MUC1* ANTIBODIES
20230295340 · 2023-09-21
Inventors
Cpc classification
A61K39/001117
HUMAN NECESSITIES
C07K2317/24
CHEMISTRY; METALLURGY
C07K2317/73
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
Abstract
The present describes monoclonal antibodies.
Claims
1-50. (canceled)
51. An isolated monoclonal antibody that binds to Mucin-1 * (MUC1*), comprising: (a) a heavy chain (HC) variable region comprising a complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO: 172, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 198, a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 224, a HC-framework region 1 (FWR1) comprising an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 362, a HC-FWR2 comprising an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 363, and a HC-FWR3 comprising an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 364; and (b) a light chain (LC) variable region comprising a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 108, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 129, a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 149, a LC-FWR1 comprising an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 365, a LC-FWR2 comprising an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 366, and a LC-FWR3 comprising an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 367.
52. The antibody of claim 51, wherein: the HC-FWR1 comprises the amino acid sequence of SEQ ID NO: 362, the HC-FWR2 comprises the amino acid sequence of SEQ ID NO: 363, and the HC-FWR3 comprises the amino acid sequence of SEQ ID NO: 364; and the LC-FWR1 comprises the amino acid sequence of SEQ ID NO: 365, the LC-FWR2 comprises the amino acid sequence of SEQ ID NO: 366, and the LC-FWR3 comprises the amino acid sequence of SEQ ID NO: 367.
53. The antibody of claim 51, wherein the heavy chain variable region comprises an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 11 and the light chain variable region comprises an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 13.
54. The antibody of claim 51, wherein the heavy chain variable region comprises an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 11 and the light chain variable region comprises an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 13.
55. The antibody of claim 51, wherein the heavy chain variable region comprises an amino acid sequence with at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 11 and the light chain variable region comprises an amino acid sequence with at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 13.
56. The antibody of claim 51, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 11 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13.
57. The antibody of claim 51, wherein the antibody is monovalent for MUC1* and specifically binds to the MUC1* amino acid sequence of SEQ ID NO: 1.
58. The antibody of claim 57, wherein the antibody inhibits MUC1*-mediated cell growth or proliferation.
59. The antibody of claim 51, wherein the antibody binds specifically to a cancer cell.
60. The antibody of claim 51, wherein the antibody is a Fab, F(ab′), F(ab′).sub.2, or a single chain variable fragment antibody (scFv).
61. The antibody of claim 60, wherein the Fab comprises a heavy chain comprising an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 47 and/or a kappa chain comprising an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 48.
62. The antibody of claim 60, wherein the Fab comprises a heavy chain comprising an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 47 and/or a kappa chain comprising an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 48.
63. The antibody of claim 60, wherein the Fab comprises a heavy chain comprising an amino acid sequence with at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 47 and/or a kappa chain comprising an amino acid sequence with at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 48.
64. The antibody of claim 60, wherein the Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 47 and/or a kappa chain comprising the amino acid sequence of SEQ ID NO: 48.
65. The antibody of claim 60, wherein the scFv comprises an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 17.
66. The antibody of claim 60, wherein the scFv comprises an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 17.
67. The antibody of claim 60, wherein the scFv comprises an amino acid sequence with at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 17.
68. The antibody of claim 60, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 17.
69. The antibody of claim 51, wherein the antibody is bivalent for MUC1*.
70. The antibody of claim 51, wherein the antibody is bispecific or trispecific.
71. The antibody of claim 70, wherein a first portion of the antibody binds to MUC1* and a second portion binds to an immune cell or a tumor-specific antigen on a cell surface.
72. The antibody of claim 51, wherein the antibody is fused to a label, a tag, a toxin, a radioactive substance, a targeting motif, a leader sequence peptide, a toxic material, a protein, or a peptide.
73. The antibody of claim 72, wherein the protein is a toxin, or a cytokine.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] The present invention will become more fully understood from the detailed description given herein below, and the accompanying drawings which are given by way of illustration only, and thus are not limitative of the present invention, and wherein;
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DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0058] In the present application, “a” and “an” are used to refer to both single and a plurality of objects.
[0059] As used herein, “MUC1 Growth Factor Receptor” (MGFR) is a functional definition meaning that portion of the MUC1 receptor that interacts with an activating ligand, such as a growth factor or a modifying enzyme such as a cleavage enzyme. The MGFR region of MUC1 is that extracellular portion that is closest to the cell surface and is defined by most or all of the PSMGFR, as defined below. The MGFR is inclusive of both unmodified peptides and peptides that have undergone enzyme modifications, such as, for example, phosphorylation, glycosylation and so forth.
[0060] As used herein, “Primary Sequence of the MUC1 Growth Factor Receptor” (PSMGFR) refers to peptide sequence that defines most or all of the MGFR in some cases, and functional variants and fragments of the peptide sequence. The PSMGFR is defined as SEQ ID NO:1, and all functional variants and fragments thereof having any integer value of amino acid substitutions up to 20 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) and/or any integer value of amino acid additions or deletions up to 20 at its N-terminus and/or C-terminus. A “functional variant or fragment” in the above context refers to such variant or fragment having the ability to specifically bind to, or other ways specifically interact with, ligands that specifically bind to, or otherwise specifically interact with, the peptide of SEQ D NO:1, while not binding strongly to identical regions of other peptide molecules identical to themselves, such that the peptide molecules would have the ability to aggregate (i.e., self-aggregate) with other identical peptide molecules. One example of a PSMGFR that is a functional variant of the PSMGFR peptide of SEQ NO:1 is SEQ ID NO:2, which differs from SEQ ID NO:1 by including an -SPY- sequence instead of the -SRY-.
[0061] As used herein, “MUC1*” refers to the MUC1 protein with the N-terminus truncated such that the extracellular domain is essentially comprised of the PSMGFR (SEQ ID NO: 1).
Sequence Listing Free Text
[0062] As regards the use of nucleotide symbols other than a, g, c, t, they follow the convention set forth in WIPO Standard ST.25, Appendix 2, Table 1, wherein k represents t or g; n represents a, c, t or g; m represents a or c; r represents a or g; s represents c or g; w represents a or t and y represents c or t.
TABLE-US-00001 GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO:1)
describes the membrane proximal extracellular region of MUC1 from amino acid 1110 to 1155.
TABLE-US-00002 GTINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO:2)
describes a variant of the membrane proximal extracellular region of MUC1 from amino acid 1110 to 1155.
TABLE-US-00003 5′-ggaaagcttatagacagatgggggtgtcgttttggc-3′ (SEQ I D NO:3)
describes HindIII restriction site containing PCR primer, IgG1 constant region reverse primer.
TABLE-US-00004 5′-ggaaagcttcttgaccaggcatcctagagtca-3′ (SEQ ID NO :4)
describes HindIII restriction site containing PCR primer, IgG2A constant region reverse primer.
TABLE-US-00005 5′-ggaaagcttaggggccagtggatagactgatgg-3′ (SEQ ID N O:5)
describes HindIII restriction site containing PCR▫primer, IgG2B▫constant region reverse primer.
TABLE-US-00006 5′-cttccggaattcsargtnmagctgsagsagtc-3′ (SEQ ID NO :6)
describes EcoRI restriction site containing PCR primer, heavy chain FR1 region forward primer 1.
TABLE-US-00007 5′-cttccggaattcsargtnmagctgsagsagtcwgg-3′ (SEQ ID NO:7)
describes EcoRI restriction site containing PCR primer, heavy chain FR1 region forward primer 2.
TABLE-US-00008 5′-ggtgtcgacggatacagttggtgcagcatc-3′ (SEQ ID NO:8 )
describes SalI restriction site containing PCR primer, kappa chain constant region reverse primer.
TABLE-US-00009 5′-gggagctcgayattgtgmtsacmcarwctmca-3′ (SEQ ID NO :9)
describes SacI restriction site containing PCR primer, kappa chain FR1 region universal degenerate forward primer.
TABLE-US-00010 gaggtccagctggaggagtcagggggaggcttagtgaagcctggagggt ccctgaaactctcctgtgcagcctctggattcactttcagtggctatgcc atgtcttgggttcgccagactccggagaagaggctggagtgggtcgcaac cattagtagtggtggtacttatatctactatccagacagtgtgaaggggc gattcaccatctccagagacaatgccaagaacaccctgtacctgcaaatg agcagtctgaggtctgaggacacggccatgtattactgtgcaagacttgg gggggataattactacgaatacttcgatgtctggggcgcagggaccacgg tcaccgtctcctccgccaaaacgacacccccatctgtctat (SEQ ID NO:10)
describes MIN-C2 Heavy chain variable region.
TABLE-US-00011 EVQLEESGGGLVKPGGSLKLSCAASGFTFSGYAMSWVRQTPEKRLEWVA TISSGGTYIYYPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAMYYCARL GGDNYYEYFDVWGAGTTVTVSSAKTTPPSVY (SEQ ID NO:11)
describes MIN-C2 Heavy chain variable region.
TABLE-US-00012 gacattgtgatcacacagtctacagcttccttaggtgtatctctggggc agagggccaccatctcatgcagggccagcaaaagtgtcagtacatctggc tatagttatatgcactggtaccaacagagaccaggacagccacccaaact cctcatctatcttgcatccaacctagaatctggggtccctgccaggttca gtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggag gaggaggatgctgcaacctattactgtcagcacagtagggagcttccgtt cacgttcggaggggggaccaagctggagataaaacgggctgatgctgcac caactgtatcc (SEQ ID NO:12)
describes MIN-C2 Kappa chain variable region.
TABLE-US-00013 DIVITQSTASLGVSLGQRATISCRASKSVSTSGYSYMHWYQQRPGQPPK LLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELP FTFGGGTKLEIKRADAAPTVS (SEQID NO:13)
describes▫MIN-C2▫Kappa▫chain▫variable▫region.
TABLE-US-00014 ggtggaggcggatcaggtggaggcggatcaggtggaggcggatca (SEQ ID NO:14)
describes Linker.
TABLE-US-00015 (SEQ ID NO: 15)gaggtccagctggaggagtcagggggaggcttag tgaagcctggagggtccctgaaactctcctgtgcagcctctggattcact ttcagtggctatgccatgtcttgggttcgccagactccggagaagaggct ggagtgggtcgcaaccattagtagtggtggtacttatatctactatccag acagtgtgaaggggcgattcaccatctccagagacaatgccaagaacacc ctgtacctgcaaatgagcagtctgaggtctgaggacacggccatgtatta ctgtgcaagacttgggggggataattactacgaatacttcgatgtctggg gcgcagggaccacggtcaccgtctcctccgccaaaacgacacccccatct gtctatggtggaggcggatcaggtggaggcggatcaggtggaggcggatc agacattgtgatcacacagtctacagcttccttaggtgtatctctggggc agagggccaccatctcatgcagggccagcaaaagtgtcagtacatctggc tatagttatatgcactggtaccaacagagaccaggacagccacccaaact cctcatctatcttgcatccaacctagaatctggggtccctgccaggttca gtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggag gaggaggatgctgcaacctattactgtcagcacagtagggagcttccgtt cacgttcggaggggggaccaagctggagataaaacgggctgatgctgcac caactgtatcc
describes MIN-C2 Heavy chain-linker-light chain.
TABLE-US-00016 (SEQ ID NO: 16)gaggtccagctggaggagtcagggggaggcttag tgaagcctggagggtccctgaaactctcctgtgcagcctctggattcact ttcagtggctatgccatgtcttgggttcgccagactccggagaagaggct ggagtgggtcgcaaccattagtagtggtggtacttatatctactatccag acagtgtgaaggggcgattcaccatctccagagacaatgccaagaacacc ctgtacctgcaaatgagcagtctgaggtctgaggacacggccatgtatta ctgtgcaagacttgggggggataattactacgaatacttcgatgtctggg gcgcagggaccacggtcaccgtctcctccgccaaaacgacacccccatct gtctatggtggaggcggatcaggtggaggcggatcaggtggaggcggatc agacattgtgatcacacagtctacagcttccttaggtgtatctctggggc agagggccaccatctcatgcagggccagcaaaagtgtcagtacatctggc tatagttatatgcactggtaccaacagagaccaggacagccacccaaact cctcatctatcttgcatccaacctagaatctggggtccctgccaggttca gtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggag gaggaggatgctgcaacctattactgtcagcacagtagggagcttccgtt cacgttcggaggggggaccaagctggagataaaacgggctgatgctgcac caactgtatcctgt
describes MIN-C2 Heavy chain-linker-light chain-Cys.
TABLE-US-00017 EVQLEESGGGLVKPGGSLKLSCAASGFTFSGYAMSWVRQTPEKRLEWVA TISSGGTYIYYPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAMYYCARL GGDNYYEYFDVWGAGTTVTVSSAKTTPPSVYGGGGSGGGGSGGGGSDIVI TQSTASLGVSLGQRATISCRASKSVSTSGYSYMHWYQQRPGQPPKLLIYL ASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPFTFGG GTKLEIKRADAAPTVSVD (SEQ ID NO:17)
describes MIN-C2 Heavy chain-linker-light chain.
TABLE-US-00018 EVQLEESGGGLVKPGGSLKLSCAASGFTFSGYAMSWVRQTPEKRLEWVA TISSGGTYIYYPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAMYYCARL GGDNYYEYFDVWGAGTTVTVSSAKTTPPSVYGGGGSGGGGSGGGGSDIVI TQSTASLGVSLGQRATISCRASKSVSTSGYSYMHWYQQRPGQPPKLLIYL ASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPFTFGG GTKLEIKRADAAPTVSC (SEQ ID NO: 18)
describes MIN-C2 Heavy chain-linker-light chain-Cys.
TABLE-US-00019 gaggttaagctggaggagtctgggggagacttagtgaagcctggagggt ccctgaaactctcctgtgcagcctctggattcactttcagtagatatggc atgtcttgggttcgccagactccagacaagaggctggagtgggtcgcaac cattagtagtggtggtacttacatctactatccagacagtgtgaaggggc gattcaccatctccagagacaatgccaagaacaccctgtacctgcaaatg agcagtctgaagtctgaggacacagccatgtattactgtgcaagggataa ctacggtagtagctacgactatgctatggactactggggtcaaggaacct cagtcaccgtctcctcagccaaaacaacagccccatcggtctat (SEQ ID NO:19)
describes MIN-E6 Heavy chain-7 variable region.
TABLE-US-00020 EVKLEESGGDLVKPGGSLKLSCAASGFTFSRYGMSWVRQTPDKRLEWVA TISSGGTYIYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARD NYGSSYDYAMDYWGQGTSVTVSSAKTTAPSVY (SEQ ID NO:20)
describes MIN-E6 Heavy chain-7 variable region.
TABLE-US-00021 gaggtaaagctggaggagtctgggggagacttagtgaagcctggagggt ccctgaaactctcctgtgtagtctctggattcactttcagtagatatggc atgtcttgggttcgccagactccaggcaagaggctggagtgggtcgcaac cattagtggtggcggtacttacatctactatccagacagtgtgaaggggc gattcaccatctccagagacaatgccaagaacaccctgtacctgcaaatg agcagtctgaagtctgaggacacagccatgtatcactgtacaagggataa ctacggtaggaactacgactacggtatggactactggggtcaaggaacct cagtcaccgtctcctcagccaaaacaacagccccatcggtctatccactg gcccctgtgtgtggagatacaactggctcctcggtgactctaggatgcct ggtcaag (SEQ ID NO:21)
describes MIN-E6 Heavy chain-8 variable region.
TABLE-US-00022 EVKLEESGGDLVKPGGSLKLSCVVSGFTFSRYGMSWVRQTPGKRLEWVA TISGGGTYIYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYHCTRD NYGRNYDYGMDYWGQGTSVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGC LVK (SEQ ID NO:22)
describes MIN-E6 Heavy chain-8 variable region.
TABLE-US-00023 gatattgtgatcacccagactacagcaatcatgtctgcatctccagggg aggaggtcaccctaacctgcagtgccacctcaagtgtaagttacatacac tggttccagcagaggccaggcacttctcccaaactctggatttatagcac atccaacctggcttctggagtccctgttcgcttcagtggcagtggatatg ggacctcttactctctcacaatcagccgaatggaggctgaagatgctgcc acttattactgccagcaaaggagtagttccccattcacgttcggctcggg gacaaagttggaaataaaacgggctgatgctgcaccaactgtatcc (SE Q ID NO:23)
describes MIN-E6 Kappa chain variable region.
TABLE-US-00024 DIVITQTTAIMSASPGEEVTLTCSATSSVSYIHWFQQRPGTSPKLWIYS TSNLASGVPVRFSGSGYGTSYSLTISRMEAEDAATYYCQQRSSSPFTFGS GTKLEIKRADAAPTVS (SEQ IDNO:24)
describes MIN-E6 Kappa chain variable region.
TABLE-US-00025 (SEQ ID NO: 25)gaggttaagctggaggagtctgggggagacttag tgaagcctggagggtccctgaaactctcctgtgcagcctctggattcact ttcagtagatatggcatgtcttgggttcgccagactccagacaagaggct ggagtgggtcgcaaccattagtagtggtggtacttacatctactatccag acagtgtgaaggggcgattcaccatctccagagacaatgccaagaacacc ctgtacctgcaaatgagcagtctgaagtctgaggacacagccatgtatta ctgtgcaagggataactacggtagtagctacgactatgctatggactact ggggtcaaggaacctcagtcaccgtctcctcagccaaaacaacagcccca tcggtctatccactggcccctgtgtgtggagatacaactggctcctcggt gactctaggatgcctggtcaagggtggaggcggatcaggtggaggcggat caggtggaggcggatcagatattgtgatcacccagactacagcaatcatg tctgcatctccaggggaggaggtcaccctaacctgcagtgccacctcaag tgtaagttacatacactggttccagcagaggccaggcacttctcccaaac tctggatttatagcacatccaacctggcttctggagtccctgttcgcttc agtggcagtggatatgggacctcttactctctcacaatcagccgaatgga ggctgaagatgctgccacttattactgccagcaaaggagtagttccccat tcacgttcggctcggggacaaagttggaaataaaacgggctgatgctgca ccaactgtatcc
describes MIN-E6 scFv: Heavy chain 7-linker-light chain (VH+linker+VL).
TABLE-US-00026 (SEQ ID NO: 26)gaggttaagctggaggagtctgggggagacttag tgaagcctggagggtccctgaaactctcctgtgcagcctctggattcact ttcagtagatatggcatgtcttgggttcgccagactccagacaagaggct ggagtgggtcgcaaccattagtagtggtggtacttacatctactatccag acagtgtgaaggggcgattcaccatctccagagacaatgccaagaacacc ctgtacctgcaaatgagcagtctgaagtctgaggacacagccatgtatta ctgtgcaagggataactacggtagtagctacgactatgctatggactact ggggtcaaggaacctcagtcaccgtctcctcagccaaaacaacagcccca tcggtctatccactggcccctgtgtgtggagatacaactggctcctcggt gactctaggatgcctggtcaagggtggaggcggatcaggtggaggcggat caggtggaggcggatcagatattgtgatcacccagactacagcaatcatg tctgcatctccaggggaggaggtcaccctaacctgcagtgccacctcaag tgtaagttacatacactggttccagcagaggccaggcacttctcccaaac tctggatttatagcacatccaacctggcttctggagtccctgttcgcttc agtggcagtggatatgggacctcttactctctcacaatcagccgaatgga ggctgaagatgctgccacttattactgccagcaaaggagtagttccccat tcacgttcggctcggggacaaagttggaaataaaacgggctgatgctgca ccaactgtatcctgt
describes MIN-E6 scFv-Cys: Heavy chain 7-linker-light chain-Cys (VH+linker+VL+Cys).
TABLE-US-00027 EVKLEESGGDLVKPGGSLKLSCAASGFTFSRYGMSWVRQTPDKRLEWVA TISSGGTYIYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARD NYGSSYDYAMDYWGQGTSVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGC LVKGGGGSGGGGSGGGGSDIVITQTTAIMSASPGEEVTLTCSATSSVSYI HWFQQRPGTSPKLWIYSTSNLASGVPVRFSGSGYGTSYSLTISRMEAEDA ATYYCQQRSSSPFTFGSGTKLEIKRADAAPTVS (SEQ ID NO:27)
describes MIN-E6 scFv Heavy chain 7-linker-light chain (VH+linker+VL).
TABLE-US-00028 EVKLEESGGDLVKPGGSLKLSCAASGFTFSRYGMSWVRQTPDKRLEWVA TISSGGTYIYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARD NYGSSYDYAMDYWGQGTSVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGC LVKGGGGSGGGGSGGGGSDIVITQTTAIMSASPGEEVTLTCSATSSVSYI HWFQQRPGTSPKLWIYSTSNLASGVPVRFSGSGYGTSYSLTISRMEAEDA ATYYCQQRSSSPFTFGSGTKLEIKRADAAPTVSC (SEQ ID NO:28)
describes MIN-E6 scFv- Cys Heavy chain 7-linker-light chain-Cys (VH+linker+VL+Cys).
TABLE-US-00029 (SEQ ID NO: 29)gaggtaaagctggaggagtctgggggagacttag tgaagcctggagggtccctgaaactctcctgtgtagtctctggattcact ttcagtagatatggcatgtcttgggttcgccagactccaggcaagaggct ggagtgggtcgcaaccattagtggtggcggtacttacatctactatccag acagtgtgaaggggcgattcaccatctccagagacaatgccaagaacacc ctgtacctgcaaatgagcagtctgaagtctgaggacacagccatgtatca ctgtacaagggataactacggtaggaactacgactacggtatggactact ggggtcaaggaacctcagtcaccgtctcctcagccaaaacaacagcccca tcggtctatccactggcccctgtgtgtggagatacaactggctcctcggt gactctaggatgcctggtcaagggtggaggcggatcaggtggaggcggat caggtggaggcggatcagatattgtgatcacccagactacagcaatcatg tctgcatctccaggggaggaggtcaccctaacctgcagtgccacctcaag tgtaagttacatacactggttccagcagaggccaggcacttctcccaaac tctggatttatagcacatccaacctggcttctggagtccctgttcgcttc agtggcagtggatatgggacctcttactctctcacaatcagccgaatgga ggctgaagatgctgccacttattactgccagcaaaggagtagttccccat tcacgttcggctcggggacaaagttggaaataaaacgggctgatgctgca ccaactgtatcc
describes MIN-E6 Heavy chain 8-linker-light chain.
TABLE-US-00030 (SEQ ID NO: 30)gaggtaaagctggaggagtctgggggagacttag tgaagcctggagggtccctgaaactctcctgtgtagtctctggattcact ttcagtagatatggcatgtcttgggttcgccagactccaggcaagaggct ggagtgggtcgcaaccattagtggtggcggtacttacatctactatccag acagtgtgaaggggcgattcaccatctccagagacaatgccaagaacacc ctgtacctgcaaatgagcagtctgaagtctgaggacacagccatgtatca ctgtacaagggataactacggtaggaactacgactacggtatggactact ggggtcaaggaacctcagtcaccgtctcctcagccaaaacaacagcccca tcggtctatccactggcccctgtgtgtggagatacaactggctcctcggt gactctaggatgcctggtcaagggtggaggcggatcaggtggaggcggat caggtggaggcggatcagatattgtgatcacccagactacagcaatcatg tctgcatctccaggggaggaggtcaccctaacctgcagtgccacctcaag tgtaagttacatacactggttccagcagaggccaggcacttctcccaaac tctggatttatagcacatccaacctggcttctggagtccctgttcgcttc agtggcagtggatatgggacctcttactctctcacaatcagccgaatgga ggctgaagatgctgccacttattactgccagcaaaggagtagttccccat tcacgttcggctcggggacaaagttggaaataaaacgggctgatgctgca ccaactgtatcctgt
describes MIN-E6 Heavy chain 8-linker-light chain-Cys.
TABLE-US-00031 EVKLEESGGDLVKPGGSLKLSCVVSGFTFSRYGMSWVRQTPGKRLEWVA TISGGGTYIYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYHCTRD NYGRNYDYGMDYWGQGTSVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGC LVKGGGGSGGGGSGGGGSDIVITQTTAIMSASPGEEVTLTCSATSSVSYI HWFQQRPGTSPKLWIYSTSNLASGVPVRFSGSGYGTSYSLTISRMEAEDA ATYYCQQRSSSPFTFGSGTKLEIKRADAAPTVS (SEQ ID NO:31)
describes MIN-E6 Heavy chain 8-linker-light chain.
TABLE-US-00032 EVKLEESGGDLVKPGGSLKLSCVVSGFTFSRYGMSWVRQTPGKRLEWVA TISGGGTYIYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYHCTRD NYGRNYDYGMDYWGQGTSVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGC LVKGGGGSGGGGSGGGGSDIVITQTTAIMSASPGEEVTLTCSATSSVSYI HWFQQRPGTSPKLWIYSTSNLASGVPVRFSGSGYGTSYSLTISRMEAEDA ATYYCQQRSSSPFTFGSGTKLEIKRADAAPTVSC (SEQ ID NO:32)
describes MIN-E6 Heavy chain 8-linker-light chain-Cys.
TABLE-US-00033 5′-gggaattccaccatggratgsagctgkgtmatsctctt-3′ (SEQ ID NO:33)
describes EcoRI restriction site containing PCR primer, Gamma forward-1.
TABLE-US-00034 5′-gggaattccaccatgracttcgggytgagctkggtttt-3′(SEQ ID NO:34)
describes EcoRI restriction site containing PCR primer, Gamma forward-2.
TABLE-US-00035 5′-gggaattccaccatggctgtcttggggctgctcttct-3′ (SEQ ID NO:35)
describes EcoRI restriction site containing PCR primer, Gamma forward-3.
TABLE-US-00036 5′-gggaattccaccatggrcagrcttacwtyy-3′ (SEQ ID NO:3 6)
describes EcoRI restriction site containing PCR primer, Gamma forward-4.
TABLE-US-00037 5′-ctacctcgagckyggtsytgctggcygggtg-3′ (SEQ ID NO: 37)
describes XhoI restriction site containing PCR primer, Gamma reverse.
TABLE-US-00038 5′-gggaattccaccatggagacagacacactcctgctat-3′ (SEQ ID NO:38)
describes EcoRI restriction site containing PCR primer, Kappa forward-1.
TABLE-US-00039 5′-gggaattccaccatggattttcaggtgcagattttcag-3′ (SEQ ID NO:39)
describes EcoRI restriction site containing PCR primer, Kappa forward-2.
TABLE-US-00040 5′-gggaattccaccatgragtcacakacycaggtcttyrta-3′ (SE Q ID NO:40)
describes EcoRI restriction site containing PCR primer, Kappa forward-3.
TABLE-US-00041 5′-gggaattccaccatgaggkccccwgctcagytyctkggr-3′ (SE Q ID NO:41)
describes EcoRI restriction site containing PCR primer, Kappa forward-4.
TABLE-US-00042 5′-gggaattccaccatgaagttgcctgttaggctgttg-3′ (SEQ I D NO:42)
describes EcoRI restriction site containing PCR primer, Kappa forward-5.
TABLE-US-00043 5′-gggaattccaccatgatgagtcctgcccagttcc-3′ (SEQ ID NO:43)
describes EcoRI restriction site containing PCR primer, Kappa forward-6.
TABLE-US-00044 5′-ctacctcgagttaacactcattcctgttgaagc-3′ (SEQ ID N O:44)
describes XhoI restriction site containing PCR primer, Kappa reverse.
TABLE-US-00045 gaggtccagctggaggagtctgggggaggcttagtgaagcctggagggt ccctgaaactctcctgtgcagcctctggattcactttcagtggctatgcc atgtcttgggttcgccagactccggagaagaggctggagtgggtcgcaac cattagtagtggtggtacttatatctactatccagacagtgtgaaggggc gattcaccatctccagagacaatgccaagaacaccctgtacctgcaaatg agcagtctgaggtctgaggacacggccatgtattactgtgcaagacttgg gggggataattactacgaatacttcgatgtctggggcgcagggaccacgg tcaccgtctcctccgccaaaacgacacccccatctgtctatccactggcc cctggatctgctgcccaaactaactccatggtgaccctgggatgcctggt caagggctatttccctgagccagtgacagtgacctggaactctggatccc tgtccagcggtgtgcacaccttcccagctgtcctgcagtctgacctctac actctgagcagctcagtgactgtcccctccagcacctggcccagcgagac cgtcacctgcaacgttgcccacccagccagcaggaccgcg (SEQ ID N O:45)
describes MIN-C2 Fab Heavy chain.
TABLE-US-00046 gacattgtgatcacacagtctacagcttccttaggtgtatctctggggc agagggccaccatctcatgcagggccagcaaaagtgtcagtacatctggc tatagttatatgcactggtaccaacagagaccaggacagccacccaaact cctcatctatcttgcatccaacctagaatctggggtccctgccaggttca gtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggag gaggaggatgctgcaacctattactgtcagcacagtagggagcttccgtt cacgttcggaggggggaccaagctggagataaaacgggctgatgctgcac caactgtatccatcttcccaccatccagtgagcagttaacatctggaggt gcctcagtcgtgtgcttcttgaacaacttctaccccaaagacatcaatgt caagtggaagattgatggcagtgaacgacaaaatggcgtcctgaacagtt ggactgatcaggacagcaaagacagcacctacagcatgagcagcaccctc acgttgaccaaggacgagtatgaacgacataacagctatacctgtgaggc cactcacaagacatcaacttcacccattgtcaagagcttcaacaggaatg agtgt (SEQ ID NO:46)
describes MIN-C2 Fab Kappa chain.
TABLE-US-00047 EVQLEESGGGLVKPGGSLKLSCAASGFTFSGYAMSWVRQTPEKRLEWVA TISSGGTYIYYPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAMYYCARL GGDNYYEYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCL VKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSE TVTCNVAHPASRTA (SEQ ID NO:47)
describes MIN-C2 Fab Heavy chain.
TABLE-US-00048 DIVITQSTASLGVSLGQRATISCRASKSVSTSGYSYMHWYQQRPGQPPK LLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELP FTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDIN VKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCE ATHKTSTSPIVKSFNRNEC (SEQ ID NO:48)
describes MIN-C2 Fab Kappa chain.
TABLE-US-00049 RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQ NGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIV KSFNRNEC (SEQ ID NO:49)
describes MIN-C2 light CL region amino acid sequence.
TABLE-US-00050 FDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPE PVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVA HPASRTA (SEQ ID NO:50)
describes▫MIN-C2▫heavy▫chain▫CH1▫region▫amino▫acid▫sequence.
TABLE-US-00051 DIVLTQSTEIMSASPGEKVTITCSASSSISYIHWFQQKPGTSPKLWIFG TSNLASGVPARFSGSGSGTSYSLTVSRMEAEDTATYYCQQRSNYPFTFGS GTKLQIKRADAAPTVS (SEQ IDNO:51)
describes MIN-A2-1 light chain variable region amino acid sequence.
TABLE-US-00052 DIVMTQSPAIMSASPGEKVTMTCSASSSVSYMHWFQQKPGTSPKLWIYS TSNLASGAPARFSGSGSGTSYSLTVSRMESEDAATYYCQQRSSYPSTFGG GTKLEIKRADAAPTVS (SEQ IDNO:52)
describes MIN-A2-2 light chain variable region amino acid sequence.
TABLE-US-00053 DIVLTQTTAIMSASPGEKVTITCSASSSVSYMYWFQQKPGTSPKLWIYS TSNLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSYPSTFGG GTKLEIKRADAAPTVS (SEQ IDNO:53)
describes MIN-C9-1 light chain variable region amino acid sequence.
TABLE-US-00054 DIVITQSTAIMSASPGEKVTITCSASSSVSYTYWFQQKPGTSPKLWIYS TSNLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSYPSTFGG GTKLEIKRADAAPTVS (SEQ IDNO:54)
describes MIN-C9-2 light chain variable region amino acid sequence.
TABLE-US-00055 DIVITQTPAIMSASPGEKVTMTCSASSSVSYMHWFQQKPGTSPKLWIYS TSNLASGVPARFSGSGSGTSYSLTVSRMESEDAATYYCQQRSSYPSTFGG GTKLEIKRADAAPTVS (SEQ IDNO:55)
describes MIN-D7-1 light chain variable region amino acid sequence.
TABLE-US-00056 DIVLTQSTAIMSASPGEKVTMTCSASSSVSYMHWFQQKPGTSPKLWIYS TSNLASGVPARFSGSGSGTSYSLTVSRMESEDAATYYCQQRSSYPSTFGG GTKLEIKRADAAPTVS (SEQ IDNO:56)
describes MIN-D7-2 light chain variable region amino acid sequence.
TABLE-US-00057 DIVMTQSPEIMSASPGEKVTITCSASSSISYIHWFQQKPGTSPKLWIFG TSNLASGVPARFSGSGSGTSYSLTVSRMEAEDTATYYCQQRSNYPFTFGS GTKLQIKRADAAPTVS (SEQ IDNO:57)
describes MIN-F2-1 light chain variable region amino acid sequence.
TABLE-US-00058 DIVITQSTEIMSASPGEKVTITCSASSSISYIHWFQQKPGTSPKLWIFG TSNLASGVPARFSGSGSGTSYSLTVSRMEAEDTATYYCQQRSNYPFTFGS GTKLQIKRADAAPTVS (SEQ IDNO:58)
describes MIN-F2-2 light chain variable region amino acid sequence.
TABLE-US-00059 EVKLQESGPELKKPGETVEISCKASGYTFTNYGMNWVKQAPGKGLKWMG WINTYTGEPTYAGDFKGRFAFSLETSASTAYLQINTLKNEDTATYFCARS GDGYWYYAMDYWGQGTSVTVSSAKTTPPSVY (SEQ ID NO:59)
describes MIN-A2-1 heavy chain variable region amino acid sequence.
TABLE-US-00060 EVQLQQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMG WINTYTGEPTYAGDFKGRFAFSLETSASTAYLQINTLKNEDTATYFCARS GDGYWYYAMDYWGQGTSVTVSSAKTTPPSVY (SEQ ID NO:60)
describes MIN-A2-2 heavy chain variable region amino acid sequence.
TABLE-US-00061 QVQLQESGPELKQPGETVKISCKASGYTFTNNGMNWVKQAPGKGLKWMG WINTYTGEPTYADDFKGRFAFSLDTSASTAYLQINNLKNEDMATYFCART GTARAFYAMDYWGQGTSVTVSSTKTTAPSVY (SEQ ID NO:61)
describes MIN-C9-1 heavy chain variable region amino acid sequence.
TABLE-US-00062 QVQLQQSGPELKQPGETVKISCKASGYTFTNNGMNWVKQAPGKGLKWMG WINTYTGEPTYADDFKGRFAFSLGTSASTAYLQINNLKNEDMATYFCART GTARAFYAMDYWGQGTSVTVSSTKTTAPSVY (SEQ ID NO:62)
describes MIN-C9-2 heavy chain variable region amino acid sequence.
TABLE-US-00063 EVQLEQSGPELKKPGETVKISCKASGYTFINYGMNWVKQAPGKGLKWMG WINTYTGEPTYVDDFKGRFAFSLETSARTAYLQINNLKNEDMATYFCART GTTAILNGMDYWGQGTSVTVSSAKTTPPSVY (SEQ ID NO:63)
describes MIN-D7-1 heavy chain variable region amino acid sequence.
TABLE-US-00064 EVQLQQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMG WINTYTGEPTYAGDFKGRFAFSLETSASTAYLQINTLKNEDTATYFCARS GDGYWYYAMDYWGQGTSVTVSSAKTTPPSVY (SEQ ID NO:64)
describes MIN-D7-2 heavy chain variable region amino acid sequence.
TABLE-US-00065 EVKLEESGPELKKPGETVKISCKASGYTFINYGMNWVKQAPGKGLKWMG WINTYTGEPTYVDDFKGRFAFSLETSARTAYLQINNLKNEDMATYFCART GTTAILNGMDYWGQGTSVTVSSAKTTPPSVY (SEQ ID NO:65)
describes MIN-F2-1 heavy chain variable region amino acid sequence.
TABLE-US-00066 EVQLEQSGAELVRPGASVKLSCKALGYTFTDYEMHWVKQTPVHGLEWIG AIHPGSGGTAYNQKFKGKATLTADKSSSTAYMELSSLTSEDSAVYYCTNY GSFAYWGQGTLVTVSAAKTTPPSVY (SEQ ID NO:66)
describes MIN-F2-2 heavy chain variable region amino acid sequence.
TABLE-US-00067 RCRLQQSGPELKKPGETVKISCKASGYTFINYGMNWVKQAPGKGLKWMG WINTYTGEPTYVDDFKGRFAFSLETSARTAYLQINNLKNEDMATYFCART GTTAILNGMDYWGQGTSVTVSSAKTTPPSCL (SEQ ID NO:67)
describes MIN-F2-3 heavy chain variable region amino acid sequence.
TABLE-US-00068 EVQLEQSGPELKKPGETVKISCKASGYTFINYGMNWVKQAPGKGLKWMG WINTYTGEPTYVDDFKGRFAFSLETSARTAYLQINNLKNEDMATYFCART GTTAILNGMDYWGQGTSVTVSSAKTTPPSVY (SEQ ID NO:68)
describes MIN-F2-4 heavy chain variable region amino acid sequence.
TABLE-US-00069 DIQMTQSPSSLSASLGERVSLTCRASQDIGSSLNWLQQEPDGTIKRLIY ATSSLDSGVPKRFSGSRSGSDYSLTISSLESEDFVDYYCLQYASSPHVRC WDQAGAETGCCTNC (SEQ IDNO:69)
describes MIN-14 light chain variable region amino acid sequence.
TABLE-US-00070 DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPR LLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELT RSEGGPSW (SEQ ID NO:70)
describes▫MIN-17-1▫light▫chain▫variable▫region▫amino▫acid▫sequence.
TABLE-US-00071 DIQMTQSPASQSASLGESVTITCLASQTIGTWLAWYQQKPGKSPQLLIY AATSLADGVPSRFSGSGSGTKFSFKISSLQAEDFVSYYCQQLYSTPWTFG GGTKLEIKRADAAPTV (SEQ IDNO:71)
describes MIN-17-2 light chain variable region amino acid sequence.
TABLE-US-00072 DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPR LLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELT RSEGGPSW (SEQ ID NO:72)
describes MIN-29 light chain variable region amino acid sequence.
TABLE-US-00073 DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPR LLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELT RSEGGPSW (SEQ ID NO:73)
describes MIN-34 light chain variable region amino acid sequence.
TABLE-US-00074 DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVGWYQQKPGQSPKALIY SASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNNYPYTFG GGTKLEIKRADAAPTV (SEQ IDNO:74)
describes MIN-42 light chain variable region amino acid sequence.
TABLE-US-00075 DIQMTQPPASLSASVGETVTITCRASGNIHNFLAWYQQKQGKSPQLLVY NAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPWTFG GGTKLEIKRADAAPTV (SEQ IDNO:75)
describes MIN-45 light chain variable region amino acid sequence.
TABLE-US-00076 QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIG EINPSNGRTNYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCATY GNYWYF (SEQ ID NO:76)
describes MIN-14 heavy chain variable region amino acid sequence.
TABLE-US-00077 QITLKESGPGIVQPSQPFRLTCTFSGFSLSTSGIGVTWIRQPSGKGLEW LATIWWDDDNRYNPSLKSRLTVSKDTSNNQAFLNIITVETADTAIYYCAQ STMVTA (SEQ ID NO:77)
describes MIN-17-2 heavy chain variable region amino acid sequence.
TABLE-US-00078 QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIG EINPSNGRTNYNEK-FKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCAT YGNYWYF (SEQ ID NO:78)
describes MIN-17-1 heavy chain variable region amino acid sequence.
TABLE-US-00079 DVKLVESGGDLXKLTEGEDIWEGLTLCRDSDQSPLAPVSKPGRVVRPQR SCTVIQGCVLRLQTAHLQVQGVLGIVSGDGESALHSVWIVGATTITINGC DQLQPLLWSLANPRHVIATESESRGCTG (SEQ ID NO:79)
describes MIN-29 heavy chain variable region amino acid sequence.
TABLE-US-00080 QVQLKQSGPGLVQPSQSLSITCTVSGFSLTSYGVHWVRQSPGKGLEWLG VIWGGGSTDYNAAFISRLSISKDNSKSQVFFKMNSLQANDTAIYYCARND YPAWF (SEQ ID NO:80)
describes MIN-34 heavy chain variable region amino acid sequence.
TABLE-US-00081 EVQLVESGGDLVKPGRSLKLSCAASGFTFSSFGMSWVRQTPDKRLEWVA TISSGGTYTYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCSRR FYYDYD (SEQ ID NO:81)
describes MIN-42 heavy chain variable region amino acid sequence.
TABLE-US-00082 cgggctgatgctgcaccaactgtatccatcttcccaccatccagtgagc agttaacatctggaggtgcctcagtcgtgtgcttcttgaacaacttctac cccaaagacatcaatgtcaagtggaagattgatggcagtgaacgacaaaa tggcgtcctgaacagttggactgatcaggacagcaaagacagcacctaca gcatgagcagcaccctcacgttgaccaaggacgagtatgaacgacataac agctatacctgtgaggccactcacaagacatcaacttcacccattgtcaa gagcttcaacaggaatgagtgt (SEQ ID NO:82)
describes MIN-C2 light chain CL nucleotide sequence.
TABLE-US-00083 Ttcgatgtctggggcgcagggaccacggtcaccgtctcctccgccaaaa cgacacccccatctgtctatccactggcccctggatctgctgcccaaact aactccatggtgaccctgggatgcctggtcaagggctatttccctgagcc agtgacagtgacctggaactctggatccctgtccagcggtgtgcacacct tcccagctgtcctgcagtctgacctctacactctgagcagctcagtgact gtcccctccagcacctggcccagcgagaccgtcacctgcaacgttgccca cccagccagcaggaccgcg_(SEQ ID NO:83)
describes MIN-C2 Heavy chain CH1 nucleotide sequence.
TABLE-US-00084 EVQLQQSGPELVKPGASVKISCKASGYSFTGYFMSWVMQSHGKSLEWIG RINPYNGDTFYNQKFKGKATLTVDKSSTTAHIELRSLASEDSAVYYCARK GLYG (SEQ ID NO:84)
describes MIN-45 heavy chain variable region amino acid sequence.
TABLE-US-00085 gacatccagatgacccagtctccatcctccttatctgcctctctgggag aaagagtcagtctcacttgtcgagcaagtcaggacattggtagtagctta aactggcttcagcaggaaccagatggaactattaaacgcctgatctacgc cacatccagtttagattctggtgtccccaaaaggttcagtggcagtaggt ctgggtcagattattctctcaccatcagcagccttgagtctgaagatttt gtagactattactgtctacaatatgctagttctcctcacgttcggtgctg ggaccaagctggagctgaaacgggc (SEQ ID NO:85)
describes MIN-14 light chain variable region nucleotide sequence.
TABLE-US-00086 gacattgtgctgacacagtctcctgcttccttagctgtatctctggggc agagggccaccatctcatacagggccagcaaaagtgtcagtacatctggc tatagttatatgcactggaaccaacagaaaccaggacagccacccagact cctcatctatcttgtatccaacctagaatctggggtccctgccaggttca gtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggag gaggaggatgctgcaacctattactgtcagcacattagggagcttacacg ttcggaggggggaccaagctggaa (SEQ ID NO:86)
describes MIN-17-1 light chain variable region nucleotide sequence.
TABLE-US-00087 gacattcagatgacccagtctcctgcctcccagtctgcatctctgggag aaagtgtcaccatcacatgcctggcaagtcagaccattggtacatggtta gcatggtatcagcagaaaccagggaaatctcctcagctcctgatttatgc tgcaaccagcttggcagatggggtcccatcaaggttcagtggtagtggat ctggcacaaaattttctttcaagatcagcagcctacaggctgaagatttt gtaagttattactgtcaacaactttacagtactccgtggacgttcggtgg aggcaccaagctggaaatcaaacgggctgatgctgcaccaactgta (SE Q ID NO:87)
describes MIN-17-2 light chain variable region nucleotide sequence.
TABLE-US-00088 gtgacattgtgctgacacagtctcctgcttccttagctgtatctctggg gcagagggccaccatctcatacagggccagcaaaagtgtcagtacatctg gctatagttatatgcactggaaccaacagaaaccaggacagccacccaga ctcctcatctatcttgtatccaacctagaatctggggtccctgccaggtt cagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtgg aggaggaggatgctgcaacctattactgtcagcacattagggagcttaca cgttcggaggggggaccaagctgg (SEQ ID NO:88)
describes MIN-29 light chain variable region nucleotide sequence.
TABLE-US-00089 gacattgtgctgacacagtctcctgcttccttagctgtatctctggggc agagggccaccatctcatacagggccagcaaaagtgtcagtacatctggc tatagttatatgcactggaaccaacagaaaccaggacagccacccagact cctcatctatcttgtatccaacctagaatctggggtccctgccaggttca gtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggag gaggaggatgctgcaacctattactgtcagcacattagggagcttacacg ttcggaggggggaccaagctgg (SEQ ID NO:89)
describes MIN-34 light chain variable region nucleotide sequence.
TABLE-US-00090 gacattgtgatgacccagtctcaaaaattcatgtccacatcagtaggag acagggtcagcgtcacctgcaaggccagtcagaatgtgggtactaatgta ggttggtatcaacagaaaccagggcaatctcctaaagcactgatttactc ggcatcctaccggtacagtggagtccctgatcgcttcacaggcagtggat ctgggacagatttcactctcaccatcagcaatgtgcagtctgaagacttg gcagagtatttctgtcagcaatataacaactatccgtacacgttcggagg ggggaccaagctggaaataaaacgggctgatgctgcaccaactgta (SE Q ID NO:90)
describes MIN-42 light chain variable region nucleotide sequence.
TABLE-US-00091 gacatccagatgactcagcctccagcctccctatctgcatctgtgggag aaactgtcaccatcacatgtcgagcaagtgggaatattcacaatttttta gcatggtatcagcagaaacagggaaaatctcctcagctcctggtctataa tgcaaaaaccttagcagatggtgtgccatcaaggttcagtggcagtggat caggaacacaatattctctcaagatcaacagcctgcagcctgaagatttt gggagttattactgtcaacatttttggagtactccgtggacgttcggtgg aggcaccaagctggaaatcaaacgggctgatgctgcaccaactgta (SE Q ID NO:91)
describes MIN-45 light chain variable region nucleotide sequence.
TABLE-US-00092 caggtccaactgcagcagcctggggctgaactggtgaagcctggggctt cagtgaagctgtcctgcaaggcttctggctacaccttcaccagctactgg atgcactgggtgaagcagaggcctggacaaggccttgagtggattggaga gattaatcctagcaacggtcgtactaactacaatgagaagttcaagagca aggccacactgactgtagacaaatcctccagcacagcctacatgcaactc agcagcctgacatctgaggactctgcggtctattactgtgcaacctatgg taactactggtacttc (SEQ ID NO:92)
describes MIN-14 heavy chain variable region nucleotide sequence.
TABLE-US-00093 caggtccaactgcagcagcctggggctgaactggtgaagcctggggctt cagtgaagctgtcctgcaaggcttctggctacaccttcaccagctactgg atgcactgggtgaagcagaggcctggacaaggccttgagtggattggaga gattaatcctagcaacggtcgtactaactacaatgagaagttcaagagca aggccacactgactgtagacaaatcctccagcacagcctacatgcaactc agcagcctgacatctgaggactctgcggtctattactgtgcaacctatgg taactactggtacttc (SEQ ID NO:93)
describes MIN-17-1 heavy chain variable region nucleotide sequence.
TABLE-US-00094 cagattactctgaaagagtctggccctgggatagttcagccatcccagc ccttcagacttacttgcactttctctgggttttcactgagcacttctggt ataggtgtaacctggattcgtcagccctcagggaaaggtctggagtggct ggcaacgatttggtgggatgatgataaccgctacaacccatctctaaaga gcaggctcacagtctccaaagacacctccaacaaccaagcattcctgaat atcatcactgtggaaactgcagatactgccatatactactgtgctcagtc tactatggttacggcggga (SEQ ID NO:94)
describes MIN-17-2 heavy chain variable region nucleotide sequence.
TABLE-US-00095 ccaggtgcagctgaagcagtcaggacctggcctagtgcagccctcacag agcctgtccatcacctgcacagtctctggtttctcattaactagctatgg tgtacactgggttcgccagtctccaggaaagggtctggagtggctgggag tgatatggggtggtggaagcacagactataatgcagctttcatatccaga ctgagcatcagcaaggacaattccaagagccaagttttctttaaaatgaa cagtctgcaagctaatgacacagccatatattactgtgccagaaatgact atccggcctggttt (SEQ ID NO:95)
describes MIN-34 heavy chain variable region nucleotide sequence.
TABLE-US-00096 gtgaggtgcaactggtggagtctgggggagacttagtgaagcctggaag gtccctgaaactctcctgtgcagcctctggattcactttcagtagctttg gcatgtcttgggttcgccagactccagacaagaggctggagtgggtcgca accattagtagtggtggtacttacacctactatccagacagtgtgaaggg gcgattcaccatctccagagacaatgccaagaacaccctgtacctgcaaa tgagcagtctgaagtctgaggacacagccatgtattactgttcaagaagg ttctactatgattacgac (SEQ ID NO:96)
describes MIN-42 heavy chain variable region nucleotide sequence.
TABLE-US-00097 gaggttcagctgcagcagtctggacctgagctggtgaagcctggggctt cagtgaagatatcctgcaaggcttctggttactcatttactggctacttt atgagctgggtgatgcagagccatggaaagagccttgagtggattggacg tattaatccttacaatggtgatactttctacaaccagaagttcaagggca aggccacattgactgtagacaaatcctctaccacagcccacatagagctc cggagcctggcatctgaggactctgcagtctattattgtgcaagaaaggg cctctatggg (SEQ ID NO:97)
describes MIN-45 heavy chain variable region nucleotide sequence.
TABLE-US-00098 DIVITQSTASLGVSLGQRATISC (SEQ ID NO:98)
describes MIN-C2 light chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00099 DIVITQTTAIMSASPGEEVTLTC (SEQ ID NO:99)
describes MIN-E6 light chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00100 DIVLTQSTEIMSASPGEKVTITC (SEQ ID NO:100)
describes MIN-A2-1 light chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00101 DIVMTQSPAIMSASPGEKVTMTC (SEQ ID NO: 101)
describes MIN-A2-2 light chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00102 DIVLTQTTAIMSASPGEKVTITC (SEQ ID NO:102)
describes MIN-C9-1 light chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00103 DIVITQSTAIMSASPGEKVTITC (SEQ ID NO:103)
describes MIN-C9-2 light chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00104 DIVITQTPAIMSASPGEKVTMTC (SEQ ID NO:104)
describes MIN-D7-1 light chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00105 DIVLTQSTAIMSASPGEKVTMTC (SEQ ID NO:105)
describes MIN-D7-2 light chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00106 DIVMTQSPEIMSASPGEKVTITC (SEQ ID NO:106)
describes MIN-F2-1 light chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00107 DIVITQSTEIMSASPGEKVTITC (SEQ ID NO:107)
describes MIN-F2-2 light chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00108 RASKSVSTSGYSYMH (SEQ ID NO:108)
describes MIN-C2 light chain variable complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00109 SATSSVSYIH (SEQ ID NO:109)
describes MIN-E6 light chain variable complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00110 SASSSISYIH (SEQ ID NO:110)
describes MIN-A2-1 light chain variable complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00111 SASSSVSYMH (SEQ ID NO:112)
describes MIN-A2-2 light chain variable complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00112 SASSSVSYMY (SEQ ID NO:113)
describes MIN-C9-1 light chain variable complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00113 SASSSVSYTY (SEQ ID NO:114)
describes MIN-C9-2 light chain variable complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00114 SASSSVSYMH (SEQ ID NO:115)
describes MIN-D7-1 light chain variable complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00115 SASSSVSYMH (SEQ ID NO:116)
describes MIN-D7-2 light chain variable complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00116 SASSSISYIH (SEQ ID NO:117)
describes MIN-F2-1 light chain variable complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00117 SASSSISYIH (SEQ ID NO:118)
describes MIN-F2-2 light chain variable complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00118 WYQQRPGQPPKLLIY (SEQ ID NO:119)
describes MIN-C2 light chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00119 WFQQRPGTSPKLWIY (SEQ ID NO:120)
describes MIN-E6 light chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00120 WFQQKPGTSPKLWIF (SEQ ID NO:121)
describes MIN-A2-1 light chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00121 WFQQKPGTSPKLWIY (SEQ ID NO: 122)
describes MIN-A2-2 light chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00122 WFQQKPGTSPKLWIY (SEQ ID NO:123)
describes MIN-C9-1▫light▫chain▫variable▫framework region 2 (FWR2) amino acid sequence.
TABLE-US-00123 WFQQKPGTSPKLWIY (SEQ ID NO:124)
describes MIN-C9-2 light chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00124 WFQQKPGTSPKLWIY (SEQ ID NO: 125)
describes MIN-D7-1 light chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00125 WFQQKPGTSPKLWIY (SEQ ID NO: 126)
describes MIN-D7-2 light chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00126 WFQQKPGTSPKLWIF (SEQ ID NO:127)
describes MIN-F2-1 light chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00127 WFQQKPGTSPKLWIF (SEQ ID NO:128)
describes MIN-F2-2 light chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00128 LASNLES (SEQ ID NO:129)
describes MIN-C2 light chain variable complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00129 STSNLAS (SEQ ID NO:130)
describes MIN-E6 light chain variable complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00130 GTSNLAS (SEQ ID NO:131)
describes MIN-A2-1 light chain variable complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00131 STSNLAS (SEQ ID NO:132)
describes MIN-A2-2 light chain variable complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00132 STSNLAS (SEQ ID NO:133)
describes MIN-C9-1 light chain variable complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00133 STSNLAS (SEQ ID NO:134)
describes MIN-C9-2 light chain variable complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00134 STSNLAS (SEQ ID NO:135)
describes MIN-D7-1 light chain variable complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00135 STSNLAS (SEQ ID NO:136)
describes MIN-D7-2 light chain variable complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00136 GTSNLAS (SEQ ID NO:137)
describes MIN-F2-1 light chain variable complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00137 GTSNLAS (SEQ ID NO:138)
describes MIN-F2-2 light chain variable complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00138 GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC (SEQ ID NO:139)
describes MIN-C2 light chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00139 GVPVRFSGSGYGTSYSLTISRMEAEDAATYYC (SEQ ID NO:140)
describes MIN-E6 light chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00140 GVPARFSGSGSGTSYSLTVSRMEAEDTATYYC (SEQ ID NO:141)
describes MIN-A2-1 light chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00141 GAPARFSGSGSGTSYSLTVSRMESEDAATYYC (SEQ ID NO:142)
describes MIN-A2-2 light chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00142 GVPARFSGSGSGTSYSLTISRMEAEDAATYYC (SEQ ID NO:143)
describes MIN-C9-1 light chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00143 GVPARFSGSGSGTSYSLTISRMEAEDAATYYC (SEQ ID NO:144)
describes MIN-C9-2 light chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00144 GVPARFSGSGSGTSYSLTVSRMESEDAATYYC (SEQ ID NO:145)
describes▫MIN-D7-1 light chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00145 GVPARFSGSGSGTSYSLTVSRMESEDAATYYC (SEQ ID NO:146)
describes MIN-D7-2 light chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00146 GVPARFSGSGSGTSYSLTVSRMEAEDTATYYC (SEQ ID NO:147)
describes MIN-F2-1 light chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00147 GVPARFSGSGSGTSYSLTVSRMEAEDTATYYC (SEQ ID NO:148)
describes MIN-F2-2 light chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00148 QHSRELPFT (SEQ ID NO:149)
describes MIN-C2 light chain variable complementarity determining region 3 (CDR3) amino acid sequence.
TABLE-US-00149 QQRSSSPFT (SEQ ID NO:150)
describes MIN-E6 light chain variable complementarity determining region 3 (CDR3) amino acid sequence.
TABLE-US-00150 QQRSNYPFT (SEQ ID NO:151)
describes MIN-A2-1 light chain variable complementarity determining region 3 (CDR3) amino acid sequence.
TABLE-US-00151 QQRSSYPST (SEQ ID NO:152)
describes MIN-A2-2 light chain variable complementarity determining region 3 (CDR3) amino acid sequence.
TABLE-US-00152 QQRSSYPST (SEQ ID NO:153)
describes MIN-C9-1 light chain variable complementarity determining region 3 (CDR3) amino acid sequence.
TABLE-US-00153 QQRSSYPST (SEQ ID NO:154)
describes MIN-C9-2 light chain variable complementarity determining region 3 (CDR3) amino acid sequence.
TABLE-US-00154 QQRSSYPST (SEQ ID NO:155)
describes MIN-D7-1 light chain variable complementarity determining region 3 (CDR3) amino acid sequence.
TABLE-US-00155 QQRSSYPST (SEQ ID NO:156)
describes MIN-D7-2 light chain variable complementarity determining region 3 (CDR3) amino acid sequence.
TABLE-US-00156 QQRSNYPFT (SEQ ID NO:157)
describes MIN-F2-1 light chain variable complementarity determining region 3 (CDR3) amino acid sequence.
TABLE-US-00157 QQRSNYPFT (SEQ ID NO:158)
describes MIN-F2-2 light chain variable complementarity determining region 3 (CDR3) amino acid sequence.
TABLE-US-00158 EVQLEESGGGLVKPGGSLKLSCAASGFTFS (SEQ ID NO:159)
describes MIN-C2 heavy chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00159 EVKLEESGGDLVKPGGSLKLSCAASGFTFS (SEQ ID NO:160)
describes MIN-E6-7▫heavy chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00160 EVKLEESGGDLVKPGGSLKLSCVVSGFTFS (SEQ ID NO:161)
describes MIN-E6-8 heavy chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00161 EVKLQESGPELKKPGETVEISCKASGYTFT (SEQ ID NO:162)
describes MIN-A2-1 heavy chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00162 EVQLQQSGPELKKPGETVKISCKASGYTFT (SEQ ID NO:163)
describes MIN-A2-2 heavy chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00163 QVQLQESGPELKQPGETVKISCKASGYTFT (SEQ ID NO:164)
describes MIN-C9-1 heavy chain variable frame work region 1 (FWR1) amino acid sequence.
TABLE-US-00164 QVQLQQSGPELKQPGETVKISCKASGYTFT (SEQ ID NO:165)
describes MIN-C9-2 heavy chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00165 EVQLEQSGPELKKPGETVKISCKASGYTFI (SEQ ID NO:166)
describes MIN-D7-1 heavy chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00166 EVQLQQSGPELKKPGETVKISCKASGYTFT (SEQ ID NO:167)
describes MIN-D7-2 heavy chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00167 EVKLEESGPELKKPGETVKISCKASGYTFI (SEQ ID NO:168)
describes MIN-F2-1 heavy chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00168 EVQLEQSGAELVRPGASVKLSCKALGYTFT (SEQ ID NO:169)
describes MIN-F2-2 heavy chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00169 RCRLQQSGPELKKPGETVKISCKASGYTFI (SEQ ID NO:170)
describes MIN-F2-3 heavy chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00170 EVQLEQSGPELKKPGETVKISCKASGYTFI (SEQ ID NO:171)
describes MIN-F2-4 heavy chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00171 GYAMS (SEQ ID NO:172)
describes MIN-C2 heavy chain variable complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00172 RYGMS (SEQ ID NO:173)
describes MIN-E6-7 heavy chain variable complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00173 RYGMS (SEQ ID NO:174)
describes MIN-E6-8 heavy chain variable complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00174 NYGMN (SEQ ID NO:175)
describes MIN-A2-1 heavy chain variable complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00175 NYGMN (SEQ ID NO:176)
describes MIN-A2-2 heavy chain variable complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00176 NNGMN (SEQ ID NO:177)
describes MIN-C9-1 heavy chain variable complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00177 NNGMN (SEQ ID NO:178)
describes MIN-C9-2 heavy chain variable complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00178 NYGMN (SEQ ID NO:179)
describes MIN-D7-1 heavy chain variable complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00179 NYGMN (SEQ ID NO:180)
describes MIN-D7-2 heavy chain variable complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00180 NYGMN (SEQ ID NO:181)
describes MIN-F2-1 heavy chain variable complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00181 DYEMH (SEQ ID NO:182)
describes MIN-F2-2 heavy chain variable complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00182 NYGMN (SEQ ID NO:183)
describes MIN-F2-3 heavy chain variable complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00183 NYGMN (SEQ ID NO:184)
describes MIN-F2-4 heavy chain variable complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00184 WVRQTPEKRLEWVA (SEQ ID NO:185)
describes MIN-C2 heavy chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00185 WVRQTPDKRLEWVA (SEQ ID NO:186)
describes MIN-E6-7 heavy chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00186 WVRQTPGKRLEWVA (SEQ ID NO:187)
describes MIN-E6-8 heavy chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00187 WVKQAPGKGLKWMG (SEQ ID NO:188)
describes MIN-A2-1 heavy chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00188 WVKQAPGKGLKWMG (SEQ ID NO:189)
describes MIN-A2-2 heavy chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00189 WVKQAPGKGLKWMG (SEQ ID NO:190)
describes MIN-C9-1 heavy chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00190 WVKQAPGKGLKWMG (SEQ ID NO:191)
describes MIN-C9-2 heavy chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00191 WVKQAPGKGLKWMG (SEQ ID NO:192)
describes MIN-D7-1 heavy chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00192 WVKQAPGKGLKWMG (SEQ ID NO:193)
describes MIN-D7-2 heavy chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00193 WVKQAPGKGLKWMG (SEQ ID NO:194)
describes MIN-F2-1 heavy chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00194 WVKQTPVHGLEWIG (SEQ ID NO: 195)
describes MIN-F2-2 heavy chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00195 WVKQAPGKGLKWMG (SEQ ID NO:196)
describes MIN-F2-3 heavy chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00196 WVKQAPGKGLKWMG (SEQ ID NO:197)
describes MIN-F2-4 heavy chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00197 TISSGGTYIYYPDSVKG (SEQ ID NO:198)
describes MIN-C2 heavy chain variable complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00198 TISSGGTYIYYPDSVKG (SEQ ID NO:199)
describes MIN-E6-7 heavy chain variable complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00199 TISGGGTYIYYPDSVKG (SEQ ID NO:200)
describes MIN-E6-8 heavy chain variable complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00200 WINTYTGEPTYAGDFKG (SEQ ID NO:201)
describes MIN-A2-1 heavy chain variable complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00201 WINTYTGEPTYAGDFKG (SEQ ID NO:202)
describes MIN-A2-2 heavy chain variable complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00202 WINTYTGEPTYADDFKG (SEQ ID NO:203)
describes MIN-C9-1 heavy chain variable complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00203 WINTYTGEPTYADDFKG (SEQ ID NO:204)
describes MIN-C9-2 heavy chain variable complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00204 WINTYTGEPTYVDDFKG (SEQ ID NO:205)
describes MIN-D7-1 heavy chain variable complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00205 WINTYTGEPTYAGDFKG (SEQ ID NO:206)
describes MIN-D7-2 heavy chain variable complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00206 WINTYTGEPTYVDDFKG (SEQ ID NO:207)
describes MIN-F2-1 heavy chain variable complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00207 AIHPGSGGTAYNQKFKG (SEQ ID NO:208)
describes MIN-F2-2 heavy chain variable complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00208 WINTYTGEPTYVDDFKG (SEQ ID NO:209)
describes MIN-F2-3 heavy chain variable complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00209 WINTYTGEPTYVDDFKG (SEQ ID NO:210)
describes MIN-F2-4 heavy chain variable complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00210 RFTISRDNAKNTLYLQMSSLRSEDTAMYYCAR (SEQ ID NO:211)
describes MIN-C2 heavy chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00211 RFTISRDNAKNTLYLQMSSLKSEDTAMYYCAR (SEQ ID NO:212)
describes MIN-E6-7 heavy chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00212 RFTISRDNAKNTLYLQMSSLKSEDTAMYHCTR (SEQ ID NO:213)
describes MIN-E6-8 heavy chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00213 RFAFSLETSASTAYLQINTLKNEDTATYFCAR (SEQ ID NO:214)
describes MIN-A2-1 heavy chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00214 RFAFSLETSASTAYLQINTLKNEDTATYFCAR (SEQ ID NO:215)
describes MIN-A2-2 heavy chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00215 RFAFSLDTSASTAYLQINNLKNEDMATYFCAR (SEQ ID NO:216)
describes MIN-C9-1 heavy chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00216 RFAFSLGTSASTAYLQINNLKNEDMATYFCAR (SEQ ID NO:217)
describes MIN-C9-2 heavy chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00217 RFAFSLETSARTAYLQINNLKNEDMATYFCAR (SEQ ID NO:218)
describes MIN-D7-1 heavy chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00218 RFAFSLETSASTAYLQINTLKNEDTATYFCAR (SEQ ID NO:219)
describes MIN-D7-2 heavy chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00219 RFAFSLETSARTAYLQINNLKNEDMATYFCAR (SEQ ID NO:220)
describes MIN-F2-1 heavy chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00220 KATLTADKSSSTAYMELSSLTSEDSAVYYCTN (SEQ ID NO:221)
describes MIN-F2-2 heavy chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00221 RFAFSLETSARTAYLQINNLKNEDMATYFCAR (SEQ ID NO:222)
describes MIN-F2-3 heavy chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00222 RFAFSLETSARTAYLQINNLKNEDMATYFCAR (SEQ ID NO:223)
describes MIN-F2-4 heavy chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00223 LGGDNYYEY (SEQ ID NO:224)
describes MIN-C2 heavy chain variable complementarity determining region 3 (CDR3) amino acid sequence.
TABLE-US-00224 DNYGSSYDYA (SEQ ID NO:225)
describes MIN-E6-7 heavy chain variable complementarity determining region 3 (CDR3) amino acid sequence.
TABLE-US-00225 DNYGRNYDYG (SEQ ID NO:226)
describes MIN-E6-8 heavy chain variable complementarity determining region 3 (CDR3) amino acid sequence.
TABLE-US-00226 SGDGYWYYA (SEQ ID NO:227)
describes MIN-A2-1 heavy chain variable complementarity determining region 3 (CDR3) amino acid sequence.
TABLE-US-00227 SGDGYWYYA (SEQ ID NO:228)
describes MIN-A2-2 heavy chain variable complementarity determining region 3 (CDR3) amino acid sequence.
TABLE-US-00228 TGTARAFYA (SEQ ID NO:229)
describes MIN-C9-1 heavy chain variable complementarity determining region 3 (CDR3) amino acid sequence.
TABLE-US-00229 TGTARAFYA (SEQ ID NO:230)
describes MIN-C9-2 heavy chain variable complementarity determining region 3 (CDR3) amino acid sequence.
TABLE-US-00230 TGTTAILNG (SEQ ID NO:231)
describes MIN-D7-1 heavy chain variable complementarity determining region 3(CDR3) amino acid sequence.
TABLE-US-00231 SGDGYWYYA (SEQ ID NO:232)
describes MIN-D7-2 heavy chain variable complementarity determining region 3 (CDR3) amino acid sequence.
TABLE-US-00232 TGTTAILNG (SEQ ID NO:233)
describes MIN-F2-1 heavy chain variable complementarity determining region 3 (CDR3) amino acid sequence.
TABLE-US-00233 YGSFA (SEQ ID NO:234)
describes MIN-F2-2 heavy chain variable complementarity determining region 3(CDR3) amino acid sequence.
TABLE-US-00234 TGTTAILNG (SEQ ID NO:235)
describes MIN-F2-3 heavy chain variable complementarity determining region 3 (CDR3) amino acid sequence.
TABLE-US-00235 TGTTAILNG (SEQ ID NO:236)
describes MIN-F2-4 heavy chain variable complementarity determining region 3(CDR3) amino acid sequence.
TABLE-US-00236 DIQMTQSPSSLSASLGERVSLTC (SEQ ID NO:237)
describes MIN-14 light chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00237 DIVLTQSPASLAVSLGQRATISY (SEQ ID NO:238)
describes MIN-17-1 light chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00238 DIQMTQSPASQSASLGESVTITC (SEQ ID NO:239)
describes MIN-17-2 light chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00239 DIVLTQSPASLAVSLGQRATISY (SEQ ID NO:240)
describes MIN-29 light chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00240 DIVLTQSPASLAVSLGQRATISY (SEQ ID NO:241)
describes MIN-34 light chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00241 DIVMTQSQKFMSTSVGDRVSVTC (SEQ ID NO:242)
describes MIN-42 light chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00242 DIQMTQPPASLSASVGETVTITC (SEQ ID NO:243)
describes MIN-45 light chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00243 RASQDIGSSLN (SEQ ID NO:244)
describes MIN-14 light chain variable complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00244 RASKSVSTSGYSYMH (SEQ ID NO:245)
describes MIN-17-1 light chain variable complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00245 LASQTIGTWLA (SEQ ID NO:246)
describes MIN-17-2 light chain variable complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00246 RASKSVSTSGYSYMH (SEQ ID NO:247)
describes MIN-29 light chain variable complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00247 RASKSVSTSGYSYMH (SEQ ID NO:248)
describes MIN-34 light chain variable complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00248 KASQNVGTNVG (SEQ ID NO:249)
describes MIN-42 light chain variable complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00249 RASGNIHNFLA (SEQ ID NO:250)
describes MIN-45 light chain variable complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00250 WLQQEPDGTIKRLIY (SEQ ID NO:251)
describes MIN-14 light chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00251 WNQQKPGQPPRLLIY (SEQ ID NO:252)
describes MIN-17-1 light chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00252 WYQQKPGKSPQLLIY (SEQ ID NO:253)
describes MIN-17-2 light chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00253 WNQQKPGQPPRLLIY (SEQ ID NO:254)
describes MIN-29 light chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00254 WNQQKPGQPPRLLIY (SEQ ID NO:255)
describes MIN-34 light chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00255 WYQQKPGQSPKALIY (SEQ ID NO:266)
describes MIN-42 light chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00256 WYQQKQGKSPQLLVY (SEQ ID NO:267)
describes MIN-45 light chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00257 ATSSLDS (SEQ ID NO:268)
describes MIN-14 light chain variable complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00258 LVSNLES (SEQ ID NO:269)
describes MIN-17-1 light chain variable complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00259 AATSLAD (SEQ ID NO:270)
describes MIN-17-2 light chain variable complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00260 LVSNLES (SEQ ID NO:271)
describes MIN-29 light chain variable complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00261 LVSNLES (SEQ ID NO:272)
describes MIN-34 light chain variable complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00262 SASYRYS (SEQ ID NO:273)
describes MIN-42 light chain variable complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00263 NAKTLAD (SEQ ID NO:274)
describes MIN-45 light chain variable complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00264 GVPKRFSGSRSGSDYSLTISSLESEDFVDYYC (SEQ ID NO:275)
describes MIN-14 light chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00265 GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC (SEQ ID NO:276)
describes MIN-17-1 light chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00266 GVPSRFSGSGSGTKFSFKISSLQAEDFVSYYC (SEQ ID NO:277)
describes MIN-17-2 light chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00267 GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC (SEQ ID NO:288)
describes MIN-29 light chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00268 GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC (SEQ ID NO:289)
describes MIN-34 light chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00269 GVPDRFTGSGSGTDFTLTISNVQSEDLAEYFC (SEQ ID NO:290)
describes MIN-42 light chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00270 GVPSRFSGSGSGTQYSLKINSLQPEDFGSYYC (SEQ ID NO:291)
describes MIN-45 light chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00271 QVQLQQPGAELVKPGASVKLSCKASGYTFT (SEQ ID NO:292)
describes MIN-14 heavy chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00272 QVQLQQPGAELVKPGASVKLSCKASGYTFT (SEQ ID NO:293)
describes MIN-17-1 heavy chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00273 QITLKESGPGIVQPSQPFRLTCTFSGFSLSTS (SEQ ID NO:294)
describes MIN-17-2 heavy chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00274 DVKLVESGGDLXKLTEGEDIWEGLTLCRDSDQSPLAPVSKPGRVVRPQR SCTVIQGCVL (SEQ ID NO:295)
describes MIN-29 heavy chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00275 QVQLKQSGPGLVQPSQSLSITCTVSGFSLT (SEQ ID NO:296)
describes MIN-34 heavy chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00276 EVQLVESGGDLVKPGRSLKLSCAASGFTFS (SEQ ID NO:298)
describes MIN-42 heavy chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00277 EVQLQQSGPELVKPGASVKISCKASGYSFT (SEQ ID NO:299)
describes MIN-45 heavy chain variable framework region 1 (FWR1) amino acid sequence.
TABLE-US-00278 SYWMH (SEQ ID NO:300)
describes MIN-14 heavy chain complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00279 SYWMH (SEQ ID NO:301)
describes MIN-17-1 heavy chain complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00280 GIGVT (SEQ ID NO:302)
describes MIN-17-2 heavy chain complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00281 SYGVH (SEQ ID NO:303)
describes MIN-34 heavy chain complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00282 SFGMS (SEQ ID NO:304)
describes MIN-42 heavy chain complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00283 GYFMS (SEQ ID NO:305)
describes MIN-45 heavy chain complementarity determining region 1 (CDR1) amino acid sequence.
TABLE-US-00284 WVKQRPGQGLEWIG (SEQ ID NO:306)
describes MIN-14 heavy chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00285 WVKQRPGQGLEWIG (SEQ ID NO:307)
describes MIN-17-1 heavy chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00286 WIRQPSGKGLEWLA (SEQ ID NO:308)
describes MIN-17-2 heavy chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00287 RLQTAHLQVQGVL (SEQ ID NO:309)
describes MIN-29 heavy chain variableframework region 2 (FWR2) amino acid sequence.
TABLE-US-00288 WVRQSPGKGLEWLG (SEQ ID NO:310)
describes MIN-34 heavy chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00289 WVRQTPDKRLEWVA (SEQ ID NO:311)
describes MIN-42 heavy chain variable framework region 2 (FWR2) amino acid sequence.
TABLE-US-00290 WVMQSHGKSLEWIG (SEQ ID NO:312)
describes MIN-45 heavy chain variable framework region 2 (FWR1) amino acid sequence.
TABLE-US-00291 EINPSNGRTNYNEKFKS (SEQ ID NO:313)
describes MIN-14 heavy chain complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00292 EINPSNGRTNYNEKFKS (SEQ ID NO:314)
describes MIN-17-1 heavy chain complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00293 TIWWDDDNRYNPSLKS (SEQ ID NO:315)
describes MIN-17-2 heavy chain complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00294 GIVSGDGESALHSVWIVG (SEQ ID NO:316)
describes MIN-29 heavy chain complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00295 VIWGGGSTDYNAAFIS (SEQ ID NO:317)
describes MIN-34 heavy chain complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00296 TISSGGTYTYYPDSVKG (SEQ ID NO:318)
describes MIN-42 heavy chain complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00297 RINPYNGDTFYNQKFKG (SEQ ID NO:319)
describes MIN-45 heavy chain complementarity determining region 2 (CDR2) amino acid sequence.
TABLE-US-00298 KATLTVDKSSSTAYMQLSSLTSEDSAVYYCAT (SEQ ID NO:320)
describes MIN-14 heavy chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00299 KATLTVDKSSSTAYMQLSSLTSEDSAVYYCAT (SEQ ID NO:321)
describes MIN-17-1 heavy chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00300 RLTVSKDTSNNQAFLNIITVETADTAIYYCAQ (SEQ ID NO:322)
describes MIN-17-2 heavy chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00301 ATTITINGCDQLQPLLWSLANPRHVIATES (SEQ ID NO:323)
describes MIN-29 heavy chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00302 RLSISKDNSKSQVFFKMNSLQANDTAIYYCAR (SEQ ID NO:324)
describes MIN-34 heavy chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00303 RFTISRDNAKNTLYLQMSSLKSEDTAMYYCSR (SEQ ID NO:325)
describes MIN-42 heavy chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00304 KATLTVDKSSTTAHIELRSLASEDSAVYYCAR (SEQ ID NO:326)
describes MIN-45 heavy chain variable framework region 3 (FWR3) amino acid sequence.
TABLE-US-00305 DIVITQSTASLGVSLGQRATISCRASKSVSTSGYSYMHWYQQRPGQPPK LLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELP FTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDIN VKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCE ATHKTSTSPIVKSFNRNECSGGGSGGGSEGGGSEGGGSEGGGSEGGGSGG GSGEVQLEESGGGLVKPGGSLKLSCAASGFTFSGYAMSWVRQTPEKRLEW VATIS SGGTYIYYPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAMYYC ARLGGDNYYEYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTL GCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTW PSETVTCNVAHPASRTA (SEQ ID NO:327)
describes MIN-C2 single chain Fab (light chain-linker-heavy chain; VL+CL+linker+VH+CH1) amino acid sequence.
TABLE-US-00306 METDTLLLWVLLLWVPGSTGD (SEQ ID NO:328)
describes Ig kappa-chain leader sequence.
TABLE-US-00307 EQKLISEEDL (SEQ ID NO:329)
describes Myc tag.
TABLE-US-00308 NYGMN (SEQ ID NO:330)
describes VH-CS-CDR1.1: CDR1 Consensus sequence for variable heavy chain for IgG anti-MUC1* antibodies.
TABLE-US-00309 GYAMS (SEQ ID NO:331)
describes VH-CS-CDR1.2: CDR1 Consensus sequence for variable heavy chain for IgG anti-MUC1* antibodies.
TABLE-US-00310 R/GYA/GMS (SEQ ID NO:332)
describes VH-CS-CDR1.3: CDR1 Consensus sequence for variable heavy chain for IgG anti-MUC1* antibodies.
TABLE-US-00311 WINTYTGEPTYA/VG/DDFKG (SEQ ID NO:333)
describes VH-CS-CDR2.1: CDR2 Consensus sequence for variable heavy chain for IgG anti-MUC1* antibodies.
TABLE-US-00312 TISSGGTYIYYPDSVKG (SEQ ID NO:334)
describes VH-CS-CDR2.2: CDR2 Consensus sequence for variable heavy chain for IgG anti-MUC1* antibodies.
TABLE-US-00313 S/TGT/DT/A--Y/FYA (SEQ ID NO:335)
describes VH-CS-CDR3.1: CDR3 Consensus sequence for variable heavy chain for IgG anti-MUC1* antibodies.
TABLE-US-00314 TGTTAILNG (SEQ ID NO:336)
describes VH-CS-CDR3.2: CDR3 Consensus sequence for variable heavy chain for IgG anti-MUC1* antibodies.
TABLE-US-00315 SGDGYWYYA (SEQ ID NO:337)
describes VH-CS-CDR3.3: CDR3 Consensus sequence for variable heavy chain for IgG anti-MUC1* antibodies.
TABLE-US-00316 DNYG--YDYG/A (SEQ ID NO:338)
describes VH-CS-CDR3.4: CDR3 Consensus sequence for variable heavy chain for IgG anti-MUC1* antibodies.
TABLE-US-00317 SASSSV/ISYM/IH/Y (SEQ ID NO:339)
describes VL-CS-CDR1.1: CDR1 Consensus sequence for variable light chain for IgG anti-MUC1* antibodies.
TABLE-US-00318 RASKSVSTSGYSYMH (SEQ ID NO:340)
describes VL-CS-CDR1.2: CDR1 Consensus sequence for variable light chain for IgG anti-MUC1* antibodies.
TABLE-US-00319 S/GTSNLAS (SEQ ID NO:341)
describes VL-CS-CDR2.1: CDR2 Consensus sequence for variable light chain for IgG anti-MUC1* antibodies.
TABLE-US-00320 LASNLES (SEQ ID NO:342)
describes VL-CS-CDR2.2: CDR2 Consensus sequence for variable light chain for IgG anti-MUC 1* antibodies.
TABLE-US-00321 QQRSS/NYPS/FT (SEQ ID NO:343)
describes VL-CS-CDR3.1: CDR3 Consensus sequence for variable light chain for IgG anti-MUC 1* antibodies.
TABLE-US-00322 QHSRELPFT (SEQ ID NO:344)
describes VL-CS-CDR3.2: CDR3 Consensus sequence for variable light chain for IgG anti-MUC 1* antibodies.
TABLE-US-00323 DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:345)
describes Framework Region 1 Human IgG1 Light Chain Amino Acid SEQ.
TABLE-US-00324 5′ gat atc cag atg acc cag tcc ccg agc tcc ctg tcc gcc tct gtg ggc gat agg gtc acc atc acc tgc cgt gcc 3′ (SEQ ID NO:346)
describes Framework Region 1 Human IgG1 Light Chain DNA SEQ.
TABLE-US-00325 WYQQKPGKAPKLLIY (SEQ ID NO:347)
describes Framework Region 2 Human IgG1 Light Chain Amino Acid SEQ.
TABLE-US-00326 5′ tgg tat caa cag aaa cca gga aaa get ccg aaa cta ctg att tac 3′ (SEQ ID NO:348)
describes Framework Region 2 Human IgG1 Light Chain DNA SEQ.
TABLE-US-00327 GVPSRFSGSRSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:349)
describes Framework Region 3 Human IgG1 Light Chain Amino Acid SEQ.
TABLE-US-00328 5′ gga gtc cct tct cgc ttc tct gga tcc aga tct ggg acg gat ttc act ctg acc atc agc agt ctg cag ccg gaa gac ttc gca atc tat tac 3′ (SEQ ID NO:350)
describes Framework Region 3 Human IgG1 Light Chain DNA SEQ.
TABLE-US-00329 FGQGTKVEIK (SEQ ID NO:351)
describes Framework Region 4 Human IgG1 Light Chain Amino Acid SEQ.
TABLE-US-00330 5′ ttc gga cag ggt acc aag gtg gag atc aaa 3′ (SEQ ID NO:352)
describes Framework Region 4 Human IgG1 Light Chain DNA SEQ.
TABLE-US-00331 EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:353)
describes Framework Region 1 Human IgG1 Heavy Chain Amino Acid SEQ.
TABLE-US-00332 5′ gag gtt cag ctg gtg gag tct ggc ggt ggc ctg gtg cag cca ggg ggc tca ctc cgt ttg tcc tgt gca gct tct 3′ (SEQ ID NO:354)
describes Framework Region 1 Human IgG1 Heavy Chain DNA SEQ.
TABLE-US-00333 WVRQAPGKGLEWVA (SEQ ID NO:355)
describes Framework Region 2 Human IgG1 Heavy Chain Amino Acid SEQ.
TABLE-US-00334 5′ tgg gtg cgt cag gcc ccg ggt aag ggc ctg gaa tgg gtt gca 3′ (SEQ ID NO:356)
describes Framework Region 2 Human IgG1 Heavy Chain DNA SEQ.
TABLE-US-00335 RFTISADTSKNTAYLQMNSLRAEDTAVYYCSR (SEQ ID NO:357)
describes▫Framework Region 3 Human IgG1 Heavy Chain Amino Acid SEQ.
TABLE-US-00336 5′ cgt ttc act ata agc gca gac aca tcc aaa aac aca gcc tac ctg cag atg aac agc ctg cgt gct gag gac act gcc gtc tat tat tgt tct aga 3′ (SEQ ID NO:358)
describes Framework Region 3 Human IgG1 Heavy Chain DNA SEQ.
TABLE-US-00337 WGQGTLVTVSS (SEQ ID NO:359)
describes Framework Region 4 Human IgG1 Heavy Chain Amino Acid SEQ.
TABLE-US-00338 5′ tgg ggt caa gga acc ctg gtc acc gtc tcc tcg 3′ (SEQ ID NO:360)
describes Framework Region 4 Human IgG1 Heavy DNA SEQ.
TABLE-US-00339 SGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSG (SEQ ID NO:361 )
describes Linker sequence amino acid.
TABLE-US-00340 EVQLVESGGGLVKPGGSLRLSCA ASGFTFS (SEQ ID NO:362)
describes IGHV3 (name from Igblast): FWR1: Human IgG antibody framework region sequence with 84.7% homology (249/294) to variable heavy chain region of MIN-C2.
TABLE-US-00341 WVRQAPGKGLEWVS (SEQ ID NO:363)
describes IGHV3 (name from Igblast): FWR2: Human IgG antibody framework region sequence with 84.7% homology (249/294) to variable heavy chain region of MIN-C2.
TABLE-US-00342 RFTISRDNAKNSLYLQMNSLRAEDTAV (SEQ ID NO:364)
describes IGHV3 (name from Igblast): FWR3: Human IgG antibody framework region sequence with 84.7% homology (249/294) to variable heavy chain region of MIN-C2.
TABLE-US-00343 DIVLTQSPASLAVSPGQRATITC (SEQ ID NO:365)
describes IGkV7 (name from Igblast): FWR1: Human IgG antibody framework region sequence with 76.4% homology (226/296) to variable light chain region of MIN-C2.
TABLE-US-00344 WYQQKPGQPPKLLIY (SEQ ID NO:366)
describes IGkV7 (name from Igblast): FWR2: Human IgG antibody framework region sequence with 76.4% homology (226/296) to variable light chain region of MIN-C2.
TABLE-US-00345 GVPARFSGSGSGTDFTLTINPVEANDTANYY (SEQ ID NO:367)
describes IGkV7 (name from Igblast): FWR▫3: Human IgG antibody framework region sequence with 76.4% homology (226/296) to variable light chain region of MIN-C2.
TABLE-US-00346 EVQLVESGGGLVKPGGSLRLSCAASGFTFS (SEQ ID NO:368)
describes IGHV3 (name from Igblast): FWR1: Human IgG antibody framework region sequence with 84.1% homology (249/296) to variable heavy chain region of MIN-E6.
TABLE-US-00347 WVRQAPGKGLEWVS (SEQ ID NO:369)
describes IGHV3 (name from Igblast): FWR2: Human IgG antibody framework region sequence with 84.1% homology (249/296) to variable heavy chain region of MIN-E6.
TABLE-US-00348 RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO:370)
describes IGHV3 (name from Igblast): FWR3: Human IgG antibody framework region sequence with 84.1% homology (249/296) to variable heavy chain region of MIN-E6.
TABLE-US-00349 EIVMTQSPATLSVSPGERATLSC (SEQ ID NO:371)
describes IGkV3 (name from Igblast): FWR1: Human IgG antibody framework region sequence with 69.5%▫homology▫(187/269)▫to▫variable light chain region of MIN-E6.
TABLE-US-00350 WFQQRPGTSPK LLIY (SEQ ID NO:372)
describes IGkV3 (name from Igblast): FWR2: Human IgG antibody framework region sequence with 69.5% homology (187/269) to variable light chain region of MIN-E6.
TABLE-US-00351 GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC (SEQ ID NO:373)
describes IGkV3 (name from Igblast): FWR3: Human IgG antibody framework region sequence with 69.5% homology (187/269) to variable light chain region of MIN-E6.
Monoclonal Antibody
[0063] Cloning monoclonal antibodies is useful for a variety of reasons. Hybridoma cells that produce a single antibody provide a method for generating large numbers of antibodies that are identical and exert identical effect. This is useful for antibody destined for therapeutic uses. Determining the sequences of monoclonal antibodies, especially of the variable regions of the light and heavy chains are particularly useful because they enable many forms of protein engineering that will allow, for example, the generation of antibody variants like single chain antibodies (e.g., scFv) or recombinant monovalent antibodies (e.g., Fabs). Determining the sequence of monoclonals, and in particular of the variable regions, importantly enables the generation of “humanized” or partially humanized (chimeric) antibodies that are sometimes preferred for human therapies because they are better able to avoid the host immune system by appearing human.
[0064] There are many methods for generating monoclonal antibodies and for generating antibody variants that are disclosed here, e.g., single chain antibodies, bispecific antibody, recombinant Fabs, humanized and particularly humanized antibodies and the like. Methods described here are meant to be exemplary and the invention is directed to monoclonal antibodies and derivations of those antibodies generated by other methods will have the same effects as those produced by the methods described herein.
[0065] Monoclonal anti-MUC1* antibodies were produced and screened as described below. Mice were immunized with synthetic peptides corresponding to the extracellular domain of MUC1* (GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO:1)) or a peptide variant containing a single amino acid substitution (GTINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGA (SEQ ID NO:2)). Hybridomas were generated, according to standard practice in the field. Individual hybridomas that produced anti-MUC1* antibodies were identified by virtue of their ability to bind to MUC1* peptide in an ELISA assay. Supernatants from hybridomas, identified in the previous selection round, were then added to MUC1*-positive as well as MUC1-negative cells and FACS was used to identify those hybridomas that secreted antibodies capable of recognizing MUC1* on live cells. Larger quantities of the identified MUC1* cognate antibodies were then obtained by either injecting mice with the hybridomas (ascites) or produced by large scale culture according to standard methods. The resultant antibodies were assayed as previously described. In addition, antibody clones were tested for their ability to stimulate the growth of MUC1*-positive cells by binding to and dimerizing the extracellular domain of MUC1*. Antibodies that effectively stimulated the growth of MUC1*-positive cells (pluripotent stem cells, cancer cells and transfected cells) were then biochemically cleaved to generate monovalent antibodies which should block MUC1* dimerization and thus inhibit the growth of MUC1* expressing cells. In some instances, antibodies were papain digested to generate monovalent Fabs, which were assayed for their ability to inhibit the growth of MUC1*-positive cells. In some cases, the Fab was PEGylated according to published methods and it was observed that pegylation increased the stability of the antibody fragments. The sequences of the heavy and light chains of the selected antibodies were obtained by standard methods and various constructs for making bivalent and monovalent MUC1* antibodies and antibody derivatives were made.
[0066] Single chain constructs that contain the variable regions of both heavy and light chain connected by a linker are monovalent and therefore are useful for the inhibition of MUC1*-mediated cell growth. Portions of the constant regions may also be incorporated to facilitate the recruitment of complement (ADCC) and methods for engineering such constructs are known to those skilled in the art. To discourage dimerization of the constant regions, which would result in a bivalent antibody derivative, mutations such as Cyteine replacements and the like could be incorporated. Pegylation is a commonly used method for extending the half-life of monovalent antibody fragments and was used to increase the stability of the Fabs and single chain antibodies described herein.
[0067] The anti-MUC1* antibodies of the invention may be single-chain variable fragment antibodies (scFv). Recombinant approaches have led to the development of single chain variable fragment antibody (scFv). A monomeric scFv has a molecular mass of only about 30 kDa, which is expressed in a variety of systems as a single VL-VH pair linked by a Gly/Ser-rich synthetic linker (Berezov A. et al., 2001, J Med Chem 44:2565). When expressed in bacteria or eukaryotic cells, the scFv folds into a conformation similar to the corresponding region of the parental antibody. It was shown to retain comparable affinity to that of a Fab (Kortt et al., 1994, Eur J Biochem 221:151). ScFvs are amenable to various genetic modifications such as humanization and the production of fusion proteins to enhance their potential as therapeutic agents.
[0068] Phage display method may be used to produce anti-MUC1* scFv. In this method, large repertoires of antibody variable region cDNAs are collected from the B cells and combinations of VHs and VLs are expressed in the form of scFvs on the surface of filamentous bacteriophage. The phages that express scFvs are to be panned from antigen-coated plates. The affinity of the anti-MUC1* scFv may be improved by mutating the CDRs of the construct and then repeating the panning procedure.
[0069] The anti-MUC1* antibodies of the invention may be Fab, Fab2 bispecific antibodies, Fab3 trispecific antibodies, bivalent minibody, trivalent triabody, or tetravalent tetrabodies.
[0070] One of the uses of a monovalent anti-MUC1* antibody is for the treatment of cancer cells that often overexpress MUC1*. To avoid the inhibition of non-cancerous cells that also present MUC1*, bispecific antibody may be made wherein one portion of the hybrid antibody binds to and blocks MUC1 * while the other binds to another tumor-specific, or similar, antigen. For example, one portion would recognize MUC1*, while the other would bind to HER2, CEA, transferrin, EGFR, TF (tissue factor), DLLs (delta-like ligand), DLL-4, jagged, notch receptor ligands, portions of the notch receptor, and the like. The increased avidity of cooperative binding over monomeric binding would preferentially retain the antibodies on the targeted tumor cells.
[0071] The anti-MUC1* antibodies of the invention may be bispecific antibodies. Bispecific antibodies are monoclonal antibodies, preferably human or humanized antibodies that have dual-targeting specificities. Bispecific antibodies are derived from the recombination of variable domains of two antibodies with different specificities; bispecific antibodies are thus capable of binding both antigens of their parental antibodies. In the case of anti-MUC1*, one of the binding specificities could be for MUC1* and the other may be for another protein, or any other cell surface protein, for example. These bispecific anti-MUC1* antibodies may function as antagonistic or agonistic antibodies.
[0072] Methods for making bispecific antibodies are well known (Traunecker et al., EMBO J, 1991, 10:3655; WO 93/08829; Suresh et al., Methods in Enzmology, 1986, 121:210; Milstein and Cuello, 1983, Nature, 305:537). Briefly, antibody variable domains with the desired binding specificities are fused to immunoglobulin constant domain. This fusion contains an immunoglobulin heavy-chain constant domain (part of the hinge, CH2 and CH3 regions) and preferably contains the first heavy chain constant region (CH1). DNAs encoding the immunoglobulin heavy chain fusions and the immunoglobulin light chain are inserted into separate expression vectors and are cotransfected.
[0073] In the case of MUC1*, a bispecific antibody would act as a cell growth inhibitor by blocking dimerization of the MUC1* ligand binding site. If one variable region recognized MUC1* and the other recognized another tumor-specific antigen, then unwanted side effects of inhibiting MUC1*-mediated growth would be avoided. The bispecific antibody would preferentially bind to tumor cells which displayed both antigens. Such an approach has been successful with a HER2-HER3 bispecific antibody that has an enhanced specificity and anti-tumor effect (Robinson, et al. 2008), as well as an EGFR-IGFR bispecific antibody that blocks signaling from both receptors (Lu, et al. 2004). Also, the second “arm” of the bispecific antibody can target, recruit and engage cells of the immune system, such as CD3 to recruit T cells (Baeuerle and Reinhardt, 2009, Bortoletto, et al 2002), or CD16, to recruit Neutrophils, Natural Killer cells, or other monocytes (Bruenke, et al. 2005, McCall, et al. 1999). Both arms of a therapeutic antibody can be cloned in tandem as a single chain in multiple formats, such as tandem scFv molecules and the similar tandem diabodies (Chames and Baty 2009, Holliger P, 1993). Fab fragments from different antibodies can also be chemically conjugated to form bispecific Fabs (Chames and Baty 2009).
[0074] Conversely, there are many instances in which it is desirable to enhance the growth of MUC1 *-positive cells. For example, pluripotent stem cells and some progenitor cells express MUC1*. Bivalent MUC1*-antibodies dimerize the MUC1* receptor on the primitive cells and enhance cell growth while maintaining the cells in a pluripotent state. Bivalent anti-MUC1* antibodies may be added exogenously or genetically added to the cells. For example, the gene that encodes a bivalent anti-MUC1* antibodies is added to primitive cells, even at times before egg and sperm fusion is complete. In this way, the resultant stem cell and its progeny will produce and secrete the ligand that stimulates its unlimited proliferation. Alternatively, a plasmid encoding the antibody is transfected into primitive cells to maintain their pluripotency by activating the MUC1* receptor or rescue stem cells that have entered the differentiation process.
[0075] Similarly, pluripotency is induced in cells by the introduction of a gene encoding MUC1*, which may be introduced alone or in combination with other genes, including those that enhance signaling through MUC1* such as genes that encode MUC1 cleavage enzymes like MMP-14 and TACE, or genes that encode NM23, the natural ligand of MUC1*.
[0076] Bivalent MUC1* antibodies and antibody derivatives are administered to patients suffering from the effects of chemotherapy and other conditions in which it is desirable to enhance proliferation of hematopoietic stem cells and precursor cells. In these instances, the patient may be administered systemically or via local injection. Various forms of stem cell transplant therapy would benefit from in situ injection of MUC1*-stimulating antibodies to establish the vitality of the transplanted cell population before the onset of differentiation.
[0077] In yet another aspect of the invention, there are many instances in which it is desirable to command the anti-apoptotic properties of MUC1* expression and growth stimulating signaling. One such example is in cases is sepsis when agents that support cell survival would stave off the disastrous effects of the condition. Transient, systemic treatment with a bivalent MUC1* antibody or antibody derivative would enhance cell survival and in doing so control the life-threatening symptoms until suitable antibiotics or other curative treatments could be administered or have time to take effect. Similarly, virulent strains of bacteria ravage areas of flesh and induce massive cell death through toxic effects. Here also, administering MUC1*-stimulating antibodies would render the cells more resistant to cell death and provide caretakers with more time to control the infection. Thus, the MUC1*-stimulating antibody may be administered locally or systemically.
[0078] Other situations lend themselves to MUC1*-mediated growth stimulation. For example, animals that spontaneously produce tumors or produce tumors that grow faster could be generated via methods for making transgenic animals and in these cases, the animals would be generated such that their cells would make and secrete the MUC1* stimulating antibody. The gene for the MUC1*-stimulating antibody in this case, is positioned downstream of a tissue-specific promoter. For example, to generate a mouse that spontaneously produced breast tumors the gene for the MUC1* antibody ligand would be placed downstream of a promoter for a mammary specific protein. The gene for the MUC1* antibody that is inserted downstream of a tissue specific promoter is injected into a pronucleus or similar just prior to the complete fusion of egg and sperm to generate a transgenic animal, e.g., a mouse model, that spontaneously forms tumors or enhances tumor formation. The antibody gene may be injected separately or in combination with other genes that enhance MUC1 * cleavage, or signaling, including but not limited to MMP-14, TACE, NM23 and the like.
[0079] In another aspect, a plasmid encoding a MUC1* antibody is added exogenously or transfected into an antibody-producing hybridoma in order to increase antibody production by increasing cell growth rate and rendering the cells resistant to cell death through the stimulation of MUC1*. In this aspect of the invention, the growth of antibody-producing cells is enhanced without contaminating the product which is the desired antibody. Antibody-producing cells are typically grown in media that contains reduced serum because the serum component contains many antibodies itself. Reducing the amount of serum in the cell culture media minimizes the carry over contamination but also reduces cell growth and overall yield of the desired antibody. The addition of exogenous anti-MUC1* or the transfection of the plasmid that codes for the anti-MUC1* increases cell growth and survival, while the single, known antibody can be selectively purified away from the product which is the desired antibody.
[0080] Having the sequence of a monoclonal antibody also allows one to make fusion proteins wherein one part is derived from the antibody and the other part may be derived from another protein, such as a toxin, a cytokine, like IL-2 and others, or a protein, like GFP (green fluorescent protein) that can act as a label. Antibodies and antibody variants of the invention can be biochemically fused to, or genetically engineered to be fused to labels, tags, toxins, radioactive substances, targeting motifs, leader sequence peptides, toxic materials, proteins or peptides. Similarly, unnatural amino acids can be incorporated into a recombinant antibody or antibody derivative wherein the unnatural amino acid may facilitate coupling, may have a signaling capability, may render the recombinant protein protease sensitive or insensitive, and the like.
[0081] The invention also provides for humanization or partial humanization of some or all of the anti-MUC1* antibodies and antibody variants described here. Cloning monoclonal antibodies and determining their sequences, especially of the variable regions of the light and heavy chains, enables the generation of a humanized and partially humanized (chimeric) antibodies. As is known in the art, humanized and chimeric antibodies are characterized by fewer side effects and higher efficacy because humanized antibodies are better able to evade detection by the human immune system (Lonberg 2005).
[0082] As those skilled in the art are familiar, a common method for generating a humanized antibody involves exchanging non-human sequences in non-recognition areas such as the framework regions (FWRs), for human sequences as described in Muzard, et al. 2009. Similarly, the recognition regions like the complementarity determining regions (CDRs) of a non-human monoclonal antibody are exchanged for the CDRs of a human antibody (Carter P, et al. Humanization of an anti-p185.sup.HER2 antibody for human cancer therapy Proc. Natl Acad. Sci 89:4285-4289). Human framework regions such as those listed in SEQ ID NOS:345-360 are human framework regions that could be exchanged for the mouse FWRs in the monoclonal sequences disclosed here. Searches of the data bases for human antibody sequences are also used to identify human antibodies that are homologous to a desirable monoclonal antibody from a non-human source. For example, the amino acid sequences of SEQ ID NOS:362-367 are framework regions (FWRs) from a human antibody that is highly homologous to MIN-C2. These sequences or the sequences can be used in conjunction with the CDRs of MIN-C2 (SEQ ID NOS:331, 334, 374, 340, 342, and 344).
[0083] In another example, the amino acid sequences shown as SEQ ID NOS:368-373 are framework regions (FWRs) from a human antibody that is highly homologous to MIN-E6. These sequences can be used in conjunction with the CDRs of MIN-E6 (SEQ ID NOS:332, 334, 338, 339, 341, and 343). The nucleic acids that encode these amino acid sequences may be used to genetically engineer such chimeric, partially humanized antibodies and antibody variants.
[0084] To replace the murine framework regions with the chosen human framework regions for making a chimeric antibody construct, a combination of synthetic DNA, and DNA generated by overlap PCR is used. For constructing humanized MIN-C2, human framework region nucleotide sequences that encode amino acid sequences of SEQ ID NOS:362, 363 and 364 are used to replace the murine heavy chain framework regions bearing the SEQ ID NOS: 159, 185 and 211 respectively. Further, nucleotide sequences that encode amino acid sequences of SEQ ID NOS:365, 366 and 367 are used for replacing the MIN-C2 light chain framework regions bearing the SEQ ID NOS:98, 119 and 139 respectively. For constructing humanized MIN-E6, human framework region nucleotide sequences that encode amino acid sequences of SEQ ID NOS:368, 369 and 370 are used to replace the heavy chain framework regions bearing the SEQ ID NOS:160, 186 and 212 respectively. Further, nucleotide sequences that encode amino acid sequences of SEQ ID NOS:371, 372 and 373 are used for the replacing the MIN-E6 light chain framework regions bearing the SEQ ID NOS:99, 120 and 140 respectively. Constructs of many designs including but not limited to scFab, recombinant Fabs, bispecific recombinant antibodies and scFvs are generated using these methods.
[0085] The anti-MUC1* antibodies and antibody variants disclosed herein can be humanized in this way. Following the humanization process, antibody affinity can be improved by a number of methods including by using phage display methods. After humanizing antibodies and antibody variants, some will lose affinity and require processes of “affinity maturation”. One method involves molecular evolution accomplished by mutagenesis followed by selection of those that show enhanced affinity for their cognate antigen (Razai, et al. 2005). Alternatively, humanized antibodies or antibody fragments can be isolated from recombinant libraries (Nahary and Benhar, 2009, Rothe C, et al.) Another method of obtaining fully humanized antibodies is to immunize transgenic mice engineered to produce human antibodies (Jakobovitz, et al. 2007, Lonberg, 2005). The present invention anticipates the use of some or all of these methods along with the sequences of the antibody regions (CDRs) that recognize MUC1* to generate antibodies that bind to the PSMGFR sequence (SEQ ID NO:1) for in vitro as well as in vivo and therapeutic uses.
[0086] Razai, et al report that after affinity maturation, also known as molecular evolution, 5 out of 60 total CDR residues were mutated, which corresponds to about an 8% rate of CDR mutagenesis. This means that that after affinity maturation about 92% of the CDR regions remain unchanged. Others have reported similar percent change after affinity maturation (Juárez-González, et al 2005; and Finlay, et al (2009).
[0087] Production of any of the novel antibodies and antibody variants disclosed herein can be carried out in a wide variety of cells from different organisms, including but not limited to bacteria, such as E. coli, with bacterial vectors (Zheng, et al.2009), yeast cells, such as Pichia pastoris with yeast vectors (Schoonooghe, et al. 2009), insect cells, such as S2 cells using insect vectors (Johannson, et al.), mammalian cells, such as Chinese hamster ovary cells (CHO), using mammalian vectors (Majors, et al. 2009) and the like. Vectors and cells available for antibody synthesis are numerous and well known to those skilled in the art.
[0088] In another embodiment, antibodies and antibody variants of the invention are produced by host animals. One can make transgenic animals that, for example, make a pre-determined antibody and secreted it in their milk (Zhang, et al. 2009) or other bodily fluid. Other scenarios for manipulating methods of protein engineering to develop novel and useful antibodies and antibody derivatives are possible using the sequences of the monoclonal antibodies disclosed herein.
[0089] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. The following examples are offered by way of illustration of the present invention, and not by way of limitation.
EXAMPLES
Example 1 - Generation of Anti-MUC1* Monoclonal Antibodies
[0090] Monoclonal antibodies were generated to MUC1* peptide using standard hybridoma generation and subsequent screening, which is familiar to those skilled in the art. A MUC1* peptide (GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA, SEQ ID NO:1) or peptide variant bearing a single amino acid substitution (GTINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGA, SEQ ID NO:2) was synthesized with an additional cysteine residue at the Carboxy-terminal end to allow conjugation to KLH. The conjugated peptide was used to immunize mice. Supernatants from wells containing fused cells were screened for recognition of the MUC1* peptide by ELISA. Out of these, several clones were selected based on their high titer for their ability to recognize MUC1* peptide in ELISA binding assay. Results of such binding of supernatants from six (6) hybridoma clones are shown in
Example 2 - Analysis of Binding of Anti-MUC1* Monoclonal Antibodies to MUC1* on the Cell Surface
[0091] Recognition of MUC1* on the cell surface was analyzed by surface staining of the cells using FACS. For MIN-C2 antibody 50 .Math.l of a 10 .Math.g/ml solution of purified antibody was bound individually to MUC1-negative HCT116 cells transfected with empty vector (HCT116-VEC8) or transfected with MUC1* expressing vector (HCT-MUC1*-10) and MUC1 positive ZR-75-1 cells at 4° C. for 30 minutes. Cells were washed twice and treated with 10 .Math.g/ml anti-mouse-PE at 4° C. for 30 minutes. Cells were washed twice, fixed in 2% formaldehyde in PBS, and analyzed using a BD FACS Cantoll flow cytometer (
Example 3 - Anti-MUC1* Monoclonal Antibodies (bivalent) MIN-C2 and MIN-E6 Induce Proliferation of MUC1 Expressing Breast Cancer Cell Line
[0092] To measure the ability of anti-MUC1* monoclonal antibodies, MUC1 expressing breast cancer cell line T47D was used. 7,500 cells were plated per well of a 96 well plate in media containing 10% serum. The following day cells in three wells were trypsinized, resuspended in media and counted using a hemocytometer to obtain the zero-day count. Media in the cells was changed to that containing 3% FBS and anti-MUC1* antibody was added in various concentrations. 48 hours later media was changed which was followed by a second addition of antibody. After another 48 hours the cells were counted, and stimulation of cell growth estimated (
Example 4 - Fab Fragment Generation and PEGylation
[0093] To prepare a monovalent Fab fragment of MUC1* antibody, a papain digestion method was employed (Pierce). MIN-C2 and MIN-E6 antibody were effectively digested with papain and purified through Protein A-agarose affinity chromatography (
[0094] Those Fab fragments were tested along with their parental mAb in a competitive ELISA assay to confirm that the Fab fragment is capable of binding to its antigen, MUC1* peptide. As shown in
Example 5 - Testing for the Effect of the Monovalent MIN-C2 and MIN-E6 Fab Fragments for Inhibition of Proliferation of Cells Overexpressing MUC1*
[0095] MUC1* expressing breast cancer cell line T47D was plated in 96 well plates (5,000 cells/well) in complete media (RPMI containing 10% FBS) and allowed to attach overnight. Next day the media was changed to that containing 3% FBS. Cells in three wells were counted following trypsinization and resuspension using hemocytometer to obtain the zero-day count. Next, MIN-C2 or MIN-E6 Fab protein was added in various concentrations to individual plates. After 48 hours the media was replaced, and the Fab protein was added for a second time. After a further 48 hours the cells in all the wells were counted. Inhibition of cell growth was calculated relative to the growth of cells containing no Fab protein. In the case of MIN-E6 a PEGylated form of the protein was also tested (
Example 6 - Construction and Expression of MUC1* Binding Polypeptides as Single Chain Fv Fragments Using Polypeptide Linker
[0096] A scFv fragment of MIN-C2 (a monoclonal antibody that recognizes a 45 amino acid membrane proximal extracellular region of MUC1 from amino acid 1110 to 1155: PSMGFR) was designed, constructed and expressed in E. coli. The construct was designed without any secretion signal using the vector pET21b (Novagen) that results in a protein with a carboxy-terminal tag containing six histidine residues. The design of the construct of MIN-C2 and MIN-E6 scFv are shown in
[0097] To sequence the monoclonal antibodies, hybridoma cells producing a monoclonal antibody were grown in RPMI media containing G418 as suggested. Then, total RNA was prepared using Trizol reagent and its first strand cDNA was generated using SuperScript III first strand cDNA synthesis kit (Invitrogen, CA). To clone the variable region of heavy and light chains, degenerate primers were used according to the literature (Wang et al., 2000). For heavy chain cloning, PCR was set up using SEQ ID NOS:3-5 (reverse) with SEQ ID NO:6 or SEQ ID NO:7 (forward). For kappa chain, SEQ ID NO:8 (reverse) with SEQ ID NO:9 (forward) were used. All forward and reverse PCR primers contain EcoRI and HindIII restriction site for heavy and SacI and SalII sites for kappa chains for downstream cloning purpose. The PCR condition is as follows: 94° C. for 1 min, 45° C. for 1 min, 72° C. for 2 min with 30 cycles, and 72° C. for 10 min. Then, the PCR products were run on 1% agarose gels and a prominent PCR band .sup.~500 bp in size for heavy and light chains were detected.
[0098] The products were purified from the agarose gel and restriction digested with corresponding enzymes and then cloned into pET21b bacterial expression vector for sequencing and simple bacterial expression to validate its full-length expression.
[0099] Plasmid DNA was sequenced. DNA and amino acid sequences for the variable regions (CDRs: complementarity determining regions) for preferred monoclonal antibody MIN-C2 are given in SEQ ID NOS: 10 and 11 for the variable heavy chain, VH, and SEQ ID NOS: 12 and 13 for the variable light or kappa chains, VL. The DNA and amino acid sequences of the constant regions of both the heavy and light chains of MIN-C2 were also determined and are shown as SEQ ID NOS:49 and 50. Recombinant antibody fragments that have greater stability and binding affinity are generated by including these constant regions into the design. A recombinant Fab is comprised of the variable regions of the heavy (VH) and light (VL) chains plus the constant regions (CH1 and CL).
[0100] DNA and amino acid sequences for another preferred monoclonal antibody MIN-E6 were similarly determined and are given in SEQ ID NOS: 19-22 for the heavy chain and SEQ ID NOS:23-24 for the light or kappa chain. The sequences of other monoclonal anti-MUC1* antibodies are shown in
[0101] To generate a single chain antibody fragment (scFv) comprised essentially of the variable regions of a monoclonal antibody, a linker sequence (SEQ ID NO: 14) with 15 amino acids (Gly-Gly-Gly-Gly-Ser)3 was introduced in between heavy and kappa variable sequences using standard PCR. scFvs of MIN-C2 and MIN-E6 were generated. The sequence of the MIN-C2 scFv generating final heavy-linker-kappa construct is shown as (SEQ ID NOS: 15 and 17). A variant of MIN-C2 scFv having a carboxyl-terminal cysteine was also generated: MIN-C2 scFv-Cys (SEQ ID NOS: 16 and 18).
[0102] A single chain antibody fragment (scFv) form of monoclonal antibody MIN-E6 and an scFv-Cys variant were similarly generated. The DNA and amino acid sequences of the resultant MIN-E6 scFv and MIN-E6 scFv-Cys are shown as SEQ ID NO:25 and 27 and SEQ ID NOS:26 and 28 (Cys). The assembled DNA containing the heavy and light chain variable region sequences joined by a linker was digested with restriction enzymes and cloned into the pET-21b bacterial expression vector and used to transform bacterial cultures.
[0103] Next, bacterial cultures were induced by adding IPTG (isopropyl thio galactoside) to a concentration of 1 mM and allowing induction to proceed for 8 hours. Tests showed that all of the scFv protein was localized in the inclusion bodies. The scFv protein for MIN-C2, MIN-E6-7 was purified by solubilization of the E. coli inclusion body fraction with 8 M urea followed by Ni-NTA column chromatography performed according to standard procedures. SDS-PAGE analysis of the eluted protein showed a single species (approximately 95% purity) with the expected size of about 28 kDa. The concentration of the eluted protein was determined by measuring absorbance at 280 nm. The protein was diluted to 150 ug/ml with binding buffer (0.02 M Tris. HC1 pH 8.0, 0.5 M NaCl, 8 M urea, 5 mM Imidazole). To the protein was added GSH and GSSG to a final concentration of 5 mM and 0.5 mM respectively and stirred for 16 hours at 4° C. The eluted protein was then subjected to a refolding procedure. Refolding was done by transferring the protein into a dialysis membrane and dialyzing against 0.4 M L-arginine containing buffer (0.05 M Tris. HC1 pH8.0, 0.4 M L-arginine). Final dialysis was done in 50 mM pH 8.0 and 5% glycerol. The protein was removed from dialysis membrane and spun down to remove precipitate and concentrated using centrifugal filtration with 10,000 MW cutoff membrane (Amicon Ultra, Millipore). A portion of the protein was further purified by affinity purification column. The recombinant scFv of MIN-C2 was expressed, purified (
Example 7 - Binding of the scFv to Target MUC1* Peptide and Its Ability to Compete With Parent MIN-C2 Antibody
[0104] MUC1* peptide was bound to the wells of a 96 well ELISA plate by overnight incubation. This was followed by blocking with superblock (Pierce) also by overnight incubation. Next, the superblock was removed and the wells were blocked with DMEM media containing 3% FBS for 30 minutes. The scFv protein was added in various concentrations using eight serial dilutions with the highest concentration being 50 ug/ml. To determine nonspecific binding of the scFv protein, the protein was added in same dilutions to another set of wells not containing bound MUC1* peptide. The incubation was continued for 1 hour. Next, the wells were washed thrice with 0.3 ml PBST (phosphate buffered saline containing 0.02% Tween 20). This was followed by addition of rabbit anti-His antibody (AbCam) in a dilution of 1:40,000 in a media prepared by a 100-fold dilution of DMEM containing 3% FBS. This was allowed to incubate for 1 hour followed by three washes with 0.3 ml pf PBST. Next 0.1 ml of the substrate tetra methyl benzidine was added to the wells and color was allowed to develop for 15 minutes in the dark, following which 0.1 ml of 2 N HCl was added. These experiments confirmed that the recombinant scFv effectively competed with the intact, parent antibody for binding to the PSMGFR (MUC1* extracellular domain) peptide.
Example 8 - Cell Surface Binding of MIN-C2 scFv to MUC1 Expressing Breast Cancer Cells
[0105] Breast cancer cell line ZR-75-1 was used to test the ability of the MIN-C2 scFv to recognize MUC1* on cell surface. Cells were incubated with 1:2 or 1:10 dilutions of 1.5 ug/ml scFv stock or without any scFv protein as control for 30 minutes at 4° C. After two washes the cells were incubated with the secondary antibody anti-penta-His conjugated to Alexa 488 (Qiagen) at dilutions of 1:200, 1:50 or 1:10 to detect the 6X Histidine tag on the scFv. Flow cytometric analysis revealed a concentration-dependent shift of a subset of cells indicating specific binding which is not seen in the absence of MIN-C2 scFv. (
Example 9 - Testing for the Effect of the MIN-C2 scFv Protein on Proliferation of Cancer Cells Overexpressing MUC1*
[0106] Because the MUC1* receptor stimulates cell growth when it is dimerized by its activating ligand, monomeric antibodies and antibody fragments are expected to inhibit the growth of MUC1*-positive cells. MUC1* expressing breast cancer cell line ZR-75-1 (aka 1500) was plated in 96 well plates (10,000 cells/well) in complete media (RPMI containing 10% FBS) and allowed to attach overnight. Next day the media was changed to that containing 3% FBs. As a control another cell line HCT116, colon carcinoma derived that does not express MUC1* was also plated (2,000 cells/well) in complete media (DMEM containing 10% FBS), allowed to attach overnight and media changed to that contained 3% FBS. For each cell line, cells in three wells were counted following trypsinization and resuspension using hemocytometer to obtain the zero-day count. Next, The MIN-C2 scFv antibody variant was added in various concentrations. After 48 hours the media was replaced and scFv protein was added for the second time. After a further 48 hours the cells in all the wells are counted. Inhibition of cell growth was calculated relative to the growth of cells containing no scFv protein. It was observed that while the scFv protein inhibited the growth of the MUC1* expressing ZR-75-1 cells, they did not have this inhibitory effect on the control cell line HCT 116 that does not express MUC1 * (
Example 10 - Construction and Expression of Recombinant Fab Antibody
[0107] Recombinant Fabs were also generated. In some circumstances, Fabs are preferred over scFv designs. Compared to the scFv, a recombinant Fab has a longer half-life in serum and its binding affinity is usually comparable to those of the whole parent antibody. Expression in mammalian systems provides enhanced activity via an increase in the amount of protein that is properly folded. Antibody variants produced from mammalian cells are generated on a large scale. To produce the recombinant antibody for mammalian expression, variable plus constant regions were cloned from monoclonal antibody producing hybridoma cells. Recombinant Fabs are produced using a number of antibody designs. They are essentially comprised of the VH, VL, CH and CL regions. In some cases, a linker is used to join heavy chain to light chain. In other cases, heavy and light chains are carried on separate vectors and expressed in the same cell where they self-associate to form a functional Fab. Fabs of both designs were made.
[0108] Hybridoma cells (MIN-C2 and MIN-E6 clones) producing monoclonal MUC1* antibody were grown in RPMI media containing G418 according to standard practice. Then, total RNA was prepared using Trizol reagent and first strand cDNA was generated using SuperScript III first strand cDNA synthesis kit (Invitrogen, CA). To clone heavy and light chains containing its own secretion signal sequence, degenerate PCR primers were used according to the literatures (Morrison, 2002; Kettleborough, 1993). For heavy chain cloning, PCR was set up using SEQ ID NOS:33, 34, 35, or 36 (forward) with 37 (reverse). For kappa chain, SEQ ID NOS:38, 39, 40, 41, 42 or 43 (forward) with 44 (reverse) were used. All forward and reverse PCR primers contain EcoRI and XhoI restriction site, respectively, for downstream cloning purposes. The PCR condition used was as follows: 94° C. for 60 sec, 55° C. for 60 sec, 72° C. for 60 sec with 30 cycles, and 72° C. for 10 min. Then, the PCR products were run on 1% agarose gels to determine which primers generated the desired product.
[0109] The products were purified from the agarose gel and restriction digested with corresponding enzymes and then cloned into pET21b bacterial expression vector for sequencing and simple bacterial expression to validate its full-length expression. The plasmid DNA was sequenced and was found to be identical to that of the MIN-C2 scFv variant that we generated, confirming that even though different primers and different enzymatic digestion was performed, the resultant variants bear the same variable regions from the parent monoclonal antibody. The heavy (SEQ ID NOS:45 and 47) and light (SEQ ID NOS:46 and 48) chain sequences for the recombinant MIN-C2 Fab are given.
[0110] The expression of the individual Fab constructs in bacteria was tested. BL21 transformant harboring pET21b-heavy or -light chain was inoculated into 5 ml LB media containing carbenicillin (100 .Math.g/ml) and incubated overnight at 37° C. Then, 50 ml of LB media containing carbenicillin (100 .Math.g/ml) was inoculated with overnight bacterial culture (2.5 ml) and further incubated at 30° C. until its OD600 nm reached 0.5. The rest of the culture was spun down and frozen. IPTG was added into the 50 ml culture in final concentration of 1 mM. The culture was further incubated for 5 hours and then pelleted down. Then, bacterial pellets (before and after IPTG induction) were analyzed on SDS-PAGE and Western blotting. A prominent ~25 kDa band corresponding to the heavy or light chain insert was produced, suggesting that those heavy and light chains were properly expressed (
[0111] Recombinant Fabs generated as described above function essentially the same as the Fabs that were enzymatically cleaved from the intact parent antibody.
[0112] For mammalian recombinant protein expression, those heavy and light inserts were cloned into mammalian expression vectors, pOptiVec and pcDNA3.3 using TOPO cloning kits (Invitrogen, CA).
Example 11 - Generation of Recombinant Anti-MUC1* Single Chain Fab (scFab)
[0113] To further increase the efficiency of refolding and ultimately to increase the specific activity of the antibody variant, single chain Fabs (scFab) were generated. Recombinant single chain Fab constructs corresponding to anti-MUC1* antibodies MIN-C2 and MIN-E6 are generated using the design shown in
[0114] After correct assembly of the light chain, the heavy chain fragment and the linker, the DNA is cloned in frame into a mammalian expression vector (psecTag, Invitrogen, Carlsbad Calif.). This results in the in-frame addition of a N-terminal Ig-k leader sequence and C-terminal myc and polyhistidine tag to facilitate purification. From this construct the DNA fragment which includes the Ig-k leader sequence, and the purification tags is subcloned via PCR and by using appropriate restriction sites into another expression vector pCEP4 which allows high-copy replication of the plasmid DNA and uses the high expression CMV promoter. Construct thus generated is used for transient expression in mammalian cells. For this purpose, human embryonic kidney cells (HEK-293) adapted to grow in suspension culture (Invitrogen, Carlsbad Calif.) is used and the secreted Fab is purified using anti-myc tag affinity chromatography.
Example 12 - Generation of a Humanized or Chimeric (Framework Regions Are Human and CDRs Are Not) Anti-MUC1* Antibody
[0115] The anti-MUC1* monoclonal antibodies of the invention may be humanized monoclonal antibodies or human monoclonal antibodies. An entirely antigenic murine mAb becomes human friendly when small parts of the murine antibodies are engrafted onto human immunoglobulin molecules creating either chimeric antibodies where only the Fc part of the immunoglobulin molecule is human, or humanized antibodies where only the complementarity determining regions (CDR) of the immunoglobulin are murine and 90 to 95% of the molecule is human. In one respect, fully human monoclonal antibodies may be generated in transgenic mice by employing conventional methods such as HuMAb-Mouse (GenPharm-Medarex) or XenoMouse (Abgenix, Inc.) technology. Humanized antibodies include human immunoglobulins in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species such as mouse, rat or rabbit having the desired specificity, affinity and biological function.
[0116] Human antibodies also can be produced using techniques such as phage display libraries (Hoogenboom and Winter, J. Mol. Biol, 1991, 227:381, Marks et al., J. Mol. Biol. 1991, 222:581). Methods for humanizing non-human antibodies are well known. Humanization can be performed following the method of Winter et al. as disclosed in Jones et al., Nature, 1986, 321:522; Riechmann et al., Nature, 1988, 332:323; and Verhoeyen et al., Science, 1988, 239:1534 by substituting rodent CDR sequences or CDRs for the corresponding sequences of a human antibody. Such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567). Typically, humanized antibodies are antibodies where CDR residues are substituted by residues from analogous sites in rodent antibodies.
[0117] Therapeutic antibodies derived from non-human species may in some cases trigger immune responses when injected into humans, thereby limiting their utility. Therefore, humanization or partial humanization of antibodies for therapeutic use in humans is preferred. There are many methods for generating humanized or partially humanized antibodies and antibody variants. The present invention is directed to the use of any of these processes is suitable for humanizing compositions of the invention. In one process, a mouse having human antibody loci is used to generate fully humanized antibodies (Jakobovits et al.). In another method, DNA from B cells from humans are selected for binding to a selected target, then re-inserted into the context of the full antibody.
[0118] In a common method, the framework regions of an antibody generated in a non-human host are exchanged for human framework regions, preserving the original CDR sequences. These are called chimeric antibodies. In this process only the six complementary determining regions (CDRs) three each forming the heavy and light chains of the murine antibody are retained and the sequences of the framework regions are substituted for those from a closely related human antibody (method described in Muzard et al., 2009). First, homology searches are performed to independently align the VH and VL amino acid sequences of anti-MUC1* antibody (MIN-C2 or MIN-E6) against a repertoire of human antibody sequences registered in protein Data Bank. Among the variable region sequences derived from different human antibodies that best match the anti-MUC1* VH and VL, that particular antibody sequence is chosen which has the highest combined homology to anti-MUC1* VH and VL sequences. This is necessary in order to preserve the inter domain contacts that occur in a natural antibody. Optionally, sequence identity in the range of 65-70% when calculated only over the framework region are preferred. Using a combination of chemical synthesis of DNA and PCR, humanized anti-MUC1* scFv are constructed, where the framework region sequences are swapped for those from the human antibody. The resultant DNA fragment is inserted into an expression vector and tested for expression and for binding affinity upon refolding.
[0119] Affinities are improved by testing adding the constant regions (CL and CH1) sequences from the same human antibody to the humanized VL and VH sequences of the anti-MUC1* scFv. The resulting chimeric heavy and light chains are linked through a 34 amino acid flexible linker SGGGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSG (SEQ ID NO:361) to produce the single chain humanized anti-MUC1* scFab. The assembled DNA molecule is cloned into a high-copy replication and high expression mammalian expression vector pCEP4 (Invitrogen, Carlsbad Calif.) after in-frame addition of IG-k signal sequence at the N-terminus and the myc and polyhistidine tags at the C-terminus. Construct thus generated is used for expression in mammalian cells, such as human embryonic kidney cells (HEK-293) which have been adapted to grow in suspension culture (Invitrogen, Carlsbad Calif.). The secreted Fab is then purified, for example, using anti-myc tag affinity chromatography.
[0120] To further improve affinity, the in silico 3D structure of the original non-human monoclonal antibody or the chimeric antibody variant is compared to many 3D structures of human antibodies available in the protein structure data base. Further variations in the framework regions are then incorporated to make the chimeric variant more closely resemble a human antibody.
[0121] Affinity enhancement can also be achieved by using phage display methods (Finlay et al 2009) which are known to those skilled in the art.
Example 13 - Comparison of Rabbit Polyclonal Antibody Generated With SEQ ID NO:1 and Monoclonal Mouse Antibody MIN-C2 for the Stimulation of Growth of Pluripotent Stem Cells
[0122] Rabbit polyclonal anti-MUC1* antibody (generated by immunization with peptide of SEQ ID NO: 1) was compared to monoclonal anti-MUC1* antibody MIN-C2 for their ability to enable the growth of stem cells in the absence of bFGF or feeder cell extracts, while maintaining their pluripotency; this is accomplished by dimerizing the extracellular domain of MUC1* comprising essentially the PSMGFR sequence (SEQ ID NO: 1).
[0123] Human embryonic stem cells were grown on Matrigel-like substrate and cultured in minimal stem cell media: a) alone; b) with 30% fibroblast conditioned medium plus 4 ng/ml bFGF (state of the art); c) with 50 ng/ml polyclonal anti-MUC1* antibody; or d) with 50 ng/ml monoclonal anti-MUC1* MIN-C2. The pluripotency of the resultant cells was assessed based on colony morphology and on their continued expression of OCT4. Pluripotent stem cell colonies grow in discrete, flat and round colonies with even, well-defined borders while those that have entered differentiation have ragged borders and begin to grow vertically. Pluripotent stem cells express OCT4 in their nuclei. Cells that have lost OCT4 expression have entered into the differentiation process. Embryonic stem cells that were engineered to express GFP (green fluorescent protein) off of the OCT4 promoter were used, so that the pluripotent stem cells, i.e., OCT4-positive cells, would fluoresce green and therefore would be easily distinguished from those that had started to differentiate.
[0124]
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[0150] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention specifically described herein. Such equivalents are intended to be encompassed in the scope of the claims.