Dendritic cell inhibitory proteins from ticks

Abstract

The present invention provides a dendritic cell modulatory protein which modulates, and preferably inhibits, the differentiation and/or maturation of mammalian dendritic cells. The invention also provides proteins comprising conserved motifs found in such proteins as well as pharmaceutical compositions comprising the dendritic cell modulatory protein and homologs and active fragments thereof, antibodies thereto and methods of treatment which utilize such proteins, homologs, fragments and antibodies.

Claims

1. A method of inhibiting the differentiation and/or maturation of dendritic cells comprising contacting said dendritic cells with a purified protein, wherein said protein comprises the amino acid sequence of SEQ ID NO:24 or SEQ ID NO:27.

2. The method of claim 1, wherein the protein is a recombinant protein.

3. The method of claim 1, wherein the protein is recovered from a cultured host cell.

4. A method of inhibiting the differentiation and/or maturation of dendritic cells in a human or an animal comprising administering to said human or animal a pharmaceutical composition comprising a pharmaceutically effective amount of an isolated protein comprising the amino acid sequence of SEQ ID NO:24 or SEQ ID NO:27.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1 shows the DNA and amino acid sequence of RaA (SEQ ID NOs: 1 & 2).

(2) FIG. 2 shows the alignment of RaA (SEQ ID NO: 2) with Japanin (SEQ ID NO: 8). The alignment of RaA with Japanin was obtained using clustalW with the following options: gap extension penalty-0.1; gap opening penalty-10.0; hydrophilic residues=G, P, S, N, D, Q, E, R or K; matrix=gonnet. Of the 131 residues that form the alignment, 61 (46.56%) are identical [indicated by *], 26 (19.85%) are strongly similar [:] and 22 (16.79%) are weakly similar H.

(3) FIG. 3 shows the DNA and amino acid sequence of RaB (SEQ ID NOs: 3 & 4).

(4) FIG. 4 shows the alignment of RaB (SEQ ID NO: 4) with Japanin (SEQ ID NO: 8). The alignment of RaB with Japanin was obtained using clustalW with the following options: gap extension penalty=0.1; gap opening penalty=10.0; hydrophilic residues=G, P, S, N, D, Q, E, R or K; matrix=gonnet. Of the 138 residues that form the alignment, 73 (52.9%) are identical [indicated by *], 30 (21.74%) are strongly similar [:] and 12 (8.7%) are weakly similar [.].

(5) FIG. 5 shows the DNA and amino acid sequence of Rs1 (SEQ ID NOs: 5 & 6).

(6) FIG. 6 shows the alignment of Rs1 (SEQ ID NO: 6) with Japanin (SEQ ID NO: 8). The alignment of Rs1 with Japanin was obtained using clustalW with the following options: gap extension penalty=0.1; gap opening penalty=10.0; hydrophilic residues=G, P, S, N, D, Q, E, R or K; matrix=gonnet. Of the 131 residues that form the alignment, 105 (80.15%) are identical [indicated by *], 12 (9.16%) are strongly similar [:] and 7 (5.34%) are weakly similar [.].

(7) FIG. 7 shows the alignment of RaA (SEQ ID NO: 2), RaB (SEQ ID NO: 4), Rs1 (SEQ ID NO: 6) and Japanin (SEQ ID NO: 8) and indicates the location of the conserved motifs and features. The CXXW (SEQ ID NO:33) motif is shown in italics, the conserved cysteine residues are shown in bold and the asparagine residue of the glycosylation site containing sequence is underlined. The following options were used: gap extension penalty=0.2; gap opening penalty=10.0; hydrophilic residues=G, P, S, N, D, Q, E, R or K; matrix=gonnet.

(8) FIG. 8 shows the alignment of RaA (SEQ ID NO: 2), RaB (SEQ ID NO: 4), Rs1 (SEQ ID NO: 6), Japanin (SEQ ID NO: 8) and DA (SEQ ID NO: 10) and indicates the location of the conserved motifs and features. The CXXW SEQ ID NO:33) motif is shown in italics, the conserved cysteine residues are shown in bold and the asparagine residue of the glycosylation site containing sequence is underlined. The following options were used: gap extension penalty=0.2; gap opening penalty=10.0; hydrophilic residues=G, P, S, N, D, Q, E, R or K; matrix=gonnet.

(9) FIG. 9 shows Rs, RaA and RaB are present in CHO transfectant supernatants

(10) FIG. 10A shows the effect of Rs1, RaA and RaB transfectant supernatants on CD86 expression in dendritic cells that have not been treated with LPS.

(11) FIG. 10B shows that Rs1, RaA and RaB transfectant supernatants inhibit CD86 expression in LPS treated dendritic cells

(12) FIG. 11 shows the enhancement of B7-H1 upregulation in LPS treated dendritic cells in response to Rs1, RaA and RaB transfectant supernatants

DESCRIPTION OF THE SEQUENCES

(13) SEQ ID NO: 1 is the nucleotide sequence of RaA

(14) SEQ ID NO: 2 is the amino acid sequence of RaA

(15) SEQ ID NO: 3 is the nucleotide sequence of RaB

(16) SEQ ID NO: 4 is the amino acid sequence of RaB

(17) SEQ ID NO: 5 is the nucleotide sequence of Rs1

(18) SEQ ID NO: 6 is the amino acid sequence of Rs1

(19) SEQ ID NO: 7 is the nucleotide sequence of Japanin

(20) SEQ ID NO: 8 is the amino acid sequence of Japanin

(21) SEQ ID NO: 9 is the nucleotide sequence of DA

(22) SEQ ID NO: 10 is the amino acid sequence of DA

(23) SEQ ID NO: 11 is the nucleotide sequence of RM

(24) SEQ ID NO: 12 is the amino acid sequence of RM

(25) SEQ ID NO: 13 is the nucleotide sequence of AM

(26) SEQ ID NO: 14 is the amino acid sequence of AM

(27) SEQ ID NO: 15 is the nucleotide sequence of RaC

(28) SEQ ID NO: 16 is the amino acid sequence of RaC

(29) SEQ ID NO: 17 is a degenerate Japanin-derived forward primer

(30) SEQ ID NO: 18 is a vector specific reverse primer

(31) SEQ ID NO: 19 is a Japanin-specific reverse primer

(32) SEQ ID NO: 20 is a Rs1 specific reverse primer

(33) SEQ ID NO: 21 is a RaA specific reverse primer

(34) SEQ ID NO: 22 is a RaB specific reverse primer

(35) SEQ ID NO: 23 is a vector specific forward primer

(36) SEQ ID NO: 24 is the full amino acid sequence of Rs1

(37) SEQ ID NO: 25 is the full length amino acid sequence of RaA

(38) SEQ ID NO: 26 is the full length amino acid sequence of RaB

(39) SEQ ID NO: 27 is the predicted mature peptide sequence of Rs1

(40) SEQ ID NO: 28 is the predicted mature peptide sequence of RaA

(41) SEQ ID NO: 29 is the predicted mature peptide sequence of RaB

(42) SEQ ID NO: 30 is the nucleotide sequence of a Rs1 synthetic gene

(43) SEQ ID NO: 31 is the nucleotide sequence of a RaA synthetic gene

(44) SEQ ID NO: 32 is the nucleotide sequence of a RaB synthetic gene

(45) SEQ ID NOs: 33-40 represent CXXW (SEQ ID NO:33) motifs

(46) SEQ ID NO: 41 is Rs1 full length DNA/coding sequence (including the stop codon)

(47) SEQ ID NO: 42 is RaA full length DNA/coding sequence (including the stop codon)

(48) SEQ ID NO: 43 is RaB full length DNA/coding sequence (including the stop codon)

(49) SEQ ID NO: 44 is a Rs1 5 DNA sequence

(50) SEQ ID NO: 45 is a RaA 5 DNA sequence

(51) SEQ ID NO: 46 is a RaB 5 DNA sequence

(52) SEQ ID NO: 47 is a Rs1 specific forward primer

(53) SEQ ID NO: 48 is a RaA specific forward primer

(54) SEQ ID NO: 49 is a RaB specific forward primer

EXAMPLES

Example 1

Identification of RaA, RaB and Rs1

(55) The incomplete amino acid sequence of the proteins RaA, RaB and Rs1, which are related to Japanin were obtained by amplifying Rhipicephalus cDNAs in expression libraries which were prepared in Lambda Zap II (Stratagene). Amplification was performed by means of the polymerase chain reaction (PCR) using a degenerate, Japanin-derived forward primer (SEQ ID NO: 17) in combination with either a vector specific reverse primer (SEQ ID NO: 18) in the case of RaB or a Japanin-specific reverse primer (SEQ ID NO: 19) in the case of RaA and Rs1.

Example 2

Alignment of Japanin with RaA, RaB and Rs1, Respectively

(56) The amino acid sequences of RaA (SEQ ID NO: 2), RaB (SEQ ID NO: 4) and Rs1 (SEQ ID NO: 6) were each aligned with Japanin (SEQ ID NO: 8) using clustalW with the following options: gap extension penalty=0.1; gap opening penalty 10.0; hydrophilic hydrophilic residues=G, P, S, N, D, O, E, R or K; matrix=gonnet. The results of these alignments are shown in FIGS. 2, 4 and 6, respectively.

(57) For RaA, of the 131 residues that form the alignment, 61 (46.56%) are identical, 26 (19.85%) are strongly similar and 22 (16.79%) are weakly similar.

(58) For RaB, of the 138 residues that form the alignment, 73 (52.9%) are identical, 30 (21.74%) are strongly similar and 12 (8.7%) are weakly similar.

(59) For Rs1, of the 131 residues that form the alignment, 105 (80.15%) are identical, 12 (9.16%) are strongly similar and 7 (5.34%) are weakly similar.

Example 3

Alignment of Japanin, RaA, RaB and Rs1

(60) The amino acid sequences of RaA (SEQ ID NO: 2), RaB (SEQ ID NO: 4), Rs1 (SEQ ID NO: 6) and Japanin (SEQ ID NO: 8) were aligned using clustalW with the following options: gap extension penalty=0.2; gap opening penalty=10.0; hydrophilic residues=G, P, S, N, D, Q, E, R or K; matrix=gonnet. The result of this alignment is shown in FIG. 7.

Example 4

Alignment of Japanin, RaA, RaB, Rs1 and DA

(61) The amino acid sequences of RaA (SEQ ID NO: 2), RaB (SEQ ID NO: 4), Rs1 (SEQ ID NO: 6), Japanin (SEQ ID NO: 8) and DA (SEQ ID NO: 10) were aligned using clustalW with the following options: gap extension penalty=0.2; gap opening penalty 10.0; hydrophilic residues=G, P, S, N, D, Q, E, R or K; matrix=gonnet. The result of this alignment is shown in FIG. 8.

(62) FIG. 8 indicates that these five proteins form a distinct cluster within the tick lipocalin family. Their evolutionary relatedness and structural similarity suggest conserved functions.

(63) The inventors have provided the amino acid and nucleotide sequences of three previously unidentified DC modulatory proteins. Based on sequence homology and alignment studies the inventors have been able to determine a number of motifs and features which may be important for the structure and function of such proteins. These results provide an insight into the workings of such molecules which is likely to be useful in designing further DC modulatory molecules for use in therapy.

(64) The following general experimental techniques were employed for the experiments described in Examples 5-8:

(65) Cell culture media and supplements were, unless otherwise stated, from PAA. LPS was atrichloroacetic acid-extracted preparation from Escherichia coli 055:B5 (Sigma, product code L4005). Human dendritic cells (DC) were generated from peripheral blood monocytes isolated from healthy adult donors. Briefly, Leucocyte cones (National Blood Service, Oxford) were mixed 1:3 (v/v) with Ca2+/Mg2+-free Phosphate-buffered salt solution (PBS), carefully layered on to Lymphoprep (Axis Shield) and centrifuged at 800 g for 30 minutes (at 22 C.). The peripheral blood mononuclear cell (PBMC) layer formed at the interface between the PBS/Buffy coat mixture and Lymphoprep was carefully collected and washed three times with PBS to remove platelets (each time centrifuging for 10 minutes at 4 C., initially at 400 g, then 300 g, and finally 200 g).

(66) Monocytes were isolated from PBMC by negative selection using the Easysep Human Monocyte Enrichment kit (Stemcell technologies). PBMC were resuspended in Ca.sup.2+/Mg.sup.2+-free PBS, and the kit was then used in accordance with the manufacturer's instructions. Purified monocytes were resuspended at 510.sup.5/ml in DC-RPMI: RPMI 1640 supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, 1000 U/ml human granulocyte macrophage colony-stimulating factor (GM-CSF) (Gentaur) and 250 ng/ml human interleukin 4 (IL-4) (Peprotech EC). After 3 days, one third of the media was removed and spun down, and the pellet resuspended in the same volume of fresh media containing 1000 U/ml GM-CSF and 750 ng/ml IL-4, then returned to the culture. After 5 days, the cells were either used immediately, or frozen. To freeze the cells, they were washed with HBSS/2% FCS, and resuspended in 5.5% hetastarch (Voluven from John Radcliffe Hospital pharmacy)/4.8% dimethyl sulfoxide (DMSO) (Hybrimax grade from Sigma)/3.8% FCS in isotonic saline, then placing into a 80 C. freezer in a controlled freezing device (1 C./minute).

Example 5

PCR Cloning of 5 Sequence of Homologues

(67) The missing N-terminal amino acid sequences of Rs1, RaA and RaB were obtained by amplifying cDNAs from the Rhipicephalus expression libraries which were used to obtain the partial sequences represented by SEQ ID NOs: 2, 4 and 6.

(68) Amplification was performed by means of the polymerase chain reaction (PCR) using a gene-specific reverse primer (SEQ ID NOs: 20, 21 or 22) in combination with a vector specific forward primer (SEQ ID NO: 23), and employing the Phusion DNA polymerase (Finnzymes) in accordance with the manufacturer's instructions. PCR products were cloned into the pCR Blunt II TOPO cloning vector (Invitrogen) in accordance with the manufacturer's instructions, and ligated plasmid used to transform TOP10 E. coli cells (Invitrogen), which were then plated on to LB agar. Individual colonies were then picked for growth in liquid LB culture, and plasmid DNA prepared from them using the QIAprep system (Qiagen). Sequencing of these plasmids provided the 5 cDNA sequence of Rs1, RaA and RaB, (given in SEQ ID NOs: 44, 45 and 46 respectively).

Example 6

PCR Cloning of 3 Sequence of Homologues

(69) The missing C-terminal amino acid sequences of Rs1 and RaA were obtained by amplifying cDNAs from the Rhipicephalus expression libraries which were used to obtain the partial sequences represented by SEQ ID NOs: 2, 4 and 6. The same approach was used to confirm the C-terminal sequence of RaB (previously determined; see example 1, SEQ ID NO:4).

(70) Amplification was performed by means of the polymerase chain reaction (PCR) using a gene-specific forward primer (SEQ ID NOs: 47, 48 or 49) in combination with a vector specific reverse primer (SEQ ID NO: 23), and employing the Phusion DNA polymerase (Finnzymes) in accordance with the manufacturer's instructions. PCR products were cloned into the pT7Blue cloning vector using the Perfectly Blunt cloning kit (Novagen) in accordance with the manufacturer's instructions, and ligated plasmids used to transform TOP10 E. coli cells (Invitrogen), which were then plated on to LB agar. Individual colonies were picked for growth in liquid LB culture, and plasmid DNA prepared from them using the QIAprep system (Qiagen). Sequencing of these plasmids provided the 3 cDNA sequence of Rs1, RaA and RaB, and this was combined with the previously generated sequences to give full nucleotide cDNA sequences (represented by SEQ ID NOs: 41, 42 and 43 respectively), and thus the encoded peptide sequences (SEQ ID NOs: 24, 25 and 26 respectively). SignaIP was used to identify likely signal peptide portions of the proteins, suggesting that the mature secreted proteins will have the amino acid sequences shown in SEQ ID NOs: 27, 28 and 29.

Example 7

Expression of Homologues in Chinese Hamster Ovary (CHO) Cells

(71) In order to test the activity of the homologues, they were expressed in CHO cells. Synthetic genes encoding each of them were subcloned into a mammalian expression vector (pcDNA3.1), these expression constructs used to transfect CHO cells, and the presence of recombinant proteins in transfectant supernatants was confirmed by Western blotting.

(72) a. Synthetic Genes

(73) Synthetic genes optimised for CHO cell expression were generated by DNA2.0 (SEQ ID NOs: 30, 31 and 32). They encode the full peptide sequences of the homologues, as well as a C-terminal extension encoding a linker portion (GG) and a polyhistidine tag (HHHHHH; SEQ ID NO:73), to facilitate detection and purification. They also include non-coding DNA comprising a 5-Kozak sequence (to allow eukaryotic expression), and restriction enzyme sites to facilitate subcloning: a 5 BamHI site and a 3 NotI site.

(74) b. Subcloning into pcDNA3.1

(75) As the synthetic genes were provided in a cloning vector, it was necessary to transfer them into an expression vector to produce recombinant protein. For expression in CHO cells, we selected the pcDNA3.1 mammalian expression vector, in which expression is driven by the i.e. CMV promoter.

(76) pcDNA3.1 was prepared from parental pcDNA3.1-v5-his C (Invitrogen) by excising the multiple cloning region with BamHI and NotI restriction enyzmes (New England Biolabs), treating with FastAP alkaline phosphatase (Fermenatas) to reduce cell ligation of the vector, and gel purifying the vector backbone using the QIEx II system (Qiagen). Synthetic genes were prepared from their carrier plasmids by excising the homologue-encoding regions (including the Kozak sequence and polyhistidine tag) with BamHI and NotI restriction enyzmes, and gel purifying them using the QIEx II system. pDNA3.1-homologue constructs were generated by ligation of excised synthetic genes with prepared pDNA3.1 using T4 DNA Ligase (Fermentas). Ligated DNA was used to transform competent E. coli which were then plated on to LB agar to allow the selection of individual colonies for liquid culture. Plasmid DNA was extracted from these cultures using the QIAprepsystem (Qiagen), and its identity confirmed by sequencing. DNA incorporating the synthetic gene sequences was then used for transfection.

(77) c. Transfection of CHO Cells

(78) CHO cells were grown in RPMI supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin and 100 g/ml streptomycin under standard cell culture conditions. For transfection, cells were seeded at an estimated 30-40% into 24 well plates, and cultured overnight. Wells with 80-95% confluency were then selected for transfection. Cells were transfected with pcDNA3.1-homologue constructs, pcDNA3.1-japanin-his (as a positive control), pcDNA3.1-lacZ-his (as a positive control for transfection and a negative control for DC-modulatory activity) or with the transfection reagent only (mock transfection control). Transfections were performed with the TransIT2020 reagent (Minis) in accordance with the manufacturer's instructions, using 1.5 l of reagent and 500 ng of plasmid DNA per well. Supernatants were harvested after 72 hours, and either analysed or frozen immediately.

(79) d. Western Blotting to Confirm Expression

(80) Expression of polyhistidine-tagged proteins was confirmed by Western blotting. Lithium dodecyl sulphate (LDS) loading buffer (Invitrogen) was added 6.5 l transfectant supernatant, along with dithiothreitol (DTT) (50 mM final concentration), and proteins denatured by heating to 70 C. for 10 minutes. Prepared samples were run on a 4-20% acrylamide Pierce Protein precast gel (Thermofisher), and then transferred to nitrocellulose membrane by wet transfer in Towbin buffer, with a constant current of 400 mA for one hour.

(81) The membrane was blocked with StartingBlock T20 (PBS) Blocking Buffer (Thermofisher) for 30 minutes at room temperature, rinsed with PBS/0.1% Tween 20, and then incubated overnight at 6 C. in anti-pentahis-biotin (Qiagen) diluted 1/1000 in PBS/3% BSA/0.1% Tween 20. It was then washed extensively in PBS/0.1% Tween 20, and incubated for 30 minutes at room temperature in streptavidin-HRP (Jackson Immunoresearch) diluted 1/20000 in PBS/0.1% Tween 20. Further PBS/0.1% Tween 20 washes were then performed, and ECL substrate (GE Lifesciences) applied, prior to exposure of X-ray film to the membrane.

(82) The developed film clearly reveals the presence of each polyhistine-tagged Japanin homologue in transfectant supernatant (see FIG. 9).

Example 8

Dendritic Cell Modulating Activity of Homologue Transfectant Supernatants

(83) a Inhibition of CD86 Upregulation in Response to Stimuli

(84) Screening for DC modulatory activity was performed using human monocyte-derived DC generated as previously described. DC were cultured in 96-well flat-bottomed tissue culture plates (Corning) in DC-RPMI supplemented with the samples to be screened. Transfectant supernatants were added to 10% of total volume, while purified japanin was used at 500 ng/ml. After 24 hours of culture LPS (100 ng/ml) were added to stimulate the DC. After another 18 to 24 hours of culture levels of CD86 expression were assessed by flow cytometry, following staining with anti-human CD86-PE, clone IT2.2 (eBioscience) at a 1/50 dilution. Flow cytometry was performed using a FACSCanto flow cytometer (Becton Dickinson). Supernatants from cells transfected with each of the three homologues were found to reduce CD86 expression in both LPS-treated and untreated DC, to an extent comparable to supernatant from japanin-transfectants (see FIG. 10).

(85) b Enhancement of B7-H1 Upregulation in Response to Stimuli

(86) DC were treated with transfectant supernatants and LPS as described in 4.1 above. Levels of B7-H1 (CD274) were then assessed by flow cytometry, following staining with anti-human B7H1-PECy7, clone MIH1 (eBioscience) at a 1/100 dilution. Flow cytometry was performed using a FACSCanto flow cytometer (Becton Dickinson). Supernatants from cells transfected with each of the three homologues, or with japanin, were found to enhance levels of B7-H1 expression over those induced by LPS alone (see FIG. 11).

(87) This suggests that the homologues (and japanin) are driving the DC into a regulatory-type phenotype, reducing levels of (T cell costimulatory) CD86, while enhancing levels of (T cell coinhibitory) B7-H1.

REFERENCES

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