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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
[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
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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
U.S. Patent Jun. 6,2000 Sheet 2 0f 73 6,071,515
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
Nmmm mmhm mmbm mbmm mmmm mmwm mmwm
HBBBBUBHUU
H MHU
U.S. Patent Jun. 6,2000 Sheet 8 0f 73 6,071,515
mmov mNov mmmm
090 009 004 099 004 099 049 009 000 U09 090 000 040 404 040 400 040 U04 009 044 U09 U44 U94 U04 000 004 04m 000 090 909 090 900 dam Hwm Hwm mhq who ~54 wHH Han. M44 94 5.6 9.8 ?wq Hwm ._..m> m._u4
040 004 090 900 404 090 009 049 090 009 040 400 909 040 004 094 SQH Hww .mm4 oum .nmm GHQ H89 #02 0904094490 0404490499 4940494440 0409049940 0909940944 9490949990 9490904999 4449990099 9094094094 4940409444 9994440044 4049049049 9004449409 0499499900 4409494909 4009994904 0944944400 9904949404 TV NZHAEE
5090444404 0909000409 9999449900 B040 0 404 U49 004 040 044 040 404 0mm 9B 95 uuu 0.2 30 mo 95am mam .54 98 www 05. uuw aw wmuma 2.5 03 $4 3w 05.. m2 5
9949404000 4494909000 9949440904 4494990409 9449999999 4994444444 9090904049 4040409094 0040494499 0090949944 9499909404 4944404909 4944990444 9499440999 0049004949 0094009494 Hm E?m 0404949940 0909494490
momm 4040999909 0404000904 4444994400 4090 0 909 094 009 090 099 090 909
couucH w Hmm mHH mun. :wA ?wq ?mq Hwm
U.S. Patent Jun. 6,2000 Sheet 11 0f 73 6,071,515
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)
U.S. Patent Jun. 6,2000 Sheet 12 0f 73 6,071,515
Nmm wmm mmN mmN 00H wm mw
0g mmq £00 95 BEG UUH. Own. who HUB uwm UUU
>000 I> 900.5% 00 000203000 Q0< oz=>_< Qz< <20
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004 000 004 0.00 000 E5. 34 P3 30 30
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000 000 mow 004 0.00 m?? m?? MHG .HSH. OHm $2 0.5 $2 $0 Em 000 E6 5m 00¢ .50 H00 H40 52 000 000 0.5 0.04 52 5E 5E .50 40a 60 004 004 .000 Em m2. 400 .56 H moom H 30 0450K UBO 9mg 0E4 002 BUB “mm 000 0H4
H 002 UBH. UB4 U64 H04 04B $0 HRH 00¢ 40H. UHF HUB UBO BUG
6B0 BUG Hm> MUN GUU 5&0 Gum GHQ DANE g HMH. 90H Haw .UBU BUB HANG HUB 4GB 444 BBB wUU UUH. 40% man.
UBU 50A UUM. MA“ 0.5 002 NNI B40 B00 0BR. BUB DUB Hww 0H BUG mjw HBO BBB mg EHO H6 408
UHU HwA EH4 UBO UHH. HUB 004 BED 0.0m.
U.S. Patent Jun. 6,2000 Sheet 15 0f 73 6,071,515
HmNH vmNH OmHH No.3“ 40H. 40H 40H. “mm 845 aha
404 000 404 004 .HQB 00H. 40H. 080 $6 mam 400 .24 0mm
.mlu 08H. 0B4 BB4 440 CH0 BOB uwm
000 H 38mm BBB 0B4 0B4 00H. Bum m5 B40 mum
000 044 840 00H. mum. 040 5H0 EH4 004 4BR. B00 B40 04H. HF“... BOB uwm
004 040 B0B 840
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050E B0B 000 BBB H00 H44 064 004 umm H40 0B0 0BR. BBO ‘CI. 080 5mg 04 cmm 0BR. B00 BBB 40H. 40H. Hwm 00H. Hwm 0H0 50A 000 084 BBO 044 00H. uwm 404 mu4 040 50 0mm H44 000 4B4 080 Hm> 000 000 80H. 04H. 004 HSH. OmN BOB mhU 04H .18.. 04B 044 00H. B0B 080 0BR. $03 000 24
U.S. Patent Jun. 6,2000 Sheet 16 0f 73 6,071,515
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