Vectors for molecule delivery to CD11b expressing cells
10004794 ยท 2018-06-26
Assignee
Inventors
- Claude Leclerc (Paris, FR)
- Pierre Guermonprez (Cachan, FR)
- Daniel Ladant (Cachan, FR)
- Nicole Guiso (Paris, FR)
- Nadia Khelef (Paris, FR)
- Cecile Bauche (Paris, FR)
- Catherine Fayolle (Epinay sur Orge, FR)
- Mohammed El-Azami El-Idrissi (Paris, FR)
Cpc classification
A61P29/00
HUMAN NECESSITIES
A61P31/00
HUMAN NECESSITIES
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A61K47/646
HUMAN NECESSITIES
A61P43/00
HUMAN NECESSITIES
A61K2039/6037
HUMAN NECESSITIES
A61K2039/57
HUMAN NECESSITIES
International classification
Abstract
The invention relates to a novel use of a Bordetella adenylcyclase toxin in the manufacturing of vectors for targeting in vivo a molecule of interest, specifically to CD11b expressing cells. The invention also relates to an immunogenic composition that primes immune responses, to pharmaceutical compositions and to a new vector for molecule delivery to CD11b expressing cells.
Claims
1. A proteinaceous vector comprising: a) a fragment of a Bordetella species adenylcyclase (CyaA) comprising amino acid residues 373-1706 and lacking all or part of residues 1-372; and b) a molecule of interest coupled to the CyaA fragment; wherein the proteinaceous vector binds to CD11b expressing dendritic cells such that the molecule of interest is delivered into the endocytic pathway of the CD11b-expressing cells.
2. The proteinaceous vector according to claim 1, wherein the Bordetella species adenylcyclase is a recombinant adenylcyclase.
3. The proteinaceous vector according to claim 1, wherein the Bordetella species is Bordetella pertussis.
4. The proteinaceous vector according to claim 1, wherein the Bordetella species adenylcyclase (CyaA) fragment is the Bordetella pertussis adenylcyclase (CyaA) lacking residues 1-372.
5. The proteinaceous vector according to claim 1, wherein the molecule of interest is genetically coupled to the adenylcyclase (CyaA) fragment.
6. The proteinaceous vector according to claim 1, wherein the molecule of interest is chemically coupled to the adenylcyclase (CyaA) fragment.
7. The proteinaceous vector according to claim 1, wherein the Bordetella species adenylcyclase (CyaA) fragment has been genetically detoxified.
8. The proteinaceous vector according to claim 1, wherein the molecule of interest comprises a peptide antigen.
9. The proteinaceous vector according to claim 8, wherein the proteinaceous vector binds to CD11 b expressing dendritic cells such that the peptide antigen is translocated to the cytosolic pathway for MHC class I presentation.
10. The proteinaceous vector according to claim 9, wherein administration of the proteinaceous vector to a subject induces a cytotoxic T lymphocyte (CTL) response against the peptide antigen.
11. The proteinaceous vector according to claim 10, wherein the CTL response is a CD8+ response.
12. The proteinaceous vector according to claim 10, wherein the CTL response is a CD4+ response.
13. A pharmaceutical composition comprising the proteinaceous vector of claim 1 and a pharmaceutically acceptable carrier.
14. An immunogenic composition comprising the pharmaceutical composition of claim 13.
15. The immunogenic composition of claim 14, wherein the immunogenic composition does not contain an adjuvant.
16. A CD11b-expressing cell bound to the proteinaceous vector of claim 1.
Description
LEGENDS TO FIGURES
(1)
(2) A: CyaA binding at the surface of macrophages (J774A.1), B cells (LB27.4), and T cells (EL4) was performed at 37 C. for 20 minutes. Surface-bound CyaA was detected with a biotinylated anti-CyaA polyclonal antibody, revealed by streptavidin-PE and detected by flow cytometry on living cells, as described in the material and methods section. Binding is expressed as MFI=(mean fluorescence intensity value of cells incubated with CyaA)(mean fluorescence intensity of cells without CyaA).
(3) B, C, D, E: Surface expression of .sub.2 integrins on J774A.1, LB27.4 and EL4 cells. CD11a (B), CD11b (C), CD11c (D), and CD18 (E) expression were determined by flow cytometry using specific mAbs coupled to PE. Integrin expression is expressed as MFI=(mean fluorescence intensity value of cells stained with specific mAb)(mean fluorescence intensity of cells stained with an isotype control mAb).
(4)
(5) Cells were preincubated at 4 C. for 15 minutes with or without 20 g/ml of specific mAbs and then incubated at 4 C. for 20 minutes with 5 g/ml CyaA and with 10 g/ml of specific mAbs if present during the preincubation. Surface-bound CyaA was detected with a biotinylated anti-CyaA polyclonal, revealed by streptavidin-PE and detected by flow cytometry on living cells, as described in the material and methods section.
(6) A, B: Effect of the M1/70 anti-CD11b mAb on the binding of various doses of CyaA. FSDC dendritic cells (a) or J774A.1 macrophages (b) were preincubated with medium alone () or with M1/70 anti-CD11b mAb (.circle-solid.) and then incubated with CyaA with or without M1/70 anti-CD11b mAb. Bmax was determined by fitting experimental points obtained from experiments performed without mAbs to MFI=Bmax*[CyaA]/(K.sub.d+[CyaA]). Binding of CyaA is plot as a % of Bmax plot against CyaA concentration.
(7) C, D: Effect of specific mAbs on a fixed dose of CyaA binding. FSDC (c) or J774A.1 (d) cells were preincubated with or without specific mAbs (anti-CD11a, 2D7, antiCD11b, M1/70 and 5C6, anti-CD11c, HL3, anti-CD18, C17/16, control A95-1) and incubated with CyaA at the fixed concentration of 5 g/ml. Values of MFI obtained for CyaA binding on cells treated with specific mAbs were normalized as MFI values obtained for CyaA binding without mAb.
(8)
(9) A, B, C: Fluorescence histograms of freshly purified neutrophils were preincubated with medium alone (A), the 44 anti-CD11b mAb (B) or an isotype-matched control mouse mAb (C) and then incubated with (gray) or without (blank) biotinylated CyaA and revealed by streptavidine-PE. Cell number is plotted against log of PE fluorescence.
(10) D: Effect of specific mAbs on CyaA binding to neutrophils (anti-CD11b, 44, M1/70, anti-CD18, TS/18, control mouse IgG2a, control rat IgG2b, A95-1). Freshly purified neutrophils were preincubated with or without specific mAbs and incubated with CyaA. Values of MFI obtained for CyaA binding on cells treated with specific mAbs were normalized as a % of the MFI values obtained for CyaA binding without mAb.
(11)
(12) A: Effect of specific mAbs on intracellular cAMP accumulation. J774A.1 cells were preincubated at 4 C. for 1 h with or without 10 g/ml of specific mAbs (anti-CD11b, M1/70, anti-CD18, C17/16) and then incubated at 37 C. for 20 min with 5 g/ml CyaA and with 10 g/ml mAbs if present during the preincubation. Intracellular cAMP contents were determined as described in the materials and methods section.
(13) B: Effect of specific mAbs on CyaA mediated cell death. J774A.1 cells were preincubated at 4 C. for 1 h with medium alone or with 10 g/ml of specific mAbs (anti-CD11a, 2D7 anti-CD11b, M1170, anti-CD11c, HL3, anti-CD18, C71/16, control, 2.4G2). Then they were incubated at 37 C. for 2 h with 0.5 g/ml CyaA and with 10 g/ml of specific mAbs when present during the preincubation. Cell lysis was determined by LDH release using the Cytotox 96 assay.
(14)
(15) A, B: CyaA binding at the surface of CHO transfectants. CHO cells transfected with human CD11b/CD18 (.circle-solid.), human CD11c/CD18 () or mock-transfected () were incubated with various doses of CyaA for 20 min at 37 C. (a) or 4 C. (b). Surface-bound CyaA was detected with a biotinylated anti-CyaA polyclonal antibody, revealed by streptavidin-PE and detected by flow cytometry on living cells, as described in the material and methods section. Binding is expressed as MFI=(mean fluorescence intensity value of cells incubated with CyaA)(mean fluorescence intensity of cells without CyaA).
(16) C: Intracellular cAMP accumulation in CHO transfectants. CHO cells transfected with human CD11b/CD18 (.circle-solid.), human CD11c/CD18 () or mock-transfected () were incubated with or without CyaA for 20 min at 37 C. Intracellular cAMP contents were determined as described in the materials and methods section.
(17) D: Cell lysis in CHO transfectants. CHO cells transfected with human CD11b/CD18, human CD11c/CD18 or mock-transfected were incubated with 5 g/ml CyaA for 4 h at 37 C. Cell lysis was determined by LDH release using the Cytotox 96 assay.
(18)
(19) C57BL/6 WT+/+ (A), CD4/ (B), CD40/ (C) or IgM/ (D) mice were intravenously immunized with 50 g of CyaAOVA, a genetically detoxified form of CyaA carrying the H-2K.sup.b restricted SIINFEKL epitope from OVA (.circle-solid., ) or with CyaAE5, a control detoxified toxin without the OVA epitope (.box-tangle-solidup., ). Seven days after, animals were sacrificed and splenocytes were restimulated in vitro for 5 days with 10 g/ml of the pOVA synthetic peptide in the presence of irradiated C57BL/6 splenocytes. CTL activity was assessed in a 4 hours chromium.sup.51 release assay against H-2K.sup.b+ EL4 cells previously pulsed (.circle-solid., .box-tangle-solidup.) or not (, ) with pOVA at 10 g/ml.
(20)
(21) The low density fraction of splenocytes presents CyaAOVA to a specific anti-OVA CD8+ T cell hybridoma (A, E):
(22) In vitro assay (A): Low (LDF, .circle-solid.) and high density (HDF, .box-tangle-solidup.) fractions or unfractionated total splenocytes from naive mice (TSC, .square-solid., ) were cultured with B3Z, a CD8.sup.+ T cell hybridoma specific for the pOVA peptide in the context of H-2K.sup.b. After 18 hours of coculture in the presence of the recombinant detoxified CyaA carrying the OVA peptide (CyaAOVA, .circle-solid., .box-tangle-solidup., .square-solid.) or a control peptide (CyaALCMV, ) at various concentrations, IL-2 released in supernatants was measured in a CTLL proliferation assay. Results are expressed in cpm and plotted against CyaA concentration during the assay cpm=[cpm+CyaA][cpmCyaA]).
(23) Ex vivo assay (E): Spleen cells were obtained from mice previously immunized iv (6-12 hours) with 50 g of CyaAOVA (.circle-solid., .box-tangle-solidup., .square-solid.) or CyaALCMV () and fractionated in LDF and HDF. Various numbers of cells recovered from TSC (U), LDF (.circle-solid., ) or HDF (A) or unfractionated splenocytes (.square-solid.) were directly put in culture with B3Z without addition of recombinant CyaA. IL-2 release was assessed after 18 hours of culture as described before. Results, expressed in cpm, are plotted against the number of APC present in each well.
(24) Dendritic Cells (CD11c.sup.+) are More Efficient APC for CyaAOVA than the CD11b.sup.high+ CD11c.sup. Cells or B Cells (CD45R.sup.+) (B, F):
(25) In vitro assay (B): CD11c.sup.+ (.circle-solid.) sorted cells from LDF, CD11b.sup.high+CD11c.sup. (0) and CD45R.sup.+ (.square-solid.) sorted cells from TSC, were put in culture with B3Z for 18 hours in the presence of various concentrations of CyaAOVA. IL-2 was assessed as above.
(26) Ex vivo assay (F): Cells sorted by flow cytometry from C57BL/6 mice previously (6-12 hours) immunized with 50 g of CyaAOVA were used as APC. CD11c+ (.circle-solid.), CD11b.sup.high+CD11c.sup. (), CD45R.sup.+ (.square-solid.) sorted cells from low density splenocytes were directly put in culture for 18 hours with B3Z at various numbers of cells per well, without addition of CyaAOVA. IL-2 was assessed as above.
(27) The CD8.sup. Myeloid Dendritic Cell Subset is a More Efficient APC for CyaA than the CD8.sup.+ Lymphoid Dendritic Cell Subset (C, G):
(28) CD11c+ low density cells from naive mice (C) or mice previously (6-12 hours) immunized iv with 50 g CyaAOVA (G) were fractionated in myeloid dendritic cells (CD11c.sup.+CD8.sup., .circle-solid.) and lymphoid dendritic cells (CD11c.sup.+CD8.sup.+, ) by flow cytometry and used as APC in in vitro (C) and ex vivo (G) assays for B3Z stimulation. IL-2 was assessed as above.
(29) B Cell Genetic Depletion does not Impair CyaAOVA Presentation by Splenocytes (D, H):
(30) TSC (.square-solid., ), LDF (.circle-solid., ) or HDF (.box-tangle-solidup., ) from C57BL/6 WT mice (.square-solid., .circle-solid., .box-tangle-solidup.) or B cell deficient (, , ) were used as APC in an in vitro assay (D, .square-solid., ) or an ex vivo assay (H, .square-solid., .circle-solid., .box-tangle-solidup., , , ) for B3Z stimulation as in a. Mice were either from naive (D) or previously (1.5 hours) immunized iv with 50 g CyaAOVA (H). IL-2 was assessed as above.
(31)
(32) In vitro assay (A): TSC (.box-tangle-solidup., ) or CD11e (.circle-solid., ) sorted cells from control C57BL6 TAP+/+ (.box-tangle-solidup., .circle-solid.) or TAP/ mice (, ) were cultured with B3Z in the presence or not of various doses of CyaAOVA. IL-2 was assessed as described in
(33) Ex vivo assay (B): TSC (.box-tangle-solidup., ) or CD11c.sup.+ (.circle-solid., ) sorted cells from control C57BL6 TAP+/+ (.box-tangle-solidup., .circle-solid.) or TAP/ mice (, ) previously iv immunized with 50 g of CyaAOVA were cultured with B3Z for 18 hours. IL-2 was assessed as described in
(34)
(35) Binding of CyaAOVA-biotine to TSC is blocked by anti-CD11b (A-E): TSC suspensions were incubated at 4 C. with 10 g/ml of the anti-CD11b M1/70 mAb or an isotype control mAb or nothing. Then, CyaAOVA-biotine at 2 g/ml (left panels) or various concentrations (right panel) was added to the cells for 30 nm at 4 C. After a wash, CyaAOVA-biotine was revealed with streptavidine-PE for 30 nm (Strep-PE). Then, after washing, cells were resuspended in PBS containing propidium iodide. The size (FSC) of living cells gated by propidium iodide exclusion was plotted against the Strep-PE fluorescence. The percentage of leukocytes positive for CyaAOVA-biotine was plotted against CyaAOVA-biotine concentration during the staining.
(36) Binding of CyaAOVA-biotine to low density cells correlates with the expression of CD11b (F-J) LDF were triple stained for CD11c, CD8 and CyaOVAbiotine (or medium) or, in separate experiments with CD11c, CD8, and CD11b (or a control mAb). After a wash, cells were stained for 30 nm with Strep-PE to reveal CyaAOVA-biotine, anti-CD11c-FITC and anti-CD8-APC. Gates were done on lymphoid DC (CD11c.sup.+CD8.sup.+), myeloid DC (CD11c.sup.+CD8.sup.), CD8+ T cells (CD11c.sup.CD8.sup.+) and other cells (CD11c.sup.+CD8.sup.). For each gate, CyaAOVA-biotine staining or CD11b staining is plotted against cell number in separate histograms. Left histograms: LDF suspensions were incubated with 0 (histograms filled in grey), 2.5 (narrow, open histograms) or 10 g/ml (thick, open histograms) of CyaAOVA-biotine for 30 minutes at room temperature. Right histograms: isotype control-PE (histogram filled in grey), CD11b-PE (thin, open histograms).
(37)
(38) In vitro antigen presenting assay with TSC (A, B): The same experiments than in a, b were performed with TSC from naive C57BL/6 mice as APC. The stimulation of B3Z was assessed by IL-2 release in coculture supernatants measured in a CTLL proliferation assay. Results are plotted in cpm against CyaAOVA or pOVA concentration.
(39) Ex vivo antigen presenting assay with TSC or CD11b.sup.+ and CD11b.sup. fractions: C57BL/6 mice were intravenously immunized with 50 g of CyaAOVA (c) or 10 g of pOVA (d). CD11b.sup.+ (.circle-solid.) and CD11b.sup. () cells were sorted by flow cytometry from TSC () and put in culture at various cell number per well with B3Z. After 18 hours of coculture, the stimulation of B3Z was assessed by IL-2 release. Results, expressed in cpm, are plotted against the numbers of APC from immunized animals present in each well.
(40)
(41)
(42)
(43) 3.Math.10.sup.5 spleen cells from C57Bl/6 mice were cocultured for 18 h with 105B3Z cells in the presence of various concentrations of cyclases. The IL2 release by B3Z was measured in a CTLL proliferation assay.
(44)
(45) C57BL/6 mice were iv injected with 50 g of the various CyaA. Seven days later, spleen cells were in vitro stimulated with OVA peptide. The cytotoxic activity was measured on 51 Cr-labelled target cells.
(46)
(47)
(48) CHO cells transfected with CD11b/CD18 were preincubated on ice for 1 hour with different concentrations of CyaA-E5 (black triangle), CyaA 1-383 (black square) or CyaA-373-1706 (Black diamond) and then incubated on ice for 30 min with 5 g/ml of biotinylated CyaA-E5. Surface bound cyclase was revealed using streptavidin-PE and analyzed by flow cytometry on living cells. Results are expressed as mean fluorescence intensity (A), percentage of positive cells (B) and percentage of inhibition (C).
(49)
(50) The large arrows represent the open reading frames of -lactamase (bla), the thermosensitive repressor cl.sup.857 of phage lambda (cl.sup.857), the cyaC gene and truncated cyaA gene (the arrows are pointing to the direction of translation of the corresponding genes). The ColE1 origin (Oil), the Pr promoter (APr), and some relevant restriction sites are also indicated. The intergenic region between the cyaC and truncated cyaA genes is detailed in the lower part. It shows the new C-terminus extension of cyaC (downstream to Arg182 of wild-type CyaC), the stop codon (underlined), the initiator Met and first codons of CyaA373-1706 (upstream to Ser373 of wild-type CyaA).
EXAMPLES
A. The Bordetella Adenylate Cyclase Toxin Interacts Specifically with the M2 Integrin (CD11b/CD18
(51) A.1 Materials and Methods
(52) A.1.1 Recombinant Toxins and Antibodies
(53) Protocol for CyaA production has already been described elsewhere [Karimova, et al, 1998]. CyaA toxins were produced in E. coli BLR strain harboring an expression plasmid, pCACT3, which carries the cyaA structural gene CyaA under the lacUV5 promoter and the cyaC accessory gene required for activation of the protoxin. After solubilization in 8M urea, Hepes-Na 20 mM, pH 7.5, CyaA was purified to more than 95% homogeneity (as judged by SDS-gel analysis, not shown) by sequential DEAE-Sepharose and Phenyl-Sepharose. A recombinant detoxified CyaA toxin, CACTE5-Cys-Ova, harboring a unique cysteine inserted within the genetically inactivated catalytic domain was constructed by inserting an appropriate double strand oligonucleotide between the Bsiwl and KpnI sites of pCACT-Ova-E5 [Guermonprez et al, 2000]. In the resulting protein CACTE5-Cys-Ova, the amino acid sequence ASCGSIINFEKLGT is inserted between residues 224 and 225 of CyaA. The recombinant toxin was expressed and purified as previously described. The purified protein was labeled on its unique Cys with the highly specific sulfhydryl reagent N-(6-(Biotinamido)hexyl))-3-(2-pyridyldithio)propionam ide (Biotin-HPDP, PIERCE) according to the manufacturers instructions. The biotinylated-CyaA was re-purified on DEAE-Sepharose to remove the unreacted Biotin-HPDP reagent. Toxin concentrations were determined spectrophotometrically from the adsorption at 280 nm using a molecular extinction coefficient of 142 M.sup.1cm.sup.1 (binding studies) or using the Biorad protein assay system (cAMP accumulation and cell death studies).
(54) Purified mAbs specific for murine CD11a (2D7, Rat IgG2a, ), murine and human CD11b (M1/70, Rat IgG2b, ), murine CD11c (HL3, Hamster 1, ), murine CD18 (C71/16, Rat IgG2a, ), control (A95-1, or anti-CD16/32, 2.4G2, Rat IgG2b, ) originated from Pharmingen (San Diego, USA). Supernatants from anti-human CD11b (44, Mouse IgG2a, ), and anti-human CD18 (TS/18, Mouse IgG1, ) hybridoma were a kind gift and were used at 1/2 dilution in blocking experiments. Supernatants from an anti-murine CD11b (5C6, Rat IgG2b, ) were a kind gift from G. Milon (Pasteur Institute, Paris) and were used at 1/2 final dilution in binding inhibition assays. Anti-CyaA polyclonal antibodies were obtained from a rabbit immunized subcutaneously with purified CyaA.
(55) Sera were precipitated from immune serum by ammonium sulfate (33%). After centrifugation the pelleted proteins were resuspended in 20 mM Hepes-Na, 150 mM NaCl, pH 7.5 (buffer C) and extensively dialysed against the same buffer. The antibodies were then biotinylated by incubation with Biotin-amidocaproate N-Hydroxysuccinimide ester (SIGMA, dissolved in dimethyl sulfoxide) for 130 min at room temperature. Then, 100 mM ethanolamine, pH 9.0 were added and after 30 min of additional incubation, the mixture was extensively dialysed at 4 C. against buffer C. Biotinylated antibodies were stored at 20 C.
(56) A.1.2 Cells and Culture Media
(57) EL4, J774A.1, LB27.4, THP-1 were obtained from the American Type Culture Collection (ATCC) and were cultured in RPMI 1640 supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 g/ml streptomycin, 2 mM L-glutamine, with or without 510.sup.5 M 2-mercaptoethanol (complete medium). FSDC [Girolomoni et al, 1995] were cultured in complete medium. CHO cells transfected for human CD11b/CD18 or CD11c/CD18 or transfected with the vector only were obtained from D. Golenbock (Boston, USA) and cultured in the presence of neomycin as previously described [Ingalls et al, 1998]. Human neutrophils were purified as previously described [Rieu et al, 1992].
(58) A.1.3 CyaA Binding Assays
(59) All binding assays were performed in DMEM 4.5 mg/ml glucose (Life Technologies) without serum in 96 well culture plates (Costar). 210.sup.5 cells/well were incubated for 20 minutes (at 4 C. or 37 C. depending on the experiments) in a 200 l final volume. In some experiments, cells were preincubated for 20 min at 4 C. in the presence of blocking mAbs in 100 l final volume. The toxin solution was added to the wells in the continuous presence of the mAbs in a total volume of 200 l at 4 C. Then, plates were centrifuged at 1500 rpm for 5 min and supernatants were removed. Cells were incubated at 4 C. for 25 minutes with biotinylated anti-CyaA rabbit polyclonal antibodies (1/400 in DMEM, 50 l/well) in the presence of a control (non-immune or pre-immune) rabbit serum as a saturating agent (1/50).
(60) After centrifugation and supernatant removal, cells were stained with streptavidin-phycoerythrin (PE) (Pharmingen) at 1/300 dilution (50 pt/well). After washing, cells were analyzed by flow cytometry on a FACStar (Becton-Dickinson, Mountain View, USA) in the presence of 5 g/ml propidium iodide. Gatings were done to exclude cells aggregates and dead cells by propidium iodide exclusion. Experimental points were fitted to a hyperbolic model MFI=Bmax*[CyaA]/(K.sub.d+[CyaA]), with Bmax=% of maximal binding, using the prism software.
(61) A.1.4 cAMP Assay
(62) cAMP accumulation was measured by an antigen competition immunoassay [Karimova et al, 1998] in which the incubation medium was composed of DMEM without serum but containing 4.5 mg/ml glucose and 20 U/ml hexokinase. Hexokinase, which catalyzes the ATP-dependent phosphorylation of glucose, was added deplete the extracellular medium for any traces of ATP, thus preventing the extracellular synthesis of cAMP. Therefore, the amount of cAMP measured is representative of the accumulation of strictly intracellular cAMP. 510.sup.5 cells were preincubated in 96 well plates in 100 l/well with or without 10 g/ml of specific mAbs at 4 C. for 1 h and then incubated at 37 C. for 20 min with 0.05, 0.5 or 5 g/ml CyaA and with 10 g/ml of specific mAbs when present during the preincubation. For the dose response effect of CyaA, cells were directly incubated with the toxin for 20 min at 37 C. After intoxication, cells were centrifuged at 2.500 rpm for 5 min. Samples were lysed with 100 l of HCl 0.1 N, boiled for 5 min at 120 C., and neutralized with 100 l of Tris 0.125 M, NaCl 0.2 M. Microtiter plates were coated with cAMP-BSA conjugates diluted at 1/4,000 in Na.sub.2CO.sub.3 0.1 M pH 9.5. They were washed twice in PBS-Tween 0.1%, saturated for 1 h in PBS-BSA 2% and washed five times with PBS-Tween 0.1%. The samples and the CAMP standard (Sigma) were directly added to the plates coated with cAMP-BSA conjugates and serially diluted with a 1/1 mixture of HCl 0.1 N and Tris 0.125 M-NaCl 0.2 M. Anti-cAMP rabbit antibody was added at 1/2,500 in PBS-BSA 2% and incubated at 37 C. for 3 h. Plates were washed five times with PBS-Tween 0.1%. Anti-rabbit antibodies coupled to horseradish peroxidase (Amersham) were added at 1/2,500 in PBS-BSA 2%, incubated at 37 C. for 1 h and revealed using the classical peroxidase reaction. Experimental points of the standard curve were fitted to a sigmoid model using the Prism software.
(63) A.1.5 Toxicity Assay
(64) Cell death was evaluated as previously described [Khelef et al, 1993; Khelef et al, 1995]. Briefly, 10.sup.5 cells were incubated for 24 hours in a 96 well plate in complete medium and washed once with serum free medium. All cell incubations were further performed in serum free medium. Dose response effects were evaluated by directly applying various concentrations of CyaA to CHO cells at 37 C. for 4 h. For cytotoxicity inhibition, cells were preincubated at 4 C. for 1 h with or without 10 g/ml of specific mAbs and then incubated at 37 C. with 0.5 g/ml CyaA for 2 h for J774A.1A.1 cells or with 5 g/ml for 4 h for CHO cells and with 10 g/ml of specific mAbs when present during the preincubation. Cell lysis was evaluated using the Cytotox 96 assay (Promega) which quantifies the amount of lactate dehydrogenase (LDH) released in the medium by dying cells.
(65) A.2 Results
(66) A.2.1 Saturable Binding of CyaA Correlates with the Presence of CD11b at the Surface of Target Cells
(67) To characterize CyaA cellular specificity toward a population of leukocytes, we choose three representative murine cell lines expressing various combination of .sub.2 integrins: J774A.1, a tumoral macrophage; EL4, a T cell thymoma, and LB27.4, a B cell lymphoma. After a 20 minutes of incubation with CyaA at 37 C., binding of CyaA to the cell surface of these cells was monitored by flow cytometry using biotinylated anti-CyaA antibodies and streptavidine-PE. Under these conditions, we observed an efficient, dose-dependent and saturable binding of CyaA on J774A.1 cell line. The affinity of CyaA for J774A.1 cells was high since the apparent K.sub.d were 9.24.5 nM and 3.21.9 nM, respectively. A low binding of CyaA to EL4 and LB27.4 cells was observed but it was not saturable at the concentrations tested.
(68) In order to determine if the binding of CyaA to J774 cell lines was correlated to the expression of one of the members of the .sub.2 integrin family, we performed a phenotypic analysis of these cells by flow cytometry using monoclonal antibodies (mAbs) specific for the three a chains of the well characterized .sub.2 integrins (CD11a, Cd11b and CD11c) and for the common chain (CD18) (
(69) A.2.2 CyaA Saturable Binding is Specifically Blocked by Anti-CD11b mAbs
(70) We next examined if CD11b/CD18 could be directly involved in the binding of CyaA to the cells expressing this integrin. We performed a quantitative analysis of inhibition obtained with anti-CD11b M1/70 mAb by calculating percentage of mean fluorescence values in the absence of mAbs at a fixed or varying concentrations of CyaA (
(71) To examine whether CyaA could similarly interact with human CD11b, CyaA binding studies were performed on human neutrophils, whose high expression of CD11b is well established. Since high background fluorescence was obtained following incubation of human myeloid cells with the anti-CyaA rabbit antibodies (data not shown), we set up an alternate binding assay. A detoxified form of CyaA was specifically biotinylated on unique cysteine residues, genetically introduced within its catalytic domain. Using this system, we were able to detect CyaA binding to neutrophils (
(72) In conclusion, CyaA binding to the surface of three myeloid cell lines from both murine and human origin (J774A.1, FSDC, THP-1) as well as freshly purified human neutrophils appears to be mainly mediated through the CD11b/CD18 integrin.
(73) A.2.3 CyaA-Mediated cAMP Increase and Toxicity are Specifically Blocked by an Anti-CD11b mAb
(74) To evaluate the physiological relevance of CD11b/CD18-dependent CyaA binding, we studied the effect of blocking mAbs on the cytotoxicity of CyaA. We first measured the amount of cAMP produced in J774A.1 cells exposed to CyaA in the presence of various mAbs. As shown in
(75) Taken together, these data indicate that CyaA binding through CD11b is strictly required for CyaA mediated toxicity in J774A.1 cells.
(76) A.2.4 Transfection of CHO Cells with CD11b/CD18 Confers Sensitivity to CyaA
(77) To confirm the role of CD11b in CyaA binding, we used CHO cells transfected with the human integrins CD11b/CD18 or CD11c/CD18 or mock transfected (vector alone). As shown in
(78) Since we found that CD11b was required for CyaA mediated toxicity in J774A.1, we then determined if CD11b expression was sufficient to confer a CyaA-sensitive phenotype to CHO transfected cells. In line with previous reports [Gordon et al, 1988], CyaA induced a notable amount of intracellular cAMP in CHO cells transfected with CD11c/CD18 or in control mock-transfected cells, but only at high concentrations of toxin (5 g/ml,
(79) We also evaluated the role of CD11b/CD18 in CyaA-mediated cell death. As shown in
(80) Altogether, these results thus clearly established that expression of human CD11b/CD18 integrin is sufficient to create a high affinity receptor for CyaA in CHO cells.
(81) A.3 Discussion: A Receptor for CyaA
(82) Unlike other toxins, CyaA has been considered for a long time, as independent of any receptor binding. This is based on the observations that CyaA can intoxicate in vitro a wide variety of model cell lines from various origin [Ladant et al, 1999] ii) CyaA binds to Jurkat cells and sheep erythrocytes in a non saturable fashion [Gray, et al 1999]. In fact, these observations established that non-specific adsorption of CyaA to lipid membranes leads to some translocation of the catalytic domain into the cytosol. However, they did not rule out the existence of a specific receptor. We showed in this study on myeloid cell lines that the binding and the toxic properties of CyaA are dependent on its interaction with the integrin CD11b/CD18. Efficient and saturable binding correlates with the expression of CD11b and is fully and specifically blocked by anti-CD11b mAbs. Moreover, expression of CD11b/CD18 in CHO cells dramatically enhances the binding of CyaA, resulting in an increased sensitivity to intoxication by this toxin. Our results are the first evidence supporting the interaction of CyaA with a cell-surface molecule specifically expressed on leukocytes. The nearly complete blockade of CyaA binding by anti-CD11b mAbs suggests that CD11b is the main receptor for CyaA in the cell lines tested. The lack of efficient binding to CD11c/CD18 transfectants, or CD11a/CD18 expressing cells such as EL4 or LB27.4 suggest that CD11b/CD18 is the only integrin of the 2 family involved in the binding of CyaA to target cells.
(83) In line with previous studies, we observed a detectable binding of CyaA to all cell lines tested. Furthermore, CyaA at high concentrations triggered a small but detectable cAMP increase in mock-transfected CHO cells, that is not associated to cell death. Thus, at high concentration, CyaA can bind and translocate to a wide variety of cell lines but efficient and saturable binding, translocation and killing is a hallmark of CD11b expressing cells.
(84) Binding of CyaA to a member of the 2 integrin family is reminiscent of the behavior of other RTX toxins which were recently found to interact with these molecules [Lally et al, 1997; Li et al, 1999; Ambagala et al, 1999; Jeyaseelan et al, 2000]. The E. coli HlyA, that shares a strong homology with CyaA, forms cationic pores at the plasma membrane. HlyA exhibits a specificity for leukocytes but only at low concentration [Welch et al, 1991]. This relative specificity was shown to be mediated by its interaction with the integrin CD11a/CD18 [Lally et al, 1997]. Similarly, A. actinomycetemcomitans and P. haemolytica leukotoxin (LtxA and LktA, respectively), which are less promiscuous RTX toxins specific for human and bovine leukocytes, respectively, also interact with CD11a/CD18 [Lally et al, 1997; Li et al, 1999; Ambagala et al, 1999; Jeyaseelan et al, 2000]. Despite its strong homology with HlyA, CyaA recognizes another .sub.2 integrin (CD11b/CD18) whose cellular distribution is different. Indeed, CD11b is expressed mostly on macrophages, neutrophils and dendritic cells, but not on the majority of T and B cells, whereas CD11a is expressed on all leukocytes including T and B lymphocytes.
(85) This specific targeting of CyaA to CD11b expressing cells is exploited in the present invention to specifically target this particular subset of cells. Detoxified mutants of CyaA remaining invasive could be used for the delivery of pharmacologically active molecules to CD11b positive cells, without noticeably affecting other cell types.
B. Targeted Antigen Delivery to the Cytosol of Myeloid Dendritic Cells and Selective CTL Priming
(86) B.1 Materials and Methods
(87) B.1.1 Recombinant Adenylate Cyclase Toxins and Peptide
(88) The pOVA synthetique peptide (SIINFEKL) originated from NEOSYSTEM and was diluted in PBS at 1 mg/ml.
(89) B.1.2 Immunization and Assay for the Detection of Cytotoxic T Cells
(90) Female C57BL/6 (H-2.sup.b) from Iffa Credo (L'arbresle, France) were used between 6 and 8 weeks of age. TAP1/ (Van Kaer et al., 1992), CD4/ (Killeen et al., 1993), CD40/ (Kawabe et al, 1994) and B cell deficient NMT (Kitamura et al, 1991) bred onto a C57BL/6 background originated from the CDTA facility (CNRS, Orleans, France) and were bred in the Institut Pasteur facilities. Animals were intravenously immunized with Ag in PBS. Seven days post-injection, animals were sacrificed and the spleen was removed. Single cell suspensions of splenocytes (2.510.sup.7 cells) were restimulated in 10 ml CM (see below) with irradiated spleen cells (2.510.sup.7 cells) for 5 days in the presence of 1 g/ml pOVA. Cytotoxicity assay was performed exactly as previously described (Fayolle, et al., 1999).
(91) B.1.3 Cell Lines
(92) B3Z (Karttunen et al., 1992), a CD8+ T cell hybridoma specific for the OVA 257-264 peptide (SIINFEKL) in the context of H-2K.sup.b was a generous gift from Dr N. Shastri (University of California, Berkeley, USA).
(93) B.1.4 Antigen Presentation Assays
(94) All antigen presentation assays were performed by coculture of APC with B3Z in 96 well culture microplates (0.2 ml final volume) in RPMI 1640 supplemented with 10% Fetal Calf Serum, 100 U/ml penicillin, 100 g/ml streptomycin, 2 mM L-glutamine, and 510.sup.5M 2-mercaptoethanol (complete medium, CM). The stimulation of B3Z cells (10.sup.5 cells/well) was monitored by IL-2 release in the supernatants of 18-24 hours cultures in the presence of APC. IL-2 was measured in CTLL assay as previously described (Guermonprez et al., 1999). In some experiments (see figure legends), B3Z stimulation was assessed using the NF-AT lacZ reporter assay. LacZ activity in cell lysates was assessed with the CPRG substrate as previously described (Karttunen et al., 1992). Two antigen presentation assays were performed: i) In vitro assay: APC originated from naive mice were cocultured (10.sup.5/well) with B3Z in the presence of Ag at various concentrations. In some experiments, APC were preincubated or not with mAbs at 10 g/ml for 40 minutes at 4 C., then Ag was added to the cells in a 100 l final volume in the continuous presence of the mAbs. After a 4 hours pulse, APC were washed twice and put in coculture with B3Z. Purified mAbs used were against CD11b (M1/70 ratIgG2b, K) or isotype-matched control and originated from Pharmingen (San Diego, USA). ii) Ex vivo assay: APC originated from mice previously intravenously immunized with various Ags and were put in culture with B3Z in a 0.2 ml final volume at various numbers of APC per well.
(95) B.1.5 Antigen Presenting Cells and Sortings
(96) Total spleen cells (TSC), low density (LDF) and high density (HDF) fractions were prepared according to the protocol of Steinman modified by Vremec et al. (Vremec et al., 1992). Briefly, spleens were digested with collagenase for 40 minutes at 37 C. and then dilacerated and prepared in the continuous presence of EDTA 5 mM. Cells were centrifugated on a dense BSA solution. Supernatant and pellet cells were collected apart and termed low density and high density fractions. CD11c staining was performed at 4 C. in PBS supplemented with 5% of Fetal Calf Serum and 2 mM EDTA (PBS-FACS) with the hamster HL3 mAb coupled to phycoerythrin (PE), fluoresceine isothyocyanate (FITC) or biotinylated and then revealed by Streptavidine-PE. CD8a staining was performed with the 53-6.7 mAb coupled to PE. CD11b staining was performed with the M1/70 mAbs coupled to PE or FITC. CD54R staining was performed with the B220 mAb coupled to PE or biotinylated and revealed by streptavidine-PE. All mAbs originated from Pharmingen. After two washes, cells were sorted using a FACStar (Beckton Dickinson, Mountain View, USA). Cells were aseptically recovered in CM. Purity of the sorted cells was checked on an aliquot of the sorted cells analyzed on a FACScan apparatus (Beckton Dickinson, Mountain View, USA). Purity of the sorted cells was typically between 80 and 98%. In other experiments (as mentioned in figure legends), CD11c+ cells were sorted directly from spleen collagenase digests using the CD11c Micro Beads and the Magnetic Cell Sorting technology following the supplier protocole (MACS, MiltenyiBiotec, Bergish Gladbach, Germany). Purity of the sorted cells ranged around 80% with this technique.
(97) B.2 Results
(98) B.2.1 CD4- and CD40-Independent CTL Priming after Systemic Immunization with CyaAOVA in the Absence of Adjuvant
(99) The chicken ovalbumin, H-2K.sup.b restricted, SIINFEKL epitope was used as an experimental model epitope. It was genetically inserted in the catalytic domain of a detoxified, still invasive mutant CyaA. C57BL/6 (H-2.sup.b) mice were immunized iv once with 50 g of the recombinant toxin or control saline solution. Seven days after immunization, CTL activity specific for pOVA was detected within splenocytes of CyaAOVA-immunized C57BL/6 mice but not in mice injected with saline or a control CyaA (
(100) B.2.2 In Vitro and In Vivo Targeting of CyaAOVA Presentation to CD11b-Expressing Cells
(101) In order to better understand the immunogenicity of CyaAOVA, we intended to determine the APC involved in its presentation to CD8+ T cells. Using the IL-2 secretion as a readout for stimulation, we show that B3Z, an H-2K.sup.b-restricted, anti-OVA T cell hybridoma, is stimulated in vitro by bulk splenocytes in the presence of CyaAOVA but not CyaALCMV (
(102) To detect K.sup.b-OVA complexes formed in vivo after immunization, the APC prepared from mice iv immunized 8-15 hrs before with 50 g CyaAOVA were cocultured with B3Z in vitro without Ag addition (ex vivo assay). As for in vitro assays, APC responsible for CyaAOVA presentation were present exclusively in the DC-enriched low density fraction of splenocytes (
(103) To further characterize APC involved in CyaAOVA, we performed subfractionation of the splenic low density CD11c.sup.+ DC in CD11c.sup.+CD8.sup. myeloid subset and CD11c.sup.+CD8.sup.+ lymphoid subset. In vitro and ex vivo assays
(104) In vitro and ex vivo assays performed with splenocytes from control (C57BL/6) or B cell-deficient mice (IgM/) confirmed the poor contribution of B cells to CyaAOVA presentation in vitro and in vivo (
(105) B.2.3 MHCI-Restricted Presentation of CyaAOVA by Dendritic Cells Depends on the Cytosolic Delivery of the Recombinant Toxin Both in Vitro and In Vivo
(106) To determine whether CyaAOVA presentation depends on the cytosolic delivery of the OVA epitope or extracellular loading, we performed Ag presentation assays with total splenocytes or CD11c.sup.+ DC from TAP/ or control TAP+/+ splenic purified from naive animals (in vitro,
(107) B.2.4 Interaction of CyaA with CD11b is Required for Cell Binding and Delivery of the Inserted Antigen to the Cytosolic Pathway for Antigen Presentation by MHC I
(108) We showed on part A that saturable and efficient binding of CyaA WT to CD11b.sup.+ cells can be blocked specifically by anti-CD11b mAbs. Moreover, CD11b transfection specifically conferred saturable binding and sensitivity to CyaA WT to otherwise CD11b.sup. cells resistant to CyaA WT. Cell binding blockade by anti-CD11b mAbs inhibited subsequent intracellular delivery of the catalytic adenylate cyclase domain, cAMP elevation and cell death induced by CyaA WT. Since this results have been obtained on cell lines, it remained to determine if CyaA binds to splenocytes. We set up a flow cytometric assay to detect the fixation of a biotinylated, detoxified form of CyaA carrying the OVA peptide (CyaAOVAbiotine) to total splenocyte suspensions with streptavidin coupled to phycoerythrin. Using this assay, we observed that CyaAOVA binds to a subset of leukocytes within the total splenocyte suspension (5-7%). Preincubation with the anti-CD11b M1/70 mAb but not a control mAb abrogated this binding (
(109) In vitro, we showed that the anti-CD11b mAb M1/70 blocks the CyaAOVA presentation by TSC cells to B3Z (
(110) Taken together, these results clearly establish that the presentation of the OVA peptide from CyaOVA is dependent on cell binding and thus on interaction with CD11b.
(111) B.3 Discussion
(112) In the present study, using the detoxified adenylate cyclase of Bordetella pertussis as an epitope-delivery vector, we established a strategy for immunization that primes CTL responses after a single injection, bypassing the need for adjuvant requirement. We identified mechanisms that contribute to the high efficiency of detoxified CyaA as a vector:
(113) B.3.1 CyaA Target Myeloid Dendritic Cells Through its Interaction with the CD11b Integrin
(114) Antigen presenting assay to a specific CD8+ T cell hybridoma using in vitro- or in vivo-loaded APC (in vitro and ex vivo assay, respectively) demonstrated that the most efficient APC for CyaAOVA are CD11c+CD11b.sup.high+ DC. Indeed, all the Ag presenting ability for CyaAOVA belonged to the low density fraction of splenocytes that retains DC. Cell sorting of defined cell types revealed that CD11c.sup.+ CD11b.sup.high+ DC cells are much more efficient than CD11c.sup.CD11b.sup.high+ cells to present CyAOVA. The minor contribution observed for B cells (CD45R.sup.+) was confirmed by efficient presentation of CyaAOVA (in vitro and ex vivo) and CTL responses in B cell-deficient mice.
(115) B.3.2 CyaA Delivers Ag to the Cytosolic Pathway for MHC Class I Presentation In Vivo
(116) Here we show the dependence of CyaAOVA presentation to total splenocytes in vitro. Strikingly, we also show that in vivo presentation of CyaAOVA takes also place according a TAP-dependent pathway. This lead to the conclusion that CyaAOVA presentation in vivo resulted effectively from cytosolic delivery and not from an eventual extracellular degradation.
(117) B.3.3 CTL Priming Bypass CD4+ T Cell Help and is Independent on CD40 Signaling
(118) Maturation from an immature stage toward a mature stage is characterized by i) a decrease in Ag capture ability, ii) an increase in T cell priming ability, iii) a migration from Ag sampling sites (marginal zone in the spleen) toward T cell area (peri arteriolar sheets in the spleen) were they maximize the probability of encounter with Ag-specific T cells (De smedt et al., 1996). In addition to Ag presentation by DC, the maturation phase is now widely assumed as a prerequisite for T cell priming. In vitro studies have highlighted the role of CD4.sup.+ T cells in signalling DC maturation, notably via CD40L-CD40 interaction (Bell et al., 1999). In the case of CD8+ T cells priming after the cross priming of cellular Ag, CD4.sup.+ T cells dispensate their help to CD8+ T cells in a CD40-dependent mechanism (Schuurhuis et al., 2000; Bennett et al., 1998; Schoenberger et al., 1998; Ridge et al., 1998). Since CyaAOVA primes CTL in a CD4 and CD40 independent way, it is tempting to speculate that detoxified CyaA could be endowed of intrinsic adjuvanticity.
(119) B.3.4 Conclusion
(120) The present study represents the first characterization, to our knowledge, of a proteinaceous vaccine vector that fits both targeting to professional APC, cytosolic delivery of the vectorized Ag and adjuvant-free CTL priming. Moreover, we elucidated the mechanism of cell targeting by demonstrating that Ag presentation is dependent on the interaction between CyaA and CD11b, its receptor. Hence, the cellular specificity of CyaA is serendipituously adapted to the Ag delivery purpose. Finally, the cellular specificity of CyaA or other bacterial toxins may serve the cytosolic delivery of a wide set of pharmaceutically-relevant molecules whose effects should be targeted on a restricted set of cells.
C. Use of Adenylcyclase to Deliver Chemically Coupled Antigens to Dendritic Cells In Vivo
(121) C.1 Methods for Coupling Molecules of Interest to CyaA-Derived Vectors
(122) A general methodology is here described to create recombinant CyaA toxins by grafting molecules of interest to CyaA by means of disulfide bonds.
(123) As an illustration, a synthetic 12 amino-acid long peptide corresponding to a CD8.sup.+ T-cell epitope from ovalbumin was chemically crosslinked through a disulfide bond to a cysteine residue genetically introduced into the CyaA catalytic domain at position 235 (wild type CyaA has no cysteine residues). The expected advantages of this novel architecture are:
(124) (i) versatility: a single CyaA carrier protein can be easily coupled to any desired synthetic peptides;
(125) (ii) immunogenicity: upon delivery into the APC cytosol, the epitopes chemically coupled to CyaA should be released from the vector (due to the reducing intracellular conditions) and introduced directly into the MHC class I presentation pathway, thus bypassing the potentially limiting step of proteolytic processing by proteasome.
(126) The general procedure to couple synthetic peptides to CyaA by disulfide bonds is outlined in
(127) In a first step, a recombinant CyaA toxin that contains a single cysteine residue genetically inserted within the catalytic domain of CyaA (wild type CyaA has no cysteine residue) is produced.
(128) The recombinant CyaA toxin, ACTM235, has been previously characterized (Heveker and Ladant, 1997). In particular, ACTM235 harbors a Cys-Ser dipeptide inserted between amino-acid 235 and 236 and is fully cytotoxic. This toxin was expressed and purified to homogeneity as previously described in A.1.1.
(129) In a second step a synthetic peptide corresponding to a CD8.sup.+ T cell epitope from ovalbumin was designed: in addition to the SIINFEKL (one letter code for amino acid) sequence that is the precise epitope sequence, a cysteine residue with an activated Nitro-pyridin-sulfonyl thiol group (Cys-NPys) was added at the N-terminus of the peptide during chemical synthesis. The activated Npys-cysteine was separated from the SIINFEKL sequence by a flexible GGA motif to facilitate further proteolytic processing of the peptide within APC. The peptide (Cys-Npys-OVA, molecular weight: 1405 da) was synthesized by Neosystem (Strasbourg, France).
(130) In a third step the Cys-Npys-OVA peptide was coupled to ACTM235 using the following procedure.
(131) Ten mg of purified ACTM235 in 8 M urea, 20 mM Hepes-Na, pH 7.5, were reduced by incubation for 12 hrs in the presence of 10 mM Dithiothreitol (DTT). The efficiency of reduction was checked by an SDS-Page analysis under non reducing conditions: essentially all the ACTM235 protein migrated as a single band of 200 kDa corresponding to the monomeric species without evidence of any dimeric species (molecular weight of about 400 kDa). The reduced protein was then loaded on a DEAE-sepharose (Amersham Pharmacia Biotech) column (5 ml packed resin) equilibrated in 8 M urea, 20 mM Hepes-Na, pH 7.5. The ACTM235 protein was fully retained on the DEAE-sepharose resin that was then extensively washed with 8 M urea, 20 mM Hepes-Na, pH 7.5, 0.1 M NaCl (>100 ml) to remove any traces of DTT (the absence of residual DTT was checked using the classical Ellman's reaction with 5,5-Dithio-bis-(2-nitrobenzoic acid), DTNB). The reduced ACTM235 protein was then eluted from the DEAE-sepharose resin in 7 ml of 8 M urea, 20 mM Hepes-Na, pH 7.5, 0.5 M NaCl. Three mg (about 2 mol) of Cys-Npys-OVA peptide were then added to the reduced ACTM235 protein (8 mg, about 45 nmol) and the mixture was incubated for 16 hrs at room temperature. Then, 25 ml of 20 mM Hepes-Na, pH 7.5 and 8 ml of 5 M NaCl were added and this diluted mixture was then loaded on a Phenyl-Sepharose (Amersham Pharmacia Biotech) column (10 ml packed resin) equilibrated in 20 mM Hepes-Na, pH 7.5, 1 M NaCl. The Phenyl-Sepharose resin was washed with 50 ml of 20 mM Hepes-Na, pH 7.5, 1 M NaCl and then with 50 ml of 20 mM Hepes-Na, pH 7.5. The derivatized ACTM235 protein was then eluted in 8 M urea, 20 mM Hepes-Na, pH 7.5. The toxin concentration was determined spectrophotometrically from the absorption at 280 nm using a molecular extinction coefficient of 142,000 M.sup.1.Math.cm.sup.1.
(132) The Cys-Npys-OVA peptide was coupled using the same procedure to a second recombinant CyaA toxin, CyaAE5-LCMVgp, which is a detoxified variant (i.e. lacking the enzymatic activity as a result of the genetic insertion of 2 amino acid LQ between residues 188 and 189). This toxin also contains a 15 amino acid long polypeptide sequence (PASAKAVYNFATCGT) inserted between residues 224 and 225 of CyaA and that contains a single Cys residues. The plasmid encoding this recombinant toxin is a derivative of pCACT-ova-E5 (Guermonprez et al. 2000) modified by the insertion between the StuI and KpnI restriction sites of an appropriate synthetic double stranded oligonucleotide encoding the PASAKAVYNFATCGT sequence. The CyaAE5-LCMVgp protein was expressed and purified as described previously in A.1.1.
(133) The peptides shown in table 1 were also coupled similarly to another detoxified recombinant CyaA toxin, CyaAE5-CysOVA, which contains the same LQ dipeptide insertion in the catalytic site and a 14 amino acid sequence inserted between residues 224 and 225 of CyaA. This insert contains a Cys residue adjacent to the OVA epitope as shown in
(134) The CyaAE5-CysOVA can be considered as a general detoxified vector for chemical coupling of CTL epitopes by disulfide bridges. The presence of the OVA epitope within CyaAE5-CysOVA allows for an easy in vitro assay for functionality in epitope delivery by measuring the presentation of the OVA epitope to specific T-cell hybridoma as described previously in B.1.4.
(135) TABLE-US-00001 TABLE1 NPysCTLpeptidescoupledtoCyaAE5-CysOVA Nameof epitopes Amino-acidsequenceofpeptides CEA571 Cys(NPys)-GGYLSGANLNL Gp100 Cys(NPys)-GGITDQVPFSV MelanA Cys(NPys)-GGEAAGIGILTV Tyrosinase Cys(NPys)-GGYMDGTMSQV
(136) Alternatively, the thiol groups of recombinant CyaA toxins can be activated with 2,2-dithiodipyridine (Sigma) and derivatised with peptides containing a reduced cysteine (the procedure to reduce the Cys in synthetic peptides is provided by the manufacturer). This would be especially appropriate if the desired peptide contains an internal cysteine residue.
(137) C.2 Analysis of the In Vitro and In Vivo Immunogenicity of the OVA Epitope Chemically Coupled to CyaA
(138) In vitro delivery of the OVA epitope to MHC class I molecules by CyaA after chemical (CyaA-gp-S-S-OVA E5) or genetic coupling (CyaA-OVA E5) was first analysed by studying the presentation of these molecules to the anti-OVA B3Z CD8.sup.+ T cell hybridoma by splenocytes. 3.Math.10.sup.5 spleen cells from C57BL/6 mice were co-cultured for 18 h with 10.sup.5 B3Z cells in the presence of various concentrations of the recombinant CyaA. The IL-2 release by B3Z was measured in a CTLL proliferation assay.
(139) As shown on the
(140) In a second step, in vivo capacity of these molecules to induce OVA-specific CTL responses was then analysed. C57BL/6 mice (2 per group) were immunized by i.v. injection of 50 g of the various CyaA. Seven days later, 25.Math.10.sup.6 spleen cells of individual mice were restimulated with 0.1 g of OVA peptide in the presence of 25.Math.10.sup.6 irradiated syngenic spleen cells for five days. The cytotoxic activity of the effector cells was measured on 51 Cr-labeled EL4 target cells pulsed or not with 50 M of the OVA peptide.
(141) As shown in
(142) In conclusion, these results clearly demonstrate that a CD8.sup.+ T cell epitope chemically linked to CyaA-derived proteinaceous vectors is very efficiently delivered to the cytosolic pathway for MHC class I presentation and induces strong CTL response in vivo.
D. Identification of a CyaA-Derived Fragment that Binds to CD11b/CD18 and can be Used as Efficient Molecule Delivery Vectors
(143) As an attempt to map the region that is involved in the interaction between CyaA and the CD11b/CD18 receptor, various subfragments of CyaA were constructed and tested for their ability to compete with a biotinylated CyaA toxin for binding to CD11b/CD18 on the surface of transfected CHO cells, using methods previously described in paragraph A.
(144) D.1 Expression of CyaA373-1706
(145) CyaA373-1706 protein was produced in E. coli by using a novel expression vector pTRAC-373-1706 (
(146) The pTRAC-373-1706 also encodes the thermosensitive repressor CI.sup.857 that strongly represses gene transcription at the Pr promoter at temperatures below 32 C., the origin of replication of colE1 and the beta-lactamase gene that confers ampicillin resistance.
(147) Expression of the CyaA373-1706 protein was carried out in E. coli strain BLR. Cells transformed with pTRAC-373-1706 were grown at 30 C. in LB medium containing 150 mg/L of ampicillin until mid-log phase and then synthesis of CyaC and of the truncated CyaA was induced by increasing the growth temperature to 42 C. Bacteria were harvested after 3-4 hrs of further growth at 42 C. CyaA373-1706 protein was then purified as described for the wild type CyaA (Guermonprez et al. 2000). CyaA373-1706 is devoid of cAMP synthesizing activity, it exhibits hemolytic activity of sheep erythrocytes and contains also a unique cysteine residues in its MGCGN N-terminal sequence.
(148) D.2 Inhibition of CyaA-E5 Binding to CD11b by CyaA-E5 and CyaA Fragments: 1-383 (Catalytic Domain) and 373-1706 (Hydrophobic and Repeat Domains)
(149) As shown on
(150) Thus, these results clearly demonstrate that the fragment of CyaA that encompasses residues 373 to 1706, CyaA373-1706, contains the structures essentially required for the interaction of CyaA with the CD11b/CD18 receptor.
E. Conclusions
(151) Since MHC class I molecules usually present peptides derived from endogenously synthesized proteins, epitopes must be delivered to the cytosol of antigen presenting cells to elicit CTL responses. It has been previously established that the CTL priming activity of recombinant ACT protein relies at least in part on its ability to deliver CD8.sup.+ T cell epitopes into the cytosol of antigen-presenting cells (APC). In one approach, epitopes were genetically inserted within the catalytic domain of CyaA as it is known that, for the wild-type toxin, this part of the polypeptide reaches the cytosol of target cells where it exerts its toxic effect (i.e. cAMP synthesis). After entering the cells, the CyaA catalytic domain harboring the epitope insert must be proteolytically processed to release the matured CD8+ T cell epitope which then will be translocated to the endoplasmic reticulum to associate with MHC class I molecules.
(152) This proteolytic processing, carried out by the proteasome, might be a limiting step in numerous cases (Pamer and Cresswell, 1998; Cascio et al., 2001), leading to a significant decrease of the overall yield of the mature epitope. The alternative approach, described here, that consist in the linkage of the epitopes to the catalytic domain of CyaA by means of a disulfide bond, offers a significant advantage in that, with this design, matured epitopes (requiring only N-terminal triming) will be quantitatively released from CyaA inside the cytosol of APC. In addition, this should be a very versatile system as a single CyaA carrier protein could be easily and rapidly coupled to any desired synthetic peptide. Furthermore, it will be easy to introduce by genetic engineering, several cysteine residues within the catalytic domain (such as in previously mapped permissive sites, WO 93/21324 (INSTITUT PASTEUR)) of a recombinant detoxified CyaA toxin so that multiple peptides can be coupled on the same CyaA molecule. This should increase the overall epitope delivery and immunogenicity of the recombinant protein.
(153) Deletion mapping allowed to identify the C-terminal part of CyaA (aa 373-1706) as the region that is involved in the interaction of the toxin with its CD11b/CD18 receptor. The truncated protein CyaA373-1706 can be therefore used as a protein module to target CD11b.sup.+ cells in vivo. In particular, polypeptides or proteins corresponding to antigens of interest can be genetically fused to CyaA373-1706 to be delivered to dendritic cells in order to elicit specific immune responses. Similar coupling can be performed on the recombinant CyaA373-1706 protein that also contained a unique cysteine at its N terminal end.
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