PEPTIDES, ESPECIALLY POLYPEPTIDES, PHAGE DISPLAY SCREENING METHOD AND ASSOCIATED MEANS, AND THEIR USES FOR RESEARCH AND BIOMEDICAL APPLICATIONS
20210107951 · 2021-04-15
Inventors
Cpc classification
C07K2319/01
CHEMISTRY; METALLURGY
International classification
Abstract
Disclosed are peptides, hosts expressing such peptides and a process for producing and screening such peptides or hosts. Also disclosed is use of the peptide or host expressing such peptide in the detection of disease, to a method for constructing a library of hosts, in particular of phages expressing peptides. Also disclosed is a library of hosts expressing peptides and its use for example for detecting molecules and/or cells in a sample, in the treatment of disease. These find application in the therapeutic and diagnostic medical technical fields.
Claims
1-55. (canceled)
56. A polypeptide having a length from 55 to 85 amino-acid residues: a) whose polypeptidic sequence comprises or consists essentially of or consists of the consensus sequence: XaPDCVTGKVEYTKYNXbXcXdTFXeVKVGDKXfXgXhXiXjXkXlXmLQSLLLSAQITGMTVTI KXnXoXpCHNXqGXrXsXtEVIFR (SEQ ID NO: 2) where Xa is selected among: T, A or S, and Xb, Xc, Xd, Xf, Xm are independently selected among: D, E or N, and Xe, Xi, Xn, Xp, Xt are independently selected among: T, A or S, and Xg is selected among: L, I or V, and Xh is selected among: F, Y, W or A, and Xj, is selected among: N, E or S, and Xk is selected among: R, K or E, and Xl is selected among: W, F, Y or A, and Xo is selected among: N, E, D or S, and Xq is selected among: G A or S, and Xr is selected among: G, A, S or T and Xs is selected among: F, L or Y, provided that when Xa is T or A, Xb, Xc, Xd are not D, Xe is not T, Xf is not E, Xg is not L, Xh is not F, Xi is not T, Xj is not N, Xk is not R, Xl is not W, Xm is not N, Xn is not T, Xo is not N, Xp is not A, Xq is not G, Xr is not G, Xs is not F and Xt is not S, and/or, b) whose polypeptidic sequence comprises or consists essentially of or consists of the consensus sequence: XaPDCVTGKVEYTKYNXbDDTFXeVKVGDKEXgXhTXjXkWNLQSLLLSAQITGMTVTIKXnNXp CHNGGXrXsXtEVIFR (SEQ ID NO: 37) where Xa, Xb, Xe, Xg, Xh, Xj, Xk, Xn, Xp, Xr, Xs, Xt are as defined in point a), and/or, c) whose polypeptidic sequence comprises or consists essentially of a sequence having for structure Xa(S1)XbXcXd(S2)Xe(S3)XfXgXhXiXjXkXlXm(S4)XnXoXp(S5)Xq(S6)XrXsXt(S7) in which S1, S2, S3, S4, S5, S6 and S7, in this order from the N-terminus to the C-terminus of the polypeptide, are defined as follows: S1 represents the amino-acid sequence PDCVTGKVEYTKYN (SEQ ID NO: 38), S2 represents the amino-acid sequence TF S3 represents the amino-acid sequence VKVGDK (SEQ ID NO: 39), S4 represents the amino-acid sequence LQSLLLSAQITGMTVTIK (SEQ ID NO: 40), S5 represents the amino-acid sequence CHN S6 represents amino-acid residue G, and S7 represents the amino-acid sequence EVIFR (SEQ ID NO: 41), and wherein Xa, Xb, Xc, Xd, Xe, Xf, Xg, Xh, Xi, Xj, Xk, Xl, Xm, Xn, Xo, Xp, Xq, Xr, Xs, Xt are amino-acids as defined in point a) above, and the polypeptidic sequence of the polypeptide keeps at least 80% identity with SEQ ID NO: 1, and/or differs from SEQ ID NO: 1 by one or several conservative amino acid substitution(s), and/or, d) whose polypeptidic sequence comprises or consists essentially of or consists of a fragment of contiguous amino-acid residues of at least 55 amino-acid residues, of any one of the sequences defined in a), b) or or c), or comprises or consists essentially of or consists of a portion of any one of the sequences defined in a), b) or c) over a length of at least 55 amino-acid residues; to the proviso that the polypeptide does not consists of SEQ ID NO: 1, or SEQ D NO: 32, or SEQ ID NO: 36 or SEQ ID NO: 43, or SEQ ID NO: 44, or SEQ ID NO: 45, or SEQ ID NO: 46, or SEQ ID NO: 47, or SEQ ID NO: 48, or SEQ ID NO: 49, or SEQ ID NO: 50, or SEQ ID NO: 51, or SEQ ID NO: 52, or SEQ ID NO: 53, in particular wherein said polypeptide has the capability, when found under a pentameric form, to bind with at least one glycosphingolipid(s) selected from the group consisting of: Gb3, Gb4, Forsmann like iGb4, fucosyl-GM1, GM1, GM2, GD2, Globo-H, NeuAc-GM3, NeuGc-GM3, GD1a, O-acetyl-GD3, O-acteyl-GD2, O-acetyl-GT3, GD3.
57. The polypeptide of claim 56, which has one or several of the following property(ies) when found under a pentameric form associating five polypeptides as defined in claim 56: a. the property to bind to a glycosphingolipid selected from the group consisting of: Gb3, Gb4, Forsmann like iGb4, fucosyl-GM1, GM1, GM2, GD2, Globo-H, NeuAc-GM3, NeuGc-GM3, GD1a, O-acetyl-GD3, O-acteyl-GD2, O-acetyl-GT3, GD3, and mixtures thereof and/or b. an affinity for its target equal or superior to 10.sup.2M as measured by ITC and/or c. an apparent affinity for a membrane displaying its target equal or superior to 10.sup.6 M.sup.−1 as measured by measured by SPR.
58. The polypeptide according to claim 56, which comprises or consists essentially of or consists of any one of the sequence selected among: SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or fragments thereof.
59. A chimeric protein comprising a polypeptide as defined in claim 56 and a compound fused at one of its end.
60. A pentameric assembly of polypeptides as defined in claim 56.
61. A fusion protein comprising a polypeptide of claim 56 or a polypeptide consisting of SEQ ID NO: 1, or a polypeptide whose polypeptidic sequence comprises or consists essentially of or consists in a sequence having at least 80% identity with SEQ ID NO: 1, and/or differing from SEQ ID NO: 1 by one or several conservative amino acid substitution(s), wherein said polypeptide is fused to a coat protein of a virus or a portion of a coat protein of a virus.
62. The fusion protein according to claim 61, which comprises or consists essentially of or consists of any one of the sequence selected among: SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28 and SEQ ID NO: 33 or fragments thereof.
63. A nucleic acid molecule encoding a polypeptide of claim 56 optionally whose nucleotide sequence comprises a stop codon at the end of the sequence encoding the polypeptide according to claim 56 or the polypeptide consisting of SEQ ID NO: 1.
64. A nucleic acid molecule encoding: a polypeptide of claim 56 optionally whose nucleotide sequence comprises a stop codon at the end of the sequence encoding the polypeptide according to claim 56 or the polypeptide consisting of SEQ ID NO: 1 which is a fusion gene encompassing, from its 3′ to its 5′ extremities: a. a first nucleic acid sequence encoding polypeptide according to claim 56 or the polypeptide consisting of SEQ ID NO: 1, and b. a second nucleic acid sequence encoding at least a portion of a pIII filamentous phage coat protein, wherein said fusion gene comprises between the first and second nucleic sequences at least one stop codon.
65. The nucleic acid molecule according to claim 64, wherein the first nucleic acid sequence comprises or consist essentially of or consists of any one of the sequence selected among: SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 8 and SEQ ID NO: 12, or comprises or consist essentially of or consists of a nucleic acid sequence having at least 70%, or at least 80%, preferably 85%, more preferably 90% or 95% identity with any one of these sequences and the second nucleic acid sequence comprises or consist essentially of or consists of SEQ ID NO: 19, or a portion of it over a length of 300 bp.
66. The nucleic acid molecule according to claim 64, which consists of any one of SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29 and SEQ ID NO: 34, or a variant thereof encoding polypeptides of any one of SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28 and SEQ ID NO: 33, respectively, or fragments thereof corresponding to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 32, respectively.
67. A nucleic acid molecule encompassing a nucleic acid molecule as defined in claim 63, which is a vector selected amongst a plasmid, a phagemid and phage vector.
68. The vector of a nucleic acid molecule encoding a polypeptide of claim 56 optionally whose nucleotide sequence comprises a stop codon at the end of the sequence encoding the polypeptide according to claim 56 or the polypeptide consisting of SEQ ID NO: 1, wherein the nucleic acid is a vector selected amongst a plasmid, a phagemid and phage vector, and which is a pHEN2 phagemid comprising a nucleic acid molecule comprising or consisting essentially of or consisting of: (1) at least one first nucleic acid sequence or a variant thereof, wherein the first nucleic acid molecule encodes: a polypeptide of claim 56 optionally whose nucleotide sequence comprises a stop codon at the end of the sequence encoding the polypeptide according to claim 56 or the polypeptide consisting of SEQ ID NO: 1 which is a fusion gene encompassing, from its 3′ to its 5′ extremities: a. a first nucleic acid sequence encoding polypeptide according to claim 56 or the polypeptide consisting of SEQ ID NO: 1, and b. a second nucleic acid sequence encoding at least a portion of a pIII filamentous phage coat protein, wherein said fusion gene comprises between the first and second nucleic sequences at least one stop codon, and wherein the first nucleic acid sequence comprises or consist essentially of or consists of any one of the sequence selected among: SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 8 and SEQ ID NO: 12, or comprises or consist essentially of or consists of a nucleic acid sequence having at least 70%, or at least 80%, preferably 85%, more preferably 90% or 95% identity with any one of these sequences and the second nucleic acid sequence comprises or consist essentially of or consists of SEQ ID NO: 19, or a portion of it over a length of 300 bp, (2) at least one stop codon selected among TAG, TAA and TGA, and (3) a second nucleic acid sequence in the order of (1), (2) and (3), or a variant thereof, wherein the second nucleic acid molecule encodes: a polypeptide of claim 56 optionally whose nucleotide sequence comprises a stop codon at the end of the sequence encoding the polypeptide according to claim 56 or the polypeptide consisting of SEQ ID NO: 1 which is a fusion gene encompassing, from its 3′ to its 5′ extremities: a. a first nucleic acid sequence encoding polypeptide according to claim 56 or the polypeptide consisting of SEQ ID NO: 1, and b. a second nucleic acid sequence encoding at least a portion of a pIII filamentous phage coat protein, wherein said fusion gene comprises between the first and second nucleic sequences at least one stop codon, and wherein the first nucleic acid sequence comprises or consist essentially of or consists of any one of the sequence selected among: SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 8 and SEQ ID NO: 12, or comprises or consist essentially of or consists of a nucleic acid sequence having at least 70%, or at least 80%, preferably 85%, more preferably 90% or 95% identity with any one of these sequences and the second nucleic acid sequence comprises or consist essentially of or consists of SEQ ID NO: 19, or a portion of it over a length of 300 bp.
69. An expression system comprising: a) a nucleic acid encoding a polypeptide having a length from 55 to 85 amino-acid residues, whose polypeptidic sequence comprises or consists essentially of or consists of the consensus sequence XaPDCVTGKVEYTKYNXbXcXdTFXeVKVGDKXfXgXhXiXjXkXlXmLQSLLLSAQ ITGMTVTIKXnXoXpCHNXqGXrXsXtEVIFR (SEQ ID NO: 2) where Xa is selected among: T, A or S, and Xb, Xc, Xd, Xf, Xm are independently selected among: D, E or N, and Xe, Xi, Xn, Xp, Xt are independently selected among: T, A or S, and Xg is selected among: L, I or V, and Xh is selected among: F, Y, W or A, and Xj, is selected among: N, E or S, and Xk is selected among: R, K or E, and Xl is selected among: W, F, Y or A, and Xo is selected among: N, E, D or S, and Xq is selected among: G A or S, and Xr is selected among: G, A, S or T and Xs is selected among: F, L or Y, or, b) a nucleic acid encoding a polypeptide having a length from to 85 amino-acid residues, whose polypeptidic sequence comprises or consists essentially of or consists in a sequence having at least 75% identity with sequence TPDCVTGKVEYTKYNDDDTFTVKVGDKELFTNRWNLQSLLLSAQITGMTVTIKTNACHNGGGFSE VIFR (SEQ ID NO: 1), and/or differing from SEQ ID NO: 1 by one or several conservative amino acid substitution(s), or, c) a nucleic acid encoding a polypeptide having a length from to 85 amino-acid residues, whose polypeptidic sequence comprises or consists essentially of or consists of a fragment of contiguous amino-acid residues of at least 55 amino-acid residues, of any one of the sequences defined in a) or b), or comprises or consists essentially of or consists of a portion of any one of the sequences defined in a) or b) over a length of 55 amino-acid residues, wherein the expression system is at least one of a plasmid, a phagemid or an expression vector.
70. A host comprising a polypeptide as defined in claim 56.
71. A virus or a library of viruses displaying at its surface a polypeptide as defined in claim 56.
72. A filamentous bacteriophage: displaying a polypeptide as defined in claim 56, the genome of the filamentous bacteriophage comprising a fusion gene in the form of a nucleic acid molecule encoding a polypeptide of claim 56 optionally whose nucleotide sequence comprises a stop codon at the end of the sequence encoding the polypeptide according to claim 56 or the polypeptide consisting of SEQ ID NO: 1.
73. The filamentous bacteriophage according to claim 72, wherein the displayed STxB-subunit or variant thereof is under the form of one STxB monomer or variant thereof in fusion with a pIII page coat protein, the STxB monomer or variant thereof being assembled with four other free STxB monomers.
74. A bacterial cell comprising a nucleic acid molecule as defined in claim 63.
75. A method of production of filamentous phage(s), or a library thereof, comprising the steps of: a. Introducing one or several vector(s) as defined in claim 67 into bacterial cell(s), and b. Culturing the bacterial cell(s) of step (a), optionally in the presence of helper phage(s), and c. Optionally, recovering the produced filamentous phage(s) or library thereof and/or isolating a particular species of produced filamentous phages or library thereof.
76. In vitro use of a polypeptide as defined in claim 56 for detecting a molecule and/or a cell in a sample.
77. Method for determining the specificity of a virus to glycosphingolipids comprising the following steps: a) Putting into contact a virus as defined in claim 71 and a support comprising glycosphingolipids on its surface, b) Incubating the virus and the support comprising glycosphingolipids present on its surface to let the virus bind to the glycosphingolipids, c) Washing the incubated surface to eliminate the non-bounded virus, and d) Recovering virus bounded to the glycosphingolipids.
78. A method to identify one or several filamentous phage(s) displaying an STxB-subunit or a variant thereof, which bind to a particular glycosphingolipid or a variant thereof, or to a mix of several glycosphingolipids or variants thereof as a target, said method comprising: d. Contacting under conditions enabling the binding with the target, a library of filamentous bacteriophages comprising a plurality of filamentous bacteriophages as defined in claim 71, with one or several glycosphingolipid(s) or variant thereof displayed on a support, such as cells expressing one or several glycosphingolipid(s) or variant thereof at their surface, or unilamellar vesicles or liposomes presenting one or several glycosphingolipid(s) or variant thereof, and e. Separating the filamentous bacteriophages that bind to the target from those that do not bind, for example through washing, and f. Recovering the filamentous phage(s) bound to the target, and g. Optionally, analyzing the filamentous phage(s) bound to the target and/or determining the sequence of at least a part of the nucleic acid content of the recovered filamentous phage(s) and/or the sequence of at least a part of the STxB-subunit or a variant thereof displayed by said recovered filamentous phage(s).
79. A method of using a filamentous bacteriophage displaying an STxB-subunit or a variant thereof at its surface as defined in claim 56 to treat one or several neoplasic condition, selected among: ovarian cancer, breast carcinoma, colon cancer, gastric adenocarcinoma, Burkitt's lymphoma, colon carcinoma, melanoma, small cell lung cancer (SCLC), renal carcinoma, neuroblastoma, cervical carcinoma, glioblastoma, renal carcinoma, glioma, retinoblastoma, neuroectodermal cancer, non-small cell lung cancer (NSCLC), Wilms tumor, osteosarcoma, and t-All condition, said method comprising administering to a patient in need thereof a filamentous bacteriophage displaying an STxB-subunit or a variant thereof at its surface as defined in claim claim 56.
80. Use of labelled filamentous bacteriophage displaying an STxB-subunit or a variant thereof at its surface as defined in claim 56, as a probe or marker for in vitro detection of glycosphingolipid(s) or variant(s) thereof.
81. Use of a polypeptide as defined in claim 56 in an in vitro or ex vivo diagnostic method.
Description
BRIEF DESCRIPTION OF THE FIGURES
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[0575]
[0576]
[0577]
[0578]
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[0580]
[0581]
[0582]
[0583]
[0584]
5A. Five STxB monomers, each of them fused with one pIII protein of the phage, were able to pentamerize. Only one pentamer of STxB could be then displayed on a phage particle. 5B. One STxB monomer in fusion with the pIII protein was able to pentamerize with 4 others free STxB monomer in the periplasm of the bacteria during the assembly of the phage particles. One to five STxB pentamer could then be displayed on a phage particle.
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SEQUENCES
[0593] The amino acid sequence of B-subunit of Shiga toxin used is TPDCVTGKVEYTKYNDDDTFTVKVGDKELFTNRWNLQSLLLSAQITGMTVTIKTNACH NGGGFSEVIFR (SEQ ID NO: 1)
[0594] The consensus sequence defined in SEQ ID NO: 2 is: XaPDCVTGKVEYTKYNXbXcXdTFXeVKVGDKXfXgXhXiXjXkXIXmLQSLLLSAQITGMT VTIKXnXoXpCHNXqGXrXsXtEVIFR wherein Xa to Xt are as defined in instant description herein.
[0595] The amino acid sequence of STxB polypeptide of clones A3-D10-H3 is:
TABLE-US-00003 (SEQ ID NO: 3) SPDCVTGKVEYTKYNNDDTFTVKVGDKELWTEKWNLQSLLLSAQ ITGMTVTIKSNACHNGGSFAEVIFR
[0596] Nucleic acid sequence encoding SEQ ID NO: 3 is:
TABLE-US-00004 (SEQ ID NO: 4) TCTCCTGATTGTGTAACTGGAAAGGTGGAGTATACAAAATATAATAAC GACGACACCTTTACTGTTAAAGTGGGTGATAAAGAACTGTGGACTGAA AAATGGAACCTTCAGTCTCTTCTTCTCAGTGCGCAAATTACGGGGATG ACTGTAACCATTAAATCTAACGCATGTCATAATGGTGGGTCTTTTGCA GAAGTTATTTTTCGT
[0597] The amino acid sequence of STxB polypeptide of clones B12-C03-D12-G05-G11-H11 is:
TABLE-US-00005 (SEQ ID NO: 5) SPDCVTGKVEYTKYNNDDTFTVKVGDKELWTEKWNLQSLLLSAQITGM TVTIKSNACHNGGSFAEVIFR
[0598] Nucleic acid sequence encoding SEQ ID NO: 5 is:
TABLE-US-00006 (SEQ ID NO: 6) GCACCTGATTGTGTAACTGGAAAGGTGGAGTATACAAAATATAATAAC GACGACACCTTTTCTGTTAAAGTGGGTGATAAAGAACTGTGGACTGAA AAATGGAACCTTCAGTCTCTTCTTCTCAGTGCGCAAATTACGGGGATG ACTGTAACCATTAAAACTAACGCATGTCATAATGGTGGGGCACTGTCT GAAGTTATTTTTCGT
[0599] The amino acid sequence of STxB polypeptide of clones A06-C06 is:
TABLE-US-00007 (SEQ ID NO: 7) SPDCVTGKVEYTKYNNDDTFSVKVGDKEIYTSKWNLQSLLLSAQ ITGMTVTIKSNTCHNGGAFSEVIFR
[0600] Nucleic acid sequence encoding SEQ ID NO: 7 is:
TABLE-US-00008 (SEQ ID NO: 8) TCTCCTGATTGTGTAACTGGAAAGGTGGAGTATACAAAATATAATAAC GACGACACCTTTTCTGTTAAAGTGGGTGATAAAGAAATCTACACTTCT AAATGGAACCTTCAGTCTCTTCTTCTCAGTGCGCAAATTACGGGGATG ACTGTAACCATTAAATCTAACACTTGTCATAATGGTGGGGCATTTTCT GAAGTTATTTTTCGT
[0601] The amino acid sequence of STxB polypeptide of clone B02 is:
TABLE-US-00009 (SEQ ID NO: 9) SPDCVTGKVEYTKYNDEDTFSVKVGDKEVWTNRCKLQSLLLSAQ ITGMTVTIKTSSCHNAGGLTEVIFR
[0602] Nucleic acid sequence encoding SEQ ID NO: 9 is:
TABLE-US-00010 (SEQ ID NO: 10) TCTCCTGATTGTGTAACTGGAAAGGTGGAGTATACAAAATATAATGAC GAAGACACCTTTTCTGTTAAAGTGGGTGATAAAGAAGTGTGGACTAAC CGTTGCAAACTTCAGTCTCTTCTTCTCAGTGCGCAAATTACGGGGATG ACTGTAACCATTAAAACTTCTTCTTGTCATAATGCAGGGGGTTTGACT GAAGTTATTTTTCGT
[0603] The amino acid sequence of STxB polypeptide of clone B05 is:
TABLE-US-00011 (SEQ ID NO: 11) APDCVTGKVEYTKYNDDNTFSVKVGDKELYTNRWNLQSLLLSAQITGM TVTIKTNSCHNGGGFAEVIFR
[0604] Nucleic acid sequence encoding SEQ ID NO: 11 is:
TABLE-US-00012 (SEQ ID NO: 12) GCACCTGATTGTGTAACTGGAAAGGTGGAGTATACAAAATATAATGAC GACAACACCTTTTCTGTTAAAGTGGGTGATAAAGAACTGTACACTAAC CGTTGGAACCTTCAGTCTCTTCTTCTCAGTGCGCAAATTACGGGGATG ACTGTAACCATTAAAACTAACTCTTGTCATAATGGTGGGGGTTTTGCA GAAGTTATTTTTCGT
[0605] SEQ ID NO: 13 (MKKTLLIAASLSFFSASALA) corresponds to a signal peptide.
[0606] SEQ ID NO: 14 is the concatenation of SEQ ID NO: 13 and SEQ ID NO: 1:
TABLE-US-00013 MKKTLLIAASLSFFSASALATPDCVTGKVEYTKYNDDDTFTVKVGDKE LFTNRWNLQSLLLSAQITGMTVTIKTNACHNGGGFSEVIFR
[0607] Nucleic acid sequence encoding the signal peptide of SEQ ID NO: 13:
TABLE-US-00014 (SEQ ID NO: 15) ATGAAAAAAACATTATTAATAGCTGCATCGCTTTCATTTTTTTCAGCA AGTGCGCTGGCG
[0608] Exemplary M13 pIII sequence:
TABLE-US-00015 (SEQ ID NO: 16) TVESCLAKPHTENSFTNVWKDDKTLDRYANYEGCLWNATGVVVC TGDETQCYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPP EYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTF MFQNNRFRNRQGALTVYTGTVTQGTDPVKTYYQYTPVSSKAMYD AYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPVNAGGGSG GGSGGGSEGGGSEGGGSEGGGSEGGGSGGGSGSGDFDYEKMANA NKGAMTENADENALQSDAKGKLDSVATDYGAANGDA
[0609] Example of STxB—PIII fusion protein:
TABLE-US-00016 (SEQ ID NO: 17) MATPDCVTGKVEYTKYNDDDTFTVKVGDKELFTNRWNLQSLLLSAQITGM TVTIKTNACHNGGGFSEVIFRAAAHHHHHHGAAEQKLISEEDLNGAAEQK
[0610] Legend for SEQ ID NO: 17 is as follows:
[0611] Restriction Sites (NcoI) Hang Over [0612] STxB
[0613] Histidine Tag
[0614] Myc Tag (3 Repeats)
[0615] Linkers [0616] position of amber stop codon, which is expressed as a Q in TG1 (amber-suppressor Host) bacteria: it allows for co-expression of STxB monomer and STxB_pIII fusion for pentameric assembly in the perisplasm of non amber-suppressor Host bacteria. In non amber-suppressor Host, it is expressed as a stop codon, in amber-suppressor Host, as Q.
[0617] PIII Fragment
The legend for SEQ ID NO: 17 applies similarly, in all correspondence, to all of SEQ ID NO: 20, 22, 24, 26, 28, respectively.
[0618] SEQ ID NO: 18 is the nucleic acid sequence encoding SEQ ID NO: 17:
TABLE-US-00017 atgGCGACGCCTGATTGTGTAACTGGAAAGGTGGAGTATACAAAATATAA TGATGACGATACCTTTACGTTAAAGTGGGTGATAAAGAATTATTTACCAA CAGATGGAATCTTCAGTCTCTTCTTCTCAGTGCGCAAATTACGGGGATGA CTGTAACCATTAAAACTAATGCCTGTCATAATGGAGGGGGATTCAGCGAA GTTATTTTTCGTGCggccGCACATCATCATCACCATCACGGGGCCGCgGA ACAAAAACTCATCTCAGAAGAGGATCTGAATGGGGCCGCAgagcaaaagc taatatctgaagaagatctcaacGGGGCCGCAgaacagaaacttatcagt
(Please note that to remove amber stop codon , it can be replaced by codon CAG encoding a Q residue, thereby producing a fully fused protein). The legend for SEQ ID NO: 18 follows that of SEQ ID NO: 17 described above and applies similarly, in all correspondence, to all of SEQ ID NO: 21, 23, 25, 27, 29, respectively.
[0619] SEQ ID NO: 30 is the nucleic acid sequence encoding SEQ ID NO: 1:
TABLE-US-00018 ACGCCTGATTGTGTAACTGGAAAGGTGGAGTATACAAAATATAA TGATGACGATACCTTTACAGTTAAAGTGGGTGATAAAGAATTAT TTACCAACAGATGGAATCTTCAGTCTCTTCTTCTCAGTGCGCAA ATTACGGGGATGACTGTAACCATTAAAACTAATGCCTGTCATAA TGGAGGGGGATTCAGCGAAGTTATTTTTCGT
[0620] SEQ ID NO: 31 represent the nucleic acid sequence encoding STxB D18E, G62T mutant of SEQ ID NO: 32 (Bold nucleotides are mutated positions):
TABLE-US-00019 ACGCCTGATTGTGTAACTGGAAAGGTGGAGTATACAAAATATAA TGATGAAGATACCTTTACAGTTAAAGTGGGTGATAAAGAATTAT TTACCAACAGATGGAATCTTCAGTCTCTTCTTCTCAGTGCGCAA ATTACGGGGATGACTGTAACCATTAAAACTAATGCCTGTCATAA TGGAGGGACATTCAGCGAAGTTATTTTTCGT
[0621] SEQ ID NO: 32 is the STxB D18E, G62T mutant sequence (Bold residues are mutated positions):
TABLE-US-00020 TPDCVTGKVEYTKYNDEDTFTVKVGDKELFTNRWNLQSLLLSAQ ITGMTVTIKTNACHNGGTFSEVIFR
[0622] SEQ ID NO: 33 corresponds to the STxB mutant of SEQ ID NO: 31 fused to pIII:
TABLE-US-00021 MATPDCVTGKVEYTKYNDEDTFTVKVGDKELFTNRWNLQSLLLSAQITGM TVTIKTNACHNGGTFSEVIFRAAAHHHHHHGAAEQKLISEEDLNGAAEQK
[0623] Legend for SEQ ID NO: 33 is as follows:
[0624] Restriction Sites (NcoI) Hang Over [0625] D18E, G62T STxB mutant
[0626] Histidine Tag
[0627] Myc Tag (3 Repeats)
[0628] Linkers [0629] position of amber stop codon, which is expressed as a Q in TG1 (amber-suppressor Host) bacteria: it allows for co-expression of STxB monomer and STxB_pIII fusion for pentameric assembly in the perisplasm of non amber-suppressor Host bacteria. In non amber-suppressor Host, it is expressed as a stop codon, in amber-suppressor Host, as Q.
[0630] PIII Fragment
[0631] SEQ ID NO: 34 is nucleic acid sequence corresponding to SEQ ID NO: 33 (same legend applies):
TABLE-US-00022 ATGGCGACGCCTGATTGTGTAACTGGAAAGGTGGAGTATACAAAATATAA TGATGAAGATACCTTTACAGTTAAAGTGGGTGATAAAGAATTATTTACCA ACAGATGGAATCTTCAGTCTCTTCTTCTCAGTGCGCAAATTACGGGGATG ACTGTAACCATTAAAACTAATGCCTGTCATAATGGAGGGACATTCAGCGA AGTTATTTTTCGTGCggccGCACATCATCATCACCATCACGGGGCCGCgG AACAAAAACTCATCTCAGAAGAGGATCTGAATGGGGCCGCAgagcaaaag ctaatatctgaagaagatctcaacGGGGCCGCAgaacagaaacttatcag
[0632] SEQ ID NO: 35 is a 4804 bp nucleic acid sequence as described in present description.
[0633] SEQ ID NO: 36 corresponds to SEQ ID NO: 1 with the first amino-acid residue being A:
TABLE-US-00023 APDCVTGKVEYTKYNDDDTFTVKVGDKELFTNRWNLQSLLLSAQ ITGMTVTIKTNACHNGGGFSEVIFR
This sequence is incidentally described in “Functional analysis of the Shiga toxin and Shiga-like toxin type II variant binding subunits by using site-directed mutagenesis.” Jackson M. P., Wadolkowski E. A., Weinstein D. L., Holmes R. K., O'Brien A. D. J. Bacteriol. 172:653-658 (1990).
[0634] The consensus sequence defined in SEQ ID NO: 37 is: XaPDCVTGKVEYTKYNXbDDTFXeVKVGDKEXgXhTXjXkWNLQSLLLSAQITGMTVTIK XnNXpCHNGGXrXsXtEVIFR where Xa, Xb, Xe, Xg, Xh, Xj, Xk, Xn, Xp, Xr, Xs, Xt are as defined in instant description herein.
[0635] SEQ ID NO: 38 corresponds to so-called Scaffold section 51 of Table 1: PDCVTGKVEYTKYN.
[0636] SEQ ID NO: 39 corresponds to so-called Scaffold section S3 of Table 1: VKVGDK.
[0637] SEQ ID NO: 40 corresponds to so-called Scaffold section S4 of Table 1: LQSLLLSAQITGMTVTIK.
[0638] SEQ ID NO: 41 corresponds to so-called Scaffold section S7 of Table 1: EVIFR.
[0639] SEQ ID NO: 42 is wild-type pIII protein having 424 amino-acids residues:
TABLE-US-00024 MKKLLFAIPLVVPFYSHSAETVESCLAKPHTENSFTNVWKDDKT LDRYANYEGCLWNATGVVVCTGDETQCYGTWVPIGLAIPENEGG GSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGT EQNPANPNPSLEESQPLNTFMFQNNRFRNRQGALTVYTGTVTQG TDPVKTYYQYTPVSSKAMYDAYWNGKFRDCAFHSGFNEDPFVCE YQGQSSDLPQPPVNAGGGSGGGSGGGSEGGGSEGGGSEGGGSEG GGSGGGSGSGDFDYEKMANANKGAMTENADENALQSDAKGKLDS VATDYGAAIDGFIGDVSGLANGNGATGDFAGSNSQMAQVGDGDN SPLMNNFRQYLPSLPQSVECRPFVFSAGKPYEFSIDCDKINLFR GVFAFLLYVATFMYVFSTFANILRNKES
[0640] Several mutant sequences are known in the art, which are not part of instant invention, only as far as isolated polypeptides are considered. For instance, Jackson M. P., Wadolkowski E. A., Weinstein D. L., Holmes R. K., O'Brien A. D. describe in “Functional analysis of the Shiga toxin and Shiga-like toxin type II variant binding subunits by using site-directed mutagenesis.” J. Bacteriol. 172:653-658 (1990), D16N SEQ ID NO: 43), D17N (SEQ ID NO: 44), D17E (SEQ ID NO: 45), D16N D17N (SEQ ID NO: 46), D18N (SEQ ID NO: 47) mutants.
TABLE-US-00025 (D16N): SEQ ID NO: 43 TPDCVTGKVEYTKYNNDDTFTVKVGDKELFTNRWNLQSLLLSAQ ITGMTVTIKTNACHNGGGFSEVIFR (D17N): SEQ ID NO: 44 TPDCVTGKVEYTKYNDNDTFTVKVGDKELFTNRWNLQSLLLSAQ ITGMTVTIKTNACHNGGGFSEVIFR (D17E): SEQ ID NO: 45 TPDCVTGKVEYTKYNDEDTFTVKVGDKELFTNRWNLQSLLLSAQ ITGMTVTIKTNACHNGGGFSEVIFR (D16N D17N): SEQ ID NO: 46 TPDCVTGKVEYTKYNNNDTFTVKVGDKELFTNRWNLQSLLLSAQ ITGMTVTIKTNACHNGGGFSEVIFR (D18N): SEQ ID NO: 47 TPDCVTGKVEYTKYNDDNTFTVKVGDKELFTNRWNLQSLLLSAQ ITGMTVTIKTNACHNGGGFSEVIFR
[0641] Clark C., Bast D. J., Sharp A. M., St Hilaire P. M., Agha R., Stein P. E., Toone E. J., Read R. J., Brunton J. L disclose in “Phenylalanine 30 plays an important role in receptor binding of verotoxin-1” Mol. Microbiol. 19:891-899 (1996) mutant F30A (SEQ ID NO: 48).
TABLE-US-00026 (F30A): SEQ ID NO: 48 TPDCVTGKVEYTKYNDDDTFTVKVGDKELATNRWNLQSLLLSAQ ITGMTVTIKTNACHNGGGFSEVIFR
[0642] Perera L. P., Samuel J. E., Holmes R. K., O'Brien A. D. “Identification of three amino acid residues in the B subunit of Shiga toxin and Shiga-like toxin type II that are essential for holotoxin activity.” J. Bacteriol. 173:1151-1160 (1991) and Jemal C., Haddad J. E., Begum D., Jackson M. P. “Analysis of Shiga toxin subunit association by using hybrid A polypeptides and site-specific mutagenesis.” J. Bacteriol. 177:3128-3132 (1995) disclose mutant R33D (SEQ ID NO: 49).
TABLE-US-00027 (R33D): SEQ ID NO: 49 TPDCVTGKVEYTKYNDDDTFTVKVGDKELFTNDWNLQSLLLSAQ ITGMTVTIKTNACHNGGGFSEVIFR
[0643] Jemal et al. above also disclose mutant W34G (SEQ ID NO: 50).
TABLE-US-00028 (W34G): SEQ ID NO: 50 TPDCVTGKVEYTKYNDDDTFTVKVGDKELFTNRGNLQSLLLSAQ ITGMTVTIKTNACHNGGGFSEVIFR
[0644] Bast D. J., Banerjee L., Clark C., Read R. J., Brunton J. L. “The identification of three biologically relevant globotriaosyl ceramide receptor binding sites on the Verotoxin 1 B subunit.” Mol. Microbiol. 32:953-960 (1999) disclose mutant W34A (SEQ ID NO: 51) and G62T (SEQ ID NO: 52).
TABLE-US-00029 (W34A): SEQ ID NO: 51 TPDCVTGKVEYTKYNDDDTFTVKVGDKELFTNRANLQSLLLSAQ ITGMTVTIKTNACHNGGGFSEVIFR (G62T): SEQ ID NO: 52 TPDCVTGKVEYTKYNDDDTFTVKVGDKELFTNRWNLQSLLLSAQ ITGMTVTIKTNACHNGGTFSEVIFR
[0645] Is also known mutant D17E G62T (SEQ ID NO: 53).
TABLE-US-00030 (D17E G62T): SEQ ID NO: 53 TPDCVTGKVEYTKYNDEDTFTVKVGDKELFTNRWNLQSLLLSAQ ITGMTVTIKTNACHNGGTFSEVIFR
A. Material and Methods
[0646] Recombinant STxB Expression and Purification
[0647] The STxB gene was cloned into the pSU108 plasmid, and expression was performed under the transcriptionnal and translational control of the thermoinducible LambdapL/PR promoter. After preparation of periplasmic extracts, these were loaded on a QFF column (Pharmacia) and eluted by a linear NaCl gradient in 20 mMTris/HCl, pH 7.5. Recombinant STxB was eluted between 120 and 400 mM. STxB-containing fractions were dialyzed against 20 mMTris/HCl, pH 7.5, reloaded on a Mono Q column (Pharmacia), and eluted as before. The resulting proteins, estimated to be 95% pure by SDS-polyacrylamide gel electrophoresis, were stored at −80° C. until use.
[0648] Generation of a Stable Gb3+CHO Cell Line
[0649] The CHO cell line was chosen to generate a cell system for which Gb3-positive and negative cells were available on the same genetic background. CHO cells normally lack expression of lactosylceramide α1,4-galactosyltransferase, the enzyme that catalyzes the conversion of lactosylceramide into Gb3 and its derivatives. To generate a Gb3-positive CHO clone, the Gb3 synthase gene under control of the cytomegalovirus promoter was stably transfected into these cells. The expression of Gb3 and its localization at the plasma membrane was then demonstrated using STxB.
[0650] pCDNA3_Gb3_synthase plasmid from J. Wiels lab (Institut Gustave Roussy UMR 8126) was transfected into CHO cells by electroporation. Briefly, 80% confluent cells were trypsinized, centrifuged at 600×g for 5 min and washed once in Phosphate Buffer Saline (PBS). 8×10.sup.6 cells were resuspended in a 240 μl mix composed of 120 μL electrobuffer mix (Cell projects), 10 μg pCDNA3_Gb3_synthase, 10 μg Salmon Sperm DNA and water. Electroporation was done in a 4 mm gap electroporation cuvette at 0.22 kV with High Cap set at 0.975 μF×1000, and cells were resuspended in 10 mL Dulbecco's modified Eagle's medium: nutrient mixture F-12 (DMEM/F12, Gibco, Life Technologies), supplemented with 10% heat-inactivated fetal bovine serum (Pan Biotech), 0.01% penicillin-streptomycin (Invitrogen), 41 mM L-glutamine and 51 mM sodium pyruvate.
[0651] Cells were seeded in a T75 dish, and grown at 37° C. in a 5% CO.sub.2/air atmosphere. After 24 hours, selection medium containing 0.5 mg/mL Geneticin (G418, ThermoFischer) was added and replaced every other day. After 2 weeks of selection, single cell were selected by limited dilution in 96 well plates.
[0652] Final selection was performed by FACS sorting, using binding of fluorescently labeled STxB, and intracellular retrograde trafficking of STxB in the selected cell line was controlled by immunofluorescence microscopy. 7×10.sup.4 Gb3.sup.+CHO and GB3.sup.−CHO cells were seeded in P6 plates, and grown overnight at 37° C. under 5% CO.sub.2. After 3 times washes with DMEM/F12 medium at 4° C., cells were incubated for 30 min at 4° C. with 0.1 μM A488-labelled STxB, washed, and either fixed (binding experiments), or incubated for 45 min at 37° C. (retrograde transport experiments). When indicated, the Golgi apparatus was labeled for GM130 (BD transduction laboratories). Images were acquired on an epifluorescence microscope (Leica DM 6000B), and processed with ImageJ software.
[0653] GSLs Synthesis Inhibition in HeLa C2TA Cells
[0654] DL-threo-1-Phenyl-2-palmitoylamino-3-morpholino-1-Propanol (PPMP) is a glucosylceramide synthetase inhibitor, which was used for the depletion of GSLs.
[0655] HeLa cells express the glycosphingolipids Gb3. The HeLa cell clone C2TA homogenously expresses Gb3. HeLa C2TA cells were cultured for 12 days at 37° C. under 5% CO2 in DMEM medium containing 5 μM PPMP (Enzo LifeSciences) with splitting of cells every 3 days. Inhibition of glycosphingolipid synthesis was confirmed by binding of fluorescently labeled STxB on C2TA treated cells analyzed by immunofluorescence and flow cytometry.
[0656] Gb3 Synthase Gene Transfection and Selection of a Stable Gb3+CHO Cell Line
[0657] Gb3 synthase gene transfection and selection of a stable Gb3+CHO cell line pCDNA3_Gb3_synthase plasmid from J. Wiels lab (Institut Gustave Roussy UMR 8126) was transfected into CHO cells CHO-K1 (ATCC® CCL-61 (trademark)) from Sigma aldrich ref: 85051005-1VL by electroporation. Briefly, 80% confluent cells were trypsinized, centrifuged at 600×g for 5 min and washed once in Phosphate Buffer Saline (PBS). 8×106 cells were resuspended in a 240 μl mix composed of 120 μL electrobuffer mix (cell projects), 10 μg pCDNA3_Gb3_synthase, 10 μg Salmon Sperm DNA and water.
[0658] Electroporation was done in a 4 mm gap electroporation cuvette at 0.22 kV with High Cap set at 0.975 μF×1000, and cells were resuspended in 10 mL Dulbecco's modified Eagle's medium: nutrient mixture F-12 (DMEM/F12, Invitrogen), supplemented with 10% heat-inactivated fetal bovine serum (Pan Biotech), 0.01% penicillin-streptomycin (Invitrogen), 41 mM L-glutamine and 51 mM sodium pyruvate. Cells were seeded in a T75 dish, and grown at 37° C. in a 5% CO.sub.2/air atmosphere. After 24 hours, selection medium containing 0.5 mg/mL Geneticin (G418, ThermoFischer) was added and replaced every other day. After 2 weeks of selection, single cell were selected by limited dilution in 96 well plates. Final selection was performed by FACS sorting, using binding of fluorescently labeled STxB, and intracellular retrograde trafficking of STxB in the selected cell line was controlled by immunofluorescence microscopy.
[0659] Glycosphingolipids Synthesis Inhibition by PPMP Treatment of C2TA Cells
[0660] C2TA cells were cultured for 12 days at 37° C. under 5% CO2 in DMEM medium containing 5 μM PPMP with splitting of cells every 3 days. Inhibition of glycosphingolipid synthesis was confirmed by binding of fluorescently labeled STxB on C2TA treated cells analyzed by immunofluorescence and flow cytometry.
[0661] Immunofluorescence Experiment to Confirm Binding and Retrograde Transport of STxB in Stable Gb3.sup.+CHO Cells
[0662] 7×10.sup.4 Gb3.sup.+CHO and GB3.sup.−CHO cells were seeded in P6 plates, and grown overnight at 37° C. under 5% CO.sub.2. After 3 times washes with DMEM/F12 medium at 4° C., cells were incubated for 30 min at 4° C. with 0.1 μM A488-labelled STxB, washed, and either fixed (binding experiments), or incubated for 45 min at 37° C. (retrograde transport experiments). When indicated, the Golgi apparatus was labelled for GM130 (BD transduction laboratories). Images were acquired on an epifluorescence microscope (Leica DM 6000B), and processed with ImageJ software.
[0663] pHEN2_STxB Phagemid and pHEN2_STxB_Mutant Design & Cloning
[0664] The pHEN2_STxB phagemids were designed for the expression of STxB or STxB mutant in fusion with the pIII capside coat protein of bacteriophage M13. These constructs were obtained using the Gibson assembly technique, by recombination between the STxB inserts and the commercially available pHEN2 phagemid. The restriction sites NcoI and Not1 were introduced at the 5′ and 3′ ends of the STxB genes, respectively. The first step consisted in 2 PCRs using appropriate primers to create overhangs of 15 base pairs shared by the plasmid and the insert.
[0665] Briefly, these PCR amplifications were done in 50 μL total volume using 10 ng of templates plasmids, 2.5 ng of each primer, 0.5 μl of Phusion (trade mark) High-Fidelity DNA polymerase with appropriate buffer and reagents as described by the manufacturer (New England BioLabs). The PCR program consisted in 5 cycles at 54° C. annealing, followed by 25 cycles at 72° C. annealing temperature. PCR products were purified using a commercial DNA gel extraction kit Cat No 28106 (Qiagen), and were then assembled according to the one-step isothermal DNA assembly method: 0.025 pmol of each DNA fragment were pooled in 5 μl, and 15 μl of home-made assembly master mixture according to Gibson's protocol (500 mM Tris-HCl pH 7.5, 50 mM MgCl.sub.2, 1 mM dGTP, 1 mM dATP, 1 mM dTTP, 1 mM dCTP, 50 mM DTT, 25% PEG-8000 and 5 mM NAD) were added. The mixture was incubated at 50° C. for 1 hour in a thermocycler. 3 μl of Gibson assembly reaction were used for the transformation of DH5alpha thermocompetent E. coli cells, according to the manufacturer's instructions (Invitrogen). Bacteria were cultured on LB plates containing antibiotics. 6 clones were sequenced, and 1 was selected, grown in 2×YT medium with antibiotics, and bacterial plasmid DNA extraction was performed using the QIAprep Spin Miniprep Kit (Qiagen). 50 ng were used for transformation of thermocompetent TG1 E. coli cells (Lucigen) grown in 50 mL 2×YT, 100 ug/mL ampicillin, 1% glucose.
[0666] Amber Mutation
[0667] The pHEN2_STxB_noAmb phagemid, where the TAG amber stop codon is replaced by a CAG codon was obtained by site-directed mutagenesis using GeneArt Site-directed mutagenesis kit (ThermoFisher Scientific). Appropriate primers were designed and ordered from Eurofins. After transformation of mutagenesis products, 8 clones were sequenced (GATC). One was selected, grown in 2×YT medium containing 100 μg/mL ampicillin. Bacterial plasmid DNA extraction was performed using GIAprep Spin Miniprep Kit (Giagen).
[0668] STxB Expression on Phages
[0669] 50 ng of each phagemids were used for transformation of thermocompetent TG1 E. coli cells (Lucigen) grown in 50 mL 2×YT, 100 μg/mL ampicillin, 1% glucose. Overnight culture of TG1 cells transformed with pHEN2_STxB, pHEN2_STxB_noAmb or pHEN2_STxB_mut were diluted in 10 mL of 2×YT medium, 100 μg/mL ampicillin, 1% glucose, grown from an OD600 of 0.1 to 0.5, infected with 4 μL of 10.sup.13 Helper phages M13KO7 (NEB) or 40 uL of 10.sup.12 Hyperphage M13 K07ΔpIII (Progen), and incubated for 30 min at 37° C. in a water bath.
[0670] Bacteria were then centrifuged for 10 min at room temperature at 3.200×g, and resuspended in 50 mL 2×YT (powder from sigma Aldrich Y2377-250G) without glucose, but containing ampicillin 100 μg/mL and kanamycin at 50 μg/mL. After an overnight growth at 30° C., the cultures were centrifuged 15 min at 3,200×g, and the phage-containing supernatant was collected.
[0671] Further isolation of phage particles was obtained by PEG precipitation. 40 mL of supernatant were incubated with 8 mL PEG 8000 30% 2.5M NaCl for 1 hr at 4° C. After 30 min centrifugation at 10,800×g, the pellets were resuspended in 2 mL PBS, and centrifuged once more for 10 min at 13,000×g to remove remaining bacterial residues.
[0672] Phage Displaying STxB (Φ_STxB) and STxB_Mut_D18E; G62T (Φ_STxB_Mut) Expression
[0673] An overnight culture of TG1 cells transformed with pHEN2_STxB or pHEN2_STxB_mut, was diluted in 10 mL of 2×YT medium, 100 μg/mL ampicillin, 1% glucose, grown from an OD600 of 0.1 to 0.5, infected with 4 μL of 1013 helper phages M13KO7 (NEB), and incubated for 30 min at 37° C. in a water bath. Bacteria were then centrifuged for 10 min at room temperature at 3,200×g, and resuspended in 50 mL 2×YT (powder from sigma Aldrich Y2377-250G) without glucose, but containing ampicillin 100 μg/mL and kanamycin at 50 μg/mL. After overnight growth at 30° C., Φ_STxB or Φ_STxB_mut were harvested by centrifugation for 15 min at 3200×g. Supernatants containing phages were stored for few days at 4° C.
[0674] Immunoblotting of Φ_STxB/φ_STxB_noAmb/Φ_STxB_mut
[0675] 30 μl of TG1 supernatant containing Φ_STxB, Φ_STxB_noAmb or Φ_STxB_mut were heated to 90° C. with 4× denaturing blue loading dye, and loaded on 4-15% gradient polyacrylamide gels (Mini-Protean TGX precast gel, Biorad). After 40 min migration at 150V, and transfer on a nitrocellulose membrane, anti_pIII mouse antibody (1/1,000 dilution, New England Biolabs (NEB)) was used with appropriate anti-mouse HRP secondary antibodies (Beckman Coulter).
[0676] Mass spectrometry analysis of STxB_mut_PIII fusion. 30 μl of TG1 supernatant containing Φ_STxB_mut were heated to 90° C. with 5× blue loading dye, and loaded on 4-15% gradient polyacrylamide gels (Mini-Protean TGX precast gel, Biorad). After 40 min migration at 150V, the gel was stained with LabSafe Gel Blue (Biosciences). The corresponding band was cut, and the sample was trypsinized for de novo peptide sequencing.
[0677] Immunoblotting of Φ_STxB and Φ_STxB_mut
[0678] 30 μl of TG1 supernatant containing Φ_STxB or Φ_STxB_mut were heated at 90° C. with 5× blue loading dye, and loaded on 4-15% gradient polyacrylamide gels (Mini-Protean TGX precast gel, Biorad). After 40 min migration at 150V, and transfer on a nitrocellulose membrane anti_pIII mouse antibody (1/1000 dilution, New England Biolabs (NEB)) was used with appropriate anti-mouse HRP secondary antibodies, i.e. from Jackson immunoresearch ref 715-035-151.
[0679] Mass Spectrometry Analysis of STxB_Mut_PIII Fusion
[0680] 30 μl of TG1 supernatant containing Φ_STxB_mut were heated at 90° C. with 5× blue loading dye, and loaded on 4-15% gradient polyacrylamide gels (Mini-Protean TGX precast gel, Biorad). After 40 min migration at 150V, the gel was stained with LabSafe Gel Blue (Biosciences). The corresponding band was cut, and the sample was trypsinized for de novo peptide sequencing.
[0681] Binding of Φ_STxB/Φ_STxB_noAmb/STxB_mut to Cells
[0682] 20 μl of precipitated phages diluted in 200 μl PBS BSA 2% were incubated for 45 min at 4° C. on a wheel for blocking.
For immunofluorescence experiments, 70,000 Gb3.sup.+CHO, GB3.sup.−CHO, C2TA, or C2TA_PPMP cells were seeded on coverslips in P24 plates, and grown overnight at 37° C. under 5% CO.sub.2. After 3 washes with DMEM/F12 medium at 4° C., cells were incubated for 45 min at 4° C. in PBS BSA 2% for blocking. After removal of the blocking solution, 200 uL phage solution were added and incubated on cells for 45 min at 4° C. The cells were washed 3 times with PBS BSA 2%, again 3 times with PBS′, and fixed with a solution of 1% PFA for 15 min at room temperature. After neutralization with a solution of 50 mM NH4Cl, cells were washed 3 times with PBS BSA 2% and labeled with appropriate M13 phage coat protein antibody (ThermoFisher), washed again 3 times, labeled with anti-mouse A488-modified antibody, and washed 3 times. Images were acquired on an epifluorescence microscope (Leica DM 6000B), and processed with ImageJ software.
For flow cytometry experiments, 100,000 cells per conditions (Gb3.sup.+CHO, GB3.sup.−CHO, C2TA, C2TA_PPMP) were incubated for 45 min at 4° C. in PBS BSA 2%. After this saturation step, cells were centrifuged for 5 min at 600×g, incubated for 45 min at 4° C. with Φ_STxB, Φ_STxB_noAmb or Φ_STxB_mut plus appropriate controls, washed 3 times, and then incubated with mouse anti-M13 antibody (GE) and anti-mouse_488 antibody (Molecular Probes, Invitrogen). STxB was directly labeled with Alexa Fluor 488 NHS ester dyes (ThermoFisher Scientific). Cells were fixed, and flow cytometry was performed. Gating was done on control cells, and readings were recorded in order to get 5,000 events in the gate at fast speed with multiple resuspensions of cells using BD Accuri C6 Cytometer. Data were analyzed using Flowjo software.
[0683] Binding of Φ_STxB and Φ_STxB_mut on Cells and Flow Cytometry
[0684] 20 μl of phages were incubated for 30 min at 4° C. in 100 μl PBS BSA 2%. 100,000 cells per conditions (Gb3.sup.+CHO, GB3.sup.−CHO, C2TA, C2TA_PPMP) were incubated for 30 min in PBS-BSA 2%. After this saturation step, cells were centrifuged for 5 min at 600×g, incubated for 45 min at 4° C. with Φ_STxB or Φ_STxB_mut plus appropriate controls, washed 3 times, and then incubated with mouse anti-M13 antibody (GE) and anti-mouse_488 antibody (Molecular Probes, Invitrogen). STxB was directly labeled with Alexa Fluor (registered trade mark) 488 NHS ester dyes (ThermoFisher Scientific). Cells were fixed, and flow cytometry was performed. Gating was done on control cells, and readings were recorded in order to get 5,000 events in the gate at fast speed with multiple resuspensions of cells using BD Accuri (trade mark) C6 Cytometer. Data were analyzed using Flowjo software.
[0685] Preparation of Magnetic Liposomes
[0686] To generate Gb3-containing liposomes, 150 μL of 5 mg/mL of 1,2-dioleoyl-sn-glycero-3-phosphocholine, 18:1 (8,9-Cis) PC, so-called DOPC (Avanti) were mixed with 100 μL of 1 mg/mL of ceramide trihexosides, or Gb3, (Matreya) in a glass tube. Solvents were removed by evaporation using nitrogen or argon to generate an homogenous lipid film on the wall of the glass tube. Remaining solvents were removed by drying under vacuum for 2 hrs.
[0687] The lipid mix was then rehydrated with a solution of 1 mL PBS at 65° C. containing 10 μL iron (II, III) oxide magnetic fluid (7% stock concentration—PlasmaChem). The solution was vortexed for 5 min. 3 cycles of freezing in ethanol/dry ice mix, thawing in water bath at 65° C. and 1 min mixing were performed.
[0688] The liposome mixture was then passed 17 times using 1 μm filters through an extruder (Avanti) that was also pre-heated to 65° C. Liposomes were then washed 3 times by recruitment on a magnet, removal of the supernatant and resuspension with a solution of PBS-BSA 2%. Liposomes were directly used or stored at 4° C. for a couple of days maximum. The same procedure was used to generate control liposomes without Gb3.
[0689] Characterization of Magnetic Liposomes by Electron Microscopy
[0690] Different dilution of magnetic liposomes preparation were made into water and deposed on carbon-coated copper grids that were ionized by glow discharge (at 1-2 mA for 30 s). After drying of the sample, negative staining was performed using uranyl acetate at 2% for 1 min. The grids were washed with water and dried. Images were captures using Tecnai Spirit electron microscope.
[0691] STxB Recruitment onto Liposomes
[0692] For blocking, a 1 mL solution of PBS-BSA 2%-500 nM STxB was incubated 1 hr at 4° C. on a wheel. This solution was added onto a 200 μL of magnetic liposomes preparations (see below), and incubated on a wheel for 45 min at 4° C. 5 washes were performed in a 15 mL tubes with PBS BSA 2% by collecting the magnetic liposomes on a magnet. 5 additional washes were performed in a new 15 mL tube with PBS. STxB recruitment was analyzed by immunobloting and FACS.
[0693] In the first case, liposomes were resuspended in 150 uL PBS to which 50 μL denaturing blue loading dye was added. The solution was boiled for 10 min at 90° C., and 50 μL were loaded on a 4-15% gradient polyacrylamide gel (Mini-Protean TGX precast gel, Biorad). After 40 min migration at 150V, and transfer on a nitrocellulose membrane, anti-STxB (13C4) antibody was used with appropriate anti-mouse HRP secondary antibodies.
[0694] For the FACS analysis, Alexa_488 labeled STxB was used. The liposomes were resuspended after washed in 300 μL and passed through a flow cytometer (BD Accuri C6, BD Biosciences).
[0695] Phage Recruitment onto Magnetic Liposomes
[0696] For blocking, 100 μL of freshly produced and precipitated phages (around 10.sup.12 phages) were diluted into 1 mL final volume of PBS-BSA 2% and incubated 1 hr at 4° C. on a wheel. This solution was added onto 200 uL of magnetic liposomes preparations (see below), and incubated on the wheel for 45 min at 4° C. 5 washes were performed in a 15 mL tubes with PBS BSA 2% by collecting the magnetic liposomes on a magnet. 5 additional washes were performed in a new 15 mL tube with PBS. The phage recruitment was analyzed by immunobloting and FACS.
[0697] In the first case, the liposomes were resuspended in 150 μL PBS to which 50 μL denaturing blue loading dye was added. The solution was boiled for 10 min at 90° C., and 50 uL were loaded on a 4-15% gradient polyacrylamide gel (Mini-Protean TGX precast gel, Biorad). After 40 min migration at 150V, and transfer on a nitrocellulose membrane, anti_pIII mouse antibody (1/1000 dilution, New England Biolabs (NEB)) was used with appropriate anti-mouse HRP secondary antibodies.
[0698] For the FACS analysis, the liposomes were resuspended 1 mL solution of PBS-BSA 2% with anti-M13 antibody and incubated on a wheel for 45 min at 4° C. 3 washes were performed before incubation with Alexa488 labeled anti-mouse antibody. After 3 more washes, the liposome solution was passed through a flow cytometer (BD Accuri C6, BD Biosciences).
[0699] Simulation of Phage Display Selection on Magnetic Liposomes
[0700] 10.sup.12 Φ_STxB_mut were mixed with 10.sup.8 Φ_STxB (ratio of 1/10 000) in 1 mL PBS-2% BSA, incubated for blocking 1 hr at 4° C. on a wheel. The solution was added to 200 uL of magnetic liposomes preparations (see below) and incubated on a wheel for 45 min at 4° C. 10 washes were performed in a 15 mL tubes with PBS BSA 2% by collecting the magnetic liposomes on a magnet. 10 additional washes were performed in a new 15 mL tube with PBS.
[0701] Phages were eluted using 1 mL of a solution of 50% Trypsin in PBS at 37° C. for 10 min. After addition of 500 uL SVF, 750 uL of the solution was used to infect 10 mL TG1 bacteria (DO=0.5) for 30 min at 37° C. without agitation. 100 uL was used to prepare several dilutions of the bacterial solution (10-1, 10-2 and 10-3), which were then seeded on 2×TY agar ampicillin 1% glucose plates which were incubated overnight at 37° C.
[0702] The next day, 24 clones were collected, and grown overnight at 37° C. in 5 mL 2×TY ampicillin 1% glucose solution. Bacterial plasmid DNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen) and sequenced (GATC).
[0703] Design of the STxB Variant Library
[0704] The twenty positions—Thr1, Asp16, Asp17, Asp18, Thr21, Glu28, Leu29, Phe30, Thr31, Asn32, Arg33, Trp34, Asn35, Thr54, Asn55, Ala56, Gly60, Gly62, Phe63, Ser64—involved in the binding of STxB to Gb3 (Ling et al., 1998) were chosen for the creation of the combinatorial library.
[0705] To reach a total population of 1.5×10.sup.10 variants, three to four alternative amino acids are possible at each of the twenty positions, as described herein. The alternative amino acids were selected with the help of the pfam platform website for sequence alignment. For this, 286 STxB homologues from Uniprot database, and 211 homologues from NCBI database were aligned, and results compiled with the Hidden Markov Model (HMM) logo generation software. The most represented amino acids were chosen in order to maximize the chance of getting properly folded pentameric STxB variants.
[0706] The library was then synthetized by the timer oligonucleotide synthesis (TRIM technology, by GeneArt), amplified, and sub-cloned into the proper phen2 expression system (GeneArt), to reach a final diversity of 10.sup.9 clones.
[0707] Characterization of the Library
[0708] The content of the library was characterized by sequencing by both, by the GeneArt company and in the laboratory (not shown). Different dilutions of the clones obtained from GeneArt were plated on 2×YT 100 μg/mL ampicillin, glucose 1%, agar plate. 96 clones were picked and sequenced using appropriate primers (GATC). Sequences were processed and aligned using CLC Workbench software.
[0709] Phage Library Amplification
[0710] 100 μL of TG1 bacteria from GeneArt (1.68×10.sup.11 clones/mL) were grown in 250 mL 2×YT, 100 μg/mL Ampicillin, 1% glucose at 37° C. to reach DO=0.5. 75 mL of culture were infected with 6×10.sup.11 M13 helper phage and incubated 30 min at 37° C. without agitation. The solution was then centrifuged for 20 min at 3,200×g at room temperature. The pellet was resuspended in 1.5 L of 2×YT, 100 μg/mL ampicillin, 100 μg/mL kanamycin and grown overnight at 30° C.
[0711] 500 mL were centrifuged at 10,800×g at 4° C. ⅕.sup.th volume of 30% PEG, 2.5MNaCl solution were added to the supernatant and incubated 1 hr at 4° c. without agitation. The solution was then centrifuged for 30 min at 10,800×g at 4° C., and the pellet was resuspended in 40 mL sterilized deionized water. 8 mL of 30% PEG, 2.5M NaCl were added, and the solution was incubated again for 30 min at 4° C. The solution was finally centrifuged for 30 min at 10,800×g at 4° C., and the pellet was resuspended in 16.5 mL PBS 15% glycerol.
[0712] After a last centrifugation step at 13,000×g at 4° C., the solution was aliquoted and stored at −80° C. 5 μL were used to titer the phage concentration by infection TG1 bacteria with different dilutions of the phage stock.
[0713] Phage Display Selection of Gb3 Binders
[0714] Day 1: Magnetic Liposomes Preparation
1 mL of 1 μm Gb3.sup.+ and Gb3.sup.− magnetic liposome solution was produced as described previously. Liposomes were then washed 3 times by recruitment on a magnet, removal of the supernatant and resuspension with a solution of PBS-BSA 2%. The liposomes solution was divided in two, resuspended in 1.5 mL PBS-2% BSA, and incubated overnight at 4° C. on the wheel.
[0715] TG1 bacteria were grown in 50 mL M9 minimal medium complemented with 2 μM MgSO.sub.4, 1% glucose, 0.1% thiamine, overnight at 37° C. This culture was kept at 4° C., and used for a maximum of 3 weeks.
[0716] Day 2: Phage Display Selection on Liposomes
One aliquot of the STxB library stock was thawed. 100 μL was diluted in 900 μL PBS-2% BSA for 1 hr at 4° C. on a wheel. Two 15 mL tubes were coated with PBS-2% BSA on ice. Gb3-liposomes were recruited on a magnet for 10-15 min at 4° C., and the 1 mL phage solution was added and incubated on a wheel at 4° C. for 1 hr.
In parallel, 3 solutions of 15 mL 2×YT 1% glucose inoculated with 1/50, 1/100 and 1/200 TG1 stock solution were incubated at 37° C. with agitation.
[0717] The liposomes were then collected, and the supernatant was added to the second solution of Gb3.sup.− liposomes, incubated 1 hr at 4° C. on a wheel. Liposomes were collected, and the supernatant was removed. After these two depletion steps, the phage supernatant was added to Gb3.sup.+ magnetic liposomes, and incubated 1 hr at 4° C. on a wheel. The liposomes were then collected on a magnet, and resuspended in 10 mL PBS-2% BSA in the first pre-coated 15 mL tube. 10× washes were performed alternating 5 min recruitment on a magnet at 4° C. and resuspension in 10 mL cold PBS-2% BSA solution. The liposomes were transferred to the second pre-coated 15 mL tube and 5× washes in cold PBS-2% BSA and 5× washes in cold PBS were performed.
[0718] Finally, the liposomes were collected on a magnet, and phages were eluted using 1 mL of a solution of 50% Trypsin in PBS at 37° C. for 10 min. After addition of 500 uL SVF, 750 uL of the solution was used to infect 10 mL TG1 bacteria (D0=0.5) for 30 min at 37° C. without agitation. 100 μL was used to make several dilutions of the bacterial solution (10.sup.−1, 10.sup.−2 and 101. Bacteria were plated on 2×TY agar plates with 100 μg/mL ampicillin 1% glucose, which were incubated overnight at 37° C. to calculate the output concentration of phages.
[0719] The remaining 10 mL TG1 solutions was centrifuged and resuspended in 1.8 mL 2×YT. 600 μL were plated on 3 large 2×YT agar plates containing 100 μg/mL ampicillin 1% glucose, and grown overnight at 37° C.
[0720] Day 3: Amplification of Selected Phages
[0721] The output and the input concentration of phages were calculated and the clones on the three large agar plates were collected in 10 mL 2×YT, 30% glycerol, the bacterial concentration was measured and the solution was stored at −20° C. consisting in the Bacterial stock R1.
[0722] To amplify the phages, an aliquot of bacterial stock R1 was diluted in 100 mL 2×YT 100 μg/mL ampicillin 1% glucose to reach DO=0.05, and grown to reach DO=0.5. 10 mL were infected with 8×10.sup.10 helper phages, and incubated for 30 min at 37° C. without agitation. The 10 mL solution was then centrifuged for 20 min at 3,200×g at room temperature. The pellet was resuspended in 50 mL of 2×YT, 100 μg/mL ampicillin, 100 μg/mL kanamycin, and grown overnight at 30° C.
40 mL were centrifuged at 10,800×g at 4° C. ⅕.sup.th volume of 30% PEG, 2.5 MNaCl solution were added to the supernatant, and incubated 1 hr at 4° C. without agitation. The solution was then centrifuged for 30 min at 10,800×g at 4° C., and the pellet was resuspended in 2 mL cold PBS. After a last centrifugation step at 13,000×g at 4° C., 100 μL were used for the second round of selection, and 5 uL for the calculation of the input concentration.
[0723] Following the same procedure, 3 rounds of selection on liposomes were performed. A final round of selection was performed on CHO cells.
[0724] R4 Selection on CHO Cells:
[0725] 20×10.sup.6 Gb3.sup.−CHO and 10×10.sup.6 Gb3+CHO cells were trypsinized, and incubated in 10 mL PBS 2% BSA for 1 hr at 4° C. on a wheel. 100 uL of amplified phages from R3 were diluted in 1 mL PBS 2% BSA, and incubated for 1 hr at 4° C. on a wheel. 10×10.sup.6 Gb3.sup.− cells were centrifuged for 5 min at 600×g at 4° C., resuspended in 1 mL phage solution, and incubated for 1 hr at 4° C. on a wheel. Cells were centrifuged for 5 min at 600×g at 4° C. The supernatant was used to resuspended the second half of the Gb3-CHO for a second step of depletion of 1 hr at 4° C. on a wheel.
[0726] Cells were centrifuged at 600×g at 4° C., and the supernatant was collected. The 10 mL solution of Gb3+CHO cells were centrifuged at 600×g at 4° C., resuspended with the 1 mL solution of depleted phages, and incubated 1 hr at 4° C. on a wheel. 10× washes were performed consisting of cycles of centrifugation at 600×g at 4° C. and resuspension in 10 mL cold PBS 2% BSA.
[0727] A final wash in PBS was performed, and phages were eluted and used to infect TG1 bacteria as described previously. One day later, the bacteria were collected from the agar plates and stored in 2×YT 30% glycerol at −20° C. (Bacterial stock R4 CHO).
[0728] Characterization of Gb3 Binders
[0729] 50 μL of Bacterial stock R4 CHO were centrifuged, and phamegid DNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen). 5 ng of DNA preparation were used to transform competent TG1 cells, which were seeded on 2×YT agar plates containing 100 μg/mL ampicillin, 1% glucose, and grown overnight at 37° C. 96 clones were picked and inoculated in 400 μl 2×YT 100 μg/mL ampicillin, 1% glucose, grown overnight at 37° C. The 96 clones were stored in 400 uL 2×YT 30% glycerol at −20 C.
[0730] 5 μL were used for sequencing (GATC). The sequences were analyzed and aligned using CLC workbench software.
[0731] a) Phage Candidate Screening by Flow Cytometry on HeLA C2TA Cells
Expression and Production:
[0732] In 96 deep well plates, 2 μL of each of the 96 clones were used to inoculate 200 μL of 2×YT solution containing 100 μg/mL ampicillin 1% glucose, and grown to reach DO=0.5. Two wells were used to grow appropriate controls (Φ_STxB and Φ_STxB_mut). 1.5×10.sup.9 helper phages were used to infect each well, and the plates were incubated at 37° C. for 30 min without agitation. Plates was then centrifuged, and the bacteria were resuspended with 600 uL 2×YT 100 μg/mL ampicillin, 50 μg/mL Kanamycin, and grown overnight at 30° C. with agitation. Plates were centrifuged at 3,200×g for 30 min at 4° C.
Flow Cytometry Experiment:
[0733] 200 μL of supernatant was used for the binding experiment. 100,000 C2TA, C2TA_PPMP cells per conditions were incubated at 4° C. for 45 min in PBS BSA 2%. After this saturation step, cells were centrifuged for 5 min at 600×g, incubated for 45 min at 4° C. with 200 μL of phage supernatant, washed 3 times, and then incubated with mouse anti-M13 antibody (GE) and anti-mouse_488 antibody (Molecular Probes, Invitrogen). Cells were fixed, and flow cytometry was performed. Gating was done on control cells, and readings were recorded in order to get >5,000 events in the gate at fast speed using BD Accuri C6 Cytometer. Data were analyzed using Flowjo software.
[0734] b) Binding of Phage Candidates and Characterization by Immunofluorescence on CHO Cells
Expression and Production:
[0735] In 24 deep well plates, 2 μL of each of the 14 selected clones were used to inoculate 200 μL of 2×YT solution containing 100 μg/mL ampicillin 1% glucose, and grown to reach DO=0.5. Two wells were used to grow appropriate controls (Φ_STxB and Φ_STxB_mut). 1.5×10.sup.9 helper phages were used to infect each well, and plates were incubated at 37° C. for 30 min without agitation. Plates were then centrifuge, and the bacteria were resuspended with 600 μL 2×YT 100 μg/mL ampicillin, 50 μg/mL Kanamycin, and grown overnight at 30° C. with agitation. Plates were centrifuged at 3,200×g for 30 min at 4° C.
Immunoblotting:
[0736] 25λL of 4× denaturing blue loading dye was added to 75 μL of supernatant of each phage candidate, and the solution was boiled for 10 min at 90° C. 50 μL were loaded on a 4-15% gradient polyacrylamide gel (Mini-Protean TGX precast gel, Biorad). After 40 min migration at 150V, and transfer on a nitrocellulose membrane, anti_pIII mouse antibody (1/1000 dilution, New England Biolabs (NEB)) was used with appropriate anti-mouse HRP secondary antibodies.
Immunofluorescence:
[0737] 200 μL of supernatant were used for the binding experiment. 70,000 Gb3.sup.+CHO and GB3.sup.−CHO cells were seeded on coverslips in P24 plates, and grown overnight at 37° C. under 5% CO.sub.2. After 3 times washes with cold PBS 2% BSA, cells were incubated for 45 min at 4° C. in PBS BSA 2% for blocking. After removal of the blocking solution, the 200 μL phage supernatant solution was added and incubated on cells for 45 min at 4° C. The cells were washed 3 times with PBS BSA 2% and again 3 times with PBS′, and fixed with a solution of 1% PFA for 15 min at room temperature. After neutralization with a solution of 50 mM NH4Cl, cells were washed 3 times with PBS BSA 2%, labeled with appropriate M13 Phage coat protein antibody (ThermoFisher), washed again 3 times, labeled with anti-mouse A488-labeled antibody, and washed 3 more times. Images were acquired on an epifluorescence microscope (Leica DM 6000B), and processed with ImageJ software.
B. Results
M13 Bacteriophages Displaying STxB (Φ_STxB) are Able to Specifically Bind Gb3 Positive Cells
[0738] Displaying STxB and STxB mutant (STxB_mut_D18E; G62T) on M13 bacteriophages The STxB gene was fused to the one coding for the coat protein pIII of M13 bacteriophage to drive the expression of a corresponding fusion protein (
[0739] A mutant of STxB (STxBmut-D18E; G62T)-SEQ ID NO: 32, which is not able to bind Gb3 anymore, was also presented on M13 bacteriophage. Correct expression into TG1 supernatant was also confirmed by immunoblotting and mass spectrometry analysis which revealed 13 matching peptides confirming the presence of the mutations and the fusion to the PIII protein (
[0740] The concentration and the infection properties of those phages were tested by a titration assay, where different dilutions of phage preparation were used to infect TG1 bacteria.
[0741] Stable and functional expression of globotriaosylceramide (Gb3) at the plasma membrane of Chinese Hamster Ovarian (CHO) cells
[0742] The CHO cell line was chosen to generate a cell system for which Gb3-positive and negative cells were available on the same genetic background. CHO cells normally lack expression of lactosylceramide α1,4-galactosyltransferase, the enzyme that catalyzes the conversion of lactosylceramide into Gb3 and its derivatives. To generate a Gb3-positive CHO clone, the Gb3 synthase gene under control of the cytomegalovirus promoter was stably transfected into these cells. The expression of Gb3 and its localization at the plasma membrane was then demonstrated using a natural Gb3 ligand, the B-subunit of Shiga Toxin (STxB).
[0743] Clear binding of STxB was observed by immunofluorescence when the protein was incubated with CHO cells that had been transfected with the Gb3 synthase gene (Gb3.sup.+CHO), when compared to non-Gb3 synthase-transfected control cells (Gb3.sup.−CHO) (
Specific Binding of Phage Displaying STxB on Gb3 Positive Cells
[0744] Gb3.sup.+CHO and Gb3.sup.−CHO cells were incubated with the phage-STxB conjugate (Φ_STxB) for 45 min on ice (no endocytosis). A clear binding was observed to Gb3.sup.+CHO cells, but not to Gb3.sup.−CHO cells, when analyzed by immunofluorescence microscopy (
[0745] The binding Φ_STxB to cells was further analyzed by flow cytometry. After incubation with Φ_STxB, a shift in the mean fluorescence intensity was observed between Gb3+CHO and Gb3.sup.−CHO cells, demonstrating that STxB was functionally expressed at the surface of the phages (
[0746] In order to confirm the specific binding of this Φ_STxB on Gb3, the same binding experiments were performed on C2TA cells, which naturally expressed Gb3. The loss of binding by treatment with PPMP, a specific inhibitor of glycosphyngolipids synthesis, strongly suggests the specific recognition of the Gb3 targets (
[0747] Finally, the binding of of STxB mutant (STxB_mut-D18E; G62T) presenting phages was analyzed by immunofluorescence microscopy and flow cytometry (
[0748] These data demonstrate that STxB is functional at the phage surface, and its binding activity is unperturbed. Inventors proposed to exploit this configuration to produce screening libraries in which the STxB gene is systematically mutated and the phages express peptides of the invention that gain binding activity against glycosphingolipids to which commonly known STxB moieties do not bind naturally (
Conformational Study of STxB Presented on the M13 Bacteriophage
[0749] Part of the conception of the present invention, the actual required conformation of STxB on a phage was investigated. Indeed, STxB molecules are only found in solution as a pentamer. Each phage particles are composed of five pIII proteins that are used for the display of the protein.
[0750] The inventors considered how monomers could be presented on phages. Two hypothesis were envisioned (
[0753] Indeed, the presence of an amber stop codon between the STxB gene and the one of the pIII has been designed to allow for the expression of either free STxB protein or STxB_pIII fusion protein with a ratio of approximately 50% each (see Experimental Section herein). Furthermore, to determine the physical rationale underlying the possibility to preform the present invention, two types of helper phage have been used. Standard helper phage possesses in their genome the gene encoding for pIII. The production of pIII protein in the bacteria results from both the expression of the viral pIII gene and the bacterial gene. A phage variant called hyperphage doesn't have this viral pIII gene (Rondot, Koch, Breitling, & Dübel, 2001). The expression of the pIII protein in this case results only from the expression of the bacterial pIII gene. Where the use of the amber stop codon could results in the expression of non-fused STxB proteins, the use of standard helper phage could results in the presentation of non-fused pIII protein.
[0754] By using a combination of expression systems were STxB monomers could be expressed either at all time (without the amber stop codon), either only from time to time (presence of both fused and non-fused forms) (with the amber stop codon) in fusion with the pIII protein and a combination of helper phage particles that could or could not present non-fused pIII protein on the phage capsid, the inventors have been able to show that the second hypothesis was correct. Indeed, the production of fully fused STxB_pIII resulted in a phage preparation, both with the use of hyper and helper phages, which was not able to bind Gb3 positive cells anymore (The correct expression of phage particles was confirmed by immunoblotting and the binding by immunofluorescence
[0755] These data demonstrate the an ingenious design from the inventors to enable production of STxB properly folded and functional when displayed on the M13 bacteriophage, potentially driving the binding of the phage particle to Gb3 positive cells.
Magnetic Liposome-Based Phage Display
[0756] As a proof of concept, a strategy increasing the chances that GSLs are presented in their “physiological environment of the lipid bilayer has been devised, using magnetic liposomes.
Generation of GSL-Containing Magnetic Liposomes
[0757] Magnetic DOPC-based liposomes of 1 μm diameter containing Gb3 (or not) were generated. By electron microscopy, rounded and electron dense structures could be observed (
Specific Recruitment of STxB and Φ_STxB onto Liposomes
[0758] In order to confirm the potential of Gb3-containing magnetic liposomes for phage display selection, STxB or Φ_STxB recruitment was analyzed by immunobloting or flow cytometry. STxB and Φ_STxB were only recruited onto Gb3-containing liposomes, which was demonstrated by the presence of a pIII_STxB band on gels (
Simulation of Phage Display Selection on Magnetic Liposomes
[0759] To finally assess the power of magnetic liposomes for phage display selection, a single round of selection was performed with a mixture of Φ_STxB and Φ_STxB_mut at a ratio of 1 to 10,000. After 2 depletion steps on Gb3-negative magnetic liposomes, the remaining phages were applied to the Gb3-positive magnetic liposomes. After extensive washes, phages were collected and used to infect TG1 bacteria. Sequencing was performed on the clones obtained after selection and a ratio of 1 to 24 between Φ_STxB and Φ_STxB_mut has been assessed. This demonstrated the potential of magnetic liposomes for phage display selection of GSL binders from a protein library.
Selection of Gb3-Specific STxB Mutants by Phage Display
[0760] As a further step in the proof of concept exploration of our phage display selection strategy of STxB variants, a complete screening was performed on Gb3-containing magnetic liposomes, using a library of 1.46×10.sup.10 variants of STxB.
Design of a STxB Variant Library
[0761] STxB contains 3 Gb3 binding sites per B-fragment monomer. Twenty positions out of the sixty-nine (28.9% of the sequence) of the STxB monomer were previously shown to be involved in the binding of the Gb3 (Ling et al., 1998). These 20 positions were chosen for the creation of a combinatorial library. Three to four amino acids can be chosen at each of the twenty positions for a total number of 1.46×10.sup.10 variants, as described herein. The alternative amino acids were selected with the help of the pfam platform website (http://pfam.xfam.org/) for sequence alignment. For this, 286 STxB homologues from Uniprot database, and 211 homologues from NCBI database were aligned, and results compiled with the Hidden Markov Model (HMM) logo generation tools from the platform. The most represented amino acids were chosen in order to maximize the chance of obtaining properly folded pentameric STxB variants. The library was then synthetized by the trimer oligonucleotide synthesis (TRIM technology, by GeneArt), amplified, and sub-cloned into the proper phen2 expression system to obtain a library of fusion proteins between STxB variant and the pIII coat protein of the M13 phage. The total number of transformants was 1.03×10.sup.9 cfu. The content of the library was characterized by sequencing (not shown). The library of phages was produced and stored in 30% glycerol at −80° C. The phages were produced at a final concentration of around 10.sup.12 phages/m L.
Library Characterization
[0762] The quality and the diversity of the library were validated by Sanger sequencing. 96 colonies from transformation plates were picked and sequenced (GeneArt). 71 of the 96 clones (73%) contained correct sequences. In the 71 sequences, all the desired mutations were found with a minimum of 8% occurrence for the amino acid F30. The remaining clones were either not showing clean sequencing data, or incorrect sequences (insertions, deletions and substitutions). To confirm these data, we also sequenced 96 clones ourselves. 76 out of the 96 clones (79%) contained correct sequences. All the desired mutations were also found with a minimum of 3% occurrence for the amino acid G62, all the other mutations showing an occurrence over 10%. The remaining clones were either not showing clean sequencing data, or incorrect sequences (insertions, deletions or substitutions).
Phage Display Selection of Gb3 Binders
[0763] The first selection was performed against Gb3 in order to assess the potential for selecting non natural sequences of STxB that keep their ability to bind Gb3. Three rounds of selection were performed on magnetic liposomes, where each round consisted in two steps of depletion on Gb3-negative liposomes, to remove unspecific binders, followed by 1 step of selection on Gb3-positive liposomes (
[0764] 96 clones were picked, sequenced and analyzed for their specific binding to GSLs by flow cytometry on HeLa C2TA cells and HeLa C2TA cells treated with PPMP (inhibition of GSL synthesis).
[0765] Of the 96 clones, 21 (A03, A06, A08, B02, B05, B12, C02, C03, C06, D04, D07, D10, D12, E7, F12, G05, G11, H03, H07, H11) showed completely “homologous” sequences with wildtype STxB. Of these 21, 13 showed significant GSL-specific binding by FACS (
[0766] Of these 13 clones, 5 unique STxB variant sequences were identified (
[0767] The occurrence of each amino acids at each position was analyzed. Interestingly, 8 positions (D17, D18, E28, T31, W34, N35, N55, G60) were never mutated.
[0768] Of note, by presenting those phages to Gb3 positive liposomes and after extensive washing, the remaining pulled phages population was highly enriched in relevant phages (efficient selection). It will be appreciated that the skilled person is aware that liposomes containing any commercial glycosphingolipids could be in principle generated and used to screen a library of STxB variants, whose target(s) potentially differ from Gb3, using the protolcol disclosed herein. It is therefore contemplated that the screening method using GSLs presenting magnetic liposomes of the present invention and described herein can be performed, according to particular embodiments: [0769] Through possibly parallel screenings of STxB libraries carried out separately on liposomes batches, each distinct liposome batch specifically presenting one particular purified GSL, or [0770] by performing a screening on liposomes containing a mix of GSLs, in particular a mix of GSLs that do not contain Gb3 to preselect a sub-population of STxB mutants that bind other GSLs.
Characterization of Gb3 Binders
[0771] The 5 unique potential Gb3 binders were further characterized by immunofluorescence on CHO cells. Either the phages displaying the STxB variants, or the STxB variants themselves were produced in TG1 bacteria. The proper expression was characterized by immunobloting. Each clone was tested for binding on Gb3+CHO and Gb3-CHO cells. Each of them showed significant binding on Gb3+CHO, and not on Gb3-CHO, whatever they are displayed on the phage (
Ongoing Experiments
Phage Display Selection of Binders of Other GSLs (for Instance GM3)
[0772] The selection is being performed against another GSL than Gb3, as for instance GM3 in order to assess the potential for selecting STxB variants with another binding specificity. Several rounds of selection (from two to five) are performed on magnetic liposomes, where each round consisted in two steps of depletion on GM3-negative liposomes, to remove unspecific binders, followed by 1 step of selection on GM3-positive liposomes (
[0773] Accordingly, this can be implemented for a large diversity of GSL as disclosed herein, with the possibility to perform the selection on liposomes, using the purified GSL species, on cells which express the GSL of interest, or also on patient sample taking from biopsy.