United States Patent [19] Mezes et al. US006071515A 6,071,515 Jun. 6, 2000 [11] Patent Number: [45] Date of Patent: [54] DIMER AND MULTIMER FORMS OF SINGLE CHAIN POLYPEPTIDES [75] Inventors: Peter S. Mezes; Ruth A. Richard; Joseph A. A?holter; Nicolas J. Kotite, all of Midland, Mich. [73] Assignee: The Dow Chemical Company, Midland, Mich. [21] Appl. No.: 08/463,903 [22] Filed: Jun. 5, 1995 Related US. Application Data [63] Continuation of application No. 07/935,695, Aug. 21, 1992. 1 m. . ...................... .. 5; G01N 33/ 3; 5 I C]7 A61K39/39 5 G01N 33/574 [52] U.S.Cl. ................................... ..424/130.1;424/1331; 424/1381; 424/141.1;424/1521; 424/1551; 424/1581; 424/1721; 424/1741; 424/1781; 424/1811; 435/71; 435/7.2; 435/7.23 [58] Field of Search .................... .. 530/3881; 424/1301, 424/1781, 1.11, 277.1, 133.1, 138.1, 141.1, 152.1, 155.1, 158.1, 172.1, 174.1, 181.1; 435/71, 7.2, 7.23 [56] References Cited U.S. PATENT DOCUMENTS 4,474,893 10/1984 Reading et al. ...................... .. 436/547 4,642,334 2/1987 Moore et al. 530/388 4,714,681 12/1987 Reading ......... .. 435/24027 4,946,778 8/1990 Ladner et al. .. 435/69.6 5,001,225 3/1991 Taylor ............ .. 530/387 5,130,297 7/1992 Sharma et al. 514/8 5,132,405 7/1992 Huston et al. ..................... .. 530/387.3 FOREIGN PATENT DOCUMENTS 8809344 12/1988 WIPO . OTHER PUBLICATIONS Filpula et al (Antibody Engineering, Eds McCafferty et al., IRL Press, Oxford, UK, pp. 253—268), 1996. Hunkapiller et al., “Implications of the Diversity of the Immunoglobulin Gene Superfamily”, Cold Spring Harbor Symposia on Quantitative Biology, LIV, 1989, pp. 15—29, Cold Spring Harbor Laboratory Press. Wraith et al., “T—cell Recognition as the Target for Immune Intervention in Autoimmune Disease”, Cell, vol. 57, pp. 709—715, Jun. 2, 1989. Adorini, et al., “New Perspectives on Immunointervention in Autoimmune Diseases”, Immunology Today, vol. 11, No. 11, pp. 383—386 (1990). Bird et al., “Single—Chain Antigen—Binding Proteins”, Sci ence, vol. 242, pp. 423—426, Oct. 21, 1988. Skerra et. al., “Assembly of a Functional Immunoglobulin, Fv Fragment in Escherichia coli”, Science, vol. 240, pp. 1038—1041, May 20, 1988. Takkinen et al., “An Active Single—Chain Antibody Con taining a Cellulase Linker Domain is Secreted by Escheri chia coli”, Protein Engineering, vol. 4, No. 7, pp. 837—841 (1991). Traunecker et al., “Bispeci?c Single Chain Molecules (Jan usins) Target Cytotoxic Lymphocytes on HIV Infected Cells”, The EMBO Journal, vol. 10, No. 12, pp. 3655—3659 (1991). Huston et al., “Protein Engineering of Antibody Binding Sites: Recovery of Speci?c Activity in an Anti—digoxin Single—Chain Fv Analogue Produced in Escherichia coli”, Proc. Natl. Acad. Sci., Biochemistry, vol. 85, pp. 5879—5883, Aug. 1988. Larson, “Improved Tumor Targeting With Radiolabeled Recombinant, Single—Chain, Antigen—Binding Protein”, Journal ofthe National Cancer Institute, vol. 82, No. 14, pp. 1173—74, Jul. 18, 1990. Colcher et al., In Vivo Tumor Targeting of a Recombinant Single—Chain Antigen—Binding Protein, Journal of the National Cancer Institute, vol. 82, No. 14, pp. 1191—1197, Jul. 18, 1990. BedZyk et al., “Immunological and Structural Characteriza tion of a High Af?nity Anti—?uorescein Single—chain Anti body”, The Journal ofBiological Chemistry, V. 265, No. 30, pp. 18615—18620, Oct. 25, 1990. Winter et al., “Man—made Antibodies”, Nature, vol. 349, pp. 293—299, Jan. 24, 1991. Davis, “Single Chain Antibody (SCA) Encoding Genes: One Step Construction and Expression in Eukaryotic Cells”, Biotechnology, vol. 9, pp. 165—169, Feb. 1991. WhitloW et al., “Single—Chain Fv Proteins and Their Fusion Proteins”, Methods." A Companion to Methods in Enzymol ogy, vol. 2, No. 2, pp. 97—105, Apr. 1991. Skerra et al., “The Functional Expression of Antibody Fv Fragments in Escherichia coli: Improved Vectors and a Generally Applicable Puri?cation Technique”, Biotechnol ogy, vol. 9, pp. 273—278, Mar. 1991. Gibbs et al., “Construction and Characterization of a Sin gle—Chain Catalytic Antibody”, Proc. Natl. Acad. Sci., USA, vol. 88, pp. 4001—4004, May 1991. Hodgson, “Making Monoclonals in Microbes”, Biotechnol ogy, vol. 9, pp. 421—425, May 1991. Pluckthun, “Antibody Engineering: Advances From the Use of Escherichia coli Expression Systems”, Biotechnology, vol. 9, pp. 545—550, Jun. 1991. Bird et al., “Single Chain Antibody Variable Regions”, Trends in Biotechnology, vol. 9, pp. 132—137 (1991). Glockshuber et al., “A Comparison of Strategies to Stabilize Immunoglobulin Fv—Fragments”, Biochemistry, vol. 29, pp. 1362—1367 (1990). (List continued on next page.) Primary Examiner—Paula K. HutZell Assistant Examiner—Susan Ungar Attorney, Agent, or Firm—Karen L. Kimble; Mark S. Scott [57] ABSTRACT The present invention discloses novel proteins Which are dimers and multimers of single chain polypeptides. The single chain polypeptides having tWo domains derived from the immunoglobulin superfamily Which are joined by a peptide linker. The dimers and multimers being formed by non-covalent linking of the single chain polypeptides. 5 Claims, 73 Drawing Sheets
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Dimer and multimer forms of single chain polypeptides
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United States Patent [19] Mezes et al.
US006071515A
6,071,515 Jun. 6, 2000
[11] Patent Number:
[45] Date of Patent:
[54] DIMER AND MULTIMER FORMS OF SINGLE CHAIN POLYPEPTIDES
[75] Inventors: Peter S. Mezes; Ruth A. Richard; Joseph A. A?holter; Nicolas J. Kotite, all of Midland, Mich.
[73] Assignee: The Dow Chemical Company, Midland, Mich.
[21] Appl. No.: 08/463,903
[22] Filed: Jun. 5, 1995
Related US. Application Data
[63] Continuation of application No. 07/935,695, Aug. 21, 1992.
1 m. . ...................... .. 5; G01N 33/ 3; 5 I C]7 A61K39/39 5 G01N 33/574
4,474,893 10/1984 Reading et al. ...................... .. 436/547 4,642,334 2/1987 Moore et al. 530/388 4,714,681 12/1987 Reading ......... .. 435/24027 4,946,778 8/1990 Ladner et al. .. 435/69.6 5,001,225 3/1991 Taylor ............ .. 530/387 5,130,297 7/1992 Sharma et al. 514/8 5,132,405 7/1992 Huston et al. ..................... .. 530/387.3
FOREIGN PATENT DOCUMENTS
8809344 12/1988 WIPO .
OTHER PUBLICATIONS
Filpula et al (Antibody Engineering, Eds McCafferty et al., IRL Press, Oxford, UK, pp. 253—268), 1996. Hunkapiller et al., “Implications of the Diversity of the Immunoglobulin Gene Superfamily”, Cold Spring Harbor Symposia on Quantitative Biology, LIV, 1989, pp. 15—29, Cold Spring Harbor Laboratory Press. Wraith et al., “T—cell Recognition as the Target for Immune Intervention in Autoimmune Disease”, Cell, vol. 57, pp. 709—715, Jun. 2, 1989. Adorini, et al., “New Perspectives on Immunointervention in Autoimmune Diseases”, Immunology Today, vol. 11, No. 11, pp. 383—386 (1990). Bird et al., “Single—Chain Antigen—Binding Proteins”, Sci ence, vol. 242, pp. 423—426, Oct. 21, 1988. Skerra et. al., “Assembly of a Functional Immunoglobulin, Fv Fragment in Escherichia coli”, Science, vol. 240, pp. 1038—1041, May 20, 1988. Takkinen et al., “An Active Single—Chain Antibody Con taining a Cellulase Linker Domain is Secreted by Escheri chia coli”, Protein Engineering, vol. 4, No. 7, pp. 837—841 (1991).
Traunecker et al., “Bispeci?c Single Chain Molecules (Jan usins) Target Cytotoxic Lymphocytes on HIV Infected Cells”, The EMBO Journal, vol. 10, No. 12, pp. 3655—3659 (1991). Huston et al., “Protein Engineering of Antibody Binding Sites: Recovery of Speci?c Activity in an Anti—digoxin Single—Chain Fv Analogue Produced in Escherichia coli”, Proc. Natl. Acad. Sci., Biochemistry, vol. 85, pp. 5879—5883, Aug. 1988. Larson, “Improved Tumor Targeting With Radiolabeled Recombinant, Single—Chain, Antigen—Binding Protein”, Journal ofthe National Cancer Institute, vol. 82, No. 14, pp. 1173—74, Jul. 18, 1990. Colcher et al., In Vivo Tumor Targeting of a Recombinant Single—Chain Antigen—Binding Protein, Journal of the National Cancer Institute, vol. 82, No. 14, pp. 1191—1197, Jul. 18, 1990. BedZyk et al., “Immunological and Structural Characteriza tion of a High Af?nity Anti—?uorescein Single—chain Anti body”, The Journal ofBiological Chemistry, V. 265, No. 30, pp. 18615—18620, Oct. 25, 1990. Winter et al., “Man—made Antibodies”, Nature, vol. 349, pp. 293—299, Jan. 24, 1991. Davis, “Single Chain Antibody (SCA) Encoding Genes: One Step Construction and Expression in Eukaryotic Cells”, Biotechnology, vol. 9, pp. 165—169, Feb. 1991. WhitloW et al., “Single—Chain Fv Proteins and Their Fusion Proteins”, Methods." A Companion to Methods in Enzymol ogy, vol. 2, No. 2, pp. 97—105, Apr. 1991. Skerra et al., “The Functional Expression of Antibody Fv Fragments in Escherichia coli: Improved Vectors and a Generally Applicable Puri?cation Technique”, Biotechnol ogy, vol. 9, pp. 273—278, Mar. 1991. Gibbs et al., “Construction and Characterization of a Sin gle—Chain Catalytic Antibody”, Proc. Natl. Acad. Sci., USA, vol. 88, pp. 4001—4004, May 1991. Hodgson, “Making Monoclonals in Microbes”, Biotechnol ogy, vol. 9, pp. 421—425, May 1991. Pluckthun, “Antibody Engineering: Advances From the Use of Escherichia coli Expression Systems”, Biotechnology, vol. 9, pp. 545—550, Jun. 1991. Bird et al., “Single Chain Antibody Variable Regions”, Trends in Biotechnology, vol. 9, pp. 132—137 (1991). Glockshuber et al., “A Comparison of Strategies to Stabilize Immunoglobulin Fv—Fragments”, Biochemistry, vol. 29, pp. 1362—1367 (1990).
(List continued on next page.)
Primary Examiner—Paula K. HutZell Assistant Examiner—Susan Ungar Attorney, Agent, or Firm—Karen L. Kimble; Mark S. Scott
[57] ABSTRACT
The present invention discloses novel proteins Which are dimers and multimers of single chain polypeptides. The single chain polypeptides having tWo domains derived from the immunoglobulin superfamily Which are joined by a peptide linker. The dimers and multimers being formed by non-covalent linking of the single chain polypeptides.
5 Claims, 73 Drawing Sheets
6,071,515 Page 2
OTHER PUBLICATIONS
DenZin et al., “Single—chain Site—speci?c Mutations of F1uorescein—Amino Acid Contact Residues in High Affinity Monoclonal Antibody 4—4—20”, Journal of Biological Chemistry, vol. 266, No. 21, pp. 14095—14103 (1991). Pantoliano et al., “Conformational Stability, Folding, and Ligand—Binding Af?nity of Single—Chain Fv Immunoglo bulin Fragments Expressed in Escherichia coli”, Biochem istry, vol. 30, pp. 10117—10125 (1991). Gregoire et al., “Engineered Secreted T—Cell Receptor (x6 Heterodimers”, Proc. Natl. Acad. Sci., USA, vol. 88, pp. 8077—8081, Sep. 1991. Pack et a1., “Miniantibodies: Use of Amphipathic Helices to Produce Functional, FleXibly Linked Dimeric Fv Fragments With High Avidity in Escherichia coli”, Biochemistry, vol. 31, No. 6, pp. 1579—1584 (1992). S00 Hoo et a1., “Characterization of a Single—chain T—ce1l Receptor Expressed in Escherichia coli ”, Proc. Natl. Acad. Sci., U.SA., vol. 89, pp. 4759—4763, May 1992. Novotny et al., “A Soluble, Single—chain T—ce1l Receptor Fragment EndoWed With Antigen—combining Properties”, Proc. Natl. Acad. Sci., USA, vol. 88, pp. 8646—8650, Oct. 1991.
Williams et al., “The Immunoglobulin Superfami1y—Do mains for Cell Surface Recognition”, Ann. Rev. Immunol, vol. 6, pp. 381—405 (1988).
Williams et al., “Structural Diversity in Domains of the Immunoglobulin Superfamily”, Cold Spring Harbor Sym posia on Quantitative Biology, LIV, pp. 637—647, 1989, Cold Spring Harbor Lab. Press.
BradWel et a1 (Monoclonal Antibodies for Cancer Detection & Therapy, 1985, Academic Press, London pp. 65—85.
Waldman, Science, 1991, 252:1657—1662.
Sheer et al, Cancer Res, 48: 6811—6818, 1988.
RodWe1l et a1, Biotechnology, 1985, 3: 889—894.
Hird et a1 (Genes and Cancer, John Wiley & Sons, 1990, Chichester, pp. 183—189.
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