Dimer and multimer forms of single chain polypeptides

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

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.

Kramer (4’11 Annual Symposium, Jan. 23—26, 1992, Diag nostic & Therapeutic Applications in Benign & Malignant Disorders Key Biscayne FL, pp. 52—54, Meeting Abstract.

U.S. Patent Jun. 6,2000 Sheet 1 0f 73 6,071,515

CONSTRUCTION OF PLASMID pSCFV31 A

K \

o

I 1‘ enP

Eco RI I ‘\ BC“ '- Barn HI

SaI I

' PCR with penP1 + penP2

Digest with H1LndIII+ Sal I

Hind III Nco I M B

BcI I Hind III Eco RI

Ncol ‘ ’ penP

Eco RI Camr

Digest with BcI I

Hind III BcI I BcI I SaI I

* Im__I rnru- \\\\\\\\\\\\\\\\\\\\\\\\\\\\\

C


FIG. 2 CONSTRUCTION OF PLASMID pSCFV31

A

E00 Rl Hind |||

Nde | Apr

LEU 2 pCGS515/SCFV1

URA 3 11.5Kbp

Hind Ill Sa | Hind m Aat "

PCR with oligos penP3 + penP6(-)

Nclol l}atll Elicll Hind||| Ncol Aatll Bcll I IVAI ’

VL PCR/SOE with fragment B VL D using oligos penP1 + penP6(-) E

Ligate with fragments A + C

U.S. Patent Jun. 6, 2000 Sheet3 0f 73

F|G.3A

Met AAAAACTAT AAGCTCCA'I'G ATG

Leu Gln Ala Phe 'I'TG CAA GCT TTC

Gly Phe Ala Ala GGT TT'I‘ GCA GCC

Ile Val Met Thr ATC G'I'G ATG ACC

Leu Ala Val Ser CTG GC'I' G'I‘G TC'I‘

'I‘hr Ile Asn Cys ACC ATC AAC TGC

Leu Phe Leu Leu CTT TTC CTT TTG

Lys Ile Ser Ala AAA ATA TCT GCA

Gln Ser Pro Asp CAG 'I'CT CCA GAC

Leu Gly Glu Arg CTG GGC GAG AGG

DRlL —

Lys Ser Ser Gln G TCC AGC 'I'GC AAG

6,071,515

Leu

CT'I'

Ala GCT

Asp GAC

Ser

TCC

Ala GCC

Ser

Val Leu 'I'yr Ser GTT T'I'A 'I'AC AGC

Tyr Leu Ala ‘Trp TAC TTA GC'I' 'I'GG

Gly Gln Pro Pro GGA CAG CCT CCT

DR2L \

I'I'rp Ala Ser Thr TGG GCA TC'I‘ ACC CGG GAA TCC

Pro Asp Arg Phe CCT GAC CGA TTC

Ser Asn Asn Lys TCC AAC AAT AAG

Tyr Gln Gln Lys 'I'AC CAG CAG AAA

Lys Leu Leu Ile AAG CTG C'I'C A'I'T

Arg Glu Ser Gly GGG

Ser Gly Ser Gly AG'I‘ GGC AGC GGG

Asn

AAC

Pro CCA

Val

GTC

Ser TCT

U.S. Patent

Thr ACA

Gly GGG

Leu

CTG Ser

AGC

Tyr TAT

Tyr TAC

Pro Leu CCT CTC

Val GTG

Lys AAG

LINKER Glu Gln GAA CAA

Asp ‘Val GAC GTC

Glu Leu GAG TTG

Lys Ile AAG ATT

Thr Phe

ACC TTC

Val Lys GTG AAA

Jun. 6, 2000 Sheet 4 0f 73

F|G.3B

Asp Phe Thr Leu Thr Ile GAT TTC ACT CTC ACC ATC

Gln Ala Glu Asp Val Ala CAG GCT GAA GAT GTG GCA

Gln CAG

Cys TGT

Thr ACT

Phe 'I'TC

Glu Ser GAG TCA

Gln Tyr CAA TAT

Gly Gly GGC GGA

Gly Ser GGT TCG

+——-—————-CDR3L Tyr Ser TAT AGT

Gly Thr GGG ACC

Val Ser GTC TCC

6,071,515

Ser AGC

Val GTT

Tyr TAT

Lys AAG

Ser TCA

Ala GCC

Leu

TTG

Gln CAG

Leu

TTG

Val GTG

Gly AAA

Ser

TCC Cys TGC

Thr

ACT

Gln

Gln Phe CAA TT'I'

Gln Gln CAG CAG

Pro Gly CCT GGG

Lys Ala AAG GCT

DRlH Asp His Ala Ile His‘ GAC CAT GCA ATT CAC TGG

Arg Ser CGT TCC

Ser Asp TCT GAC

Ala Ser GCT TCA

Ser Gly TCT GGC

Leu

TTA

Ala GCT

Val GTG

Tyr TAC

TrP

Asn Pro Glu Gln Gly Leu CAG AAC CCT GAA CAG GGC CTG

U.S. Patent

Glu GAA

Trp TGG

Jun. 6, 2000 Sheet 5 0f 73

F|G.3C

Gly Tyr Phe GGA TAT 'I'TT

Ile ATT

Asn AA'I'

Phe TTC

Asp GAC

Gln CAG

Ser TCT

Asp GAT

Lys AAG

Lys AAA

Leu

CTC

Ala GCA

Ser

TCT Pro

CCC

6,071,515

Gly GGA

(JDRZH

Asp Phe Lys Tyr GAT TTT AAA TAC

Al

Gly Lys Ala GGC AAG GCC

Thr ACA

Thr ACT

Ser Ser Ser 'I‘CC TCC AGC

Thr ACA

Asn Ser Leu

AAC AGC CTG

Val Tyr Phe G'I'G 'I'A'I' TTC

Cys TGT

———-—CDR3H

Leu Asn Met Ala Tyr ‘Tarp CTG AAT ATG GCC TAC TGG

Thr Ser Val Thr Val Ser ACC TCA GTC ACC GTC TCC

Asn

AAT

Leu

CTG

Ala GCC

Ser

TCT

Thr ACA

Gly GGT

TAG

Glu

Thr ACT

'I‘yr TAC

Glu GAG

Arg AGA

Gln CAA

Arg AGG

Ala GCA

Val GTG

Asp GAT

Ser TCC

Gly GGA

AGCTTGGAAC ACCACACAAA CCA'I'ATCCAA A

U.S. Patent Jun. 6, 2000 Sheet 7 0f 73 6,071,515

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ANTI TAG-72 SINGLE CHAIN ANTIBODY EXPRESSION PLASMIDS AND PRODUCTS

Xho' FIG. 6C

pSCFV UHH &

pSCFV UHM(X) 6.0 kbp

Sal | VL

COOH

SINGLE CHAIN Fv MONOMER

(scFv) COOH

\ SINGLE CHAIN Fv Dimer (Fv2)


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Documents

Dimer and multimer forms of single chain polypeptides