SCF antibody compositions and methods of using the same Number:7,144,731 from the United States Patent and Trademark Office (PTO) owispatent Novel stem cell factors, oligonucleotides encoding the same, and methods of production, are disclosed. Pharmaceutical compositions and methods of treating disorders involving blood cells are also disclosed.<H compositions, antibody, SCF, antibody, co, 7144731.html, Patent, pto, 7144731

Title: SCF antibody compositions and methods of using the same

Abstract: Novel stem cell factors, oligonucleotides encoding the same, and methods of production, are disclosed. Pharmaceutical compositions and methods of treating disorders involving blood cells are also disclosed.

Patent Number: 7,144,731 Issued on 12/05/2006 to Zsebo, et al.

Inventors: Zsebo; Krisztina M. (Thousand Oaks, CA), Bosselman; Robert A. (Thousand Oaks, CA), Suggs; Sidney V. (Newbury Park, CA), Martin; Francis H. (Thousand Oaks, CA)
Assignee: Amgen Inc. (Thousand Oaks, CA)

Appl. No.: 10/353,783

Filed: January 28, 2003

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
09643659	Nov., 2002	6967029
08448729	May., 1995
08172329	Apr., 2001	6218148
07982255	Mar., 2001	6204363
07684535	Apr., 1991
07589701	Oct., 1990
07573616	Aug., 1990
07537198	Jun., 1990
07422383	Oct., 1989

Current U.S. Class: 435/326 ; 530/387.1; 530/387.9; 530/388.1; 530/388.15; 530/388.23

Current International Class: C12N 5/12 (20060101); C07K 16/22 (20060101)

Field of Search: 530/387.1,388.1,388.24,389.1,389.2 424/130.1,139.1,141.1,143.1,145.1,158.1 435/326,334,335

References Cited [Referenced By]

U.S. Patent Documents


3898837	August 1975	Boege
4275056	June 1981	Takaku et al.
4338397	July 1982	Gilbert et al.
4353242	October 1982	Harris et al.
4438032	March 1984	Golde et al.
4634665	January 1987	Axel et al.
4695542	September 1987	Yokota et al.
4703008	October 1987	Lin
4714680	December 1987	Civin
4721096	January 1988	Naughton et al.
4722998	February 1988	Cantor et al.
4808611	February 1989	Cosman
4810643	March 1989	Souza
4845078	July 1989	Masaoka
4847325	July 1989	Shadle et al.
4877729	October 1989	Clark et al.
4879227	November 1989	Clark et al.
4959314	September 1990	Mark et al.
4959455	September 1990	Clark et al.
4965204	October 1990	Civin
5087570	February 1992	Weissman et al.
5119315	June 1992	Kemp et al.
5121337	June 1992	Brown
5489516	February 1996	Broudy et al.
5545533	August 1996	Bartke et al.
5599703	February 1997	Davis et al.
5620685	April 1997	Nishi et al.
5641670	June 1997	Treco et al.
5767074	June 1998	Besmer et al.
5772992	June 1998	Bauer et al.
5786323	July 1998	Nakahata
5808002	September 1998	Buhring
5919911	July 1999	Broudy et al.
5935565	August 1999	Besmer et al.

Foreign Patent Documents


0 232 707	Aug., 1987	EP
0 845 268	Jun., 1998	EP
62-223126	Oct., 1987	JP
8325294	Dec., 1996	JP
62-107796	May., 1997	JP
09154578	Jun., 1997	JP
WO 89/04452	May., 1989	WO
WO 92/03459	Mar., 1992	WO
WO 92/07074	Apr., 1992	WO
WO 93/21936	Nov., 1993	WO
WO 95/06112	Mar., 1995	WO
WO 95/28479	Oct., 1995	WO
WO 98/18924	May., 1998	WO
WO 98/23773	Jun., 1998	WO

Other References

Broudy et al. Isolation and characterization of a monoclonal antibody that recognizes the human c-kit receptor. Blood 79(2): 338-345, 1992. cited by examiner .
Lerner et al. Monoclonal antibody YB5.B8 identifies the human c-kit protein product. Blood 77(9): 1876-1883, 1991. cited by examiner .
Moore, G. Genetically engineered antibodies. Clin Chem 35(9): 1849-1853, 1989. cited by examiner .
Ballow et al. Immunopharmacology, immunomodulation and immunotherapy. J Am Med Assoc 278(22: 2008-2017, 1997. cited by examiner .
Rico-Vargas et al. c-kit expression by B cell precursors in mouse bone marrow. J Immunol 152: 2845-2852, 1994. cited by examiner .
Cuthbert et al. Anti-interleukin-2 receptor monoclonal antibody (BT 563) in the treatment of severe acute GVHD refractory to systemic corticosteroid therapy. Bone Marrow Transplant. 10(5):451-455, 1992 (abstract only). cited by examiner .
Scheinberg et al. A phase I trial of monoclonal antibody M195 in acute myelogenous leukemia: specific bone marrow targeting and internalization of radionuclide. J Clin Oncol. 9(3):478-490, 1991. cited by examiner .
Kokai, Oct. 1, 1987, "Human hematopoietic stem cell growth factor-purification and characterization," JP 62-223126, abstract. cited by other .
Bodine et al., "Combination of Interleukins 3 and 6 Preserves Stem Cell Function in Culture and Enhances Retrovirus-Mediated Gene Transfer Into Hematopoietic Stem Cells," Proc. Nat'l Acad. Sci., USA, 86:8897-8901 (1989). cited by other .
Boswell et al., "A Novel Mast Cell Growth Factor (MCGF-3) Produced by Marrow-adherent Cells that Synergizes with Interleukin 3 and Interleukin 4," Exp. Hematol., 18:794-800 (1990). cited by other .
Bowie et al., "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science, 247:1306-1310 (1990). cited by other .
Broxmeyer et al., "Cell-Free Granulocyte Colony Inhibiting Activity Derived from Human Polymorphonuclear Neutrophils," Exp. Hemat., 5:87-102 (1977). cited by other .
Dexter, T.M., "Haemopoietic Growth Factors," I, 45(2):337-349 (1989). cite- d by other .
Dick et al., "Introduction of New Genes into Hematopoietic Stem Cells," J. Cell. Biol., Supplement 11A, p. 187 (1987) (Abstract D 026). cited by oth- er .
Dick et al., "Introduction of Selectable Gene into Primitive Stem Cells Capable of Long-Term Reconstitution of the Hemopoietic Systme of W/W.sup.v Mice," Cell, 42:71-79 (1985). cited by other .
Frommel et al., "An Estimate of the Effect of Point Mutation and Natural Selection on the Rate of Amino acid Replacement in Proteins," J. Mol. Evol., 21:233-257 (1985). cited by other .
Fung et al., "Molecular cloning of cDNA for murine interleukin-3," Nature, 307:233-237 (1984). cited by other .
Gough et al., "Molecular Cloning of cDNA Encoding a Murine Haematopoietic Growth Regulator, Granulocyte-Macrophage Colony Stimulating Factor," Nature, 309:763-767 (1984). cited by other .
Grabbe, J. et al., "Stem Cell Factor, a Novel Cutaneous Growth Factor for Mast Cells and Melanocytes," Arch. Dermatol. Res., 287:78-84 (1994). cite- d by other .
Grichnik, J.M. et al., "The SCF/KIT Pathway Plays a Critical Role in the Control of Normal Human Melanocyte Homeostasis," J. Invest. Dermatol., 111:233-238 (1998). cited by other .
Hiraoka et al., "Further Characterization of the Biological Properties of Human Hematopoietic Survival and Growth Factor," Expl. Cell. Biol., 57:27-34 (1989). cited by other .
Hiraoka et al., "Production of Human Hematopoietic Survival and Growth Factor by a Myeloid Leukemia Cell Line (KPB-M15) and Placenta as Detected by a Monoclonal Antibody," Cancer Research, 47:5025-5030 (1987). cited by other .
Hiraoka et al., "Human Hematopoietic Survival and Growth Factor," Cell Biology and International Reports, 10(5):347-355 (1986). cited by other .
Hiraoka et al., "Monoclonal Antibodies Against Human Hematopoietic Survival and Growth Factor," Biomed. Biochim. Acta, 46(5):419-427 (1987). cited by other .
Hiraoka et al., "In Vitro Effect of Murine Peritoneal Exudate Cells Activated with a Streptococcal Preparation, OK-432, on Hematopoietic Stem Cells," ACTA Hematol. Japan, 45:82-90 (1982). cited by other .
Hiraoka et al., Cancer Research, 47(19):5025-5030 (1987); Abstract. cited by other .
Hollands, "Differentiation of Embryonic Haemopoietic Stem Cells from Mouse Blastocysts Growth in Vitro," Development, 102:135-141 (1988). cited by other .
Humphries et al., "Aplastic Anemia and Stem Cell Biology," in Aplastic Anemia: Stem Cell Biology and Advances in Treatment, Alan R. Liss, Inc., New York, New York, pp. 3-12 (1984). cited by other .
Jones et al., "Cyclic Hematopoiesis: Animal Models," Exp. Hematol., 11:571-580 (1983). cited by other .
Kamamoto et al., "Establishment of Two Ph.sup.1 Chromosome-Positive Cell Lines, KPB-M8 and KPB-M15," Jpn. J. Clin. Oncol., 16:107-115 (1986). cite- d by other .
Kawasaki et al., "Molecular Cloning of a Complementary DNA Encoding Human Macrophage-Specific Colony-Stimulating Factor (CSF-1)," Science, 230:291-296 (1985). cited by other .
King, R.C. et al., in A Dictionary of Genetics, Fifth Edition, Oxford University Press, p. 138 (Nov. 1996). cited by other .
Kriegler et al., "Sources of Murine Macrophage Colony-Stimulating Factor that also Contain Growth Factors for Primitive Macrophage Progenitor Cells," Exp. Hematol., 12:844-849 (1984). cited by other .
Kriegler et al., "Partial Purification and Characterization of a Growth Factor for Macrophage Progenitor Cells with a High Proliferative Potential in Mouse Bone Marrow," Blood, 60:503-508 (1982). cited by other .
Langley et al., "Soluble Stem Cell Factor in Human Serum," Blood, 81(3):656-660 (1993). cited by other .
Lee et al., "Isolation of cDNA for a Human Granulocyte-Macrophage Colony-Stimulating Factor by Functional Expression in Mammalian Cells," Proc. Natl. Acad. Sci., USA, 82:4360-4364 (1985). cited by other .
Lev, S. et al., "Steel Factor and c-kit Protooncogene: Genetic Lessons in Signal Transduction," Critical Reviews in Oncogenesis, 5:141-168 (1994). cited by other .
Lewin, ed. Genes II, John Wiley & Sons, New York, p. 681 (1985). cited by other .
Lyman et al., "Molecular Cloning of a Ligand for the flt3/flk-2 Tyrosine Kinase Receptor: A Proliferative Factor for Primitive Hematopoietic Cells," Cell, 75:1157-1167 (1993). cited by other .
Lyman et al., "Molecular Cloning of a Ligand for the flt3/flk-2 Tyrosine Kinase Receptor: A Proliferative Factor for Primitive Hematopoietic Cells," Abstract 335, American Society of Hematology (1993). cited by oth- er .
MacKenzie, M.A.F. et al., "Activation of the Receptor Tyrosine Kinase Kit is Required for the Proliferation of Melanoblasts in the Mouse Embryo," Developmental Biology, 192:99-107 (1997). cited by other .
March et al., "Cloning, Sequence and Expression of Two Distinct Human Interleukin-1 Complementary DNAs," Nature, 315:641-647 (1985). cited by other .
Martin et al., "Primary Structure and Functional Expression of Rat and Human Stem Cell Factor DNAs," Cell, 63:203-211 (1990). cited by other .
Martin, P., "HEL Cells: A New Human Erythroleukemia Cell Line with Spontaneous and Induced Globin Expression," Science, 216:1233-1235 (1982). cited by other .
Mayer, T.C. et al., "An Experimental Analysis of the Pigment Defect Caused by Mutations at the W and SI Loci in Mice," Developmental Biology, 18:62-75 (1968). cited by other .
McDermott et al., "Inhibition of Cell Proliferation in Renal Failure and Its Significance to the Uraemic Syndrome: A Review," Scot. Med. J., 20:317-327 (1975). cited by other .
McNeice et al., "A Growth Factor Produced by WEHI-3 Cells for Murine High Proliferative Potential GM-Progenitor Colony Forming Cells," Cell Biol. Int. Rep., 6:243-251 (1982). cited by other .
McNiece et al., "Studies on the Myeloid Synergistic Factor From 5637: Comparison with Interleukin-1 Alpha," Blood, 73:919-923 (1987). cited by other .
McNiece et al., "Detection of Muring Hemopoietin-1 in Media Conditioned by EMT6 Cells," Exp. Hematol., 15:854-858 (1987). cited by other .
McNiece et al., "Synergistic Internactions Between Hematopoietic Growth Factors as Detected by in vitro Mouse Bone Marrow Colony Formation," Exp. Hematol., 16:383-388 (1988). cited by other .
Moore, M.A.S., "Clinical Implications of Positive and Negative Hematopoietic Stem Cell Regulators," Blood, 78:1-19 (1991). cited by othe- r .
Mori et al., "Myeloproliferative Sarcoma Virus Stimulates Pluripotent Hematopoietic Stem Cells and Provokes Tumoral Transformation of the Hematopoietic Microenvironment in Vitro," Leukemia Research, 7:77-86 (1983). cited by other .
Nabel et al., "Inducer T Lymphocytes Synthesize a Factor That Stimulates Proliferation of Cloned Mast Cells," Nature, 291:332-334 (1981). cited by other .
Nagata, S., et al., "Molecular cloning and expression of cDNA for human granulocyte colony-stimulating factor," Nature, 319:415-418 (1986). cited by other .
Ngo et al., "Computational Complexity Protein Structure Prediction, and the Levinthal Paradox," In The Protein Folding Problem and Tertiary Structure Prediction, Merz et al., (eds.), 1994 pp. 433-495. cited by oth- er .
Qiu et al., "Primary Structure of c-kit: Relationship with the CSF-1/PDGF Receptor Kinase Family-Oncogenic Activation of v-kit Involves Deletion of Extracellular Domain and C Terminus," EMBO J., 7(4):1003-1011 (1988). cit- ed by other .
Ross, M.H. et al., In Histology A Text and Atlas, Coryell, P.A. (Ed.), Third Edition Williams & Wilkins, Baltimore, MD, USA, pp. 54-55, 58-59, 106-107, 200-202, 206 and 375 (1995). cited by other .
Sarvella. P.A. et al., "Steel, A New Dominant Gene in the House Mouse, With Effects on Coat Pigment and Blood," The Journal of Histology, 47:123-128 (1956). cited by other .
Schrader, "Role of a Single Haemopoietic Growth Factor in Multiple Proliferative Disorders of Haemopoietic and Related Cells," Lancet, 2:133-137 (1984). cited by other .
Song et al., "Hematopoietic Factor Production by a Cell Line (TC-1) Derived from Adherent Murine Marrow Cells," Blood, 66:273-281 (1985). cit- ed by other .
Spangrude et al., "Purification and Characterization of Mouse Hematopoietic Stem Cells," Science, 214:58-62 (1988). cited by other .
Stanley et al., "Regulation of Very Primitive, Multipotent, Hemopoietic Cells by Hemopoietin-1," Cell, 45:667-674 (1986). cited by other .
Watson et al., "Molecular Cloning of the Polypeptide Factors That Stimulate Growth, Maturation, and Function of Blood Cells.sup.60-66," Molecular Biology of the Gene, Fourth Edition, pp. 983-992 (1987). cited by other .
Webster's Third New International Dictionary of the English Language Unabridged, Gove, P.B. et al. (Eds.), Merriam-Webster Inc., Springfield, MA, USA, p. 1827 (1986). cited by other .
Welte et al., "Recombinant Human Granulocyte Colony-Stimulating Factor," J. Exp. Med., 165:941-948 (1987). cited by other .
Wong et al., "Human GM-CSF: Molecular Cloning of the Complementary DNA and Purification of the Natural Recombinant Proteins," Science, 228:810-814 (1985). cited by other .
Yarden et al., "Human Proto-Oncogene c-kit: a New Cell Surface Receptor Tyrosine Kinase for an Unidentified Ligand," EMBO J., 6(11):3341-3351 (1987). cited by other .
Yokota et al., "Isolation and Characterization of a Mouse cDNA Clone that Expresses Mast-Cell Growth-Factor Activity in Monkey Cells," Proc. Natl. Acad. Sci., USA, 81:1070-1074 (1984). cited by other .
Young et al., "Efficient Isolation of Genes by Using Antibody Probes," Proc. Natl. Acad. Sci., USA, 80:1194-1195 (1983). cited by other .
Zajicek, "Inflammation Initiates Cancer by Depleting Stem Cells," Medical Hypotheses, 18:207-219 (1985). cited by other .
Zsebo et al. "Effects of Hematopoietin-1 and Interleukin 1 Activities on Early Hematopoietic Cells of the Bone Marrow," Blood, 71(4):962-968 (1988). cited by other .
Zsebo et al., "Stem Cell Factor Is Encoded at the SI Locus of the Mouse and Is the Ligand for the c-kit Tyrosine Kinase Receptor," Cell, 63:213-224 (1990). cited by other.

Primary Examiner: Brumback; Brenda
Assistant Examiner: Bunner; Bridget E.
Attorney, Agent or Firm: Marshall, Gerstein & Borun LLP

Parent Case Text

This is a continuation of U.S. application Ser. No. 09/643,659, filed Aug. 21, 2000, now U.S. Pat. No. 6,967,029, issued Nov. 22, 2005, which is a continuation application of U.S. application Ser. No. 08/448,729, filed May 24, 1995, now abandoned, which is a divisional application of U.S. application Ser. No. 08/172,329 filed Dec. 21, 1993, now U.S. Pat. No. 6,218,148 issued Apr. 17, 2001, which is a continuation of U.S. application Ser. No. 07/982,255 filed Nov. 25, 1992, now U.S. Pat. No. 6,204,363 issued Mar. 20, 2001, which is a continuation of U.S. application Ser. No. 07/684,535 filed Apr. 10, 1991, now abandoned, which is a continuation-in-part of U.S. application Ser. No. 07/589,701 filed Oct. 1, 1990, now abandoned, which is a continuation-in-part application of U.S. application Ser. No. 07/573,616 filed Aug. 24, 1990, now abandoned, which is a continuation-in-part application of U.S. application Ser. No. 07/537,198 filed Jun. 11, 1990, now abandoned, which is a continuation-in-part application of U.S. application Ser. No. 07/422,383 filed Oct. 16, 1989, now abandoned, each of which are hereby incorporated by reference.

Claims

What is claimed is:

1. A purified antibody that is specifically immunoreactive with a stem cell factor (SCF).

2. The purified antibody of claim 1, wherein said antibody is a monoclonal antibody.

3. The purified antibody of claim 1, wherein said antibody is specifically immunoreactive with human SCF.

4. The purified antibody of claim 1, wherein said antibody is specifically immunoreactive with a non-human vertebrate SCF.

5. The purified antibody of claim 1, wherein said antibody is specifically immunoreactive with an SCF factor comprising the sequence of SEQ ID NO:44, SEQ ID NO: 46, or SEQ ID NO: 61.

6. The purified antibody of claim 1, wherein said antibody is specifically immunoreactive with a non-human SCF factor comprising the sequence of any of the sequences set out in SEQ ID NOs: 49 57.

7. A hybridoma cell line producing a monoclonal antibody that is specifically immunoreactive with a stem cell factor protein.

8. A composition comprising: i) an antibody specifically immunoreactive with an SCF polypeptide; and ii) an acceptable carrier excipient or diluent.

Description

The present invention relates in general to novel factors which stimulate primitive progenitor cells including early hematopoietic progenitor cells, and to DNA sequences encoding such factors. In particular, the invention relates to these novel factors, to fragments and polypeptide analogs thereof and to DNA sequences encoding the same.

BACKGROUND OF THE INVENTION

The human blood-forming (hematopoietic) system is comprised of a variety of white blood cells (including neutrophils, macrophages, basophils, mast cells, eosinophils, T and B cells), red blood cells (erythrocytes) and clot-forming cells (megakaryocytes, platelets),

It is believed that small amounts of certain hematopoietic growth factors account for the differentiation of a small number of "stem cells" into a variety of blood cell progenitors for the tremendous proliferation of those cells, and for the ultimate differentiation of mature blood cells from those lines. The hematopoietic regenerative system functions well under normal conditions. However, when stressed by chemotherapy, radiation, or natural myelodysplastic disorders, a resulting period during which patients are seriously leukopenic, anemic, or thrombocytopenic occurs. The development and the use of hematopoietic growth factors accelerates bone marrow regeneration during this dangerous phase.

In certain viral induced disorders, such as acquired autoimmune deficiency (AIDS) blood elements such as T cells may be specifically destroyed. Augmentation of T cell production may be therapeutic in such cases.

Because the hematopoietic growth factors are present in extremely small amounts, the detection and identification of these factors has relied upon an array of assays which as yet only distinguish among the different factors on the basis of stimulative effects on cultured cells under artificial conditions.

The application of recombinant genetic techniques has clarified the understanding of the biological activities of individual growth factors. For example, the amino acid and DNA sequences for human erythropoietin (EPO), which stimulates the production of erythrocytes, have been obtained. (See, Lin, U.S. Pat. No. 4,703,008, hereby incorporated by reference). Recombinant methods have also been applied to the isolation of cDNA for a human granulocyte colony-stimulating factor, G-CSF (See, Souza, U.S. Pat. No. 4,810,643, hereby incorporated by reference), and human granulocyte-macrophage colony stimulating factor (GM-CSF) [Lee, et al., Proc. Natl. Acad. Sci. USA, 82, 4360 4364 (1985); Wong, et al., Science, 228, 810 814 (1985)], murine G- and GM-CSF [Yokota, et al., Proc. Natl. Acad. Sci. (USA), 81, 1070 (1984); Fung, et al., Nature, 307, 233 (1984); Gough, et al., Nature, 309, 763 (1984)], and human macrophage colony-stimulating factor (CSF-1) [Kawasaki, et al., Science, 230, 291 (1985)].

The High Proliferative Potential Colony Forming Cell (HPP-CFC) assay system tests for the action of factors on early hematopoietic progenitors [Zont, J. Exp. Med., 159, 679 690 (1984)]. A number of reports exist in the literature for factors which are active in the HPP-CFC assay. The sources of these factors are indicated in Table 1. The most well characterized factors are discussed below.

An activity in human spleen conditioned medium has been termed synergistic factor (SF). Several human tissues and human and mouse cell lines produce an SF, referred to as SF-1, which synergizes with CSF-1 to stimulate the earliest HPP-CFC. SF-1 has been reported in media conditioned by human spleen cells, human placental cells, 5637 cells (a bladder carcinoma cell line), and EMT-6 cells (a mouse mammary carcinoma cell line). The identity of SF-1 has yet to be determined. Initial reports demonstrate overlapping activities of interleukin-1 with SF-1 from cell line 5637 [Zsebo et al., Blood, 71, 962 968 (1988)]. However, additional reports have demonstrated that the combination of interleukin-1 (IL-1) plus CSF-1 cannot stimulate the same colony formation as can be obtained with CSF-1 plus partially purified preparations of 5637 conditioned media [McNiece, Blood, 73, 919 (1989)].

The synergistic factor present in pregnant mouse uterus extract is CSF-1. WEHI-3 cells (murine myelomonocytic leukemia cell line) produce a synergistic factor which appears to be identical to IL-3. Both CSF-1 and IL-3 stimulate hematopoietic progenitors which are more mature than the target of SF-1.

Another class of synergistic factor has been shown to be present in conditioned media from TC-1 cells (bone marrow-derived stromal cells). This cell line produces a factor which stimulates both early myeloid and lymphoid cell types. It has been termed hemolymphopoietic growth factor 1 (HLGF-1). It has an apparent molecular weight of 120,000 [McNiece et al., Exp. Hematol., 16, 383 (1988)].

Of the known interleukins and CSFs, IL-1, IL-3, and CSF-1 have been identified as possessing activity in the HPP-CFC assay. The other sources of synergistic activity mentioned in Table 1 have not been structurally identified. Based on the polypeptide sequence and biological activity profile, the present invention relates to a molecule which is distinct from IL-1, IL-3, CSF-1 and SF-1.

TABLE-US-00001 TABLE 1 Preparations Containing Factors Active in the HPP-CFC Assay Source.sup.1 Reference Human Spleen CM [Kriegler, Blood, 60, 503(1982)] Mouse Spleen CM [Bradley, Exp. Hematol. Today Baum, ed., 285 (1980)] Rat Spleen CM [Bradley, supra, (1980)] Mouse lung CM [Bradley, supra, (1980)] Human Placental CM [Kriegler, supra (1982)] Pregnant Mouse Uterus [Bradley, supra (1980)] GTC-C CM [Bradley, supra (1980)] RH3 CM [Bradley, supra (1980)] PHA PBL [Bradley, supra (1980)] WEHI-3B CM [McNiece, Cell Biol. Int. Rep., 6, 243(1982)] EMT-6 CM [McNiece, Exp. Hematol., 15, 854 (1987)] L-Cell CM [Kriegler, Exp. Hematol., 12, 844 (1984)] 5637 CM [Stanley, Cell, 45, 667 (1986)] TC-1 CM [Song, Blood, 66, 273 (1985)] .sup.1CM = Conditioned media.

When administered parenterally, proteins are often cleared rapidly from the circulation and may therefore elicit relatively short-lived pharmacological activity. Consequently, frequent injections of relatively large doses of bioactive proteins may be required to sustain therapeutic efficacy. Proteins modified by the covalent attachment of water-soluble polymers such as polyethylene glycol, copolymers of polyethylene glycol and polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline are known to exhibit substantially longer half-lives in blood following intravenous injection than do the corresponding unmodified proteins [Abuchowski et al., In: "Enzymes as Drugs", Holcenberg et al., eds. Wiley-Interscience, New York, N.Y., 367 383 (1981), Newmark et al., J. Appl. Biochem. 4:185 189 (1982), and Katre et al., Proc. Natl. Acad. Sci. USA 84, 1487 1491 (1987)]. Such modifications may also increase the protein's solubility in aqueous solution, eliminate aggregation, enhance the physical and chemical stability of the protein, and greatly reduce the immunogenicity and antigenicity of the protein. As a result, the desired in vivo biological activity may be achieved by the administration of such polymer-protein adducts less frequently or in lower doses than with the unmodified protein.

Attachment of polyethylene glycol (PEG) to proteins is particularly useful because PEG has very low toxicity in mammals [Carpenter et al., Toxicol. Appl. Pharmacol., 18, 35 40 (1971)]. For example, a PEG adduct of adenosine deaminase was approved in the United States for use in humans for the treatment of severe combined immunodeficiency syndrome. A second advantage afforded by the conjugation of PEG is that of effectively reducing the immunogenicity and antigenicity of heterologous proteins. For example, a PEG adduct of a human protein might be useful for the treatment of disease in other mammalian species without the risk of triggering a severe immune response.

Polymers such as PEG may be conveniently attached to one or more reactive amino acid residues in a protein such as the alpha-amino group of the amino-terminal amino acid, the epsilon amino groups of lysine side chains, the sulfhydryl groups of cysteine side chains, the carboxyl groups of aspartyl and glutamyl side chains, the alpha-carboxyl group of the carboxyl-terminal amino acid, tyrosine side chains, or to activated derivatives of glycosyl chains attached to certain asparagine, serine or threonine residues.

Numerous activated forms of PEG suitable for direct reaction with proteins have been described. Useful PEG reagents for reaction with protein amino groups include active esters of carboxylic acid or carbonate derivatives, particularly those in which the leaving groups are N-hydroxysuccinimide, p-nitrophenol, imidazole or 1-hydroxy-2-nitrobenzene-4-sulfonate. PEG derivatives containing maleimido or haloacetyl groups are useful reagents for the modification of protein free sulfhydryl groups. Likewise, PEG reagents containing amino, hydrazine or hydrazide groups are useful for reaction with aldehydes generated by periodate oxidation of carbohydrate groups in proteins.

It is an object of the present invention to provide a factor causing growth of early hematopoietic progenitor cells.

SUMMARY OF THE INVENTION

According to the present invention, novel factors, referred to herein as "stem cell factors" (SCF) having the ability to stimulate growth of primitive progenitors including early hematopoietic progenitor cells are provided. These SCFs also are able to stimulate non-hematopoietic stem cells such as neural stem cells and primordial germ stem cells. Such factors include purified naturally-occurring stem cell factors. The invention also relates to non-naturally-occurring polypeptides having amino acid sequences sufficiently duplicative of that of naturally-occurring stem cell factor to allow possession of a hematopoietic biological activity of naturally occurring stem cell factor.

The present invention also provides isolated DNA sequences for use in securing expression in procaryotic or eukaryotic host cells of polypeptide products having amino acid sequences sufficiently duplicative of that of naturally-occurring stem cell factor to allow possession of a hematopoietic biological activity of naturally occurring stem cell factor. Such DNA sequences include:

(a) DNA sequences set out in FIGS. 14B, 14C, 15B, 15C, 15D, 42 and 44 or their complementary strands;

(b) DNA sequences which hybridize to the DNA sequences defined in (a) or fragments thereof; and

(c) DNA sequences which, but for the degeneracy of the genetic code, would hybridize to the DNA sequences defined in (a) and (b).

Also provided are vectors containing such DNA sequences, and host cells transformed or transfected with such vectors. Also comprehended by the invention are methods of producing SCF by recombinant techniques, and methods of treating disorders. Additionally, pharmaceutical compositions including SCF and antibodies specifically binding SCF are provided.

The invention also relates to a process for the efficient recovery of stem cell factor from a material containing SCF, the process comprising the steps of ion exchange chromatographic separation and/or reverse phase liquid chromatographic separation.

The present invention also provides a biologically-active adduct having prolonged in vivo half-life and enhanced potency in mammals, comprising SCF covalently conjugated to a water-soluble polymer such as polyethylene glycol or copolymers of polyethylene glycol and polypropylene glycol, wherein said polymer is unsubstituted or substituted at one end with an alkyl group. Another aspect of this invention resides in a process for preparing the adduct described above, comprising reacting the SCF with a water-soluble polymer having at least one terminal reactive group and purifying the resulting adduct to produce a product with extended circulating half-life and enhanced biological activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an anion exchange chromatogram from the purification of mammalian SCF.

FIG. 2 is a gel filtration chromatogram from the purification of mammalian SCF.

FIG. 3 is a wheat germ agglutinin-agarose chromatogram from the purification of mammalian SCF.

FIG. 4 is a cation exchange chromatogram from the purification of mammalian SCF.

FIG. 5 is a C.sub.4 chromatogram from the purification of mammalian SCF.

FIG. 6 shows sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) (SDS-PAGE) of C.sub.4 column fractions from FIG. 5.

FIG. 7 is an analytical C.sub.4 chromatogram of mammalian SCF.

FIG. 8 shows SDS-PAGE of C.sub.4 column fractions from FIG. 7.

FIG. 9 shows SDS-PAGE of purified mammalian SCF and deglycosylated mammalian SCF.

FIG. 10 is an analytical C.sub.4 chromatogram of purified mammalian SCF.

FIG. 11 shows the amino acid sequence (SEQ ID NO.: 1) of mammalian SCF derived from protein sequencing.

FIG. 12 shows

A. oligonucleotides for rat SCF cDNA (SEQ ID NOS.: 2 19)

B. oligonucleotides for human SCF DNA (SEQ ID NOS.: 20 30)

C. universal oligonucleotides. (SEQ TD NOS.: 31 38).

FIG. 13 shows

A. a scheme for polymerase chain reaction (PCR) amplification of rat SCF cDNA

B. a scheme for PCR amplification of human SCF cDNA.

FIG. 14 shows

A. sequencing strategy for rat genomic DNA

B. the nucleic acid sequence of rat (SEQ ID NOS.: 39 and 40) genomic DNA.

C. the nucleic acid sequence of rat SCF cDNA and amino acid sequence of rat SCF protein. (SEQ ID NOS.: 41 and 42).

FIG. 15 shows

A. the strategy for sequencing human genomic DNA

B. the nucleic acid sequence of human (SEQ ID NO.: 43 and 44) genomic DNA.

C. the composite nucleic acid sequence of (SEQ ID NOS.: 45 and 46) human SCF cDNA and amino acid sequence of SCF protein.

D. the nucleic acid sequence of genomic DNA and amino acid sequence of human SCF protein, including (SEQ ID NOS.: 47 and 48) flanking regions and introns.

FIGS. 16A and B shows the aligned amino acid sequences of human, monkey, dog, mouse, and rat (SEQ ID NOS.: 49 57) SCF protein.

FIG. 16C shows an elution profile of hSCF.sup.1-248 from CHO cells after AspN peptidase digestion and HPLC.

FIG. 16D shows the nucleotide sequence of the 221 base pair EcoRI-BamHI fragment constructed from (SEQ ID NOS.: 58 and 59) oligonucleotides that were used in creating the plasmid for human [Met.sup.-1] SCF.sup.1-165. Uppercase letters below the nucleotide sequence represent the amino acid sequence. Lowercase letters above the nucleotide sequence show nucleotides in the original hSCF.sup.1-183 sequence that were altered to generate codons most frequently used by E. coli.

FIG. 16E shows the 39 base pair BstEII-BamHI fragment used in creating the plasmid for human [Met.sup.-1] SCF.sup.1-165 with optimized C-terminal codons.

FIG. 17 shows the structure of mammalian cell expression vector V19.8 SCF.

FIG. 18 shows the structure of mammalian CHO cell expression vector pDSVE.1.

FIG. 19 shows the structure of E. coli expression vector pCFM1156.

FIG. 20 shows

A. a radioimmunoassay of mammalian SCF

B. SDS-PAGE of immune-precipitated mammalian SCF.

FIG. 21 shows Western analysis of recombinant human SCF.

FIG. 22 shows Western analysis of recombinant rat SCF.

FIG. 22A shows radioimmune assay determination of SCF in Human Serum. The percent inhibition of .sup.125I-human SCF binding produced was determined for various doses of an unlabeled standard of CHO HuSCF.sup.1-248 (solid circles); a sample of NHS Lot 500080713 (open circles); and NHS Lot 500081015 (solid triangle).

FIG. 23 is a bar graph showing the effect of COS-1 cell-produced recombinant rat SCF on bone marrow transplantation.

FIG. 24 shows the effect of recombinant rat SCF on curing the macrocytic anemia of Steel mice, as assessed by hematocrit analysis (24A) or mean red blood cell volume (24B).

FIG. 25 shows the peripheral white blood cell count (WBC) of Steel mice treated with recombinant rat SCF.

FIG. 26 shows the platelet counts of Steel mice treated with recombinant rat SCF.

FIG. 27 shows the differential WBC count for Steel mice treated with recombinant rat SCF.sup.1-164 PEG25.

FIG. 28 shows the lymphocyte subsets for Steel mice treated with recombinant rat SCF.sup.1-164 PEG25.

FIG. 29 shows the effect of recombinant human sequence SCF treatment of normal primates in increasing WBC count.

29A. expressed as white blood cells in [K/cmm]

29B. expressed as peripheral blood cells in [K/cmm].

FIG. 30 shows the effect of recombinant human sequence SCF treatment of normal primates in increasing hematocrits (30B) and platelet numbers (30A).

FIG. 31 shows photographs of

A. human bone marrow colonies stimulated by recombinant human SCF.sup.1-162

B. Wright-Giemsa stained cells from colonies in FIG. 31A.

FIG. 31C shows proliferation of the UT-7 cell line by E. coli derived SCFs. Open squares are human [Met.sup.-1]SCF.sup.1-164, crosses and open diamonds are human [Met.sup.-1]SCF.sup.1-165.

FIG. 32 shows SDS-PAGE of S-Sepharose column fractions from chromatogram shown in FIG. 33

A. with reducing agent

B. without reducing agent.

FIG. 33 is a chromatogram of an S-Sepharose column of E. coli derived recombinant human SCF.

FIG. 34 shows SDS-PAGE of C.sub.4 column fractions from chromatogram showing FIG. 35

A. with reducing agent

B. without reducing agent.

FIG. 35 is a chromatogram of a C.sub.4 column of E. coli derived recombinant human SCF.

FIG. 36 is a chromatogram of a Q-Sepharose column of CHO derived recombinant rat SCF.

FIG. 37 is a chromatogram of a C.sub.4 column of CHO derived recombinant rat SCF.

FIG. 38 shows SDS-PAGE of C.sub.4 column fractions from chromatogram shown in FIG. 37.

FIG. 39 shows SDS-PAGE of purified CHO derived recombinant rat SCF before and after de-glycosylation.

FIG. 40 shows

A. gel filtration chromatography of recombinant rat pegylated SCF.sup.1-164 reaction mixture

B. gel filtration chromatography of recombinant rat SCF.sup.1-164, unmodified.

FIG. 41 shows labelled SCF binding to fresh leukemic blasts.

FIGS. 42A 42D show human SCF cDNA sequence (SEQ ID NOs: 60 and 61) obtained from the HT1080 fibrosarcoma cell line.

FIG. 43 shows an autoradiograph from COS-7 cells expressing human SCF.sup.1-248 and CHO cells expressing human SCF.sup.1-164.

FIGS. 44A 44C show human SCF cDNA sequence (SEQ ID NOs: 62 and 63) obtained from the 5637 bladder carcinoma cell line.

FIG. 45 shows the enhanced survival of irradiated mice after SCF treatment.

FIG. 46 shows the enhanced survival of irradiated mice after bone marrow transplantation with 5% of a femur and SCF treatment.

FIG. 47 shows the enhanced survival of irradiated mice after bone marrow transplantation with 0.1 and 20% of a femur and SCF treatment.

FIG. 48 shows radioprotection effects of rat SCF on survival of mice after irradiation.

FIG. 49 shows radioprotection effects of rat SCF on survival of mice after irradiation.

FIG. 50 shows a single coinjection of rrSCF plus G-CSF causes an increase in circulating neutrophils that is approximately additive as compared to the rrSCF alone- and G-CSF alone-induced neutrophilia. The kinetics of rrSCF plus G-CSF-induced neutrophilia reflect the combined effect of the differing kinetics of rrSCF-induced neutrophilia peaking at 6 hours and G-CSF-induced neutrophilia peaking at 12 hours.

FIG. 51 shows daily coinjection of rrSCF and G-CSF for one week caused a highly synergistic increase in circulating neutrophils with a marked linear increase between day 4 and day 6.

FIG. 52 shows a kinetic study of rrSCF plus G-CSF-induced neutrophilia after the seventh daily injection shows that the peak of circulating neutrophils occurs at 12 hours and reaches a level of 69.times.10.sup.3 PMN/mm.sup.3.

FIG. 53 shows in vivo administration of SCF-platelet counts.

FIG. 54 shows dose response of rratSCF-PEG on platelet counts.

FIG. 55 shows effect of 5-FU on platelet levels.

FIG. 56 shows 5-FU effect on ACH+ cells in marrow (56A) and spleen (56B).

FIG. 57 shows mean platelet volume after 5-FU treatment.

FIG. 58 shows SCF mRNA levels after 5-FU treatment. The data in this figure were generated from the same marrow samples collected in FIG. 56. Data points are the values determined from individual mice.

FIG. 59 shows the effects of HuSCF and zidovudine on peripheral blood BFU-E in normal donors. Light density cells were plated in duplicate in the presence of (A) 1 U/ml or (B) 4 U/ml of erythropoietin, four concentrations of zidovudine (0, 10.sup.-7 M, 10.sup.-6 M and 10.sup.-5 M) and four concentrations of HuSCF (0, 10 ng/ml, 100 ng/ml and 1000 ng/ml). The bars represent the mean.+-.S.E.M. for the duplicate determinations of both normal donors. All of the increases for HuSCF are statistically significant (independent t-test, 2-tailed, p<0.01).

FIG. 60 shows the effects of HuSCF and zidovudine on peripheral blood BFU-E in normal and HIV-infected donors. Light density cells were plated in duplicate in the presence of 1 U/ml of erythropoietin and four concentrations of HuSCF (0, 10 ng/ml, 100 ng/ml and 1000 ng/ml). The bars represent the mean for the duplicate determinations.

FIG. 61 shows alteration of the BFU-E ID.sub.50 of zidovudine by HuSCF. The 50% inhibitory concentration for BFU-E for each level of HuSCF was calculated as described in the text. The bars represent the mean for the two normal donors.

FIG. 62 shows effects of HuSCF on AZT suppression of bone marrow culture as measured by BFU-E.

FIG. 63 shows effect of HuSCF on AZT suppression of bone marrow culture as measured by CFU-GM.

FIG. 64 shows effects of HuSCF on gancyclovir suppression of bone marrow culture as measured by BFU-E.

FIG. 65 shows effect of HuSCF on gancyclovir suppression of bone marrow culture as measured by CFU-GM.

FIG. 66 shows effect of rat SCF alone and in combination with CFU-S number in a pre-CFU-S assay.

FIG. 67 shows effect of SCF alone and in combination on the recovery of hemaglobin.

FIG. 68 shows fluorescence emission spectra of human SCF.sup.1-164. Emission intensity is shown for CHO cell derived [Met.sup.-1]SCF.sup.1-162 (dotted line) and E. coli derived [Met.sup.-1]SCF.sup.1-164 (solid line).

FIG. 69 shows circular dichroism of SCF. The far ultraviolet spectra (A) and near ultraviolet spectra (B) are shown for CHO cell-derived [met.sup.-1]SCF.sup.1-162 (dotted line) and E. coli derived [Met.sup.-1]SCF.sup.1-164 (solid line).

FIG. 70 shows second derivative infrared spectra of SCF. The second derivative infrared spectra in the amide I region (1700 1620 cm.sup.-1) for E. coli derived [Met.sup.-1]SCF.sup.1-164 (A) and CHO cell derived (Met.sup.-1SCF.sup.1-162 (B) are shown.

Numerous aspects and advantages of the invention will be apparent to those skilled in the art upon consideration of the following detailed description which provides illustrations of the practice of the invention in its presently-preferred embodiments.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, novel stem cell factors and DNA sequences coding for all or part of such SCFs are provided. The term "stem cell factor" or "SCF" as used herein refers to naturally-occurring SCF (e.g. natural human SCF) as well as non-naturally occurring (i.e., different from naturally occurring) polypeptides having amino acid sequences and glycosylation sufficiently duplicative of that of naturally-occurring stem cell factor to allow possession of a hematopoietic biological activity of naturally-occurring stem cell factor. Stem cell factor has the ability to stimulate growth of early hematopoietic progenitors which are capable of maturing to erythroid, megakaryocyte, granulocyte, lymphocyte, and macrophage cells. SCF treatment of mammals results in absolute increases in hematopoietic cells of both myeloid and lymphoid lineages. One of the hallmark characteristics of stem cells is their ability to differentiate into both myeloid and lymphoid cells [Weissman, Science, 241, 58 62 (1988)]. Treatment of Steel mice (Example 8B) with recombinant rat SCF results in increases of granulocytes, monocytes, erythrocytes, lymphocytes, and platelets. Treatment of normal primates with recombinant human SCF results in increases in myeloid and lymphoid cells (Example 8C).

There is embryonic expression of SCF by cells in the migratory pathway and homing sites of melanoblasts, germ cells, hematopoietic cells, brain and spinal chord.

Early hematopoietic progenitor cells are enriched in bone marrow from mammals which has been treated with 5-Fluorouracil (5-FU). The chemotherapeutic drug 5-FU selectively depletes late hematopoietic progenitors. SCF is active on post 5-FU bone marrow.

The biological activity and pattern of tissue distribution of SCF demonstrates its central role in embryogenesis and hematopoiesis as well as its capacity for treatment of various stem cell deficiencies.

The present invention provides DNA sequences which include: the incorporation of codons "preferred" for expression by selected nonmammalian hosts; the provision of sites for cleavage by restriction endonuclease enzymes; and the provision of additional initial, terminal or intermediate DNA sequences which facilitate construction of readily-expressed vectors. The present invention also provides DNA sequences coding for polypeptide analogs or derivatives of SCF which differ from naturally-occurring forms in terms of the identity or location of one or more amino acid residues (i.e., deletion analogs containing less than all of the residues specified for SCF; substitution analogs, wherein one or more residues specified are replaced by other residues; and addition analogs wherein one or more amino acid residues is added to a terminal or medial portion of the polypeptide) and which share some or all the properties of naturally-occurring forms. The present invention specifically provides DNA sequences encoding the full length unprocessed amino acid sequence as well as DNA sequences encoding the processed form of SCF.

Novel DNA sequences of the invention include sequences useful in securing expression in procaryotic or eucaryotic host cells of polypeptide products having at least a part of the primary structural conformation and one or more of the biological properties of naturally-occurring SCF. DNA sequences of the invention specifically comprise: (a) DNA sequences set forth in FIGS. 14B, 14C, 15B, 15C, 15D, 42 and 44 or their complementary strands; (b) DNA sequences which hybridize (under hybridization conditions disclosed in Example 3 or more stringent conditions) to the DNA sequences in FIGS. 14B, 14C, 15B, 15C, 15D, 42, and 44 or to fragments thereof; and (c) DNA sequences which, but for the degeneracy of the genetic code, would hybridize to the DNA sequences in FIGS. 14B, 14C, 15B, 15C, 15D, 42, and 44. Specifically comprehended in parts (b) and (c) are genomic DNA sequences encoding allelic variant forms of SCF and/or encoding SCF from other mammalian species, and manufactured DNA sequences encoding SCF, fragments of SCF, and analogs of SCF. The DNA sequences may incorporate codons facilitating transcription and translation of messenger RNA in microbial hosts. Such manufactured sequences may readily be constructed according to the methods of Alton et al., PCT published application WO 83/04053.

According to another aspect of the present invention, the DNA sequences described herein which encode polypeptides having SCF activity are valuable for the information which they provide concerning the amino acid sequence of the mammalian protein which have heretofore been unavailable. The DNA sequences are also valuable as products useful in effecting the large scale synthesis of SCF by a variety of recombinant techniques. Put another way, DNA sequences provided by the invention are useful in generating new and useful viral and circular plasmid DNA vectors, new and useful transformed and transfected procaryotic and eucaryotic host cells (including bacterial and yeast cells and mammalian cells grown in culture), and new and useful methods for cultured growth of such host cells capable of expression of SCF and its related products.

DNA sequences of the invention are also suitable materials for use as labeled probes in isolating human genomic DNA encoding SCF and other genes for related proteins as well as cDNA and genomic DNA sequences of other mammalian species. DNA sequences may also be useful in various alternative methods of protein synthesis (e.g., in insect cells) or in genetic therapy in humans and other mammals. DNA sequences of the invention are expected to be useful in developing transgenic mammalian species which may serve as eucaryotic "hosts" for production of SCF and SCF products in quantity. See, generally, Palmiter et al., Science 222, 809 814 (1983).

The present invention provides purified and isolated naturally-occurring SCF (i.e. purified from nature or manufactured such that the primary, secondary and tertiary conformation, and the glycosylation pattern are identical to naturally-occurring material) as well as non-naturally occurring polypeptides having a primary structural conformation (i.e., continuous sequence of amino acid residues) and glycosylation sufficiently duplicative of that of naturally occurring stem cell factor to allow possession of a hematopoietic biological activity of naturally occurring SCF. Such polypetides include derivatives and analogs.

In a preferred embodiment, SCF is characterized by being the product of procaryotic or eucaryotic host expression (e.g., by bacterial, yeast, higher plant, insect and mammalian cells in culture) of exogenous DNA sequences obtained by genomic or cDNA cloning or by gene synthesis. That is, in a preferred embodiment, SCF is "recombinant SCF." The products of expression in typical yeast (e.g., Saccharomyces cerevisiae) or procaryote (e.g., E. coli) host cells are free of association with any mammalian proteins. The products of expression in vertebrate [e.g., non-human mammalian (e.g. COS or CHO) and avian] cells are free of association with any human proteins. Depending upon the host employed, polypeptides of the invention may be glycosylated with mammalian or other eucaryotic carbohydrates or may be non-glycosylated. The host cell can be altered using techniques such as those described in Lee et al. J. Biol. Chem. 264, 13848 (1989) hereby incorporated by reference. Polypeptides of the invention may also include an initial methionine amino acid residue (at position -1).

In addition to naturally-occurring allelic forms of SCF, the present invention also embraces other SCF products such as polypeptide analogs of SCF. Such analogs include fragments of SCF. Following the procedures of the above-noted published application by Alton et al. (WO 83/04053), one can readily design and manufacture genes coding for microbial expression of polypeptides having primary conformations which differ from that herein specified for in terms of the identity or location of one or more residues (e.g., substitutions, terminal and intermediate additions and deletions). Alternately, modifications of cDNA and genomic genes can be readily accomplished by well-known site-directed mutagenesis techniques and employed to generate analogs and derivatives of SCF. Such products share at least one of the biological properties of SCF but may differ in others. As examples, products of the invention include those which are foreshortened by e.g., deletions; or those which are more stable to hydrolysis (and, therefore, may have more pronounced or longer-lasting effects than naturally-occurring); or which have been altered to delete or to add one or more potential sites for O-glycosylation and/or N-glycosylation or which have one or more cysteine residues deleted or replaced by, e.g., alanine or serine residues and are potentially more easily isolated in active form from microbial systems; or which have one or more tyrosine residues replaced by phenylalanine and bind more or less readily to target proteins or to receptors on target cells. Also comprehended are polypeptide fragments duplicating only a par