Patent Application
20240034757
The invention relates to tick chemokine binding polypeptides (tick CKBPs, typically tick Evasins) including hybrid CKBPs based on sequences from two or more tick CKBPs, and the uses of such polypeptides in inhibition of chemokines or detection of chemokine expression and inflammation.
Chemokine-driven inflammation plays a major role in several disorders, including myocardial infarction[1], myocarditis[2], atherosclerotic plaque[3], and stroke[4], pulmonary inflammation and fibrosis, multiple sclerosis, rheumatoid arthritis, psoriasis, atopic dermatitis, inflammatory bowel disease, and cancer (reviewed in [5]).
Chemokines are a group of 45-50 secreted small extracellular proteins, classified as CC, CXC or CX3C based on the arrangement of cysteine residues at the N-terminus, that function via 19 G-protein coupled receptors, to recruit inflammatory and immune cells to injured or diseased tissues[6,7]. Properties of the chemokine network that make it robust to attack are the expression of multiple receptors on inflammatory cells[8], expression of several chemokines in diseased tissues[9], polyvalent chemokine-receptor interactions—with chemokines typically targeting more than one receptor, and receptors typically being activated by more than one chemokine[6], synergistic and cooperative interactions between chemokines[10] and chemokine receptors[11], and feed-forward loops that amplify the network response[12]. The robustness of the chemokine network is clearly demonstrated by the observation that targeting individual chemokines or receptors has failed as a strategy to develop effective therapeutics for inflammatory disorders[9,13].
Both CC and CXC chemokines are important mediators of inflammation in human disease. This is indicated in
A number of pathogens, including viruses, helminths and ticks, produce structurally unrelated chemokine binding proteins (CKBPs) that polyvalently target multiple chemokines disrupting the chemokine network (reviewed in [13]). Viral and helminth CKBPs described to date do not appear to discriminate between CC and CXC chemokines[14,15]. Tick CKBPs identified to date fall into two structurally unrelated classes. These were originally identified from the brown dog tick Rhipicephalus sanguineus by Proudfoot and colleagues[16] as Evasin-1 and Evasin-4 which solely bind a subset of CC chemokines, and Evasin-3 which binds only a subset of CXC chemokines. Additional tick CKBP polypeptides have also been identified, PCT/GB2017/050563, [17,18].
Pre-clinical trials have indicated potential therapeutic efficacy of viral [19], helminth[15] and tick[13,16,20-34] CKBPs in inflammatory disease providing proof-of-concept of polyvalent targeting of the chemokine network as a therapeutic approach for inflammatory disease. Like other CKBPs, the ability of polyvalent tick CKBPs to disrupt the chemokine network provides an advantage over monoclonal antibodies that target single chemokines.
Properties of tick CKBPs which indicate that, like other naturally occurring tick peptides such as COVERSIN®[35], they could be clinically translated include a), systemic anti-chemokine effects following parenteral administration, b), ability to inhibit inflammation in a diverse range of pre-clinical animal models and c), lack of significant immunogenicity or toxicity in such studies[16].
The preferential binding of tick CKBPs to discrete subsets of chemokines (unlike viral CKBPs), could provide a method to precisely target the disease-relevant chemokine network without unnecessarily targeting all chemokines. The inadvertent targeting of chemokines that are not involved in the disease process however could increase the likelihood of off-target effects. Indeed several chemokines may play a beneficial role in the disease process, and targeting these may have undesirable effects. For instance, chemokines such as CCL19, CXCL5 and CXCL12 are known to be atheroprotective [3]. Loss of XCL1 leads to inflammation in the heart and other organs [36], and loss of CXCL10 leads to increased susceptibility to experimental autoimmune encephalitis [37].
There is a need to provide additional CKBPs for use in inhibition and detection of chemokines.
The inventors provide CKBPs having previously undescribed chemokine binding properties. The CKBPs are based on sequences from tick salivary polypeptides. The CKBPs may be hybrid polypeptides representing sequences from two (or more) different CKBPs, or polypeptides comprising sequences derived from newly isolated CKBPs binding to CXC chemokines.
The inventors have unexpectedly demonstrated the ability to combine sequences from different tick CKBPs to form a hybrid polypeptide having unique chemokine binding properties. The hybrid polypeptide may combine different chemokine binding properties from two or more tick CKBPs together in a single polypeptide. The hybrid polypeptide may represent a specific chemokine binding activity derived from a first tick CKBP in the context of a second tick CKBP. The flexibility in combination of sequences from different CKBPs identified by the inventors provides the ability to specifically engineer desired chemokine binding properties for a CKBP. This advantageously allows for a CKBP to be matched as precisely as possible to the chemokine expression pattern of a given disease, and/or to avoid targeting of chemokines not involved in that disease. The hybrid polypeptides may also provide both CC and CXC binding functions in a single CKBP, which is not previously described for any tick CKBP polypeptide to the inventors' knowledge, and advantageously caters for the discussion of both CC and CXC chemokines in human disease.
Additionally, the inventors have isolated novel tick CKBP polypeptides with unique CXC binding functions, which are of further utility in provision of CKBPs with novel chemokine binding properties.
The invention therefore provides a hybrid polypeptide comprising an amino acid sequence of a first tick CKBP polypeptide or a variant thereof and an amino acid sequence of a second tick CKBP polypeptide or a variant thereof, wherein said hybrid polypeptide has an altered chemokine binding profile compared to the first or second tick CKBP polypeptide.
The invention further provides a polypeptide comprising (a) all or part of an amino acid sequence shown in any one of SEQ ID NOs 45-72 or (b) all or part of an amino acid sequence having at least 70% homology or identity to a sequence of (a) over its entire length, wherein said polypeptide binds at least one CXC chemokine.
The invention also provides a polypeptide comprising (a) all or part of an amino acid sequence shown in SEQ ID NO: 88, 89, 103 to 109 or (b) all or part of an amino acid sequence having at least 70% homology or identity to a sequence of (a) over its entire length, wherein said polypeptide binds at least one chemokine selected from CCL8, CCL7 and CCL18, preferably wherein said polypeptide binds all said chemokines.
The invention additionally provides a combination of two or more of the above polypeptides of the invention. References to polypeptides of the invention herein include both the hybrid polypeptide and polypeptide described above.
The invention also provides a polynucleotide which encodes a polypeptide of the invention.
The invention additionally provides a combination of two or more polynucleotides each of which encodes a polypeptide of the invention.
The invention further provides a vector which comprises a polynucleotide of the invention or a combination of two or more polynucleotides of the invention.
The invention also provides a host cell which comprises a polynucleotide of the invention, a combination of two or more polynucleotides of the invention or a vector of the invention.
The invention additionally provides a pharmaceutical composition comprising (a) a polypeptide of the invention, a combination of two or more polypeptides of the invention, a polynucleotide of the invention, a vector of the invention or a host cell of the invention and (b) a pharmaceutically acceptable carrier or diluent.
The invention further provides a method of producing a polypeptide of the invention or a combination of two or more polypeptides of the invention comprising, culturing a host cell of the invention under conditions which produce the polypeptide or the combination.
The invention also provides a method of inhibiting the signalling of one or more chemokines in an in vitro culture, the method comprising contacting the culture with a polypeptide of the invention, a combination of two or more polypeptides or polynucleotides of the invention, a polynucleotide of the invention, a vector of the invention or a host cell of the invention.
The invention additionally provides a method of inhibiting the signalling of one or more chemokines in a subject, the method comprising administering to the subject a polypeptide of the invention, a combination of two or more polypeptides or polynucleotides of the invention, a polynucleotide of the invention, a vector of the invention or a host cell of the invention.
The invention further provides a method of treating or preventing in a subject one or more diseases associated with one or more chemokines, the method comprising administering to the subject a polypeptide of the invention, a combination of two or more polypeptides or polynucleotides of the invention, a polynucleotide of the invention, a vector of the invention or a host cell of the invention.
The invention also provides a polypeptide of the invention, a combination of two or more polypeptides or polynucleotides of the invention, a polynucleotide of the invention, a vector of the invention or a host cell of the invention for use in a method of inhibiting the signalling of one or more chemokines in a subject
The invention further provides a polypeptide of the invention, a combination of two or more polypeptides or polynucleotides of the invention, a polynucleotide of the invention, a vector of the invention or a host cell of the invention for use in a method of treating in a subject one or more diseases associated with one or more chemokines.
The invention additionally provides an antibody or a fragment thereof which specifically binds a polypeptide of the invention.
The invention further provides a method of detecting one or more chemokines in a tissue, comprising contacting the tissue with a detectably-labelled polypeptide of the invention or a detectably-labelled combination of two or more polypeptides of the invention and detecting the binding of the polypeptide or the combination to one or more chemokines.
The invention also provides a detectably-labelled polypeptide of the invention or a detectably-labelled combination of two or more polypeptides of the invention for use in a method of detecting one or more chemokines in a tissue.
Literature references are as follows: Myocarditis, including giant cell, viral, Chagas and lymphocytic myocarditis: [42-50]; myocardial infarction: [51,52], atherosclerosis: [53-67], vasculitis, including Takayasu disease, ANCA vasculitis, and giant cell arteritis: [68-73], stroke: [4], multiple sclerosis [74-76], Alzheimer's disease [77], primary biliary cirrhosis [78-84], primary sclerosing cholangitis [81,85], liver fibrosis [86,87], nonalcoholic steatohepatitis [88,89], paracetamol liver injury [90], alcoholic liver injury [91], idiopathic pulmonary fibrosis [92-102], acute lung injury [103,104], sarcoidosis [105-111], influenza [112-122], kidney fibrosis [86], inflammatory bowel disease [123-134], pancreatitis [135,136], rheumatoid arthritis [9], psoriasis [137-141], skin fibrosis [86], atopic dermatitis [137,142-147], breast cancer [148-154], colorectal cancer [155-159].
Alignment was performed using the MUSCLE algorithm in DNASTAR. The mature peptide sequences of Evasin-1 (EVA1_RHISA) and 4 (EVA4_RHISA) are published [20,21]. Other tick CKBP sequences were disclosed previously in PCT/GB2017/050563 and published in Singh et al[17]. Peptide sequence prefix indicates the identity, and suffix indicate the tick species as follows: RHISA and RHIPU—Rhipicephalus sanguineus and pulchellus respectively, and AMBPA, AMBCA, AMBMA, AMBTR—Amblyomma parvum, cajennense, maculatum, triste) respectively. Amino acid residues shaded as black are identical to EVA1_RHISA. Disulfide bonds (DSB) in Evasin-1 are indicated, and were taken from the analysis of the Evasin-1:CCL3 structure 3FPU provided in PDBSum[22]. The positions of the 8 conserved cysteines are indicated by arrows, and are conserved in all CC-chemokine binding tick CKBPs, which we term “8-Cys” tick chemokine binding proteins. The arrangement of Cys residues is C-x(14,17)-C-x(3)-C-x(11,16)-C-x(17,20)-C-x(4)-C-x(4)-C-x(8)-C, with numbers in parentheses indicating spacing between Cys residues. CKBPs in
The following peptides were used in this experiment:
The following peptide was used in this experiment:
A. Surface representation (top) and ribbon diagram (bottom) of a homology model of P672 (darker grey) and CCL8 (lighter grey) complex. P672 and CCL8 in 1:1 ratio was pre-incubated for 1 h, then diluted in D2O containing buffer and quenched at different time intervals (5 s, 30 s, 5 min, 60 min).
B. Surface representation in light grey (top) and ribbon diagram (bottom) of P672 and CCL8 complex at the time points indicated. Residues with statistically significant increased HDX rates, (exposed residues) are shown in darkest grey. Regions with statistically significant decreased HDX rates (protected residues) are shown in mid-greys for P672 and CCL8. All analyses were performed in triplicate.
C. Surface representations in light grey (top) and ribbon diagrams (bottom) of P672 (−90° rotated view along the y-axis of B). Residues protected at 5 s and 30 s time points (E22-F32) are indicated in mid-grey in the top panel. Exposed residues (G87-C94) are indicated in darkest grey in the bottom panel. The surface of the protected residues (E22-F32) is also shown in the bottom panel.
D. Surface representation in light grey (top) and ribbon diagram (bottom) of CCL8, with residues protected at all time points (R18-S27) indicated in mid-grey. Disulfide bonds are indicated in lightest grey in the bottom panel. The surface of the N-loop (residues C12-R24) is also shown in the bottom panel to show the overlap with protected residues.
E. Spectra of two representative peptides from the Y21-F32 region in P672 (mid-grey and black bars) that are protected from deuterium uptake upon complex formation. H/D exchange mass spectra was measured at t=5 s. These peptides display reduced relative deuterium uptake upon complex formation. Other peptides from this region are indicated as gray bars. Mass spectra is shown for control non-deuterated peptides (c-i, iv), unbound P672 deuterated peptides (c-ii, v), and P672 deuterated peptides when in complex with CCL8 (c-iii, vi).
A. Alignment of EVA1 (SEQ ID NO: 32), P672 (SEQ ID NO: 3) and EVA1/P672 (EVA1 containing P672E22-E32; SEQ ID NO: 95) hybrid protein using MUSCLE algorithm. Amino acids are color-coded according to physicochemical properties: aromatic (F, W, and Y); acidic (D and E); basic (R, H, and K); nonpolar aliphatic (A, G, I, L, M, P, and V); polar neutral (C, N, Q, and T). Amino acids that were protected from deuterium uptake in P672 are indicated with the right black box. The N-terminal acidic region is enclosed in the left black box.
B. Biolayer interferometry sensorgram obtained when either P672, EVA1/P672 or EVA1 is loaded onto the BLI sensor and exposed to 600 nM CCL8. Plots display wavelength shift (Y-axis, nm) versus time (X axis, seconds).
C. Biolayer interferometry sensorgram for EVA1(P672E22-E32) hybrid binding to CCL8. Dotted lines indicate collected data, solid lines indicate modelled data. Plots display wavelength shift (Y-axis, nm) versus time (X axis, seconds).
A. Design of a P672 peptide tiling array to identify CCL8-binding peptides. Positions of each residue within P672 are indicated, and the gray box indicates the CCL8 binding region identified by HDX-MS. P672 residues are shaded according to CCL8 binding affinity from the Ala-scanning mutagenesis (see text and Table 9). Y21A, E22A, F25A, P27A, V28A, and Y31A mutants lead to either complete or highly significant loss of activity (P<0.0001), D18A and F32A mutants lead to moderately significant loss of activity (P<0.05). Peptides synthesized (BK1.1-BK8) are indicated as gray bars.
B. Fluorescent peptides BK1.1-BK6 (50 nM) were incubated with CCL8 (1 μM) and the resulting anisotropy determined. A scrambled peptide (S, SCRFITC) was used as a negative control. The anisotropy of each peptide after being incubated with CCL8 was compared to scrambled peptide using one-way ANOVA with Sidak's correction for multiple comparisons. **** indicates P≤0.0001.
C. Fluorescent polarization assay to determine binding of BK1.1FITC to CCL8. The Y axis shows anisotropy, and X axis the dose of CCL8. Individual data points are indicated and the curve was generated as a non-linear fit with 3 parameters to estimate KD.
D. Fluorescent polarization assay to assess effect of alanine-scanning mutagenesis of BK1.1FITC on CCL8 binding. KD values for each BK1.1FITC Ala mutant are shown as mean±s.e.m of three biological replicates, which are individually indicated as points. Data for each mutant was compared to wild-type (WT) BK1.1, using a one-way ANOVA with Sidak's correction for multiple comparisons. ****=P≤0.0001, *=P≤0.05. The mutant P27A showed no detectable binding.
E. Mass spectrometry (MS) to assess effect of BK1.1 on CCL8 Top panel: Native MS of CCL8 homodimer. Mid panel: In-solution dissociation of CCL8 dimer and further binding of CCL8 to one and two BK-1. Confirmation of CCL8/BK-1 complex by HCD gas-phase dissociation of isolated precursor ions: Bottom panel, left: 2217 m/z corresponding to CCL8/BK-1 (1:1) and Bottom panel, left: 2555 m/z corresponding to CCL8/BK-1 (1:2). Buffers contained up to 0.5% DMSO. All analyses were performed in triplicate.
F. Fluorescent polarization assay to assess the binding of BK1.1FITC against a CC-chemokine panel. Data are presented as mean±s.e.m of three biological replicates, which are individually indicated as points. Each biological replicate was performed as technical duplicate. CXCL1 was used as a negative control. CC-chemokine binding compared to the negative control using a one-way ANOVA with Sidak's correction for multiple comparisons. ****=P≤0.0001, * P<0.05.
G-I. Fluorescence polarization competition assay for BK1.1FITC and CC-chemokine interactions. BK1.1FITC (50 nM) was incubated with the indicated chemokine (1 μM) with or without unlabeled BK1.1 or SCR (BK1.1 scrambled) peptides (50 μM) for 30 min and the resulting anisotropy was measured. Data are presented as mean±s.e.m of three biological replicates, which are individually indicated as points. Each biological replicate was performed as technical duplicate. Statistical significance of differences (SCR versus BK1.1) were calculated using a one-way ANOVA. ****=P≤0.0001, ***=P≤0.001.
A. Sequences of peptides studied, with disulfide bond (BK1.3) or thioether cyclization (BK1.2, BK1.4) indicated by lines. SCR is a scrambled peptide based on the sequence of BK1.1 (SEQ ID NO: 88). Y21F32, C30A (SEQ ID NO: 103); Y21F32 (SEQ ID NO: 104); BK1.2 (SEQ ID NO: 105); BK1.3 (SEQ ID NO: 106); BK1.4 (SEQ ID NO: 107); SCR (SEQ ID NO: 91).
B-D. Effect of indicated peptides at a concentration of 100 μM on a His-tagged P672-biotinylated CCL8, CCL2 or CCL3 interaction respectively using an AlphaScreen assay. In each panel. Y axis shows intensity counts, and X axis the peptide. Data are presented as mean±s.e.m. of three independent experiments, shown as individual data points. Statistically significant differences (compared to chemokine+P672), using a one-way ANOVA with Sidak's multiple comparisons test are indicated by asterisks. ****=P≤0.0001, ***=P≤0.001, **=P≤0.01.
E-G. Representative dose-response AlphaScreen assay curves showing disruption of His-tagged P672 interactions with biotinylated human CCL8, CCL2 and CCL3 respectively by each member of the BK1.1 derived series. Y axis shows intensity counts, and X axis the peptide concentration (Log10 Molar). Data are shown as mean of two technical replicates. Curves were fitted with 4 parameters to estimate IC50.
H-J. Summary IC50 values for inhibition of His-tagged P672 binding to human CCL8, CCL2 and CCL3 respectively by each member of the BK1.1 derived series, where these could be calculated. Y axis shows IC50 (M). Data are presented as mean±s.e.m. of three independent experiments, each shown as individual data points. Each independent experiment was conducted as two technical replicates. Statistically significant differences (compared to BK1.1), using a one-way ANOVA with Sidak's multiple comparisons test, are indicated by black asterisks. Statistically significant differences (pairwise comparisons of BK1.2, BK1.3, BK1.4 and BK1.5) using one-way ANOVA with Tukey's multiple comparisons test are indicated with blue asterisks (comparisons to BK1.2), or green asterisks (comparisons to BK1.3). ***=P≤0.001, **=P≤0.01, *=P≤0.05.
A-D. Inhibition of human chemokine induced THP-1 cell migration by BK1.1, BK1.2, BK1.3, SCR (BK1.1 scrambled, negative control) peptides, each at 10 μM, and by P672 protein (positive control, 300 nM). Y axis in each panel shows % migration of THP-1 cells normalized to chemokine alone which was set at 100%. All experiments were performed at EC80 doses of CCL8 (5.8 nM), CCL7 (7.2 nM), CCL3 (3.5 nM), and CCL2 (1.2 nM), respectively. Data are shown as mean±s.e.m. of three independent biological replicates, shown as individual data points. Each biological experiment was performed as three technical replicates. Statistically significant differences (compared to SCR), using a one-way ANOVA with Sidak's correction for multiple comparisons, are indicated by asterisks: ****=P≤0.0001, ***=P≤0.001, **=P≤0.01, *=P≤0.05.
E. Representative dose-response curves showing inhibition of human CCL8 induced THP-1 cell migration by BK1.1 (black), BK1.2 (darker grey), BK1.3 (lighter grey) peptides and by P672 protein (positive control, magenta). Y axis shows % migration of THP-1 cells normalized to CCL8 alone which was set at 100%. Data are shown as mean±s.e.m. of three technical replicates. X-axis shows inhibitor concentration (Log10 Molar). Curves were fitted with 4 parameters to estimate IC50.
F. Summary IC50 values for inhibition of human CCL8 induced THP-1 cell migration by BK1.1, BK1.2, BK1.3, and P672 protein. Y axis shows IC50 (M). Data are shown as mean s.e.m. of three biological replicates. Statistically significant differences (compared to BK1.1) using a one-way ANOVA with Sidak's correction for multiple comparisons, are indicated by asterisks: ****=P≤0.0001, ***=P≤0.001, **=P≤0.01, *=P≤0.05.
G. I. Representative dose-response curves showing inhibition of human CCL8-647 (g) and human CCL2-647 (i) induced THP-1 cell fluorescence by BK1.1 (black), BK1.2 (darker grey), BK1.3 (lighter grey), SCR (scrambled, negative control, light-grey without line) peptides and by P672 protein (positive control, mid grey). Y axis shows fluorescence (arbitrary units). Data are shown as mean of two technical replicates. X axis shows inhibitor concentration (Log10 Molar). Curves were fitted with 4 parameters to estimate IC50.
H. J. Summary IC50 values for inhibition of human CCL8-647 (h) or CCL2-647 (i) induced THIP-1 cell fluorescence by BK1.1, BK1.2, BK1.3, and P672 protein. Y axis shows IC50 (M). Data are shown as mean±s.e.m. of three biological replicates, shown as individual data points. Each biological experiment was conducted as two technical replicates. Statistically significant differences (compared to BK1.1) using a one-way ANOVA with Sidak's correction for multiple comparisons, are indicated by asterisks: ****=P≤0.0001, ***=P≤0.001, **=P≤0.01, *=P≤0.05.
A. Experimental design to characterize the zymosan air-pouch model. A dorsal air-pouch (a.p.) was created by subcutaneous (s.c.) injection of air on day 0 and day 3. Zymosan or PBS (control) was injected into the air-pouch (a.p.) on day 6. Air-pouch exudate was collected and analysed at 4 and at 24 hours by a membrane assay.
B. Top: Images of membranes used to analyse air-pouch fluid chemokines at different time points and conditions. See example 19 for details. Bottom: arrangement of chemokines, positive (PC) and negative (NC) controls.
C. Chemokine expression relative to the positive control which was set at 100, at different time points and conditions (mean of two spots. Indicated in shades of grey) and fold change (FC) in chemokine expression at 4 and 24 hours compared to PBS control (indicated in shades of grey).
A. Experimental design to assess efficacy of locally administered peptide. A dorsal air-pouch (a.p.) was created by subcutaneous (s.c.) injection of air on day 0 and day 3. Zymosan or PBS (control) was injected into the air-pouch (a.p.) on day 6. Peptide or protein (blue) was injected into the air-pouch on day 6 at the time of zymosan injection and repeated 9 hours later. Air-pouch exudate was collected and analysed on day 7 by flow cytometry (FC). Nine mice were studied in each of 5 study arms: PBS alone (PBS), zymosan (zymo), zymosan+scrambled peptide (SCR), zymosan+P672 (P672), and zymosan+BK1.3 (BK1.3).
B-F. Summary data for flow cytometry analysis for locally administered peptide. Y axis shows cell counts of total leucocytes (B), neutrophils (C), eosinophils (D), monocytes (E) and T-cells (F). Data are presented for each arm as mean±s.e.m. and with individual data points. Statistically significant differences (compared to zymosan) using a one-way ANOVA with Dunnett's correction for multiple comparisons, are indicated by asterisks: ****=P≤0.0001, ***=P≤0.001, **=P≤0.01, *=P≤0.05.
G. Experimental design to assess efficacy of intraperitoneally administered peptide. This is identical to that used for locally administered peptide (above) except that peptide or protein was administered intraperitoneally (i.p.). Nine mice were studied in each of 3 study arms: zymosan+SCR, zymosan+P672 (P672), and zymosan+BK1.3 (BK1.3).
H-L. Summary data for flow cytometry analysis for intraperitoneally administered peptide. Y axis shows cell counts of total leucocytes (H), neutrophils (I), eosinophils (J), monocytes (K) and T-cells (L). Data are presented for each arm as mean±s.e.m. and with individual data points. Statistically significant differences (compared to SCR) using a one-way ANOVA with Dunnett's correction for multiple comparisons, are indicated by asterisks: ****=P≤0.0001, ***=P≤0.001, **=P≤0.01, *=P≤0.05.
A. CCL8 protein (1-97)(SEQ ID NO: 110) aligned against the 44 peptides generated by peptic digestion of CCL8, yielding 96.9% coverage, and 8.0 redundancy (calculated as the average number of peptides in which each residue is found.
B. P672 protein (1-130)(SEQ ID NO: 111) aligned against the 38 peptides generated by peptic digestion of P672, yielding 100% sequence coverage, and 4.95 redundancy.
These peptides passed the identification criteria outlined example 19, and were used for further H/D exchange measurements. Adequate overlap is observed, except for CCL8 P2-N14 and A40-C52 regions for which only one peptide was identified.
Percentage of H/D exchange measured at four incubation time points (5 s, 30 s, 5 min, 60 min). Regions with increased HDX rates (exposed residues) are shown in green and regions with decreased HDX rates (protected residues) are shown as darker shades. Regions with no significant exchange are indicated as lighter grey. Protein tags are highlighted in grey box on sequence. All analyses were performed in triplicate. CCL8 (SEQ ID NO: 112); P672 (SEQ ID NO: 111).
A. Mass spectrum of 20 μM BK1.3 after incubation for 4 h in RPMI media at 37° C. mimicking cell assay. Note that RPMI contains a mix of amino acids including Cys-SH (200 μM). The mass M-S—S-Cys is that of the peptide bonded to a cysteine.
B. Mass spectrum of 20 μM BK1.3 after incubation for 1 h in AlphaScreen buffer (50 mM HEPES, 150 mM NaCl, 0.1% BSA, 0.01% Tween20, pH7.5) at room temperature mimicking AlphaScreen assay.
Representative flow cytometry data of zymosan air-pouch exudate from different arms of peptide in vivo efficacy experiments. Panel series from the top to bottom show scrambled (SCR) peptide, isotype control, SCR, P672 protein, BK1.3 peptide (see
SEQ ID NOs: 1 to 72 are shown in Tables 1, 4 and 5 below and in the electronic sequence listing.
SEQ ID NOs: 73 to 109 are shown above and in the Detailed Description and electronic sequence listing.
It is to be understood that different applications of the disclosed products and methods may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.
In addition as used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a polypeptide” includes two or more such polypeptides, or reference to “a polynucleotide” includes two or more such polynucleotides and the like.
All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. The disclosure of PCT/GB2017/050563 in relation to SEQ ID NOs 1-31 is specifically incorporated by reference, including each of Tables 1-5 and
Table 1. Tick CKBPs described in PCT/GB2017/050563 and [17]. Tick peptide sequences isolated in yeast surface display fluorescent-activated cell sorting (FACS) screens using a labelled chemokine. Identity with Evasin-1, 4 or 3 was calculated using BLAST. Abbreviations: IXORI—Ixodes ricinus, RHISA—Rhipicephalus sanguineus, AMBMA—Amblyomma maculatum, AMBPA—Amblyomma parvum, AMBTR—Amblyomma triste, AMBAM—Amblyomma americanum, AMBCA—Amblyomma cajennense, RHIPU—Rhipicephalus pulchellus.
Table 2A-C. Binding characteristics of tick CKBPs previously disclosed (PCT/GB2017/050563 and [17]).
All members of the human chemokine family[164] are listed in column 1. Binding to human chemokines was determined for 14 tick CKBPs using biolayer interferometry (BLI) [17,165] with calculated Kd shown as Molar (Moles/Litre). Binding data for 17 other novel tick CKBPs was assayed using yeast surface display[17,166], with positive binding results shown as “YES”. For biolayer interferometry, His-tagged purified tick CKBP was bound to a Ni-NINTA sensor on an OctetRed® 384 system, and then binding to each chemokine listed (with the exception of CCL25, CCL26, CXCL16, CXCL17, CXCL4L1, XCL2) was assayed in a cross-binding screen at a chemokine concentration of 300 nM as described[17]. For those chemokines showing binding to a tick CKBP in the cross-binding screen, binding assays were repeated using different doses of chemokine. Association, equilibrium and dissociation data were analysed using Octet software to create corresponding fitted curves, and used to calculate Kd. For yeast surface display, (YSD) background fluorescence was controlled for by using either an empty vector or by omitting the chemokine (i.e. using streptavidin-Alexa647 alone). An arbitrary threshold of >3 fold over background mean fluorescence intensity was chosen to describe confirmed re-tests. Where binding was detected data are indicated as “YES”. For biolayer interferometry (BLI) data, empty cells in
Table 3: Neutralisation of human chemokines by tick CKBPs previously disclosed (PCT/GB2017/050563 and [17]). Neutralising activity was determined using a quantitative THP1 cell migration assay in a 96-well Boyden chamber with chemokine in the bottom chamber as described[17]. Cells migrating through to the bottom chamber at 4 hours were counted using flow cytometry in a 96 well plate format. IC50 for neutralisation was determined at the chemokine EC80 dose as determined using a range of tick CKBP concentrations. Data was analysed using GraphPad Prism to determine IC50, which is shown as Molar (Moles/Litre). Empty cells represent experiments not done.
Table 4—Other previously described tick CKBPs [18,20-22]. Tick CKBPs that were also described in PCT/GB2017/050563 and Singh et al. [17] are indicated in “Notes”.
Table 5.—Novel tick CKBPs of the invention.
Table 6. Binding characteristics of new CXC chemokine binding tick CKBPs shown in Table 3. Column 1 shows sequence ID, column 2 the name of the tick peptide. The peptide sequence prefix indicates the identity, and suffix indicate the tick species as follows: AMBCA, Amblyomma cajennense and IXORI, Ixodes ricinus. Column 3 shows the identity of the chemokine that was used in the yeast surface display screen to isolate the yeast clone displaying the peptide from a yeast library. Certain tick peptides e.g. P1074_IXORI were recovered from screens performed with more than one chemokine. Individual yeast clones recovered from the library were re-tested using a FACS (fluorescence activated cell sorting), experiment. The binding of the peptide to the chemokine was assayed by measuring the percent (%), of yeast cells expressing the given peptide that exceeded a background threshold set by measuring the fluorescence of the yeast library pool treated with streptavidin-Alexa467 alone.
When more than one independent yeast clone was isolated for a given peptide-chemokine combination, the mean percent shift was calculated and reported in column 3. Note that as binding of other chemokines to the indicated tick peptide have not yet been determined, the data in column 3 is necessarily incomplete. Column 4 shows the percent identity of each tick peptide to prior art tick CKBPs (EVA1, EVA4 or EVA3). This was calculated using the BLAST algorithm using default parameters, and is reported together with the alignment length in residues. A blank cell indicates that no homology was identified by BLAST.
Table 7. Potential disease applications of tick CKBPs in Table 1. Table based on binding and inhibition data shown above and on published chemokine expression in disease states as shown in
Table 8. Potential disease applications of tick CKBPs in Table 5. Table based on binding and inhibition data shown above and on published chemokine expression in disease states as shown in
The invention provides a hybrid polypeptide representing amino acid sequences derived from two or more different tick CKBPs. The hybrid polypeptide typically has different chemokine binding properties compared to any single tick CKBPs from which it is derived. The hybrid polypeptide may have different chemokine binding properties compared to any single tick CKBP.
The invention typically provides a hybrid polypeptide comprising an amino acid sequence of a first tick CKBP or a variant thereof and an amino acid sequence of a second tick CKBP or a variant thereof, wherein said hybrid polypeptide has an altered chemokine binding profile compared to the first or second tick CKBP. The first and second tick CKBP polypeptides are not identical.
The hybrid polypeptide comprises at least an amino acid sequence of a first tick CKBP polypeptide or variant thereof, and an amino acid sequence of a second tick CKBP polypeptide or variant thereof, but may also comprise amino acid sequences from one or more other tick CKBP polypeptides or variants thereof. Thus, the hybrid polypeptide may be derived from three, four, five or more different tick CKBP polypeptides. The discussion herein of selection of second tick CKBP polypeptides by comparison with first tick CKBP polypeptides for provision of sequences for a hybrid polypeptide is also applicable to selection of any additional tick CKBP polypeptide to be represented in the hybrid polypeptide. Thus, an additional sequence to be provided from a further (for example, third) tick CKBP polypeptide may be selected to provide an additional chemokine-binding activity for the hybrid polypeptide compared to those provided by sequences derived from the other (for example, first and second) tick CKBP polypeptides.
Chemokine Binding
The altered chemokine binding profile for the hybrid polypeptide comprises the ability to bind a different selection of chemokines as compared to those bound by the first or second tick CKBP polypeptide individually. The hybrid polypeptide may thus be able to bind one or more chemokines not bound by the first or second tick CKBP polypeptide individually. The hybrid polypeptide may not exhibit binding to one or more chemokines that are bound by the first or second tick CKBP polypeptide. It should be understood that the altered chemokine binding profile for the hybrid polypeptide is by comparison to that of any single tick CKBP polypeptide from which it is derived, taken individually. Thus, the hybrid polypeptide displays an altered chemokine binding profile compared to any single tick CKBP polypeptide whose sequence it represents. In some aspects, the chemokine binding profile of the hybrid polypeptide may in contrast be substantially identical or identical to the cumulative (combined) chemokine binding profile of each of the individual tick CKBP polypeptides whose sequences it represents.
The hybrid polypeptide may bind at least one additional chemokine compared to a first tick CKBP polypeptide from which it is derived. The additional chemokine binding for the hybrid polypeptide is provided by the presence of at least one chemokine binding sequence derived from a different (second) tick CKBP polypeptide to the first tick CKBP polypeptide. The second tick CKBP polypeptide thus binds one or more different chemokines compared to the first tick CKBP polypeptide. The hybrid polypeptide may bind at least two, at least three, at least four, at least five, at least six, or at least eight additional chemokines as compared to the first tick CKBP polypeptide.
The hybrid polypeptide may bind in total at least two, at least three, at least four, at least five, at least six, at least eight, at least ten, at least twelve, at least fourteen or more different chemokines, The hybrid polypeptide may bind up to five, up to ten, up to twelve, up to fifteen or up to twenty different chemokines. The hybrid polypeptide may bind two to five, two to eight, two to ten, two to twelve, two to fifteen, or two to twenty different chemokines. The hybrid polypeptide may bind five to ten, five to fifteen, or five to twenty different chemokines.
The hybrid polypeptide may bind all chemokines bound by the two or more different tick CKBP polypeptides from which it is derived.
The hybrid polypeptide may alternatively bind a reduced number of chemokines compared to the total number of chemokines that are bound by the two or more different tick CKBP polypeptides from which it is derived. The reduced chemokine binding for the hybrid polypeptide may be provided by the loss of one or more chemokine binding sequences present in the two or more different tick CKBP polypeptides from which it is derived. In this aspect, the hybrid polypeptide may not bind at least one, at least two, at least three, at least four, at least five, at least six or at least eight of the chemokines that are bound (jn combination) by the two or more different tick CKBP polypeptides from which it is derived. In some aspects, the hybrid polypeptide may have reduced chemokine binding (bind to a reduced number of different chemokines) compared to any individual tick CKBP from which it is derived. Thus, it may only bind one chemokine, two chemokines, three chemokines, four chemokines, or five different chemokines. It may bind up to two, up to three, up to four or up to five different chemokines.
The chemokines may be selected from any known chemokines or chemokines newly identified in the future which are bound by tick CKBP polypeptides. The chemokines are preferably human chemokines. However chemokines may also be selected from other animals of veterinary importance (e.g. dog, cat, pig, sheep, cow, horse) and scientific importance (e.g. mouse, rat, monkey).
It is preferred that a hybrid polypeptide bind at least one CC chemokine and at least one CXC chemokine, i.e. at least one chemokine of the CC class and at least one chemokine of the CXC class. The known human CC and CXC chemokines are indicated in Table 2 and the hybrid polypeptide may bind any of the CC and/or CXC chemokines shown in Table 2. Certain CC and CXC chemokines are not known to be bound by tick CKBPs described to date (including ones detailed here). These include: CCL28, CXCL13, CXCL14, CXCL16, CXCL17, CXCL4, CXCL4L1.
Binding of at least one CC and at least one CXC chemokine is of particular utility in matching to chemokine expression in disease where both CC and CXC chemokines are expressed. A CC chemokine may be selected from any of the disease expressed CC chemokines shown in
Ideally a hybrid polypeptide should bind CC and CXC chemokines expressed and relevant to a particular disease. The hybrid polypeptide may be designed to bind CX3C and CC chemokines or CX3C and CXC chemokines, or CX3C, CC and CXC chemokines if the CX3C chemokine is expressed in the disease, and thought to be relevant to the disease.
Tick CKBPs
The tick CKBP polypeptides from which the hybrid polypeptide is derived may be selected from any tick CKBP polypeptides, including currently described tick CKBPs and tick CKBPs identified in the future. A tick CKBP polypeptide may be derived from any tick species, preferably a tick species that infects humans. The tick species may be selected from any of Amblyomma, Anomalohimalaya, Bothriocroton, Cosmiomma, Cornupalpatum, Compluriscutula, Dermacentor, Haemaphysalis, Hyalomma, Ixodes, Margaropus, Nosomma, Rhipicentor, Rhipicephalus, Nuttalliella, Antricola, Argas, Nothoaspis, Ornithodoros, and Otobius genera. A tick CKBP polypeptide binds one or more chemokines, preferably one or more human chemokines. A tick CKBP polypeptide typically binds multiple chemokines, such as at least two different chemokines.
The tick CKBP family is characterised by low sequence identity between members (although more closely related tick CKBPs may display greater sequence identity). Conserved structural features though exist allowing for ready classification of chemokine-binding proteins as tick CKBPs. A tick CKBP is typically a tick Evasin polypeptide. A tick CKBP may be a previously described tick Evasin or tick Evasin variant or a tick Evasin or tick Evasin variant identified in the future. An example of a previously described tick Evasin variant is provided by the sequence having the accession number EZ406190.1, which may be used in place of native Evasin-1 (SEQ ID NO: 32) in any sequence combination based on SEQ ID NO: 32 described herein.
A tick CKBP polypeptide may thus display a conserved set of eight cysteine residues (typically forming four disulphide bonds), which can be aligned with corresponding cysteine residues in known tick CKBP having a set of eight cysteines. Tick CKBPs of this type are typically CC binding tick CKBPs. Examples include Evasin-1 and Evasin-4 (SEQ ID NOs 32 and 34) and SEQ ID NOs 1-3, 6-9, 20-23, 29, and 35-44.
An illustration of a sequence alignment of tick CKBPs of this type against Evasin-1, showing the conserved eight cysteine positions, is provided in
Alternatively, a tick CKBP polypeptide may display a conserved set of six cysteine residues which can be aligned against sequences of known tick CKBPs also having a corresponding set of six cysteines. Tick CKBPs of this type are typically CXC binding CKBPs. Examples of CXC-binding tick CKBPs include Evasin-3 (SEQ ID NO: 33) and SEQ ID NOs 5, 18, 19, and 45-72. An illustration of a sequence alignment of tick CKBPs of this type against Evasin-3, showing the conserved six cysteine positions, is provided in
Tick CKBP polypeptides may also be selected from any of polypeptides comprising the amino acid sequence of any one of SEQ ID NOs 1-72 or naturally occurring homologues thereof, including homologues present in any tick species discussed above. Such naturally occurring homologues may comprise an amino acid sequence having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% homology or identity to the amino acid sequence of any one of SEQ ID NOs 1-72. Preferably, the above homology or identity is measured over the full length of the homologue.
As discussed above, a first tick CKBP polypeptide represented in the hybrid polypeptide is selected to differ from the second tick CKBP polypeptide represented in the hybrid polypeptide (and any additional tick CKBP polypeptides represented in the hybrid polypeptide are also selected to differ from other tick CKBPs represented). However, the first and second (and additional) tick CKBP polypeptides may otherwise be selected from any tick CKBP polypeptides discussed above. Each tick CKBP polypeptide selected as a basis for provision of the hybrid polypeptide typically has at least one differing chemokine-binding activity. Thus, for example a first tick CKBP polypeptide may be a CC-binding tick CKBP and the second tick CKBP polypeptide a CXC-binding tick CKBP. Alternatively, the first and second tick CKBP polypeptides may both be CC-binding tick CKBPs, but with at least one differing CC-binding activity between them. Thus, for example the first tick CKBP may bind CCL8 and the second tick CKBP CCL5, or the first tick CKBP may bind CCL8 and the second tick CKBP CCL5 and CCL8. In another aspect, the first and second tick CKBP polypeptides may both be CXC-binding tick CKBPs, but with at least one differing CXC-binding activity between them. Thus, for example the first tick CKBP may bind CXCL8 and the second tick CKBP CXCL12, or the first tick CKBP may bind CXCL1 and CXCL8 and the second tick CKBP CXCL1, CXCL3 and CXCL8.
Examples of differing chemokine-binding activities for individual tick CKBP polypeptides are provided in Tables 2-4 and 6. Combinations of two or more tick CKBPs may accordingly be selected together to provide a desired combination of chemokine-binding activities in a hybrid polypeptide, starting from consideration of the individual binding activities displayed by each tick CKBP. The combination of chemokine-binding activities may be selected to reflect chemokine expression in particular disease, such as any chemokine disease expression pattern shown in
Particular CC-binding tick CKBPs are provided by SEQ ID NOs 1-3, 6-9, 20-23, 29. 32, and 34-44. Particular CXC-binding tick CKBPs are provided by SEQ ID NOs 5, 18, 19, 33 and 45-72. Thus a hybrid polypeptide may comprise (i) an amino acid sequence of a first tick CKBP polypeptide selected from any one of SEQ ID NOs 1-3, 6-9, 20-23, 32, and 34-44, or a variant of any thereof, and (ii) an amino acid sequence of a second tick CKBP polypeptide selected from any one of SEQ ID NOs 5, 18, 19, 33 and 45-72 or a variant of any thereof. A preferred variant of SEQ ID NO: 3 is the peptide of SEQ ID NO: 89 (EDEDYEDFFKPVTCYF) or a variant thereof, such as SEQ ID NO: 88 (EDEDYEDFFKPVTAYF). A variant of SEQ ID NO: 89 typically binds CCL8, CCL7 and CCL18. SEQ ID NO: 89 or a variant thereof as above may be used in place of SEQ ID NO: 3 in any hybrid polypeptide described herein including an amino acid sequence of a tick CKBP polypeptide selected from SEQ ID NO: 3 or a variant thereof.
A hybrid polypeptide may alternatively comprise first and second tick CKBP amino acid sequences or variants thereof each selected from group (i) above, or first and second tick CKBP amino acid sequences each selected from group (ii) above.
Specific examples of hybrid polypeptides based on the above tick CKBP sequences are provided by the hybrid polypeptides comprising amino acid sequences as shown in SEQ ID NOs: 7-74 and 80-81 shown below.
GGS
ADDDNELFTVQYCGMNCTKDEGGTWTGCTGKKEGCKCYHESGKNYGL
SEQ ID NO: 73 comprises a first (CC-binding) tick CKBP amino acid sequence shown in SEQ ID NO: 9 and a second (CXC-binding) tick CKBP amino acid sequence shown in SEQ ID NO: 19. SEQ ID NO: 80 comprises these two tick CKBP amino acid sequences in the alternative order. More generally, a hybrid polypeptide may comprise the amino acid sequence of SEQ ID NO: 9 or a variant thereof and the amino acid sequence of SEQ ID NO: 19 or a variant thereof.
SEQ ID NO: 74 comprises a first (CC-binding amino acid sequence shown in SEQ ID NO: 29 and a second (CXC-binding) tick CKBP amino acid sequence shown in SEQ ID NO: 19. SEQ ID NO: 81 comprises these two tick CKBP amino acid sequences in the alternative order. A hybrid polypeptide may more generally comprise the amino acid sequence of SEQ ID NO: 29 or a variant thereof and the amino acid sequence of SEQ ID NO: 19 or a variant thereof.
Hybrid polypeptides comprising first, second and third (or more) chemokine binding sequences are also provided herein, including the 3-warhead evasins described below. SEQ ID NO: 92 comprises a first chemokine-binding sequence shown in SEQ ID 65, a second chemokine-binding sequence shown in SEQ ID 19, and a third chemokine-binding sequence shown in SEQ ID NO: 1, with intervening GGGGS linkers. A related hybrid polypeptide may more generally comprise the amino acid sequence of SEQ ID NO: 65 or a variant thereof, the amino acid sequence of SEQ ID NO: 19 or a variant thereof, and the amino acid sequence of SEQ ID NO: 1 or a variant thereof. The three amino acid sequences may be present in any order and may be fused contiguously or separated by any suitable linkers.
SEQ ID NO: 93 comprises a first chemokine-binding sequence shown in SEQ ID 65, a second chemokine-binding sequence shown in SEQ ID 19, and a third chemokine-binding sequence shown in SEQ ID NO: 9, with intervening GGGGS linkers. A related hybrid polypeptide may more generally comprise the amino acid sequence of SEQ ID NO: 65 or a variant thereof, the amino acid sequence of SEQ ID NO: 19 or a variant thereof, and the amino acid sequence of SEQ ID NO: 9 or a variant thereof. The three amino acid sequences may be present in any order and may be fused contiguously or separated by any suitable linkers.
Specific diseases that could be suitable for targeting with a hybrid polypeptide, and additional tick CKBP sequence combinations are described in the section “therapeutic methods of the invention” below.
Engineering of Hybrid Polypeptides
A hybrid polypeptide may be engineered from first and second tick CKBP polypeptides in any manner. A hybrid polypeptide may comprise a fusion of an amino acid sequence of a first tick CKBP polypeptide or a variant thereof and an amino acid sequence of a second tick CKBP polypeptide or a variant thereof. The amino acid sequences or variants thereof may be fused directly or separated by a suitable linker. Suitable linkers include, but are not limited to, chemical crosslinkers and peptide linkers. Peptide linkers are preferred if the polypeptide of the invention and second peptide or polypeptide are genetically fused. Preferred linkers are amino acid sequences (i.e. peptide linkers). A peptide linker may be of any amino acid sequence composition or length. A linker may be at least three, at least four, or at least five amino acids in length. The length, flexibility and hydrophilicity of the peptide linker are typically designed such that it does not to disturb the functions of the polypeptide of the invention. A linker is preferably selected to be conformationally flexible and may comprise one or more glycine residues, and optionally one or more serine residues. A linker may comprise in sequence at least two, at least three or least four glycine residues. A linker may consist essentially of or consist of glycine residues. Preferred flexible peptide linkers are stretches of 2 to 20, such as 4, 6, 8, 10 or 16, serine and/or glycine amino acids. Other preferred flexible linkers include (SG)1, (SG)2, (SG)3, (SG)4, (SG)5 and (SG)8 wherein S is serine and G is glycine. A particularly preferred linker sequence is GGGGS (SEQ ID NO: 75).
A hybrid polypeptide may comprise a fusion of a first tick CKBP amino acid sequence of SEQ ID NOs 1-72 or variant thereof and a second, different amino acid sequence selected from any one of SEQ ID NOs 1-72 or a variant thereof. A hybrid polypeptide may comprise a fusion of (i) an amino acid sequence selected from any one of SEQ ID NOs 1-3, 6-9, 20-23, 29, 32, and 34-44 or a variant thereof and (ii) an amino acid sequence selected from any one of SEQ ID NOs 5, 18, 19, 33 and 45-72. The amino acid sequence or variant of (i) and the amino acid sequence or variant of (ii) may be in either orientation; thus the amino acid sequence or variant of (i) may be N-terminal or C-terminal to the amino acid sequence or variant of (ii). Examples of hybrid polypeptides which are fusions of first and second tick CKBP amino acid sequences are provided by SEQ ID NOs 73, 74 and 80-81 described above.
Alternatively, a hybrid polypeptide may comprise the amino acid sequence of a second tick CKBP polypeptide or a variant thereof substituted into the amino acid sequence of a first tick CKBP polypeptide or variant thereof. Such a hybrid polypeptide comprises a substituted derivative of the amino acid sequence of the first tick CKBP polypeptide or variant thereof.
The substitution may introduce a chemokine-binding sequence provided by the amino acid sequence of the second tick CKBP polypeptide or variant thereof into the amino acid sequence of the first tick CKBP polypeptide or variant thereof. Alternatively or additionally, the substitution may remove a chemokine-binding sequence present in the amino acid sequence of the first tick CKBP polypeptide or variant thereof. The introduced chemokine-binding sequence may bind one or more chemokines. The chemokine binding sequence may bind at least one CC chemokine and/or at least one CXC chemokine.
The substitution may introduce a CXC chemokine-binding sequence from a first tick CKBP polypeptide or variant thereof into an amino acid sequence of a second tick CKBP polypeptide or variant thereof. The second tick CKBP polypeptide may not previously have any CXC-chemokine binding activity. Alternatively, the substitution may introduce an additional CXC-chemokine binding activity. The CXC chemokine-binding sequence may bind one or more of CXCL1-14, 16.
Alternatively, the substitution may introduce a CC chemokine-binding sequence from a first tick CKBP polypeptide or variant thereof into an amino acid sequence of a second tick CKBP polypeptide or variant thereof. The second tick CKBP polypeptide may not previously have any CC-chemokine binding activity. Alternatively, the substitution may introduce an additional CC-chemokine binding activity. The CC chemokine-binding sequence may bind one or more of CCL1-24, 28.
The substitution may result in a hybrid polypeptide only having chemokine-binding activity from the introduced chemokine-binding sequence. The substitution may introduce a single chemokine-binding activity. The substitution may introduce a single chemokine-binding activity and reduce or remove the original chemokine-binding activity of the tick CKBP amino acid sequence or variant thereof into which the substitution is made. The substitution may result in a hybrid polypeptide binding a reduced number of chemokines compared to the second tick CKBP polypeptide or variant thereof.
The substitution may comprise exchange of any sequence region in the amino acid sequence of the second tick CKBP polypeptide or variant thereof for any sequence region in the amino acid sequence of the first tick CKBP polypeptide or variant thereof. The substitution may be of a chemokine-binding sequence in the amino acid sequence of the first tick CKBP polypeptide or variant thereof for a chemokine-binding sequence in the amino acid sequence of the second tick CKBP polypeptide or variant thereof. Alternatively, the substitution may introduce an additional chemokine-binding sequence provided by the first tick CKBP polypeptide or variant thereof into a region of the amino acid sequence of the second tick CKBP polypeptide or variant thereof not comprising a chemokine-binding sequence.
A hybrid polypeptide may comprise a substitution of a first tick CKBP amino acid sequence of SEQ ID NOs 1-72 or variant thereof into a second, different amino acid sequence selected from any one of SEQ ID NOs 1-72 or a variant thereof. A hybrid polypeptide may comprise a substitution of (i) an amino acid sequence selected from any one of SEQ ID NOs 1-3, 6-9, 20-23, 29, 32, and 34-44 or a variant thereof into (ii) an amino acid sequence selected from any one of SEQ ID NOs 5, 18, 19, 33 and 45-72, or vice-versa. Such a hybrid polypeptide may comprise a substitution of a chemokine-binding sequence from an amino acid sequence of (i) into an amino acid sequence of (ii) or a variant thereof. Alternatively, a chemokine-binding sequence from an amino acid sequence of (ii) may be substituted into an amino acid sequence of (i) or a variant thereof.
A specific example of a hybrid polypeptide comprising a substitution of an amino acid sequence of a second tick CKBP polypeptide into the amino acid sequence of a first tick CKBP polypeptide is provided by a polypeptide comprising the amino acid sequence of SEQ ID NO: 76, shown below.
VCEVSEQEGVGEDNATEDEDYEDFFKPVTCYFANSTVGPLRPPN
CKQD
CNGTTETAPNGTRCFSIGDEGLRRMTANLPYDCPLGQCSNGDCIPKET
YEVCYRRNWRDEKN.
The introduced chemokine binding sequence comprised in SEQ ID NO: 76 is derived from SEQ ID NO: 3 and shown below as SEQ ID NO: 77. SEQ ID NO:77 provides a chemokine-binding sequence binding CCL8. The full-length recipient tick CKBP sequence substituted to create SEQ ID NO: 76 is a variant sequence of Evasin-1 of SEQ ID NO: 32 shown in EZ406190.1 (as discussed above), with the residual recipient sequence remaining after the substitution shown below as SEQ ID NO: 78. The sequence removed from SEQ ID NO: 32 by the substitution is shown below as SEQ ID NO: 79. A chemokine-binding sequence comprising SEQ ID NO: 79 may be used to provide one or more chemokine-binding functions of Evasin-1.
The hybrid polypeptide of SEQ ID NO: 76 binds CCL8 by virtue of the introduced chemokine-binding sequence from the tick CKBP of SEQ ID NO: 3, whereas Evasin-1 natively does not have CCL8-binding activity. Accordingly, the inventors have shown the ability to isolate an independent binding function from a first tick CKBP and transport this into a second tick CKBP, resulting in a hybrid tick CKBP polypeptide with an altered chemokine binding profile.
Also provided is a hybrid polypeptide comprising the amino acid sequence of SEQ ID NO: 76 or a variant thereof substituted into the amino acid sequence of any tick CKBP polypeptide. A variant of SEQ ID NO: 76 is selected to have CCL8-binding activity. The tick CKBP polypeptide may be selected from any tick CKBP described above. The tick CKBP amino acid sequence into which the amino acid sequence of SEQ ID NO: 76 or a variant thereof is substituted may be selected from any of SEQ ID Nos 1-72 or variants thereof. The tick CKBP amino acid sequence is typically one which does not have CCL8-binding activity, such as SEQ ID NO: 32, 39, 41. The tick CKBP amino acid sequence may be selected from one having a conserved set of eight cysteines as described above, for example any one of SEQ ID NOs 1-3, 6-9, 20-23, 29, 32 and 34-44.
Also provided is a hybrid polypeptide comprising the amino acid sequence of P672_RHIPU E22-F32 (SEQ ID NO: 108) or of any one of SEQ ID NOs 88, 89 and 103-107, or a variant of any thereof substituted into the amino acid sequence of any tick CKBP polypeptide described herein. The tick CKBP polypeptide may be SEQ ID NO: 32. In one aspect, the hybrid polypeptide comprises the amino acid sequence of SEQ ID NO: 95 or a variant thereof.
Further provided is a hybrid polypeptide comprising a chemokine binding sequence of a first tick CKBP amino acid sequence or a variant thereof fused (directly or by a linker as described above) to SEQ ID NO: 78 or a variant thereof. A variant of SEQ ID NO: 78 (or of any other recipient sequence fragment derived from Evasin-1 described herein) includes the corresponding sequence fragment from Evasin-1 of SEQ ID NO: 32 (without the K92E substitution discussed herein). SEQ ID NO: 78 represents an amino acid sequence derived from Evasin-1 able to functionally accommodate a chemokine-binding sequence from another tick CKBP. The chemokine-binding sequence to be provided upstream of SEQ ID NO: 78 or a variant thereof typically binds one or more chemokines that are not bound by SEQ ID NO: 32. The chemokine-binding sequence may be derived from a first tick CKBP amino acid sequence selected from any one of SEQ ID NOs 1-3, 6-9, 20-23, 29, 32 and 34-44.
The inventors have also shown that a greater extent of sequence may be introduced from the tick CKBP of SEQ ID NO: 3 into Evasin-1, and a lesser extent of recipient sequence retained, while providing a hybrid polypeptide binding CCL8. This demonstrates flexibility in substitution of chemokine-binding sequences from one tick CKBP into another tick CKBP. Thus, the additional substituted hybrid polypeptides of SEQ ID NOs 82 and 83 are provided, as shown below. The first (introduced) tick CKBP sequence is bold and underlined; and the residual recipient second tick CKBP sequence in italics): The introduced and recipient tick CKBP sequences are shown below as SEQ ID NOs 84-85 (derived from SEQ ID NO: 82) and SEQ ID NOs: 86-87 (derived from SEQ ID NO: 83).
VCEVSEQEGVGEDNATEDEDYEDFFKPVTCYFANSTVGPLRPPNCTVV
CTNNTAWWNDTKSDGGHCYSEYRPEKRTHSREIYNCTIGVCGNG
DCIP
KETYEVCYRRNWRDEKN
VCEVSEQEGVGEDNATEDEDYEDFFKPVTCYFANSTVGPLRPPNCTVV
CTNNTAWWNDTKSDGGHC
FSIGDEGLRRMTANLPYDCPLGQCSNGDCI
PKETYEVCYRRNWRDEKN
Also provided herein is a hybrid polypeptide comprising the amino acid sequence of SEQ ID NO: 84 or 86 or a variant of either thereof substituted into the amino acid sequence of any tick CKBP polypeptide. A variant of SEQ ID NO: 84 or 86 is selected to have CCL8-binding activity. Additionally described herein is a hybrid polypeptide comprising a chemokine binding sequence of a first tick CKBP amino acid sequence or a variant thereof fused (directly or by a linker as described above) to SEQ ID NO: 85 or 87 or a variant of either thereof.
Thus, a range of hybrid polypeptides may be provided based on substitution of a chemokine-binding sequence of a first tick CKBP polypeptide into the amino acid sequence of a second tick CKBP polypeptide. Hybrid polypeptides may also be provided which comprise one or more substituted tick CKBP amino acid sequences as described above (comprising a chemokine-binding sequence derived from a first tick CKBP amino acid sequence) fused directly or via a linker region with one or more additional tick CKBP amino acid sequences or variant thereof. Thus, a hybrid polypeptide may comprise a chemokine-binding sequence derived from a first tick CKBP polypeptide and additional tick CKBP amino acid sequences, for example one or two additional tick CKBP amino acid sequences or variants thereof. Such additional tick CKBP amino acid sequences may be selected from any one of SEQ ID NOs 1-72. The combination of one or more chemokine-binding sequences (which may be specific for a single chemokine) and one or more additional tick CKBP amino acid sequences may assist provision of a specific chemokine-binding profile of interest.
Chemokine-Binding Sequences
Identification of a suitable chemokine-binding sequence and selection of a region for substitution may be performed by various means. The inventors have identified that discrete contiguous sequence regions of tick CKBP polypeptides encode chemokine-binding activity. Thus, a tick CKBP polypeptide may be truncated N- or C-terminally and a series of truncated polypeptides then screened for binding activity for one or more chemokines bound by the full-length tick CKBP. Where the tick CKBP polypeptide has a conserved set of eight cysteine residues as discussed above, the inventors' analysis in relation to polypeptides of this group (SEQ ID Nos 3 and 32) indicates that one or more chemokine binding activities are typically present in an N-terminal region. Thus, C-terminal truncations of SEQ ID NOs 1-3, 6-9, 20-23, 29, 32 and 34-44 may be preferable when providing a chemokine-binding sequence based on any of the above tick CKBP polypeptides.
The chemokine-binding sequence may be identified by hydrogen-deuterium exchange mass spectrometry (HDX-MS). The amino acid residues involved in the interface between a chemokine and a tick CKBP polypeptides can be identified and a chemokine-binding sequence inferred. Thus, a tick CKBP polypeptide may result from N- and/or C-terminal truncations of a tick CKBP polypeptide. A tick chemokine binding polypeptide may be truncated N- and/or C-terminally and/or be chemically modified. A series of truncated and/or modified polypeptides may be screened for binding activity for one or more chemokines bound by the full-length tick CKBP. A tick chemokine binding polypeptide may be extended N- and/or C-terminally and/or chemically modified.
The chemokine binding sequence may be at least 10, at least 11, at least 12, at least 15, at least 16, at least 17, at least 20, at least 30, at least 40, at least 50, at least 80 or at least 90 amino acids in length, depending on the particular tick CKBP. The chemokine binding sequence may be of 20-100, 20-90, 20-70, 20-60, 20-50, 10-100, 11-100, 12-100, 16-100, 11-70, 11-60, 11-50, 11-17 amino acids in length. Corresponding N- or C-terminal truncations may be made to any tick CKBP polypeptide described herein to provide a chemokine-binding sequence, and also a recipient sequence able to accommodate a chemokine-binding sequence derived from another tick CKBP.
A chemokine-binding sequence may thus be identified by performing a chemokine binding assay on truncation variants of a tick CKBP polypeptide, such as the biointerferometry assay described in Table 2A, and also in [17]. Other suitable binding assays include HDX-MS, mass spectrometry (MS) dimerization, Alphascreen, surface plasmon resonance, microscale thermophoresis, fluorescent polarization, and FRET based assays. The tick CKBP polypeptide may be truncated to provide a minimal chemokine-binding sequence (for one or more chemokines of interest), and not include other sequence of the tick CKBP polypeptide not essential for the relevant chemokine-binding activity. Truncation variants of a first tick CKBP amino acid sequence that comprise chemokine-binding sequences may also be screened for their ability to inhibit or neutralize chemokine activity, for example by performing a chemokine-induced cell migration assay, for example the assay as described in Table 3, or as described in
A variant of SEQ ID NOs: 88, 89 or 105 to 107 preferably retains one or more, two or more, three or more, preferably four N-terminal acidic amino acid residues. A variant of SEQ ID NOs: 88, 89 or 103 to 107 preferably does not substitute Pro27 for another amino acid (numbering according to the P672_RHIPU parental evasin, SEQ ID NO: 3). A variant of SEQ ID NOs: 89 or 104 to 107 preferably does not substitute Cys30 for alanine (numbering according to the P672_RHIPU parental evasin, SEQ ID NO: 3). A variant of SEQ ID NOs: 88, 89 or 103 to 107 may comprise a substitution of one or more other amino acid residues to alanine or a similar amino acid residue. Such changes do not significantly affect the ability of the peptide to specifically bind CCL8 (see
A truncated tick CKBP polypeptide such as SEQ ID NO: 89 may also be extended N- and/or C-terminally and variants screened for their ability to inhibit or neutralize chemokine activity, for example by performing suitable binding assays such as those described above. For example, the fluorescent polarization, MS dimerization, Alphascreen and cell migration assays described in
Examples of functional substituted variants are provided by peptide BK1.1 (SEQ ID NO: 88). Peptide BK1.1 retained the ability of the parental (BK1.5, SEQ ID NO: 89) peptide to bind chemokines CCL7, CCL8 and CCL18, which is retained with or without an N-terminal FITC molecule (
Also provided is a method of treating or preventing in a subject one or more diseases associated with CCL2, CCL3, CCL7, CCL8 and/or CCL18, the method comprising administering to the subject a polypeptide selected from any of SEQ ID NOs: 88, 89, 103 to 109, or a variant thereof. The disease is preferably a disease associated with CCL8. The polypeptide is preferably SEQ ID NO: 106.
A tick CKBP polypeptide may also be modified. Examples of modification include addition of an N-terminal tyrosine residue or presence of an intra- or inter-polypeptide disulfide bond (see for example BK1.3 peptide,
A consensus sequence for a functional variant peptide based on SEQ ID NO: 88, 89 or 103 to 107 is also provided herein. A variant of SEQ ID NO: 88, 89 or 105 to 107 may retain one or more N-terminal acidic amino acid residues, such as EDED. A variant of SEQ ID NO: 88, 89 or 103 to 107 may retain Pro27 and/or Cys30 (numbering according to the parental P672_RHIPU evasin, SEQ ID NO: 3). Other amino acid residues may be removed, added and/or substituted, such as for alanine or a similar amino acid residue. Said variant preferably specifically binds CCL8.
SEQ ID NOs: 103-107, and more preferably SEQ ID NO: 106 may be used alone as a chemokine-binding agent or as a chemokine-binding sequence in any hybrid polypeptide described herein.
The hybrid polypeptide having a chemokine-binding sequence introduced from the first tick CKBP polypeptide can be confirmed as having the transferred chemokine-binding activity by performing similar binding, inhibition and/or neutralization assays for the relevant chemokine(s).
Additionally, chemokine-binding sequences may be identified based on sequence alignment and structural modelling of tick CKBPs.
Structural modelling may also be used to assist determination of chemokine-binding sequences. A published structure is available for Evasin-1 (3FPU, SEQ ID NO: 32)[22]), in complex with CCL3. As described in
Structural analysis may also identify residues that make inter-chain hydrogen or salt bridges and suitable points of transfer that do not disrupt structural folds or motifs, assisting selection of a discrete chemokine-binding sequence and a position for introduction of a chemokine-binding sequence in a recipient tick CKBP amino acid sequence. The models shown in
The invention further provides a polypeptide comprising (a) all or part of an amino acid sequence shown in any one of SEQ ID NOs 45-72 or (b) all or part of an amino acid sequence having at least 70% homology or identity to a sequence of (a) over its entire length, wherein said polypeptide binds at least one CXC-class chemokine. SEQ ID NOs 45-72 represent tick CKBP amino acid sequences newly identified and functionally characterised as binding CXC chemokines by the inventors.
The sequence of (a) may be an amino acid sequence shown in any one of SEQ ID NOs 45-60 and 64-65. In such an embodiment, the polypeptide binds one or more human chemokines selected from CXCL7, CXCL9, CXCL10, CXCL11 and CXCL12.
The sequence of (a) may be an amino acid sequence shown in any one of SEQ ID NOs 45-48, 51-53, 56, 59, 60, and 65. In this embodiment, the polypeptide binds one or more human chemokines selected from CXCL7, CXCL9, and CXCL11.
The polypeptide can be any length. The polypeptide is preferably at least 40 amino acids in length, such as at least 50, at least 60, at least 70 or at least 80 amino acids in length. The polypeptide is preferably 250 amino acids or fewer in length, such as 200 amino acids or fewer, 150 amino acids or fewer or 100 amino acids or fewer in length. The length of the polypeptide typically depends on the length of any one of SEQ ID NOs 45-72. Deletions and/or extension are allowable in accordance with the invention as discussed in detail below. The polypeptide is typically from 40 to 250 amino acids in length, such as from 45 to 200 amino acids in length or from 50 to 160 amino acids in length.
The polypeptide is typically formed from naturally-occurring amino acids. The polypeptide may contain non-naturally-occurring amino acids. The polypeptide typically comprises L-amino acids. The polypeptide may comprise D-amino acids.
The selection of variants of SEQ ID NOs: 45 to 72 as discussed below is also applicable to selection of variants of any of SEQ ID NOs 1-44, 73-74 and 76-94. A variant of any one of SEQ ID NOs: 45 to 72 is a polypeptide that has an amino acid sequence which varies from that of any one of SEQ ID NOs: 45 to 72 and has the ability to bind to one or more chemokines. A variant of any one of SEQ ID NOs: 45 to 72 may be a polypeptide that has an amino acid sequence which varies from that of any one of SEQ ID NOs: 45 to 72 and has the ability to bind to and inhibit one or more chemokines.
The variant preferably binds and preferably inhibits one or more or all of the same chemokines as the sequence on which the variant is based. For instance, a variant of SEQ ID NO: 45 is a polypeptide that has an amino acid sequence which varies from that of SEQ ID NO: 45 and has the ability to bind to the chemokine shown in SEQ ID NO: 45's row in Table 6 (CXCL9). The same is true for any of SEQ ID NOs: 46 to 72. Thus, variants of the tick CKBPs as described above preferably bind to and preferably inhibit the same chemokines as the sequence on which the variant is based.
The ability of a variant to bind to and preferably inhibit a chemokine can be assayed using any method known in the art. Suitable methods are described in the Examples and Figures, and include yeast surface display and biolayer interferometry (for binding) and chemotaxis assays (for inhibition).
The variant may be a naturally occurring variant which is expressed naturally, for instance in ticks. Alternatively, the variant may be expressed in vitro or recombinantly as discussed below. Variants also include non-naturally occurring variants produced by recombinant technology.
Over the entire length of the amino acid sequence of any one of SEQ ID NOs: 45 to 72 (or SEQ ID NOs 1-44, 73-74 and 76-94), a variant will preferably be at least 70% homologous or identical to that sequence. More preferably, the variant may have at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% homology or amino acid identity to the amino acid sequence of any one of SEQ ID NOs: 35 to 62 over the entire sequence. There may be at least 80%, for example at least 85%, 90% or 95%, homology or amino acid identity over a stretch of 20 or more, for example 30, 40, 50, 60, 70, or more, contiguous amino acids (“hard homology” or “hard identity”).
Standard methods in the art may be used to determine homology. For example the UWGCG Package provides the BESTFIT program, which can be used to calculate homology, for example used on its default settings (Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (such as identifying equivalent residues or corresponding sequences (typically on their default settings)), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S. F et al (1990) J Mol Biol 215:403-10. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov).
Amino acid substitutions may be made to the amino acid sequences of SEQ ID NOs: 45 to 72 (or SEQ ID NOs 1-44, 73-74 and 76-107), for example up to 1, 2, 3, 4, 5, 10, 20, 30 or 50 substitutions. Conservative substitutions replace amino acids with other amino acids of similar chemical structure, similar chemical properties or similar side-chain volume. The amino acids introduced may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge to the amino acids they replace. Alternatively, the conservative substitution may introduce another amino acid that is aromatic or aliphatic in the place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well-known in the art and may be selected in accordance with the properties of the 20 main amino acids as defined in Table 9 below. Where amino acids have similar polarity, this can also be determined by reference to the hydropathy scale for amino acid side chains in Table 10.
One or more amino acids of the amino acid sequence of any one of SEQ ID NOs: 45 to 72 may additionally be deleted from the polypeptides described above. Up to 1, 2, 3, 4, 5, 10, 20 or 30 amino acids may be deleted, or more.
Variants may include fragments of any one of SEQ ID NOs: 45 to 72. Such fragments typically retain a chemokine-binding sequence (for one or more chemokines) of any one of SEQ ID NOs: 45 to 72. Fragments may be at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids in length.
One or more amino acids may be alternatively or additionally added to the polypeptides described above. Put another way, the polypeptide may comprise a sequence consisting of any one of SEQ ID NOs: 45 to 72 or a variant thereof having an N-terminal and/or C-terminal extension of a number of amino acids. The N-terminal and/or C-terminal extension may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids or more, such as 15, 20, 30, 40, 50 or 100 amino acids.
Variants of other tick CKBP amino acid sequences described herein (such as SEQ ID NOs 1-44) are typically selected according to the same principles described above for variants of SEQ ID NOs 45-72. Thus, a variant of a first tick CKBP amino acid sequence selected for inclusion in a hybrid polypeptide, and a variant of a second tick CKBP polypeptide also selected for inclusion in the hybrid polypeptide (as described above) may comprise (a) part of the relevant tick CKBP amino acid sequence or (b) all or part of an amino acid sequence having at least 70% homology or identity to the relevant tick CKBP amino acid sequence over its entire length. The variant may comprise any extent of length of the tick CKBP amino acid sequence as described above. The variant may comprise any degree of homology or identity to the relevant tick CKBP amino acid sequence as described above, such as at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% homology or amino acid identity to the amino acid sequence of the relevant tick CKBP amino acid sequence over the entire sequence. The variant may comprise substitutions or represent a fragment or extension of the tick CKBP amino acid sequence as described above. Typically the variant binds to and preferably inhibits one or more of the same chemokines as the tick CKBP amino acid sequence on which it is based. Chemokine binding for SEQ ID NOs 1-72 is shown in Tables 2, 4 and 6 above. Thus, a variant of a given tick CKBP amino acid sequence selected from SEQ ID NOs 1-72 may bind to (and preferably inhibit) one or more of, or all of the chemokines shown to be bound by the relevant tick CKBP polypeptide in Tables 2, 4 and 6.
The invention additionally provides variants of the hybrid polypeptides of SEQ ID NOs 73, 74, 76, 80-82 and 92-95, or of the chemokine-binding and recipient sequences of SEQ ID NOs 76-78, 84-89 and 103-107 selected accorded to similar principles to those described above. Such variants may be selected to have the same chemokine binding as the above hybrid polypeptides or chemokine-binding sequences as described herein, or the same ability to act as recipient for a chemokine binding sequence, and for example to comprise a degree of identity or homology to SEQ ID NOs 73, 74, 76-78, 80-89, 92-94 and 103-107 as described above.
Any references to polypeptides herein encompass the hybrid polypeptides discussed above, and the novel tick CKBP polypeptides described above, unless indicated otherwise.
The invention encompasses any pharmaceutically acceptable salt of a polypeptide described herein. Said pharmaceutically acceptable salts include, for example, mineral acid salts such as chlorides, hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like; and salts of monocationic metal ions such as sodium and potassium and the like; and salts of bases such as ammonia. A hydrochloride salt or an acetate salt is preferred.
The polypeptide may be labelled with a detectable label. The detectable label may be any suitable label which allows the polypeptide to be detected. Suitable labels include, but are not limited to, fluorescent molecules, radioisotopes, e.g. 125I, 35S, enzymes, antibodies, antigens, polynucleotides and ligands such as biotin. The label is preferably a tracer that is suitable for positron emission tomography (PET), such as fluorine (18F). The label is preferably a tracer suitable for magnetic resonance imaging (MRI), such as fluorine (19F). The label may be a Fluorescein isothiocyanate (FITC) moiety.
The polypeptides of the invention may be made in any way. They may be made in accordance with the invention as discussed in more detail below.
The polypeptides described herein can be prepared by any suitable technique.
Alternatively, the polypeptide may be made by solid-phase peptide synthesis (SPPS) is a preferred technique. This involves formation of the peptide on small solid beads. Using SPPS, the polypeptide remains covalently attached to a bead during synthesis. The polypeptide is synthesised using repeated cycles of coupling-washing-deprotection-washing. In particular, the free N-terminal amine of a solid-phase attached polypeptide is coupled to a single N-protected amino acid unit. This unit is then deprotected, revealing a new N-terminal amine to which a further protected amino acid is attached. These steps are repeated until the polypeptide is complete. The polypeptide is then cleaved from the beads using a suitable reagent.
Suitable protecting groups, reagents, solvents and reaction conditions for SPPS are well known to those skilled in the art and as such conditions can be determined by one skilled in the art by routine optimization procedures.
Pharmaceutically acceptable salts of polypeptides can be prepared by any suitable technique. Typically, salification involves reaction of the polypeptide or a salt thereof with a suitable reagent, typically acid, to obtain the pharmaceutically acceptable salt selected.
For example, a hydrochloride salt of a polypeptide can be prepared by initially cleaving the polypeptide from the solid phase using trifluoroacetic acid. The polypeptide will thus initially be a trifluoroacetate salt. The trifluoroacetate salt can then be converted into a hydrochloride salt by any known technique, such as ion exchange on a suitable column using hydrochloric acid as an eluent.
The polypeptide or polypeptide salt products can be purified, where required, by any suitable technique. High pressure liquid chromatography (HPLC) can be used, for example.
The term “polypeptide” includes not only molecules in which amino acid residues are joined by peptide (—CO—NH—) linkages but also molecules in which the peptide bond is reversed. Such retro-inverso peptidomimetics may be made using methods known in the art, for example such as those described in Meziere et al (1997) J. Immunol. 159, 3230-3237. This approach involves making pseudopolypeptides containing changes involving the backbone, and not the orientation of side chains.
Similarly, the peptide bond may be dispensed with altogether provided that an appropriate linker moiety which retains the spacing between the carbon atoms of the amino acid residues is used; it is particularly preferred if the linker moiety has substantially the same charge distribution and substantially the same planarity as a peptide bond. It will also be appreciated that the peptide may conveniently be blocked at its N-or C-terminus so as to help reduce susceptibility to exoproteolytic digestion. For example, the N-terminal amino group of the polypeptides may be protected by reacting with a carboxylic acid and the C-terminal carboxyl group of the peptide may be protected by reacting with an amine. Other examples of modifications include glycosylation and phosphorylation. Another potential modification is that hydrogens on the side chain amines of R or K may be replaced with methylene groups (—NH2→
—NH(Me) or —N(Me)2). Other potential modifications include thioether cyclization and intra- and/or inter-peptide disulphide bonds.
Polypeptides according to the invention may also include peptide variants that increase or decrease the polypeptide's half-life in vivo. Examples of analogues capable of increasing the half-life of polypeptides used according to the invention include peptoid analogues of the peptides, D-amino acid derivatives of the peptides, and peptide-peptoid hybrids. A further embodiment of the variant polypeptides used according to the invention comprises D-amino acid forms of the polypeptide. The preparation of polypeptides using D-amino acids rather than L-amino acids greatly decreases any unwanted breakdown of such an agent by normal metabolic processes, decreasing the amounts of agent which needs to be administered, along with the frequency of its administration.
The polypeptides may also be derived from amino acid mutants, glycosylation variants and other covalent derivatives of the parent polypeptides. Exemplary derivatives include molecules wherein the polypeptides of the invention are covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid. Further included are naturally occurring variant amino acid sequences of the parent polypeptides. Such a variant amino acid sequence may be encoded by an allelic variant or represent an alternative splicing variant.
Modifications as described above may be prepared during synthesis of the peptide or by post-production modification, or when the polypeptide is in recombinant form using the known techniques of site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and/or ligation of nucleic acids.
The polypeptides described herein may also be modified to improve physicochemical characteristics. Thus, for example, original amino acid sequences may be altered to improve their solubility, and accordingly a polypeptide of the invention having a variant sequence will preferably be more soluble than a polypeptide having the corresponding original amino acid sequence under equivalent conditions. Methods for evaluating the solubility of polypeptides are well known in the art.
The present invention also provides a fusion polypeptide comprising fusion polypeptide comprising a polypeptide of the invention linked to a second peptide or polypeptide. The polypeptide of the invention may be any of those discussed above. The polypeptide of the invention is typically covalently linked to the second peptide or polypeptide. The polypeptide of the invention is typically genetically fused to the second peptide or polypeptide. The polypeptide of the invention is genetically fused to the second peptide or polypeptide if the whole construct is expressed from a single polynucleotide sequence. The coding sequences of the polypeptide of the invention and the second peptide or polypeptide may be combined in any way to form a single polynucleotide sequence encoding the construct. They may be genetically fused in any configuration. They are typically fused via their terminal amino acids. For instance, the amino terminus of the polypeptide of the invention may be fused to the carboxy terminus of the second peptide or polypeptide and vice versa.
The polypeptide of the invention may be attached directly to the second peptide or polypeptide. The polypeptide of the invention is preferably attached to the second peptide or polypeptide using one or more linkers. The one or more linkers may be designed to constrain the mobility of the polypeptides. Suitable linkers include, but are not limited to, chemical crosslinkers and peptide linkers. Peptide linker are preferred if the polypeptide of the invention and second peptide or polypeptide are genetically fused. Preferred linkers are amino acid sequences (i.e. peptide linkers). The length, flexibility and hydrophilicity of the peptide linker are typically designed such that it does not to disturb the functions of the polypeptide of the invention. Preferred flexible peptide linkers are stretches of 2 to 20, such as 4, 6, 8, 10 or 16, serine and/or glycine amino acids. More preferred flexible linkers include (SG)1, (SG)2, (SG)3, (SG)4, (SG)5 and (SG)8 wherein S is serine and G is glycine. Preferred rigid linkers are stretches of 2 to 30, such as 4, 6, 8, 16 or 24, proline amino acids. More preferred rigid linkers include (P)12 wherein P is proline. The polypeptide of the invention may be attached to the second peptide or polypeptide via the side chains of the amino acid residues. Such attachments include thioether and disulphide bonds.
The polypeptide of the invention may be transiently attached to the second peptide or polypeptide by a hex-his tag or Ni-NTA. They may also be modified such that they transiently attach to each other. The polypeptide of the invention may also be attached to the second peptide or polypeptide via cysteine linkage. This can be mediated by a bi-functional chemical linker or by a polypeptide linker with a terminal presented cysteine residue.
The second peptide or polypeptide may be any peptide or protein. The second protein is preferably a fragment crystallizable region (Fc region). The Fc region may be from any of the types of subject discussed below. Fc region is preferably human. The Fc region may derived from any isotype of antibody, such as IgA, IgD, IgG, IgE or IgM.
The second peptide or polypeptide may be an epitope tag or purification tag or cell-surface display tag or a tag that enables or facilitates systemic peptide delivery or delivery and targeting to a specific organ or to a tumour, or facilitates transfer across a barrier such as skin or gut or blood brain barrier. Suitable tags are known in the art. Suitable tags include, but are not limited to, AviTag, calmodulin-tag, polyglutamate tag, E-tag, FLAG-tag, HA-tag, His-tag, Myc-tag, S-tag, SBP-tag, Softag 1, Softag 3, Strep-tag, TC tag, V5 tag, VSV-tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, BCCP (Biotin Carboxyl Carrier Protein), Glutathione-S-transferase-tag, Green fluorescent protein-tag, Halo-tag, Maltose binding protein-tag, Nus-tag, Thioredoxin-tag, Strep-tag, Skin permeating and cell entering (SPACE)-tag, TD1-tag, magainin tag, TAT-tag, penetratin-tag, cell penetrating peptide (CPP)-tag, Fc tag. The second peptide or polypeptide may be a signal peptide, such as an IgK peptide.
The fusion polypeptide may be labelled with a detectable label. The detectable label may be any of those discussed above.
The invention also provides a combination of two or more polypeptides of the invention, i.e. two or more different polypeptides of the invention. The combination may comprise two or more polypeptides of the invention, two or more fusion polypeptides of the invention or a two or more of both types of polypeptide.
The combination may comprise any number of different polypeptides of the invention. For instance, the combination may comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 different polypeptides of the invention. The combination may comprise 10 or more, 20 or more, 30 or more, 40 or more or 50 or more polypeptides of the invention.
One or more of, such as all of, the polypeptides in the combination may be labelled with a detectable label. The label may be any of those discussed above. Different polypeptides in the combination may be labelled with the same detectable label or different detectable labels.
The invention also provides a polynucleotide which encodes a polypeptide of the invention. The polypeptide may be any of those discussed above.
The invention also provides a polynucleotide which encodes a fusion polypeptide of the invention. The fusion polypeptide is preferably genetically fused as discussed above.
The invention also provides a polynucleotide which encodes a combination of the invention. The coding sequences for the two or more polypeptides in the combination may be present in a single polynucleotide of the invention. This is typically the case when the combination is encoded by a single vector of the invention.
A polynucleotide, such as a nucleic acid, is a polymer comprising two or more nucleotides. The nucleotides can be naturally occurring or artificial. A nucleotide typically contains a nucleobase, a sugar and at least one linking group, such as a phosphate, 2′O-methyl, 2′ methoxy-ethyl, phosphoramidate, methylphosphonate or phosphorothioate group. The nucleobase is typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines and more specifically adenine (A), guanine (G), thymine (T), uracil (U) and cytosine (C). The sugar is typically a pentose sugar. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The nucleotide is typically a ribonucleotide or deoxyribonucleotide. The nucleotide typically contains a monophosphate, diphosphate or triphosphate. Phosphates may be attached on the 5′ or 3′ side of a nucleotide.
Nucleotides include, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), 5-methylcytidine monophosphate, 5-methylcytidine diphosphate, 5-methylcytidine triphosphate, 5-hydroxymethylcytidine monophosphate, 5-hydroxymethylcytidine diphosphate, 5-hydroxymethylcytidine triphosphate, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP) and deoxycytidine triphosphate (dCTP), 5-methyl-2′-deoxycytidine monophosphate, 5-methyl-2′-deoxycytidine diphosphate, 5-methyl-2′-deoxycytidine triphosphate, 5-hydroxymethyl-2′-deoxycytidine monophosphate, 5-hydroxymethyl-2′-deoxycytidine diphosphate and 5-hydroxymethyl-2′-deoxycytidine triphosphate. The nucleotides are preferably selected from AMP, TMP, GMP, UMP, dAMP, dTMP, dGMP or dCMP.
The nucleotides may contain additional modifications. In particular, suitable modified nucleotides include, but are not limited to, 2′amino pyrimidines (such as 2′-amino cytidine and 2′-amino uridine), 2′-hyrdroxyl purines (such as, 2′-fluoro pyrimidines (such as 2′-fluorocytidine and 2′fluoro uridine), hydroxyl pyrimidines (such as 5′-α-P-borano uridine), 2′-O-methyl nucleotides (such as 2′-O-methyl adenosine, 2′-O-methyl guanosine, 2′-O-methyl cytidine and 2′-O-methyl uridine), 4′-thio pyrimidines (such as 4′-thio uridine and 4′-thio cytidine) and nucleotides have modifications of the nucleobase (such as 5-pentynyl-2′-deoxy uridine, 5-(3-aminopropyl)-uridine and 1,6-diaminohexyl-N-5-carbamoylmethyl uridine).
One or more nucleotides in the polynucleotide can be oxidized or methylated. One or more nucleotides in the polynucleotide may be damaged. For instance, the polynucleotide may comprise a pyrimidine dimer. Such dimers are typically associated with damage by ultraviolet light.
The nucleotides in the polynucleotide may be attached to each other in any manner. The nucleotides may be linked by phosphate, 2′O-methyl, 2′ methoxy-ethyl, phosphoramidate, methylphosphonate or phosphorothioate linkages. The nucleotides are typically attached by their sugar and phosphate groups as in nucleic acids. The nucleotides may be connected via their nucleobases as in pyrimidine dimers.
The polynucleotide can be a nucleic acid, such as deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). The polynucleotide may be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA), morpholino nucleic acid or other synthetic polymers with nucleotide side chains. The polynucleotide may be single stranded or double stranded.
The polynucleotide sequence encodes the relevant polypeptide(s) on the basis of the genetic code, including its degeneracy.
The polynucleotide may be a ribonucleic acid modified to reduce immunogenicity and increase stability for instance by substitution of uridine and cytidine with 1-methylpseudouridine and 5-methylcytidine, and/or placing an Anti-Reverse Cap Analog (ARCA) cap at the 5′ end. Such modified ribonucleic acids can be delivered using nanoparticles and other transfection reagents ([38-41]).
Polynucleotide sequences may be derived and replicated using standard methods in the art, for example using PCR involving specific primers. It is straightforward to generate polynucleotide sequences using such standard techniques. These are discussed in more detail below.
The invention also provides a combination of two or more polynucleotides each of which encodes a polypeptide of the invention, i.e. each of which encodes a different polypeptide of the invention. The combination may encode two or more polypeptides of the invention, two or more fusion polypeptides of the invention or a two or more of both types of polypeptide.
The combination may comprise any number of different polynucleotides. For instance, the combination may comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 different polynucleotide of the invention. The combination may comprise 10 or more, 20 or more, 30 or more, 40 or more or 50 or more polynucleotides of the invention.
The invention also provides a vector comprising a polynucleotide of the invention or a combination of two or more polynucleotides of the invention.
The vector may be a cloning vector. The amplified sequences may be incorporated into a recombinant replicable vector such as a cloning vector. The vector may be used to replicate the polynucleotide in a compatible host cell. Thus polynucleotide sequences may be made by introducing the polynucleotide into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector.
The vector may be recovered from the host cell. Suitable host cells for cloning of polynucleotides are known in the art and described in more detail below. The vector may be an expression vector. The polynucleotide sequence may be cloned into any suitable expression vector. In an expression vector, the polynucleotide of the invention or the combination of the invention is typically operably linked to a control sequence which is capable of providing for the expression of the polynucleotide or the combination by the host cell. Such expression vectors can be used to express one or more polypeptides of the invention.
The term “operably linked” refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. Multiple copies of the same or different polynucleotide may be introduced into the vector.
The term “control sequence” is intended to include promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g. transcription termination signals, such as polyadenylation signals and poly-U sequences). Such control sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif (1990). Control sequences include those that direct constitutive expression of a nucleotide sequence in many types of brain cell and those that direct expression of the nucleotide sequence only in certain brain cells. A non-limiting example of a suitable neuron-specific promoters include the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477.
Control sequences may also direct expression in a temporal-dependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific. In some embodiments, a vector comprises one or more pol III promoter (e.g. 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g. 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g. 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof. Examples of pol III promoters include, but are not limited to, U6 and H1 promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter. Also encompassed by the term “control sequence” are enhancer elements, such as WPRE; CMV enhancers; the R-U5′ segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, etc. With regards to control sequences, mention is made of U.S. patent application Ser. No. 10/491,026. With regards to promoters, mention is made of PCT publication WO 2011/028929 and U.S. application Ser. No. 12/511,940.
The expression vector may then be introduced into a suitable host cell. Thus, polypeptide of the invention can be produced by inserting a polynucleotide or a combination into an expression vector, introducing the vector into a compatible bacterial host cell, and growing the host cell under conditions which bring about expression of the polynucleotide or combination. The vectors may be for example, plasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide or combination and optionally a regulator of the promoter. The vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene. Promoters and other expression regulation signals may be selected to be compatible with the host cell for which the expression vector is designed. A T7, trc, lac, ara or λL promoter is typically used.
The vector may be used to administer a polynucleotide of the invention or a combination of two or more polynuclelotides to a subject as discussed in more detail below. Conventional viral and non-viral based gene transfer methods can be used to introduce the polynucleotide or combination into cells. Non-viral vector delivery systems include DNA plasmids, RNA, naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Methods of non-viral delivery of nucleic acids include lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Felgner, WO 91/17424; WO 91/16024. The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).
Conventional viral based expression systems could include retroviral, lentivirus, adenoviral, adeno-associated (AAV) and herpes simplex virus (HSV) vectors for gene transfer. Methods for producing and purifying such vectors are know in the art.
Exemplary vector systems for using the invention are a virus, such as rAAV, that comprises or consists essentially of an exogenous polynucleotide encoding the polypeptide, fusion polypeptide or polypeptide combination of the invention, e.g., a cassette comprising or consisting essentially of a promoter, a polynucleotide encoding the polypeptide, fusion polypeptide or polypeptide combination of the invention and a terminator.
Since AAV is a DNA virus, the polynucleotides used in AAV or rAAV are advantageously DNA.
The vector may be delivered using nanoparticle delivery systems. Such delivery systems include, but are not limited to, lipid-based systems, liposomes, micelles, microvesicles, exosomes, and gene gun. With regard to nanoparticles that can deliver RNA, see, e.g., Alabi et al., Proc Natl Acad Sci USA. 2013 Aug. 6; 110(32):12881-6; Zhang et al., Adv Mater. 2013 Sep. 6; 25(33):4641-5; Jiang et al., Nano Lett. 2013 Mar. 13; 13(3):1059-64; Karagiannis et al., ACS Nano. 2012 Oct. 23; 6(10):8484-7; Whitehead et al., ACS Nano. 2012 Aug. 28; 6(8):6922-9 and Lee et al., Nat Nanotechnol. 2012 Jun. 3; 7(6):389-93. Lipid Nanoparticles, Spherical Nucleic Acid (SNA™) constructs, nanoplexes and other nanoparticles (particularly gold nanoparticles) are also contemplated as a means for delivery of a polynucleotide or a polynucleotide of the invention. The invention provides any of these deliver systems comprising a vector of the invention, a polynucleotide of the invention or a polynucleotide combination of the invention.
In some embodiments, the vector may form a component of an inducible system. The inducible nature of the system would allow for spatiotemporal control of expression of a polypeptide of the invention or a combination of such polypeptides using a form of energy. The form of energy may include but is not limited to electromagnetic radiation, sound energy, chemical energy and thermal energy. Examples of inducible system include tetracycline inducible promoters (Tet-On or Tet-Off), small molecule two-hybrid transcription activations systems (FKBP, ABA, etc), or light inducible systems (Phytochrome, LOV domains, or cryptochrome).
As will be clear from below, the polynucleoide of the invention or a polynucleotide combination of the invention or any expression vector containing these components may be present in a population of cells. The cells may be administered to the subject. Suitable ways of modifying and administering cells are known in the art.
The invention also provides a host cell which comprises a polynucleotide of the invention, a polynucleotide combination of the invention or a vector of the invention. The host cell may be used to replicate the polynucleotide, combination or vector. The host cell may be used to express a polypeptide of the invention or a combination of polypeptides of the invention in vitro. The host cell may be used to deliver the polynucleotide, combination or vector to a subject in need thereof as discussed below.
Host cells will be chosen to be compatible with the cloning or expression vector used to transform the cell. Suitable conditions are known in the art (see, for instance, Sambrook, J. and Russell, D. supra).
Suitable cells for use in the invention include prokaryotic cells and eukaryotic cells. The prokaryotic cell is preferably a bacterial cell. Suitable bacterial cells include, but are not limited to, Escherichia coli, Corynebacterium and Pseudomonas fluorescens. Any E. coli cell with a DE3 lysogen, for example C41 (DE3), BL21 (DE3), JM109 (DE3), B834 (DE3), TUNER, Origami and Origami B, can express a vector comprising the T7 promoter.
Suitable eukaryotic cells include, but are not limited to, Saccharomyces cerevisiae, Pichia pastoris, filamentous fungi, such as Aspergillus, Trichoderma and Myceliophthora thermophila C1, baculovirus-infected insect cells, such as Sf9, Sf21 and High Five strains, non-lytic insect cells, Leishmania cells, plant cells, such as tobacco plant cells, and mammalian cells, such as Bos primigenius cells (Bovine), Mus musculus cells (Mouse), Chinese Hamster Ovary (CHO) cells, Human Embryonic Kidney (HEK) cells, Baby Hamster Kidney (BHK) cells and HeLa cells. Other preferred mammalian cells include, but are not limited to, PC12, HEK293, HEK293A, HEK293T, CHO, BHK-21, HeLa, ARPE-19, RAW264.7 and COS cells.
The host cell is preferably HEK293T.
If the cell is being administered to a subject, the cell is preferably derived from the subject or a subject of the same species. For instance, a human cell is typically administered to a human subject. The host cell is preferably autologous. In other words, the cell is preferably derived from the subject into which the cell will be administered. Alternatively, the host cell is preferably allogeneic. In other words, the cell is preferably derived from a patient that is immunologically compatible with the patient into which the cell will be administered.
The cell may be isolated, substantially isolated, purified or substantially purified. The cell is isolated or purified if it is completely free of any other components, such as culture medium or other cell types. The cell is substantially isolated if it is mixed with carriers or diluents, such as culture medium and others discussed above and below, which will not interfere with its intended use. Alternatively, the host cell of the invention may be present in a growth matrix or immobilized on a surface as discussed below.
The invention also provides a pharmaceutical composition comprising (a) a polypeptide of the invention, a polypeptide combination of the invention, a polynucleotide of the invention, a vector of the invention or a host cell of the invention and (b) a pharmaceutically acceptable carrier or diluent. The carrier or diluent may be any of those discussed above with reference to the vectors of the invention.
The carrier(s) or diluent(s) present in the pharmaceutical composition must be “acceptable” in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof. Typically, carriers for injection, and the final formulation, are sterile and pyrogen free. Preferably, the carrier or diluent is water. A pharmaceutically acceptable carrier or diluent may comprise as one of its components thioglycerol or thioanisole.
Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like, may be present in the excipient or vehicle. These excipients, vehicles and auxiliary substances are generally pharmaceutical agents that do not induce an immune response in the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethyleneglycol, hyaluronic acid, glycerol, thioglycerol and ethanol. Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients, vehicles and auxiliary substances is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).
The active agents are typically present at 0.1% to 50% by weight in the pharmaceutical composition, more preferably at 0.1% to 5% by weight. They may be present at less than 0.1% by weight in the pharmaceutical composition.
The pharmaceutically acceptable carrier or diluent is typically present at 50% to 99.9% by weight in the pharmaceutical composition, more preferably at 95% to 99.9% by weight. The pharmaceutically acceptable carrier or diluents may be present at more than 99.9% by weight in the pharmaceutical composition.
Pharmaceutical compositions include, but are not limited to pharmaceutically acceptable solutions, lyophilisates, suspensions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable compositions. Such pharmaceutical compositions may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. A lyophilisate may comprise one or more of trehalose, thioglycerol and thioanisole. In one embodiment of a pharmaceutical composition for parenteral administration, the active ingredient is provided in dry form (e.g., a lyophilisate, powder or granules) for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted pharmaceutical composition.
The pharmaceutical composition may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable compositions may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono-or di-glycerides.
Other parenterally-administrable pharmaceutical compositions which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Pharmaceutical compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
For example, solid oral forms may contain, together with the active substance, diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, gum arabic, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical compositions. Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film-coating processes.
Liquid dispersions for oral administration may be syrups, emulsions or suspensions. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.
Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. The suspensions or solutions for intramuscular injections may contain, together with the active substance, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.
Solutions for intravenous administration or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.
For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%.
Oral compositions include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release compositions or powders and contain 10% to 95% of active ingredient, preferably 25% to 70%. Where the pharmaceutical composition is lyophilised, the lyophilised material may be reconstituted prior to administration, e.g. a suspension. Reconstitution is preferably effected in buffer.
Capsules, tablets and pills for oral administration to an individual may be provided
with an enteric coating comprising, for example, Eudragit “S”, Eudragit “L”, cellulose acetate, cellulose acetate phthalate or hydroxypropylmethyl cellulose.
Polynucleotides may be present in combination with cationic lipids, polymers or targeting systems.
Uptake of polynucleotide or oligonucleotide constructs may be enhanced by several known transfection techniques, for example those including the use of transfection agents. Examples of these agents include cationic agents, for example, calcium phosphate and DEAE-Dextran and lipofectants, for example, lipofectamine and transfectam. The dosage of the polynucleotide or oligonucleotide to be administered can be altered.
Alternatively, the active agent may be encapsulated, adsorbed to, or associated with, particulate carriers. Suitable particulate carriers include those derived from polymethyl methacrylate polymers, as well as PLG microparticles derived from poly(lactides) and poly(lactide-co-glycolides). See, e.g., Jeffery et al. (1993) Pharm. Res. 10:362-368. Other particulate systems and polymers can also be used, for example, polymers such as polylysine, polyarginine, polyornithine, spermine, spermidine, as well as conjugates of these molecules.
The composition will depend upon factors such as the nature of the active agent and the method of delivery. The pharmaceutical composition may be administered in a variety of dosage forms. It may be administered orally (e.g. as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules), topically, parenterally, subcutaneously, by inhalation, intravenously, intramuscularly, intralymphatically (such as to lymph nodes in the groin), intrasternally, transdermally, intradermally, epidermally, sublingually, intranasally, buccally or by infusion techniques. The administration may be intratonsillar. The administration may be as suppositories. The administration may be made by iontophoresis. Preferably, the administration is intradermal, epidermal or transdermal. The administration may be made by a patch, such as a microtine patch. Administration is discussed in more detail below.
A physician will be able to determine the required route and means of administration for each particular individual.
The pharmaceutical compositions of the invention are preferably provided sealed in a container. The pharmaceutical compositions are typically provided in unit dose form, for example single dose form. They may alternatively be provided in multi-dose form. Where the pharmaceutical composition is a pharmaceutically acceptable solution, the solution may be provided in an ampoule, sealed vial, syringe, cartridge, flexible bag or glass bottle. Where the pharmaceutical composition is a lyophilisate, it is preferably provided in a sealed vial.
The pharmaceutical compositions of the invention will comprise a suitable concentration of each agent to be effective without causing adverse reaction. Where the pharmaceutical composition is for example a lyophilisate, the relevant concentration will be that of each polypeptide following reconstitution. Typically, the concentration of each agent in the pharmaceutical composition when in solution will be in the range of 0.03 to 200 nmol/ml. The concentration of each agent may be more preferably in the range of 0.3 to 200 nmol/ml, 3 to 180 nmol/ml, 5 to 160 nmol/ml, 10 to 150 nmol/ml, 50 to 200 nmol/ml or 30 to 120 nmol/ml, for example about 100 nmol/ml. The pharmaceutical composition should have a purity of greater than 95% or 98% or a purity of at least 99%.
In an embodiment where the invention involves combines therapy, the other therapeutic agents or adjuvants may be administered separately, simultaneously or sequentially. They may be administered in the same or different pharmaceutical compositions. A pharmaceutical composition may therefore be prepared which comprises an agent of the invention and also one or more other therapeutic agents or adjuvants. A pharmaceutical composition of the invention may alternatively be used simultaneously, sequentially or separately with one or more other therapeutic compositions as part of a combined treatment.
The invention also provides a method of inhibiting the signalling of one or more chemokines in an in vitro culture, the method comprising contacting the culture with a polypeptide of the invention, a combination of the invention, a polynucleotide of the invention, a vector of the invention or a host cell of the invention.
The method may comprise inhibiting any number of chemokines, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 chemokines. The chemokines may be selected from any of those in Tables 2, 4, and 6. When inhibiting the one or more chemokines in a particular row in the above Tables, a polypeptide, combination, polynucleotide, vector or host cell of the invention based on the tick CKBP in the same row is preferably used in the method of the invention. For instance, when inhibiting CCL8 employing a chemokine-binding sequence from a CCL8-binding tick CKBP, a polypeptide, combination, polynucleotide, vector or host cell of the invention based on the sequence shown in SEQ ID NO: 8 may be used. A hybrid polypeptide comprising the amino acid sequence of SEQ ID NO: 76, 82, 83 or 95 may also be used.
Similarly, when inhibiting CCL2 or CCL1/CCL2/CCL3/CCL5 employing applicable chemokine-binding sequences from tick CKBPs described herein, a polypeptide, combination, polynucleotide, vector or host cell of the invention based on the sequence shown in SEQ ID NO: 1 is preferably used. When inhibiting one or more of CCL2, CC13 and/or CCL20 employing applicable chemokine binding sequences from tick CKBPs described herein, a polypeptide, combination, polynucleotide, vector or host cell of the invention based on the sequence shown in any one of SEQ ID NOs 1-3, 6-9, 20-23 and 29 is preferably used. When inhibiting one or more of CXCL3, CXCL10 and/or CXCL12 employing applicable chemokine binding sequences from tick CKBPs described herein, a polypeptide, combination, polynucleotide, vector or host cell of the invention based on the sequence shown in SEQ ID NO 5 or 19 is preferably used.
When inhibiting one or more of CXCL1, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11 or CXCL12, a polypeptide, combination, polynucleotide, vector or host cell of the invention based on the sequence shown in any one of SEQ ID NOs: 45-72 and indicated in Table 6 as binding the relevant chemokine(s) may be used. For example, products of the invention as above based on SEQ ID NO: 45 may be used to inhibit CXCL9. Products of the invention as above based on one of SEQ ID NOs 45-48, 51-53, 56, 59-60 and 65 may be used to inhibit one or more of CXCL7, CXCL9 and CXCL11, as indicated in Table 6.
The in vitro culture is preferable a culture of cells capable of undergoing chemotaxis. The in vitro culture is preferably a chemotactic assay. The culture may be present in a culture flask or the wells of a flat plate, such as a standard 96 or 384 well plate. Such plates are commercially available Fisher scientific, VWR suppliers, Nunc, Starstedt or Falcon. Conditions for culturing cells are known in the art.
The polypeptide, combination, polynucleotide, vector or host cell of the invention may be administered in any of the forms discussed above.
The invention also provides a method of inhibiting the signalling of one or more chemokines in a subject, the method comprising administering to the subject a polypeptide of the invention, a combination of the invention, a polynucleotide of the invention, a vector of the invention or a host cell of the invention. The invention also provides a polypeptide of the invention, a combination of the invention, a polynucleotide of the invention, a vector of the invention or a host cell of the invention for use in a method of inhibiting the signalling of one or more chemokines in a subject. The invention also provides use of a polypeptide of the invention, a combination of the invention, a polynucleotide of the invention, a vector of the invention or a host cell of the invention in the manufacture of a medicament for use in inhibiting the signalling of one or more chemokines in a subject.
The method may comprise inhibiting any number of chemokines, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 chemokines. The chemokines may be selected from any of those in Tables 2, 4 and 6. When inhibiting the one or more chemokines in a particular row in the above Tables, a polypeptide, combination, polynucleotide, vector or host cell of the invention based on the tick CKBP in the same row is preferably used in the method of the invention. The examples of selection of particular tick CKBP amino acid sequences for in vitro inhibition of particular chemokines provided above are also applicable to selection of tick CKBP amino acid sequences for in vivo inhibition of the same chemokines.
The skilled person can design combinations of tick CKBPs to inhibit specific combinations of chemokines.
The invention also provides a method of treating or preventing in a subject one or more diseases associated with one or more chemokines, the method comprising administering to the subject a polypeptide of the invention, a combination of the invention, a polynucleotide of the invention, a vector of the invention or a host cell of the invention. The invention also provides a polypeptide of the invention, a combination of the invention, a polynucleotide of the invention, a vector of the invention or a host cell of the invention for use in a method of treating or preventing in a subject one or more diseases associated with one or more chemokines. The invention also provides use of a polypeptide of the invention, a combination of the invention, a polynucleotide of the invention, a vector of the invention or a host cell of the invention in the manufacture of a medicament for treating or preventing in a subject one or more diseases associated with one or more chemokines.
A disease is associated with one or more chemokines if the disease has a chemokine component. In other words, one or more symptoms of the disease may be treated or prevented by inhibiting one or more chemokines. Any number of chemokines may be involved as discussed above. The chemokines are preferably selected from those shown in any of Tables 2, 4 and 6 and also from those shown in
The method may comprise treating or preventing any number of diseases associated with one or more chemokines, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 diseases. The chemokines may be selected from any of those in Tables 2, 4 and 6. The one or more diseases may be as identified in Table 7 or 8 or
As seen from
Diseases that may be treated or prevented by polypeptides representing amino acid sequences derived from SEQ ID NOs 5 or 19 (and related polynucleotides/combinations/host cells) include diseases where CXCL3 is known to be expressed, including, idiopathic pulmonary fibrosis and breast cancer, where CXCL10 is known to be expressed, including atherosclerosis, inflammatory bowel disease, rheumatoid arthritis, liver fibrosis, idiopathic pulmonary fibrosis, multiple sclerosis, psoriasis, Alzheimer disease, myocarditis, primary biliary cirrhosis, autoimmune hepatitis, vasculitis, non-alcoholic steatohepatitis, myocardial infarction, and alcohol liver injury, or where CXCL12 is expressed, as in atherosclerosis, inflammatory bowel disease, rheumatoid arthritis, idiopathic pulmonary fibrosis, multiple sclerosis, colorectal cancer, myocarditis, primary biliary cirrhosis, primary sclerosing cholangitis, and autoimmune hepatitis.
A hybrid polypeptide of the invention may be used to treat or prevent a disease comprising expression of multiple chemokines, such as five or more, eight or more or ten or more chemokines. The multiple chemokines may preferably comprise both CC and CXC chemokines. A hybrid polypeptide binding both a CC chemokine and a CXC chemokine may be used to inhibit chemokine signalling in, and to treat or prevent, any of myocarditis, myocardial infarction, atherosclerosis, vasculitis, stroke, multiple sclerosis, Alzheimer's disease, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, liver fibrosis, non-alcoholic steatohepatitis, paracetamol liver injury, alcohol liver injury, idiopathic pulmonary fibrosis, acute lung injury, cardiac allograft vasculopathy, sarcoidosis, influenza, inflammatory bowel disease, pancreatitis, rheumatoid arthritis, psoriasis, skin fibrosis, breast cancer and colorectal cancer, which all comprise expression of both CC and CXC chemokines, as shown in
Exemplary therapeutic indications suitable for the hybrid polypeptide of SEQ ID 74, as seen in
First and second tick CKBP amino acid sequences selected in combination for provision of a hybrid polypeptide for binding chemokines expressed in a particular disease may be selected to individually bind multiple chemokines expressed in that disease. For instance, when treating rheumatoid arthritis, a hybrid polypeptide, combination, polynucleotide, vector or host cell of the invention representing a tick CKBP amino acid sequence shown in SEQ ID NO: 3 may be used. Similarly, when treating or preventing one or more of atherosclerosis, rheumatoid arthritis, inflammatory bowel disease, liver fibrosis, lung fibrosis, kidney fibrosis, skin fibrosis, multiple sclerosis, breast cancer, or Alzheimer's disease, a hybrid polypeptide, combination, polynucleotide, vector or host cell of the invention representing a tick CKBP amino acid sequence shown in SEQ ID NO: 1 may be used.
Where the disease to be treated or prevented is myocarditis, giant cell myocarditis, myocardial infarction, stroke or idiopathic pulmonary fibrosis, a hybrid polypeptide, combination, polynucleotide, vector or host cell of the invention representing a tick CKBP amino acid sequence shown in SEQ ID NO: 29 and/or a tick CKBP amino acid sequence shown in SEQ ID NO: 9 may be used. A hybrid polypeptide, combination, polynucleotide, vector or host cell of the invention representing a tick CKBP amino acid sequence shown in SEQ ID NO: 1 may also be used for treatment or prevention of the above diseases.
Where the disease comprises expression of one or more of CXCL1, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11 and CXCL2, a polypeptide, combination, polynucleotide, vector or host cell of the invention representing a tick CKBP amino acid sequence shown in any one of SEQ ID NOs 45-72 or a variant thereof may be used for treatment or prevention of the disease. The polypeptide may be a hybrid polypeptide comprising a binding sequence for one or more of the above CXC chemokines derived from any one of SEQ ID NOs 45-72 or a variant thereof. Alternatively, the polypeptide may comprise the full-length sequence of any one of SEQ ID NOs 45-72 or a variant thereof. Where the disease comprises expression of one or more of CXCL7, CXCL9, CXCL10, CXCL11 and CXCL12, a chemokine-binding sequence comprised in the hybrid polypeptide may be derived from, or the polypeptide may comprise, the amino acid sequence of any one of SEQ ID NOs 45-60 and 64-65 or a variant thereof. Where the disease comprises expression of one or more of CXCL7, CXCL9 and CXCL11 a chemokine-binding sequence comprised in the hybrid polypeptide may be derived from, or the polypeptide may comprise, the amino acid sequence of any one of SEQ ID NOs 45-48, 51-53, 56, 59, 60 and 65 or a variant thereof. The disease to be treated or prevented by one or more of the above CXC-binding chemokines may be one in which multiple CXC chemokines are expressed, such as rheumatoid arthritis, atherosclerosis or pancreatitis.
The skilled person can provide hybrid tick CKBPs having appropriate combinations of chemokine-binding activities from first and second tick CKBP amino acid sequences or variants thereof, or select novel tick CKBP polypeptides described herein to treat or prevent specific diseases or combinations of diseases. The skilled person can further provide hybrid tick CKBPs having chemokine-binding activities from three different tick CKBP amino acid sequences or variants thereof, as illustrated by the 3-warhead tick CKBPs described herein.
The 3-warhead tick CKBPs of SEQ ID NOs 92 and 93 or variants thereof as described herein are particularly suitable for treatment of diseases in which one or more, preferably three or more of CCL2, CCL5, CCL8, CXCL8, CXCL10 and CXCL1 are expressed, including any such disease described above. For example, acute lung injury (also referred to as acute respiratory distress syndrome) occurs in the context of smoke inhalation, toxins, aspiration, severe burns, pneumonia, sepsis, pancreatitis, trauma, transplant donor ischemia, and cardiopulmonary bypass. CC and CXC chemokines (e.g. CCL2, CCL5, CCL7, CXCL1, CXCL3, CXCL5, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11) are expressed in the lung following acute injury.[103,104,170-177]. The three-warhead evasins described above would be predicted to be of therapeutic benefit in acute lung injury.
A major complication of heart transplantation is cardiac allograft vasculopathy (CAV) which reduces graft and recipient survival. Chemokines that drive CAV include CXC chemokines (CXCL1, CXCL2, CXCL5, CXCL8, CXCL9) and CC-chemokines (CCL1, CCL2, CCL3, CCL4, CCL5), which drive the influx of neutrophils, NK cells and monocyte/macrophages [178,179]. The three-warhead evasins described above would also be predicted to be of therapeutic benefit in CAV.
The skilled person can also provide truncated forms of evasins retaining chemokine-binding activity for use in treatment of diseases, such as the truncated peptide of SEQ ID NO: 89 or a variant thereof. The truncated peptide or variant may be modified for example by cyclisation, or an inter-peptide disulphide bond or be in a stapled form, and/or may be bound or fused to a carrier, such as albumin. Such a truncated peptide or variant thereof may also be used to provide CCL8, CCL7 and CCL18 binding activity in a hybrid evasin polypeptide of the invention. SEQ ID NO: 89 and variants thereof are useful for treatment of diseases comprising expression of one or more of CCL8, CCL7 and CCL18, including any such disease described above. A peptidomimetic of SEQ ID NO: 89 or a variant thereof may also be provided and used in the above treatments. Suitable variants include SEQ ID NOs: 88, 103-109, comprising truncations and/or other modifications as described above. In preferred embodiments, the peptide variant is BK1.2 or BK1.3 (SEQ ID NOs: 105 or 106), preferably BK1.3. The truncated variant peptide may be modified for example by cyclisation, inter-peptide disulphide bond or be in a stapled form, and/or may be bound or fused to a carrier, such as albumin. The variant peptide may comprise SEQ ID NO: 106 fused to a second variant peptide via a disulphide bond at Cys 30 of each peptide. Particular diseases in which the above chemokines are expressed are also as follows: CCL18—atherosclerosis, rheumatoid arthritis, myocarditis, sarcoidosis, idiopathic pulmonary fibrosis, vasculitis, atopic dermatitis, breast cancer, influenza; CCL7—acute lung injury, stroke, idiopathic pulmonary fibrosis, Psoriasis, colorectal cancer, skin fibrosis, rheumatoid arthritis, inflammatory bowel disease; CCL8—rheumatoid arthritis, inflammatory bowel disease, idiopathic pulmonary fibrosis.
Any subject may be treated in accordance with the invention. The subject is typically human. However, the subject can be another animal or mammal, such as a research animal, such as a rat, a mouse, a rabbit or a guinea pig, a commercially farmed animal, such as a horse, a cow, a sheep or a pig, or a pet, such as a cat, a dog or a hamster.
The subject may be asymptomatic. A prophylactically effective amount of the polypeptide, combination, polynucleotide, vector or host cell is administered to such a subject. A prophylactically effective amount is an amount which prevents the onset of one or more, preferably all of, symptoms of the one or more diseases.
Alternatively, the subject may be in need thereof. That is, the subject may exhibit one or more symptoms of the one or more diseases. A therapeutically effective amount of the polypeptide, combination, polynucleotide, vector or host cell is administered to such a subject. A therapeutically effective amount is an amount which is effective to ameliorate one or more of, preferably all of, the symptoms of the one or more diseases.
The polypeptide, combination, polynucleotide, vector or host cell may be administered to the subject in any appropriate way. In the invention, the polypeptide, combination, polynucleotide, vector or host cell may be administered in a variety of dosage forms. Thus, it can be administered orally, for example as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules. It may also be administered by enteral or parenteral routes such as via buccal, anal, pulmonary, intravenous, intra-arterial, intramuscular, intraperitoneal, intraarticular, topical or other appropriate administration routes. A physician will be able to determine the required route of administration for each particular subject.
The polypeptide, combination, polynucleotide, vector or host cell may be in any of the forms discussed above with reference to the pharmaceutical composition of the invention.
Methods for gene delivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859 and 5,589,466. The nucleic acid molecule or a modified nucleic acid molecule can be introduced directly into the recipient subject, such as by standard intramuscular or intradermal or intravenous or intra coronary artery or intramyocardial injection; transdermal particle delivery; inhalation; topically, or by oral, intranasal or mucosal modes of administration. The molecule alternatively can be introduced ex vivo into cells that have been removed from a subject. For example, a polynucleotide, expression cassette or vector of the invention may be introduced into APCs of an individual ex vivo. Cells containing the nucleic acid molecule of interest are re-introduced into the subject such that an immune response can be mounted against the peptide encoded by the nucleic acid molecule. The nucleic acid molecules used in such immunization are generally referred to herein as “nucleic acid vaccines.”
The dose may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the subject to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular subject. A typical daily dose is from about 0.1 to 50 mg per kg of body weight, such as 5 mg per kg of body weight, according to the activity of the specific inhibitor, the age, weight and conditions of the subject to be treated and the frequency and route of administration. The dose may be provided as a single dose or may be provided as multiple doses, for example taken at regular intervals, for example 2, 3 or 4 doses administered hourly. Preferably, dosage levels of inhibitors are from 5 mg to 2 g.
Typically polynucleotide or oligonucleotide inhibitors are administered in the range of 1 pg to 1 mg, preferably to 1 pg to 10 μg nucleic acid for particle mediated delivery and 10 μg to 1 mg for other routes.
The polypeptide, the combination, the polynucleotide, the vector or the host cell is preferably administered in combination with another therapy
The inhibitor may be used in combination with one or more other therapies intended to treat the same subject. By a combination is meant that the therapies may be administered simultaneously, in a combined or separate form, to the subject. The therapies may be administered separately or sequentially to a subject as part of the same therapeutic regimen. For example, the polypeptide, the combination, the polynucleotide, the vector or the host cell be used in combination with another therapy intended to treat the one or more disease. The other therapy may be a general therapy aimed at treating or improving the condition of the subject. For example, treatment with methotrexate, glucocorticoids, salicylates, nonsteroidal anti-inflammatory drugs (NSAIDs), analgesics, other DMARDs, aminosalicylates, corticosteroids, and/or immunomodulatory agents (e.g., 6-mercaptopurine and azathioprine) may be combined with the inhibitor. The other therapy may be a specific treatment directed at the one or more diseases. Such treatments are known in the art. For instance in the treatment of rheumatoid arthritis this may include anti-TNFα [180] or other biologics targeting other cytokines (e.g. IL7, IL17, IL17) or their receptors (e.g. IL1-R, IL-6R), that are in clinical use or development [181]. In the treatment of inflammatory bowel disease we may use biologics such as vedolizumab [182]. For atherosclerosis simvastatin or other statins may be used.
The invention also provides an antibody or a fragment thereof which specifically binds a polypeptide comprising (a) an amino acid sequence shown in any one of SEQ ID NOs: 45 to 72 or (b) an amino acid sequence having at least 70% homology or amino identity to a sequence of (a) over its entire length. The antibody or fragment thereof preferably specifically binds a polypeptide comprising an amino acid sequence shown in any one of SEQ ID NOs: 45 to 72.
An antibody “specifically binds” to a polypeptide when it binds with preferential or high affinity to that polypeptide but does not substantially bind, does not bind or binds with only low affinity to other polypeptides. For instance, an antibody “specifically binds” to SEQ ID NO: 45 or a variant thereof when it binds with preferential or high affinity to SEQ ID NO: 45 or a variant thereof but does not substantially bind, does not bind or binds with only low affinity to other polypeptides. The same applies to any one of SEQ ID NOs: 46 to 72.
An antibody binds with preferential or high affinity if it binds with a Kd of 1×10-7 M or less, more preferably 5×10-8 M or less, more preferably 1×10-8 M or less or more preferably 5×10-9 M or less. An antibody binds with low affinity if it binds with a Kd of 1×10-6 M or more, more preferably 1×10-5 M or more, more preferably 1×10-4 M or more, more preferably 1×10-3 M or more, even more preferably 1×10-2 M or more. A variety of protocols for competitive binding or immunoradiometric assays to determine the specific binding capability of compounds, such as antibodies or antibody constructs and oligonucleotides are well known in the art (see for example Maddox et al, J. Exp. Med. 158, 1211-1226, 1993).
The antibody may be, for example, a monoclonal antibody, a polyclonal antibody, a single chain antibody, a chimeric antibody, a CDR-grafted antibody or a humanized antibody. The antibody may be an intact immunoglobulin molecule or a fragment thereof such as a Fab, F(ab′)2 or Fv fragment. Furthermore, the antibodies and fragment thereof may be chimeric antibodies, CDR-grafted antibodies or humanised antibodies.
Antibodies of the invention can be produced by any suitable method. Means for preparing and characterising antibodies are well known in the art, see for example Harlow and Lane (1988) “Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. For example, an antibody may be produced by raising an antibody in a host animal against the whole polypeptide or a fragment thereof, for example an antigenic epitope thereof, hereinafter the “immunogen”. The fragment may be any of the fragments mentioned herein (typically at least 10 or at least 15 amino acids long).
A method for producing a polyclonal antibody comprises immunising a suitable host animal, for example an experimental animal, with the immunogen and isolating immunoglobulins from the animal's serum. The animal may therefore be inoculated with the immunogen, blood subsequently removed from the animal and the IgG fraction purified. A method for producing a monoclonal antibody comprises immortalising cells which produce the desired antibody. Hybridoma cells may be produced by fusing spleen cells from an inoculated experimental animal with tumour cells (Kohler and Milstein (1975) Nature 256, 495-497).
An immortalized cell producing the desired antibody may be selected by a conventional procedure. The hybridomas may be grown in culture or injected intraperitoneally for formation of ascites fluid or into the blood stream of an allogenic host or immunocompromised host. Human antibody may be prepared by in vitro immunisation of human lymphocytes, followed by transformation of the lymphocytes with Epstein-Barr virus.
For the production of both monoclonal and polyclonal antibodies, the experimental animal is suitably a goat, rabbit, rat, mouse, guinea pig, chicken, sheep or horse. If desired, the immunogen may be administered as a conjugate in which the immunogen is coupled, for example via a side chain of one of the amino acid residues, to a suitable carrier. The carrier molecule is typically a physiologically acceptable carrier. The antibody obtained may be isolated and, if desired, purified.
The invention also provides a method of detecting one or more chemokines in a tissue, comprising contacting the tissue with a detectably-labelled polypeptide of the invention or a detectably-labelled polypeptide combination of the invention and detecting the binding of the polypeptide or the combination to one or more chemokines in the tissue. The polypeptide may be a fusion polypeptide of the invention. The tissue may be in vitro or in vivo. The invention also provides a detectably-labelled polypeptide of the invention or a detectably-labelled combination of the invention for use in a method of detecting one or more chemokines in a tissue. The invention also provides use of a detectably-labelled polypeptide of the invention or a detectably-labelled combination in the manufacture of medicament for detecting one or more chemokines in a tissue.
Any method of detecting binding may be used. The method may be positron emission tomography (PET) or magnetic resonance imaging (MRI).
The tissue may be any tissue. The tissue is preferably in a subject. The subject may be any those discussed above. The polypeptide or combination may be administered to the subject in any of the forms discussed above.
Any of the polypeptides of the invention or combinations of the invention discussed above may be used. Suitable detectable labels are also discussed above. The label is preferably a tracer that is suitable for positron emission tomography (PET), such as fluorodeoxyglucose (18F). The label is preferably a tracer suitable for magnetic resonance imaging (MRI), such as fluorine (19F).
The method may comprise detecting any number of chemokines, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 chemokines. The chemokines may be selected from any of those in Table 2, 4 and 6. When detecting one or more of CXCL1, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11 and CXCL2, a hybrid polypeptide comprising a binding sequence for one or more of the above CXC chemokines derived from any one of SEQ ID NOs 45-72 or a variant thereof may be used. Alternatively, a polypeptide comprising the full length sequence of any one of SEQ ID NOs 45-72 or a variant thereof may be used. When detecting both one or more CC and one or more CXC chemokines, a hybrid polypeptide of the invention binding a CC and a CXC chemokine may be used.
The method is preferably for diagnosing or prognosing one or more diseases associated with one or more chemokines. The method may comprise diagnosing or prognosing any number of diseases associated with one or more chemokines, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 diseases. The one or more diseases may as be identified in Table 7 and 8 or
Particular selections of tick CKBP amino acid sequences for diagnosis or prognosis of particular diseases may be made according to the same criteria discussed above in relation to medical uses. Thus, a hybrid polypeptide binding both a CC chemokine and a CXC chemokine may be used to diagnose or prognose any of myocarditis, myocardial infarction, atherosclerosis, vasculitis, stroke, multiple sclerosis, Alzheimer's disease, autoimmune hepatitis, primary biliary cirrhosis, primary schlerosing cholangitis, liver fibrosis, non-alcoholic steatohepatitis, paracetamol liver injury, alcohol liver injury, idiopathic pulmonary fibrosis, acute lung injury, sarcoidosis, influenza, inflammatory bowel disease, pancreatitis, rheumatoid arthritis, psoriasis, skin fibrosis, breast cancer and colorectal cancer, which all comprise expression of both CC and CXC chemokines, as shown in
The skilled person can provide hybrid tick CKBP polypeptides having appropriate combinations of chemokine-binding activities from first and second tick CKBP amino acid sequences or variants thereof, or select novel tick CKBP polypeptides described herein or combinations of the above to diagnose or prognose specific diseases or combinations of diseases.
1. Identification of Chemokine-Binding Tick CKBPs.
To identify protein-protein interactions between extracellular proteins we adapted yeast surface display technology, originally developed for the identification of single chain antibodies[17,183]. Here candidate proteins are expressed in yeast and displayed on the cell wall. Fluorescent-activated cell sorting (FACS) is used to select a desired yeast cell that bind a fluorescent-labelled target. To identify tick CKBPs we created yeast surface display libraries that express mature peptides identified in tick salivary transcriptomes, we systematically screened the above libraries using the chemokines CCL1, CCL2, CCL3, CCL4, CCL5, CCL8, CCL11, CCL15, CCL17, CCL18, CCL19, CCL20, CCL22, CCL25, CX3CL1, CXCL8, CXCL10, CXCL11, CXCL12, CXCL13. We obtained interacting clones that were retested and bound to one or more chemokines confirmed using FACS (SEQ ID NOs: 1 to 31) Table 1). This method can be used also to alter affinities and binding characteristics of a tick CKBP e.g. through mutagenesis and FACS selection.
2. Characterisation of Tick CKBP Binding to Chemokines.
Characterisation of binding of certain tick CKBPs identified in Example 1 against all known human chemokines (with exception of CCL25, CCL26, CXCL16, CXCL17, CXCL4L1, XCL2) was carried out using biolayer interferometry. The data for their binding properties are shown in Table 2, alongside published Kd data in relation to binding of human chemokines for previously described tick CKBPs (Evasins 1, 3 and 4). Other binding data for the tick CKBPs obtained using yeast surface display is also summarised. From this data three classes of novel tick CKBPs were identified, as shown in Table 2. Class I tick CKBPs bind CC-class chemokines CCL2, CCL13 or CCL20 in addition to other CC chemokines as indicated. Class III tick CKBPs bind CXC-class chemokines CXC-chemokines CXCL3, CXCL10 or CXCL12 in addition to other CXC chemokines as indicated. Class III tick CKBPs have other chemokine-binding characteristics.
3. Characterisation of Inhibition of Chemokine Function by a Tick CKBP.
Evaluation of the neutralisation activity of certain tick CKBPs identified in Example 1 against particular human chemokines was carried out using a THP1 transwell cell migration assay, with results (IC50 data) shown in Table 3. The results illustrate neutralisation of function of multiple chemokines by certain tick CKBPs.
4. Isolation and Characterisation of Novel Tick CKBP Polypeptides
28 novel CXC-binding tick CKBP polypeptides (SEQ IDS 45-72) were isolated in an additional yeast surface display screening carried out in accordance with Example 1, with results shown in Tables 5 and 6.
5. Hybrid Tick CKBP Comprising a Substituted Chemokine-Binding Sequence.
Alignment of P672_RHIPU to EVA1_RHISA (
We exchanged the N-terminal 29 residues of Evasin-1, with the N-terminal 44 residues of P672_RHIPU to generate a hybrid tick CKBP having the amino acid sequence shown in SEQ ID NO: 76. The nucleotide sequence used here is EZ406190.1, which encodes the Evasin-1 peptide variant K92E. Evasin-1 (EVA1_RHISA) binds CCL3, CCL3L1, CCL4, CCL4L1, CCL14 and CCL18 but not CCL8[20,21]. We have confirmed using biolayer interferometry that the Evasin-1 peptide encoded by EZ406190.1 also does not bind CCL8 at a concentration of 300 nM CCL8. P672_RHIPU binds CCL8 with Kd=3.7 nM. The hybrid tick CKBP (P672:EVA1) binds CCL8 with Kd=223 nM (
6. Two-Warhead Tick CKBPs
We genetically engineered P991_AMBCA and P1243_AMBAM (CC binding tick CKBPs) to link each via a flexible GGGGS linker to P1156_IXORI (CXC binding tick CKBP) to create “2-warhead” tick CKBPs, shown respectively in SEQ ID NO: 73, and 74, We show that the “2-warhead” tick CKBPs retain some of the properties of each of the parental tick CKBPs by binding and neutralizing both CC and CXC chemokines as shown in the results in
The results obtained for a 2-warhead tick CKBP indicate, that two (or more) tick CKBPs can be physically linked e.g. via a flexible linker or linkers, of variable length or design, to create a non-natural peptide that that retains the properties of the two parent tick CKBPs. Novel artificial chemokine binding peptides with desired properties that match CC and CXC chemokine expression patterns in disease, can thus be created by mixing and matching a number of CC or CXC binding natural tick CKBPs.
2-warhead evasins (SEQ ID NOs 74 and 81) were further investigated for their ability to functionally inhibit CC and CXC chemokines as compared to individual (parental) evasins represented in the 2-warhead molecules. Results are shown in
2-warhead evasins (SEQ ID NOs 73 and 80) were also further analysed for their binding to human chemokines compared to parental evasins, with summary data shown in
The ability of 2-warhead evasins to engage in polyvalent binding of CC and CXC chemokines was further determined. Results are shown in
7. CXC Chemokine Binding Evasins
The various CXC chemokine binding evasins described in the application were analysed in more detail for their chemokine binding activity using biolayer interferometry. This permitted identification of two functional classes of CXC chemokine binding evasins. Results are shown in
The functional inhibition of CXC chemokines by P1142_AMBCA was also further investigated. Results from chemokine-induced cell migration assays are shown in
8. Analysis of a Truncated Evasin Peptide with Chemokine-Binding Activity
A truncated peptide P672 PEP (SEQ ID NO: 88, peptide BK1.1) was generated consisting of residues E17 to E32 of P672_RHIPU, within the CCL8-binding region of this evasin. Residue C30 was mutated to A to avoid an unpaired cysteine residue. The peptide was N-terminally labelled with FITC (P672_PEP-FITC) to allow for characterisation of chemokine binding by fluorescence polarisation. Results are shown in
The binding specificity of the truncated peptide was further investigated in displacement assays, with results shown in
Additionally, functional inhibition by the truncated evasin peptide in cell migration assays was investigated, with results shown in
9. 3-Warhead Evasins
The ability to design 3-warhead evasins representing sequences from three different individual parental evasins was investigated.
Three warhead evasin P1820 was created by genetically fusing P1142_AMBCA (SEQ ID 65), P1156_IXORI (SEQ ID 19), and P467_RHIPU (SEQ ID 1) with intervening GGGGS linkers (bold and underlined in sequence below) to create P1142:G4S:P1156:G4S:P467 (SEQ ID NO: 92, P1820).
Three warhead evasin P1821 was created by genetically fusing P1142_AMBCA (SEQ ID 65), P1156_IXORI (SEQ ID 19), and P991_AMBCA (SEQ ID 9) with intervening GGGGS linkers (bold and underlined in sequence below) to create P1142:G4S:P1156:G4S:P991 (SEQ ID NO: 93, P1821).
The sequences of the three warhead evasins are shown below:
The binding affinities (Kd, M) of immobilized purified 3 warhead evasins P1820 (P1142:G4S:P1156:G4S:P467) and P1821 (P1142:G4S:P1156:G4S:P991) to exemplar human CC and CXC-chemokines were then determined using biolayer interferometry, with results shown below.
The 3-warhead evasins thus were found to bind CCL2, CCL5 and CCL8 (bound by each of the parental evasins P467_RHIPU and P991_AMBCA), CXCL8 (bound by the parental evasin P1156_IXORI), CXCL10 (bound by the parental evasin P1142_AMBCA) and CXCL1, bound by both P1156_IXORI and P1142_AMBCA. Based on the ability to combine each of the individual binding activities of the parental evasins in a single 3-warhead evasin, the potential therapeutic indications for each 3-warhead evasin represent a combination of the individual indications for the parental evasins. The use of a 3-warhead evasin thus extends the therapeutic application of the parental evasins. Additionally, increasing the molecular weight (e.g. in 2, 3 or multi-warheads) in comparison to each parental evasin and may be expected to have advantageous pharmacokinetic effects such as reduced renal clearance resulting in prolonged half-life [184,185]. This would be expected to result in reduction of dose required to be therapeutically effective and resulting also in a prolongation of intervals between doses which would enhance patient acceptability.
10. Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) of the P672:CCL8 Complex Interface
Peptide-resolution HDX-MS was performed to characterise the interaction between P672_RHIPU (referred to hereinafter as “P672”) and CCL8. Deuterium uptake of free P672, free CCL8 and of each protein upon complex formation was measured. Sequence mapping and coverage of each protein (100% for P672, 96.9% for CCL8,
Spectra of two representative P672 peptides showing a reduction in deuterium incorporation for this protected region upon complex formation are shown in
In this study we used HDX-MS and identified a 11-residue region (E22-F32) of P672 that was protected from deuterium uptake upon complexing with CCL8. The HDX-MS result also indicated that CCL8 residues R18-S27, which overlap the N-terminal loop (C12-R24), interact with P672. A key function of the N-terminal loop of CC chemokines is receptor binding, and it is targeted by several pathogenic chemokine-binding proteins. For example, the viral chemokine binding protein VV-35 kDa targets K19 and R24 of CCL2 and the viral chemokine binding protein vCCI targets R18 and R24 of CCL2. This common mechanism suggests the convergent evolution of these proteins to target the residues found in this region. The binding of P672 to this region would competitively prevent CCL8 binding to its receptor, explaining how CCL8 function is neutralised. The N-terminal loop of CCL8 and other CC chemokines is also part of the homodimerization interface, and binding to this loop explains the prevention of CCL8 dimerization by P672 reported previously.
11. Residues E22-F32 in P672 Contain a Transferable CCL8 Binding Activity
The P672 (E22-F32) region was swapped with the corresponding segment of EVA1_RHISA, which is a related CC-chemokine binding evasin that does not bind CCL8 (Eaton, J. R. O. et al., JBC [2018]) (
Taken together with the HDX-MS analysis, these experiments indicate that P672(E22-F32) is involved in forming protein-protein interactions with CCL8, and that this function can be transferred to another evasin.
Swapping the P67222-32 region into EVA1, an evasin that does not bind CCL8, transferred CCL8 binding activity to the hybrid protein. These results indicate that this 11-residue region binds CCL8.
12. Development of BK1.1, a CCL8 Binding Peptide
A number of tiled peptide fragments spanning the E17-F32 region in P672 were tested for CCL8 binding (
13. BK1.1 Disrupts CCL8 Homodimerization
As shown by native mass spectrometry CCL8 exists as a homodimer (
14. BK1.1 Promiscuously Binds Three CC Class Chemokines
BK1.1FITC was screened for binding against the 13 CC-chemokines known to bind to P672 (Eaton, J. R. O. et al., JBC [2018])(
15. Engineering of Peptides with Improved Potency and Promiscuous CC-Chemokine Binding
The role of the four acidic residues N-terminal to Y21, and the impact of C30A mutation introduced into BK1.1, were explored. Two shorter peptides, Y21-F32 with or without the C30A mutation (
The addition of four acidic N-terminal residues and Tyr21 was necessary to be able to detect binding under these conditions, suggesting these acidic residues may be needed for increased affinity, or that the shorter peptides were sterically hindered from binding by the FITC moiety. Alanine scanning mutagenesis of the 16-residue peptide BK1.1 indicated that binding to CCL8 was mediated by Tyr and Phe residues, and also by the acidic residues at the N-terminus. Notably, Tyr and Phe are both found in protein interaction “hot-spots”, and complementarity in surface charge mediated by acidic residues can modulate protein interactions. A notable finding was that the Pro residue is critical for binding. Pro residues are found in turns, and can undergo cis-trans isomerisation, making it likely that the Pro residue is of structural importance for BK1.1. BK1.1 was observed to prevent CCL8 homodimerization, suggesting that it likely employs a similar mechanism as P672 in binding CCL8, i.e. to the N-loop region. The fluorescent polarisation studies reported indicate that BK1.1 also binds the chemokines CCL7, and CCL18, but not several others.
Given the role of Pro in protein conformation, cyclisation was employed as a strategy for restricting conformational flexibility. A surprising finding was that the addition of an N-terminal Tyr residue, introduced for the purpose of thioether cyclisation, enhanced affinity. The role of the added N-terminal Tyr is supported by the increased affinity of the peptides (BK1.2, BK1.3) that carry it compared to the ones that do not (BK1.1, BK1.4, BK1.5). The role of the re-introduced Cys30 is supported by BK1.5, which differs by a single residue in comparison to BK1.1, and has marked improvement in affinity. The substantial enhancement of activity of BK1.3 thus likely arises from addition of Tyr and re-introduction of Cys30. In addition, it is likely that the unexpected formation of a Cys linked dimer in BK1.3 enhances the functional affinity or avidity of the molecule. Cyclisation in these experiments did not appear to enhance affinity, as evidenced by the lack of improvement of BK1.4 in comparison to BK1.5 or BK1.2 in comparison to BK1.3. This may be, in part, due to non-optimised cyclisation points and/or forced constraint.
16. Engineered Peptides Promiscuously Neutralize Chemokine Function
The effect of BK1.1, 1.2 and 1.3 on CCL8, CCL7, CCL3 and CCL2 induced cell migration was explored. P672 has previously been shown to neutralise these chemokines in analogous experiments (Eaton, J. R. O. et al., JBC [2018]). THP-1 cells express CCR1, CCR2 and CCR5 (Parker, L. C. et al., Journal of immunology [2004])(Achour, L. et al., Blood [2009]), which are activated by CCL8, CCL7 and CCL2, while CCL3 activates CCR1 and CCR5 (Harding, S. D. et al., Nucleic Acids Res [2018]). The experiments were performed with a single concentration of peptide (10 μM,
17. Engineered Peptides Prevent Cellular Chemokine Binding
To explore the effect of BK1.1 and derivatives on chemokine ligand-cell interactions, a fluorescent-chemokine cell binding assay was developed. Fluorescent chemokine (conjugated to AlexaFluor-647) binding to THP-1 cells results in an increase in the cellular fluorescence intensity, which is quantitatively measured using flow cytometry. In dose-response assays, increasing doses of peptide suppressed CCL8-647 and CCL2-647 induced cellular fluorescence (
The improvement in binding to CCL8 observed in the BK1.1-BK1.3 peptide series, as well as their ability to inhibit P672:CC-chemokine interactions (CCL8, CCL2, CCL3), correlated with increased chemokine neutralization potency and promiscuity. In cell-based chemotaxis assays, it was found that the improvement in binding affinity for CCL8 translated into increased potency for inhibiting CCL8-induced cell migration, as evidenced by the reduced IC50. In addition to neutralizing CCL8 and CCL7, which was predicted by the BK1.1 fluorescent polarisation binding study, the peptides BK1.2 and BK1.3 were also able to neutralize CCL2 and CCL3 induced chemotaxis. The inhibition of chemokine binding to cells indicate that the mechanism of neutralization is the prevention of chemokine binding to the cells, likely by preventing chemokine-receptor interactions.
18. Engineered Peptide BK1.3 has In Vivo Anti-Inflammatory Activity
The results suggested that the chemokine-neutralizing properties of the engineered peptides may translate into anti-inflammatory activity in vivo. To study this, the lead peptide BK1.3 was tested in a mouse short-term inflammation model. In this model, zymosan, a yeast cell wall derived PAMP, activates cytokine and chemokine production, and leucocyte infiltration, when injected into an artificially created subcutaneous air-pouch (Coates, N. J. et al., Journal of immunology [2001])(El-Achkar, G. A. et al., PLoS One [2019]). Characterization of this model showed that Ccl9 is expressed at a high basal level but is not induced by zymosan. Ccl2, 5, 11, 12, 20, 22, 24, and Cxcl1, 2, 4, 5, 11, 13, 16 are expressed (>3 fold) at 4 hours following zymosan, and Ccl2, 5, 12, and Cxcl2, 4, 13, 16 are expressed (>3 fold) at 24 hours (
Systemic administration of BK1.3 peptide would have anti-inflammatory activity. BK1.3, control SCR peptides, and the positive control P672 were injected intraperitoneally at 0 and 9 h following zymosan injection, and the air-pouch exudate characterized at 24 hours after zymosan injection. Both BK1.3 and P672 showed a substantial and significant reduction in the number of neutrophils, eosinophils, monocytes and T-cells recruited to the air-pouch (
A critical step in the clinical translation of novel anti-inflammatory therapeutics is the demonstration of efficacy in vivo, using a model where many components of the immune-inflammatory network are activated. A short-term inflammation model using the well characterized PAMP, zymosan, which activates TLR2 signalling, was used, and resulted in the production of cytokines, chemokines, and complement. The results indicate that zymosan-induced inflammation is significantly inhibited by both local as well as systemic administration of BK1.3. It is likely that the in vivo mechanism of action of BK1.3 includes the inhibition of CC-class chemokines which not only are chemoattractants for leucocyte recruitment, but also heterodimerize and synergise with certain CXC-class chemokines. Our work indicates that peptides with promiscuous chemokine-binding and anti-inflammatory activity can be developed from tick evasins. Such peptides could provide a route to the development of new anti-inflammatory therapeutics.
19. Materials and Methods for Examples 10 to 18
Reagents
All chemokines, unless otherwise stated, were purchased from Peprotech (UK). Fluorescent chemokines were purchased from Almac (UK). THP-1 cells (ECACC 88081201) were maintained in RPMI-1640 media supplemented with 10% fetal calf serum and 4 mM L-glutamine. Cultures were maintained between 3×105 and 1×106 cells/ml in a 37° C. incubator with 5% CO2. HEK 293F cells (Thermo Fisher) were maintained between 3×105 and 1×106 cells/ml in a 37° C. incubator with 8% CO2 and 130 RPM agitation in FreeStyle™ 293 Expression Medium.
Plasmids
Evasins were cloned in the expression vector pHLSec. P672 (N-terminal 8×His-StrepII tag) expression vector and EVA1 (C-terminal Strep-8×HisII tag) have been described previously (Eaton, J. R. O. et al., JBC [2018]). The expression vector EVA1(P67221-32) was constructed using PCR and infusion cloning as described15, and has a N-terminal 8×His-StrepII tag. Plasmid sequences were confirmed by Sanger sequencing (Source Bioscience, UK). The CCL8 expression plasmid in vector pNIC-BIO3 has been described previously (Eaton, J. R. O. et al., JBC [2018]). Plasmids and sequences are available on request.
Protein Expression
Evasin proteins were expressed as described previously using a mammalian expression system (Eaton, J. R. O. et al., JBC [2018]). Briefly, HEK293F cells were transiently transfected with expression vectors using polyethylenimine and incubated for five days. The supernatant was collected and the recombinant proteins isolated using nickel affinity chromatography followed by size exclusion chromatography. Fractions showing absorbance at 280 nm were analyzed on SDS-PAGE and protein containing fractions were combined for future use. Recombinant CCL8 was expressed as described previously as a SUMO fusion protein from Escherichia coli RosettaGami™ 2 (DE3) cells (Novagen)(Eaton, J. R. O. et al., JBC [2018]). To produce biotinylated CCL8 the same protocol was followed except the cells were also transformed with a plasmid encoding for BirA and 500 μM biotin was also added at the same time as IPTG51. Following IPTG induction, the over-expressed protein was isolated from the soluble fraction using nickel-charged IMAC Sepharose 6 Fast Flow resin (GE Healthcare). SUMO protease was added to partially purified protein and left overnight at 30° C. The SUMO tag was separated from CCL8 using cation exchange chromatography and the chemokine purified further using size exclusion chromatography. Fractions showing absorbance at 280 nm were analyzed on SDS-PAGE and protein containing fractions were combined for future use.
Hydrogen Deuterium Exchange Analysis
Working solutions of CCL8 and P672 were prepared at a concentration of 35 μM in 50 mM ammonium bicarbonate buffer pH=6.5. For estimation of HDX in the heterodimer state, solutions of CCL8 and P672 were mixed in a (1:1) ratio to reach a final concentration of 17.5 μM and incubated at 4° C. for 1 h (Eaton, J. R. O. et al., JBC [2018]). For estimation of HDX in the unbound state, working solution were diluted to 17.5 μM with 50 mM ammonium bicarbonate pH=6.5. Aliquots of 4.3 μL of heterodimer or unbound proteins were mixed with 48.2 μL of D2O containing 50 mM ammonium bicarbonate buffer adjusted to pH=6.5 with DCl (final content of D2O of 91.8%) and incubated for 5 s, 30 s, 5 min and 60 min at RT. HDX was quenched by adding 22.5 μL of 10% formic acid to reach a final volume of 75 μL and pH=2.5, corresponding to a final concentration of 1 μM. Samples were then rapidly flash frozen in liquid nitrogen and stored at −80° C. for up to 5 days before analysis.
An Acquity M class ultra-high performance liquid chromatographer with a nanoAcquity HDX manager coupled to a Synapt G2-Si time-of-flight mass spectrometer (Waters) was used and controlled using the MassLynx 4.1 software. Samples were loaded at 200 μL/min into an Enzymate pepsin column (2.1 mm×30 mm, 5 μm particle size) where the proteins were quickly digested at 20° C. Peptides were then captured for 2 min into a BEH C18 trap column (300 μm×30 mm, 1.7 μm particle size) at 0° C. and then separated in a BEH C18 analytical column (2.1 mm×50 mm, 1.7 μm particle size) at 40 μL/min and 0° C. under a 12 minutes linear gradient from 4 to 85% of acetonitrile with 0.1% formic acid. The MSE approach was used for peptide mapping of non-deuterated proteins with trap collision energies of 15 to 35 V. Deuterated samples were analysed in scan mode only. Source parameters included: cone voltage 30V, capillary voltage 2.8 KV, source temperature 80° C., desolvation temperature 150° C., gas cone flow rate 80 L/h and desolvation gas 250 L/h.
The ProteinLynx Global Server 3.0.2 software was used for peptide mapping. Spectra were searched against a custom database containing the protein sequence of interest, requiring a non-specific digestion enzyme and allowing for variable modifications (i.e. N-terminus pyroglutamic acid from glutamine and deamidation or HexNAc (N-acetylhexosamine) of asparagine present in a N-X-S/T motif). Peptide identification required at least 3 fragment ion matches, the peptide presence in 4 out of 5 replicates, a retention time relative standard deviation of ≤5%, a precursor ion mass tolerance of 10 ppm and peptide maximum length of 30 residues. Relative deuterium uptakes % at the peptide-level were estimated using Dynamix 3.0 as the difference between the uptake (Da) observed for the complex species and the free species divided by the maximum possible uptake of the peptide. Manual check of peptide retention time, charge state and possible peak overlap were also performed. Statistical analysis included a t-Student test and HDX rate differences ≥5% with a p-value ≤0.05 were considered significant. Residues with statistically significant increased or decreased HDX rates were mapped on to a previously described homology model of the P672:CCL8 complex (Eaton, J. R. O. et al., JBC [2018])(generated using the EVA1:CCL3 complex 3FPU (Gault, J. et al., Nature methods [2016]) as template) in which the CCL8 homology structure was replaced by the CCL8 x-ray crystal structure (1ESR)(Kawamura, A. et al., Nat Comms. [2017]). Note that in the case of overlapping peptides, Dynamix 3.0 displays the % Relative Uptake for any given residue as the % Relative Uptake of the shortest peptide. Additionally, in the particular case of overlapping peptides of equal length, the % Relative Uptake refers to that of the peptide in which the residue is closest to the peptide C-terminus. Structural models were created using PyMol2.3.
Biolayer Interferometry
This was carried out as described previously using an OctetRed® system (Singh, K. et al., Sci Rep [2017]). Briefly, affinity determination was evaluated with chemokine concentrations typically ranging from 300 to 0.4 nM, using a non-interacting reference protein to allow for nonspecific binding to the sensor. We used ForteBio Data Analysis 9 software to process the data and calculate association (kon), dissociation (koff), and affinity (Kd) constants. Data with poor curve fits (R2<0.9) were excluded. All biolayer interferometry experiments were performed at least three times.
Fluorescent Peptides
All fluorescent peptides and scrambled (SCR) peptide were purchased from GL Biochem (Shanghai) and were synthesized using standard Fmoc solid phase synthesis to give peptides with a C-terminal amide. The scrambled peptide sequence EFTEVYEFDFKYDAPD is based on BK1.1. All were deemed to be >90% pure by HPLC analysis and verified by LC-MS. Peptides were dissolved in DMSO and the concentration determined using NMR with TSP as an internal standard (Larive, C. K., et al., Applied Spectroscopy [1997]). All peptides were analysed using a Bruker Microflex LRF MALDI-TOF mass spectrometer.
Peptide Synthesis In-House
Amino acids were purchased from CEM. Peptides were purified by HPLC using a Waters SFO system with a Kinetex® 5 mm EVO C18 100 Å (150×21.2 mm) column. All peptides were synthesized with a C-terminal amide on a 50 mmol scale using standard Fmoc protection chemistry on a CEM Liberty Blue automated peptide synthesiser. We used NovaPEG Rink Amide resin (Merck) with N,N′-diisopropylcarbodiimide as a coupling reagent. Following the final Fmoc deprotection step, the resin was washed with N,N-dimethylformamide (5 ml, twice) and dichloromethane (5 ml, twice). The peptides were deprotected and cleaved from the resin by incubation of the beads with a mixture of trifluoroacetic acid/1,3 dimethoxybenzene/triisopropylsilane/water (92.5:2.5:2.5:2.5) at room temperature for 3 h. The cleaved peptides were then precipitated through the addition of 50 mL ice cold diethyl ether, the solution centrifuged for 10 min at 750 g and the resulting pellet resuspended in acetonitrile/water. The peptides were purified by reverse-phase preparative HPLC (5 to 50% B, A: solution of ammonium bicarbonate 0.1 M at pH 8 with 5% acetonitrile in water; B: solution of ammonium bicarbonate 0.1 M at pH 8 with 5% water in acetonitrile). Fractions were analysed using a MALDI-TOF mass spectrometer and peptide containing fractions were combined, dried under vacuum using a Genevac EZ-2 Elite system, and characterised using High Resolution mass spectroscopy. For cyclic peptides, the linear precursor was prepared as described above except following the final Fmoc deprotection step, on-bead chloroacetylation of the N-terminus was achieved through incubating the beads with a 2 mL of a solution of 0.3 M chloroacetic anhydride in N,N-dimethylformamide for 3 h at room temperature. After washing, cleaving and precipitating as described above the N-terminally chloroacetylated linear peptides were dissolved in no more than 2 mL of an adequate mixture of 1 M triethylammonium acetate pH 9.6 buffer and acetonitrile, where the pH was maintained at >9 through the addition of KOH. The cyclic peptide precursors were incubated at 42° C. for 1 h and the cyclisation reaction was monitored using MALDI-TOF mass spectrometry. Once the reaction appeared to have gone to completion the cyclic peptides were purified as above. Peptides were dissolved in DMSO-d6 to form 100 mM stocks. Accurate concentrations were measured by 1H-qNMR in D2O accounting for aromatic protons with TSP as standard and using a Bruker AVII 500 instrument.
Fluorescence Polarization Assays
Fluorescence polarization assays were performed using a Clariostar (BMG Tech) plate reader with the supplied FITC excitation and emission filters using 96 half area well plates (Corning). The buffer used (FP assay buffer) was 50 mM HEPES, 150 mM NaCl, 0.1% BSA, 0.002% TWEEN-20, 0.2% DMSO, pH 7.4 and the final volume in each well was 30 μL. Polarization was converted to anisotropy using the equation A=(2*P)/(3−P) where P is polarization and A is anisotropy. For each peptide tested the gain was set to 35mP and adjusted to a well containing fluorescent peptide only. The polarization of the emitted light in the FITC emission channel was then determined. Experiments were performed as two technical and three biological replicates. Screening of P672 peptide fragments was achieved through incubation of each peptide (50 nM) with 1 μM CCL8 (Peprotech) for 30 minutes in FP assay buffer and the resulting anisotropy of the emitted light determined as above. The chemokine cross binding screen was performed by incubating 1 μM chemokine (Peprotech) with 50 nM BK1.1FITC for half an hour in FP assay buffer and the resulting anisotropy of the emitted light determined as above. To monitor BK1.1FITC Ala mutants of binding to CCL8, 50 nM labelled peptide was incubated with varying concentrations of recombinant CCL8 (0-25 μM, final) in 30 μL FP assay buffer for 30 minutes and the resulting anisotropy of the emitted light determined as above. The anisotropy was plotted as a function of CCL8 concentration and fitted to the equation: Y=Bmax*X/(KD+X)+NS*X+Background, where Y is the measured anisotropy, X is the concentration of CCL8 added, Bmax is the maximum binding, KD is the equilibrium dissociation constant, NS is the slope of the nonlinear regression and Background is the anisotropy when no CCL8 is present, in Graph Pad Prism. Displacement assays were carried out with CCL7, CCL8, and CCL18 (Peprotech, 1 μM). The chemokines were incubated with BK1.1 (50 μM) or SCR (50 μM) and BK1.1FITC (50 nM) for 30 minutes in FA buffer and the resulting anisotropy of the emitted light determined as above. For all FP assays, the experiments were carried out as two technical and three biological replicates.
Native Mass Spectrometry Analyses
Samples were analysed using a modified Q-Exactive mass spectrometer (Thermo Fisher Scientific) for high-mass range measurements (Gault, J. et al., Nature methods [2016]). CCL8 was buffered exchanged into 200 mM ammonium acetate solution (pH=6.5). BK1.1 obtained in dimethyl sulfoxide (DMSO) was then added to the CCL8 homodimer solution in a 1:1 (CCL8 monomer:BK1.1) ratio. In all cases no more than 0.5% DMSO was present in the final mixture, and a control sample of CCL8 homodimer containing 0.5% DMSO was also analysed. Instrumental parameters were set to: capillary voltage of 1.2 KV, source temperature of 50° C. and 60 V of source induced dissociation (SID). Gas phase dissociation was carried out by applying 35 and 55 V of higher-energy collisional dissociation (HCD) to the most intense charge state after isolation (25 m/z window). Spectra was acquired using a mass resolution of 60,000 for both precursor and dissociated product ions. All measurements were done in triplicates.
AlphaScreen Assay
AlphaScreen® Histidine detection kit was purchased from PerkinElmer (6760619M lot: 2457886) and the assay was set up in white bottom Proxiplate™ 384 Plus microplates (PerkinElmer) following the manufacturer's instructions. The assay buffer used was 50 mM HEPES, 150 mM NaCl, 0.1% BSA, 0.01% Tween20, 1% DMSO, pH 7.5 and the final volume in each well was 20 μL. Briefly, biotinylated chemokine (recombinant, final concentration 1.25 nM (CCL8), 5 nM (CCL2) and 2.5 nM (CCL3)) was pre-incubated at room temperature for 15 min with different concentrations of each peptide. His-tagged P672 (final concentration 2.5 nM (CCL8), 5 nM (CCL2) and 1.25 nM (CCL3)) was then added to each well and the plate was incubated at room temperature for 30 min. Finally, acceptor and donor beads were added as a 1:1 suspension in buffer to each well and the plate was further incubated at RT for 1 h. Data was obtained by reading the plate using a Pherastar FSX plate reader (excitation 680 nm, emission 570 nm) and was analysed using GraphPad Prism.
Fluorescent Chemokine Receptor Blocking Assay
CCL8-647 (2.5 nM, final) or CCL2-657 (1.2 nM, final) was incubated for 30 minutes with varying doses of peptide (0-100 μM, final concentration) in 50 μL assay buffer (RPMI-1640+L-glutamine (4 mM)+10% heat treated fetal bovine serum+0.2% DMSO) at 37° C. This mixture was then added to 50,000 THP-1 cells in a 96-well v-bottomed plated to give a final volume of 100 μL, and everything incubated together for a further 30 minutes at 37° C. Following this time, the plate was centrifuged, the supernatant flicked off, and the cells resuspended in 150 μL ice cold PBSA. This was repeated twice more and the cells were finally resuspended in 150 μL ice cold PBSA. The median fluorescence intensity of 10,000 cells on the RL-1 channel was determined using an ATTUNE flow cytometer and plotted as function of peptide concentration and the data fitted to an inhibitor response curve with 4 parameters using Graph Pad prism 6. Experiments were performed as two technical and three biological replicates.
THP-1 Cell Migration Assays
THP-1 monocyte cell migration assays were carried out as described (Deruaz, M. et al., J Exp Med [2008]). Briefly, effective chemokine concentration (EC) EC80 was determined using a 96-transwell migration plate (5 μM pore size, Corning), with THP-1 cells (300,000) in the top chamber and varying chemokine doses (0-100 nM) in the bottom chamber. The migration buffer used was RPMI 1640+0.5% FCS and 4 mM L-glutamine. This was incubated for four hours and the number of migrating cells in the bottom chamber counted using an ATTUNE flow cytometer. Data were analysed by fitting an agonist response curve with 4 parameters in GraphPad Prism. Neutralization assays were performed using the above system, using an EC80 chemokine dose, and varying evasin or peptide doses in the bottom chamber for 30 min at 37° C. before beginning the assay. In experiments involving peptides, 0.2% DMSO was maintained in the migration buffer to ensure peptide solubility. IC50 was calculated by fitting an inhibitor response curve with 4 parameters in GraphPad Prism. Experiments were performed as 3 technical and 3 biological replicates.
Subcutaneous Dorsal Air Pouch Model
C57BL/6J male mice (25-30 g, 8-10-week-old) were obtained from Charles River (UK). They were group housed in temperature and humidity-controlled rooms, kept on a 12-hr light-dark cycle, and provided with food and water ad libitum. Air pouches were established at the dorsal side of the mice as described (Duarte, D. B. et al., Curr Protoc Pharmacol [2016]). Briefly, mice were anesthetized using isoflurane inhalation and air pouches were produced on day 0 by subcutaneously injecting 4 ml of sterile air into the back of the mice. On day 3, pouches were re inflated with 3 ml of sterile air. On day 6, 0.5 ml of sterile saline solution or 0.5 ml of 25 μg of zymosan (Cat #Z4250, Sigma Aldrich) in saline (w/v) was injected into the air pouch. Five minutes prior to the injection of zymosan, mice were either injected into the air pouch (local) or intra peritoneally (systemic) with 100 μL of 5 mg/kg body weight of either P672 protein or the indicated peptide which was repeated 9 hours later. Mice were sacrificed 24 hours following zymosan injection by isoflurane inhalation followed by cervical dislocation and the air-pouch exudates were collected in 2 ml of saline containing 2 mM EDTA to prevent cell aggregation. Total numbers of leucocytes, neutrophils, monocytes, T-cells and eosinophils were counted using an Attune N×T flow cytometer following staining with cell specific antibodies. Supernatants were kept at −80° C. for chemokine profiling. All animal procedures were approved and carried out in accordance with the UK Home Office Animals (Scientific Procedures) Act 1986, under project licence PPL P973A60F5.
Flow Cytometry Analysis
To characterize the inflammatory subsets in the pouch, multi-colour fluorescence cell staining was conducted using the combination of the following antibodies for various immune cells. Leucocytes (CD45-PE, Cat #130-110-797, Miltenyi Biotec), T-Cells (CD3-FITC, Cat #130-119-798, Miltenyi Biotec), neutrophils (Ly6G-PE.Vio 770, Cat #130-121-438, Miltenyi Biotec), monocytes (Ly-6C-APC, Cat #130-111-779, Miltenyi Biotec) and eosinophils (SigF-APC.Cy7, Cat #565527, BD-Pharmingen). Isotype control antibodies were run parallel (REA-PE, Cat #130-113-450, REA-PE.Vio770, Cat #130-113-452, REA-APC, Cat #130-113-446, REA-FITC, Cat #130-113-449, Miltenyi Biotec, Iso-APC.Cy7, Cat #5527770, BD-Pharmingen). One sample from each group was chosen randomly and labelled for all isotype control antibodies. Briefly, 200 μL cell suspension was pelleted down and before staining with cell surface markers, cells were incubated with an Fc receptor block (2.5 μg/106 cells; BD Pharmingen, cat #553142) to reduce non-specific binding. Then, cells were suspended in 100 μL FACS buffer (0.5% BSA/PBS/2 mM EDTA) containing fluorophore conjugated antibodies (1:50 dilution, Miltenyi Biotec antibodies and 1:100 for BD-Pharmingen antibodies) and stained for 15 minutes on ice in the dark. After washing with FACS buffer, samples were run and analysis was performed on Attune N×T flow cytometer (Life Technologies, USA). Samples were run on an Attune N×T flow cytometer. Data were analysed using FlowJo by an observer blinded to the treatment received. Neutrophils were defined as CD45+Ly-6G+, monocytes/macrophages were defined as CD45+Ly-6C+, T cells were defined as CD45+CD3+ and eosinophils were defined as CD45+Ly-6G− SigF+ (
Chemokine Expression in Air Pouch Fluid
Air pouch exudate supernatants were screened for mouse chemokine profile using a mouse chemokine antibody array (RayBiotech C1 array), according to the manufacturer's instructions. The array consists of 25 different mouse chemokine antibodies spotted in duplicate, three positive controls, two negative controls and two blanks. Membranes were incubated overnight with air pouch exudate, washed and incubated with streptavidin-horseradish peroxidase and chemiluminescent reagent mix and then imaged on a BioRad ChemiDoc MP system following the manufacturer's instructions. The intensity of each chemokine spot was determined by Image Lab software (Version 5.0). Background intensity (from blanks) were subtracted from each measurement. Relative intensity to the positive control (set at 100) was calculated (see
Statistical Analysis
All statistical analyses were performed using GraphPad Prism8. The statistical significance was evaluated by one-way analysis of variance (ANOVA) and P value (probability of a type I error) was adjusted for multiple comparisons with threshold (alpha) for a type I error was P≤0.05. Unless otherwise indicated all data are represented as the mean±s.e.m. of three independent experiments.
2E−10
6E−11
8E−09
2E−08
9E−11
7E−10
7E−09
2E−08
8E−09
9E−09
3E−11
1E−08
1E−11
2_HUMAN
_HUMAN
E−09
E−09
E−10
E−08
CL
_HUMAN
_HUMAN
_HUMAN
_HUMAN
_HUMAN
9E−08
5E−08
7E−08
E−08
8
CL
_HUMAN
_HUMAN
_HUMAN
_HUMAN
indicates data missing or illegible when filed
2E−10
6E−11
9E−11
7E−10
3E−11
2E−10
6E−11
9E−11
7E−10
3E−11
A., Frade, A., Donadi, E., Dias, F., Saba, B., Wang, H. T., Fragata, A., Sampaio, M., Hirata, M. H., Buck, P., Mady, C., Bocchi, E. A., Stolf, N. A., Kalil, J., and Cunha-Neto, E. (2012) Myocardial chemokine expression and intensity of myocarditis in Chagas cardiomyopathy are controlled by polymorphisms in CXCL9 and CXCL10. PLoS neglected tropical diseases 6, e1867
| Number | Date | Country | Kind |
|---|---|---|---|
| 1713284.6 | Aug 2017 | GB | national |
This application is a continuation of U.S. patent application Ser. No. 16/784,920 (filed Feb. 7, 2020, which is a continuation-in-part of PCT/GB2018/052331 (filed Aug. 16, 2108), which claims the benefit of priority to international application GB1713284.6 (filed Aug. 18, 2017). Each of these applications is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16784920 | Feb 2020 | US |
| Child | 18153944 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/GB18/52331 | Aug 2018 | US |
| Child | 16784920 | US |
