletion or attenuated retrovirus or another viral vector (see U.S. Patent No. 4,980,286) Or by direct injection of DNA or by the use of microparticle collision (e.g., genetic gun; Biolistic, Dupont) to allow the nucleic acid sequence to be present in the cell, by coating with a lipid or cell-surface receptor or transfectant, , By incorporation into microparticles or microcapsules, or by linking nucleic acid sequences to peptides known to be introduced into the nucleus, or by administering the receptor-mediated cells to water absorption (see, e.g., Wu and Wu, J. Biol. Chem. 262: 4429- 4432 < / RTI > (1987) (this method can be used to target cell types that specifically express the receptor) In another embodiment, the ligand may comprise a soluble viral peptide to disrupt the endosome, thereby forming a nucleic acid-ligand complex in which the nucleic acid is not lysosomal degraded. In another embodiment , Nucleic acids can be targeted in vivo for cell specific uptake and expression by targeting specific receptors (see for example WO 92/06180; WO 92/22635; WO 92/20316; WO 93/14188 and WO 93/20221 ) Alternatively, the nucleic acid may be introduced into the cell by homologous recombination and incorporated for expression in host cell DNA (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86: 8932-8935 (1989) ; Zijlstra et al., Nature 342: 435-438 (1989)).

In certain embodiments, a viral vector containing a nucleic acid sequence encoding an antibody of the invention is used. For example, retroviral vectors can be used (Miller et al., Meth. Enzymol. 217: 581-599 (1993)). These retroviral vectors contain components necessary for accurate packaging of the viral genome and integration of host cell DNA. Nucleic acid sequences encoding antibodies to be used in gene therapy are cloned into one or more vectors, which facilitate gene transfer into the patient. Further details of retroviral vectors can be found in Boesen et al., Biotherapy 6: 291-302 (1994), which discloses that hematopoietic stem cells are more resistant to chemotherapy than mdr1 The use of retroviral vectors to transfer genes into hematopoietic liver cells has been described. Other references illustrating the use of retroviral vectors in gene therapy include: Clowes et al., J. Clin. Invest. 93: 644-651 (1994); Kiem et al., Blood 83: 1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4: 129-141 (1993); And Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3: 110-114 (1993).

Adenoviruses are other viral vectors that can be used in gene therapy. Adenovirus is a particularly suitable vehicle for delivering a responder to the respiratory epithelium. Adenoviruses naturally infect respiratory epithelium and cause mild illness here. Other targets of the adenovirus-basic delivery system are liver, central nervous system, endothelial cells and muscle. Adenovirus has the advantage of infecting mitotic cells. Adenovirus-based gene therapy has been reviewed in Kozarsky and Wilson, Current Opinion in Genetics and Development 3: 499-503 (1993). Bout et al., Human Gene Therapy 5: 3-10 (1994) demonstrates the use of adenoviral vectors to deliver genes to the respiratory epithelium of rhesus monkeys. Other examples of using adenovirus for gene therapy are described in Rosenfeld et al., Science 252: 431-434 (1991); Rosenfeld et al., Cell 68: 143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91: 225-234 (1993); WO 94/12649; And Wang, et al., Gene Therapy 2: 775-783 (1995). In a preferred embodiment, an adenovirus vector is used.

In addition, the use of adeno-associated virus (AAV) in gene therapy has been proposed (Walsh et al., Proc. Soc. Exp. Biol. Med., 204: 289-300 (1993), U.S. Patent No. 5,436,146).

Other methods of gene therapy include transferring genes to cells in tissue culture by methods such as electroporation, lipofection, calcium phosphate-mediated transfection or viral infection. Usually, the delivery method involves delivery of a selectable marker to the cell. The cells are then subjected to a screening procedure to separate cells expressing the expressed gene. The separated cells are then delivered to the patient.

In this embodiment, the nucleic acid is introduced into the cell prior to in vivo administration of the resulting recombinant cell. Such introduction may include, but is not limited to, transfection, electroporation, microinjection, infection with a virus or bacteriophage vector containing nucleic acid sequences, cell fusion, chromosome-mediated gene delivery, microcell- ≪ RTI ID = 0.0 > spirofast < / RTI > fusion, and the like. Numerous techniques for introducing foreign genes into cells are known in the art (see for example Loeffler and Behr, Meth. Enzymol. 217: 599-618 (1993); Cohen et al., Meth. Enzymol. 217: 644 (1993); Clin., Pharmac. Ther. 29: 69-92m (1995)) and can be used according to the invention unless the necessary development and physiological function of the recipient cell is destroyed. This technique should provide a stable delivery of the nucleic acid into the cell so that the nucleic acid can be expressed by the cell and preferably is inheritably expressed by the cell progeny.

The resulting recombinant cells can be delivered to the patient by various methods known in the art. Recombinant blood cells (e.g., hematopoietic liver or progenitor cells) are preferably administered intravenously. The intended use of the cells depends on the desired effect, the condition of the patient, etc., and can be measured by a person skilled in the art.

Cells into which nucleic acid can be introduced for the purpose of gene therapy include all cell types available, including, but not limited to, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes, blood cells Various blood or progenitor cells obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver and the like, particularly hematopoietic liver or progenitor cells, are included do.

In a preferred embodiment, the cells used for gene therapy are autologous to the patient.

In embodiments in which recombinant cells are used in gene therapy, the nucleic acid sequences encoding the antibodies are introduced into the cells such that they can be expressed by the cells or their progeny, and then the recombinant cells are administered in vivo for therapeutic effect. In certain embodiments, liver or progenitor cells are used. Any of the liver and / or progenitor cells that can be isolated and maintained can potentially be used according to aspects of the invention (see, e.g., PCT Publication WO 94/08598; Stemple and Anderson, Cell 71: 973-985 (1992) Rheinwald, Meth. Cell. Bio. 21A: 229 (1980) and Pittelkow and Scott. Mayo Clinic Proc. 61: 771 (1986)).

In certain embodiments, for purposes of gene therapy, the nucleic acid to be administered comprises an inducible promoter operably linked to the coding region, whereby expression of the nucleic acid can be regulated by modulating the presence or absence of a suitable inducer of transcription.

Proof of therapeutic and prophylactic activity

The compounds or pharmaceutical compositions according to the present invention are preferably tested in vitro for the desired therapeutic or prophylactic activity prior to use in humans and then tested in vivo. For example, in vitro assays to demonstrate therapeutic or prophylactic use of a compound or pharmaceutical composition include the effects of compounds on cell lines or patient tissue samples. The effects of compounds or compositions on cell lines and / or tissue samples can be determined using techniques known to those skilled in the art, including, but not limited to, rosette formation assays and cell lysis assays. In accordance with the present invention, in vitro assays that can be used to determine whether administration of a particular compound is indicated include those in which the patient tissue sample is grown in a culture and exposed to the compound or administered the compound, Included are in vitro cell culture assays to be observed.

Therapeutic and / or prophylactic administration and composition

The present invention provides a method for treating, inhibiting and preventing an effective amount of a compound or pharmaceutical composition according to the present invention, preferably an antibody of the present invention, by administering to the patient. In a preferred embodiment, the compound is substantially purified (e. G., Substantially free of a substance that confers the effect of the compound or causes undesirable side effects). The patient is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs and the like, and is preferably a mammal, most preferably a human.

Formulations and methods of administration that may be employed when the compound comprises a nucleic acid or immunoglobulin are described above. Additional suitable formulations and routes of administration may be selected from those described below.

Various known delivery systems can be used to administer the compounds of the present invention and include, for example, liposomes, microparticles, encapsulation in microcapsules, recombinant cells capable of expressing the compound, water-absorbing cells (see, for example, Wu and Wu, J. Biol. Chem. 262: 4429-4432 (1987)), constructing nucleic acids as part of retrovirus or other vectors, and the like. Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compound or composition can be administered by conventional routes, for example by infusion or bolus injection, by absorption through the epithelial or mucosal system (e.g., oral mucosa, rectal mucosa and intestinal mucosa, etc.) and other biologically active agents ≪ / RTI > Administration may be systemic or local. It may also be desirable to administer the pharmaceutical compounds or compositions of the present invention into the central nervous system by a suitable route, including intracisternal and intrathecal injection. In-depth injections may be facilitated by an intraventricular catheter attached to a reservoir, for example an omaya reservoir. Pulmonary administration can also be used, for example, using inhalers or formulations with an atomizer and an aerosol.

In certain embodiments, it may be desirable to administer the pharmaceutical compounds or compositions of the invention topically to the site where treatment is desired. This includes, but is not limited to, local infusion during surgery, local application (e.g., in conjunction with post-surgical wound dressing), injection, catheter means, suppository means or implanted means, Porous or gelatinous material comprising a membrane, such as a sialastic membrane or a fiber. Care should be taken to use a material that does not absorb the protein when administering the protein of the invention, preferably comprising an antibody.

In another embodiment, the compound or composition can be delivered in a vehicle, particularly a liposome (see Langer, Science 249: 1527-1533 (1990); Treat et al., In Liposomes in the Infectious Disease and Cancer, Lopez Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989), Lopez-Berestein, pp. 317-327).

In another embodiment, the compound or composition may be delivered to a controlled release system. Buchwald et al., Surgery 88: 507 (1980); Saudek et al., ≪ RTI ID = 0.0 > N. Engl., J. Med., 321: 574 (1989)). In another embodiment, polymeric materials can be used (see: Medical Applicaion of Controlled Release, Langer and Wise (eds.), CRC Press, Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Chem. 23: 61 (1983); Levy et al., Science 228: 190 (1983); Ranger and Peppas, J., Macromol. Sci. 1985); For et al., J. Neurosurg., 71: 105 (1989)). In another embodiment, the controlled release system can be set near the brain requiring only a therapeutic target, i. E., A fraction of the body's capacity (see e.g., Goodson, in Medical Applications of Controlled Release, vol. 138 (1984)).

Other controlled release systems are described in Langer, Science 249: 1527-1533 (1990).

In certain embodiments in which the compound of the present invention is a nucleic acid encoding a protein, the nucleic acid is constructed as part of a suitable nucleic acid expression vector to facilitate expression of the encoded protein, and the expression vector is expressed by a retroviral vector U.S. Patent No. 4,980,286) or by direct injection or by the use of microparticle collision (e.g., gene gun; Biolistic, Dupont) to cause the nucleic acid sequence to be present in the cell, resulting in a lipid or cell-surface receptor or transfection agent Alternatively, the nucleic acid sequence may be linked to a homobox-like peptide known to be introduced into the nucleus. Alternatively, the nucleic acid may be introduced into the cell by homologous recombination for expression and incorporated into the host cell DNA have.

The present invention also provides a pharmaceutical composition. Such compositions comprise a therapeutically effective amount of a compound and a pharmaceutically acceptable carrier. In certain embodiments, the term " pharmaceutically acceptable " means that it may be used in animals, particularly humans, and has been approved by a federal or state government agency or contained in the United States Pharmacopoeia or other generally approved pharmacopoeia. The term " carrier " refers to a diluent, adjuvant, excipient or vehicle to be administered with the therapeutic agent. Such pharmaceutical carriers may be sterile liquids, examples of which include vegetable, synthetic or animal oils (including petroleum) and water, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is the preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, especially for injection solutions. Suitable pharmaceutical excipients include but are not limited to starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, hull, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk, glycerol, Ethanol and the like. The composition may also contain minor amounts of wetting, emulsifying or pH adjusting agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, fatty preparations and the like. The composition may be formulated as a suppository with traditional binders and carriers (e.g., triglycerides). Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in E.W. Martin, " Remington ' s Pharmaceutical Sciences ". Such compositions preferably contain a purified form of the compound in a therapeutically effective amount in association with a suitable amount of carrier to provide a form for administration appropriate to the patient. The formulation should be appropriate to the mode of administration.

In a preferred embodiment, the composition is formulated according to conventional procedures as a pharmaceutical composition suitable for intravenous administration to a human. Typically, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If desired, the composition may also contain a solubilizing agent and a local anesthetic such as lignocaine to lessen pain at the injection site. In general, the ingredients are mixed together or separately in unit dosage form as anhydrous concentrate or anhydrous lyophilized powder in a sealed container such as, for example, a chasey or ampoule indicating the amount of active agent. When the composition is administered by injection, the composition may be dispensed into an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of injectable sterile water or salt water may be provided so that the ingredients can be mixed prior to administration.

The compounds of the present invention may be prepared in neutral or salt form. Pharmaceutically acceptable salts include salts formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid and the like, and salts formed with sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, - salts formed with cations such as salts derived from ethylamino ethanol, histidine, procaine and the like.

The amount of a compound according to the present invention effective in the treatment, inhibition and prevention of diseases or disorders associated with abnormal expression and / or activity of the polypeptides of the present invention can be measured by standard clinical techniques. In addition, in vitro assays can optionally be used to assist in determining the optimal dosage range. The exact dose to be used in the formulation will also depend on the route of administration and the severity of the disease or disorder and should be measured taking into account the judgment of the physician and the condition of each patient. Effective doses can be determined from dose-response curves derived from in vitro or animal model test systems.

For antibodies, the dose administered to a patient is typically from 0.1 mg to 100 mg per kg body weight of the patient. Preferably, the dose administered to the patient is from 0.1 mg to 20 mg, more preferably from 1 mg to 10 mg, per kg body weight of the patient. In general, human antibodies exhibit a longer half-life in the body than antibodies from other species due to their response to foreign polypeptides. Thus, administration of a lower dose and frequency of human antibodies is often possible. Further, the dose and frequency of administration according to the present invention can be reduced by, for example, augmentation of the antibody by absorption, such as lipidation, and tissue infiltration (e.g., into the brain).

The present invention also provides pharmaceutical packs or kits comprising one or more containers filled with one or more components of a pharmaceutical composition according to the present invention. Such containers may be adhered to at any time by government authorities that control the manufacture, use, or sale of pharmaceutical or biological products, and this notice reflects the government's approval for manufacture, use or sale for human consumption do.

Diagnosis and Imaging

Labeled antibodies and derivatives and analogues thereof that specifically bind to the polypeptide of interest may be used for diagnostic purposes to detect, diagnose or monitor diseases and / or disorders associated with abnormal expression and / or activity of a polypeptide according to the invention . The present invention relates to a method of detecting the expression of a polypeptide of interest in a cell or body fluid of a subject using (a) one or more antibodies specific for the polypeptide of interest, and (b) comparing the level of gene expression with a standard gene expression level And detecting the abnormal expression of the polypeptide. Here, an increase or decrease in the level of the polypeptide polypeptide gene expression as compared to the standard expression level is indicative of abnormal expression.

The present invention relates to a method of detecting the expression of a polypeptide of interest in a cell or body fluid of a subject using (a) one or more antibodies specific for the polypeptide of interest, and (b) comparing the level of gene expression with a standard gene expression level Thereby providing a diagnostic test method for diagnosing the disease. Here, an increase or decrease in the level of the polypeptide polypeptide gene expression compared to the standard expression level is indicative of a particular disease. In the context of cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may be indicative of a disease outbreak or may provide a means of detecting disease prior to the appearance of substantial clinical symptoms. With this type of more accurate diagnosis, doctors may be able to curtail the development or ongoing progression of cancer by taking preventive measures or active treatment earlier.

The antibodies of the present invention can be used to test protein levels in biological samples using conventional immunohistological methods known to those skilled in the art (see, for example, Jalkanen, et al., J. Cell Biol. 101: 976-985 1985); Jalkanen, et al., J. Cell Biol. 105: 3087-3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays such as enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs). Suitable antibody black labels are known in the art and include enzyme labels such as glucose oxidase; Iodine ^{(131 I, 125 I, 123} I, 121 I), carbon ⁽¹⁴ C), sulfur ⁽³⁵ S), 3 tritium ⁽³ H), indium ^{(115m In, 113m In, 112} In, 111 In) and technetium ⁽⁹⁹ Tc, ^99m Tc), thallium ⁽²⁰¹ Ti), gallium ⁽⁶⁸ Ga, ⁶⁷ Ga), palladium ⁽¹⁰³ Pd), molybdenum ⁽⁹⁹ Mo), xenon ⁽¹³³ Xe), fluorine ⁽¹⁸ F), ¹⁵³ Sm Radioactive isotopes such as ¹⁷⁷ Lu, ¹⁵⁹ Gd, ¹⁴⁹ Pm, ¹⁴⁰ La, ¹⁷⁵ Yb, ¹⁶⁶ Ho, ⁹⁰ Y, ⁴⁷ Sc, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁴² Pr, ¹⁰⁵ Rh, ⁹⁷ Rh; An emission marker such as luminol; And fluorescent labels such as fluorescein and rhodamine and biotin.

Techniques known in the art can be used to label the antibodies of the present invention. Such techniques include, but are not limited to, the use of bifunctional binders (see, e.g., U.S. Patents 5,756,065; 5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003). The entire contents of each of the above patents are incorporated herein by reference.

One aspect of the invention is the detection and diagnosis of diseases or disorders associated with abnormal expression of the polypeptide of interest in an animal, preferably a mammal, most preferably a human. In one embodiment, the diagnosis is made by (a) administering to a patient an effective amount of a labeled molecule that specifically binds to the polypeptide of interest (eg, parenterally, subcutaneously or intraperitoneally), (b) (C) measuring the background level, and (d) determining the concentration of the labeled molecule (s) in the patient so that the concentration of the labeled molecule . Here, the detection of molecules labeled above the background level indicates that the patient has a particular disease or disorder associated with abnormal expression of the polypeptide. Background levels can be measured by a number of methods, including comparing the amount of detected labeled molecule with a standard value previously measured for a particular system. As described herein, certain embodiments of the invention relate to quantifying or qualifying B cell lineage or mononuclear cell lineage cells using the antibody of the invention.

As described herein, antibodies of the invention can be used to treat, diagnose, or prognose patients with immunodeficiency. In certain embodiments, the antibodies of the invention are used for the diagnosis, prognosis, treatment, or prevention of diseases characterized by defective serum immunoglobulin production, recurrent infection, and / or immune system disorders. In another specific embodiment, the antibodies of the invention are used for the diagnosis, prognosis, treatment, or prevention of patients suffering from rheumatoid arthritis or sub-diseases thereof.

It will be understood in the art that the size of the patient and the imaging system used will measure the amount of imaging moiety required to produce a diagnostic image. In the case of radioactive isotope residues, the amount of radioactivity injected into a human normally ranges from about 5 to 20 milli-squares of ⁹⁹ mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of the cell containing the specific protein. In vivo tumor imaging has been described in SW Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments" (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, SW Burchiel and BA Rhodes, eds., Masson Publishing Inc. ).

Depending on several variables, including the type of label and mode of administration, the time interval at which the labeled molecule is preferentially enriched at the site within the patient and the unbound labeled molecule is removed at the background level is 6 to 48 hours or 6 to 24 hours. In another embodiment, the time interval after administration is 5 to 20 days or 5 to 10 days.

In one embodiment, the disease or disease is monitored by, for example, repeating the method of diagnosing the disease or disease by 1 month after the initial diagnosis, 6 months after the initial diagnosis, and 1 year after the initial diagnosis.

The presence of the labeled molecule can be detected in a patient using methods known in the art for in vivo scanning. These methods depend on the type of label used. Methods and equipment that may be used in the diagnostic methods of the present invention include, but are not limited to, computed tomography (CT), whole body scanning (e.g., Imaging), magnetic resonance imaging (MRI) and ultrasound imaging.

In certain embodiments, molecules are labeled with radioactive isotopes and detected in patients using radiation reactive surgical guidelines (Thurston et al., U.S. Patent No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and detected in the patient using fluorescent reactive scanning instructions. In another embodiment, the molecule is labeled with a positron emitting metal and detected using positron emission tomography. In another embodiment, the molecule is labeled with a paramagnetic label and detected in the patient using magnetic resonance imaging (MRI).

Kit

The present invention provides a kit that can be used in the above method. In one embodiment, the kit comprises an antibody of the invention, preferably a purified antibody, in one or more containers. In certain embodiments, the kit of the present invention contains a substantially isolated polypeptide comprising an epitope that specifically immunoreacts with an antibody contained within the kit. Preferably, the kit of the invention further comprises a control antibody that does not react with the polypeptide of interest. In another particular embodiment, a kit of the invention comprises two or more antibodies (monoclonal and / or polyclonal) that recognize the same and / or different sequences or regions of a polypeptide according to the invention. In another specific embodiment, the kit of the invention contains means for detecting binding of the antibody to the polypeptide (e. G., The antibody binds to a detectable substrate such as a fluorescent compound, an enzyme substrate, a radioactive compound or a luminescent compound Or to a second antibody that recognizes a first antibody that can bind to a detectable substrate).

In another specific embodiment of the invention, the kit is a diagnostic kit for use in screening sera containing antibodies specific for proliferative and / or carcinogenic polynucleotides and polypeptides. Such a kit may comprise a control antibody that does not react with the polypeptide of interest. Such a kit may comprise a substantially separate polypeptide antigen containing an epitope that specifically shows an immune response to one or more anti-polypeptide antigen antibodies. In addition, such a kit comprises means for detecting binding of the antibody to the antigen (e. G., The antibody may be coupled to a fluorescent compound such as fluorescein or rhodamine, which may be detected by flow cytometry) . In certain embodiments, the kit may comprise recombinantly produced or chemically synthesized polypeptide antigens. Polypeptide antigens of the kit can also be linked to a solid support.

In a more specific embodiment, the detection means of the kit described above comprise a solid support to which the polypeptide antigen is attached. Such kits may also include non-combined reporter-labeled anti-human antibodies. In this embodiment, binding of the antigen to the polypeptide antigen can be detected by binding of the reporter-labeled antibody.

In a further aspect, the invention comprises a diagnostic kit for use in screening sera containing an antigen of a polypeptide according to the invention. The diagnostic kit comprises a substantially separate antibody specifically immunoreactive with the polypeptide or polynucleotide and means for detecting binding of the polynucleotide or polypeptide antigen to the antibody. In one embodiment, the antibody binds to a solid support. In certain embodiments, the antibody may be a monoclonal antibody. The detection means of the kit comprise a second labeled monoclonal antibody. Alternatively or additionally, the detection means may comprise a labeled competitive antigen.

In one diagnostic configuration, the test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the method of the present invention. After binding and washing the specific antigen antibody to the reagent to remove unbound serum components, the reagent reacts with the reporter-labeled anti-human antibody and the reporter binds to the reagent in proportion to the amount of bound anti-antigen antibody on the solid support . The reagent is washed again to remove unbound labeled antibody and the amount of the reporter associated with the reagent is measured. Typically, a reporter is an enzyme that is detected by incubating a solid phase in the presence of a suitable fluorometric, luminescent, or colorimetric substrate (Sigma, St. Louis, Mo.).

In this assay, solid surface reagents are prepared by well known techniques that attach proteins to solid support materials (e.g., polymeric beads), dip sticks, 96-well plates or filter materials. These attachment methods generally involve nonspecific adsorption of the protein to the support or covalent attachment of the protein to a chemical reaction group (e.g., an activated carboxyl, hydroxyl or aldehyde group) on a solid support, typically via a free amine group. Alternatively, streptavidin-coated plates can be used in combination with biotinylated antigens.

Thus, the present invention provides an assay system or kit for conducting such diagnostic methods. Kits generally include a support with a surface-bound recombinant antigen and a reporter-labeled anti-human antibody for detecting surface-bound anti-antigen antibodies.

The invention also relates to an antibody which acts as an agonist or antagonist of the polypeptide according to the invention. For example, the invention includes antibodies that partially or totally disrupt receptor / ligand interactions with the polypeptides of the invention. Both receptor-specific antibodies and ligand-specific antibodies are included. Receptor-specific antibodies that do not inhibit ligand binding but inhibit receptor activation. Receptor activation (i. E. Signal transmission) may be measured by techniques described herein or by techniques known in the art. Also included are receptor-specific antibodies that inhibit both ligand binding and receptor activation. Similarly, antibodies that bind to a ligand and neutralize antibodies that inhibit binding of the ligand to the receptor, as well as antibodies that bind with the ligand thereby inhibiting receptor activation but inhibiting the ligand from binding to the receptor. Also included are antibodies that activate the receptor. These antibodies can act as agonists for all or a portion of the biological activity that is affected by the ligand-mediated receptor activation. The antibody may be specified as an agonist or antagonist for a biological activity comprising the specific activity described herein. In addition, antibodies that bind neurotoxin-alpha and / or neurotoxin-alpha SV, whether or not neurotoxin-alpha or neurotoxin-alpha SV are bound to the neurotoxin-alpha receptor, are included. These antibodies act as neurotoxin-alpha and / or neurotoxin-alpha SV agonists, as evidenced by increased cell proliferation in response to binding of neurotoxin-alpha and / or neurotoxin-alpha SV in the presence of these antibodies do. Such antibody agonists can be prepared using methods known in the art (see, for example, PCT Publication WO 96/40281; U.S. Patent 5,811,097; Deng, B. et al., Blood 92 (6): 1981-1988 (1998); Chen, Z, et al., Cancer Res. 58 (16): 3668-3678 (1998); Harrop, J.A. et al., J. Immunol. 161 (4): 1786-1794 (1999); Zhu. Z. et al., Cancer Res. 58 (15): 3209-3214 (1998); Yoon, D, Y. et al., J. Immunol. 160 (7): 3170-3179 (1998); Prat. M. et al., J. Cell. Sci. 111 (Pt 2): 237-247 (1998); Pitard. V. et al., J. Immunol. Methods 205 (2): 177-190 (1997); Liautard, j. et al., Cytokine 9 (4): 233-241 (1997), Carlson, N.G. et al., J. Biol. Chem. 272 (17): 11295-11301 (1997); Taryman, R.E. et al., Neuron 14 (4): 755-762 (1995); Muller, Y.A. et al., Structure 6 (9): 1153-1167 (199); Bartunek. P. et al., Cytokine 8 (1): 14-20 (1996)]. The entire contents of each of the above documents are incorporated herein by reference.

More than 14 monoclonal antibodies were generated against neurotoxin-alpha. These monoclonal antibodies were named as: 12D6, 2E5, 9B6, 1B8, 5F4, 9A5, 10G12, 11G12, 16B4, 3D4, 16C9, 13D5, 15C10 and 12C5. A preliminary analysis of these antibodies revealed that each antibody binds to the neurotoxin-alpha protein in Western blot analysis and when the neurotoxin-alpha protein is bound to the ELISA plate. However, further analysis of antibodies 12D6, 2E5, 9B6, 1B8, 5F4, 9A5, 10G12, 11G12 and 16B4 showed that only antibodies 12D6, 9B6, 2E5, 10G12, 9A5 and 11G12 bind with the membrane- Respectively. Thus, a subset of monoclonal antibodies raised against neurotoxin-alpha may bind only to membrane-associated forms of neurotoxin-alpha, which is largely limited to expression in monocytes and dendritic cells as discussed herein (I.e., this subset does not bind to the soluble form of the neurotoxin-alpha corresponding to amino acids 134-285 of SEQ ID NO: 2).

Antibody 9B6 was found to bind specifically to the membrane-bound form of neurotoxin-alpha but not to the soluble form of neurotoxin-alpha.

From the epitope mapping of antibody 9B6, it was shown that this antibody specifically binds to the amino acid sequence contained in the amino acid residues Ser-171 to about Phe-194 of SEQ ID NO: 2. More specifically, epitope mapping revealed that antibody 9B6 specifically binds to a peptide comprising the amino acid residues Lys-173 to Lys-188 of SEQ ID NO: 2.

In contrast, antibodies 16C9 and 15C10 were found to bind to the soluble form of neurotoxin-alpha (amino acids 134-285 of SEQ ID NO: 2) and inhibit neurotoxin-alpha-mediated proliferation of B cells (see Example 10 ). In addition, 15C10 antibody was found to inhibit the binding of neurotoxin-alpha to its receptor. From the epitope map of antibody 15C10, it was found that this antibody specifically binds to the amino acid sequence contained in amino acid residues about Glu-223 to about Tyr-246 of SEQ ID NO: 2. More specifically, as a result of epitope mapping, it was shown that antibody 15C10 specifically binds to a peptide containing the amino acid residues Val-227 to Asn-242 of SEQ ID NO: 2. In addition, antibody 15C10 specifically binds to a peptide comprising the amino acid residues Phe-230 to Cys-245 of SEQ ID NO: 2.

As described above, anti-neutrophil-alpha monoclonal antibodies were prepared. Hybrids producing antibodies 9B6 and 15C10 were deposited with the ATCC and assigned accession numbers PTA-1158 and PTA-1159, respectively. In one embodiment, an antibody of the invention has one or more of the same biological characteristics as one or more antibodies secreted by a hybridoma cell line deposited with accession numbers PTA-1158 or PTA-1159. &Quot; Biological feature " means an in vitro or in vivo activity or property of an antibody, such as a neurotoxin-alpha (e.g., a polypeptide of SEQ ID NO: 2, a mature form of neurotoxin- Alpha / neurotoxin-alpha receptor binding, the ability to bind to the soluble form of neurotoxin-alpha, the soluble form of neurotoxin-alpha (amino acids 134-285 of SEQ ID NO: 2) and the antigenic and / or epitope region of neurotoxin- , Or the ability to block neurotoxin-alpha mediated biological activity (e. G., Stimulation of B cell proliferation and immunoglobulin production). Optionally, an antibody of the invention will bind to the same epitope as one or more of the antibodies specifically mentioned herein. Such epitope binding can be routinely determined using assays known in the art.

Thus, in one embodiment, the invention provides an antibody that specifically binds to the membrane-bound form of neurotoxin-alpha and does not bind to the soluble form of neurotoxin-alpha. These antibodies are used as diagnostic probes for identifying and / or isolating mononuclear cell lines expressing membrane bound forms of neurotoxin-alpha, although not limited thereto. For example, the membrane-bound form of neurotoxin-alpha is increased in activated mononuclear cells so that antibodies of the invention can be used to detect and / or quantitate levels of activated mononuclear cells. In addition, antibodies that bind only to the membrane-bound form of neurotoxin-alpha may be administered to a neoplastic, pre-cancerous, and / or other cell (e.g., mononuclear cells and dendritic cells) expressing a membrane-bound form of neurotoxin- It can be used to target toxins.

In another embodiment, the antibody of the invention specifically binds only to the soluble form of neurotoxin-alpha (amino acids 134-285 of SEQ ID NO: 2). These antibodies include, but are not limited to, as diagnostic probes for testing soluble Neutrokine-alpha in biological samples and for targeting cells that express neurotoxin-alpha receptor (e.g., B cells) to toxins and / As a therapeutic agent for reducing or blocking in vivo neurotoxin-alpha mediated biological activity (e. G., Stimulation of B cell proliferation and / or immunoglobulin production).

The present invention also provides an antibody that specifically binds to a membrane-bound, soluble form of neurotoxin-alpha.

As described above, the present invention provides a method of inhibiting the ability of neurotoxin-alpha and / or neurotoxin-alpha SVs to bind neurotoxin-alpha receptors and / or neurotoxin-alpha SV receptors in vitro and / Or decreasing < / RTI > In certain embodiments, the antibodies of the invention inhibit or reduce the ability of neurotoxin-alpha and / or neurotoxin-alpha SV to bind to the neurotoxin-alpha receptor and / or neurotoxin-alpha SV receptor in the test. In another customary embodiment, the antibodies of the invention inhibit or inhibit the ability of neurotoxin-alpha and / or neurotoxin-alpha SVs to bind neurotoxin-alpha and / or neurotoxin-alpha SV receptors in vivo . Such inhibition can be assayed using techniques described herein or known in the art.

The present invention also relates to a pharmaceutical composition which specifically binds to neurotoxin-alpha and / or neurotoxin-alpha SV, but neurotoxin-alpha and / or neurotoxin-alpha SV binds to neurotoxin-alpha receptor and / Or an antibody that does not inhibit the ability to bind to the neurotoxin-alpha SV receptor. In certain embodiments, the antibodies of the invention do not inhibit or reduce the ability of neurotoxin-alpha and / or neurotoxin-alpha SV to bind neurotoxin-alpha receptors and / or neurotoxin-alpha SV receptors in the test. In another customary embodiment, the antibodies of the invention inhibit or inhibit the ability of neurotoxin-alpha and / or neurotoxin-alpha SVs to bind neurotoxin-alpha and / or neurotoxin-alpha SV receptors in vivo .

As described above, the present invention includes antibodies that inhibit or reduce neurotoxin-alpha and / or neurotoxin-alpha SV-mediated biological activity in vitro. In certain embodiments, the antibodies of the invention inhibit or reduce neurotoxin-alpha and / or neurotoxin-alpha SV-mediated B cell proliferation in vitro. Such inhibition can be assayed by routinely modifying the B cell proliferation assays described herein or known in the art. In other customary embodiments, the antibodies of the invention inhibit or reduce neurotoxin-alpha and / or neurotoxin-alpha SV-mediated B cell proliferation in vivo. In certain embodiments, the antibody of the invention is 15C10 or a humanized form thereof. In another preferred embodiment, the antibody is 16C9 or a humanized form thereof. Thus, in certain embodiments of the present invention, the 16C9 and / or 15C10 antibody or a humanized form thereof may bind to soluble neurotoxin-alpha and / or neurotoxin-alpha SV and / or its agonists and / In part or completely.

Alternatively, the present invention also encompasses a method of treating a neurotoxin-alpha and / or neurotoxin-alpha SVN-mediated biological activity, Lt; / RTI > stimulation) in vitro. In certain embodiments, the antibodies of the invention do not inhibit or reduce neurotoxin-alpha and / or neurotoxin-alpha SV-mediated biological activity in vitro. In other customary embodiments, the antibodies of the present invention do not inhibit or reduce neurotoxin-alpha and / or neurotoxin-alpha SV-mediated biological activity in vivo. In another specific embodiment, the antibody of the invention is 16C9 or a humanized form thereof.

As described above, the present invention includes antibodies that specifically bind in vitro and / or in vivo with the same epitope as one or more of the antibodies specifically described herein.

In certain embodiments, the antibodies of the invention specifically bind in vitro with the amino acid sequences contained in the amino acid residues Ser-171 to about Phe-194 of SEQ ID NO: 2. In another typical embodiment, the antibody of the present invention specifically binds in vivo with the amino acid sequence contained in the amino acid residues about Ser-171 to about Phe-194 of SEQ ID NO: 2. In another typical particular embodiment, the antibody of the invention specifically binds in vitro with the amino acid sequence contained in the amino acid residues Lys-173 to Lys-188 of SEQ ID NO: 2. In another customary embodiment, the antibody of the invention specifically binds in vivo with the amino acid sequence contained in amino acid residues Lys-173 to Lys-188 of SEQ ID NO: 2.

In a further particular embodiment, the antibody of the invention specifically binds in vitro with the amino acid sequence contained in amino acid residues from SEQ ID NO: 2 about Glu-223 to about Tyr-246. In another further particular embodiment, the antibody of the invention specifically binds in vivo with the amino acid sequence contained in amino acid residues about Glu-223 to about Tyr-246 of SEQ ID NO: 2. In another typical embodiment, the antibody of the present invention specifically binds in vitro with the amino acid sequence contained in the amino acid residues Val-227 to Asn-242 of SEQ ID NO: 2. In another customary embodiment, the antibody of the invention specifically binds in vivo with the amino acid sequence contained in the amino acid residues Val-227 to Asn-242 of SEQ ID NO: 2. In another customary embodiment, the antibody of the invention specifically binds in vitro with the amino acid sequence contained in the amino acid residues Phe-230 to Cys-245 of SEQ ID NO: 2. In another typical particular embodiment, the antibody of the invention specifically binds in vivo with the amino acid sequence contained in amino acid residues Phe-230 to Cys-245 of SEQ ID NO: 2.

The present invention also relates to a polypeptide of the present invention, preferably a polypeptide of SEQ ID NO: 2, more preferably a polypeptide of SEQ ID NO: 2 in SEQ ID NO: 2, more preferably a polypeptide of SEQ ID NO: 171 to < RTI ID = 0.0 > Phe-194 < / RTI > Competitive inhibition can be measured using methods known in the art, for example, competitive binding assays as described herein. In a preferred embodiment, the antibody indicates that the 9B6 monoclonal antibody binds to a polypeptide of SEQ ID NO: 2, more preferably to a polypeptide having the amino acid sequence Ser-171 to Phe-194 in SEQ ID NO: 2, Or more, 85% or more, 80% or more, 75% or more, 70% or more, 60% or more, 50% or more.

The invention also relates to a polypeptide of the invention, preferably a polypeptide of SEQ ID NO: 2, more preferably a polypeptide of SEQ ID NO: 2, more preferably a polypeptide of SEQ ID NO: 223 to < RTI ID = 0.0 > Tyr-246 < / RTI > In a preferred embodiment, the antibody indicates that the 15C10 monoclonal antibody binds to a polypeptide of SEQ ID NO: 2, more preferably to a polypeptide having the amino acid sequence of amino acid residues Glu-223 to Tyr-246 in SEQ ID NO: 2, Or more, 85% or more, 80% or more, 75% or more, 70% or more, 60% or more, 50% or more.

A further aspect of the invention relates to a 9B6 antibody and a hybridoma cell line expressing the antibody. A hybridoma cell line expressing the 9B6 antibody was deposited with the ATCC on Jan. 7, 2000 and received ATCC Deposit No. PTA-1159. In a preferred embodiment, antibody 9B6 is humanized.

A further aspect of the invention relates to a 15C10 antibody and a hybridoma cell line expressing the antibody. A hybridoma cell line expressing the 15C10 antibody was deposited with the ATCC on Jan. 7, 2000 and received ATCC Deposit No. PTA-1158. In a preferred embodiment, antibody 15C10 is humanized.

In certain embodiments, the specific antibodies described above are humanized using techniques described herein or known in the art and then used as therapeutic agents as described herein.

In another particular embodiment, any of the antibodies described above are used in soluble form.

In another specific embodiment, any of the antibodies described above are conjugated (as described below) to the toxin or label. Such conjugated antibodies are used to kill specific cell populations or to quantify specific cell populations. In a preferred embodiment, the conjugated antibody is used to kill B cells expressing a neurotoxin-alpha receptor at the surface. In another preferred embodiment, the conjugated antibody is used to quantitate B cells expressing a neurotoxin-alpha receptor at the surface.

In another particular embodiment, any of the antibodies described above are linked to a toxin or label (as described below). The antibodies thus bound are used to kill specific cell populations or to quantify specific cell populations. In a preferred embodiment, the conjugated antibody is used to kill mononuclear cells expressing a membrane-bound form of neurotoxin-alpha. In another preferred embodiment, the conjugated antibody is used to quantitate mononuclear cells expressing a membrane-bound form of neurotoxin-alpha.

The antibodies of the present invention may also be used to activate monocytes expressing a membrane-bound form of neurotoxin-alpha on the cell surface, to block monocyte activation and / or to kill mononuclear cell lines (For treating, preventing and / or diagnosing a disease or condition associated with, for example, a myelogenous leukemia, a mononuclear cell leukemia, and a lymphoma, mononucleosis, sarcoidosis, rheumatoid arthritis and other activated mononuclear cells) . In certain embodiments, the antibodies of the invention immobilize complement. In another specific embodiment, an antibody (or fragment thereof) of the invention, as described herein, is a heterologous polypeptide or nucleic acid (e. G., A toxin, a radioactive isotope such as a compound that binds and activates an endogenous cytotoxic effector system, Toxic prodrugs).

In another embodiment, one or more monoclonal antibodies that recognize or bind neurotoxin-alpha and / or muteins thereof but do not recognize or bind neurotoxin-alpha SV and / or muteins thereof, . In a related embodiment, one or more monoclonal antibodies are generated that recognize or bind neurotoxin-alpha SV and / or muteins thereof, but do not recognize or bind neurotoxin-alpha and / or its muteins .

As discussed above, antibodies to the neurotoxin-alpha and / or neurotoxin-alpha SV according to the present invention can be produced using anti-genotypic antibodies " normalized " to neurotoxin-alpha using techniques well known to those skilled in the art (See, for example, Greenspan & Bona, FASEB J. 7 (5): 437-444 (1989) and Nissinoff, J. Immunol. 147 (8): 2429-2438 (1991)). For example, an antibody that binds to neurotoxin-alpha and / or neurotoxin-alpha SV and competitively inhibits the oligomerization and / or binding of neurotoxin-alpha and / or neurotoxin-alpha SV to ligands and / Can be used to generate anti-genotypes that "neutralize" the alpha TNF multimerization and / or binding domains and consequently neutralize them by binding to neurotoxin-alpha and / or neurotoxin-alpha SV and / or its ligands. Such neutralizing anti-genotypes or Fab fragments thereof can be used for therapeutic regimens to neutralize the neurotoxin-alpha ligand. For example, such anti-genotype antibodies may be conjugated to a neurotoxin-alpha and / or neurotoxin-alpha SV receptor on the cell surface of the B cell lineage, Can be used to block neurotoxin-alpha and / or neurotoxin-alpha SV mediated B cell activation, proliferation and / or differentiation.

Immune system - diagnosis of related diseases

Neutrokine-alpha is expressed in kidney, lung, peripheral white blood cells, bone marrow, T cell lymphoma, B cell lymphoma, activated T cells, stomach cancer, smooth muscle, macrophage and cord blood tissue, especially in mononuclear cells. In addition, neurotoxin-alpha SV is expressed in primary dendritic cells. In addition, neurotoxin-alpha is expressed on the cell surface of the following non-hematopoietic tumor cell lines: colon carcinoma HCT 116 (ATCC Accession No. CCL-247) and HT-29 (ATCC Accession No. HTB-38); Colon adenocarcinoma Caco-2 (ATCC Accession No. HTB-37), COLO 201 (ATCC Accession No. CCL-224) and WiDr (ATCC Accession No. CCL-218); Breast adenocarcinoma MDA-MB-231 (ATCC Accession No. HTB-26); Bladder carcinoma SCaBER (ATCC Accession No. HTB-3); Bladder carcinoma HT-1197 (ATCC Accession No. CRL-1473); Kidney carcinoma A-498 (ATCC Accession No. HTB-44), Caki-1 (ATCC Accession No. HTB-46) and Caki-2 (ATCC Accession No. HTG-47); Kidney Wiley ' s tumor SK-NEP-1 (ATCC Accession No. HTB-48); And pancreatic carcinoma Hs 766T (ATCC Accession No. HTB-134), MIA PaCa-2 (ATCC Accession No. CRL-1420) and SU.86.86 (ATCC Accession No. CRL-1837). For many immune system-related diseases, the " standard " neurotoxin-alpha and / or neurotoxin-alpha SV gene expression levels (i. E., Neurotoxin- alpha and / Alpha] and / or neurotoxin-alpha SV gene expression relative to normal, or neurotoxin-alpha SV) may be determined by measuring the level of altered (increased or decreased) levels of expression of the neurotoxin- E. G., Serum, plasma, urine, synovial fluid or spinal fluid). Thus, the present invention provides a method for determining the level of expression of a neurotoxin-alpha and / or neurotoxin-alpha SV encoding gene in an immune system tissue or other cell or body fluids from a human and comparing the measured expression levels to standard neurotoxin-alpha and / Neu- trocin-alpha SV gene expression levels, wherein the method comprises the steps of comparing the level of expression of the immune system with that of the immune system disease or normal activation, proliferation, differentiation and / or lethality It is instructive.

In particular, certain tissues in mammals that have cancer in the cells or tissues of the immune system have a "standard" level of neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides and neurotoxin-alpha and / or neurotoxin- Is expressed at a significantly increased or decreased level compared to the peptide-encoding mRNA. In addition, when compared to plasma from a mammal not afflicted with the cancer, an increase level of the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide may be present in a particular body fluid from a mammal of the same species with the cancer, Plasma, urine, and spinal fluid) or cells or tissues.

For example, as described herein, neurotoxin-alpha is highly expressed in cells of the mononuclear cell lineage. Thus, polynucleotides of the invention (e.g., polynucleotide sequences complementary to all or part of the neurotoxin-alpha mRNA and / or neurotoxin-alpha SV mRNA) and antibodies (and antibody fragments) to the polypeptides of the invention Can be used to quantify or qualitate the concentration of mononuclear cell lines (e.g., mononuclear cell leukocyte cells) expressing neurotoxin-alpha on the cell surface. These antibodies may further be used for the detection of abnormal or neurotoxin-alpha and / or neurotoxin-alpha SV structures at the level of neurotoxin-alpha gene expression and / or abnormalities in the temporal, Lt; / RTI > These diagnostic assays can be performed in vivo or in vitro, such as blood samples, biopsy tissue or autopsy tissue.

In addition, as described herein, neurotoxin-alpha receptors are predominantly expressed in cells of the B cell lineage. Thus, the neurotoxin-alpha polypeptides of the present invention (including labeled neurotoxin-alpha polypeptide and neurotoxin-alpha fusion protein) and anti-neurotoxin-alpha antibodies (including anti- Alpha-antibody fragments) can be used to quantitate or qualitate the concentration of cells of the B-cell lineage (e.g., B-cell associated leukemia or lymphoma) expressing neurotoxin-alpha on the cell surface. These neurotrophin-alpha polypeptides and antibodies may further be used to detect abnormalities at the level of neurotoxin-alpha receptor gene expression or abnormalities in the structure of neurotoxin-alpha receptors and / or in temporal, And / or to diagnose activity / defects in the signal transduction pathway associated with neurotoxin-alpha. These diagnostic assays can be performed in vivo or in vitro using, for example, blood samples or biopsy tissue using techniques described herein or known in the art.

In one embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptides of the invention or the neurotoxin-alpha and / or neurotoxin-alpha SV agonists or antagonists (e.g., anti- Neurotoxin-alpha and / or neurotoxin-alpha SV antibodies) are used to treat, prevent, diagnose, or prognose immunodeficient persons.

Neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptides or neurotoxin-alpha and / or neurotoxin-alpha SV agonists or antagonists of the invention (e.g., Immunodeficiency (SCID) -X-associated, SCID-autosomal, adenosine deaminase deficiency (ADA deficiency), X- (XLA), Bruton's disease, congenital non-gammaglobulinemia, recurrent infantile non-gammaglobulinemia, acquired non-gammaglobulinemia, adult onset non-gammaglobulinemia, subacute non-gammaglobulinemia, selective immunoglobulinemia (CVID) (acquired), Wisdom-Aldrich ' s syndrome, < RTI ID = 0.0 > glioma < / RTI > (WAS), IgM hyperinsulinemic deficiency syndrome, IgM hyperinsulinemic deficiency syndrome, selective IgA deficiency syndrome, IgG subtype deficiency syndrome (with or without IgA deficiency), normal or elevated Ig antibody deficiency, immunodeficiency with thymoma (IgE), Ig oligosaccharide deficiency syndrome, Hypoxia nephropenia, Severe congenital leukopenia, Immunoglobulinemia, Immunoglobulinemia, Immunoglobulinemia, (XLP), Nelgelope ' s Syndrome - Molecular IgE deficiency, purinemic nucleoside phosphorylase < RTI ID = 0.0 > (PNP), MHC type II deficiency (bare lymphocyte syndrome), and severe combined immunodeficiency syndrome. Not one.

According to an embodiment of the present invention, individuals suffering from immunodeficiency develop ideally lower levels of neurotoxin-alpha and / or neurotoxin-alpha SV compared to those who are not suffering from immune deficiency. Any means described herein or known in the art may be used to detect the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptides of the invention (e. G., The neurotoxin-alpha And / or a FACS analysis or ELISA detection of a neurotoxin-alpha SV polypeptide and hybridization or PCR detection of a neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotide according to the present invention. Alpha-and / or neurotoxin-alpha SV polynucleotides or polypeptides of the invention can be used to separately measure the expression pattern of the kin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptides.

Biological samples of persons with immune deficiency are characterized by low expression levels of neurotoxin-alpha and / or neurotoxin-alpha SV compared to levels observed in persons not having immunodeficiency. Thus, the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides and / or polypeptides and / or agonists or antagonists thereof of the present invention may be used in the diagnosis and / or prognosis of immunodeficiency disorders according to the methods of the present invention have. For example, a biological sample obtained from a person suspected of having an immunodeficiency (" patient ") has a relative level of expression of the neurotoxin-alpha and / or neurotoxin- alpha SV polynucleotides and / Can be analyzed. The level of expression of one or more of these molecules according to the invention is then compared to the level of expression of the same molecule according to the invention when expressed in a person known not to be immunodeficient. Significant differences between patients at the expression levels of the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides and / or polypeptides of the present invention and / or agonists and / or antagonists thereof, and the samples obtained from the control, Suggesting that they have immunodeficiency.

In another embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides and / or polypeptides and / or agonists and / or antagonists thereof (e.g., anti- - Neutrokine-alpha SV antibody) is used for the treatment, diagnosis and / or prognosis of a person suffering from non-canine immunodeficiency syndrome (CVID) (also known as acquired non-gamma globulinosis and acquired gamma globulin hypoxia) or a subgroup of such diseases Is used. According to an aspect of the present invention, a subgroup of persons suffering from CVID or those suffering from CVID are selected from the group consisting of neurotoxin-alpha and / or anti-neurotoxin-alpha Express an abnormal level of the receptor. Any means described herein or known in the art may be used to detect a neurotoxin-alpha polynucleotide or polypeptide and / or a neurotoxin-alpha receptor polypeptide of the invention (e. G., A neurokinin- FACS analysis or ELISA detection of alpha and / or neurotoxin-alpha SV polypeptides and hybridization or PCR detection of neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides according to the present invention) Alpha and / or neurotoxin-alpha SV polynucleotides of the present invention in a sample containing at least a mononuclear cell or a portion thereof (e.g., RNA), as compared to a sample containing a nucleic acid Or may be used to separately measure the expression pattern of a polypeptide and / or a neurotoxin-alpha receptor polypeptide. A sample containing at least a mononuclear cell or a portion thereof (e.g., RNA) is measured to reflect neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotide or polypeptide expression and is at least a B cell or a component thereof (E.g., RNA) is measured to reflect less than the normal level of expression of neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptide, the sample may be a CVID (i. E., Acquired non-gamma globulin Or acquired gamma globulin depletion).

The subgroup of persons suffering from CVID is characterized by a high level of expression of the neurotoxin-alpha and neurotoxin-alpha receptors (NARs) in peripheral or circulating B cells as compared to those observed in people who do not have CVID. Conversely, people who are not suffering from CVID are typically characterized by low levels of neurotoxin-alpha expression and high levels of NAR expression in peripheral or circulating B cells. Thus, the neurotoxin-alpha, neurotoxin-alpha SV polypeptide and / or NAR polypeptide, polynucleotides and / or polypeptides of the present invention and / or agonists or antagonists thereof are specific for small subgroups of CVIDs according to the invention It can be used for diagnosis. For example, a sample of peripheral B cells obtained from a person (patient) who is presumed to be suffering from CVID may be a relative of the neurotoxin-alpha, neurotoxin-alpha SV and / or NAR polynucleotide and / Can be analyzed for expression levels. The level of expression of one or more of these molecules according to the invention is then compared to the level of expression of the same molecule according to the invention in a person known as not having CVID (" control "). Significant differences between the patients at the expression levels of the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides and / or polypeptides and / or agonists and / or antagonists thereof of the invention and the samples obtained from the control, Suggesting CVID.

In certain embodiments, the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides and / or polypeptides and / or agonists and / or antagonists thereof (e.g., anti- Neurotoxin-alpha SV antibodies) are used for the diagnosis, prognosis, treatment, or prevention of diseases characterized by defective serum immunoglobulin production, recurrent infections and / or immune system disorders. In addition, the use of neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides and / or polypeptides and / or agonists thereof and / or antagonists such as anti-neurotoxin-alpha and / or anti- Antibodies) are useful in the treatment of a variety of conditions including, but not limited to, joint, bone, skin and / or parotid infections, blood-mediated infections (eg, sepsis, meningitis, septic arthritis and / or osteomyelitis), autoimmune diseases (Including, but not limited to CVID, another primary immunodeficiency syndrome, HIV disease, CLL, recurrent bronchitis, sinusitis, and all other diseases associated with malignant tumors and / or their infections, diseases, Prophylactic, therapeutic, or prophylactic treatment of otitis media, conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster (eg, severe herpes zoster) and / or neuromuscular carcinoma.

In another embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides and / or polypeptides and / or agonists and / or antagonists thereof (e.g., anti- - neurotoxin-alpha SV antibody) is used to treat, diagnose, or prognose an individual having an autoimmune disease or disease.

The neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptides of the invention or the neurotoxin-alpha and / or neurotoxin-alpha SV agonists or antagonists of the invention (e.g., Autoimmune diseases, or diseases that can be treated, prevented, diagnosed, or prognosed with, for example, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenic purpura, (Eg, IgA nephritis), multiple sclerosis, neuritis, uveitis, eye diseases, endocrine disorders, autoimmune hemolytic anemia, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, recurrent polychondritis, rheumatic heart disease, , Purpura (e.g. Henloch-Scoenlein purpura, Reiter's disease, Stiff-Man syndrome, autoimmune pulmonary inflammation, Guillain-Ba (eg, Hashimoto's thyroiditis, systemic lupus erythematosus, Goodpasture syndrome, pemphigus, receptor autoimmunity (eg, (a) Grave's syndrome), insulin-dependent diabetes mellitus and autoimmune inflammatory diseases, autoimmune thyroiditis, hypothyroidism (B) myasthenia gravis and (c) insulin resistance), autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, rheumatoid arthritis, scleroderma with anti-collagen antibodies, (Eg, primary glomerulonephritis and IgA nephropathy), pemphigoid, Sjogren's syndrome, diabetes mellitus, and adrenergic-acting drug resistance (such as asthma, chronic obstructive pulmonary disease, Or adrenergic-acting drug resistance with cystic fibrosis), chronic active hepatitis, primary biliary cirrhosis, other endocrine disorders, albumin, vasculitis, post-MI (po st-MI), cardiac incision syndrome, urticaria, atopic dermatitis, asthma, inflammatory muscle disease and other inflammatory, granulomatous, degenerative and atrophic diseases.

According to this embodiment, an individual suffering from an autoimmune disease or disease expresses an ideally high level of neurotoxin-alpha, neurotoxin-alpha SV and / or NAR as compared to an individual not suffering from an autoimmune disease or disease . Any means described herein or known in the art may be used to detect the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptides and / or NAR polypeptides of the invention (e. G., The neurotoxin - FACS analysis or ELISA detection of alpha and / or neurotoxin-alpha SV polypeptides and hybridization or PCR detection of the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides of the invention. Alpha-and / or neurotoxin-alpha SV polynucleotides or polypeptides and / or NAR polypeptides.

A biological sample of a person suffering from an autoimmune disease or disease is characterized by a high expression level of neurotoxin-alpha, neurotoxin-alpha SV and / or NAR as compared to that observed in a person not suffering from an autoimmune disease or disease It is erased. Thus, the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides and / or polypeptides and / or agonists or antagonists thereof of the present invention may be used for the diagnosis and / or prognosis of an autoimmune disease or disease according to the methods of the present invention . For example, a biological sample obtained from a person suspected of having an autoimmune disease or disease (" patient ") may be a neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotide and / / RTI > and / or relative expression levels of the NAR polypeptide. The level of expression of one or more of these molecules according to the invention is then compared to the level of expression of the same molecule according to the invention in a person known not to have an autoimmune disease or disease. Samples obtained from patients and controls at the expression level of the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides and / or polypeptides and / or agonists and / or antagonists and / or NAR polypeptides of the present invention Significant differences between the two groups suggest that the patient has immune deficiency syndrome.

In another embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptides of the invention or agonists of the neurotoxin-alpha and / or neurokin-alpha SV and / or antagonists - neurotoxin-alpha and / or anti-neurotoxin-alpha SV antibodies) are used for the treatment, diagnosis and / or prognosis of a person suffering from systemic lupus erythematosus or a subgroup of this disease. According to this embodiment, the subgroup of patients with systemic lupus erythematosus or systemic lupus erythematosus exhibits an ideal high level of neurotoxin-alpha and / or neurotoxin-alpha SV compared to those without systemic lupus erythematosus do. Any means described herein or known in the art may be used to detect the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptides of the invention (e.g., the neurotoxin-alpha and / or FACS analysis or ELISA detection of neurotoxin-alpha SV polypeptides and hybridization or PCR detection of neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides according to the present invention. The neurotoxin-alpha And / or the expression profile of neurotoxin-alpha SV polynucleotides and / or polypeptides.

A biological sample of a person suffering from systemic lupus erythematosus is characterized by a high expression level of neurotoxin-alpha and / or neurotoxin-alpha SV as compared to that observed in a person who is not suffering from systemic lupus erythematosus. Thus, the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides and / or polypeptides and / or agonists or antagonists thereof of the present invention may be used in the diagnosis and / or prophylaxis of systemic lupus erythematosis or subtypes thereof, And / or prognosis. For example, a biological sample obtained from a person suspected of suffering from systemic lupus erythematosus ("patient") may be a relative sample of neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides and / or polypeptides of the invention Can be analyzed for expression levels. The level of expression of one or more of these molecules according to the invention is then compared to the level of expression of the same molecule according to the invention in a person known not to suffer from systemic lupus erythematosus. Significant differences between the patients at the expression levels of the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides and / or polypeptides and / or agonists and / or antagonists thereof of the invention and the samples obtained from the control, Immune deficiency syndrome and subtypes thereof.

In addition, there is a direct correlation between the concentration of neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides and / or polypeptides of the present invention with systemic lupus erythematosus or subtypes of the disease. Thus, the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides (RNA), polypeptides and / or agonists or antagonists thereof of the present invention may be administered in combination with systemic lupus erythematosus or its subtype severity . ≪ / RTI > For example, a biological sample obtained from a person suspected of suffering from systemic lupus erythematosus ("patient") may be a relative sample of neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides and / or polypeptides of the invention Can be analyzed for expression levels. The level of expression of one or more of these molecules according to the invention is then compared to the level of expression of the same molecule according to the invention in a panel of humans known to exhibit a range of the extent of systemic lupus erythematosus. According to this method, the coincidence of expression levels with the characteristic members of the panel is indicative of the severity of the disease.

In another embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptides of the invention or agonists and / or antagonists thereof (e.g., anti-neurotoxin-alpha and / or anti- Alpha SV antibody) is used to treat, diagnose, or prognose a person suffering rheumatoid arthritis or a subgroup of the disease. According to this embodiment, the subgroup of rheumatoid arthritis patients or patients with rheumatoid arthritis express an abnormally high level of neurotoxin-alpha and / or neurotoxin-alpha SV as compared to those who are not suffering from rheumatoid arthritis. Any means described herein or known in the art may be used to detect the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptides of the invention (e.g., the neurotoxin-alpha and / or FACS analysis or ELISA detection of neurotoxin-alpha SV polypeptides and hybridization or PCR detection of neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides according to the present invention. The neurotoxin-alpha And / or the expression profile of neurotoxin-alpha SV polynucleotides and / or polypeptides.

A biological sample of a person suffering from rheumatoid arthritis is characterized by high expression levels of neurotoxin-alpha and / or neurotoxin-alpha SV as compared to those observed in people who are not suffering from rheumatoid arthritis. Thus, the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides and / or polypeptides and / or agonists or antagonists thereof of the present invention may be used in the diagnosis and / or treatment of rheumatoid arthritis or sub- Or prognosis. For example, a biological sample obtained from a person suspected of suffering from rheumatoid arthritis (the " patient ") has a relative expression of the neurotoxin-alpha and / or neurotoxin- alpha SV polynucleotides and / or polypeptides of the invention ≪ / RTI > The level of expression of one or more of these molecules according to the invention is then compared to the level of expression of the same molecule according to the invention in a person known not to be suffering from rheumatoid arthritis. Significant differences between the patients at the expression levels of the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides and / or polypeptides and / or agonists and / or antagonists thereof of the invention and the samples obtained from the control, Rheumatoid arthritis and its subtypes.

Thus, the present invention provides a method of measuring the level of expression of a gene encoding a neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide in an immune system tissue or other cell or body fluids from an individual and measuring the measured gene expression level using a standard neurotoxin- There is provided a diagnostic method useful during the diagnosis of an immune system disease and / or autoimmune disease, including cancer and immunodeficiency disease of the immune system, including comparing the level of alpha and / or neurotoxin-alpha SV gene expression, By comparison, increasing or decreasing the level of gene expression indicates the disease of the immune system.

If the diagnosis of a disease of the immune system, including but not limited to the diagnosis of a tumor, the diagnosis of an immunodeficiency, and / or the diagnosis of an autoimmune disease is made according to conventional methods, the present invention is useful as a prognostic indicator, A patient exhibiting increased or decreased neurotoxin-alpha and / or neurotoxin-alpha SV gene expression will suffer from worse clinical outcomes compared to patients expressing the gene at a level closer to the standard level.

Analyzing or measuring the expression level of a gene encoding a neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide may be accomplished by measuring the level of neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide or the level of neurotoxin- alpha and / Or the neurotoxin-alpha SV polypeptide in a second biological sample directly relative to the first biological sample (e.g., by measuring absolute protein level or mRNA level) or relatively (e.g., by measuring the neurotoxin- Alpha and / or neurotoxin-alpha SV polypeptide levels or mRNA levels). Preferably, the level of neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide or mRNA in the first biological sample is measured and obtained from a second biological sample obtained from a person not suffering from the disease, Alpha < / RTI > and / or neurotoxin-alpha SV polypeptide levels or mRNA levels measured by averaging the levels from the non-treated population. As recognized in the art, once the standard neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide or mRNA levels are known, the standard levels can be used repeatedly as a comparison standard.

Biological samples refer to all biological samples obtained from human, body fluids, cell lines, tissue cultures or other sources containing neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide levels or mRNA. As mentioned, biological samples include biological fluids (e. G., Serum, plasma, urine, synovial fluid and spinal fluid) that contain the free extracellular domain of the neurotoxin-alpha and / or neurotoxin- alpha SV polypeptides, Other tissue sources that have been found to express complete or free extracellular domains of neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides are included. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. If the biological sample contains mRNA, tissue biopsy is the preferred source.

The compounds of the invention are useful for the diagnosis, prognosis or treatment of a variety of immune system-related disorders in a mammal, preferably a human. Such diseases include, but are not limited to, tumors (e.g., B cells and mononuclear cell leukemias and lymphomas) and tumor metastases, infections by bacteria, viruses and other parasites, immunodeficiency, inflammatory diseases, lymphadenopathy, autoimmune diseases Rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, mixed connective tissue diseases and inflammatory muscle diseases) and graft versus host disease.

Total cellular RNA is described in Chomczynski and Sacchi, Anal. Biochem. 162: 156-159 (1987)) using a suitable technique such as the one-step guanidinium-thiocyanate-phenol-chloroform method. The level of mRNA encoding the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide is then assayed using an appropriate method. (RT-PCR) and reverse transcription (RT-LCR) in combination with the ligase chain reaction. The RT-PCR was performed in the same manner as RT-PCR .

Assays of the levels of neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide in biological samples can be performed using antibody-based techniques. For example, expression of neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide in tissues can be studied by classical immunohistological methods (Jalkanen, M., et al., J. Cell. Biol. 101 : 976-985 (1985); Jalkanen, M., et al., J. Cell. Biol. 105: 3087-3096 (1987)). Other antibody-based methods useful for detecting neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide gene expression include immunoassays such as enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs) . Suitable antibody black labels are known in the art and include enzyme labels such as glucose oxidase; Iodine ^{(131 I, 125 I, 123} I, 121 I), carbon ⁽¹⁴ C), sulfur ⁽³⁵ S), 3 tritium ⁽³ H), indium ^{(115m In, 113m In, 112} In, 111 In) and technetium ⁽⁹⁹ Tc, ^99m Tc), thallium ⁽²⁰¹ Ti), gallium ⁽⁶⁸ Ga, ⁶⁷ Ga), palladium ⁽¹⁰³ Pd), molybdenum ⁽⁹⁹ Mo), xenon ⁽¹³³ Xe), fluorine ⁽¹⁸ F), ¹⁵³ Sm , Radioactive isotopes such as ¹⁷⁷ Lu, ¹⁵⁹ Gd, ¹⁴⁹ Pm, ¹⁴⁰ La, ¹⁷⁵ Yb, ¹⁶⁶ Ho, ⁹⁰ Y, ⁴⁷ Sc, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁴² Pr, ¹⁰⁵ Rh, ⁹⁷ Ru; An emission marker such as luminol; And fluorescent labels such as fluorescein and rhodamine, and biotin.

Techniques known in the art can be applied for labeling the antibodies of the present invention. Such techniques include but are not limited to the use of bifunctional binders (see, for example, U.S. Patent Nos. 5,756,065; 5,714,631; 5696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119,4,994,560; and 5,808,003) , The entire contents of each of which are incorporated herein by reference.

The tissue or cell type analyzed includes, but is not limited to, those known or suspected to express a neurotoxin-alpha gene, such as a cell or tissue known to express neurotoxin-alpha gene (e.g., a cell of the mononuclear cell lineage) Cells or tissues (e.g., B cell lines and spleen cells). Examples of protein separation methods used herein include the methods described in Harlow, E. and Lane, D., 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY , The entire contents of which are incorporated herein by reference. The isolated cells may be derived from the cell culture or patient. Analysis of the cells obtained from the cultures can be used as part of a cell-based gene therapy technique or to evaluate cells that can be used to test the effect of compounds on the expression of the neurotoxin-alpha gene or the neurotoxin-alpha receptor gene It can be a necessary step.

For example, an antibody or fragment thereof as described herein can be used to quantitatively or qualitatively detect the presence of a neurotoxin-alpha gene product or a conserved variant or peptide fragment thereof. This can be achieved, for example, by immunofluorescence techniques using fluorescently labeled antibodies in conjunction with optical microscopy, flow cytometry or fluorescence measurement detection.

The antibodies (or fragments thereof) or the neurotoxin-alpha polypeptides or polynucleotides of the present invention may further comprise a neurotoxin-alpha gene product or a conserved variant or peptide fragment thereof, such as in immunofluorescence, immunoelectron microscopy or non- Can be used histologically for detection in the reaction system or binding of neurotoxin-alpha to the neurotoxin-alpha receptor. Detection in the reaction system can be accomplished by removing the histological specimen from the patient and applying it to the labeled antibody or the neurotoxin-alpha polypeptide of the present invention. The antibody (or fragment) of the neurotoxin-alpha polypeptide is preferably applied by overlapping the labeled antibody (or fragment) on the biological sample. Through the use of such a method, the presence of a neurotoxin-alpha gene product or conserved variant or peptide fragment or its neurotoxin-alpha polypeptide binding as well as its distribution in a test can be determined. Those skilled in the art using the present invention will understand that any of a wide variety of histological methods (e.g., staining procedures) can be modified to achieve detection in a reaction system.

Immunoassay and nonimmunity assays for the neurotoxin-alpha gene product or its conserved variants or peptide fragments are typically carried out using a sample such as biological fluids, tissue extracts, freshly harvested cells, or lysates of cells cultured in cell cultures Incubating in the presence of a detectably labeled antibody capable of identifying a neurotoxin-alpha gene product or a conserved variant or peptide fragment thereof, and detecting the bound antibody by any of a number of techniques known in the art do.

Immunoassay and nonimmunity assays for the neurotoxin-alpha receptor gene product or conserved variants or peptide fragments thereof are typically performed on samples such as biological fluids, tissue extracts, freshly harvested cells, or lysates of cells cultured in cell cultures Is incubated in the presence of a detectable or labeled neurotoxin-alpha polypeptide capable of identifying a neurotoxin-alpha receptor gene product, or a conserved variant or peptide fragment thereof, and the bound antibody is incubated with any of a number of techniques known in the art And detecting by one.

The biological sample can be fixed in contact with a solid support or carrier such as nitrocellulose or other solid support capable of immobilizing cells, cell particles or soluble proteins. The support may then be washed with a suitable buffer and then treated with a detectably labeled anti-neutrophil-alpha antibody or a detectable neurotoxin-alpha polypeptide. The solid support may then be washed twice with buffer to remove unbound antibody or polypeptide. The antibody is then optionally labeled. The amount of label bound on the solid support can then be detected by conventional means.

&Quot; Solid phase support or carrier " means any support capable of binding an antigen or antibody. Examples of well-known supports or supports include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, gab and magnetite. The nature of the carrier may be slightly soluble or insoluble for the purposes of the present invention. The support material may have substantially any possible structural form as long as the bound molecule can bind the antigen or antibody. Thus, the support shape may be spherical, such as beads, or may be cylindrical, such as the inner surface of the test tube or the outer surface of the rod. Alternatively, the surface may be flat, such as a sheet, a test strip, or the like. A preferred support includes polystyrene beads. Those skilled in the art are aware of many other carriers suitable for conjugating antibodies or antigens and will be able to identify such carriers by using routine experimentation.

The binding activity of many of the resulting anti-neurotoxin-alpha antibodies or neurotoxin-alpha polypeptides can be determined according to methods well known in the art. Those skilled in the art will be able to determine effective and optimal assay conditions for each measurement by using routine experimentation.

In addition to assaying the level of neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide or polynucleotide in a biological sample obtained from an individual, the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide or polynucleotide may also Can be detected in vivo by an image. For example, in one embodiment of the invention, B cell lymphoma is imaged using a neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide and / or an anti-neurotoxin-alpha antibody. In another embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides and / or anti-neurotoxin-alpha antibodies and / or neurotoxin-alpha polynucleotides of the invention (e.g., Or polynucleotides complementary to all or part of the neurotoxin-alpha SV mRNA) are used to image lymphoma (e.g., mononuclear cells and B cell lymphoma).

Antibody labels or markers for in vivo imaging of neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides include labels or markers detectable by X-ray photography, NMR, MRI, CAT-scan or ESR . For X-ray photography, suitable labels include radioactive isotopes such as barium or cesium that emit detectable radiation but are not harmful to the patient.

Suitable markers for NMR and ESR include those having a detectable characteristic spin such as deuterium that can be incorporated into the antibody by labeling the nutrients to the relevant hybridomas. When in vivo imaging is used to detect elevated levels of neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides for the diagnosis of a human, the use of human antibodies or " humanized " chimeric monoclonal antibodies Lt; / RTI > Such antibodies can be prepared using techniques described herein or known in the art. For example, methods for producing chimeric antibodies are known in the art (see for example Morrison, Science 229: 1202 (1985); Oi et al., BioTechniques 4: 214 (1986); Cabilly et al. Boulianne et al., Nature 312: 643 (1984); Neuberger et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671; et al., Nature 314: 268 (1985)).

In addition, any neurotrophin-alpha polypeptide capable of detecting its presence can be administered. For example, a neurotrophin-alpha polypeptide, labeled with a radioactive contrast agent or other suitable compound, can be administered and imaged as discussed above for the labeled antibody. In addition, such neurotoxin-alpha polypeptides can be used in in vitro diagnostic procedures.

Radioactive isotopes ^{(e.g., 131 I, 112 In, 99m} Tc, (131 I, 125 I, 123 I, 121 I), carbon ⁽¹⁴ C), sulfur ⁽³⁵ S), 3 tritium ⁽³ H), indium ^{(115m In, 113m In, 112} In, 111 In) , and technetium ⁽⁹⁹ Tc, ^99m Tc), thallium ⁽²⁰¹ Ti), gallium ⁽⁶⁸ Ga, ⁶⁷ Ga), palladium ⁽¹⁰³ Pd), molybdenum ⁽⁹⁹ Mo) , ¹³³ Xe, fluorine ( ¹⁸ F), ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁵⁹ Gd, ¹⁴⁹ Pm, ¹⁴⁰ La, ¹⁷⁵ Yb, ¹⁶⁶ Ho, ⁹⁰ Y, ⁴⁷ Sc, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁴² Pr, ¹⁰⁵ Alpha, and / or neurotoxin-alpha SV polypeptide- specific antibody or antibody fragment labeled with a suitable detectable imaging moiety such as a radioisotope, Rh, ⁹⁷ Ru), a radioconjugate material, or a material detectable by nuclear magnetic resonance For example, parenterally, subcutaneously or intraperitoneally, into the mammal to be tested for immune system disease. It will be understood in the art that the size of the patient and the imaging system used will measure the amount of imaging moiety required to produce a diagnostic image. In the case of radioactive isotope residues, the amount of radioactivity injected into a human subject is generally in the range of about 5 to 20 milli-curies, ⁹⁹ mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of the cell containing the neurotoxin-alpha protein. In vivo tumor imaging has been described in SW Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments" (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, SW Burchiel and BA Rhodes, eds., Masson Publishing Inc. ).

One of the ways that an anti-neurotoxin-alpha antibody can be detectably labeled in connection with an antibody is to link the antibody to the enzyme and to detect the linked product by enzyme immunoassay (EIA) (Voller, A., " The Enzyme Linked Immunosorbent Assay (ELISA) ", 1978, Diagnostic Horizons 2: 1-7, Microbiological Associates Quarterly Publication, Walkersville, MD); Voller et al., J. Clin. Pathol. 31: 507-520 (1978); Butler, J. E., Meth. Enzymol. 73: 482-523 (1981); Maggio, E. (ed.). 1980, Enzyme Immunoassay, CRC Press. Boca Raton, FL.,; Ishikawa, E. et al., (Eds.). 1981, Enzyme Immunoassay, Kagaku Shoin, Tokyo). The enzyme bound to the antibody will react with a suitable substrate, preferably a chromogenic substrate, in such a way as to produce chemical moieties that can be detected, for example, by spectrophotometers, fluorimetry or visual means. Enzymes that can be used to detectably label an antibody include malate dehydrogenase, staphylococcus nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, Dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6- Phosphate dehydrogenase, glucoamylase, and acetylcholinesterase. In addition, detection can be accomplished by a chromogenic assay using a chromogenic substrate for the enzyme. In addition, detection can be achieved by visually comparing the degree of enzymatic reaction of the substrate with standards prepared similarly.

In addition, detection can be accomplished using a variety of different immunoassays. For example, neutrophil-alpha can be detected via radioimmunoassay (RIA) by radioactively labeling antibodies or antibody fragments (see, e.g., Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated herein by reference). Radioactive isotopes can be detected by means including, but not limited to, gamma counters, scintillation counters, or radiographic photographs.

The antibody may be labeled with a fluorescent compound. The presence of the antibody can be detected by fluorescence when the fluorescently labeled antibody is exposed to light of the appropriate wavelength. The most commonly used fluorescent labeling compounds include fluorescein isothiocyanate, rhodamine, picoerythrine, ficocyanin, allophycocyanin, offline dehydrodehyde, and fluorescamine.

The antibody can also be detectably labeled using a fluorescent emission metal such as ¹⁵² Eu or a lanthanide metal. These metals may be attached to the antibody using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

In addition, the antibody can be detectably labeled by linking it to a chemiluminescent compound. The presence of the chemiluminescent-tag antibody is then determined by detecting the presence of luminescence occurring during the course of the chemical reaction. Examples of particularly useful chemiluminescent marker compounds include luminol, isoleuminol, teematic acridinium ester, imidazole, acridinium salts and oxalate esters.

Similarly, a bioluminescent compound can be used to label an antibody of the invention. Bioluminescence is a type of chemiluminescence found in biological systems in which catalytic proteins increase the efficacy of chemiluminescent reactions. The presence of a bioluminescent protein is measured by detecting the presence of luminescence. Bioluminescent compounds that are of interest for purposes of labeling include, but are not limited to, luciferin, luciferase and acuinin.

Treatment of immune system-related diseases

As noted above, the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides and polypeptides and the anti-neurotoxin-alpha antibodies are useful in the treatment of abnormally high or low levels of neurotoxin-alpha and / or neurotoxin- It is useful for diagnosis of symptoms associated with low expression. Alpha < RTI ID = 0.0 > and / or < / RTI > neurotoxin-alpha SV is expressed and the activity is regulated by neurotoxin-alpha and / or neurotoxin- A substantially altered (increased or decreased) expression level of neurotoxin-alpha and / or neurotoxin-alpha SV is indicative of pathology associated with the body system in which neurotoxin-alpha and / or neurotoxin-alpha SV is expressed and / It is obvious that it induces.

In addition, those skilled in the art will appreciate that the extracellular domain of the individual proteins can be modified by proteolytic cleavage, such that the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides of the invention are members of the TNF family, Alpha < / RTI > and / or neurotoxin-alpha SV polypeptides (especially the soluble form of the individual extracellular domain) can be released from the source expressing the alpha SV in a soluble form, Or added to the body, the polypeptide will exert its modulatory activity on all of its individual target cells. In addition, such type II transmembrane protein expressing cells may be added to a cell, tissue or body of an individual so that the added cell binds to a cell expressing the receptor for neurotoxin-alpha and / or neurotoxin-alpha SV, Cells expressing neurotoxin-alpha and / or neurotoxin-alpha SV can initiate action (e.g., proliferation or cytotoxicity) on receptor-bearing target cells.

In one embodiment, the invention provides a composition comprising a polypeptide of the invention (eg, a heterologous polypeptide, a heterologous nucleic acid, a neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide coupled to a toxin or prodrug, Alpha) and / or neurotoxin-alpha SV antibodies) is administered to target cells (e.g., B cells expressing neurotoxin-alpha and / or neurotoxin-alpha SV receptors or neurotoxin-alpha And / or monocytes expressing a cell surface-bound form of neurotoxin-alpha SV). The neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides or anti-neurotoxin-alpha and / or anti-neurotoxin-alpha SV antibodies of the invention may be administered via hydrophobic, hydrophilic, ionic and / Heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs.

In one embodiment, the invention provides a polypeptide of the invention (e.g., a neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide or an anti-neurotoxin-alpha and / or anti- A neurotoxin-alpha SV antibody) to a cell to thereby specifically deliver the composition of the present invention to the cell. As one example, the present invention provides a method of delivering a therapeutic protein into a target cell. As another example, the present invention provides a nucleic acid molecule encoding a nucleic acid (e. G., An antisense or ribozyme) or a double stranded nucleic acid (e. G., DNA that can integrate into the genome of a cell or replicate as an epitope and can be transcribed) . ≪ / RTI >

In another embodiment, the invention provides a method of modulating the activity of a polypeptide of the invention (e.g., a neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide or an anti-neurotoxin-alpha and / or anti- (E. G., Destruction of tumor cells) by administering in combination with a toxin or cytotoxic prodrug.

In certain embodiments, the present invention provides a method of identifying a B cell lineage cell (eg, B cell-associated leukemia or lymphoma) by administering a neurotoxin-alpha and / or neurotoxin- alpha SV polypeptide in combination with a toxin or cytotoxic prodrug It provides a means of destroying enemies.

In another specific embodiment, the invention provides a method of treating a cell (e.g., mononuclear cell leukemia or lymphoma) of the mononuclear cell line by administering a neurotoxin-alpha and / or neurotoxin-alpha SV antibody in combination with a toxin or cytotoxic prodrug It provides a means of destroying enemies.

&Quot; Toxin " refers to compounds, radioactive isotopes, holotoxins, modified toxins, catalytic subunits of toxins, cytotoxins (cytotoxic agents) that bind to and act on endogenous cytotoxic agent systems, Quot; means any molecule or enzyme that causes the death of a cell or does not normally exist in the cell or on the surface of the cell. Toxins that may be used in accordance with the methods of the invention include, but are not limited to, antibodies (or complement fixations containing a moiety) that bind to radioactive isotopes, intrinsic or derived endotoxic cytotoxic agent systems known in the art, , Thymidine kinase, endonuclease, RNAse, alpha toxin, lysine, avrine, Pseudomonas exotoxin A, diphtheria toxin, saponin, momorphine, gelonin, Ginsenosides and cholera toxins. The " toxin " may also be a cell proliferation inhibitor or a cell killing agent, a therapeutic agent or a radioactive metal ion (e.g., an alpha-radiator such as ²¹³ Bi or ¹⁰³ Pd, ¹³³ Xe, ¹³¹ I, ⁶⁸ Ge, ⁵⁷ Co, ⁶⁵ Zn, ⁸⁵ Sr, Radioactive isotopes such as ³² P, ³⁵ S, ⁹⁰ Y, ¹⁵³ Sm, ¹⁵³ Gd, ¹⁶⁹ Yb, ⁵¹ Cr, ⁵⁴ Mn, ⁷⁵ Se, ¹¹³ Sn, ⁹⁰ yttrium, ¹¹⁷ tin, ¹⁸⁶ rhenium, ¹⁶⁶ holmium and ¹⁸⁸ rhenium) , Luminescent labels such as luminol and fluorescent labels such as fluorescein and rhodamine, and biotin.

Techniques known in the art can be used to label the antibodies of the present invention. Such techniques include, but are not limited to, the use of bifunctional binders (see, for example, U.S. Patents 5,756,065; 5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119; 4,994,560 and 5,808,003). The entire contents of each of the above patents are incorporated herein by reference. Cytotoxins or cytotoxic agents include all substances that are harmful to the cell. Examples include but are not limited to paclitaxel, cytosalacin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenofoside, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, Dexamethasone, glucocorticoid, proline, tetracaine, lidocaine, propranolol, and furomycin, and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (BCNU), cyclostaspamide, cyclopentamide, epidermis, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly named as daunomycin) and doxorubicin), antibiotics (such as dactinomycin (formerly actinomycin), bleomycin, (AMC) and anti-mitotic agents (e.g., vincristine and vinblastine).

&Quot; Cytotoxic prodrug " means a non-toxic compound that is normally converted to a cytotoxic compound by an enzyme present in the cell. Cytotoxic prodrugs that may be used in accordance with the methods of the present invention include but are not limited to glutamyl derivatives of benzoic acid mustard alkylating agents, phosphate derivatives of etoposide, mitomycin C, cytosine arabinoside, daunorubicin and doxorubicin Of phenoxyacetamide derivatives.

Also provided is a method of treating a condition in which an individual has an increased level of neurotoxin-alpha and / or neurotoxin-alpha SV activity above a normal or normal level, particularly where the immune system disease is a neurotoxin-alpha and / or neurotoxin- alpha SV polypeptide (E. G., In the form of a foreign domain or a cell expressing a complete protein) or antagonist (e. G., An anti-neurotoxin-alpha antibody). Thus, the present invention provides a method for reducing the level of neurotoxin-alpha and / or neurotoxin-alpha SV activity in an individual in need of a reduced level of neurotoxin-alpha and / or neurotoxin- Alpha and / or neurotoxin-alpha SV activity, including administering to said individual a pharmaceutical composition comprising a separate neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide or an antagonist thereof according to the invention, RTI ID = 0.0 > a < / RTI >

The neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptides of the invention or agonists of neurotoxin-alpha and / or neurotoxin-alpha SV can be used for the treatment of infectious bacteria. For example, infectious diseases can be treated by increasing the reaction, particularly by increasing the proliferation and differentiation of B cells. The immune response can be increased by enhancing the current immune response or by initiating a new immune response. Alternatively, the agonist of neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotide or polypeptide or neurotoxin-alpha and / or neurotoxin-alpha SV may also be administered directly to the infectious agent directly .

The virus may be infected with a neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotide or polypeptide or with a neurotoxin-alpha and / or neurotoxin-alpha SV agonist It is an example. Examples of viruses include, but are not limited to, the following DNA and RNA viruses and viruses: Abroviruses, adenoviruses, arenaviruses, arterioviruses, birnaviruses, buneaviruses, caliciviruses, (E. G., Influenza A viruses), cornaviruses, cornaviruses, dengue, EBV, HIV, flaviviruses, hepadiviruses (hepatitis), herpes viruses (e.g., paramyxoviridae, (Eg, influenza B and parainfluenza), papilloma virus, papovavirus, parvovirus, picornavirus, poxvirus (eg, tuberculosis or vulva), reovirus -II, lentivirus), and tovirus (e.g., ruby virus). These viruses can cause a variety of diseases or conditions including, but not limited to, arthritis, bronchiolitis, respiratory syncytial virus, encephalitis, eye infections (eg, conjunctivitis, keratitis) , Chronic fatigue syndrome, hepatitis (A, B, C, E, chronic active, delta), B encephalitis, Juin, Chikungunya, Rift Valley fever, yellow fever, meningitis, opportunistic infections , Pneumonia, Burkitt's lymphoma, chickenpox, hemorrhagic fever, measles, mumps, parainfluenza, rabies, cold, polio, leukemia, rubella, sexually transmitted infections, skin diseases (e.g. The agonists or antagonists of neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptides or of neurotoxin-alpha and / or neurotoxin-alpha SV may be used to treat, prevent, diagnose and / . In certain embodiments, the neurotoxin alpha polynucleotide, polypeptide or agonist is used to treat, prevent and / or diagnose meningitis, dengue fever, EBV and / or hepatitis (hepatitis B). In a further particular embodiment, the neurotoxin alpha polynucleotide, polypeptide or agonist is used to treat a patient who is unreactive with one or more other commercial hepatitis vaccines. In a further specific embodiment, the neurotoxin alpha polynucleotide, polypeptide or agonist is used for the treatment, prevention and / or diagnosis of AIDS. In a further particular embodiment, the neurotoxin-alpha and / or neurocrine-alpha SV and / or neurotoxin-alpha receptor polynucleotides, polypeptides, agonists and / or antagonists are used for the treatment, prevention and / or treatment of cryptosporidiosis patients It is used for diagnosis.

Similarly, it is possible to induce a disease or condition and may be induced by an agonist or antagonist of neurotoxin-alpha and / or neurocrine-alpha SV polynucleotide or polypeptide or neurotoxin-alpha and / or neurocrine- alpha SV Bacteria or fungi that may be cured include, but are not limited to, gram-negative and gram-positive bacteria and germs and fungi such as: Actinomycetales (e.g., Corynebacterium, (Eg anthracnose, norcadia), cryptococcus neoformans, aspergillosis, basilaceae (eg anthrax, clostridium), bacteroids, blastomases, bordetellae, borrelia Abruzzo ferry) Brucella disease, Candida disease, Campylobacter, Coccidiosis, Yeast fungus, Dermatophyceis, (E. G., Salmonella typhi and Salmonella paratyphi), Serratia, < / RTI > Yersinia), eelphi pelotrichus (Eg, Acinetobacter, gonorrhoeae, meningococcal disease), meningococcal, pustularea, psoriasis, psoriasis, psoriasis, gastritis, (Eg, Actinobacillus, Haemophilus (eg, Haemophilus influenzae type B), Pasteurella), Pseudomonas, Reticcias, Chlamydia, Staphylococcus, Syphilis, Shigella, Staphylococcus, Meningococcus, And Streptococcus (e.g., Streptococcus pneumoniae and Group B streptococcus). These bacterial or fungal pathogens can cause, but are not limited to, bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections (eg, AIDS- Meningitis, meningitis (meningitis type A and type B), menstrual cramps, meningitis, menstrual cramps, meningitis, menstrual cramps, menstrual cramps, ), Chlamydia, syphilis, diphtheria, leprosy, tuberculosis, tuberculosis, lupus, botulism, rhinotracheitis, tetanus, rheumatic fever, scarlet fever, sexually transmitted infections, skin diseases (eg, , Urinary tract infection, wound infection. The agonists or antagonists of neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptides or of neurotoxin-alpha and / or neurotoxin-alpha SV may be used to treat, prevent, diagnose and / . In certain embodiments, the neurotoxin alpha polynucleotide, polypeptide or agonist thereof is used for the treatment, prevention and / or diagnosis of tetanus, diphtheria, botulism, and / or type B meningitis.

In addition, a parasite, which can induce a disease or condition and which can be treated with neurotoxin-alpha and / or nuclcine-alpha SV polynucleotides or polypeptides or antagonists of neurotoxin-alpha and / But are not limited to, the following or classes of: amebia, Babes fever, coccidiosis, cryptosporidium, diantamoebia, trade, in vitro parasitic disease, giardiasis, parasitic disease, (Eg, Trichomoniasis, and Trichomoniasis) (eg, Trichomoniasis, Tropical Fungi, Quercus Fasciata, and Oviparous Fever). These parasites can cause a variety of diseases or conditions, including but not limited to: amelioration, thrombocytosis, eye infections, intestinal diseases (eg diarrhea, gliadiasis), liver diseases, pulmonary diseases, opportunistic infections , AIDS-associated), malaria, pregnancy complications and toxoplasmosis. The agonists or antagonists of neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptides or of neurotoxin-alpha and / or neurotoxin-alpha SV may be used to treat, prevent, diagnose and / . In certain embodiments, the neurotoxin alpha polynucleotide, polypeptide or agonist thereof is used for the treatment, prevention and / or diagnosis of malaria.

In another embodiment, agonists or antagonists of the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptides or of neurotoxin-alpha and / or neurotoxin-alpha SV are useful not only for inner ear infections (e.g., Prevention and / or diagnosis of an infection by Streptococcus pneumoniae and other pathogenic organisms.

In certain embodiments, the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides and / or polypeptides and / or agonists thereof and / or antagonists (e.g., anti-neurotoxin-alpha and / or anti- Alpha SV antibodies) are used to treat or prevent diseases characterized by defective serum immunoglobulin production, recurrent infections, and / or immune system disorders. In addition, the use of neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides and / or polypeptides and / or agonists thereof and / or antagonists such as anti-neurotoxin-alpha and / or anti- Antibodies) are useful in the treatment of a variety of conditions including, but not limited to, joint, bone, skin and / or parotid infections, blood-mediated infections (eg, sepsis, meningitis, septic arthritis and / or osteomyelitis), autoimmune diseases (Including, but not limited to CVID, other primary immunodeficiency syndromes, HIV disease, CLL, recurrent bronchitis, venous sinusitis, otitis media, and the like), all of which are associated with malignant and / or these infections, diseases, , Conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster (e.g., severe herpes zoster), and / or neuromostasis carnii).

In another embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides and / or polypeptides and / or agonists and / or antagonists thereof of the invention are one of the following diseases, disorders or conditions associated therewith: (Eg, new bone marrow transplantation in adults or children), chronic B-cell lymphoma (eg, chronic lymphocytic leukemia), chronic lymphocytic leukemia Leukemia, HIV infection (eg, HIV infection in adults or children), chronic inflammatory dehydrative polyneuropathy, and post-transfusion purpura.

In addition, the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides and / or polypeptides and / or agonists and / or antagonists thereof of the invention may comprise one or more of the following diseases, disorders or conditions associated therewith: (Eg, anemia associated with parvovirus B19, a stable multiple myeloma patient with a high risk of infection (eg, recurrent infection), autoimmune hemolytic anemia (Eg, CMV-negative recipients of cytomegalovirus (CMV) -stimulatory organs), gamma-kinase (CMV) -mediated transplantation (eg, autoimmune hemolytic anemia of the thyroid), thrombocytopenia (eg neonatal thrombocytopenia) (Eg, gamma globulin hypoxia neonates with risk factors for infection or disease), epilepsy (eg, intractable epilepsy), systemic angiitis syndrome, myasthenia gravis (eg, Dyspepsia), dermatosis, and multiple myositis.

Further preferred embodiments of the invention include, but are not limited to, the use of a neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide, a neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotide and a functional agonist thereof And the like:

Administration to animals (eg, mice, rats, rabbits, hamsters, guinea pigs, pigs, pigs, chickens, camels, goats, horses, cows, sheep, cats, non-human primates and humans, most preferably humans) (E.g., IgG, IgA, IgM, and IgE) and / or to produce an increased amount of one or more antibodies (eg, IgG, IgA, IgM and IgE) Increase the response. In certain embodiments, the neurotoxin-alpha polypeptide and / or agonist of the invention is administered to enhance the immune system and produce an increased amount of IgG. In other, customary embodiments, the neurotoxin-alpha polypeptide and / or agonist of the invention is administered to augment the immune system and produce an increased amount of IgA. In another customary embodiment, the neurotoxin-alpha polypeptides and / or agonists of the invention are administered to enhance the immune system and produce an increased amount of IgM.

An animal capable of producing a functional immunoglobulin molecule by the means of the immune system which is capable of producing a functional endogenous antibody molecule or which has an impaired endogenous immune system but which has been reconstituted from another animal or is partially reconstituted; Including animals, and also including transgenic animals (see for example WO / 9824893, WO / 9634096, WO / 9633735 and WO / 9110741).

A vaccine adjuvant that enhances immune response to a particular antigen. In a particular embodiment, the vaccine adjuvant is a neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide as described herein. In another specific embodiment, the vaccine adjuvant is a neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotide described herein (i.e., the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotide is a gene vaccine adjuvant) . As discussed herein, neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides are known in the art, including, but not limited to, liposome delivery, recombinant vector delivery, Lt; / RTI > techniques.

Adjuvants that enhance tumor-specific immune responses.

An adjuvant that enhances the anti-viral immune response. Anti-viral immune responses that can be augmented using the compositions of the present invention as adjuvants include, but are not limited to, viral and viral associated diseases or conditions as described herein or as known in the art. In certain embodiments, the compositions of the invention are used as adjuvants to enhance an immune response to a virus, disease or condition selected from the group consisting of AIDS, meningitis, dengue, EBV, and hepatitis (e.g., hepatitis B). In another specific embodiment, the compositions of the present invention are useful for the treatment of HIV / AIDS, respiratory syncytial virus, dengue disease, rotavirus, type B encephalitis, influenza A and B, parainfluenza, measles, cytomegalovirus, rabies, , Rift Valley, Herpes simplex and yellow fever. ≪ Desc / Clms Page number 2 > In another specific embodiment, the composition of the present invention is used as an adjuvant to enhance the immune response against the HIV gp 120 antigen.

An adjuvant for enhancing an anti-bacterial or anti-fungal immune response. Anti-bacterial or anti-fungal immune responses that can be augmented using an ancestor of the invention as an adjuvant include viruses and virus-associated diseases or conditions described herein or known in the art. In certain embodiments, the compositions of the present invention are used as adjuvants to enhance an immune response to a bacterium, fungus, disease or condition selected from the group consisting of tetanus, diphtheria, botulism, and type B meningitis. In another specific embodiment, the compositions of the present invention are cholera, Mycobacterium, Salmonella typhi, Salmonella paratyphoid, Myceria meningitidis, Streptococcus pneumoniae, B group streptococcus, Shigella species, enterotoxins. Coli and intestinal hemorrhage. Is used as an adjuvant to enhance the immune response against bacteria, fungi, diseases or conditions selected from the group consisting of E. coli, Borrelia burgdorferi and plasmodium (malaria).

An adjuvant that enhances the anti-parasitic immune response. Anti-parasitic immune responses that can be augmented using the compositions of the present invention as adjuvants include parasitic and parasitic related diseases or conditions described in the art or known in the art. In certain embodiments, the compositions of the present invention are used as adjuvants to enhance immune responses to parasites. In another specific embodiment, the composition of the present invention is used as an adjuvant to enhance the immune response to plasmodium (malaria).

As stimulators of B cell responses to pathogens.

As an agent that enhances the immune status of an individual before receiving immunosuppressive therapy.

As an agent for inducing a higher affinity antibody.

As an agent for increasing serum immunoglobulin concentration.

As an agent to promote the recovery of immunocompromised persons.

As an agent to increase immunoreactivity between aging populations.

As an immune system enhancer before, during, or after bone marrow transplantation and / or other transplantation (eg, allogeneic or xenogeneic immune organ transplantation). In connection with the implantation, the composition of the present invention may be administered prior to, at the same time as, and / or after, the implantation. In certain embodiments, the compositions of the invention are administered prior to the onset of recovery of the T-cell population after transplantation. In another specific embodiment, the composition of the present invention is preferentially administered after the initiation of recovery of the T cell group after transplantation but before the B cell group is completely recovered.

As an agent for enhancing immune response in B cell immunodeficiency patients, such as those receiving partial or complete splenectomy. B cell immunodeficiencies that may be alleviated or treated by administering the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide or polynucleotide of the invention include, but are not limited to, severe combined immunodeficiency (SCID) , SCID-autosomal deficiency, adenosine deaminase deficiency (ADA deficiency), refractory non-gammaglobulinemia (XLA), Bruton's disease, congenital non-gammaglobulinemia, Specific IgG, non-gammaglobulinemia, non-classifiable immune deficiency syndrome (CVID) (acquired), non-gonadal hypermagnesemia, hypogammaglobulinemia, hypogammaglobulinemia, hypogammaglobulinemia, hypogammaglobulinemia, , Wistar-Aldrich syndrome (WAS), IgM hyperreactive immunodeficiency syndrome, IgM hyperreactive immunodeficiency syndrome, selective IgA deficiency, IgG (IgA deficiency associated or unrelated), normal or elevated Ig antibody deficiency, immunodeficiency associated with thymic carcinoma, Ig oligosaccharide deficiency, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD), selective IgM immunodeficiency , Neurogenic neutropenia, severe congenital leukopenia, immunodeficiency-associated thymic lymphoid hypoplasia - amorphous or dysplasia, ataxia - capillary dilatation, meridian hypoplasia, reflex lymphoma (PNP), MHC type II deficiency (bare lymphocyte syndrome), and severe combined immunodeficiency syndrome. ≪ Desc / Clms Page number 2 >

As an agent to enhance immunoreactivity in patients with acquired B cell function loss. Symptoms due to acquired deficiencies in B cell function that can be alleviated or treated by administering the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides or polynucleotides of the invention include, but are not limited to, HIV infection, AIDS , Bone marrow transplants and B cell chronic lymphocytic leukemia (CLL).

As an agent for enhancing immune response in patients suffering from a temporary immune deficiency. Transient immune deficiency syndromes that may be alleviated or treated by administering the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides or polynucleotides or agonists of the invention include, but are not limited to, viral infections (e. , Influenza), depression associated with malnutrition, recovery from infectious mononucleosis, or stress-related symptoms, recovery from measles, recovery from transfusion, and recovery from surgery.

As modulators of antigen presentation by monocytes, dendritic cells and / or B-cells. In one embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides (in soluble, membrane-bound or dermal form) or polynucleotides can be used to enhance antigen presentation or antagonist presentation in vitro or in vivo . In addition, in related embodiments, enhancement or antagonism of antigen presentation may be useful in anti-tumor therapy or in modulating the immune system.

As a mediator of mucosal immune responses. The expression of neurotoxin-alpha by mononuclear cells and the reactivity of B cells to this factor suggests that neurotoxin alpha is involved in signal transduction between B cells and mononuclear cells or their differentiated descendants. This activity occurs in a number of ways similar to the transmission of CD40-CD154 signals between B cells and T cells. Thus, neurotoxin-alpha can be an important regulator of T cell-independent immune responses to peripheral pathogens. In particular, a particular group of B cells (CD5 +), which is responsible for many of the innate immunity in humans that are associated with mucosal sites, can respond to neurotoxin-alpha and thereby enhance the protective immune status of the individual.

As an agent that allows an individual's immune system to develop into a humoral response (ie, TH2) as opposed to a TH1 cell response.

As a means of inducing tumor proliferation and thereby making the tumor more susceptible to anti-neoplastic agents. For example, multiple myeloma is a slowly dividing disease and thus is virtually resistant to all anti-neoplastic regimens. If these cells are multiplied more rapidly, their sensitivity profile will surely change.

As a B cell specific binding protein to which a specific activator or inhibitor of cell growth can be bound. This result will focus on the active or disease-causing or neoplastic B cell population of the activator or inhibitor.

As a means for detecting B-lineage cells as their specificity. This application may require labeling of the protein with biotin or other agents (e.g., as described herein) to provide a detection means.

AIDS, chronic lymphatic disease, and / or common variable immunodeficiency.

As part of a B cell selection device capable of separating B cells from a heterogeneous mixture of cell types. Neutrokine-alpha can bind to a solid support and cells specifically bind to it. Unbound cells are washed and bound cells are sequentially eluted. The non-limiting use of this selection makes it possible to purify tumor cells from bone marrow or peripheral blood, for example, prior to transplantation.

As a therapy for the generation and / or regeneration of lymphatic tissue for surgery, trauma and genetic defects.

As a gene-based therapy for genetic disorders that cause immunodeficiency, as observed between SCID patients.

As an antigen for the generation of antibodies for inhibiting or enhancing neurotoxin-alpha mediated responses.

As a means to activate mononuclear cells / macrophages to defend parasitic diseases affecting mononuclear cells such as Leshmania.

Pretreatment of bone marrow samples prior to transplantation. This treatment increases B cell expression and thus promotes recovery.

As a means of controlling secreted cytokines induced by neurotoxin-alpha.

The neurotoxin-alpha or neurotoxin-alpha SV polypeptides or polynucleotides of the invention can be used to control IgE concentration in vitro or in vivo.

In addition, the neurotoxin-alpha or neurotoxin-alpha SV polypeptides or polynucleotides or agonists thereof of the invention can be used to treat, prevent and / or diagnose an IgE-mediated allergic response. Such allergic reactions include, but are not limited to, asthma, rhinitis, and eczema.

In certain embodiments, the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides or polynucleotides or agonists thereof of the invention are administered to treat, prevent, diagnose and / or alleviate a selective IgA deficiency.

In another specific embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides or polynucleotides or agonists thereof of the invention are administered to treat, prevent, diagnose and / or alleviate vascular ataxia.

In another specific embodiment, a neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide or polynucleotide or an agonist thereof of the invention is administered to treat, prevent, diagnose and / or alleviate a common variable immunodeficiency syndrome.

In another specific embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides or polynucleotides or agonists thereof of the invention are administered to treat, prevent, diagnose and / or ameliorate a non-gamma globulinemia .

In another specific embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides or polynucleotides or agonists thereof of the invention are used to treat, prevent, diagnose and / or ameliorate severe combined immunodeficiency (SCID) .

In another particular embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides or polynucleotides or agonists thereof of the invention are administered to treat, prevent, diagnose and / or alleviate Weissstoff-Aldrich .

In another specific embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides or polynucleotides or agonists of the invention are useful for treating, preventing, diagnosing and / or alleviating a defective refractory Ig associated with an excess of IgM Lt; / RTI >

In another specific embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides or polynucleotides or agonists or antagonists thereof (e.g., anti-neurotoxin-alpha antibodies) of the invention are useful in the treatment of chronic myelogenous leukemia, Leukemia, leukemia, histiocytic leukemia, mononuclear cell leukemia (e.g., acute mononuclear cell leukemia), leukemic paranoia, ceiling mononuclear cell leukemia and / or other mononuclear cells and / or mononuclear cell and / or tissue For the treatment, prevention, diagnosis and / or alleviation of induced leukemia.

In another particular embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides or polynucleotides or agonists of the invention are used to treat mononuclear cell leukemia-like responses, for example, as seen in tuberculosis , Prevention, diagnosis and / or alleviation.

In another specific embodiment, the neurotoxin-alpha or neurotoxin-alpha SV polypeptides or polynucleotides or agonists of the present invention are useful for the treatment of mononuclear cell leukocytosis, mononuclear cell leukopenia, mononuclear cell depletion, and / Preventing, diagnosing and / or alleviating the disease.

In certain embodiments, a neurotoxin-alpha or neurotoxin-alpha SV polypeptide or polynucleotide of the invention and / or an anti-neurotoxin-alpha antibody and / or agonist or antagonist thereof is administered to a patient with a primary B lymphoid disease and / Preventing, diagnosing and / or alleviating the symptoms associated with, and / or associated with, In one embodiment, such primary B lymphoid disease, disease and / or symptom is characterized by complete or partial loss of humoral immunity.

Primary B lymphoid diseases, diseases and / or symptoms characterized by complete or partial loss of humoral immunity and which can be prevented, treated, detected and / or diagnosed with a composition of the invention include, but are not limited to, (XLA), severe combined immunodeficiency (SCID), and selective IgA deficiency.

In a preferred embodiment, the neurotoxin-alpha or neurotoxin-alpha SV polypeptide or polynucleotide and / or agonist and / or antagonist thereof is used to treat, prevent and / or ameliorate a disease, disorder or condition associated with one or more of the mucous membranes of the body. Or diagnosis. Such diseases or disorders include, but are not limited to, mucositis, mucosal pouching, mucous mucositis, mucosal skin lesionism, such as, for example, Alzheimer's disease, Leishmaniasis americana, Mucinous adenocarcinoma, mucinous adenocarcinoma, mucinous adenocarcinoma, mucinous adenocarcinoma, mucinous metaplasia, mucinous metaplasia, myxoid metaplasia, mucinous adenocarcinoma, Mucosal dissemination (e.g., medial metamolysis), mucosal deposition (e.g., type I mucosal deposition, type II mucosal deposition, type III mucosal deposition and type IV mucosal deposition), mucolysis disease, mucosal enteritis , Type II mucopolysaccharidoses (eg, Fucoidan syndrome), IS type mucopolysaccharidoses (ie, Scheie syndrome or V mucopolysaccharidosis), type II mucopolysaccharidoses (I.e., (E.g., Hunter's syndrome), type III mucopolysaccharidosis (i.e., San Fillipo syndrome), type IV mucopolysaccharidosis (i.e., morphogenesis syndrome), type VI mucopolysaccharidosis Mucopolysacchariduria, mucocutaneous conjunctivitis, congestive mucositis, mucormycosis (i. E., Conjunctival infections), < / RTI > mucopolysaccharidosis, mucopolysaccharidoses (i. E., Mucopolysaccharidosis due to beta-glucuronidase deficiency and mucopolysaccharidosis) (Eg, bovine viral diarrhea), mucinous colitis (eg, mucositis and mucosal colitis) and mucinous adherence (eg, cystic fibrosis, pancreatic cystic fibrosis, clark-headed field syndrome, pancreatic fibrosis ≪ / RTI > disease, mucinous adhesions and tackiness). In a highly preferred embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides, polypeptides and / or antagonists and / or agonists thereof are used in particular for the treatment, prevention and / or diagnosis of mucositis in combination with chemotherapy do.

In a preferred embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides, polypeptides and / or agonists and / or antagonists thereof are used for the treatment, prevention and / or prophylaxis of diseases or disorders or conditions associated with sinusitis It is used for diagnosis.

A further symptom, disease or condition which can be treated, prevented and / or diagnosed by the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotide, polypeptide and / or agonist and / or antagonist thereof is osteomyelitis.

A further symptom, disease or condition which can be treated, prevented and / or diagnosed by the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotide, the polypeptide and / or its agonists and / or antagonists is endocarditis .

All of the uses described above can also be applied to veterinary medicine.

Antagonists of neurotoxin-alpha include binding and / or inhibitory antibodies, antisense nucleic acids, ribozymes and neurotoxin-alpha polypeptides of the present invention. They are expected to reverse many of the activities of the ligands described above and to be used clinically or substantially in the following applications:

A means of blocking outpatient pathogens or various aspects of the immune response to themselves. Examples include autoimmune diseases such as lupus and arthritis, as well as immune reactions to skin allergies, inflammation, intestinal diseases, wounds and pathogens. Although our present data directly demonstrate the potential role of neurokinin-alpha in B-cells and mononuclear cells associated with pathology, the possibility that other cell types can acquire expression or response to neurotoxin-alpha Remains. Thus, neurotoxin-alpha can be regulated by the state of the immune system, such as CD40 and its ligand, and the microenvironment in which the cell is located.

Treatment for preventing B cell proliferation and Ig secretion associated with autoimmune diseases such as idiopathic thrombocytopenic purpura, systemic lupus erythematosus and MS.

Inhibitor of graft versus host disease or graft rejection.

B cell malignancies such as ALL, Hodgkin's disease, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, myeloma, multiple myeloma, Burkitt's lymphoma and EBV-transformed disease.

Therapy for chronic gammaglobulin and hyperammonemia in diseases such as non-measurable monoclonal gammopathy (MGUS), Waldenstrom disease, associated idiopathic monoclonal gammopathy and myeloma.

Therapy to reduce cellular proliferation of large B-cell lymphoma.

Means for reducing the involvement of B cells and Ig associated with chronic myelogenous leukemia.

Immunosuppressant (s).

The neurotoxin-alpha or neurotoxin-alpha SV polypeptides or polynucleotides or antagonists of the invention may be used to modulate IgE concentration in vitro or in vivo.

In another embodiment, the administration of the neurotoxin-alpha or neurotoxin-alpha SV polypeptides or polynucleotides or antagonists of the invention includes, but is not limited to, treatment of an IgE-mediated allergic response including asthma, rhinitis and eczema , Prevention and / or diagnosis.

Inhibitors of signal transduction pathways involving ERK1, COX2 and cyclin D2 associated with neurotoxin-alpha induced B cell activation.

Such applications are applicable to a variety of hosts. Such hosts include but are not limited to humans, mice, rabbits, goats, guinea pigs, camels, horses, mice, rats, hamsters, pigs, smile-pigs, chickens, goats, cows, sheep, Human primates and humans. In certain embodiments, the host is a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig, sheep, dog or cat. In a preferred embodiment, the host is a mammal. In a most preferred embodiment, the host is a human.

Agonists and antagonists can be used as a composition with a pharmaceutically acceptable carrier as described herein.

Antagonists can be used, for example, in macrophages and their precursors and neutrophils, basophils, B lymphocytes, and some T-cell subpopulations, such as activated and CD8 cytotoxic T cells, in certain autoimmune and chronic inflammatory and infectious diseases, Alpha-mediated and / or neurotoxin-alpha SV-mediated chemotaxis and activation of natural killer cells. Examples of autoimmune diseases include multiple sclerosis and insulin-dependent diabetes mellitus. Antagonists can also be used to treat, prevent and / or diagnose infectious diseases, including silicosis, sarcoidosis, idiopathic pulmonary fibrosis, by inhibiting the proliferation and activation of mononuclear cells. They can also be used to treat, prevent, and / or diagnose idiopathic eosinophilic leukocyte hyperreactivity by inhibiting eosinophilic leukocyte generation and migration. In addition, endotoxic shock can be treated by inhibiting migration of macrophages by antagonists and their production of the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides of the present invention. Antagonists can also be used to treat arteriosclerosis by inhibiting monocyte infiltration from the arterial wall. Antagonists can also be used to treat, prevent and / or prevent histamine-mediated allergic reactions and immunological diseases, including late allergic reactions, chronic urticaria and atopic dermatitis, by inhibiting chemokine-induced mast cell and basophilic degranulation and secretion of histamine / RTI > In addition, IgE-mediated allergic reactions such as allergic asthma, rhinitis and eczema can be treated. Antagonists can also be used to treat, prevent and / or diagnose chronic and acute inflammation by inhibiting mononuclear cells from being attracted to wound sites. They can also be used to control normal lung macrophages because chronic and acute inflammatory lung diseases are associated with sequestration of mononuclear cells in the lungs. Antagonists can also be used to treat, prevent and / or diagnose rheumatoid arthritis by inhibiting the mononuclear cells from being attracted into the synovial fluid of the patient's joints. Mononuclear cell inflow and activation play an important role in the development of degenerative and inflammatory arthropathy. Antagonists can be used to block toxic cascades that are predominantly caused by IL-1 and TNF, which inhibit the biosynthesis of other inflammatory cytokines. In this way, antagonists can be used to prevent inflammation. Antagonists can also be used to inhibit prostaglandin-independent heat induced by neurotoxin-alpha and / or neurotoxin-alpha SV. Antagonists can also be used to treat, prevent and / or diagnose patients with bone marrow abnormalities (e.g., aplastic anemia and myelodysplastic syndromes). Antagonists can also be used to treat, prevent and / or diagnose asthma and allergy by preventing eosinophil accumulation in the lungs. Antagonists can also be used to treat, prevent, and / or diagnose supraventricular basement membrane fibrosis, which is a major feature of asthmatic lungs. Antagonists can also be used to treat, prevent, and / or diagnose a lymphoma (including, but not limited to, many of the various lymphomas described herein).

All of the applications described above can be applied to veterinary medicine. In addition, all uses described herein can also be applied to veterinary medicine.

The neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptides and / or agonists and / or antagonists thereof of the invention are useful in the treatment of a variety of immune-related diseases and / Can be used to treat, prevent and / or diagnose the condition. Many autoimmune diseases are caused by improper recognition of themselves as foreign substances by immune cells. This inappropriateness of recognition leads to an immune response that leads to the destruction of the host tissue. Thus, the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptides and / or agonists and / or agonists thereof of the invention which are capable of inhibiting the immune response, in particular the proliferation of B cells and / or the production of immunoglobulins, Or antagonist may be an effective therapy for the treatment and / or prevention of autoimmune diseases. Thus, in a preferred embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV antagonists of the invention (e.g., the neurotrophin-alpha and / or neurotoxin-alpha SV and the polypeptide fragment of the anti- For the treatment, prevention and / or diagnosis of autoimmune diseases.

Autoimmune diseases that can be treated, prevented and / or diagnosed by the neurotoxin-alpha polynucleotides, polypeptides and / or antagonists of the present invention (e.g., anti-neurotoxin-alpha antibodies) But are not limited to, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia, autoimmune hemolytic disease, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, recurrent polychondritis, rheumatic heart disease , Autoimmune pneumonia, autoimmune pneumonitis, autoimmune pneumonitis, autoimmune pneumonia, autoimmune pneumonitis, autoimmune pneumonitis, autoimmune pneumonitis, autoimmune pneumonitis, autoimmune pneumonitis, autoimmune pneumonitis, glomerulonephritis (e.g., IgA nephropathy), multiple sclerosis, neuritis, uveitis conjunctivitis, Barr syndrome, insulin-dependent diabetes mellitus, and autoimmune inflammatory ocular disease.

Additional autoimmune diseases (high probability) that can be treated, prevented and / or diagnosed with the compositions of the invention include, but are not limited to, autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's thyroiditis (Often characterized as an anti-basal membrane antibody), pemphigus (often characterized as an epithelial dissociated, polar cell dissociative antibody), < RTI ID = 0.0 > , A receptor autoimmune (e.g., (a) Grave's disease (often characterized as a TSH receptor antibody), (b) myasthenia gravis (often characterized as an acetylcholine receptor antibody) and (c) insulin resistance (often characterized, for example, by the phagocytosis of antibody-screening RBCs), autoimmune thrombocytopenic purpura If antibody is shown as phagocytosis of sensitization platelets) are included.

Additional autoimmune diseases (high probability) that can be treated, prevented and / or diagnosed with the compositions of the present invention include, but are not limited to, rheumatoid arthritis (often characterized as an immune complex in the joints ), Scleroderma with an anti-collagen antibody (often characterized as a nuclear body and other nuclear antibodies), mixed connective tissue disease (often characterized as an extractable nuclear antigen, e.g. ribonucleoprotein) (Often characterized as non-histone ANA), malignant anemia (often characterized as anti-wall cells, microsomes and endogenous antibody antibodies), idiopathic Edison's disease Characterized by, for example, humoral and cell-mediated adrenocortical toxicity), infertility (often characterized as an anti-sperm antibody), glomerulonephritis (e.g., (Often characteristically as IgG and complement in the basement membrane), Sjogren ' s syndrome (often characterized as primary glomerulonephritis and IgA nephritis) (often characterized as a glomerular basement membrane antibody or immune complex) Such as multiple tissue antibodies and / or specific non-histone ANA (SS-B), diabetes (often characterized as cell-mediated and humoral islet cell antibodies), and adrenergic drug resistance Adrenergic drug resistance with asthma or cystic fibrosis) (often characterized by, for example, beta-adrenergic receptor receptor antibodies).

Additional autoimmune diseases (high probability) that can be treated, prevented and / or diagnosed with the compositions of the present invention include, but are not limited to, chronic active hepatitis (often characterized as a smooth muscle antibody), primary Biliary cirrhosis (often characterized as a mitochondrial antibody), endocrine disorders (often characterized by certain tissue antibodies in some cases), albumin (often characterized as a melanocyte antibody) , Post-MI (often characterized by, for example, myocardial antibody), cardiac incision syndrome (often characterized by, for example, cardiomyopathy), vasculitis (often characteristically as Ig in blood vessels and complement and / or low serum complement) (Often characterized as IgG and IgM antibodies to IgE), atopic dermatitis (often characterized as < RTI ID = 0.0 > For example IgG and IgM antibodies to IgE), asthma (often characterized as IgG and IgM antibodies to IgE), inflammatory myopathy and other inflammatory, granulomatous, degenerative and atrophic diseases .

In a preferred embodiment, autoimmune diseases and conditions and / or symptoms associated with these diseases and diseases are treated, prevented and / or diagnosed using anti-neutrophil-alpha antibodies and / or anti-neurotoxin-alpha SVs.

In a preferred embodiment, rheumatoid arthritis is treated, prevented and / or diagnosed using anti-neutrophil-alpha antibodies and / or anti-neutrophil-alpha SV antibodies and / or other antagonists of the invention.

In a preferred embodiment, lupus is treated, prevented and / or diagnosed using anti-neurotoxin-alpha antibodies and / or anti-neurotoxin-alpha SV antibodies and / or other antagonists of the invention.

In a preferred embodiment, nephritis associated with lupus is treated, prevented and / or diagnosed using an anti-neurotoxin-alpha antibody and / or an anti-neurotoxin-alpha SV antibody and / or other antagonists of the invention.

In certain embodiments, the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptides or antagonists thereof (e.g., anti-neurokinin-alpha and / or anti- And / or a disease, disorder or condition associated therewith. A lupine-related disease, disorder, or condition that can be treated or prevented with the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptides of the invention or antagonists thereof include, but are not limited to, (Eg, anti-DNA antibodies and anti-Sm antibodies), rashes, photosensitivity, oral ulcers, arthritis, heat, fatigue, weight loss, intestinal inflammation (eg, hemolytic anemia, leukopenia, lymphocytopenia and thrombocytopenia) (Eg, epididymitis), kidney disease (eg, nephritis), neurological diseases (eg, acute seizures, peripheral neuropathy, CNS related diseases), gastrointestinal diseases, Raynaud's phenomenon and pericarditis. In a preferred embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptides or antagonists thereof (e.g., anti-neurotoxin-alpha and / or anti-neurotoxin- alpha SV antibodies) ≪ / RTI > associated renal disease. In a most preferred embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptides or antagonists thereof (e.g., anti-neurotoxin-alpha and / or anti- It is used to treat or prevent nephritis associated with lupus.

Similarly, allergic reactions and symptoms such as asthma (especially allergic asthma) or other dyspepsia may also be caused by the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptides of the invention and / / ≪ / RTI > antagonist. In addition, these molecules can be used to treat, prevent, and / or diagnose hypersensitivity, hypersensitivity to an antigenic molecule, or blood group incompatibility.

The neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptides and / or agonists and / or antagonists thereof of the invention may also be used to treat organ rejection or graft-versus-host disease (GVHD) and / Can be used to treat, prevent and / or diagnose the condition. Tracheal rejection is caused by host immune cell destruction of the transplanted tissue through an immune response. Similarly, the immune response is also implicated in GVHD, but in this case the exogenously transplanted immune cells destroy host tissue. Administration of the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptides of the present invention and / or agonists and / or antagonists thereof, which inhibit the proliferation, differentiation or chemotaxis of the immune response, particularly T- It may be an effective therapy to prevent organ rejection or GVHD.

Similarly, the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptides of the invention and / or agonists and / or antagonists thereof may also be used to control inflammation. For example, the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptides of the invention and / or agonists and / or antagonists thereof may inhibit proliferation and differentiation of cells involved in an inflammatory response. These molecules include, but are not limited to, chronic prostatitis, granulomatous prostatitis and malacoplakia, inflammation associated with infection (e.g., septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxic leprosy, (E.g. TNF or IL-1), including inflammatory bowel disease, complement-dependent superficial rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, Prevention and / or diagnosis of chronic and acute inflammatory conditions due to overproduction.

In certain embodiments, the anti-neurotoxin-alpha antibodies and / or anti-neurotoxin-alpha SV antibodies of the invention are used to treat, prevent, modulate, detect and / or diagnose inflammation.

In certain embodiments, the anti-neurotoxin-alpha antibodies and / or anti-neurotoxin-alpha SV antibodies of the invention are used for the treatment, prevention, modulation, detection and / or diagnosis of inflammatory diseases.

In another specific embodiment, the anti-neurotoxin-alpha antibodies and / or anti-neurotoxin-alpha SV antibodies of the invention are used for the treatment, prevention, modulation, detection and / or diagnosis of allergic and / or hypersensitive reactions.

Antibodies against neurotoxin-alpha and / or noclucin-alpha SV bind to and inhibit the activity of neurotoxin-alpha and / or noclucin-alpha SV and prevent neutrophil infiltration into the post- To treat, prevent and / or diagnose ARDS. The agonists and antagonists of the present invention may be used, for example, as a composition with a pharmaceutically acceptable carrier as described below.

The neurotoxin-alpha and / or neurotoxin-alpha SV and / or neurotoxin-alpha receptor polynucleotides or polypeptides and / or agonists and / or antagonists thereof of the present invention may be useful in the treatment of diseases and diseases of the lung system, Prevention and / or diagnosis of symptoms associated with such diseases and diseases and other respiratory diseases and diseases. In certain embodiments, such diseases and disorders include but are not limited to bronchial adenomas, bronchial asthma, pneumonia (e.g., bronchopneumonia, bronchitis, tuberculous bronchopneumonia), chronic obstructive pulmonary disease (COPD), bronchial polyps Bronchial hyperplasia, bronchial hyperplasia (eg, exudative bronchial hyperalgesia, fibrous obstructive bronchial hyperalgesia and proliferative bronchial hyperalgesia), bronchial hypercellular carcinoma, bronchial hyperplasia, bronchial hyperplasia, Bronchial asthma, bronchitis (e.g., asthmatic bronchitis, Castellani bronchitis, chronic bronchitis, Crohn's bronchitis, fibrosing bronchitis, hemorrhagic bronchitis, avian infectious bronchitis, obstructive bronchitis, gastritis bronchitis, pseudomembranous bronchitis, Bronchogenic carcinomas, bronchogenic carcinomas, bronchial edema, bronchogenic carcinomas, bronchogenic cysts, Bronchial asthma, bronchial asthma (eg bronchial aspergillosis), bronchial spirometry, hemorrhagic bronchitis, bronchospasm, bronchial wall hemorrhage, bronchial stenosis, biotreatment, bronchial respiration, Respiratory acidosis, respiratory alkalosis, neonatal respiratory distress syndrome, respiratory insufficiency, respiratory system sclerosis, respiratory syncytial virus, and the like.

In certain embodiments, the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptides and / or agonists and / or antagonists thereof of the invention are useful for the treatment, prevention and / or diagnosis of chronic obstructive pulmonary disease (COPD) .

In a particular embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptides of the invention and / or agonists and / or antagonists thereof are used for the treatment, prevention and / or diagnosis of fibrosis and related symptoms Examples of fibrosis include, but are not limited to, cystic fibrosis (including cystic fibrosis of the pancreas, clock-headed field syndrome, fibrocystic disease of the pancreas, mucinous adhesions and adhesions), endocardial fibrosis, Peritoneal fibrosis, aortic fibrosis, mediastinal fibrosis, subthalamic subepithelial fibrosis, peripheral fibrosis, myofascial fibrosis, pipe stem fibrosis, restorative fibrosis, subcutaneous fibrosis, and simmus clay pipstem fibrosis.

TNF family ligands are known to be one of the most multifunctional expression cytokines including many cellular responses (including cytotoxicity, antiviral activity, immunoregulatory activity and transcriptional regulation of several genes) (DV Goeddel et al., &Quot; Tumor Necorsis Factors: Gene Structure and Biological Activities. "Symp. Quant. Biol. 51: 587-609 (1986). Cold Spring Harbor: B. Beutler and A. Cerami. Annu. Rev. Biochem. WJ Fiers, FEBS Lett., 285: 199-224 (1991)). TNF family ligands, including the neurotoxin-alpha and / or neurotoxin-alpha SV of the present invention, induce the various cellular responses described above by binding to TNF family receptors. Neutrokine-alpha and / or neurotoxin-alpha SV polypeptides are believed to elicit potent cellular responses involving all genetic, phenotypic and / or morphological changes to cells, cell lines, tissues, tissue cultures or patients Loses. As mentioned, such cell-cell responses include diseases associated with normal physiological responses to TNF family ligands as well as increased inhibition of apoptosis or apoptosis. Apoptosis-programmed cell death is a physiological mechanism involved in the absence of peripheral B and / or T lymphocytes of the immune system, and abnormal control thereof can induce many other pathogenic processes (JC Ameisen, AIDS 8: 1197-1213 (1994) ; PH Krammer et al., Curr. Opin. Immunol. 6: 279-289 (1994)).

Diseases associated with increased cell survival or inhibition of apoptosis that can be diagnosed, treated or prevented by the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptides of the invention and / or their agonists and / (Including, but not limited to, colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, adenocarcinoma, testicular cancer, pancreatic cancer, (Including, for example, gastric cancer, neuroblastoma, myxoma, myoma, lymphoma, epithelioma, osteoblast, osteoclast, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi sarcoma and ovarian cancer) Viral infections (e.g., herpes viruses, poxviruses and adenoviruses), inflammation, graft versus host disease Ring, include acute transplant rejection and chronic graft're added. Thus, in a preferred embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptides of the invention and / or agonists and / or antagonists thereof are used for the treatment, prevention and / or diagnosis of autoimmune diseases (Including, for example, MLL and chronic lymphocytic leukemia (CLL)) and cancers including, but not limited to, various cancers as described herein, including, but not limited to, Power generation and / or transition. In another embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptides of the invention are used to treat cancer cells or tissues, including B-cell lineage associated cancers (e.g., CLL and MLL), lymphocytic leukemia or lymphoma, Differentiation, or proliferation, thereby making the cell more susceptible to cancer therapies (e.g., chemotherapy or radiation therapy).

In addition, in another embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptides and / or agonists and / or antagonists thereof of the invention are useful for the growth, development and / Leukemia (including acute leukemia, such as acute lymphocytic leukemia, acute myelocytic leukemia (myeloblastic leukemia, promyelocytic leukemia, myeloid leukemia, monocytic leukemia and leukemia, Including chronic myeloid leukemia and chronic lymphocytic leukemia), intrinsic erythropoiesis, lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, (Including, but not limited to, fulminant tumors including sarcoma and carcinoma (e.g., fibrosarcoma, mucinous sarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, Epithelial sarcoma, lymphangiosarcoma, lymphoepithelial sarcoma, synovial tumor, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma Pancreatic carcinoma, choriocarcinoma, choriocarcinoma, choriocarcinoma, embryonic sarcoma, Wilm's tumor, uterine carcinoma, testicular tumor, papillary carcinoma, papillary carcinoma, papillary carcinoma, papillary carcinoma, papillary carcinoma, cystic carcinoma, spinal cord carcinoma, bronchogenic carcinoma, The present invention relates to a method for treating a cancer selected from the group consisting of lung cancer, non-small cell lung cancer, bladder cancer, epithelioma, glioma, astrocytoma, hematoblastoma, craniopharyngioma, ependymoma, pineal gland, angioblastoma, .

Diseases associated with increased apoptosis that can be diagnosed, treated or prevented with the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptides of the invention and their agonists and antagonists include AIDS, neurodegenerative diseases , Myocardial infarction syndrome (eg, aplastic anemia), ischemic injury (eg, myocardial infarction, wound caused by myocardial infarction, stroke and reperfusion injury), myocardial infarction Toxin-induced liver disease (e. G., Diseases caused by alcohol), septic shock, cachexia, and loss of appetite. Thus, in a preferred embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotides or polypeptides of the invention and / or agonists and / or antagonists thereof are used for the treatment, diagnosis and / Used to prevent.

In a preferred embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides and / or agonists and / or antagonists thereof (e.g., anti-neurotoxin-alpha antibodies) of the invention bind to the human histiocytic lymphoma U-937 Inhibit the growth of cells in a dose-dependent manner. In a further preferred embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides and / or agonists and / or antagonists thereof (e.g., anti-neurotoxin-alpha antibodies) HT-29 cells, HeLa cells, MCF-7 cells and A293 cells. In a highly preferred embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides and / or agonists and / or antagonists thereof (e.g., anti-neurotoxin-alpha antibodies) of the present invention are useful in the treatment of prostate cancer, colon cancer, Development and / or metastasis of the species, breast, and breast cancers.

Thus, in a further preferred embodiment, the present invention provides a method of treating a neurotoxin-alpha and / or neurotoxin-alpha SV receptor, comprising administering to a cell expressing a neurotoxin-alpha and / or neurotoxin- Lt; RTI ID = 0.0 > TNF < / RTI > family ligand, including the administration of an agonist or antagonist thereof capable of increasing or decreasing neurotoxin-alpha SV mediated signal transduction. Prevention and / or diagnosis of a disease in which increased or decreased neurotoxin-alpha and / or neurotoxin-alpha SV mediated signal transduction is manifested by decreased apoptosis or reduced cytokine and adsorptive molecule expression . Agonists or antagonists may include monoclonal antibodies to soluble forms of neurotoxin-alpha and / or neurotoxin-alpha SV and neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides.

In a further aspect, the invention provides a method of treating a neurotoxin-alpha and / or neurotoxin-alpha SV receptor comprising administering to a cell expressing a neurotoxin-alpha and / or neurotoxin-alpha SV receptor an effective amount of neurotoxin-alpha and / Alpha < / RTI > SV mediated signal transduction, comprising administering an agonist or antagonist capable of increasing or decreasing the alpha SV mediated signal transmission. Prevention and / or diagnosis of diseases in which elevated apoptosis or NF-kappa B expression occurs, preferably by increasing or decreasing neurotoxin-alpha and / or neurotoxin-alpha SV mediated signal transduction. Agonists or antagonists may include monoclonal antibodies to soluble forms of neurotoxin-alpha and / or neurotoxin-alpha SV and neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides.

Because neurotoxin-alpha and / or neurotoxin-alpha SV belong to the TNF superfamily, polypeptides also have to regulate angiogenesis. In addition, because neurotoxin-alpha and / or neurotoxin-alpha SV inhibits immune cell function, polypeptides have a broad spectrum of anti-inflammatory activity. Neutrokine-alpha and / or neurotoxin-alpha SV can be used as neovascularization inhibitors to stimulate penetration and activation of host defense cells (e.g., cytotoxic T cells and macrophages) and inhibit tumor angiogenesis, , ≪ / RTI > can be treated, prevented and / or diagnosed. Those skilled in the art will recognize other non-cancerous conditions that do not require angiogenesis. They can also be used to enhance host defense against resistant chronic and acute infections (eg, myobacterial infections) through the induction and activation of microbial-killing white blood cells. In addition, neurotoxin-alpha and / or neurotoxin-alpha SV have been shown to inhibit IL-2 biosynthesis for the treatment of T-cell mediated autoimmune bilirubin and lymphoma (e.g., chronic lymphoid lymphoma (CLL) Can be used to inhibit T-cell proliferation. Neutrokine-alpha and / or neurotoxin-alpha SVs can also be used to promote wound healing through both connective tissue promoting inflammatory cells and recovery of debris sweeping. In the same manner, neurotoxin-alpha and / or neurotoxin-alpha SV can also be used to treat, prevent and / or diagnose other fibrosing diseases including cirrhosis, osteoarthritis and pulmonary fibrosis. Also, neurotoxin-alpha and / or neurotoxin-alpha SVs increase the presence of eosinophils with a unique function that kill larval parasites that infiltrate tissue, such as schistosomiasis, tachypnea and amygdala. In addition, neurotoxin-alpha and / or neurotoxin-alpha SV regulates the activation and differentiation of several hematopoietic progenitor cells, for example, by regulating mature leukocytes from bone marrow after chemotherapy, . Neutrokine-alpha and / or neurotoxin-alpha SV can also be used to treat, prevent and / or diagnose sepsis.

The polynucleotides and / or polypeptides of the present invention and / or agonists and / or antagonists thereof are useful for diagnosing and treating or preventing a wide range of diseases and / or conditions. Such diseases and conditions include, but are not limited to, cancer (e.g., cancer related to immune cells, breast cancer, prostate cancer, ovarian cancer, follicular lymphoma, cancer associated with mutation or deformation of p53, brain cancer, bladder cancer, cervical cancer, (Eg, HIV-1 infection, HIV-1 infection, etc.), lymphocytic leukemia (eg, lymphadenitis), colon cancer, colon cancer, non-small cell carcinoma of the lung, small cell carcinoma of the lung, gastric cancer, 2 infection, herpes virus infection (including but not limited to HSV-1, HSV-2, CMV, VZV, HHV-6, HHV-7, EBV), adenovirus infection, (Eg, HAV, HBV, HCV, etc.), Helicobacter pylori infection, infiltrating staphylococcus, etc.), parasitic infections, nephritis, bone diseases (eg, osteoporosis), arteriosclerosis, pain, cardiovascular disease Angiogenesis, or decreased circulation (e.g., ischemic diseases (Acute and chronic), graft-versus-host disease (AIDS), inflammation, neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration) (Eg, acute and chronic hepatitis, liver damage and cirrhosis), autoimmune diseases (eg, multiple sclerosis, rheumatoid arthritis), diseases caused by bone marrow disorders (eg, aplastic anemia) Diabetic complications such as diabetic nephropathy (eg, arthritis, systemic lupus erythematosus, immunocompromised glomerulonephritis, autoimmune diabetes mellitus, autoimmune thrombocytopenic purpura, Grave's disease, , Diabetic neuropathy, diabetic retinopathy), influenza, asthma, psoriasis, glomerulonephritis, sepsis shock and ulcerative colitis.

The polynucleotides and / or polypeptides and / or agonists and / or antagonists thereof of the present invention are useful for promoting angiogenesis, wound healing (e.g., wounds, burns and fractures). The polynucleotides and / or polypeptides of the present invention and / or agonists and / or antagonists thereof are also useful as adjuvants to enhance immune responses, antiviral immune responses to particular antigens.

More generally, the polynucleotides and / or polypeptides of the invention and / or agonists and / or antagonists thereof are useful for modulating (i.e., increasing or decreasing) the immune response. For example, the polynucleotides and / or polypeptides of the present invention may be useful for preparing or restoring from surgery, trauma, radiation therapy, chemotherapy and transplantation, or for promoting immune response and / or recovery in aged and immunocompromised persons Can be used. Alternatively, the polynucleotides and / or polypeptides of the present invention and / or agonists and / or antagonists thereof are useful as immunosuppressants, for example, for the treatment or prevention of autoimmune diseases. In certain embodiments, polynucleotides and / or polypeptides of the invention are used to treat or prevent chronic inflammatory, allergic or autoimmune conditions as described herein or as known in the art.

Preferably, an agonist or antagonist of a neurotoxin-alpha and / or neurotoxin-alpha SV polynucleotide or polypeptide and / or neurotoxin-alpha and / or neurotoxin-alpha SV, Antibody) can be administered to a patient by administering to the patient an effective amount of a neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide or agonist or antagonist of the invention, or by removing cells from the patient and administering the neurotoxin-alpha and / Or neurotoxin-alpha SV polynucleotide, and re-feeding the engineered cells to the patient (in-vitro treatment). In addition, as described herein, neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides or polynucleotides can be used as adjuvants in vaccines to induce an immune response against infectious diseases.

Formulation and administration

A neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide composition (preferably containing a polypeptide that is a soluble form of the neurotoxin-alpha and / or neurotoxin-alpha SV extracellular domain) Alpha, and / or neurotoxin-alpha SV polypeptide polypeptides), a delivery site of a neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide composition, a method of administration , The schedule of administration, and other factors known to the physician, in a manner consistent with good medical practice. Taking these into account, therefore, an " effective amount " of a neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide is determined for the purposes of the present application.

As a general proposition, the total pharmacologically effective amount of the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide administered parenterally per dose may vary from about 1 microgram per kilogram of patient body weight per day 10 mg / kg / day. A more preferred dose is 0.01 mg / kg / day or more, and most preferably about 0.01 to 1 mg / kg / day for a human.

In another embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides of the invention are administered to a human in a dose of 0.0001 to 0.045 mg / kg / day, preferably 0.0045 to 0.045 mg / kg / day Dose, more preferably about 45 micrograms / kg / day, and in the case of mice, about 3 mg / kg / day.

When administered sequentially, the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides are typically injected 1 to 4 times per day at a dose of about 1 microgram / kg / hour to about 50 microgram / kg / For example, subcutaneously injected continuously using a mini-pump. An intravenous cystic solution may also be used.

The duration of treatment required to observe the change and the interval at which the post-treatment response occurs varies depending on the desired effect.

In certain embodiments, the total pharmacologically effective amount of parenterally administered neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide per dose may vary from about 0.1 microgram per kilogram body weight per day 45 mg / kg / day. A more preferred dose is 0.1 mg / kg / day or more, and most preferably about 0.01 to 50 microgram / kg / day. The neurotoxin-alpha and / or neurotoxin-alpha SV may be administered by continuous infusion, multiple daily injections (eg, three or more times daily, or twice daily), one daily injection or a divided injection at regular intervals , Once daily, every other day, twice a week, once a week, once every two weeks, once a month, once every two months, once every quarter. When continuously injected, the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides are typically administered at a dose of about 0.001 microgram / kg / hr to about 10 microgram / kg / hr to about 50 microgram / kg / hr Dose by one to four injections per day, or by continuous subcutaneous injection using, for example, a mini-pump.

The effective dose of a composition according to the present invention to be administered can be determined by methods well known to those skilled in the art, taking into account variables such as biological half life, bioavailability and toxicity. Such measurements fall within the skill of those of ordinary skill in the art in view of the detailed description provided herein.

During treatment, neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides may also play an important role in determining therapeutically and / or pharmacologically effective dosing regimens. A change in dosage, such as repeated dosing of relatively low doses of the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides over a relatively long period of time, results in a relatively high dose of neurotoxin-alpha and / or neurotoxin-alpha (For example, the serum immunoglobulin level test described in Example 6). In addition, the present invention is not limited to the above-described embodiments.

Freireich, E.J. alpha] and / or neurotoxin-alpha < / RTI > in a given experimental system, using an equivalent surface area capacity conversion factor as described in U. S. et al. (Cancer Chemotheraphy Reports 50 (4): 219-44 The data obtained using SV can be conveniently converted to a precise estimate of the pharmacologically effective amount of the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide to be administered per dose in different experimental systems. Experimental data obtained through the administration of neurotoxin-alpha in mice (reference example: Example 6) was compared with the neurotoxin-alpha levels in rats, monkeys, dogs and humans via the conversion factors provided by Freireich et al. Lt; RTI ID = 0.0 > effective < / RTI > The following conversion table (Table 3) summarizes the data provided by FreyReach. Table 3 provides an appropriate factor to convert the indicated dose in mg / kg from one species to an equivalent surface area dose in mg / kg from another species.

Equivalent surface area capacity conversion factor. The mouse (20 g) Rats (159 g) Monkey (3.kg) Dog (8kg) People (60kg) mouse One 1/2 1/4 1/6 1/12 Rat 2 One 1/2 1/4 1/7 monkey 4 2 One 3/5 1/3 dog 6 4 5/3 One 1/2 Person 12 7 3 2 One

Thus, for example, when using the conversion factor provided in Table 3, the dose of 50 mg / kg in mice was 1.5 mg / kg in monkeys (50 mg / kg) x (1/4) = 12.5 mg / Lt; / RTI > As a further example, doses of 0.02, 0.08, 0.8, 2 and 8 mg / kg in mice were 1.667 micrograms / kg, 6.67 micrograms / kg, 66.7 micrograms / kg, 166.7 micrograms / kg and 0.667 microgram / The same effect can be obtained with a microgram / kg dose.

The pharmaceutical compositions containing the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides of the present invention can be administered orally, rectally, parenterally, subcutaneously, intracisternally, intravaginally, intraperitoneally, topically (including powders, Or by oral or oral or nasal spray (e.g., via aspiration of a vapor or powder). In one embodiment, " pharmaceutically acceptable carrier " means any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating agent or formulation aid. In certain embodiments, " pharmaceutically acceptable " means that it may be used in animals, particularly humans, and has been approved by a federal or state government agency or contained in the United States Pharmacopoeia or other generally approved pharmacopoeia. Non-limiting examples of suitable pharmaceutical carriers according to the embodiments are described in EW Martin, " Remington ' s Pharmaceutical Sciences ", and includes aseptic fluids such as vegetable, synthetic or animal oils (including petroleum) and water, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is the preferred carrier when the pharmaceutical composition is administered intravenously. Saline and aqueous dextrose and glycerol solutions can also be used as liquid carriers, especially for injection solutions. The composition may contain minor amounts of wetting or emulsifying agents or pH buffering if desired. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like.

The term " parenteral " as used herein refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

In a preferred embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV compositions of the invention (including polypeptides, polynucleotides and antibodies and agonists and / or antagonists) are administered subcutaneously.

In another preferred embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV compositions of the invention (including polypeptides, polynucleotides and antibodies and agonists and / or antagonists) are administered intravenously.

The neurotoxin-alpha and / or neurotoxin-alpha SV compositions of the invention are also suitably administered by a release system. Suitable examples of the sustained-release composition include suitable polymeric materials (e.g., semi-permeable polymeric matrices shaped like films or microcapsules), suitable hydrophobic materials (such as emulsions in acceptable oils) or ion exchange resins and slightly soluble derivatives (E.g., weakly soluble salts).

(US Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman, U. et al., Biopolymers 22: 547-556 12 (1983)), poly (2-hydroxyethyl methacrylate) (R. Langer et al., J. Biomed. Mater. Res. 15: 167-277 (1981) and R. Langer, 98-105 (1982)), ethylene vinyl acetate (R. Langer et al., Id.) Or poly-D - (-) - 3-hydroxybutyric acid (EP 133,988).

(Langer, Science 249: 1527-1533 (1990); Treat et al., In Liposomes in the Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 317-327 and 353-365 (1989)). Liposomes containing neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides can be prepared by per se known methods: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. (USA) 77: 4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Patent Application No. 83-118008; U.S. Patent Nos. 4,485,045 and 4,544,545 and EP 102,324. Typically, the liposomes are small (about 200-800 angstroms) thin films with a lipid content of greater than about 30 mole% cholesterol and selected ratios can be adjusted for optimal neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide therapy .

In another embodiment, the fatty composition of the present invention comprises crystalline formulations known in the art.

In another further embodiment, the compositions of the present invention are delivered via a pump (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14: 201 (1987); Buchwald et al., Surgery 88: 507 (1980); Saudek et al., N. Engl. J. Med., 321: 574 (1989)).

Other controlled release systems are described in Langer, Science 249: 1527-1533 (1990).

In the case of parenteral administration, in one embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides will generally comprise a polypeptide of the desired purity in a pharmaceutically acceptable carrier, i.e., (Liquid, suspension or emulsion) with a carrier which is non-toxic and compatible with the other ingredients of the formulation and is formulated. For example, the formulations preferably do not contain oxidants and other compounds known to be detrimental to the polypeptide.

Generally, formulations are prepared by uniformly and intimately admixing neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides with a liquid carrier or a finely divided solid carrier, or both. The product is then formed into the desired formulation, if desired. Preferably, the carrier is a parenteral carrier, more preferably a solution which is isotonic with the patient ' s blood. Examples of such carrier vehicles include water, saline, Ringgel solutions and dextrose solutions. Non-aqueous vehicles such as non-volatile oils and ethyl oleate are also useful herein, as well as liposomes.

The carrier suitably contains small amounts of adjuvants, such as substances which enhance isotonicity and chemical stability. Such materials are non-toxic to the patient at the dosages and concentrations employed and include buffers such as phosphate, citrate, succinate, acetic acid and other organic acids or salts thereof; Antioxidants such as ascorbic acid; Low molecular weight (less than about 10 residues) polypeptides (e.g., polyarginine or tripeptides); Proteins (e.g., serum albumin, gelatin or immunoglobulins); Hydrophilic polymers (e.g., polyvinylpyrrolidone); Amino acids (glycine, glutamic acid, aspartic acid or arginine); Monosaccharides, disaccharides and other carbohydrates including cellulose or its derivatives, glucose, mannose, sucrose or dextrin; Chelating agents such as EDTA; Sugar alcohols such as mannitol or sorbitol; County ions such as sodium; Preservatives such as cresol, phenol, chlorobutanol, benzyl alcohol and parabens; And / or nonionic surfactants such as polysorbate, poloxamer or PEG.

The neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides typically have a pH of about 3 to 10 or a pH of 3 to 8, more preferably a pH of 5 to 8, and most preferably 6 to 7 At a concentration of about 0.001 mg / ml to 100 mg / ml or 0.1 mg / ml to 100 mg / ml, preferably 1 to 10 mg / ml or 1 to 10 mg / ml, It will be appreciated that the use of certain of the excipients, carriers or stabilizers will result in the formation of a neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide polypeptide.

The neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides to be used for therapeutic administration should be sterile. Sterilization is easily accomplished by filtration through a sterile filter membrane (e.g., a 0.2 micron membrane). Therapeutic neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide compositions are typically placed in vials having a sterile entry port, for example, an intravenous solution sachet, or a plug having a penetrable cap with a hypodermic syringe.

The neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides are usually stored as either an aqueous solution in a single or multiple volume container, such as a sealed ampoule or vial, or as a lyophilisate for regeneration. As an example of a lyophilizate, a 10-ml vial is prepared by filling a 5 ml sterile-filtered 1% (w / v) aqueous Neutrokine-Alpha and / or Neutrokine-Alpha SV polypeptide solution and freezing the resulting mixture . The infusion solution is prepared by restoring lyophilized neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides with bacterial infusion water.

Alternatively, the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides are stored in a single dose container in lyophilized form. The infusion solution is returned to the sterile carrier for injection.

The invention also provides a pharmaceutical pack or kit comprising at least one container filled with at least one of the components of the pharmaceutical composition according to the invention. Optionally, the container may be adhered to a bill stipulated by a government agency that controls the manufacture, use, or sale of a drug or biological product, and this notice may be subject to government approval for manufacture, use, or sale for human administration. Reflect. In addition, the polypeptide of the present invention can be used in combination with other therapeutic compounds.

The composition of the present invention may be administered alone or in combination with other adjuvants. Adjuvants that may be administered with the compositions of the present invention include but are not limited to alum, alum and deoxycholate (ImmunoAg), MTP-PE (Biocine Corp), QS21 (Genentech, Inc.), BCG MPL is included. In certain embodiments, the composition of the invention is administered in admixture with QS-21. Other adjuvants that may be administered with the compositions of the present invention include but are not limited to monophosphoryl lipid immunomodulators AdjuVax 100a, QS-21, QS-18, CRL1005, aluminum salts, MF- Technology. Vaccines that may be administered with the compositions of the present invention include but are not limited to MMR (measles, mumps, rubella), poliomyelitis, chickenpox, tetanus / diphtheria, hepatitis A, hepatitis B, Haemophilus influenzae type B , Pneumonia, influenza, Lyme disease, rotavirus, cholera, yellow fever, Japanese encephalitis, asthma, rabies, typhoid fever and pertussis and / or PNEUMOVAX-23 ^TM . The formulations may be administered simultaneously (e. G. As a mixture), separately but simultaneously or sequentially or sequentially. This includes the manner in which the formulated agents are administered together as a therapeutic mixture and also the method of administering the formulated agents separately but simultaneously (e.g., via a separate intravenous line to the same person). Administration of the " combined " condition further comprises administration of one of the compounds or agents first and administration of the second one separately.

In another specific embodiment, the compositions of the invention are used in combination with PNEUMOVAX-23 ^TM to treat, prevent and / or diagnose an infection and / or any disease, disorder, and / or symptom associated therewith. In one embodiment, the compositions of the present invention are used in combination with PNEUMOVAX-23 ^TM to treat, prevent and / or diagnose Gram-positive bacterial infections and / or any diseases, diseases and / or symptoms associated therewith. In another embodiment, the compositions of the present invention are used in combination with PNEUMOVAX-23 ^TM to treat, prevent, and / or prevent infection and / or any disease, disease, and / or symptom associated with one or more bacterial members of the genus Enterococcus and / or Streptococcus And / or diagnose. In another embodiment, the compositions of the present invention are used in combination with PNEUMOVAX-23 ^TM to treat, prevent and / or diagnose an infection associated with one or more bacterial members of the B group streptococcus and / or any disease, disorder and / do. In another embodiment, the compositions of the invention are used in combination with PNEUMOVAX-23 ^TM to treat, prevent and / or diagnose an infection associated with Streptococcus pneumoniae and / or any disease, disorder, and / or symptom.

The composition of the present invention may be administered alone or in combination with other therapeutic agents. Therapeutic agents that may be administered with the compositions of the present invention include, but are not limited to, chemotherapeutic agents, antibiotics, antiviral agents, steroidal and nonsteroidal antiinflammatory agents, conventional immunotherapeutic agents, and cytokines. The formulations may be administered simultaneously (e. G. As a mixture), separately but simultaneously or sequentially or sequentially. This includes the manner in which the formulated agents are administered together as a therapeutic mixture and also the method of administering the formulated agents separately but simultaneously (e.g., via a separate intravenous line to a human). Administration of the " combined " condition further comprises administering one of the compounds or agents first and the second one separately.

In one embodiment, the compositions of the invention are administered in combination with other members of the TNF family. TNF, TNF-related or TNF-like molecules that may be administered with the compositions of the present invention include, but are not limited to, TNF-alpha, lymphotoxin-alpha (also known as LT-alpha, also TNF- , LT-beta (found in complex heterotrimer LT-alpha 2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (International Patent Publication No. WO 96/14328 AIM-I (International Patent Publication No. WO 97/33899), AIM-II (International Patent Publication No. WO 97/34911), APRIL (J. Exp. Med. 188 (6): 1185- 1190), endocaine-alpha (International Patent Publication No. WO 98/07880), OPG and Neutrokine-alpha (International Patent Publication No. WO 98/18921), OX40 and nerve growth factor (NGF) Soluble forms of CD30, CD27, CD40 and 4-IBB, TR2 (International Patent Publication No. WO 96/34095), DR3 (International Patent Publication No. WO 97/33904), DR4 (International Patent Publication No. WO 98 / 32856), TR5 (International Patent Publication No. WO 98/30693), TR6 ( TR9 (International Patent Publication No. WO 98/56892), TR10 (International Patent Publication No. WO 98/30694), TR7 (International Patent Publication No. WO 98/41629), TRANK, 98/54202), 312C2 (International Patent Publication No. WO 98/06842) and TR12.

In a preferred embodiment, the compositions of the invention comprise a CD40 ligand (CD40L), a soluble form of CD40L (e.g. AVREND ^TM ), a biologically active fragment, variant or derivative of CD40L, an anti-CD40L antibody (e.g. an efficacious antibody or antagonist ) And / or an anti-CD40 antibody (e.g., an efficacious antibody or an antagonistic antibody).

In certain embodiments, the compositions of the invention are administered in combination with an anti-retroviral agent, a nucleoside reverse transcriptase inhibitor, a non-nucleoside reverse transcriptase inhibitor, and / or a protease inhibitor. Nucleoside reverse transcriptase inhibitors that may be administered in combination with the compositions of the present invention include but are not limited to RETROVIR ^™ (Zidovudine / AZT), VIDEX ^™ (didanosine / ddI), HIVID ^™ (zalcitabine / ddC) , ZERIT ^TM (stavudine / d4T), EPIVIR ^TM (lamivudine / 3TC) and COMBIVIR ^TM (zidovudine / lamivudine). A binyu nucleoside reverse transcriptase inhibitors that may be administered in a composition of the invention and the formulation include, but are limited to, but include VIRAMUNE ^TM (nevirapine), RESCRIPTOR ^TM (delavirdine), and SUSTIVA ^TM (Fabien renjjeu a) . Protease inhibitors that may be administered in combination with the compositions of the present invention include, but are not limited to, CRIXIVAN ^™ (indinavir), NORVIR ^™ (ritonavir), INVIRASE ^™ (saquinavir), and VIRACEPT ^™ Birr). In certain embodiments, an anti-retroviral agent, a nucleoside reverse transcriptase inhibitor, a non-nucleoside reverse transcriptase inhibitor and / or a protease inhibitor is combined with a composition of the present invention to treat, prevent and / or diagnose AIDS and / Treatment, prevention and / or diagnosis.

In another embodiment, the compositions of the present invention may be administered in combination with an anti-opportunistic infectious agent. Anti-opportunistic agents that may be administered in combination with the compositions of the present invention include but are not limited to TRIMETHOPRIM-SULFAMETHOXAZOLE ^™ , DAPSONE ^™ , PENTAMIDINE ^™ , ATOVAQUONE ^™ , ISONIAZID ^™ , RIFAMPIN ^™ , PYRAZINAMIDE ^™ , ETHAMBUTOL ^™ , ^{rIFABUTIN TM, CLARITHROMYCIN TM, AZITHROMYCIN TM} , GANCICLOVIR TM, fOSCARNET TM, CIDOFOVIR TM, fLUCONAZOLE TM, iTRACONAZOLE TM, KETOCONAZOLE TM, aCYCLOVIR TM, FAMCICOLVIR TM, PYRIMETHAMINE TM, lEUCOVORIN TM, NEUPOGEN TM ( Phil Grass team / G-CSF) LEUKINE (Sarg Ramos Team / GM-CSF). In certain embodiments, the compositions of this invention are used in combination with TRIMETHOPRIM-SULFAMETHOXAZOLE ( ^TM) , DAPSONE ( ^TM) , PENTAMIDINE ( ^TM) and / or ATOVAQUONE ( ^TM) to prophylactically treat, prevent and / or diagnose opportunistic Newstomy S. pneumoniae. In another specific embodiment, the compositions of the present invention are used in combination with ISONIAZID ^TM , RIFAMPIN ^TM , PYRAZINAMIDE ^TM and / or ETHAMBUTOL ^TM for prophylactic treatment, prevention and / or diagnosis of opportunistic Mycobacterium avium complex infection do. In another specific embodiment, the compositions of the present invention are used in combination with RIFABUTIN ^TM , CLARITHROMYCIN ^TM and / or AZITHROMYCIN ^TM to prophylactically treat, prevent and / or diagnose opportunistic M. tuberculosis infection. In another specific embodiment, the compositions of the present invention are used in combination with GANCICLOVIR ( ^TM) , FOSCARNET ( ^TM) and / or CIDOFOVIR ( ^TM) to prophylactically treat, prevent and / or diagnose opportunistic cytomegalovirus infection. In another specific embodiment, compositions of the present invention is used to treat, prevent and / or ^TM FLUCONAZOLE, ITRACONAZOLE ^TM and / or KETOCONAZOLE ^TM and formulated to diagnose opportunistic fungal infection prophylactically. In another specific embodiment, the compositions of the present invention are used in combination with ACYCLOVIR ^™ and / or FAMCICOLVIR ^™ for prophylactically treating, preventing and / or diagnosing opportunistic herpes simplex virus type I and type II infections. In another specific embodiment, the compositions of the present invention are used in combination with PYRIMETHAMINE ( ^TM) and / or LEUCOVORIN ( ^TM) to prophylactically treat, prevent and / or diagnose opportunistic Toxoplasma gondii infection. In another specific embodiment, the compositions of the present invention are used in combination with LEUCOVORIN ^™ and / or NEUPOGEN ^™ to prophylactically treat, prevent and / or diagnose opportunistic bacterial infections.

In a further embodiment, the composition of the invention is administered in combination with an antiviral agent. Antiviral agents that may be administered with the compositions of the present invention include, but are not limited to, acyclovir, ribavirin, amantadine, and lemantidine.

In a further embodiment, the composition of the invention is administered in combination with an antibiotic. Antibiotics that may be administered with the compositions of the present invention include, but are not limited to, amoxicillin, aminoglycoside, beta-lactam (glycopeptide), beta-lactamase, clindamycin, chloramphenicol, cephalosporin, ciprofloxacin, Erythromycin, fluoroquinolone, macrolide, metronidazole, penicillin, quinazolone, rifampin, streptomycin, sulfonamide, tetracycline, trimethoprim, trimethoprim-sulphamethoxazole and vancomycin.

Common non-specific immunosuppressants that may be administered in combination with the compositions of the present invention include, but are not limited to, steroids, cyclosporine, cyclosporine analogs, cyclophosphamide, cyclophosphamide IV, methylprednisolone, prednisolone, Prk, FK-506, 15-deoxy spergulane, and other immunosuppressants that act by inhibiting the function of the responding T cells.

In certain embodiments, the compositions of the invention are administered in combination with an immunosuppressive agent. Immunosuppressive agents that may be administered with the compositions of the present invention include but are not limited to ORTHOCLONE ^™ (OKT3), SANDIMMUNE ^™ / NEORAL ^™ / SANGDYA ^™ (cyclosporine), PROGRAF ^™ (tacrolimus), CELLCEPT ^™ Rate), azathioprine, glucocorticosteroid, and RAPAMUNE ^TM (sirolimus). In certain embodiments, the immunosuppressive agent can be used to inhibit rejection of an organ or bone marrow transplant.

In a preferred embodiment, the composition of the present invention is administered in combination with steroid therapy. Steroids that may be administered in combination with the compositions of the present invention include, but are not limited to, oral corticosteroids, prednisone, and methylprednisolone (e.g., IV methylprednisolone). In certain embodiments, the compositions of the invention are administered in combination with prednisone. In a further particular embodiment, the composition of the invention is administered in combination with prednisone and an immunosuppressive agent. The compositions of the present invention and immunosuppressive agents that may be administered with prednisone are those described herein and include, but are not limited to, azathioprine, cyclophosphamide and cyclophosphamide IV. In another specific embodiment, the composition of the present invention is administered in combination with methylprednisolone. In a further particular embodiment, the composition of the invention is administered in combination with methyl prednisone and an immunosuppressive agent. Immunosuppressive agents that may be administered with the compositions of the present invention and methyl prednisone are those described herein and include, but are not limited to, azathioprine, cyclophosphamide and cyclophosphamide IV.

In a preferred embodiment, the composition of the present invention is administered in combination with an anti-malaria agent. Anti-malaria agents that may be administered with the compositions of the present invention include, but are not limited to, hydroxychloroquine, chloroquine, and / or quinacrine.

In a preferred embodiment, the composition of the present invention is administered in combination with an NSAID.

In a typical embodiment, the composition of the present invention is administered in combination with 1, 2, 3, 4, 5, 10 or more of the following drugs: NRD-101 (Hoechst Marion Roussel), Diclofenac Monsanto, Chiron, T-614, Eli Lilly, Abbott, Monsanto, Byk Gulden, CBP-BS2 (KS-Biomedix), CM-101 (CarboMed), ML-3000 (Merckle), CB-2431 (KS Biomedix) , IL-1Ra gene therapy (Valentis), JTE-522 (Japan Tobacco), paclitaxol (Angiotech), DW-166HC (Dong Wha), Darbupelon mesylate (Warner-Lambert), soluble TNF- Hoechman-La Roche, EF-5 (Scotia Pharmaceuticals), BIIL-284 (Boehringer Ingelheim), BIIF-1149 (Boehringer Ingelheim), LeukoVax Inflammatics, MK-663 (Merck), ST-1482 (Sigma-Tau) Propionate (Warner Lambert).

In a preferred embodiment, the compositions of the invention are administered in combination with 1, 2, 3, 4, 5 or more of the following drugs: methotrexate, sulfasalazine, sodium aurothiomalate, auranopin, cyclosporine, Min, azathioprine, antimalarial drugs (such as that described herein), cyclophosphamide, chlorambucil, gold, ENBREL ^TM (Etanercept), anti -TNF antibody and prednisolone.

In a more preferred embodiment, the composition of the present invention is administered in combination with an anti-malaria agent, methotrexate, anti-TNF antibody, ENBREL ( ^TM) and / or sulfasalazine. In one embodiment, the composition of the invention is administered in combination with methotrexate. In another embodiment, the composition of the invention is administered in combination with an anti-TNF antibody. In another embodiment, the compositions of the invention are administered in combination with methotrexate and anti-TNF antibodies. In another embodiment, the composition of the present invention is administered in combination with sulfasalazine. In another specific embodiment, the compositions of the invention are administered in combination with methotrexate, anti-TNF antibody, and sulfasalazine. In another embodiment, the composition of the present invention is administered in combination with ENBREL ^™ . In another embodiment, the composition of the present invention is administered in combination with ENBREL ( ^TM) and methotrexate. In another aspect, the present composition is administered to ENBREL ^TM, methotrexate and snowy Plastic Ala Gin formulation. In another aspect, the present composition is administered to ENBREL ^TM, methotrexate and snowy Plastic Ala Gin formulation. In another embodiment, one or more anti-malarial agents are combined with one of the formulations described above. In certain embodiments, the compositions of the present invention are administered in combination with an anti-malarial agent (eg, hydroxychloroquine), ENBREL ^™ , methotrexate, and sulfasalazine. In another specific embodiment, the composition of the present invention is administered in combination with an anti-malarial agent (e.g., hydroxychloroquine), sulfasalazine, anti-TNF antibody and methotrexate.

In a further embodiment, the composition of the invention is administered in combination with, or alone in combination with, one or more intravenous immunoglobulin preparations. Intravenous immune globulin preparations that may be administered with the compositions of the present invention is not limited to these but include ^{GAMMAR TM, IVEEGAM TM, SANDOGLOBULIN TM} , GAMMAGARD S / D TM and ^TM GAMIMUNE. In certain embodiments, the compositions of the invention are administered in combination with an intravenous immunoglobulin preparation in a graft (e.g., bone marrow transplant) regimen.

A CD40 ligand (CD40L), a soluble form of CD40L (e.g., AVREND ^™ ), a biologically active fragment, variant or derivative of CD40L, an anti-CD40L antibody (eg, an efficacious or antagonistic antibody) and / , An antagonistic or an efficacious antibody).

In a further embodiment, the composition of the present invention is administered alone or in combination with an anti-inflammatory agent. Anti-inflammatory agents that may be administered with the composition of the present invention include, but are not limited to, glucocorticoids and nonsteroidal anti-inflammatory agents, aminoarylcarboxylic acid derivatives, acrylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, Pyrazolone, salicylic acid derivatives, thiazinecarboxamide, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, ammicillin, But are not limited to, pyrazole, pyrazole, pyrazole, pyrazole, pyrazole, pyrazole, pyrazole, pyrazole, pyrazole, .

In another embodiment, the composition of the present invention is administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that may be administered with the compositions of the invention include, but are not limited to, antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin); Antiestrogens (eg, tamoxifen); Antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, phloxuridine, interferon alpha-2b, glutamic acid, placaemycin, mercaptopurine and 6-thioguanine); A cytotoxic agent such as carmustine, BCNU, rosmutin, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, proccarbazine, mitomycin, Vincristine sulfate); Hormones such as medroxy progesterone, estramustine phosphate sodium, ethinyl estradiol, estradiol, megestrol acetate, methyl testosterone, diethylstilbestrolsulfate, chlorotrienicene and testolactone; Nitrogen mustard derivatives (e.g., mephalan, corramsyl, mechlorethamine (nitrogen mustard) and thiotepa); Steroids and combinations thereof (e.g., betamethasone sodium phosphate); And other (e.g., dicarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate and etoposide).

In certain embodiments, the compositions of the invention are administered in combination with all respective combinations of the components of CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or CHOP. In another embodiment, the composition of the present invention is administered in combination with rituximab. In a further embodiment, the composition of the present invention is formulated with rituximab and CHOP or together with every other combination of components of rituximab and CHOP.

In a further embodiment, the composition of the invention is administered in combination with a cytokine. IL-10, IL-12, IL-13, IL-15, anti-CD40, IL-10, , CD40L, IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha and TNF-beta. In another embodiment, the compositions of the present invention include, but are not limited to, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL- IL-18, IL-19, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL- 20, IL-21 and < RTI ID = 0.0 > IL-22. ≪ / RTI > In a preferred embodiment, the composition of the present invention is administered in combination with IL4 and IL10. Both IL4 and IL10 have been observed by the present inventors to enhance neurotoxin-alpha mediated B cell proliferation.

In a further embodiment, the compositions of the invention are administered in combination with a chemokine. In another embodiment, the composition of the invention is administered in combination with chemokine beta-8, chemokine beta-1 and / or macrophage inflammatory protein-4. In a preferred embodiment, the composition of the invention is administered in combination with chemokine beta-8.

In a further embodiment, the composition of the invention is administered in combination with an IL-4 antagonist. IL-4 antagonists that may be administered with the compositions of the present invention include, but are not limited to, soluble IL-4 receptor polypeptides, multimeric forms of soluble IL-4 receptor polypeptides, biological An IL-4 receptor that binds to an IL-4 receptor without blocking the signal, an anti-IL4 antibody that blocks binding of IL-4 to one or more IL-4 receptors, Lt; RTI ID = 0.0 > IL-4 < / RTI > that does not conduct the biological signal induced by IL-4. Preferably, the antibody used in accordance with this method is a monoclonal antibody (including, for example, an antibody fragment as described herein).

In a further embodiment, the composition of the present invention is administered in combination with hematopoietic growth factors. Hematopoietic growth factors that may be administered with the compositions of the present invention include, but are not limited to, LEUKINE ^TM (SARGRAMOSTIM ^™ ) and NEUPOGEN ^™ (FILGRASTIM ^™ ).

In a further embodiment, the composition of the present invention is administered in combination with a fibroblast growth factor. FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7 and FGF- -8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14 and FGF-15.

In addition, the compositions of the present invention may be administered alone or in combination with other therapeutic regimens, including, but not limited to, radiation therapy. Such combination therapies may be administered sequentially and / or concurrently.

Agonists and antagonists - assays and molecules

The present invention also encompasses compounds that increase or block the action of a neurotoxin-alpha or neurotoxin-alpha SV polypeptide on a cell, such as the interaction of a receptor molecule with a neurotoxin-alpha or neurotoxin-alpha SV binding molecule There is provided a method for selecting a compound to be identified. Agonists are compounds that increase the natural biological action of neurotoxin-alpha or neurotoxin-alpha SV or act in a manner similar to neurotoxin-alpha or neurotoxin-alpha SV, whereas antagonists decrease or eliminate this effect .

In another aspect, the invention provides a method of identifying a receptor protein or other ligand-binding protein that specifically binds a neurotoxin-alpha or neurotoxin-alpha SV polypeptide. For example, membranes, such as membranes or their manufacture, can be prepared from cells expressing molecules that bind to neurotoxin-alpha or neurotoxin-alpha SV. This preparation is incubated with labeled neurotoxin-alpha or neurotoxin-alpha SV and then the neurotoxin-alpha or neurotoxin-alpha SV bound to the receptor or other binding protein is isolated according to conventional methods known in the art, Check. Alternatively, a neurotoxin-alpha or neurotoxin-alpha SV polypeptide is conjugated to a solid support, the binding molecules dissolved from the cells are bound to the column and then eluted and characterized by conventional methods.

In the assays of the invention for agonists or antagonists, cellular membranes such as membranes or preparations thereof may be conjugated to a neurotoxin-alpha or neurotransmitter such as a molecule of a signal transduction or control pathway regulated by neurotoxin-alpha or neurotoxin- Can be prepared from cells expressing a molecule that binds to a kin-alpha SV. The agent is incubated with the labeled neurotoxin-alpha or neurotoxin-alpha SV in the absence or presence of a candidate molecule which may be a neurotoxin-alpha or neurotoxin-alpha SV agonist or antagonist. The ability of a candidate molecule to bind to a binding molecule appears as a reduced binding of the labeled ligand. A molecule that binds without inducing the effect of neurotoxin-alpha on binding to neurotoxin-alpha or neurotoxin-alpha SV binding molecules is likely to be an excellent antagonist. A molecule that binds well and exhibits the same or closely related effect as neurotoxin-alpha or neurotoxin-alpha SV is an agonist.

The neurotoxin-alpha or neurotoxin-alpha SV-like effect of potential agonists and antagonists can be measured, for example, by measuring the activity of a second messenger system after interaction of the cell or a candidate cell with a candidate agent, Alpha or neurotoxin-alpha SV or neurotoxin-alpha or neurotoxin-alpha SV with the effect of a molecule inducing the same effect. In this regard, a second messenger system that may be useful includes, but is not limited to, an AMP guanylate cyclase, an ion channel, or a phosphoinositide hydratase second messenger system.

Other examples of assays for neurotoxin-alpha or neurotoxin-alpha SV antagonists include binding of neurotoxin-alpha or neurotoxin-alpha SV and a potential antagonist to a membrane-bound receptor molecule or recombinant neurotoxin-alpha or neurotoxin- alpha SV Lt; RTI ID = 0.0 > competitive < / RTI > assay with receptor molecules under appropriate conditions. The neurotoxin-alpha or neurotoxin-alpha SV can be labeled by, for example, radioactivity, thereby accurately measuring the number of neurotoxin-alpha or neurotoxin-alpha SV molecules bound to the receptor molecule and determining the potency of the potential antagonist Can be evaluated.

Potential antagonists include small organic molecules, peptides, polypeptides (e. G., IL-13) and antibodies that inhibit or abolish their activity by binding to the polypeptides of the invention. In addition, the potential antagonist binds to the same site on a binding molecule, such as a receptor molecule, without inducing neurotoxin-alpha or neurotoxin-alpha SV induced activity, thereby generating neurotoxin-alpha or neurotoxin-alpha SV May be polypeptides or antibodies, such as small organic molecules, peptides, closely related proteins, which inhibit the action of neurotoxin-alpha or neurotoxin-alpha SV.

Other potential antagonists include antisense molecules. Antisense technology can be used to regulate gene expression through antisense DNA or RNA or through triple helix formation. Antisense technology is described, for example, in Okano, J. Neurochem. 56: 560 (1991); &Quot; Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression. &Quot; CRC Press, Boca Raton, FL (1988). Antisense technology can be used to regulate gene expression through antisense DNA or RNA or through triple helix formation. Antisense technology is described, for example, in Okano, J. Neurochem. 56: 560 (1991); &Quot; Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression. &Quot; CRC Press, Boca Raton, FL (1988). Triple helix formation is described, for example, in Lee et al., Nucleic Acids Research 6: 3073 (1979); Cooney et al., Science 241: 456 (1988); And Dervan et al., Science 251: 1360 (1991). This method is based on the binding of polynucleotides to complementary DNA or RNA. For example, the 5 'coding portion of a polynucleotide encoding the extracellular domain of a polypeptide of the invention can be used to design antisense RNA oligonucleotides that are about 10 to 40 base pairs in length. The DNA oligonucleotides are designed to be complementary to the region of the gene involved in transcription, thereby inhibiting the transcription and the production of neurotoxin-alpha and / or neurotoxin-alpha SV. Antisense RNA oligonucleotides hybridize in vivo with mRNA and block the translation of mRNA molecules into neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides. The oligonucleotides described above may also be delivered to cells so that antisense RNA or DNA is expressed in vivo to inhibit the production of neurotoxin-alpha and / or neurotoxin-alpha SV.

In one embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV antisense nucleic acids of the invention are produced intracellularly by transcription from a foreign sequence. For example, a vector or a portion thereof is transcribed to generate the antisense nucleic acid (RNA) of the present invention. These vectors contain sequences encoding the neurotoxin-alpha and / or neurotoxin-alpha SV antisense nucleic acids. Such a vector may be maintained as an epitope as long as it can be transcribed to produce the desired antisense RNA or integrated into the chromosome. Such vectors may be constructed by standard recombinant DNA techniques in the art. Vectors may be plasmids, viruses, and the like known in the art that are used for replication and expression in vertebrate cells. Expression of a sequence encoding neurotoxin-alpha and / or neurotoxin-alpha SV or fragments thereof may be possible in any promoter known in the art to act in vertebrate animals, preferably human cells. Such promoters may be inducible or constitutive. These promoters include but are not limited to the SV40 early promoter region (Bernoist and Chambon, Nature 29: 304-310 (1981), promoters contained in the 3 'long terminal repeat of the sarcoma virus (Yamamoto et al. USA, Cell 22: 787-797 (1980), Herpes thymidine promoter (Wagner et al., Proc Natl Acad Sci USA 78: 1441-1445 (1981)), the regulatory sequence of the metallothionein gene (Brinster, et al., Nature 296: 39-42 (1982).

The antisense nucleic acid of the present invention comprises a sequence complementary to at least a portion of an RNA transcript of a neurotoxin-alpha and / or neurotoxin-alpha SV gene. However, absolute complementarity is desirable, but not necessarily required. As used herein, the term " complementary to at least a portion of the RNA " refers to a sequence that is sufficiently complementary to hybridize with its RNA to form a stable duplex, and includes double-stranded neurotoxin-alpha and / In the case of alpha SV antisense nucleic acids, the Japanese chain of double-stranded DNA can be tested or triple-stranded formation can be assayed. The ability to hybridize depends on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the greater the number of hybridized nucleic acids, the greater the number of base mismatches with neurotoxin-alpha and / or neurotoxin-alpha SV RNA, which can form a stable double strand (or triple strand) containing nucleic acid. Those skilled in the art will be able to determine the acceptable degree of mismatch by using standard procedures to determine the melting point of the hybridized complexes.

Oligonucleotides that are complementary to the 5 'end of the message (eg, the 5' untranslated sequence to date, including the AUG start codon), work most efficiently when inhibiting decoding. However, sequences complementary to the 3 ' -dependent sequence of mRNA have also been suggested to be effective in inhibiting the detoxification of mRNA (Wagner, R., 1994, Nature 372: 333-335). Thus, oligonucleotides complementary to the 5'- or 3'-relative non-coding regions of the neurotoxin-alpha and / or neurotoxin-alpha SV described in Figures 1A-B and 5A-B, respectively, And / or antisense methods which inhibit the detoxification of neurotoxin-alpha SV mRNA. The oligonucleotide complementary to the 5 'untranslated region of the mRNA must contain a complement of the AUG start codon. Antisense oligonucleotides that are complementary to the mRNA coding region may be used in accordance with the present invention or as less efficient detoxification inhibitors. The antisense nucleic acid should be at least 6 nucleotides in length, whether or not designed to hybridize to the 5'-, 3'- or cipher region of neurotoxin-alpha and / or neurotoxin-alpha SV mRNA, And an oligonucleotide having a length of 6 to about 50 nucleotides. In a particular aspect, the oligonucleotide consists of at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides, or at least 50 nucleotides.

The polynucleotide of the present invention may be a DNA or RNA of a single-stranded or double-stranded chain, or a chimeric mixture or derivative or variant thereof. Oligonucleotides can be modified at the base residue, sugar residue or phosphate backbone to improve stability, hybridization, etc., of the molecule, for example. Oligonucleotides may be derived from peptides (e. G., To target host cell receptors in vivo), cell membranes (see Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA 86: 6553-6556 (see PCT Publication No. WO 88/09810, published December 5, 1988), or blood-brain barrier (see, eg, April 25, 1988, (See, for example, Krol et al., BioTechniques 6: 958-976 (1988)) or septic agents (see Zon, Pharm. Res 5: 539-549 (1988)). To this end, oligonucleotides can be linked to other molecules such as peptides, cross-linking agents targeted for hybridization, transport agents, hybridization-targeted cleavage agents, and the like.

Antisense oligonucleotides include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- Carboxymethylaminomethyl uracil, dihydrouracil, beta-D-galactosylquinoxine, inosine, N6-isopentenyl adenine, 1- Methylguanidine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5- , 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylquinosine, 5-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil- 5-oxyacetic acid (v), wybutoxin, pseudouracil, quiosine, 2-thiocytosine, 5-methyl-2-thiouracil, 5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3- (3-amino- 2-carboxypropyl) uracil, (acp3) w, and 2,6-diaminopurine.

Antisense oligonucleotides may also include one or more modified sugar moieties selected from the group consisting of, but not limited to, arabinose, 2-fluoro arabinose, xylulose and hexose.

In yet another embodiment, the antisense oligonucleotides include, but are not limited to, phosphorothioate, phosphorodithioate, phosphoramidothioate, phosphoramidate, phosphorodiamidate, methylphosphonate, alkyl Phosphotriester esters and formacetals, or analogs thereof. ≪ Desc / Clms Page number 7 >

In another embodiment, the antisense oligonucleotide is an alpha-anomer oligonucleotide. Alpha-anomeric oligonucleotides form specific double-stranded hybrids with complementary RNA, and in contrast to the common beta-units, the strands are parallel to one another (Gautier et al., Nucl. Acids Res. 15: 6625-6641 1987). Oligonucleotides may be prepared by PCR using 2-0-methyl ribonucleotides (Inoue et al., Nucl. Acids Res. 15: 6131-6148 (1987)) or chimeric RNA-DNA analogs (Inoue et al., FEBS Lett. 215: 327-330 (1997)).

The polynucleotides of the present invention can be synthesized by standard methods known in the art, for example, using automated DNA synthesizers (such as those available from Biosearch, Applied Biosystems, etc.). As an example, phosphorothioate oligonucleotides can be synthesized by the method described in Stein et al., Nucl. Acids Res. 16: 3209 (1988), and methylphosphonate oligonucleotides can be prepared using a controlled-release open polymeric polymeric support or the like [Sarin et al., Proc. Natl. Acad. Sci. U.S.A. 85: 7448-7451 (1988).

Antisense nucleotides complementary to the neurotoxin-alpha and / or neurotoxin-alpha SV coding region sequences may be used, while antisense nucleotides complementary to the transcribed untranslated region are most preferred.

Potential antagonists in accordance with the present invention may also be conjugated to catalytic RNA or ribozyme (see: PCT International Publication No. WO90 / 11364; Sarver et al., Science 247: 1222-1225 (1990) ). It is preferred to use hammerhead ribozymes while ribozymes that cleave mRNAs in site-specific recognition sequences can be used to degrade neurotoxin-alpha and / or neurotoxin-alpha SV mRNAs. The hammerhead ribozyme cleaves mRNA at the designated position by adjoining a region forming a base pair that is complementary to the target mRNA. The only requirement is that the target mRNA has two base 5'-UG-3 'sequences. The construction and production of hammerhead ribozymes is well known in the art and is described in more detail in Haseloff and Gerlach, Nature 334: 585-591 (1988). There are many potential hammerhead ribozyme cleavage sites within the nucleotide sequences of neurotoxin-alpha and / or neurotoxin-alpha SV (FIGS. 1A-B and 5A-B, respectively). Preferably, the ribozyme is positioned so that the cleavage recognition site is located near the 5 'end of the neurotoxin-alpha and / or neurotoxin-alpha SV mRNA, i.e., enhances efficiency and minimizes intracellular accumulation of non- .

As with the antisense method, the ribozymes of the present invention can be composed of modified oligonucleotides (e.g., for improved stability, target targeting, etc.) and are capable of inhibiting neurotoxin-alpha and / or neurotoxin- alpha SV in vivo Lt; / RTI > DNA constructs encoding ribozymes can be inserted into cells in the same manner as described above for insertion of antisense-encoding DNA. A preferred delivery method is a DNA construct that " encrypts " the ribozyme so that the transfected cells can provide a sufficient amount of ribozyme to disrupt the endogenous neurotoxin-alpha and / or neurotoxin- , Under the control of a strong constitutive promoter (e.g. pol III or pol II promoter). Because ribozymes are catalysts, unlike antisense molecules, lower intracellular concentrations are needed for efficiency.

Endogenous gene expression can also be reduced by inactivating or " knocking out " the neurotoxin-alpha and / or neurotoxin-alpha SV gene and / or its promoter using target homologous recombination Smithies et al., Nature 317: 230-234 (1985); Thomas & Capecchi, Cell 51: 503-512 (1987); Thompson et al., Cell 5: 313-321 (1989)]. The entire contents of each of these documents are incorporated herein by reference. For example, the non-functional mutant polynucleotide of the present invention, which is flanked by DNA homologous to the endogenous polynucleotide sequence, is used in the presence or absence of a selectable marker and / or a negative selectable marker to express the polypeptide of the present invention in vivo RTI ID = 0.0 > transfected < / RTI > In another embodiment, knockout occurs within cells that contain, but do not express, the gene using techniques known in the art. Insertion of DNA constructs through target homologous recombination results in the inactivation of the target gene. This method is particularly suitable in research and agriculture fields in which transformation of fetal liver cells can be used to generate newborn animals with inactivated target genes (Thomas & Capecchi 1987 and Thompson 1989). This method, however, can be applied to humans, which are conventionally applied, provided that the recombinant DNA construct is directly administered or targeted to the required site in vivo using a suitable viral vector as is known to those skilled in the art. The entire contents of which are incorporated herein by reference.

In another embodiment, an antagonist according to the invention is a soluble form of a neurotoxin-alpha and / or neurotoxin-alpha SV (e.g., a ligand binding domain of a neurotoxin-alpha and / or neurotoxin- alpha SV, a TNF conserved domain Alpha and / or neurotoxin-alpha SV, TNF conserved domain and / or extracellular domain of the neurotoxin-alpha and / or neurotoxin-alpha SV fragments described in Figures 1A-B, Alpha < / RTI > SV described in Figures 5A-B). The soluble forms of the neurotoxin-alpha and / or neurotoxin-alpha SV, which may be naturally occurring or synthetic, include neurotoxin-alpha and / or neurotoxin-alpha SV receptors such as DR5 (see WO 98/41629 ), TR10 (see 98/54202); 312C2 (see WO 98/06842) and TR11, TR11SV1 and TR11SV2 (see U.S. Patent Application Serial No. 09 / 176,200) Alpha < / RTI > and / or neurotoxin-alpha SV mediated signal transmission by competing with the neurotoxin-alpha SV or by forming multimers that are capable of binding or not binding to the receptor but not inducing signal transduction These antagonists inhibit neurotoxin-alpha and / or neurotoxin-alpha SV mediated stimulation of lymphocyte (e.g., B-cell) proliferation, differentiation and / or activation. Antagonists of the present invention also inhibit TNF family ligands , CD30) and neurotoxin-alpha-Fc and / or neurotoxin-alpha SV-Fc fusion Lt; RTI ID = 0.0 > a < / RTI > sum protein.

&Quot; TNF family ligand " means a natural, recombinant, and synthetic ligand capable of binding members of the TNF receptor family and inducing and / or blocking the ligand / receptor signaling pathway. The TNF ligand family includes, but is not limited to, TNF-alpha, lymphotoxin-alpha (also known as LT-alpha, also known as TNF-beta), LT-beta (complex heterotrimer LT-alpha 2-beta ), FasL, CD40L, TNF-gamma (International Publication No. WO 96/14328), AIM-I (International Publication No. WO 97/33899), AIM-II (International Publication No. WO 97/34911) , APRIL (J. Exp. Med. 188 (6): 1185-1190), endocaine-alpha (WO 98/07880), neurotoxin-alpha (WO 98/18921), CD27L, CD30L , 4-1BBL, OX40L, CD27, CD30, 4-1BB, OX40 and nerve growth factor (NGF). In a preferred embodiment, the neurotoxin-alpha and / or neurotoxin-alpha SV TNF family ligands of the invention are DR5 (International Publication No. WO 98/41629), TR10 (International Publication No. WO 98/54202), 312C2 WO 98/06842) and TR11, TR11SV1 and TR11SV2 (U.S. Patent Application 09 / 176,200).

Antagonists of the invention also include antibodies specific for the TNF family receptor or the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides of the invention. Antibodies according to the present invention can be produced according to any of a variety of standard methods using the neurotoxin-alpha and / or neurotoxin-alpha SV immunogens of the present invention. As mentioned, such kin-alpha and / or neurotoxin-alpha SV immunogens include the complete neurotoxin-alpha and / or neurokinin-alpha SV immunogens described in Figures 1A-B (SEQ ID NO: 2) and Figures 5A- A neurotoxin-alpha SV polypeptide (which may or may not include the leader sequence) and, for example, a ligand binding domain, a TNF-conserved domain, an extracellular domain, a transmembrane domain, and / Alpha < / RTI > and / or neurotoxin-alpha SV polypeptide fragments.

The polyclonal and monoclonal antibody agonists or antagonists according to the present invention are described in Tartaglia and Goeddel, J. Biol. Chem. 267 (7): 4304-4307 (1992); Can be produced according to the methods described in Tartaglia et al., Cell 73: 213-216 (1993) and PCT Application WO 94/09137, and are preferably specific for the polypeptides of the invention having the amino acid sequence of SEQ ID NO: 2 (Ie, bind specifically). The term " antibody " (Ab) or " monoclonal antibody " (mAb) as used herein refers to the complete molecule capable of binding an antigen as well as its fragments (e.g. Fab and F (ab ')) fragments. The Fab, Fab ', and F (ab') fragments are more rapidly removed from the circulation and may have weaker nonspecific tissue binding of the complete antibody lacking the Fc fragment (Wahl et al., J. Nucl. Med. 24: 316-325 (1983)).

In a preferred method, the antibody according to the invention is a mAb. Such mAbs can be prepared using hybridoma technology (Kohler and Millstein, Nature 256: 495-497 (1975) and US Patent No. 4,376,110 ,; harlow et al., Antibodies: A Laboratory Manual. Cold Spring Harbor Monoclonal Antibodies ", " In " Laboratory Techiques in Biochemistry and Molecular Biology, New York, NY, 1980; Monoclonal Antibodies and Hybridomas: A New Dimension in Biological Analyzes. , Volume 13 (Burdon et al., Eds.), Elsevier, Amsterdam (1984)).

Proteins and other compounds that bind to the neurotoxin-alpha and / or neurotoxin-alpha SV domains are also candidate agonists and antagonists according to the present invention. Such binding compounds can be captured using a yeast-hybrid system (Fields and Song, Nature 340: 245-246 (1989)). A modification of the yeast-hybrid system has been disclosed by Roger Brent and colleagues (Gyuris, Cell 75: 791-803 (1993); Zervos et al., Cell 72: 223-232 (1993)). Preferably, the yeast-hybrid system utilizes in accordance with the invention to capture compounds that bind to the ligand binding domain, extracellular, intracellular, epidermal, and killing domains of neurotoxin-alpha and / or neurotoxin-alpha SV . These compounds are good candidate agonists and antagonists according to the present invention.

For example, using the e-hybrid assay described above and using extracellular or intracellular domains of the neurotoxin-alpha and / or neurotoxin-alpha SV receptors, or a portion thereof, to generate neurotoxin-alpha and / - Identify cell proteins that interact with the alpha SV receptor in vivo. This assay can also be used to identify ligands with potential efficacy or antagonistic activity of neurotoxin-alpha and / or neurotoxin-alpha SV receptor function. These screening assays have previously been used to identify proteins that interact with the cytoplasmic domain of mouse TNF-RII and as a result two receptor-bound proteins have been identified (Rothe et al., Cell 78: 681 (1994) . The protein and amino acid sequences that bind to the cytoplasmic domain of the neurotoxin-alpha and / or neurotoxin-alpha SV receptors are good candidate agonists and antagonists according to the present invention.

Other screening techniques include, but are not limited to, cells expressing the polypeptide of the invention in a system for measuring extracellular pH changes caused by receptor activation as described in, for example, Science, 246: 181-296 (1989) E.g., transfected CHO cells). As another example, a potential agonist or antagonist may be contacted with a cell expressing a polypeptide of the invention and a second messenger response (e.g., signal conduction) may be measured to determine whether the potential antagonist or agonist is effective.

Examples of potentiators according to the present invention include TNF family ligand peptide fragments, transforming growth factors, neurotransmitters (e.g., glutamate, dopamine, N-methyl-D-aspartate), tumor suppressor (p53), cytolytic T cells, And natural and synthetic compounds such as metabolites. Preferred agonists include chemotherapeutic drugs such as cisplatin, doxorubicin, bleomycin, cytosine arabinoside, nitrogen mustard, methotrexate and vincristine. Other potentiators include ethanol and - amyloid peptides (Science, 267: 1457-1458 (1995)).

Preferred agonists are fragments of the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides of the invention that stimulate lymphocyte (e.g., B cell) proliferation, differentiation and / or activation. Additional preferred agonists include polyclonal and monoclonal antibodies or fragments thereof produced against the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides of the invention. Agonist antibodies generated against TNF family receptors are described in Tartaglia et al., Proc. Natl. Acad. Sci. USA 88: 9292-9296 (1991); And Tartaglia et al., J. Biol. Chem. 267: 4304-4307 (1992)) (another reference example is International Publication No. WO 94/09137).

In a further aspect, immunomodulatory molecules such as IL2, IL3, IL4, IL5, IL6, IL7, ILlO, IL12, IL13, IL15, anti-CD40, CD40L, IFN-gamma and TNF- Alpha] and / or neurotoxin-alpha SV polypeptides of the invention that stimulate proliferation, differentiation and / or activation of the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides of the invention. In certain embodiments, IL4 and / or IL10 are used to enhance neurotoxin-alpha-and / or neurotoxin-alpha SV-mediated proliferation of B cells.

In a further aspect of the invention, a cell genetically engineered to express the polypeptide of the invention or a cell genetically engineered (e. G., Knockout) so as not to express the polypeptide of the invention is administered to the patient in vivo . Such cells can be obtained from a patient (i. E., An animal including a human) or an MHC-matched donor, including but not limited to fibroblasts, bone marrow cells, blood cells (i. E., Lymphocytes), adipocytes, muscle cells, May be included. But are not limited to, plasmids, cosmids, YACs, DNA, electroporation, transfection or transfection (using vectors that integrate viral vectors, preferably transgene into the cellular genome) To genetically engineer these cells according to recombinant DNA technology to introduce the coding sequence of the polypeptide according to the present invention into the cell or disrupt the coding sequence and / or endogenous regulatory sequence associated with the polypeptide of the present invention. The coding sequence of a polypeptide according to the present invention may be set under the control of a strong constitutive or inducible promoter or promoter / enhancer so that the expression and preferably secretion of the polypeptide according to the invention is effected. Engineered cells that express and preferably secrete the polypeptides of the present invention can be injected systemically into the patient (e. G., Circulating or intraperitoneally).

Alternatively, the cells can be introduced into the matrix or transplanted into the body, for example, genetically engineered fibroblasts can be implanted as part of a skin graft, and genetically engineered endothelial cells can be introduced into the lymph or vessel (Anderson et al., U.S. Pat. No. 5,399,349 and Mulligan & Wilson, U.S. Pat. No. 5,460,959). The entire contents of these documents are incorporated herein by reference.

If the cells to be administered are non-autologous or non-MHC compatible cells, these cells may be administered using well known techniques to inhibit the development of a host immune response to the introduced cells. For example, cells may be introduced into a capsule form, which allows the component to be exchanged for an adjacent extracellular environment, while the introduced cells are not recognized by the host immune system.

In another embodiment of the invention, the activity of the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide can be reduced using " dominant negative ". To this end, constructs encoding deletion neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides such as mutants in which all or part of the TNF-conserved domain have been deleted are used in gene therapy to generate Lt; RTI ID = 0.0 > neurotoxin-alpha < / RTI > and / or neurotoxin-alpha SV. For example, a nucleotide sequence that induces host cell expression of all or part of the TNF-conserved domain of the modified or deleted neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide may be introduced into a mononuclear cell or other cell or tissue (By in vivo or in vitro gene therapy as described herein or known in the art). Alternatively, the target homologous recombination can be used to introduce the deletion or mutation into an endogenous neurotoxin-alpha and / or neurotoxin-alpha SV gene in a patient's mononuclear cells. Engineering engineered cells will express non-functional neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides (i.e., ligands that can bind but can not induce signal transduction (e.g., multimers)).

Chromosome black

The nucleic acid molecule of the present invention is also valuable for chromosome identification. The sequence can specifically target and hybridize to a specific site on an individual's chromosome. In addition, it is necessary to identify a specific site on the present chromosome. Only a few are currently used to display chromosome positions as chromosome display reagents based on substantial sequence data (repeat polymorphism). Mapping of DNA to the chromosome according to the present invention is an important first step in revealing the correlation between the sequences and disease-related genes.

In this regard, in certain preferred embodiments, the genomic DNA of the neurotoxin-alpha and / or neurotoxin-alpha SV gene is cloned using the cDNAs and / or polynucleotides described herein. This can generally be accomplished using commercially available libraries and various known techniques. Genomic DNA is then used for chromosome mapping in the reaction system according to known techniques for this purpose.

Also, in some cases, sequences for chromosomes can be mapped by preparing PCR primers (preferably 15-25 bp) from the cDNA. By computer analysis of the 3 'untranslated region of the gene, the primers that complicate the amplification process are quickly selected by not linking one or more exons in the genomic DNA. These primers are then used for PCR screening of somatic cell hybrids containing individual chromosomes. Fluorescent in situ hybridization (FISH) of cDNA clones to mid-term chromosome dispersions can be used to provide one-step accurate chromosomal location. This technique can be used with probes from cDNA as short as 50 or 60 bp (see Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988)

Once the exact chromosomal location of the sequence is mapped, the physical location of the sequence on the chromosome may be correlated with the gene map data. Such data can be found, for example, in V. McKusick, Mendelian Inheritance in Man (available on-line from the Welch Medical Library at Johns Hopkins University). Next, the correlation between genes and diseases assigned to the same chromosomal region is identified through gene analysis (co-inheritance of physically adjacent genes).

Next, it is necessary to measure the difference in cDNA or genome sequence between the patient and the normal person. If the mutation is observed in all or part of the patient but not in any normal person, the mutation must be a causative factor.

Based on current analysis of physical mapping and genetic mapping techniques, cDNAs that are located precisely in the chromosome region associated with the disease can be one of 50 to 500 potential onset genes. (This assumes a mapping analysis of 1 me base and one gene per 20 kb).

In accordance with the technique described above, the combined use of somatic and radiation hybrids resulted in a chromosomal location of neurotoxin-alpha and neurotoxin-alpha SV as 13q34 with very high confidence.

The present invention has been described in overview and the present invention will be more readily understood by reference to the following embodiments. This embodiment is provided for the purpose of illustration and is not limited thereto. In the following Examples, most of the neurotoxin-alpha polynucleotides and polypeptides of the present invention are specifically cited and explained. Each example can also be used to produce and / or detect the neurotoxin-alpha SV polynucleotides and / or polypeptides of the present invention. Those of ordinary skill in the art will readily be able to prepare neurotoxin-alpha SV using the following examples.

Example 1a: " His-tagged " neurotoxin-alpha. Expression and purification in E. coli

In this example, the bacterial expression vector pQE9 (pD10) is used to see expression in bacteria (see QIAGEN, Inc., supra). pQE9 encodes Ampicillin antibiotic resistance (" Ampr ") and encodes the bacterial replication origin (" ori "), IPTG inducible promoter, ribosome binding site (" RBS "), QIAGEN, Six codons encoding a histidine residue that allows purification by an affinity purification method using a nickel-nitrilo-triacetic acid ("Ni-NTA") affinity resin, . These factors are arranged such that the inserted DNA fragment encoding the polypeptide can express the polypeptide in which 6 His residues (i.e., " 6 X His tag ") are covalently linked to the amino terminus of the polypeptide.

The DNA sequence coding for the target site of the neurotoxin-alpha protein comprising the extracellular domain comprises the amino terminal sequence of the target site of the protein and the PCR oligonucleotide annealing to the sequence in the deposited construct located 3 ' Amplified from the deposited cDNA clone using primers. In addition, additional nucleotides, including restriction sites that facilitate cloning in the pQE9 vector, are added to the 5 'and 3' primer sequences, respectively.

The 5 ' primer for cloning the extracellular domain of the protein consists of an underlined BamHI restriction site followed by a sequence 5 ' comprising 18 nucleotides of the amino terminal secretion sequence of the extracellular domain in the sequence shown in Figures 1A and < RTI ID = -GTG GGA TCC A GC CTC CGG GCA GAG CTG-3 '(SEQ ID NO: 10). Of course, the point in the protein coding sequence where the 5 ' primer starts can change the DNA segment encoding any target site of the entire neurotoxin-alpha protein to be shorter or longer than the extracellular domain of that form. The 3 'primer contained an underlined HindIII restriction site followed by two stop codons and a sequence 5'-GTG AAG CTT TTA containing 18 nucleotides complementary to the 3' end of the coding sequence of the DNA sequence shown in FIGS. 1A and 1B TTA CAG CAG TTT CAA TGC ACC-3 '(SEQ ID NO: 11).

The amplified DNA fragment and vector pQE9 are digested with BamHI and HindIII, and then the digested DNA is ligated together. The DNA inserted into the restriction enzyme digested pQE9 vector is placed such that the protein coding region is downstream of the IPTG-inducible promoter and inserted into the frame of the initiation AUG and six histidine codons.

The coupling mixture is then applied to the compartment using standard procedures as described in Sambrook et al., Molecular Cloning: a Laboratory Manual, 2nd Ed: Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) this. To transform E. coli cells. this. Cola strains M15 / rep4, which contain multiple copies of the plasmid pREP4 expressing the lac repressor and conferring kanamycin resistance (" Kan ^r "), are used to perform the exemplary embodiments herein. This strain, the only strain among a number of strains useful for expressing proteins, is available from Quiagen Inc. (see above). The transformants are identified by their activity of proliferating in LB plate medium in the presence of ampicillin and kanamycin. Plasmid DNA is isolated from resistant colonies and the presence of cloned DNA is confirmed by restriction enzyme analysis, PCR and DNA sequencing. Clones containing the desired construct are grown overnight (" O / N ") through liquid culture in LB medium supplemented with ampicillin (100 μg / ml) and kanamycin (25 μg / ml). The O / N culture is inoculated into the bulk culture at a dilution of about 1: 25 to 1: 250. Cells are grown to an optical density (" OD600 ") at 600 nm of 0.4 to 0.6. The isopropyl-beta-D-thiogalactopyranoside (" IPTG ") is then added at a final concentration of 1 mM to inactivate the lacI inhibitor and induce transcription from the lac inhibitor-sensitive promoter. The cells are then incubated for 3 to 4 hours. The cells are collected by centrifugation.

The cells are then agitated in 4M guanidine-HCl (pH 8) for 6 hours at 3-4 hours. The cellular debris is removed by centrifugation and the supernatant containing the protein is injected onto a nickel-nitrilo-triacetic acid ("Ni-NTA") affinity resin column (Qiagen Inc., see above). Proteins with a 6x His tag can be purified with a simple one-step procedure by binding to the Ni-NTA resin with high affinity (see QIAexpressionist, 1995 (Quijagen Inc., see above) for a detailed description). Briefly, the supernatant was injected onto a column in 6 M guanidine-HCl (pH 8), the column was washed with 10 volumes of 6 M guanidine-HCl (pH 6) and finally 6 M guanidine- To elute the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptides.

The purified protein is then recovered by dialysis against phosphate buffered saline (PBS) or 50 mM sodium acetate (pH 6 buffer) + 200 mM NaCl. Alternatively, the protein may be recovered under fixed conditions on a Ni-NTA column. As a recommended condition, a linear 6M-1M urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris / HCl (pH 7.4) containing the protease inhibitor is used. Restoration should be carried out for more than 1.5 hours. After restoration, the protein can be eluted by adding 250 mM imidazole. The imidazole is removed via dialysis to final PBS or 50 mM sodium acetate (pH 6 buffer) + 200 mM NaCl. The purified protein is stored at 4 ° C or frozen at -80 ° C.

Example 1b: Synthesis of this neurotoxin-alpha. Expression and purification in E. coli

For bacterial expression, the bacterial expression vector pQE60 is used in this example (Quiagen Inc., 9759 Eaton Avenue 91311 Charsworth, CA). pQE60 encodes the Ampicillin antibiotic resistance ("Ampr") gene and encodes a bacterial replication origin ("ori"), IPTG inducible promoter, ribosome binding site ("RBS"), Quiagen 6 codons that encode histidine residues that allow purification by an affinity purification method using a nitrilo-triacetic acid (" Ni-NTA ") affinity resin and suitable single restriction enzyme cleavage sites. These factors are arranged such that the inserted DNA fragment encoding the polypeptide is capable of expressing the polypeptide in which 6 His residues (i.e., " 6 X His tag ") are covalently linked to the carboxy terminus of the polypeptide. However, in this embodiment, the polypeptide coding sequence is inserted such that the decoding of six His codons is blocked so that the polypeptide does not contain the 6X His tag.

The DNA sequence coding for the target site of the protein comprising the extracellular domain sequence is obtained by using a PCR oligonucleotide primer annealing to the amino terminal sequence of the target site of the protein and the sequence in the deposited construct located 3 ' And amplified from the deposited cDNA clone. In addition, additional nucleotides containing restriction sites that facilitate cloning in the pQE60 vector are added to the 5 'and 3' sequences, respectively.

The 5 ' primer for cloning the extracellular domain of the protein consists of the underlined Bsp HI restriction site and then the sequence 5 < RTI ID = 0.0 > 5 < / RTI > containing 17 nucleotides of the amino terminal cipher sequence of the extracellular domain in the sequences shown in FIGS. IA and IB '-GTG TCA TGA GCC TCC GGG CAG AGC TG-3' (SEQ ID NO: 12). Of course, the point in the protein coding sequence where the 5 ' primer starts can change the target site of the whole protein to be shorter or longer than the extracellular domain of that form. The 3 'primer contained an underlined HindIII restriction site followed by two stop codons and a sequence 5'-GTG AAG CTT TTA containing 18 nucleotides complementary to the 3' end of the coding sequence of the DNA sequence shown in FIGS. 1A and 1B TTA CAG CAG TTT CAA TGC ACC-3 '(SEQ ID NO: 13).

The amplified DNA fragment and the vector pQE60 are digested with BspHI and HindIII, and then the digested DNA is ligated together. The DNA inserted into the restriction enzyme digested pQE60 vector is positioned such that the protein coding region comprising the related stop codon is downstream of the IPTG-inducible promoter and inserted into the frame of the initiation AUG.

The coupling mixture is then applied to the compartment using standard procedures as described in Sambrook et al., Molecular Cloning: a Laboratory Manual, 2nd Ed: Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) this. To transform E. coli cells. this. Cola strains M15 / rep4, which contain multiple copies of the plasmid pREP4 expressing the lac repressor and conferring kanamycin resistance (" Kan ^r "), are used to perform the exemplary embodiments herein. This strain, the only strain among a large number of strains suitable for expressing proteins, is available from Quiagen Inc. (see above). The transformants are identified by their activity of proliferating in LB plate medium in the presence of ampicillin and kanamycin. Plasmid DNA is isolated from resistant colonies and the presence of the cloned DNA is confirmed by restriction enzyme analysis, PCR and DNA sequencing.

Those skilled in the art will appreciate that any of a number of bacterial expression vectors may be used in place of pQE9 and pQE60 in the expression protocols provided in this example. For example, a novel pHE4-based bacterial expression vector, in particular a pHE4-5 vector, can be used for the expression of the bacterium of this embodiment (ATCC Accession No. 209311: and variants thereof). The plasmid DNA pHE4-5 / MPIFD23 deposited with ATCC Accession No. 209311 is a vector plasmid DNA containing an insert encoding another ORF. This construct was deposited on September 30, 1997 at the American Bacterium Culture Collection (Manassas University, Boulevard 10801, Manassas, Va., 20110-2209, USA). Substitution of the irrelevant ORF of pHE4-5 with the neurotoxin-alpha ORF of the present invention can be readily accomplished by those of ordinary skill in the art, using conventional NdeI and Asp718 restriction sites adjacent to the unrelated MPIF ORF insert, It is possible.

The pHE4-5 bacterial expression vector is a neomycin phosphotransferase gene for selection. A replication origin of E. coli, a T5 phage promoter sequence, two lac operative sequences, a Shine-Dalgano sequence and a lactose operon inhibitor gene (lacIq). These factors are arranged such that the inserted DNA fragment encoding the polypeptide can express a polypeptide in which six His residues (i.e., " 6 X His tag ") are covalently attached to the amino terminus of the polypeptide. The promoter and operative factor sequences of the pHE4-5 vector are synthesized. The synthesis of nucleic acid sequences is well known in the art (CLONETECH 95/96 catalog, p.215-216, CLONETECH, Palo Alto East, MI, USA, 94303, Dow Circle 1020).

Clones containing the desired neurotoxin-alpha construct were grown overnight (" O / N ") by liquid culture in LB medium supplemented with ampicillin (100 μg / . The O / N culture is inoculated into the bulk culture at a dilution of about 1: 25 to 1: 250. Cells are grown to an optical density (" OD600 ") at 600 nm of 0.4 to 0.6. The isopropyl-beta-D-thiogalactopyranoside (" IPTG ") is then added at a final concentration of 1 mM to inactivate the lacI inhibitor and induce transcription from the lac inhibitor-sensitive promoter. The cells are then incubated for 3 to 4 hours. The cells are collected by centrifugation.

The cells are then agitated in 4M guanidine-HCl (pH 8) for 6 hours at 3-4 hours. Cell debris is removed by centrifugation and the supernatant containing neurotoxin-alpha is dialyzed against 50 mM sodium acetate buffer (pH 6) supplemented with 200 mM NaCl. Alternatively, the protein can be successfully restored by dialysis against 500 mM NaCl, 20% glycerol, 25 mM Tris HCl (pH 7.4) containing a protease inhibitor. After reconstitution, the protein can be purified by ion exchange, hydrophobic interaction and size exclusion chromatography. Alternatively, purified proteins can be obtained using affinity chromatography, such as antibody columns. The purified protein is stored at 4 ° C or stored at -80 ° C.

In some embodiments, it is also preferable to prepare expression constructs as described in this example to mutate one or more of the three cysteine residues in the neurotoxin-alpha polypeptide sequence. The cysteine residues in the neurotoxin-alpha polypeptide sequence are located at positions 147, 232 and 245 in the sequence as shown in SEQ ID NO: 2 and at positions 213 and 226 of the neurotoxin-alpha polypeptide sequence as shown in SEQ ID NO: 19 -Alpha SV polypeptide sequence does not have a residue corresponding to 143 to 160 amino acid residues of the neurotoxin-alpha polypeptide sequence, the cysteine corresponding to Cys-147 in the neurotoxin-alpha polypeptide sequence is a Neutrokine-alpha polypeptide Lt; / RTI > sequence).

Example 2:

Cloning, expression and purification in the Baculovirus expression system of the neurotoxin-alpha protein

In this example, cloned DNA encoding the extracellular domain of the naturally associated intracellular sequence and the extracellular domain of the protein from which the epidermal sequence was removed was inserted into baculovirus and inserted into a baculovirus reader and a baculovirus reader (Summers et al., A Manual The plasmid shuttle vector pA2GP is used to express the extracellular domain of the neurotoxin-alpha protein using standard methods described in the Methods of Baculovirus Vectors and Insect Cell Culture Precedures, Texas Agricultural Experimental Station Bulletin No.1555 (1987) . This expression vector can be used in combination with a strong polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcMNPV), followed by a secretory signal peptide (leader) of the baculovirus gp67 protein and a convenient polypeptide such as BamHI, XbaI and Asp718 Restriction sites. The polyadenylation site of simian virus 40 (" SV40 ") is useful for effective polyadenylation. For easy selection of recombinant viruses, the plasmid contains a < RTI ID = 0.0 > The? -Galactosidase gene derived from E. coli is contained in the same orientation, and then the polyadenylation signal of the polyhedrin gene is arranged. The inserted gene creates a viable virus expressing the cloned polynucleotide by contacting the viral sequences on both sides for cell-mediated homologous recombination with the wild-type virus DNA.

In addition to the above vectors, various baculovirus vectors such as pAc373, pVL941, and pAcIM1, which are familiar to those of skill in the art, may be used as long as they provide signals of appropriately located transcription, translation, secretion, etc., . Such vectors are described, for example, in Luckow et al., Virology 170: 31-39 (1989).

The intracellular domain sequence and the transmembrane domain sequence that are naturally associated with the AUG initiation codon and the amino acids Gln-73 to Leu-79 (SEQ ID NO: 2) shown in Figures IA and IB are deleted, The cDNA sequence encoding the N-terminal deletion form of the extracellular domain of the alpha protein is amplified using a PCR oligonucleotide primer corresponding to the 5 ' and 3 ' sequences of this gene. The 5 'primer comprises an underlined BamHI restriction site followed by 18 nucleotides of the extracellular domain sequence of the neurotoxin-alpha protein shown in Figures 1A and 1B and starts with the indicated N-terminal end of the extracellular domain of the protein -GTG GGA TCC CCG GGC AGA GCT GCA GGG C-3 '(SEQ ID NO: 14). The 3 'primer consists of the underlined BamHI restriction site followed by two subsequent stop codons and the sequence 5'-GTG GGA TCC TTA TTA CAG CAG containing 18 nucleotides complementary to the 3' TTT CAA TGC ACC-3 '(SEQ ID NO: 15).

In other embodiments, constructs designed to express the entire putative extracellular domain of neurotoxin-alpha (i. E., Amino acid residues Gln-73 to Leu-285) are preferred. Those skilled in the art will be able to use the polynucleotide sequences and the polypeptide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, to design the polynucleotide primers that generate such clones.

In another preferred embodiment, the pA2GP expression construct encodes the amino acid residues Ser-78 to Leu-285 of the neurotoxin-alpha polypeptide sequence shown in SEQ ID NO: 2.

The amplified fragments are separated from 1% agarose gel using a commercial kit (" Geneclean " BIO 101 Inc., La Jolla, CA). The fragment is subsequently digested with BamHI and purified again on 1% agarose gel. This fragment is denoted by F1.

The plasmid can be digested with the restriction enzyme BamHI and optionally dephosphorylated according to the usual procedures known in the art using small intestinal phosphatase. The DNA is then separated from the 1% agarose gel using a commercial kit (" Geneclean " BIO 101 Inc., La Jolla, CA). The vector DNA is represented by " V1 ".

Fragment F1 and dephosphorylated plasmid V1 are ligated with T4 DNA ligase. this. Coli HB101 or other suitable XL-1 Blue (Stratagene Cloning Systems product, La Jolla, CA). The coli host cells are transformed with the above linking mixture and plated on a culture plate. BamHI is used to digest DNA from each colony and the degradation products are analyzed by gel electrophoresis to identify bacteria containing the plasmid containing the human gene. The sequence of the cloned fragment is confirmed by DNA sequencing. This plasmid is designated pA2GP-Neutrokine-alpha.

Using 5 μg of plasmid pA2GP-Neutrokine-alpha and 1 μg of commercially available linearized baculovirus DNA ("BaculoGold ™ baculovirus DNA", Pharmingen product, San Diego, CA), Felgner et al., Proc. Natl.Acad.Sci. USA 84: 7413-7417 (1987). 1 μg of BaculoGold ™ virus DNA and 5 μg of plasmid pA2GP neurotoxin-alpha are mixed in a sterile well of a microtiter plate containing 50 μl of serum-free grace medium (Life Technologies Inc., Gottsburg, MA) . Then, 10 μl of lipofectin and 90 μl of grace medium were added and mixed, followed by incubation at room temperature for 15 minutes. Thereafter, the transfection mixture is added dropwise to Sf9 insect cells (ATCC CRL1711) inoculated on a 35 mm tissue culture plate containing 1 ml of the serum-deficient grace medium. The culture plate is incubated at 27 DEG C for 5 hours. Then, the transfection solution is separated from the culture plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. The culture is then continued for 4 days at 27 < 0 > C.

After 4 days the supernatant is collected (as described in Summers and Smith, supra) and subjected to plaque analysis. Agarose gels supplemented with " Blue Gal " (Life Technologies Inc., Rockville, Md., USA) can be used to easily identify and isolate gal- expressing clones producing blue stained plaques A detailed description of " plaque analysis " is also provided in User Guides p 9-10 for Insect Cell Culture and Baculoviruses, published by Life Technologies Inc). After moderate incubation, the blue stained plaque is collected with a tip of a micropipette (eg Eppendorf). The agar containing the recombinant virus was resuspended in a microfuge tube containing 200 mu l of Grace's medium and the suspension containing the recombinant baculovirus was used to infect Sf9 cells inoculated into a 35 mm culture dish. After 4 days, the supernatant of the culture dish is collected and stored at 4 캜. This recombinant virus is called V-Neutrokine-Alpha.

To prove the expression of the neurotoxin-alpha gene, Sf9 cells are grown in Grace's medium supplemented with 10% heat-activated FBS. The cells are infected with a multiplicity of infection (" MOI ") of about 2 using recombinant baculovirus V-neurotoxin-alpha. If radioactively labeled proteins are required, the medium is removed after 6 hours and replaced with SF900 II medium (Life Technologies Inc.) which is deficient in methionine and cysteine. 42 hours after, ³⁵ S- methionine and 5μCi ³⁵ S- cysteine (Amersham product) is added 5μCi. The cells are further cultured for 16 hours and then harvested by centrifugation. Proteins in the supernatant as well as intracellular proteins are analyzed by SDS-PAGE and subsequent automatic radioactivity photographs (if radioactively labeled).

A microsequence analysis of the amino acid sequence at the amino terminus of the purified protein can be used to determine the amino terminal sequence of the extracellular domain of the purified protein and the cleavage point and length of the secretory signal peptide.

As a specific experimental example, recombinant neurotoxin-alpha is purified from baculovirus-infected Sf9 cell supernatant as follows. The insect cells are grown in EXCEL401 medium (JRH Scientific) supplemented with 1% (v / v) fetal calf serum. After 92 hours of infection, the collected supernatant is clarified by centrifugation at 18,000 x g and subsequent filtration at a height of 0.45 m. Also, a degreasing filtration step can be used to remove lipid contaminants and consequently to increase the initial yield of the neurotoxin-alpha protein.

The supernatant is injected onto a set of Poros HS-50 / HQ-50 in series. Alternatively, Toyopearl QAE, Toyopearl Super Q (Tosohass), Q-Sepharose (Pharmacia) and similar resins may be used. This step is used as a negative purification step to remove strong anion-binding contaminants. The HS / HQ effluent is adjusted to pH 7.5 with 1M Tris-HCl (pH 8), diluted with an equal volume of 50 mM Tris-HCl (pH 8) and then injected onto a Poros PI-20 or PI-50 column. The PI column was first washed with 4 column volumes of 75 mM sodium chloride in 50 mM Tris-HCl (pH 7.5) and then with 3 to 5 column volumes of 300 mM, 750 mM, 1500 mM sodium chloride in 50 mM Tris-HCl (pH 7.5) Lt; / RTI > The neurotoxin-alpha protein appears as a 17 KD band on reducing SDS-PAGE and is present in the 0.75 M to 1.5 M sodium chloride fraction.

The PI fraction is further purified through a Sephacryl S100 HR (Pharmacia) size exclusion column equilibrated with 0.15 M sodium chloride, 50 mM sodium acetate (pH 6). The S200 fraction is mixed with sodium chloride to a final concentration of 3M and injected onto a Toyopearl Hexyl 650C (Tosohass) column. The hexyl column is eluted with 5 to 15 column volumes using a linear gradient of 3M to 0.05M sodium chloride in 50 mM sodium acetate (pH 6). In addition, the sodium chloride gradient in the hexyl chromatography step may be replaced with a 1M to 0M gradient of ammonium sulfate in 50mM sodium acetate (pH 6). The fractions containing the purified neurotoxin-alpha as analyzed via SDS-PAGE are combined and dialyzed against a buffer containing 150 mM sodium chloride, 50 mM sodium acetate (pH 6).

The final purified neurotoxin-alpha protein expressed in the baculovirus system as described herein has an N-terminal sequence beginning with the amino acid residue Ala-134 described in SEQ ID NO: 2. RP-HPLC analysis shows a single peak with a purity of> 95%. Endotoxin levels are below the detection limit in LAL assays.

In another embodiment, the recombinant neurotoxin-alpha is purified from baculovirus-infected Sf9 cell supernatants containing 0.25% bovine serum as follows.

Sf9 supernatant is collected by centrifugation at 18,000xg. The supernatant is then treated with 10 mM calcium chloride in weakly alkaline conditions for 10 to 15 minutes and then centrifuged and filtered to a depth of 0.22 μm. The resulting Sf-9 cell supernatant is diluted 2-fold and injected onto a Poros PI-50 column (PE Biosystems). The column is equilibrated with 50 mM Tris (pH 7.4). The PI-50 column was washed with 50 mM Tris (pH 7.4) 1 CV and then eluted with 1.5 M NaCl 3 CV in 50 mM NaOAc (pH 6). The PI fraction is injected onto a Sephacryl S200 column equilibrated with 50 mM NaOAc (pH 6), 125 mM NaCl. The S200 fraction was mixed with the salt to a final concentration of 0.7 M ammonium sulfate and 0.6 M NaCl and loaded on a Toyopearl Hexyl 650C column (Toso Haas, Calif.) Equilibrated with buffer containing 0.6 M NaCl, 0.7 M ammonium sulfate in 50 mM NaOAc Product). The column is then washed with 2 CV of the same buffer. Thereafter, recombinant neurotoxin-alpha was eluted stepwise with 3 CV of 50 mM NaOAc (pH 6), followed by 2 CV of 20% ethanol. The recombinant neurotoxin-alpha protein was eluted at the end of the ammonium sulfate (0.3M to 0M salt) gradient. The appropriate fractions were pooled, dialyzed against buffer containing 50 mM NaOAc (pH 6) and then passed through a Poros 50 HQ column. The HQ effluent was diluted to 4ms and injected onto a Toyopearl DEAD 650M column and eluted with 25mM NaCitrate, 125mM NaCl.

In another embodiment, recombinant neurotoxin-alpha is expressed and purified using a baculovirus vector system in Sf + insect cells.

First, the amino acid residues Ser-78 through Leu-285 (the amino acid residues Ser-78 through Leu-285 of the neurotoxin-alpha polypeptide sequence shown in SEQ ID NO: 2) of the neurotoxin-alpha polypeptide shown in FIG. 1A and FIG. Exactly the same) is subcloned into the baculovirus transfer construct PSC to generate a baculovirus expression plasmid. The pA2GP transcription vector from pVL941 contains a downstream cloned lacZ gene of the gp67 signal peptide, a modified multi-cloning site and a Drosophila thermal shock promoter for selection with blue plaques. For cloning, the PSC signal peptide cipher sequence was cloned into the neurotoxin-alpha coding sequence Ala-134 (SEQ ID NO: 2 and Figs. 1A and 1B) using Neutrokine-alpha (SEQ ID NO: 2) and pA2GP vector sequences And the fusion is inserted into the PSC baculovirus transfer plasmid. In this method, a two-step polymerase chain reaction (PCR) procedure is used. First, a primer is designed to amplify the Neutrokine-alpha sequence. The 5 'primer contained the sequence encoding Ala-134 and the following residues (5'-GGT CGC CGT TTC TAA CGC GGC CGT TCA GGG TCC AGA AG-3'; SEQ ID NO: 31) and then the PSC signal peptide C- Encoding sequence. The 3 'primer (5'-CTG GTT CGG CCC AAG GTA CCA AGC TTG TAC CTT AGA TCT TTT CTA GAT C-3'; SEQ ID NO: 32) encodes the reverse complement of the pA2GP vector sequence downstream of the neurotoxin- , Followed by a KpnI restriction enzyme site and a spacer sequence (sequence for increasing cleavage efficiency by KpnI). PCR is performed with pA2GP containing the neurotoxin-alpha plasmid template and primers O-1887 and O-1888, and the PCR products obtained are purified using standard techniques.

The secondary PCR reaction is carried out using PSC baculovirus transfer plasmid pMGS 12 as a template. The pMGS12 plasmid consists of the AcNPV EcoRI " I " fragment inserted into pUC8 and the polyhedrin coding sequence followed by the polylinker site followed by the ATG start codon replaced by the PSC signal peptide. The PCR reaction contained 5 'primers (5'-CTG GTA GTT CTT CGG AGT GTG-3'; SEQ ID NO: 33) annealed to AcNPV ORF603 upstream of the pMGS12, unique NgoM IV and EcoR V sites as template and PSC signal peptide 3 'primer (5'-CGC GTT AGA AAC GGC GAC C-3'; SEQ ID NO: 34) annealed at the 3 'end of the coding sequence is used.

The PCR product is further combined with the PSC signal peptide-polyhedrin upstream region PCR product to generate a PCR product in which the PSC signal peptide is fused seamlessly to the Ala-134 of the neurotoxin-alpha coding sequence. Because the 3 'end of the PSC signal peptide PCR product (pMGS12 / O-959 / O-1044) overlaps the 5' end of the neurotoxin-alpha PCR product made with primer O-1887 / O-1888, The products were combined and duplicated elongated by PCR using primers O-959 and O-1888.

The resulting elongated PCR product containing the PSC signal peptide fused to the resulting neurotoxin-alpha sequence is then inserted into the baculovirus transfer plasmid pMGS12. The PCR product was digested with NgoM IV and Kpn I, and the fragment was purified and ligated into NgoM IV-Kpn I-cleaved pMGS12. Use a combination mixture to make the comp. After colony DH5? Cells are transformed, colonies are collected to prepare plasmid DNA micro-preparations. Plasmid DNA was analyzed by restriction enzyme digestion to identify several positive clones obtained after each ligation reaction, and three clones (pAcC9669, pAcC9671 and pAcC9672) were selected for mass plasmid purification. The resulting plasmid DNA is subjected to DNA sequencing to identify the neurotoxin-alpha insert and sequenced.

The following steps relate to methods for recovering and purifying recombinant Neutrokine-alpha from Sf + insect cells. These steps are carried out at 2-8 占 폚 unless otherwise indicated.

collection

Step 1. Treatment with CaCl ₂

Sf + cell supernatant was obtained by centrifugation at 8,000xg. Recovery buffer-1 (1M CaCl ₂ ) is added to the supernatant to a final concentration of CaCl ₂ of 10 mM. (In another preferred embodiment, 1M ZnCl ₂ is used instead of 1M CaCl _2. ) The pH of this solution is adjusted to 7.7 ± using Recovery Buffer-2 (1M Tris pH 8 (± 0.2)). The solution is incubated for 15 minutes and centrifuged at 8,000 xg.

refine

Step 1. Chromatography on a Poros PI-50 column

The Sf + cell supernatant is injected onto a Poros PI-50 column (PE Biosystem). The column is equilibrated with PI-I buffer (50 mM Tris, 50 mM NaCl, pH 7.4 (± 0.2)). The PI-50 column is washed with 1 to 2 CV of PI-1 buffer and then eluted with a 3 CV linear gradient with PI-2 buffer (50 mM sodium citrate, pH 6 (± 0.2)). Elution is monitored by ultraviolet (UV) absorbance at 280 nm. Appropriate fractions are collected according to the peak of the eluate and analyzed by SDS PAGE. The appropriate fraction is collected.

Step 2. Chromatography on a Toyopearl Hexyl 650C column

The PI collection was mixed with a salt having a final concentration of 0.7 M (NH ₄ ) ₂ SO ₄ and dissolved in HIC-1 buffer (50 mM NaOAc, 0.6 M NaCl, 0.7 M (NH ₄ ) ₂ SO ₄ pH 6 (± 0.2) Lt; RTI ID = 0.0 > Toyopearl < / RTI > Hexyl 650C (Toso Haas) column. The column is then washed with 2 CV of HIC-1 buffer. The recombinant neurotoxin-alpha was then eluted stepwise with 3 to 5 CV of HIC-2 buffer (50 mM NaOAc pH 6.0 (± 0.2)) and 2 CV 20% ethanol rinse. Elution is monitored by UV absorbance and conductivity at 280 nm. Fractions along the eluent peak are collected and analyzed by SDS-PAGE. Then, the appropriate fraction is collected.

Step 3. Chromatography on SP Sepharose FF

The hexyl fraction was dialyzed and adjusted to pH 4.5 with SP-1 buffer (50 mM sodium acetate, pH 4.5 (+ -0.2)) and diluted to 4 ms before adding SP-1 buffer (50 mM sodium acetate, pH 4.5 (Cation Exchange, Pharmacia) columns equilibrated with PBS. The recombinant neurotoxin-alpha protein is eluted from the SP column by SP-2 buffer (50 mM sodium acetate, pH 5.5 (± 0.2)) at pH 5.5. Elution is monitored by ultraviolet absorbance at 280 nm. Fractions along the eluent peaks are collected and analyzed by SDS PAGE. The appropriate fraction is collected.

Step 4. Dialysis of recombinant neurotoxin-alpha

The SP fraction is injected into a 6- to 8-kd cut-off membrane apparatus and then dialyzed overnight or dentally filtered using dialysis buffer (10 mM sodium citrate, 140 mM sodium chloride, pH 6 (± 0.2)) overnight.

Step 5. Filtration and charging

The protein concentration of the recombinant neurotoxin-alpha solution derived from step 4 is determined by non-synuclein acid (BCA) protein assay. The recombinant neurotoxin-alpha preparation is adjusted to a final protein concentration with the appropriate buffer and filtered under controlled conditions. The filtrate (bulk water) is stored in a suitable sterile container at -20 占 폚 or lower.

In a specific embodiment, the neurotoxin-alpha protein of the present invention produced as described below is adjusted to a final protein concentration of 1-5 mg / ml and is supplemented with 10 mM sodium citrate, 140 mM sodium chloride, pH 6.0 ) And stored at -20 < 0 > C or lower in a Type 1 glass bayer.

During the chromatographic treatment, the process is monitored by UV absorbance at 280 nm. Where available, the conductivity, pH, and pH are monitored using chromatographic intermediates in the process and monitored by SDS and / or RP-HPLC.

The column and the purification apparatus are cleaned and sterilized using 0.2 M or 0.5 M NaOH followed by deionized water and subsequently 0.1 M or 0.5 M acetic acid. The column device and the purification device are rinsed with deionized water and stored in a suitable storage solution, if necessary. Prior to use, the device was equilibrated with an appropriate buffer (as described herein or equilibrated as known in the art).

In another preferred embodiment, 1M ZnCl ₂ is used instead of 1M CaCl ₂ in step 1 of the abovementioned recovery process. In this embodiment, a combination of ZnCl ₂ and CaCl ₂ may also be used. Various combinations of 0.1M ZnCl ₂ and 0.9M CaCl ₂ can be used for the recovery of the recombinant neurotoxin-alpha protein, for example, a combination of 0.1M ZnCl ₂ and 0.9M CaCl ₂ , 0.2M ZnCl ₂ , A combination of 0.8M CaCl ₂ , a combination of 0.3M ZnCl ₂ and 0.7M CaCl ₂ , a combination of 0.4M ZnCl ₂ and 0.6M CaCl ₂ , a combination of 0.5M ZnCl ₂ and 0.5M CaCl ₂ , a 0.6M ZnCl 2 ₂ and 0.4 M CaCl ₂ , a combination of 0.7 M ZnCl ₂ and 0.3 M CaCl ₂ , a combination of 0.8 M ZnCl ₂ and 0.2 M CaCl ₂ , a combination of 0.9 M ZnCl ₂ and 0.1 M CaCl ₂ , and the like And not limited to this. However, the presence of EDTA interferes with the recovery process. Moreover, ZnCl ₂ and / or CaCl ₂ present in Recovery Buffer-1 may induce the formation of high molecular weight (or molecular mass) neurotoxin-alpha oligomers in large quantities.

Example 3: Cloning and expression of neurotoxin-alpha in mammalian cells

General mammalian expression vectors include promoter elements that mediate transcription initiation of mRNA, promoter coding sequences, and signals necessary for transcription termination and polyadenylation of transcripts. Other factors include the enhancer, the Kozak sequence, and the RNA splicing donor site and the intervening sequence adjacent to the receptor site. High-efficiency transcription can be achieved by early promoters of SV40-derived early and late promoters, long-term repeat (LTR) derived from retroviruses such as RSV, HTLVI, HIVI and cytomegalovirus (CMV). However, cell factors may also be used (e. G., The human actin promoter). Suitable expression vectors suitable for use in the practice of the present invention include vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC 12MI (ATCC 67109) do. Examples of host cells that can be used include mammalian HeLa, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells, Chinese hamster ovary (CHO) Cells and HEK293 cells.

Alternatively, the gene may be expressed in a stable cell line containing a gene introduced into the chromosome. In addition, co-transfection with selectable markers such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of transfected cells.

In addition, the transfected gene can be amplified to express a large amount of the encoded protein. DHFR (dihydrofolate reductase) markers are useful for developing cell lines that have hundreds or even thousands of copies of the gene of interest. Another useful selection marker is the enzyme glutamine synthetase (GS) (Murphy et al., Biochem., J 227: 277-279 (1991); Bebbington et al., Bio / Technology 10: 169-175 (1992) . Using these markers, mammalian cells are grown in selective medium and cells with the highest resistance are selected. This cell line contains an amplified gene integrated into the chromosome. Chinese hamster ovary (CHO) cells and NSO cells are also useful for protein production.

Expression vectors pC1 and pC4 were transfected with a strong promoter (LTR) of Lewis sarcoma virus (Cullen et al., Molecular and Cellular Biology, 438-447 (March, 1985)) + a fragment of CMV-enhancer (Boshart et al., Cell 41 : 521-530 (1985)). Multiple cloning sites, such as restriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate cloning of the gene. The vector further comprises a polyadenylation signal and a termination signal of the 3 ' intron, the rat preproinsulin gene.

Example 3 (a): Cloning and expression in COS cells

Expression plasmid pNutrocin-α-HA is prepared by cloning a part of the deposited cDNA encoding the extracellular domain of the protein into the expression vector pcDNAI / Amp or pcDNAIII. The extracellular domain is fused to the secretory leader sequence of the human IL-6 gene to produce the polypeptide in a soluble secretory form.

The expression vector pcDNAI / amp (1) This is effective for propagation in E. coli and other prokaryotic cells. Replication of Coli Origin; (2) ampicillin resistance gene for selecting plasmid-containing prokaryotic cells; (3) SV40 replication origin for propagation in eukaryotic cells; (4) CMV promoter, polylinker, SV40 intron; (5) Several codons encoding the hemagglutinin fragment (i. E., The " HA " tag to facilitate purification) and then cDNA are advantageously placed under the control of the expression of the CMV promoter and SV40 And include stop codons and polyadenylation signals to be operably linked to intron and polyadenylation signals. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein described in Wilson et al., Cell 37: 767 (1984). When the HA tag is fused to the target protein, the recombinant protein can be easily detected and recovered using an antibody recognizing the HA epitope. pcDNAII may also contain neomycin markers for selection.

The DNA fragment encoding the extracellular domain of the neurotoxin-alpha polypeptide is cloned into the polylinker region of the vector such that the expression of the recombinant protein is induced by the CMV promoter. The plasmid construction method is as follows. The neurotoxin-alpha cDNA of the deposited clone is shown in Fig. Amplification is carried out using a primer containing an advantageous restriction site as described above with respect to the construction of a vector for expressing neurotoxin-alpha in E. coli. Suitable primers include those used in this Example. A sequence encoding the secretory leader peptide derived from the human IL-6 gene, and an extracellular domain of the neurotoxin-alpha protein, including the underlined BamHI site, the Kozak sequence, the AUG initiation codon, the human IL- 'Primers have the following sequences: 5'-GCG GGA TCC GCC ACC ATG AAC TCC TTC TCC ACA AGC GCC TTC GGT CCA GTT GCC TTC TCC CTG GGG CTG CTC CTG GTG TTG CCT GCT GCC TTC CCT GCC CCA GTT GTG AGA CAA GGG GAC CTG GCC AGC-3 '(SEQ ID NO: 16). The 3 'primer, which contains 18 nucleotides complementary to the underlined BamHI restriction site and the 3' coded sequence immediately before the stop codon, has the sequence: 5'-GTG GGA TCC TTA CAG CAG TTT CAA TGC ACC- 3 '(SEQ ID NO: 17).

The PCR amplified DNA fragment and the vector pcDNAI / Amp are digested with BamHI and ligated. Using the connecting mixture, The coli strain SURE (product of Stratagene Cloning Systems, La Jolla North Torrey Pines Road, CA 92037, California, USA) was transformed, and the transformed culture was plated on a plate of ampicillin medium and incubated to amplify ampicillin resistant colonies . Plasmid DNA is isolated from resistant colonies and subjected to restriction analysis or other techniques for the presence of fragments encoding the neurotoxin-alpha extracellular domain.

DEAE-DEXTRAN was used to express recombinant neurotoxin-alpha as described in Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold Spring Laboratory Press, Cold Spring Harbor, New York COS cells are transfected with an expression vector as described above. Cells are cultured under conditions that allow expression of neurotoxin-alpha through this vector.

Expression of neurotoxin-alpha-HA fusion proteins is described in Harlow et al., Antibodies: A Laboratory Manual, 2nd Ed .; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988)], and then detected by radioimmunoprecipitation. Two days after transfection, cells are incubated for 8 hours in medium containing ³⁵ S-cystein and labeled. Cells and medium were collected and the cells were resuspended in RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM TRIS , pH 7.5). HA-specific monoclonal antibodies are used to precipitate proteins from cell culture media and culture media. The precipitated proteins are analyzed by SDS-PAGE and automated radiography. The expected size of expression product was observed in cell culture supernatants and not in negative control.

Example 3 (b): Cloning and Expression in CHO Cells

The vector pC4 is used to express the neurotoxin-alpha protein. Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146). A portion of the deposited cDNA encoding the extracellular domain is fused to the secretory leader sequence of the human IL-6 gene to produce soluble secreted neurokin-alpha polypeptide. The vector plasmid contains the mouse DHFR gene located under the control of the SV40 early promoter. Chinese hamster ovary cells or other cells transfected with this plasmid and deficient in dehydrofolate activity can be selected by proliferation of cells in a selection medium (alpha-MEM, Life Technologies) supplemented with methotrexate, a chemotherapeutic agent. Amplification of the DHFR gene in cells resistant to methotrexate (MTX) is well known in the literature (e.g., Alt, FW, Kellems, RM, Bertino, JR, and Schimke, RT, 1978, J. Biol. Chem. Acta, 1097: 107-143, Page, MJ and Sydenham, MA 1991, Biotechnology 9: 64-68). Cells that proliferate at increasing concentrations of MTX overexpress DHFR, the target enzyme, as a result of amplification of the DHFR gene, expressing resistance to the drug. If the second gene is bound to this DHFR gene, it is usually co-amplified and overexpressed. It is well known in the art that this method can be used to develop cell lines harboring over 1000 copies of amplified genes. As a result, when methotrexate is removed, a cell line in which an amplified gene has been introduced into one or more chromosomes of the host cell is obtained.

Plasmid pC4 expresses a strong promoter of the long terminal repeat (LTR) of Lute sarcoma virus (Cullen, et al., Molecular and Cellular Biology, March 1985: 438-447) + human cytomegalovirus (CMV) (Boshart et al., Cell 41: 521-530 (1985)). Downstream of the promoter, there is a single restriction enzyme cleavage site that can integrate the gene: BamHI, XbaI, and Asp718. In addition, the plasmid contains the 3 'intron and polyadenylation site of the rat prepro insulin gene behind this cloning site. In addition, other high efficiency promoters may be used for expression, such as the human? -Actin promoter, the SV40 early or late promoter, or other retrovirus-derived long terminal repeat, such as HIV and HTLVI. Clontech's Tet-Off and Tet-On gene expression systems and similar systems can be used to express neurotoxin-alpha in a controlled manner in mammalian cells (Gossen, M., & Bujard, H. 1992, Proc Natl. Acad. Sci USA 89: 5547-5551). For polyadenylation of mRNA, other signals from, for example, human growth hormone or globin genes may be used. Stable cell lines harboring the desired gene integrated into the chromosome may also be selected for co-transfection with selectable markers such as gpt, G418 or hygromycin. It is advantageous to first use one or more of the selection markers, such as G418 and methotrexate.

Plasmid pC4 is digested with restriction enzyme BamHI and then dephosphorylated using bovine intracellular phosphatase according to procedures known in the art. The vector is then separated from the 1% agarose gel.

The DNA sequence encoding the extracellular domain of the neurotoxin-alpha protein is amplified using a PCR oligonucleotide primer corresponding to the 5 ' and 3 ' sequences of the gene. A sequence encoding the secretory leader peptide derived from the human IL-6 gene, and a 5 'coding region of the 5' coding region of the extracellular domain of the neurotoxin-alpha protein. 'Primers have the following sequences: 5'GCG GGA TCC GCC ACC ATG AAC TCC TTC TCC ACA AGC GCC TTC GGTCCA GTT GCC TTC TCC CTG GGG CTG CTC CTG GTG TTG CCT GCT GCC TTC CCT GCC CCA GTT GTG AGA CAA GGG GAC CTG GCC AGC-3 '(SEQ ID NO: 16). The 3 'primer containing 18 nucleotides complementary to the underlined BamHI sequence and the 3' coding sequence immediately before the stop codon has the following sequence: 5'-GTG GGA TCC TTA CAG CAG TTT CAA TGC ACC-3 ' (SEQ ID NO: 17).

The amplified fragment is digested with restriction enzyme BamHI and purified again on 1% agarose gel. The isolated fragments and the dephosphorylated vector are ligated with T4 DNA ligase. Then, this. Coli HB101 or XL-1 blue cells are transformed and bacteria containing fragments inserted into the plasmid pC4 are identified using restriction enzyme analysis or the like.

Chinese hamster ovary cells deficient in active DHFR gene are used for transfection. 5 μg of the expression plasmid pC4 is cotransfected with 0.5 μg of the plasmid pSVneo using lipofectin (see Felgner et al., Supra). The plasmid pSV2-neo contains a Tn5-derived neo gene encoding an enzyme that confers resistance to a group of antibiotics, including G418, a major selectable marker. Cells are inoculated with alpha-MEM supplemented with 1 mg / ml G418. Two days later, the cells were trypsinized and inoculated into a hybridoma cloning plate (Greiner, Germany) injected with? -MEM supplemented with 10 ng / ml of methotrexate, 25 ng / ml or 50 ng / ml + G410 . After about 10 to 14 days, the monoclonal is trypsinized and inoculated into a 6-well Petri dish or 10 ml flask using various concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing in the highest concentration of methotrexate were again transferred to a new plate of 6 wells containing a higher concentration of methotrexate (1 μM, 2 μM, 5 μM, 10 μM, 20 μM). The same procedure is repeated until a clone growing at a concentration of 100-200 μM is obtained. Expression of the desired gene product is analyzed, for example, by SDS-PAGE and Western blot or reverse phase HPLC analysis.

Six or more neurotoxin-alpha expression constructs have been produced by the inventors of the present invention, and neurotrophin-alpha and / or neurotoxin-alpha SV polypeptides of various sizes have been readily generated in a variety of expression systems. Expression constructs were: (1) pNa.A71-L285 (amino acid residues Ala-71 to Leu-285 expression), (2) pNa.A81-L285 (amino acid residues Ala-81 to Leu-285 expression) (3) pNa.L112-L285 (expression of amino acid residues Leu-112 to Leu-285), (4) pNa.A134-L285 (expression of amino acid residues Ala-134 to Leu-285), (5) pNa.L147-L285 (Amino acid residues Leu-147 to Leu-285 expression), and (6) pNa.G161-L285 (amino acid residues Gly-161 to Leu-285 expression).

In a preferred embodiment, expression constructs are used to express a variety of neurotoxin-alpha muteins in bacterial systems, baculovirus systems, and mammalian systems.

In another preferred embodiment, the construct comprises a heterologous polypeptide, e. G., A signal peptide from human IL-6, a signal peptide from CK-? 8 (disclosed in published PCT application PCT / US95 / 09058, Amino acids -21 to -1 of the sequence) or a neurotoxin-alpha polypeptide fragment fused to the N-terminal and / or C-terminal to the human IgG Fc region. Other sequences known in the art may also be used.

Example 4: Tissue distribution of neurotoxin-alpha mRNA expression

To examine the expression of the neurotoxin-alpha gene in human tissues, Northern blot analysis is carried out using the method described in the above-mentioned Sambrook et al. A cDNA probe containing the entire nucleotide sequence (SEQ ID NO: 1) of the neurotoxin-alpha protein is labeled with ³² P according to the manufacturer's instructions using the redi prime ™ DNA labeling system (Amersham Life Sciences). After labeling, the probe is purified using a CHROMA SPIN-100 ™ column (Clontech Laboratories, Inc) according to the manufacturer's protocol PT1200-1. Neutrokine-alpha and / or neurotoxin-alpha mRNA is examined in a variety of human tissues using purified labeled probes.

Multi-tissue Northern (MTN) blots containing various human tissues (H) or human immune system tissues (IM) were obtained from Clontech and ExpressHyb ™ hybridization solution (Clontech) was used according to manufacturer's protocol PT1190-1 And irradiated with the labeled probe. After hybridization and washing, the blots are mounted and exposed to film at -70 < 0 > C overnight. The film is then developed according to standard procedures.

A panel of multi-tissue Northern blots is probed to determine the pattern of neurotoxin-alpha and / or neurotoxin-alpha expression. As a result, significant expression of a single 2.6 kb mRNA was observed in peripheral blood leukocytes, spleen, lymph nodes and bone marrow, and a detectable expression was observed in placenta, heart, lung, fetal liver, thymus and pancreas. Analysis of the cell line panel demonstrated high expression of neurotoxin-alpha and / or neurotoxin-alpha and detectable expression in K562 in HL60 cells and no expression in Raji, HeLa or MOLT-4 cells. In conclusion, neurotoxin-alpha and / or neurotoxin-alpha mRNA expression appears to be concentrated in the immune system.

Example 5: Gene therapy using an endogenous neurotoxin-alpha gene

Another method of gene therapy described in the present invention is described in U. S. Patent No. 5,641, 670 (July 26, 1997); International Publication No. WO 96/29411 (published Sep. 26, 1996); International Publication No. WO 94/12650 (published Aug. 4, 1994); Koller et al., Proc. Natl. Acad. Sci. USA 86: 8932-8935 (1989); And Zijlstra et al., Nature 342: 435-438 (1989)). The promoter and the endogenous neurotoxin-alpha sequence are operably linked to each other through homologous recombination. This method involves activating a gene that is present in the target cell but is not expressed in the cell or expressed at a lower concentration than is required. A polynucleotide construct comprising a target sequence and a promoter homologous to a 5'-non-coding sequence of endogenous neurotoxin-alpha adjacent to the promoter is prepared. The target sequence is sufficiently close to the 5 ' end of neurotoxin-alpha so that upon homologous recombination the promoter is operably linked to the endogenous sequence. Promoters and target sequences can be amplified using PCR. Preferably, the amplified promoter comprises a unique restriction enzyme site at the 5 'end and the 3' end. Also, the 3 'end of the first target sequence contains the same restriction enzyme site as the 5' end of the amplified promoter and the 5 'end of the second target sequence contains the same restriction site as the 3' end of the amplified promoter .

The amplified promoter and the amplified target sequence are digested with appropriate restriction enzymes and then treated with phosphatase in the digestive tract. The degraded promoter and the degraded target sequence are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions suitable for linking the two fragments. The construct is size fractionated on an agarose gel and then purified by phenol extraction and ethanol precipitation.

In this example, the polynucleotide construct is administered as a polynucleotide excreted through electroosmosis. However, polynucleotide constructs may also be administered in conjunction with transfection agents such as liposomes, viral sequences, viral particles, precipitating agents, and the like. Such delivery methods are well known in the art.

Once the cells are transfected, homologous recombination will occur that will operably bind the promoter to the endogenous neurokinin-alpha sequence. As a result, neurotoxin-alpha is expressed in the cells. Expression can be detected by immunological staining or other methods known in the art.

Fibroblasts are obtained from the subject through skin biopsy. The tissue obtained is placed in DMEM + 10% fetal calf serum. The fibroblasts of the exponential growth phase or early stasis are trypsinized and cleaned from the plastic surface using nutrient media. A certain amount of the cell suspension is collected for counting, and the remaining cells are centrifuged. The supernatant is removed by aspiration and the pellet is resuspended in 5 ml of electrophoresis buffer (20 mM HEPES pH 7.3, 137 mM NaCl, 5 mM KCl, 0.7 mM Na ₂ HPO ₄ , 6 mM Dextrose). Cells are separated by centrifugation, and supernatant is removed by aspiration. The cells are resuspended in an electrotransmission buffer containing 1 mg / ml acetylated bovine serum albumin. The final cell suspension contains about 3 x ¹⁰ cells / ml. Electroinfiltration must be performed immediately after reconstitution.

Plasmid DNA is prepared according to standard techniques. For example, the plasmid pUC18 (MBI Fermentas, Amherst, NY) is digested with HindIII to construct a plasmid targeting the neurotoxin-alpha locus. The CMV promoter is amplified by PCR using the XbaI site on the 5 'end and the BamHI site on the 3' end. The two neurotrophin-alpha non-coding sequences are amplified by PCR as follows: The first neurotrophin-alpha non-coding sequence (Neutrokin-alpha fragment 1) contains the HindIII site at the 5 ' Amplified using the Xba site; The second neurotrophin-alpha non-coding sequence (neurotoxin-alpha fragment 2) is amplified using the BamHI site at the 5 'end and the HindIII site at the 3' end. The CMV promoter and the neurotoxin-alpha fragment are digested with the appropriate enzymes (CMV promoter-XbaI and BamHI, neurotoxin-alpha fragment 1-XbaI, neurotoxin-alpha fragment 2-BamHI) and ligated together. The resulting ligation product is digested with HindIII and ligated with the HindIII-digested pUC18 plasmid.

Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap (BioRad). The final DNA concentration is generally at least 120 μg / ml. Then, 0.5 ml of the cell suspension (containing about 1.5 X 10 ⁶ cells) is added to the cuvette and the cell suspension and DNA solution are gently mixed. Electrophoresis is carried out using a Gene-Pulser device (BioRad). Capacitance and voltage are set to 960μF and 250-300V, respectively. As the voltage increases, the cell viability decreases, but the proportion of viable cells stably feeding the introduced DNA into the genome increases sharply. A pulse time of about 14 to 20 msec should be observed for these variables to be provided.

Electroinfected cells are maintained at room temperature for about 5 minutes, and then the contents in the cuvette are carefully taken with a sterile pre-pipette. The cells are directly added to 10 ml pre-warmed nutrient medium (DMEM containing 15% bovine serum) in a 10-cm culture dish and incubated at 37 ° C. The next day, the medium is aspirated off, replaced with 10 ml of fresh medium, and further cultured for 16 to 24 hours.

The thus manipulated fibroblasts are then propagated alone or in fusion state on cytodex 3 microcarrier beads and then injected into the host. This fibroblast produces a protein product. Fibroblasts can be injected into the patient as described above.

Example 6: Tumor necrosis factor acting as B lymphocyte stimulating factor A new member of the ligand system, Neutrokine-alpha

TNF-alpha (16.2%) (Pennica, D., et al., Nature 312,724 (1998)), APRIL (28.7%) (Hahne, M., et al., J. Exp. Med. Human ligand binding domain that shares significant homology within the predicted extracellular receptor-ligand binding domain for LT-alpha (-729 (1984)) and LT- alpha (14.1%) (Gray, Nature 312, 721-724 A 285 amino acid protein is identified in a monocyte-derived cDNA library (Fig. 7A). We express this as cytokine neurotoxin-alpha (also referred to as B lymphocyte stimulating factor (BLyS) on the basis of biological activity). The hydrophobic analysis of the neurotoxin-alpha protein sequence revealed a potential dermal dichroic domain between amino acid residues 47 and 73 preceding the non-hydrophobic amino acid, indicating that neurotoxin-alpha, like other members of the TNF ligand system, Binding protein (Cosman, D., Stem. Cells. 12: 440-55 (1994)). Expression of this cDNA in mammalian cells (HEK 293 and Chinese hamster ovary) and Sf9 insect cells identifies soluble forms of 152 amino acids with N-terminal sequence starting with alanine residue at amino acid 134 (arrow in FIG. 7A) . By restoring the mass to charge ratio, the mass of neurotoxin-alpha was found to be 17,038 daltons, which corresponds to the expected mass (17037.5 daltons) for the 152 amino acid proteins with single disulfide bonds.

As a result of using the human / hamster somatic hybrid and radiation-hybrid mapping panels, the gene coding for neurotoxin-alpha has previously been unbound to any component of the TNF phase and gene (Cosman, D. Stem. Cells. 12 < / RTI >: 440-55 (1994)) marker SHGC-36171, which maps to chromosome 13q34.

The expression profile of neurotoxin-alpha is measured by Northern blot (Figure 7B) and flow cytometry (Table 5 and Figure 8). Neutrokine-alpha is encoded by a single 2.6 kb mRNA found in high concentrations in peripheral blood leukocytes, spleen, lymph nodes and bone marrow. Lower expression levels are detected in the placenta, heart, lung, fetal liver, thymus and pancreas. Among the cell line panels, neurotoxin-alpha is detected in HL-60 and K562, and not in Raji, HeLa or MOLT-4 cells. These results are confirmed by flow cytometry analysis using neurotoxin-alpha-specific mAb 2E5. As shown in Table 5, neurotoxin-alpha expression was not detected in T or B lineage cells but only in cells of bone marrow origin. In addition, as a result of the analysis of normal hemocyte types, significant expression was observed in the mononuclear cells of the dormant regulator whose expression level was about 4-fold after 3 days of exposure to IFN-y (100 U / ml) (Fig. 8A). An accompanying increase in neurotoxin-alpha-specific mRNA was also observed (Figure 8B). In contrast, neurotoxin-alpha was not expressed in newly isolated peripheral blood granulocytes, T cells, B cells or NK cells.

A variety of cell-based assays, including B cells, T cells, mononuclear cells, NK cells, hematopoietic progenitor cells, and various cells of the endothelial and epithelial origin, have been activated, proliferated , ≪ / RTI > differentiation or death. Among these assays, the neurotoxin-alpha was cultured in the presence of Staphylococcus aureus Cowan I (SAC) or fixed anti-human IgM, which specifically fixed purified one-way B cells with formalin as a promoter It is found to increase B cell proliferation in standard co-stimulation assays (Sieckmann, DG, et al., J. Exp. Med. 147: 814-29 (1978); Ringden, O., et al., Scand. Immunol. 6: 1159-69 (1977)). As shown in FIG. 9A, recombinant neurotoxin-alpha induces dose dependent proliferation of tonsillar B cells. The reaction was similar to that of rIL2 in the dose range of 0.1 to 10,000 ng / ml. Neutrokine-alpha also induces B cell proliferation when cultured with cells co-stimulated with immobilized anti-IgM (FIG. 9B). A dose dependent response was readily observed when increasing the amount of cross-linker in the presence of a fixed concentration of IL2 or rNutrocin-alpha.

Purified neurotoxin-alpha was biotinylated to determine whether the specific biological activity of B cells correlated with receptor expression. The resulting biotin-neurotoxin-alpha protein retained its biological function in standard B cell proliferation assays. Human whole peripheral blood cells were determined by a systematic-specific assay and the binding of biotinylated neurotoxin-alpha was measured in T cells, monocytes, NK cells and granulocytes as measured by CD3, CD14, Cd56 and CD66b, respectively (Fig. 10A). In contrast, biotinylated neurotoxin-alpha bound to peripheral CD20 ⁺ B cells. Receptor expression was also detected in B cell tumor cell lines REH, ARH-77, Raji, Namalwa, RPMI8226 and IM-9 but not in any of the tested bone marrow derived strains, including THP-1, HL-60, K-562 and U- Lt; / RTI > cell line. A representative flow cytometric analysis profile for myeloma cell line IM-9 and histiocytic cell line U-937 is shown in FIG. 10B. Similar results are obtained when biologically active FLAG-tagged neurotoxin-alpha proteins are used instead of chemically modified biotin-neurotoxin-alpha. Taken together, these results confirm that neurotoxin-alpha exhibits clear B-cell orientation in all respects to receptor distribution and biological activity. However, it is necessary to determine whether cell activation can induce the expression of neurotoxin-alpha receptors in peripheral blood cells, other normal cells, or existing cell lines.

To investigate the species specificity of neurotoxin-alpha, B cells of mouse spleen are cultured in the presence of human Neutrokine-alpha and SAC. As a result, it was confirmed that rNutrokin-alpha induces in vitro proliferation of spleen B cells of mice and binds to cell surface receptors on the cells. Interestingly, immature surface Ig-negative B cell precursors isolated from mouse bone marrow did not proliferate in response to neurotoxin-alpha nor bind to ligands.

To evaluate the in vivo activity of neurotoxin-alpha, BALB / c mice (3 / group) were administered with buffer or ruturekin-alpha 0.08 mg / kg, 0.8 mg / kg, 2 mg / kg or 8 mg / kg (Intraperitoneally) twice a day. Mice receiving this treatment for 4 consecutive days are killed and various tissues and sera are collected for analysis. In another embodiment, BALB / c mice may be injected (intraperitoneally) with any amount of rNutrocin-alpha ranging from 0.01 to 10 mg / kg twice a day. In a preferred embodiment, BALB / c mice are injected (intraperitoneally) twice per day with any amount of r-neurotoxin-alpha ranging from 0.01 to 3 mg / kg (specific preferred dosages in this embodiment include Kg, 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / Kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1.0 mg / kg, 1.1 mg / kg , 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.6 mg / kg, 1.7 mg / kg, 1.8 mg / kg, 1.9 mg / kg, 2.0 mg / , 2.1 mg / kg, 2.2 mg / kg, 2.3 mg / kg, 2.4 mg / kg, 2.5 mg / kg, 2.6 mg / kg, 2.7 mg / kg, 2.8 mg / kg, 2.9 mg / , But are not limited thereto). In another preferred embodiment, BALB / c mice are injected (intraperitoneally) twice per day with any amount of rNeutrokine-alpha ranging from 0.02 to 2 mg / kg (in a specific preferred embodiment administration 0.04 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / kg, 0.09 mg / kg, Kg, 0.1 mg / kg, 0.1 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1.0 mg / Kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.6 mg / kg, 1.7 mg / kg, 1.8 mg / kg, 1.9 mg / kg and 2.0 mg / , But is not limited to this).

Microscopically, the effect of neurotoxin-alpha administration was observed in normal hematoxylin and eosin (H & E) stained spleen fragments and immunohistochemically observed by mAb specific for CD45R (B220) . The normal spleen structure is altered by a sudden swelling of the white water marginal zone and a unique increase in cell fidelity of the red water (Fig. 11A). The expansion of the peripheral zone appears to be the result of an increased number of lymphocytes expressing the B cell marker CD45R (B220). In addition, the appropriate number of CD45R (B220) positive cells also invade the lymphocyte (PALS) region around the T-cell-enriched arteries. This suggests that the change in white number is due to an increase in the number of B cells. Concentrated packed cell populations, often filled with red water pores, are not stained with CD45R (B220). Additional experiments are required to characterize and characterize all cell types involved in the mechanism by which neurotoxin-alpha changes the structure of the spleen.

Flow cytometry analysis of the spleen of mice treated with 2 mg / kg of neurotoxin-alpha showed that the ratio of mature (CD45R (B220) ^dull , ThB ^bright ) B cells to neurotoxin- 10-fold increase (Fig. 11B). In addition, 0.08 mg / kg, 0.8 mg / kg and 2 mg / kg of mice were treated with buffer, 0.08 mg / kg, 0.8 mg / kg, 2 mg / kg or 8 mg / kg of neurotoxin- While the ratio of mature (CD45R (B220) ^dull , ThB ^bright ) B cells was increased by about 10-fold compared to that observed in control mice, buffer and 8 mg / kg produced mature B cells at approximately the same ratio . See Table 4.

FACS analysis of B cell population of mouse spleen Neutrokine-a (mg / kg) Mature B cells% (R2) CD45R-positive cells% (R1) Control (buffer) 1.26 52.17 0.08 mg / kg 16.15 56.53 0.8 mg / kg 18.54 57.56 2 mg / kg 16.54 57.55 8 mg / kg 1.24 61.42

A possible outcome of mature B cells in vivo is a relative increase in serum Ig titer. Thus, serum IgA, IgG and IgM concentrations are compared between buffered and neurotoxin-alpha treated mice (FIG. 11C). Neutrokine-alpha administration increases the serum concentrations of IgA and IgM by 2-fold and 5-fold, respectively. Interestingly, there is no increase in blood circulation of IgG.

Moreover, dose-dependent responses were observed in serum IgA titers in mice treated with various amounts of neurotoxin-alpha over 4 days, whereas significant dose-dependency was observed when the same amount of neurotoxin-alpha was administered over 2 days It does not. In the case of administration for 4 days, administration of Neutrokine-alpha at 8 mg / kg, 2 mg / kg, 0.8 mg / kg, 0.08 mg / kg and 0 mg / kg resulted in about 800 μg / Mu] g / ml, 200 [mu] g / ml and 200 [mu] g / ml. In other words, it was found that the concentration of neurotoxin-alpha of 8 mg / kg, 2 mg / kg, 0.8 mg / kg, 0.08 mg / kg, and 0 mg / kg of neurotoxin-alpha for 4 days yielding about 4 times, 3.75 times, Administration increases the IgA serum concentration beyond the baseline concentration observed only when the buffer is administered. In another embodiment, the experiment can be carried out with any amount of r-neutrokine-alpha in the range of 0.01 to 10 mg / kg. In a preferred embodiment, the neurotoxin-alpha is administered in the range of 0.01 to 3 mg / kg. 0.0 > mg / kg, < / RTI > 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / Kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / Kg, 1.7 mg / kg, 1.8 mg / kg, 1.0 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / 2.5 mg / kg, 2.7 mg / kg, 2.8 mg / kg, 1.9 mg / kg, 2.0 mg / kg, 2.1 mg / kg, 2.2 mg / kg, 2.3 mg / Kg, 2.9 mg / kg, and 3.0 mg / kg, respectively). In another preferred embodiment, the neurotoxin-alpha is administered in the range of 0.02 to 2 mg / kg (the specific preferred dosages in this embodiment are 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / kg, 0.09 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / 0.1 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.6 mg / kg, 1.7 mg / kg, 1.8 mg / kg, 1.9 mg / kg and 2.0 mg / kg.

The data presented herein define neurotoxin-alpha as a novel component of a TNF-ligand phase that induces B cell proliferation and differentiation both in vivo and in vitro. Neutrokine-alpha inhibits other B cell growth and differentiation factors such as IL2 (Metzger, DW, et al., Res. Immunol. 146: 499-505 (1995)), IL4 (Armitage, RJ et al. Et al., Proc. Natl. Acad. Sci. USA 84: 5894-98 (1986)), IL5 (Takatsu, K., et al Bertolini, JN, et al., Eur. J. Immunol. 23: 398-402 (1993)), IL6 (Poupart, P., et al., Proc. Natl. Acad Sci USA 84: 4234-38 (1987); et al., EMBO J. 6: 1219-24 (1987); Hirano, T., et al., Nature 324: 73-76 (1986)), IL7 (Goodwin, RG, et al., Proc. Et al., Nature 333: 571-73 (1988)), IL13 (Punnonen, J., et al., Allergy. 49: 576 (1986)), IL15 (Armitage, RJ et al., J. Immunol. 154: 483-90 (1995)), CD40L (Armitage, RJ, et al., Nature 357: 80-82 Koshen, C. and Banchereau, J. Int. Arch. Allergy. Immunol. 113: 393-99 (1997)) or CD27L (CD70) (Oshima, H., et al., Int. Immunol. 10: 517-26 (1998); Lens, SM, et al., Semin. Immunol. 10: 491-99 Specific receptor distribution and biological activity. Taken together, these data suggest that neurotoxin-alpha is involved in the exchange of signals between B cells and mononuclear cells or their differentiated progeny. Although all B cells can use this type of signaling, limited expression patterns and Ig secretion suggest a role for neurokinin-alpha that is involved in the activation of CD5 ⁺ or "non-conventional" B cell responses. These B cells provide important components in the innate immune system and provide a protective effect against environmental pathogens through the secretion of multi-reactive IgM and IgA antibodies (Pennell, CA, et al., Eur. J. Immunol. 19: 1289-95 (1989); Hayakawa, K., et al., Proc. Natl. Acad. Sci. USA 81: 2494-98 (1984)). Alternatively, neurotoxin-alpha may act as a modulator of T cell independent responses in a manner similar to that of CD40 and CD40L in T cell dependent antigen activation (van den Eertwegh, AJ et al., J. Exp. Med. 178 : 1555-65 (1993); Grabstein, KH, et al., J. Immunol. 150: 3141-47 (1993)). Thus, neurotoxin-alpha, its receptor or related antagonist is useful for the treatment of B cell diseases associated with autoimmune, neoplasia and / or immunodeficiency syndrome.

Way

mouse.

BALB / cAnNCR (6-8 weeks) was purchased from Charles River Laboratories, Inc, and used in accordance with recommended standard methods (National Research Council, Guide for the Care and Use of Laboratory Animals (1999) (Harlan Sprague Dawley, Inc) and a potable water bottle in an arbitrary manner. The animal protocols used in this study were reviewed and approved by the HGS Institutional Animal Care and Use Committee.

Isolation of full length neurotoxin - alpha cDNA.

To find sequences homologous to the receptor binding domain of the TNF system, the BLAST algorithm was used to investigate the EST database of the Human Genome Cycles Ink. The full-length neurotoxin-alpha clone was identified and sequenced and deposited in GenBank (accession number AF132600). The neurotoxin-alpha open reading frame is a 5 'primer (5'-CAG ACT GGA TCC GCC ACC ATG GAT GAC TCC ACA GAA AG-3') annealing to the expected initiation codon and a 5 'primer designed to anneal to the expected downstream stop codon 3 'primer (5'-CAG ACT GGT ACC GTC CTG CGT GCA CTA CAT GGC-3'). BimHI and Asp 718 restriction sites are attached to the tail of the resulting amplicon and subcloned into the mammalian expression vector. Neutrokine-alpha is also expressed in p-CMV-1 (Sigma Chemicals).

Purification of the recombinant human neurokinin-alpha.

The full-length cDNA encoding neurotoxin-alpha is subcloned into baculovirus expression vector pA2 and transfected with Sf9 insect cells (Patel, VP, et al., J. Exp. Med. 185: 1163-72 )). Recombinant Neutrokine-Alpha was purified from cell supernatants at 92 hours post infection using a combination of anion exchange, size exclusion and hydrophobic interaction columns. The purified protein is prepared in a buffer containing 0.15 M NaCl, 50 mM NaOAc (pH 6), sterile filtered and stored at 4 ° C until needed. SDS-PAGE and RP-HPLC analysis showed that the purity of r-neurokinin-alpha was 95% or more. Endotoxin concentration is below the detection limit in LAL assays (Associates of Cape Cod, Falmouth, MA). The final purified neurotoxin-alpha protein has an N-terminal sequence of Ala-Val-Gln-Gly-Pro. This was identical to the sequence of soluble neurotoxin-alpha from the CHO cell line stably transfected with the neurotoxin-alpha gene of the battlefield.

Generation of monoclonal antibodies.

BALB / cAnNCR mice were immunized with 50 μg Suspension HisTag-Neutrokine-alpha suspended in complete Freund's adjuvant followed by two doses of the antigen suspended in incomplete Freund's adjuvant. Hybridomas and monoclonal antibodies are described in Gefter, M. L. et al., Somatic. Cell Genet. 3: 231-36 (1977); Akerstrom, B., et al., J. Immunol. 135: 2589-92 (1985).

Cell line.

All human cell lines were purchased from ATCC (American Type Culture Collection, Manassas, Va.).

FACS analysis.

Neutrokine-alpha expression was assessed for human anti-human neurokinin-alpha mAb 2E5 (IgG1) and then PE conjugated F (IgGl) against human cell lines, newly isolated normal peripheral blood nucleated cells and in vitro cultured monocytes (ab ') 2 goat antibodies (CALTAGLaboratories, Burlingame, CA). Cells were analyzed by FACScan (Becton Dickinson Immunocytometry Systems, San Jose, Calif.) Using propidium iodide to remove extracellular cells. The neurotoxin-alpha linkage was synthesized using biotinylated ruturekin-alpha and then PE-conjugated streptavidin (Dako Corp, Glostrup, Denmark) with N-hydroxysuccinimide biotin reagent (Pierce, .

Chromosome mapping.

(5 'primer: 5'-TGG TGT CTT TCT ACC AGG TGG-3' and 3 'primer: 5'-TTT CTT CTG GAC CCT GAA CGG-3 ') was used to select a single-chromosome somatic cell hybrid panel (Quantum Biotechnology, Canada) carrying each chromosome by PCR. The expected 233 bp PCR product is detected only in human chromosome 13 hybrids. As a result of using a panel of 83 radioactive hybrids (Research Genetics, St. Louis, MO) and the Stanford Human Genome Center database (http://www.shgc.stanford.edu.RH/rhserver), neurotoxin- -36171 marker. ≪ / RTI > As a result of overlapping this map with a cytogenetic map of human chromosome 13, human neurokinin-alpha corresponded to chromosome band 13q34.

B lymphocyte proliferation assay.

B cells of human tonsil were purified by the method of consuming magnetic beads (MACS) of CD3-positive cells. The resulting cell population typically has 95% or more B cells as measured by expression of CD19 and CD20. Various dilutions of human ruturkin-alpha or control protein recombinant human IL2 were incubated in a total volume of 150 μl of culture medium (10% FBS, 5 × 10 ^-5 M 2 ME, 100 U / ml penicillin, 100 μg / ml streptomycin, Are added to each well of a 96-well plate to which 10 ⁵ B cells suspended in RPMI 1640 containing 10 ^-5 dilution of pansorubin (SAC) or anti-IgM are added. Proliferation is quantified with ³ H-thymidine (6.7 Ci / mM) 20 h pulse (1 μCi / well) from 72 hours after the addition of the factor.

Histological analysis.

The spleen was fixed in 10% neutral buffered formalin, impregnated with paraffin, cut into 5 μm, mounted on a glass slide, stained with hematoxylin and eosin, or immunohistochemistry for CD45R (B220) (Hilbert, DM, et al., Eur. J. Immunol. 23: 2412-18 (1993)).

Neutrokine-alpha cell surface expression Cell line Cell morphology Neutrokine-α cell surface expression Monocyte line U-937 Lymphoma, tissue / macrophage + BL-60 Leukemia, acute botulism + K-562 Leukemia, chronic myelogenous + THP-1 Leukemia, acute monocytic + T-system Jurkat Leukemia, T lymphocyte - SUP-T13 Leukemia, T lymphocyte - MOLT-4 Leukemia, T lymphocyte - B-system Daudi Bucket bottle, lymphocyte - Namalwa Bucket bottle, lymphocyte - Raji Bucket bottle, lymphocyte - Reh Leukemia, lymphocyte - ARH-77 Leukemia, plasma cells - IM9 Myeloma - RPMI 8226 Myeloma -

Example 7: Assay for detecting stimulation or inhibition of B cell proliferation and differentiation

The production of a functional humoral immune response requires homologous signaling of solubility between B-lineage cells and their microenvironment. The signal may confer a benign stimulus that causes the B system cells to sustain natural growth or a voice stimulus that directs the B system cells to stop the current growth pathway. To date, a number of stimulatory and inhibitory signals have been shown to affect B cell responses including IL-2, IL-4, IL5, IL6, IL-7, IL-10, IL-13, IL14 and IL15. Interestingly, these signals themselves are weak effectors, but they can induce activation, proliferation, differentiation, homeostasis, resistance and death of B cell entities with various co-stimulatory proteins. Among the B cell co-stimulatory proteins, the most studied class is the TNF phase. In this superfamily, CD40, CD27 and CD30 have been shown to modulate various immune responses with their respective ligands, CD154, CD70 and CD153. An assay that can detect and / or observe the proliferation and differentiation of these B cell entities and their precursors is an important tool for measuring the effects of various proteins on the B cell entity in terms of proliferation and differentiation. Two assays designed to detect the differentiation, proliferation or inhibition of B cell populations and their precursors are described below.

In vitro method

Were evaluated for activity to induce activation, proliferation, differentiation or inhibition of B cell populations and their precursors with purified neurotoxin-alpha and / or neurotoxin-alpha SV proteins or truncated forms thereof. The activity of purified neurotoxin-alpha and / or neurotoxin-alpha SV proteins on purified human one-way B cells in the dose range of 0.1 to 10,000 ng / ml is determined by the activity of Staphylococcus aureus Was assessed by standard B lymphocyte co-stimulation assay culturing purified one-way B cells in the presence of Reus Cowan I (SAC) or fixed anti-human IgM antibody. A second signal such as IL-2 and IL-15 synergizes with SAC and IgM bridging to induce B cell proliferation as measured by the triple-hydrogenated thymidine incorporation rate. New synergists can be readily identified using this assay. This assay involves the isolation of human tonsil B cells by magnetic bead (MACS) consumption of CD3-positive cells. The resulting cell population contains more than 95% of B cells, as measured by the expression of CD45R (B220). The various dilutions of each sample were diluted in a total volume of 150 μl of culture medium (RPMI 1640 containing 10% FBS, 5 × 10 ^-5 M 2 ME, 100 U / ml penicillin, 10 μg / ml streptomycin and 10 ^-5 dilution of SAC ) ≪ / RTI > was added to each well of a 96 well plate with 10 ⁵ B cells added. Proliferation or inhibition was quantified with ³ H-thymidine (6.7 Ci / mM) 20 h pulse (1 μCi / well) from 72 hours after the addition of the factor. The positive and negative controls are IL2 and medium, respectively.

The working substance (including the neurotoxin-alpha and / or neurotoxin-alpha SV polypeptide fragment) yields increased B cell proliferation as compared to the cell proliferation observed when contacting the same B cell count with the same concentration of the promoter do. The antagonistic substances described in the present invention can be used in the form of a neurokinin-alpha-soluble form that induces an increase in B cell proliferation activity in the absence of the same B cell count, the same concentration of promoter and antagonist (e.g., - < / RTI > alpha-polypeptide of 71-285, 81-285, 112-285 or 134-285) compared to a control containing the same concentration of B cell proliferation.

In vivo analysis

BALB / c mice are injected (intraperitoneally) with buffer, neurotoxin-alpha and / or neurotoxin-alpha SV protein or truncated form thereof 2 mg / kg twice a day. Mice treated for 4 consecutive days with this treatment are killed and various tissues and sera are collected and used for analysis. Comparing the normal spleen with the H & E fragments from the spleen from the neurotoxin-alpha and / or neurotoxin-alpha SV proteins, a red region that can exhibit the proliferation of perigraft lymphocytes and / or the activation of differentiation and proliferation of B- Alpha < / RTI > and / or neurotoxin-alpha SV proteins on spleen cells, such as a significant increase in eukaryotic cell integrity of the cells. Immunohistochemical studies using anti-CD45R (B220), a B cell marker, showed that all physical changes to spleen cells, such as splenic degeneration, increased B cell appearance in loosely defined B cell regions infiltrating existing T cell regions Is used.

Flow cytometry of spleens of mice treated with neurotoxin-alpha and / or neurotoxin-alpha SV protein revealed that Neutrokine-alpha and / or neurotoxin-alpha SV proteins were more potent than ThB ⁺ , CD45R Lt; RTI ID = 0.0 > (B220) < / RTI > dull B cells.

Similarly, the expected result of increased mature B cells in vivo is a relative increase in serum Ig titers. Therefore, serum IgM and IgA concentrations are compared between neurotoxin-alpha and / or neurotoxin-alpha SV protein treated mice and mice treated with buffer alone.

Example 8 Effect of Neutrokine-Alpha and Its Working Substance on the Treatment of Gastrointestinal Disease-Related Atrophy and Dysgenesis in Mice

Analysis of the utility of neurotoxin-alpha in the treatment, prevention and / or diagnosis of graft-versus-host disease (GVHD) -related lymphoid hypoplasia / atrophy was performed using the (BALB / cxC57BL / Using a C57BL / 6 parent. These parents used in the F1 mouse model are well characterized and are reproducible animal models of GVHD in bone marrow transplant patients known in the art (Gleichemann, et al., Immunol. Today 5: 324, 1984) . Soluble neurotoxin-alpha is thought to induce the proliferation and differentiation of B lymphocytes and to treat the lymphoid hypoplasia and atrophy observed in the GVHD animal model (Piguet, et al., J.Exp.Med.166: 1280 (1987); Hattori, et al., Blood 90: 542 (1997)).

The onset of GVHD disease was induced by intravenous injection of about 1-5 x 10 ⁸ spleen cells from C57BL / 6 mice (both mice were obtained from Jackson Lab, Bar Harbor, Maine) into (BALB / cxC57BL / do. Six to eight mice were injected intraperitoneally with a dose of 20 μg / kg of the neurotoxin from the time (about 5 days), the middle stage (about 12 days) or the severe stage (about 20 days) - Alpha 0. 1 to 5.0 mg / kg or buffer control is administered intraperitoneally, intramuscularly or intradermally daily. The effect of neurotoxin-alpha on spleen's lymphoid hypoplasia and atrophy is analyzed by FACS and histopathology at several time points (3 to 4 times) between 10 and 30 days. Briefly, spleen cells were prepared from normal CBF1, GVHD, or neurotoxin-alpha treated mice and immunoprecipitated with fluorescein phycoerythrin-conjugated anti-H-2Kb, biotin conjugated anti-H2Kd and FITC conjugated anti-CD4, CD8 or anti-B220 and then cytochrome conjugated avidin. All conjugated antibodies can be purchased from PharMingen (San Diego, Calif.). The cells are then analyzed by FACScan (Becton Dickinson, San Jose, CA). The recipient and donor lymphocytes were identified as H-2Kb + Kd + and H-2Kb + Kd-cells, respectively. Cell numbers of CD4 + T, CD8 + T and B220 + B cells of recipient or donor origin are calculated from the total number of recovered splenocytes, and the proportion of each subunit is determined by tricolor assay. Histological evaluation of the relative tissue damage seen in other GVHD-related organs (liver, skin and intestines) can be performed after killing animals.

Finally, the neurotoxin-alpha and buffer treated animals undergo daily clinical evaluations to assess poor health status, weight and mortality.

Neutrokine-alpha agonists and antagonists can be investigated in an acute GVHD mouse model.

Example 9: Isolation of antibody fragments that are directed against the neurotoxin-alpha polypeptide derived from the scFvs library

The natural V-gene isolated from human PBL is constructed into a large library of antibody fragments containing the reactivity against neurotoxin-alpha and / or neurotoxin-alpha SV, which donor has never been exposed or has never been exposed See U.S. Patent No. 5,885,793, which is incorporated herein by reference).

Library Selection

The library of scFvs constructs from the RNA of human PBL as described in WO 92/01047 (incorporated herein by reference). Approximately 10 < ⁹ > of the phagemid carrying the antibody fragments expressing the phage are selected. Cola is inoculated into 50 ml of 2xTY (2xTY-AMP-GLU) containing 1% glucose and 100 占 퐂 / ml ampicillin and grown to OD 0.8 under shaking. 5 ml of this culture was inoculated into 50 ml of 2xTY-AMP-GLU, 2x10 ⁸ TU of a delta gene 3 helper (M13 delta gene III, WO92 / 01047) was added, and the culture was incubated at 37 ° C for 45 Min, and then incubated at 37 DEG C for 45 minutes under shaking. The culture was centrifuged at 4000 rpm for 10 minutes and the pellet was resuspended in 2 L TY containing 100 μg / ml ampicillin and 50 μg / ml kanamycin and grown overnight. The phage are prepared as described in WO92 / 01047.

M13 delta gene III is prepared as follows. The M13 delta gene III helper phage does not code for the gene III protein. Thus, the phage (mid) expressing antibody fragments exhibit greater binding affinity for the antigen. The infectious M13 delta gene III particle is prepared by growing the helper phage in a cell harboring a pUC19 derivative supplying the wild-type gene III protein during phage formation. The cultures are incubated at 37 ° C for 1 hour without shaking, and then cultured again at 37 ° C for 1 hour under shaking. Cells were spun down (IEC-Centra 8, 4000 revs / min, 10 min) and resuspended in 300 ml of 2xTY liquid medium (2xTY-AMP-KAN) containing 100 μg / ml ampicillin and 25 μg / ml kanamycin And allowed to proliferate overnight at 37 ° C under shaking. The phage particles were purified and concentrated from the culture medium through two PEG-precipitation (Sambrook et al., 1990), resuspended in 2 ml PBS, and passed through a 0.45 占 퐉 filter (Minisart NML; Sartorius) ¹³ transduction unit / ml (ampicillin resistant colony) concentration.

Library panning

The immunopotent (Nunc) is coated overnight in PBS supplemented with 100 μg / ml or 10 μg / ml of 4 ml of the polypeptide of the present invention. The tubes are blocked with 2% Marvel-PBS for 2 hours at 37 ° C and then washed three times with PBS. Add approximately 10 ¹³ TU of phage to this tube, incubate for 30 min at room temperature while rotating on top and bottom rotors, and allow to sit for another 1.5 h. The tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with PBS. The phage is eluted by adding 1 ml of 100 mM triethylamine and spinning for 15 minutes on the upper and lower rotating rolls, and then immediately neutralizing the solution with 0.5 ml of 1.0 M Tris-HCl (pH 7.4). The eluted phages were incubated with the bacteria at 37 ° C for 30 min. 10 ml of E. coli TG1 is infected. This. The cola is then plated on a TYE plate containing 1% glucose and 100 μg / ml ampicillin. The obtained bacterial library is selected with the delta gene 3 helper phage as described above to prepare the phage for the subsequent selection step. This procedure is repeated with a total of four affinity tablets, and the tube washing performed at this time is increased 20 times with PBS, 0.1% Tween-20, 20 times with the third and fourth PBS.

Characteristics of binder

Using the phage eluted from the 3rd and 4th screening, Coli HB2151 and produced soluble scFv from a single colony for analysis (Marks, et al., 1991). ELISA is performed on a microtiter plate coated with 10 pg / ml of the polypeptide of the present invention contained in 50 mM bicarbonate pH 9.6. Clones positive in the ELISA further analyze the characteristics by PCR fingerprinting (e.g., WO92 / 01047) and subsequent sequencing.

Example 10: Neutralization of an anti-neutrokin-alpha monoclonal antibody and a neurotoxin-alpha / neurotoxin-alpha receptor interaction

A monoclonal antibody against the neurotoxin-alpha protein was prepared according to the following method. Briefly, mice were transdermally injected (in the front part of the back) with an emulsion in which 100 μl of His-tagged neurotoxin-alpha protein prepared in Example 2 in 100 μl of PBS was emulsified in 100 μl of complete Freund's adjuvant . Twenty-five micrograms of neurotoxin-alpha in unsupported Freund's adjuvant was injected three times at intervals of two weeks. Finally, animals were rested for 1 month prior to intraperitoneal administration of 25 μg of Neutrokine-alpha in PBS. Four days later, mice were killed and spleen cells were collected for fusion.

A "fusion" method is a method in which the manufacturer has modified the method described in the prior art (Gefter, ML, et al. Somatic Cell Genet 3: 231-36 (1977); Boehringer Mannheim, PEG 1500 (Cat. No. 783641) (Boehringer Mannheim) according to the manufacturer's instructions, using 2x10E7 P3X63Ag8.653 plasma cell and spleen-derived spleen cells.

After fusion, the cells were resuspended in 400 mL of HAT medium supplemented with 20% FBS and 4% hybridoma supernatant (Boehringer Mannheim) and distributed to 96 well plates at a density of 200 [mu] l / well. Seven days after fusion, 100 쨉 l of the medium was aspirated and replaced with 100 쨉 l of the fresh medium. After 14 days of fusion, the hybridoma is selected for antibody production.

Hybridoma supernatant was screened for binding to the neutrophil-alpha protein immobilized on a plate using ELISA. Plates are incubated overnight with 100 μl per well of Neutrokine-Alpha in PBS at a concentration of 2 μg per ml. The hybridoma supernatants are diluted 1:10 with PBS and incubated with Neutrokine- - inject into each well of alpha-coated plate and incubate overnight at 4 ° C. The following day, plates were washed three times with PBS containing 0.1% Tween-20 and developed using anti-mouse IgG ABC system (Vector Laboratories). The color reaction was stopped by adding 2M H ₂ SO ₄ at 25 ml / well. The plate is read at 450 nm.

Hybridoma supernatants are examined for Ig isotype using isostrips. Cloning is carried out by the limiting dilution method of HT medium. Approximately 3x10 < 6 > cells in 0.9 ml of HBSS were injected into pre-pretreated mice. After 7 to 9 days, the plural liquids are collected by a 19 g syringe. All antibodies are purified by protein G affinity chromatography using an Acta FPLC system (Pharmacia).

After the first and second consecutive transdermal injections, a strong immune response occurred in all three mice and the serum retroperitoneum was 10E-7 as determined by ELISA on a neurotoxin-alpha coated plate.

In one experiment, spleen cells from benign mice were used to generate over 1000 primitive hybridomas. Of these, 917 are selected to produce anti-neutrokin-alpha antibodies. Screening is performed using 1: 1 diluted supernatant to detect all positive clones. Of the 917 selected hybridomas, 76 were found to be positive, 17 of which were observed to be IgG production factors. After affinity testing and cloning, 9 strains are selected for further propagation and purification.

All purified monoclonal antibodies can bind to various forms of neurotoxin-alpha (including proteins produced in the baculovirus system (Example 2) and His-tagged proteins) in Western blot analysis and ELISA. Six of the nine clones could bind to the neurotoxin-alpha present on the surface of THP-1 cells. However, none of the antibodies tested can capture neurotoxin-alpha from the solution.

Alpha-monoclonal antibody that recognizes neurotoxin-alpha expressed on the cell surface but does not recognize neurotoxin-alpha in solution is useful for in vivo neutralization studies and in vitro monocyte and B cell assays . This antibody is also useful for sensitive detection of neurotoxin-alpha in western blots.

In another experiment, spleen cells from benign mice were used to generate over 1000 primitive hybridomas. 729 of the primitive hybridomas are selected to produce anti-neutrokin-alpha antibodies. Screening is carried out under stringent conditions using a 1:10 diluted supernatant to collect only clones with higher affinity. Of the 729 selected hybridomas, 23 were positive, including 16 IgM production factors and 7 IgG production factors (four of which provided a strong IgM reference). In this experiment, the Ig type distribution of IgG was biased only to IgG2 subtype. Three of the seven IgG hybridomas produced IgG2a subtype antibodies, two IgG2b antibodies while the other two IgG1 production factors.

The supernatant from all the positive hybridomas produced in the second experiment is tested for activity inhibiting neurotoxin-alpha mediated proliferation of B cells. In a first screening experiment, two hybridomas producing IgG neutralizing antibodies were detected (antibodies 16C9 and 12C5). In another experiment, the IgG neutralizing activity of the hybridomas (i.e., 16C9 and 12C5) , A strong neutralization supernatant from the hybridoma 15C10 and 4A6 is additionally confirmed.

Three clones were then propagated in vivo (15C10 clones were propagated in a hollow fiber system) and the antibodies were purified by affinity chromatography. All three of these clones can bind to Neutrokine-alpha on the surface of THP-1 cells and can also bind (i.e., capture) Neutrokine-Alpha in solution.

Specifically, an experiment is performed using the anti-neutrokin-alpha monoclonal antibody described in the second experiment to determine whether the antibody neutralizes the neurotoxin-alpha / neurotoxin-alpha receptor binding. Briefly, the neurotoxin-alpha protein was biotinylated using EZ-link T NHS-biotin reagent (Pierce, Rockford, Ill.). Biotinylated neurotoxin-alpha is used to identify cell surface proteins that bind to neurotoxin-alpha. Preliminary results indicate that neurotoxin-alpha binds to receptors in B lymphoid cells.

The addition of the anti-neutrokin-alpha antibody generated in the second experiment described above neutralized the binding of neurotoxin-alpha to the neurotoxin-alpha receptor. In a specific embodiment, the anti-neutrokin-alpha antibody 15C10 neutralizes the binding of neurotoxin-alpha to the neurotoxin-alpha receptor.

Namely, the anti-neurotrophin-alpha monoclonal antibody (particularly, antibody 15C10) produced in the above-mentioned second experiment recognizes and binds both the membrane-bound neurotoxin-alpha protein and the soluble neurotoxin-alpha protein, Neutralizes the neurotoxin-alpha / neurotoxin-alpha receptor binding.

It is to be understood that the present invention may be practiced otherwise than as specifically described in the foregoing Detailed Description and Examples. Various modifications of the invention are possible in light of the above teachings, and are therefore intended to be within the scope of the appended claims.

The entire contents of all publications (patents, patent applications, trade journals, experiments, books or other documents) cited herein are incorporated by reference.

In addition, the sequence listing submitted with the present specification may be found in computerized form and written form in co-pending applications 09 / 005,874 (1998.1.12), US60 / 036,100 (1997.1.14) and PCT / US96 / 17957 All submitted sequence listings are incorporated herein by reference.

Information on deposited microorganisms

(PCT Rule 13bis)

A. The information given below relates to the microorganisms mentioned in paragraph 21 of the seventh aspect of the specification. B. Deposits Confirmation Additional deposits are indicated in the next section Name of Depositor American Type Culture Collection Address of the depositary (including postal code and country) University of Virginia, USA 20110-2209 Manassas University University College Boulevard 10801 Date of deposit: October 22, 1996 Access number: 97768 C. Additional information (blank when not in use) The above information is included in the next chapter □ D. The designated country to which the information is applied (if the information does not apply to all the designated countries) In connection with the designation of a European European patent, a sample of deposited microorganisms may be deposited by a person requesting the sample until a statement on the grant of the European patent is published, or until the date the application is refused or withdrawn, (RPC 28 (4) EPC) by distributing the sample to a designated expert E. Separate information (blank when not used) The information listed below will be subsequently submitted to the International Bureau (specifying the general characteristics of the information, for example, "Deposit Number of Deposits") For office International Office □ You have received this document along with the international application. □ This document was received by the International Bureau on the date. Official hall Official hall

Form PCT / RO / 134 (July 1992)

ATCC Accession No. 97768

Canada

Until the Canadian patent is granted on the basis of an application, the application is refused or can no longer be recovered or withdrawn, the Commissioner of the Patent Office shall only sell the sample of the biological deposit referred to herein to an independent expert appointed by the Commissioner The applicant must notify the International Bureau in writing prior to the completion of the technical preparations for publication of the international patent application.

Norway

Applicant hereby requests that the sale of samples be made only to the experts in the field until the final measurement is made without disclosure or disclosure by the Norwegian Patent Office as this document. This application must be filed with the Norwegian Patent Office prior to the point in time when the application is available to the general public under sections 22 and 33 (3) of the Norwegian patent provision. If such an application is filed by the applicant, it is granted that the third party can use the specialist when applying for the sale of the sample. The expert may be in the list of experts recognized and prepared by the Norwegian Patent Office or may be an individual recognized by the applicant as an individual case.

Australia

The applicant hereby declares that the sale of the microbial sample may only be made to the intended recipient without prior understanding of the invention prior to patent approval or before the expiration, rejection or withdrawal of the application (Rule 3.25 (3) of the Australian Patent Regulations).

Finland

Applicant hereby requests that the sale of samples be made only to the experts in the field until the final determination of the application is made by the National Board of Patents and Regulations without being published or made public.

England

Applicant hereby requests that the distribution of samples of microorganisms be made only to experts. This application shall be submitted by the applicant to the International Bureau before the technical preparations for the international publication of the application have been completed.

Denmark

Applicant hereby requests that the sale of samples be done only by experts in the field, until the final measurement is made without disclosure or disclosure by the Danish Intellectual Property Office. This application must be filed with the Norwegian Patent Office prior to the time the application is available by a third party under sections 22 and 33 (3) of the Danish patent provision. If such an application is filed by the applicant, it is granted that the third party can use the specialist when applying for the sale of the sample. The expert may be in the list of experts recognized and prepared by the Danish Patent Office or may be an individual recognized by the applicant as an individual case.

Sweden

Applicant hereby requests that the sale of samples be made only to the experts in the field until the final measurement is made without disclosure or disclosure by the Swedish Patent Office. This application must be submitted to the International Bureau by the applicant (preferably using the form PCT / RO / 134 copied in PCT Applicant Guide Volume 1 Appendix Z) before the expiration of 16 months from the priority date. If such an application is filed by the applicant, it is granted that the third party can use the specialist when applying for the sale of the sample. The expert may be in the list of experts recognized and prepared by the Swedish Patent Office or may be an individual recognized by the applicant as an individual case.

Netherlands

The applicant hereby requests that the microorganism distribute the sample to an expert only as provided for in Rule 31F (1) until the date of the approval of the Dutch patent or the date on which the application is refused, withdrawn or expired. This application must be filed with the Dutch industrial property office by the applicant earlier than the earlier of the two points available for the application by a third party under section 22C or section 25 of the Dutch patent provision.

Information on deposited microorganisms

(PCT Rule 13bis)

A. The information given below relates to the microorganisms mentioned in the fifth row of the eighth aspect of the specification. B. Deposits Confirmation Additional deposits are indicated in the next section Name of Depositor American Type Culture Collection Address of the depositary (including postal code and country) University of Virginia, USA 20110-2209 Manassas University University College Boulevard 10801 Date of deposit: December 10, 1998 Access number: 203518 C. Additional information (blank when not in use) The above information is included in the next chapter □ D. The designated country to which the information is applied (if the information does not apply to all the designated countries) In connection with the designation of a European European patent, a sample of deposited microorganisms may be deposited by a person requesting the sample until a statement on the grant of the European patent is published, or until the date the application is refused or withdrawn, (RPC 28 (4) EPC) by distributing the sample to a designated expert E. Separate information (blank when not used) The information listed below will be subsequently submitted to the International Bureau (specifying the general characteristics of the information, for example, "Deposit Number of Deposits") For office International Office □ You have received this document along with the international application. □ This document was received by the International Bureau on the date. Official hall Official hall

Form PCT / RO / 134 (July 1992)

ATCC Accession No. 203518

Canada

Until the Canadian patent is granted on the basis of an application, the application is refused or can no longer be recovered or withdrawn, the Commissioner of the Patent Office shall only sell the sample of the biological deposit referred to herein to an independent expert appointed by the Commissioner The applicant must notify the International Bureau in writing prior to the completion of the technical preparations for publication of the international patent application.

Norway

Applicant hereby requests that the sale of samples be made only to the experts in the field until the final measurement is made without disclosure or disclosure by the Norwegian Patent Office as this document. This application must be filed with the Norwegian Patent Office prior to the point in time when the application is available to the general public under sections 22 and 33 (3) of the Norwegian patent provision. If such an application is filed by the applicant, it is granted that the third party can use the specialist when applying for the sale of the sample. The expert may be in the list of experts recognized and prepared by the Norwegian Patent Office or may be an individual recognized by the applicant as an individual case.

Australia

The applicant hereby declares that the sale of the microbial sample may only be made to the intended recipient without prior understanding of the invention prior to patent approval or before the expiration, rejection or withdrawal of the application (Rule 3.25 (3) of the Australian Patent Regulations).

Finland

Applicant hereby requests that the sale of samples be made only to the experts in the field until the final determination of the application is made by the National Board of Patents and Regulations without being published or made public.

England

Applicant hereby requests that the distribution of samples of microorganisms be made only to experts. This application shall be submitted by the applicant to the International Bureau before the technical preparations for the international publication of the application have been completed.

Denmark

Applicant hereby requests that the sale of samples be done only by experts in the field, until the final measurement is made without disclosure or disclosure by the Danish Intellectual Property Office. This application must be filed with the Norwegian Patent Office prior to the time the application is available by a third party under sections 22 and 33 (3) of the Danish patent provision. If such an application is filed by the applicant, it is granted that the third party can use the specialist when applying for the sale of the sample. The expert may be in the list of experts recognized and prepared by the Danish Patent Office or may be an individual recognized by the applicant as an individual case.

Sweden

Applicant hereby requests that the sale of samples be made only to the experts in the field until the final measurement is made without disclosure or disclosure by the Swedish Patent Office. This application must be submitted to the International Bureau by the applicant (preferably using the form PCT / RO / 134 copied in PCT Applicant Guide Volume 1 Appendix Z) before the expiration of 16 months from the priority date. If such an application is filed by the applicant, it is granted that the third party can use the specialist when applying for the sale of the sample. The expert may be in the list of experts recognized and prepared by the Swedish Patent Office or may be an individual recognized by the applicant as an individual case.

Netherlands

The applicant hereby requests that the microorganism distribute the sample to an expert only as provided for in Rule 31F (1) until the date of the approval of the Dutch patent or the date on which the application is refused, withdrawn or expired. This application must be filed with the Dutch industrial property office by the applicant earlier than the earlier of the two points available for the application by a third party under section 22C or section 25 of the Dutch patent provision.

Information on deposited microorganisms

(PCT Rule 13bis)

A. The information given below relates to the microorganisms mentioned in paragraph 229, line 8 of the specification. B. Deposits Confirmation Additional deposits are indicated in the next section Name of Depositor American Type Culture Collection Address of the depositary (including postal code and country) University of Virginia, USA 20110-2209 Manassas University University College Boulevard 10801 Date of deposit: January 7, 2000 Accession Number: PTA-1158 C. Additional information (blank when not in use) The above information is included in the next chapter □ D. The designated country to which the information is applied (if the information does not apply to all the designated countries) In connection with the designation of a European European patent, a sample of deposited microorganisms may be deposited by a person requesting the sample until a statement on the grant of the European patent is published, or until the date the application is refused or withdrawn, (RPC 28 (4) EPC) by distributing the sample to a designated expert E. Separate information (blank when not used) The information listed below will be subsequently submitted to the International Bureau (specifying the general characteristics of the information, for example, "Deposit Number of Deposits") For office International Office □ You have received this document along with the international application. □ This document was received by the International Bureau on the date. Official hall Official hall

Form PCT / RO / 134 (July 1992)

ATCC Accession No. PTA-1158

Canada

Until the Canadian patent is granted on the basis of an application, the application is refused or can no longer be recovered or withdrawn, the Commissioner of the Patent Office shall only sell the sample of the biological deposit referred to herein to an independent expert appointed by the Commissioner The applicant must notify the International Bureau in writing prior to the completion of the technical preparations for publication of the international patent application.

Norway

Applicant hereby requests that the sale of samples be made only to the experts in the field until the final measurement is made without disclosure or disclosure by the Norwegian Patent Office as this document. This application must be filed with the Norwegian Patent Office prior to the point in time when the application is available to the general public under sections 22 and 33 (3) of the Norwegian patent provision. If such an application is filed by the applicant, it is granted that the third party can use the specialist when applying for the sale of the sample. The expert may be in the list of experts recognized and prepared by the Norwegian Patent Office or may be an individual recognized by the applicant as an individual case.

Australia

The applicant hereby declares that the sale of the microbial sample may only be made to the intended recipient without prior understanding of the invention prior to patent approval or before the expiration, rejection or withdrawal of the application (Rule 3.25 (3) of the Australian Patent Regulations).

Finland

Applicant hereby requests that the sale of samples be made only to the experts in the field until the final determination of the application is made by the National Board of Patents and Regulations without being published or made public.

England

Applicant hereby requests that the distribution of samples of microorganisms be made only to experts. This application shall be submitted by the applicant to the International Bureau before the technical preparations for the international publication of the application have been completed.

Denmark

Applicant hereby requests that the sale of samples be done only by experts in the field, until the final measurement is made without disclosure or disclosure by the Danish Intellectual Property Office. This application must be filed with the Norwegian Patent Office prior to the time the application is available by a third party under sections 22 and 33 (3) of the Danish patent provision. If such an application is filed by the applicant, it is granted that the third party can use the specialist when applying for the sale of the sample. The expert may be in the list of experts recognized and prepared by the Danish Patent Office or may be an individual recognized by the applicant as an individual case.

Sweden

Applicant hereby requests that the sale of samples be made only to the experts in the field until the final measurement is made without disclosure or disclosure by the Swedish Patent Office. This application must be submitted to the International Bureau by the applicant (preferably using the form PCT / RO / 134 copied in PCT Applicant Guide Volume 1 Appendix Z) before the expiration of 16 months from the priority date. If such an application is filed by the applicant, it is granted that the third party can use the specialist when applying for the sale of the sample. The expert may be in the list of experts recognized and prepared by the Swedish Patent Office or may be an individual recognized by the applicant as an individual case.

Netherlands

The applicant hereby requests that the microorganism distribute the sample to an expert only as provided for in Rule 31F (1) until the date of the approval of the Dutch patent or the date on which the application is refused, withdrawn or expired. This application must be filed with the Dutch industrial property office by the applicant earlier than the earlier of the two points available for the application by a third party under section 22C or section 25 of the Dutch patent provision.

Information on deposited microorganisms

(PCT Rule 13bis)

A. The information given below relates to the microorganisms mentioned in paragraph 229, line 8 of the specification. B. Deposits Confirmation Additional deposits are indicated in the next section Name of Depositor American Type Culture Collection Address of the depositary (including postal code and country) University of Virginia, USA 20110-2209 Manassas University University College Boulevard 10801 Date of deposit: January 7, 2000 Accession Number: PTA-1159 C. Additional information (blank when not in use) The above information is included in the next chapter □ D. The designated country to which the information is applied (if the information does not apply to all the designated countries) In connection with the designation of a European European patent, a sample of deposited microorganisms may be deposited by a person requesting the sample until a statement on the grant of the European patent is published, or until the date the application is refused or withdrawn, (RPC 28 (4) EPC) by distributing the sample to a designated expert E. Separate information (blank when not used) The information listed below will be subsequently submitted to the International Bureau (specifying the general characteristics of the information, for example, "Deposit Number of Deposits") For office International Office □ You have received this document along with the international application. □ This document was received by the International Bureau on the date. Official hall Official hall

Form PCT / RO / 134 (July 1992)

ATCC Accession No. PTA-1159

Canada

Until the Canadian patent is granted on the basis of an application, the application is refused or can no longer be recovered or withdrawn, the Commissioner of the Patent Office shall only sell the sample of the biological deposit referred to herein to an independent expert appointed by the Commissioner The applicant must notify the International Bureau in writing prior to the completion of the technical preparations for publication of the international patent application.

Norway

Applicant hereby requests that the sale of samples be made only to the experts in the field until the final measurement is made without disclosure or disclosure by the Norwegian Patent Office as this document. This application must be filed with the Norwegian Patent Office prior to the point in time when the application is available to the general public under sections 22 and 33 (3) of the Norwegian patent provision. If such an application is filed by the applicant, it is granted that the third party can use the specialist when applying for the sale of the sample. The expert may be in the list of experts recognized and prepared by the Norwegian Patent Office or may be an individual recognized by the applicant as an individual case.

Australia

The applicant hereby declares that the sale of the microbial sample may only be made to the intended recipient without prior understanding of the invention prior to patent approval or before the expiration, rejection or withdrawal of the application (Rule 3.25 (3) of the Australian Patent Regulations).

Finland

Applicant hereby requests that the sale of samples be made only to the experts in the field until the final determination of the application is made by the National Board of Patents and Regulations without being published or made public.

England

Applicant hereby requests that the distribution of samples of microorganisms be made only to experts. This application shall be submitted by the applicant to the International Bureau before the technical preparations for the international publication of the application have been completed.

Denmark

Applicant hereby requests that the sale of samples be done only by experts in the field, until the final measurement is made without disclosure or disclosure by the Danish Intellectual Property Office. This application must be filed with the Norwegian Patent Office prior to the time the application is available by a third party under sections 22 and 33 (3) of the Danish patent provision. If such an application is filed by the applicant, it is granted that the third party can use the specialist when applying for the sale of the sample. The expert may be in the list of experts recognized and prepared by the Danish Patent Office or may be an individual recognized by the applicant as an individual case.

Sweden

Applicant hereby requests that the sale of samples be made only to the experts in the field until the final measurement is made without disclosure or disclosure by the Swedish Patent Office. This application must be submitted to the International Bureau by the applicant (preferably using the form PCT / RO / 134 copied in PCT Applicant Guide Volume 1 Appendix Z) before the expiration of 16 months from the priority date. If such an application is filed by the applicant, it is granted that the third party can use the specialist when applying for the sale of the sample. The expert may be in the list of experts recognized and prepared by the Swedish Patent Office or may be an individual recognized by the applicant as an individual case.

Netherlands

The applicant hereby requests that the microorganism distribute the sample to an expert only as provided for in Rule 31F (1) until the date of the approval of the Dutch patent or the date on which the application is refused, withdrawn or expired. This application must be filed with the Dutch industrial property office by the applicant earlier than the earlier of the two points available for the application by a third party under section 22C or section 25 of the Dutch patent provision.

Claims (25)

(a) a nucleotide sequence encoding a neurotoxin-alpha polypeptide having the complete amino acid sequence set forth in Figures 1A and 1B (SEQ ID NO: 2) (b) a nucleotide sequence encoding a neurotoxin-alpha polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in the deposit of ATCC Accession No. 97768, (c) a nucleotide sequence encoding a neurotoxin-alpha polypeptide extracellular domain, (d) a nucleotide sequence encoding a neurotoxin-alpha polypeptide neuronal transmembrane domain, (e), a nucleotide sequence encoding the intracellular domain of the neurotoxin-alpha polypeptide, (f) a nucleotide sequence encoding an extracellular and intracellular domain but lacking a transmembrane domain and encoding a soluble neurotoxin-alpha polypeptide; and (g) having a nucleotide sequence that is at least 95% identical to a sequence selected from the group consisting of the nucleotide sequence complementary to the nucleotide sequence of (a), (b), (c), (d) An isolated nucleic acid molecule comprising a polynucleotide. The nucleic acid molecule of claim 1, wherein the polynucleotide has the complete nucleotide sequence (SEQ ID NO: 1) as set forth in Figures 1A and 1B. The nucleic acid according to claim 1, wherein the polynucleotide encodes a neurotoxin-alpha polypeptide having the complete amino acid sequence (SEQ ID NO: 2) as set forth in Figures 1A and 1B. molecule. The nucleic acid molecule of claim 1, wherein the polynucleotide has a nucleotide sequence encoding a soluble neurotoxin-alpha polypeptide comprising an extracellular domain as shown in Figures 1A and 1B (SEQ ID NO: 2). (a) a nucleotide sequence encoding a polypeptide having an amino acid sequence consisting of residue n-285 of SEQ ID NO: 2, wherein n is an integer ranging from 2 to 190, (b) a nucleotide sequence encoding a polypeptide having an amino acid sequence consisting of residue 1-m of SEQ ID NO: 2, wherein m is an integer ranging from 274 to 284, (c) a nucleotide sequence encoding a polypeptide having an amino acid sequence consisting of residues n-m of SEQ ID NO: 2, wherein n and m are as defined in each of (a) and (b) (d) a portion of the complete neurotoxin-alpha amino acid sequence encoded by the cDNA clone contained in the deposit of ATCC Accession No. 97768, wherein a portion is a full complement of the full amino acid sequence excluding 1 to 190 amino acids A polynucleotide having a nucleotide sequence that is at least 95% identical to a nucleotide sequence selected from the group consisting of a nucleotide sequence encoding a polypeptide consisting of a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: The nucleic acid molecule of claim 1, wherein the polynucleotide has the complete nucleotide sequence of a cDNA clone contained in a deposit of ATCC Accession No. 97768. 2. The nucleic acid molecule of claim 1, wherein the polynucleotide has a nucleotide sequence encoding a neurokinin-alpha polypeptide having a complete amino acid sequence that is encoded by a cDNA clone contained in a deposit of ATCC Accession No. 97768. The nucleic acid molecule of claim 1, wherein the polynucleotide encodes a soluble neurotoxin-alpha polypeptide comprising an extracellular domain encoded by a cDNA clone contained in a deposit of ATCC Accession No. 97768. Hybridizing under stringent hybridization conditions with a polynucleotide having the same nucleotide sequence as the nucleotide sequence described in (a), (b), (c), (d) Or a polynucleotide that does not hybridize under stringent hybridization conditions with a polynucleotide having a nucleotide sequence consisting only of T residues or T residues. Encoding an amino acid sequence of an epitope-containing portion of a neurotoxin-alpha polypeptide having the amino acid sequence set forth in (a), (b), (c), (d), (e) An isolated nucleic acid molecule comprising a polynucleotide. 11. The polypeptide of claim 10, which comprises a polypeptide comprising the amino acid residue (SEQ ID NO: 2) of about Phe-115 to about Leu-147, a polypeptide comprising the amino acid residue (SEQ ID NO: 2) of about Ile-150 to about Tyr- (SEQ ID NO: 2) of about Ser-171 to about Phe-194, a polypeptide comprising the amino acid residue (SEQ ID NO: 2) of about Glu-223 to about Tyr-246, and a polypeptide comprising about Ser-271 to about Phe- An isolated nucleic acid molecule encoding an epitope-containing portion of a neurotoxin-alpha polypeptide selected from the group consisting of polypeptides comprising an amino acid residue (sequence 2) of Phe-278. 9. A method for producing a recombinant vector, comprising inserting the isolated nucleic acid molecule of claim 1 into a vector. 12. A recombinant vector produced by the method of claim 12. 14. A method for producing a recombinant host cell comprising introducing the recombinant vector of claim 13 into a host cell. 14. Recombinant host cells produced by the method of claim 14. 15. A recombinant method for producing a neurotoxin-alpha polypeptide, comprising culturing the recombinant host cell of claim 15 under conditions in which the neurotoxin-alpha polypeptide is expressed and recovering the polypeptide. (a) the amino acid sequence of a neurotoxin-alpha polypeptide having the complete amino acid sequence (SEQ ID NO: 2) set forth in Figures 1A and 1B, (b) the amino acid sequence of a neurotoxin-alpha polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in the deposit of ATCC Accession No. 97768, (c) the amino acid sequence of the neurotoxin-alpha polypeptide extracellular domain, (d) the amino acid sequence of the neurotoxin-alpha polypeptide secretory domain, (e) the amino acid sequence of the intracellular domain of the neurotoxin-alpha polypeptide, (f) the amino acid sequence of the soluble neurotoxin-alpha polypeptide comprising the domain and (g) at least 95% of the sequence selected from the group consisting of the amino acid sequence of the epitope-containing portion of the polypeptide described in (a), (b), (c), (d) A separate neurotoxin-alpha polypeptide comprising the same amino acid sequence. 18. The polypeptide of claim 17, which comprises a polypeptide comprising the amino acid residue (SEQ ID NO: 2) of about Phe-115 to about Leu-147, a polypeptide comprising the amino acid residue (SEQ ID NO: 2) of about Ile-150 to about Tyr- A polypeptide comprising the amino acid residue (SEQ ID NO: 2) of about Ser-171 to about Phe-194, a polypeptide comprising the amino acid residue (SEQ ID NO: 2) of about Glu-223 to about Tyr- A isolated polypeptide comprising an epitope-containing portion of a neurotoxin-alpha protein selected from the group consisting of polypeptides comprising an amino acid residue (SEQ ID NO: 2) of Phe-278. 17. A isolated antibody that specifically binds to the neurotoxin-alpha polypeptide of claim 17. 17. A pharmaceutical composition comprising the polypeptide of claim 17 and a pharmaceutically acceptable carrier. Except for one or more conservative amino acid substitutions, (a) amino acids 1 to 285 of SEQ ID NO: 2, (b) amino acids 2 to 285 of SEQ ID NO: 2, (c) amino acids 1 to 46 of SEQ ID NO: 2, (d) amino acids 47 to 72 of SEQ ID NO: 2 and (e) an isolated polynucleotide encoding a modified neurotoxin-alpha protein having the same amino acid sequence as the sequence selected from the group consisting of amino acids 73 to 286 of SEQ ID NO: 2. Except for one or more conservative amino acid substitutions, (a) amino acids 1 to 285 of SEQ ID NO: 2, (b) amino acids 2 to 285 of SEQ ID NO: 2, (c) amino acids 1 to 46 of SEQ ID NO: 2, (d) amino acids 47 to 72 of SEQ ID NO: 2 and (e) a modified neurotoxin-alpha polypeptide molecule having the same amino acid sequence as the sequence selected from the group consisting of amino acids 73 to 286 of SEQ ID NO: 2. (a) a nucleotide sequence of SEQ ID NO: 7, (b) a nucleotide sequence of SEQ ID NO: 8, (c) a nucleotide sequence of SEQ ID NO: 9, (d) a nucleotide sequence of nucleotides 1387 to 1421, a sequence of nucleotides 9 to 382, a sequence of nucleotides 1674 to 1996, a sequence of nucleotides 1401 to 1784, a sequence of nucleotides 900 to 1237 and all fragments located within these sequences. The nucleotide sequence of a portion of the sequence (SEQ ID NO: 1) described in Figures 1A and 1B that contains more than 30 consecutive nucleotides at 2442 and (e) a polynucleotide comprising a polynucleotide having a sequence at least 95% identical to a sequence selected from the group consisting of a nucleotide sequence complementary to the nucleotide sequence described in (a), (b), (c) Nucleic acid molecule. (a) a nucleotide sequence encoding a neurotoxin-alpha SV polypeptide having the complete amino acid sequence shown in Figures 5A and 5B (SEQ ID NO: 19) (b) a nucleotide sequence encoding a neurotoxin-alpha SV polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in the deposit of ATCC Accession No. 203518, (c) a nucleotide sequence encoding a neurotoxin-alpha SV polypeptide extracellular domain, (d) a nucleotide sequence encoding a neurotoxin-alpha SV polypeptide < RTI ID = 0.0 > (e) a nucleotide sequence encoding a neurotoxin-alpha SV polypeptide polypeptide intracellular domain, (f) a nucleotide sequence encoding an extracellular and intracellular domain but lacking a transmembrane domain and encoding a soluble neurotoxin-alpha SV polypeptide; and (g) having a nucleotide sequence that is at least 95% identical to a sequence selected from the group consisting of the nucleotide sequence complementary to the nucleotide sequence of (a), (b), (c), (d) An isolated nucleic acid molecule comprising a polynucleotide. 20. The isolated antibody of claim 19, wherein the protein of SEQ ID NO: 2 inhibits binding to the neurotoxin-alpha receptor.