US20040001867A1 - Vectors for molecule delivery to CD11b expressing cells - Google Patents

Vectors for molecule delivery to CD11b expressing cells Download PDF

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US20040001867A1
US20040001867A1 US10/387,486 US38748603A US2004001867A1 US 20040001867 A1 US20040001867 A1 US 20040001867A1 US 38748603 A US38748603 A US 38748603A US 2004001867 A1 US2004001867 A1 US 2004001867A1
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adenylcyclase
cells
cyaa
molecule
antigen
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Claude Leclerc
Pierre Guermonprez
Daniel Ladant
Nicole Guiso
Nadia Khelef
Cecile Bauche
Catherine Fayolle
Mohammed El-Idrissi
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Centre National de la Recherche Scientifique CNRS
Institut Pasteur
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Assigned to INSTITUT PASTEUR, CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE reassignment INSTITUT PASTEUR ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BAUCHE, CECILE, GUERMONPREZ, PIERRE, GUISO, NICOLE, LADANT, DANIEL, EL-AZAMI EL-IDRISSI, MOHAMMED, FAYOLLE, CATHERINE, KHELEF, NADIA, LECLERC, CLAUDE
Publication of US20040001867A1 publication Critical patent/US20040001867A1/en
Priority to US11/098,404 priority Critical patent/US20050238637A1/en
Priority to US13/169,605 priority patent/US9370564B2/en
Priority to US15/177,029 priority patent/US10004794B2/en
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    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12Y406/00—Phosphorus-oxygen lyases (4.6)
    • C12Y406/01—Phosphorus-oxygen lyases (4.6.1)
    • C12Y406/01001—Aodenylate cyclase (4.6.1.1)
    • A—HUMAN NECESSITIES
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    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • 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 a new vector for molecule delivery to CD11b expressing cells.
  • Bordetella pertussis the causative agent of whooping cough, secretes several toxins including the well-known pertussis toxin (PT) and the adenylate cyclase toxin (CyaA) or also adenylcyclase.
  • CyaA is a critical virulence factor of B. pertussis in the murine respiratory model that is required for the early steps of lung colonization. Indeed, genetic deletion of this toxin dramatically decreases the pathological effects of B.
  • CyaA is an antigen protective against B. pertussis infection in the murine respiratory model [Guiso et al., 1989; Guiso et al., 1991].
  • Adenylcyclase is encoded by the cyaA gene, and its expression, like that of other virulence genes of B. pertussis , is coordinately regulated by rucal signals.
  • the cyaA gene is part of an operon that also contains genes cya B, D and E, that are required for CyaA secretion [Ladant et al., 1999].
  • the CyaA toxin is a bifunctional protein of 1706 residues that is made of a N-terminal catalytic domain of 400 amino acids and a C-terminal part of 1306 residues which is responsible for the binding of the toxin to target cell membrane and the subsequent delivery of the catalytic moiety into the cell cytosol [Sakamoto et al., 1992] [Ladant et al., 1999]. This part also exhibits a weak hemolytic activity due to its ability to form cation-selective channels in biological membranes [Benz et al., 1994] [Gray et al., 1998].
  • This region is homologous to Escherichia coli hemolysin and other members of the RTX (Repeat in ToXin) family of bacterial toxins. In particular, it contains a series of glycine and aspartate-rich nonapeptide repeats that are involved in calcium binding [Rose et al., 1995] [Coote et al., 1992].
  • CyaA polypeptide is synthesized as an inactive protoxin that is converted to an active toxin by posttranslational palmitoylation of two internal lysines (lysines 856 and 963). This modification requires the product of an accessory gene, cyaC, which is located nearby cyaA on B. pertussis chromosome.
  • CyaA has been shown to bind to and invade a variety of cell types including cells lacking membrane traffic like mammalian erythrocytes [Rogel et al., 1992]. This suggested that the catalytic domain of CyaA is directly translocated across the plasma membrane of target cells. The internalization of the catalytic domain into the cell cytosol is calcium and temperature-dependent and depends upon the plasma membrane potential [Rogel et al., 1992] [Karimova et al., 1998] [Otero et al., 1995]. However, the molecular mechanisms by which the toxin transports its N-terminus catalytic domain across the membrane remain largely unknown to date. Furthermore no specific receptor has been reported for CyaA binding.
  • CyaA The physiological consequences of cellular intoxication by CyaA were characterized in vitro in phagocytes. Confer and Eaton first showed that the Cya A extracted from B. pertussis increases the intracellular cAMP level in neutrophils or macrophages leading to an inhibition of chemotaxis and bactericidal functions such as superoxide generation and phagocytic abilities [Confer et al., 1982]. These activities were later confirmed with purified toxins or with bacterial mutants genetically deleted of CyaA [Pearson et al., 1987; Friedman et al., 1987] [Njamkepo et al., 2000].
  • CyaA i) is mainly responsible for macrophage apoptosis, ii) might be responsible for neutrophil apoptosis, but that another factor may also be responsible.
  • CyaA has been considered for a long time, as independent of any receptor binding. This is based on the observations that i) CyaA can intoxicate 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]. However, some specificity has been found in respect of cells infected by CyaA.
  • dendritic cells In line with their surface phenotype, dendritic cells (high expression of MHCI and II, costimulatory and adhesion molecules) represent the most potent APC in many in vitro assay for the priming of naive T cells [Bell et al., 1999; Viola et al., 1999]. Other APC like resting naive B cells, for example, could even be tolerogenic since injection of resting, male B cells into female hosts leads to the specific tolerization of male-specific CD8+ T cells [Fuchs et al., 1992]. In vitro, naive B cells could delete naive CD8+ T cells via a Fas dependent-mechanism [Bennett et al., 1998].
  • dendritic cells can be directly transfected at the local site of injection and then migrate to the afferent LN via afferent lymphatics [Condon et al., 1996; Casares et al., 1997; Porgador et al., 1998]. The migration is known as a key event of immunity since mechanical disruption of afferent lymphatics abrogates T cell response to skin sensitizers or skin grafts [Zinkemagel et al., 1997].
  • Applicants have been interested in studying the presentation of adenylcyclase of Bordetella species by T cell, and have identified a specific receptor molecule present on specific cells, that interacts with CyaA and opens new possibilities for the use of CyaA as a proteinaceous vector for molecules of interest.
  • the invention provides means that may at least in part, fulfil these needs and proposes new vectors that would specifically target molecules to determined populations of pAPC for example, to enable stimulating immune response.
  • molecule targeted to the pAPC and specific leukocytes would enable the manufacturing of new vectors useful to deliver biologically active molecule to the proximal environment of these cells. These molecules, for example, could modulate the functional properties of the targeted cells or those involved in the immune response or in the inflammatory response.
  • ⁇ M / ⁇ 2 (CD11b/CD18) integrin is a dimer of the ⁇ 2 integrin family, the expression of these integrins being restricted to leukocytes.
  • CD11b/CD18 ⁇ M / ⁇ 2 displays a pattern of expression in mouse and human, which is restricted to neutrophils/granulocytes, macrophages, dendritic cells, NK cells and subsets of B and T CD8+ lymphocytes (Jeyaseelan et al., 2000, Amaout et al., 1990).
  • this receptor would represent an ideal target for new vectors, designed in particular for T epitope immunization.
  • Bordetella adenylcyclase can be used to target a molecule in vivo specifically to CD11b expressing cells.
  • a peptide antigen comprised in the Bordetella pertussis adenylcyclase toxin can efficiently be targeted specifically to the surface of dendritic cells, translocated in the cytosol of said dendritic cells and prime a CTL response.
  • said response is obtained bypassing adjuvant requirement and CD4+ T-cell help.
  • genetically modified adenylcylase can be chemically coupled to a peptide of interest to target said peptide to CD11b expressing cells, especially the cytosol of dendritic cells.
  • This invention thus provides new efficient immunogenic composition as well as new drug delivery vector to CD11b expressing cells.
  • This invention relates to the use of Bordetella adenylcyclase in the manufacturing of a proteinaceous vector for targeting a molecule of interest specifically to CD11b expressing cells.
  • the invention also relates to the use of a Bordetella adenylcyclase wherein said adenylcyclase is recombined with an antigen and especially modified by insertion of a peptide of interest or modified by insertion of a molecule of interest for the preparation of a composition for the targeting of said peptide or molecule specifically to CD11b expressing cells.
  • adenylcyclase when used as a vector for a molecule of interest, is preferentially directed to CD11b expressing cells, thereby offering means to target the molecule of interest at the surface of said cells or within said cells in a selective way with respect to other cells.
  • the molecule of interest is essentially directed to CD11b expressing cells.
  • CD11b expressing cells relates to the cells that express the CD11b/CD18 ⁇ m ⁇ 2 receptor on their surface.
  • these cells are granulocytes/neutrophils, macrophages, NK cells, subsets of T CD8+ and B cells and myeloid dendritic cells.
  • the invention relates to the manufacturing of a proteinaceous vector or a composition capable of targeting a molecule or a peptide of interest to these CD11b expressing cells especially to myeloid dendritic cells, neutrophils or macrophages.
  • the targeting of said molecule or peptide is effective in vivo.
  • the invention thereby provides means appropriate for the design of compositions suitable for administration to animal or human hosts requiring targeting of certain leukocytes and in particular myeloid dendritic cells, neutrophils or macrophages.
  • Bordetella adenylcyclase is the calmodulin-dependent adenylcyclase secreted in Bordetella species or fragment thereof, said fragment retaining the function properties of the adenylcyclase, a major virulence factor mandatory for the initial phases of bacterial colonization in the lung.
  • the adenylcyclase is synthesized and secreted in the form of a polypeptide of 1706 amino acids:
  • the calmodulin-dependent catalytic activity is localized in the first 400 amino acids.
  • said adenylcyclase toxin is rendered invasive and hemolytic when post-traductionally modified by the coexpression of the cyaC gene product.
  • Bordetella adenylcyclase toxin indicate that this toxin can be used in the manufacturing of a proteinaceous vector for targeting in vivo a molecule of interest to CD11b expressing cells:
  • this adenylcyclase binds specifically to CD11b expressing cells
  • the N-terminal catalytic domain is translocated into the cytosol of those CD11b expressing cells.
  • d) epitope chemically coupled to genetically modified adenylcyclase can elicit in vivo specific CTL responses.
  • adenylcyclase encompasses, within the present invention, natural or modified adenylcyclase, including genetically or chemically modified adenylcyclase, providing the resulting product is able to target a molecule of interest specifically to CD11b expressing cells.
  • the invention thus relates to the use of Bordetella adenylcyclase as defined above and more particularly to the use of a modified or recombinant, adenylcyclase for targeting a molecule of interest specifically to CD11b expressing cells.
  • recombinant adenylclases include adenylcyclases which have been genetically modified to provide either adenyclases with peptide sequence or cysteine residues inserted within the catalytic domain, or truncated adenylcyclases lacking all or part of their catalytic domain.
  • the molecule of interest is specifically targeted at least to the surface of CD11b expressing cells.
  • the Bordetella adenylcyclase toxin is used in the manufacturing of a proteinaceous vector to deliver the molecule of interest either in the cytosol of CD11b expressing cells, at the surface of CD11b expressing cells or into the endocytic pathway of CD11b expressing cells.
  • Expression vectors for the preparation of recombinant Bordetella adenylcyclase are described in patent application WO 93/21324 (Institut Pasteur). Novel expression vectors for the preparation of genetically modified Bordetella adenylcyclase appropriate for chemical coupling of a peptide of interest are also described in the experimental part hereafter. More specifically, expression vectors may be constructed directing the expression of both the cyaA gene and the cyaC gene (Sebo et al., 1991). In parallel, a secondary plasmid carrying genes necessary for the secretion of the cytotoxic adenylcyclase in E.
  • coli such as hlyB and hlyD as described for example in Meckman et al., 1985 can be constructed.
  • the expression plasmid pCACT3 described in WO 93121324 can be used.
  • the adenyclyclase may be expressed in E. coli and possibly secreted by this bacterium in large amounts. It is also readily purified for example using affinity chromatography on CaM Affi-Gel resin or other published procedures, as those using DEAE-sepharose and phenyl-sepharose (Guermonprez et al., 2000).
  • the adenylcyclase is a recombinant genetically modified adenylcyclase.
  • mutations such as point mutations, deletions or insertions can be obtained using usual site-directed or random mutagenesis techniques, provided that the domains necessary for binding to CD11b expressing cells and, optionnaly for translocation in the cytosol are still functional.
  • Assays to evaluate specific binding of recombinant toxins and fragments thereof, to CD11b expressing cells and optionnaly subsequent translocation of the catalytic domain are described in the following experimental part.
  • the recombinant Bordetella species adenylcyclase is a fragment of the native, modified or recombinant Bordetella species adenylcyclase toxin, wherein said fragment is capable of binding the CD11b receptor.
  • fragment encompassing residues 373 to 1706 contains the structures essentially required for the interaction with the CD11b/CD18 receptor.
  • a preferred fragment of the Bordetella species adenylcyclase toxin is the Bordetella adenylcyclase toxin lacking all or part of the N-terminal catalytic domain, and more specifically Bordetella pertussis adenylcyclase lacking all or part of residues 1-373.
  • CD11b Specific binding to CD11b can be assessed in vitro with anti-CD11b monoclonal antibodies as illustrated in the examples.
  • the adenylcyclase is preferably non toxic.
  • Non toxic mutants of the adenylcyclase toxin are well described in the Art. (Betsou et al., 1993; Betsou et al., 1995.
  • the adenylcyclase is isolated from Bordetella pertussis.
  • the molecule of interest is selected in the group comprising: peptides, glycopeptides, lipopeptides, polysaccharides, oligosaccharides, nucleic acids, lipids and chemicals.
  • a molecule of interest is a heterologous antigen.
  • heterologous refers to an antigen other than the adenylcyclase which is used in the vector itself.
  • the manufacturing of the proteinaceous vector comprises the step of inserting a heterologous molecule and especially a peptide in the catalytic domain of the adenylcyclase at a permissive site.
  • the term “permissive site” relates to a site where the heterologous molecule and especially a peptide can be inserted without substantially affecting the desired functional properties of the adenylcyclase toxin, i.e. without affecting the domains necessary for the specific binding to CD11b/CD18 receptor and advantageously without affecting the process of translocation of the catalytic domain.
  • the capacity of the CyaA toxin to promote the synthesis of CAMP in the targeted cells is further maintained.
  • a permissive site is selected from the group consisting of residues 137-138, residues 224-225, residues 228-229, residues 235-236 residues 317-318 and residues 335-336 of the Bordetella pertussis adenylcyclase.
  • the manufacturing of the proteinaceous vector can also comprise a step of fusing a molecule of interest, for example a heterologous peptide, at the N-terminal extremity of a Bordetella adenylcyclase lacking all or part of its N-terminal catalytic domain, and more preferably, Bordetella pertussis adenylcyclase lacking residues 1-373.
  • a molecule of interest for example a heterologous peptide
  • the adenylcyclase according to one of the above definitions is used in the manufacturing of a proteinaceous vector or in the preparation of a composition specifically designed to prime CD8+ cytoxic T-cell response (CTL response) said response follows the targeting of the adenylcyclase modified (especially recombined or conjugated) with a molecule of interest to CD11b expressing cells, followed by the translocation of the molecule of interest to the cytosol of said CD11b expressing cells, and in particular to myeloid dendritic cells.
  • the molecule of interest is or comprises preferably an epitope or an antigen.
  • epitope refers to a heterologous molecule and especially a heterologous peptide that can induce an immune response.
  • the antigen is selected from the group consisting of an intracellular bacterial cell antigen, a tumoral cell antigen, a viral antigen, a fungus antigen or a parasite cell antigen.
  • the adenylcydase i.e., a natural, modified or recombinant adenylcyclase according to one of the above definitions is used in the manufacturing of the proteinaceous vector or in the preparation of a composition specifically designed to prime CD4+ cells response said response follows the targeting of the adenylcyclase modified (especially recombined or conjugated) with a molecule of interest to CD11b expressing cells, in particular myeloid dendritic cells.
  • the molecule of interest is or comprises preferably an epitope or an antigen.
  • a molecule of interest can be especially an antigen selected from the group consisting of: a poliovirus antigen, an HIV virus antigen, an influenza virus antigen, a choriomeningitis virus epitope, a tumor antigen.
  • the functional properties of the CD11b expressing cells define furthermore a novel use of the Bordetella adenylcyclase toxin in the manufacturing of a proteinaceous vector for drug targeting to these specific cells.
  • the so-called molecule of interest is a drug Said drug may be chemically or genetically coupled to the adenylcyclase.
  • Method for coupling a drug to a polypeptide are well known in the Art and comprise for example disulfide linkage by using N-pyridyl sulfonyl-activated sulfhydryl.
  • a molecule of interest is an anti-inflammatory drug which is, when coupled to the adenylcyclase toxin, specifically targeted to the surface of the cells involved of the inflammatory response, such as neutrophils.
  • antigen presentation for selective CD8+cytotoxic cells priming is mainly performed by myeloid dendritic cells.
  • the recombinant adenylcyclase used for the manufacturing of proteinaceous vector is a genetically modified adenylcyclase containing one or more molecule(s) chemically coupled by means of a disulfide bond to genetically inserted cysteine residue(s) located within the catalytic domain of said adenylcyclase.
  • multiple molecules can be chemically coupled to the adenylcyclase by means of a disulfide bond to different cysteine residues located at different permissive sites within the catalytic domain.
  • CTL specific for the vectorized antigen can be primed in vivo after a single intravenous injection of the recombinant toxin, especially with no need to provide an heterologous adjuvant.
  • the invention also relates to the use of a Bordetella adenylcyclase toxin recombined with a molecule and especially a peptide of interest for the preparation of a composition formulated for intravenous administration and enabling a CD8+ T cell immune response in vivo, said composition being free of a heterologous adjuvant.
  • the invention also concerns this composition as such.
  • the invention in particular also relates to a new immunogenic composition formulated for administration, especially intravenous administration, in an animal or human host, characterized in that it comprises a recombinant Bordetella adenylcyclase which comprises an antigen inserted in the catalytic domain.
  • the invention further relates to a pharmaceutical composition for administration in a human or an animal formulated for targeting a molecule of interest specifically to CD11b expressing cells characterized in that said molecule of interest is coupled to a Bordetella species adenylcyclase.
  • the molecule of interest is selected in the group comprising: peptides, glycopeptides, lipopeptides, polysaccharides, oligosaccharides, nucleic acids, lipids and chemicals.
  • the molecule of interest is an antigen.
  • the pharmaceutical or immunogenic composition comprises a nucleic acid construction encoding the recombinant Bordetella species adenylcyclase comprising a recombinant Bordetella species adenylcyclase coupled to a molecule of interest.
  • the adenylcyclase is from Bordetella pertussis toxin.
  • the adenylcyclase toxin is a genetically modified toxin.
  • the adenylcyclase is a non toxic adenylcyclase, especially a detoxified adenylcyclase.
  • the genetically modified adenylcyclase is able to translocate the molecule of interest specifically in the cytosol of CD11b expressing cells.
  • said genetically modified adenylcyclase is a Bordetella adenylcyclase lacking all or part of its catalytic N-terminal domain, and more specifically Bordetella pertussis adenylcyclase lacking residues 1-373.
  • the genetically modified adenylcyclase comprises one or more cysteine residues inserted within the catalytic domain at permissive sites.
  • Such genetically modified adenylcyclase can be coupled to one or more molecule(s) of interest by means of disulfide bond(s) at the inserted cysteine residue(s).
  • the molecule, and especially an antigen is inserted in the permissive sites selected from the group consisting of residues 137-138, residues 224-225, residues 228-229, residues 235-236 and residues 317-318, and residues 335-336 of the Bordetella pertussis adenylcyclase, or the molecule is fused to the N-terminal part of a Bordetella adenylcyclase lacking all or part of its N-terminal catalytic domain, and more specifically to the N-terminal part of a Bordetella pertussis adenylcyclase lacking residues 1-373.
  • the molecule is an antigen which is an intracellular bacterial cell antigen, a tumoral cell antigen, a viral antigen, a fungus antigen or a parasite cell antigen.
  • a molecule of interest is an antigen selected from the group consisting of: a poliovirus epitope, an HIV virus, an influenza virus antigen, a choriomeningitis virus antigen, a tumor antigen.
  • the genetically modified toxin is able to deliver the molecule of interest specifically at the surface of CD11b expressing cells or into the endocytic pathway.
  • the immunogenic compositions of the invention are capable of inducing or stimulating, in vivo or in vitro an immune cell response involving specifically dendritic cells.
  • the immunogenic or pharmaceutical composition is advantageously devoid of priming adjuvants commonly used in the Art, such as aluminium hydroxide.
  • the molecule of interest is a drug, preferably an anti-inflammatory drug.
  • the invention also relates to the use of the immunogenic composition as defined above for the preparation of a vaccine or an immunotherapeutic composition, for administration to an animal or human host.
  • immunotherapeutic composition relates to a composition which leads to an immunological response and which is associated to therapeutic treatments, such as treatment against cancers, viral infections, parasites infections or bacterial infections.
  • the invention further relates to a method to immunize an animal or human host, wherein said method comprises the steps of:
  • the invention finally relates to a proteinaceous vector for delivery of a molecule of interest, specifically to CD11b expressing cells, characterized in that said vector comprises a Bordetella species adenylclyclase and more preferably a recombinant or modified Bordetella species adenylcyclase coupled to said molecule of interest.
  • the proteinaceous vector is able to target a molecule of interest to CD11b expressing cells via the specific binding of Bordetella adenylcyclase with CD11b/CD18 ⁇ m ⁇ 2 which is present on the surface of specific cells.
  • the vector is also able to deliver the molecule of interest specifically in the cytosol of CD11b expressing cells.
  • the proteinaceous vector is able to target a molecule of interest to dendritic cells, particularly myeloid dendritic cells, or to neutrophils.
  • the molecule of interest is chemically or genetically coupled to the adenylclase, more preferably the recombinant adenylcyclase toxin.
  • Method for coupling a molecule to a polypeptide are well known in the Art.
  • the inventors showed that a biotin derivative, biotin HDPD can be selectively coupled on an unique cysteine residue genetically inserted within the catalytic domain.
  • Synthetic peptides have been coupled similarly to the cysteine containing adenylcyclase toxin, or to genetically modified adenylcyclase devoid of native cysteine residue but containing an unique genetically inserted cysteine residue.
  • a molecule of interest coupled to the adenylcyclase is an epitope chemically coupled to a genetically modified adenylcyclase devoid of native cysteine residue but containing genetically inserted cysteine residue(s) at a permissive site within its catalytic domain.
  • the proteinaceous vector comprises a genetically modified adenylcyclase.
  • the adenylcyclase is a recombinant non toxic adenylcyclase from Bordetella pertussis.
  • the invention more specifically relates to a proteinaceous vector which consists of a recombinant Bordetella adenylcyclase lacking all or part of the N-terminal catalytic domain, and more preferably a Bordetella pertussis lacking residues 1-373.
  • Another preferred proteinaceous vector of the invention is a genetically modified Bordetella adenylcyclase devoid of native cysteine residue, but containing genetically inserted cysteine residue(s) at a permissive site within the catalytic domain.
  • a drug to be delivered is an anti-inflammatory drug which is, when coupled to the adenylcyclase toxin, targeted to the surface or to the cytosol of the cells involved of the inflammatory response, such as neutrophils.
  • part A experimental data are shown that reveal the specific binding of adenylcyclase toxin to CD11b/CD18 receptor, and in particular the specific binding of adenycyclase toxin to CD11b expressing cells in vitro.
  • part B the experimental results show the possibility to target a genetically coupled molecule, more particularly an antigen in vitro and in vivo specifically to the cytosol of CD11b expressing cells, and in particular to myeloid dendritic cells.
  • the results show that the targeting is mediated by the CD11b receptor and that CTL priming is observed after systemic immunization in the absence of adjuvant.
  • FIG. 1 Saturable binding of CyaA correlates with CD11b expression
  • 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.
  • b, c, d, e Surface expression of ⁇ 2 integrins on J774A.1, LB27.4 and EL4 cells.
  • FIG. 2 CyaA binding to murine cell lines is blocked by anti-CD11b mAb.
  • 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 (O) or with M1/70 anti-CD11b mAb ( ⁇ ) and then incubated with CyaA with or without M1/70 anti-CD11b mAb.
  • 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.
  • FIG. 3 CyaA binding to the human neutrophils is blocked by anti-CD11b and anti-CD18 mAbs.
  • 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 ploted against log of PE fluorescence.
  • FIG. 4 Intracellular cAMP increase and cell death mediated by CyaA are specifically blocked by an anti-CD11b mAb in J774A.1 cells.
  • 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.
  • 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, M1/70, 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 96TM assay.
  • FIG. 5 CHO cells bind CyaA and become sensitive to CyaA when transfected with CD11b, but not with CD11c
  • a, b CyaA binding at the surface of CHO transfectants.
  • CHO cells transfected with human CD11b/CD18 ( ⁇ ), human CD11c/CD18 ( ) or mock-transfected ( ) were incubated with various doses of CyaA for 20 min at 37° C. (a) or 4° C. (b).
  • c Intracellular cAMP accumulation in CHO transfectants. CHO cells transfected with human CD11b/CD18 (O), 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.
  • 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 96TM assay.
  • FIG. 6 Intravenous immunization with CyaAOVA primes anti-OVA CTL responses in a B cell, CD4 and CD40-independent way.
  • FIG. 7 Identification of splenic antigen presenting cells involved in CyaAOVA presentation in vitro or in situ, after intravenous immunization.
  • Dendritic cells (CD11c + ) are more efficient APC for CyaAOVA than the CD11b high+ CD11c ⁇ cells or B cells (CD45R + ) (c,d):
  • Ex vivo assay (d) Cells sorted by flow cytometry from C57BL/6 mice previously (6-12 hours) immunized with 50 ⁇ g of CyaAOVA were used as APC. CD11c+( ⁇ ), CD11b high+ CD11c ⁇ ( ), CD45R + ( ⁇ ) 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.
  • the CD8 ⁇ ⁇ myeloid dendritic cell subset is a more efficient APC for CyaA than the CD8 ⁇ + lymphoid dendritic cell subset (e, f):
  • CD11c+low density cells from naive mice (e) or mice previously (6-12 hours) immunized iv with 50 ⁇ g CyaAOVA (f) were fractionated in myeloid dendritic cells (CD11c + CD8 ⁇ ⁇ , ⁇ ) and lymphoid dendritic cells (CD11c+CD8 ⁇ + , ) by flow cytometry and used as APC in in vitro (e) and ex vivo (f) assays for B3Z stimulation. IL-2 was assessed as above.
  • TSC TSC
  • LDF LDF
  • HDF ⁇ ,
  • ⁇ , ⁇ B cell deficient mice
  • mice ⁇ , ⁇ , ⁇
  • mice B cell deficient mice
  • mice ⁇ , ⁇
  • mice B cell deficient mice
  • mice were used as APC in an in vitro assay (g, ⁇ , ⁇ ) or an ex vivo assay (h, ⁇ , ⁇ , ⁇ , , , ) for B3Z stimulation as in a.
  • Mice were either from naive (g) or previously (1.5 hours) immunized iv with 50 ⁇ g CyaAOVA (h).
  • IL-2 was assessed as above.
  • FIG. 8 The presentation of CyaAOVA by dendritic cells requires the TAP transporters in vitro and in vivo after intravenous immunization.
  • FIG. 9 Role of ⁇ M ⁇ 2 integrin (CD11b) in CyaAOVA binding to cells.
  • Binding of CyaAOVA-biotine to TSC is blocked by anti-CD11b (a): TSC suspensions were incubated at 4° C. with 10 ⁇ g/ml of the anti-CD11b M1/7O mAb or an isotype control mAb or nothing. Then, CyaAOVA-biotine at 2 ⁇ g/ml (left pannels) or various concentrations (right pannel) 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 ploted against the Strep-PE fluorescence.
  • the percentage of leukocytes positive for CyaAOVA-biotine was ploted against CyaAOVA-biotine concentration during the staining.
  • Binding of CyaAOVA-biotine to low density cells correlates with the expression of CD11b (b): LDF were triple stained for CD11c, CD8 a 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.
  • FIG. 10 Role of ⁇ M ⁇ 2 integrin (CD11b) in CyaAOVA presentation by MHC I
  • Ex vivo antigen presenting assay with TSC or CD11b + and CD11b ⁇ fractions C57BL/6 mice were intravenously immunized with 50 ⁇ g of CyaAOVA (c) or 10 ⁇ g of pOVA (d).
  • CD11b + ( ⁇ ) and CD11b ⁇ ( ) 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 ploted against the numbers of APC from immunized animals present in each well.
  • FIG. 11 Summary of the methodology for chemical coupling of epitopes to recombinant CyaA through disulfide bond.
  • FIG. 12 A diagram of a vector for chemical coupling of CTL epitopes
  • FIG. 13 A graph showing IL-2 release by B3Z measured in a CTLL proliferation assay.
  • FIG. 14 A graph showing cytotoxic activity measured on 51 Cr-labelled EL4 target cells pulsed (A) or not (B) with 50 ⁇ M of the OVA peptide.
  • 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 51Cr-labelled target cells.
  • FIG. 14 shows the in vivo capacity of the proteinaceous vectors of the invention to induce OVA-specific CTL responses.
  • FIG. 15 Inhibition of CyaA binding to CD11b by CyaA-E5 and CyaA fragments
  • 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).
  • FIG. 16 Schematic representation of pTRAC-373-1706 expression vector.
  • the large arrows represent the open reading frames of ⁇ -lactamase (bla), the thermosensitive repressor cl 857 of phage lambda ( ⁇ cl 857 ), the cyaC gene and truncated ‘cyaA gene (the arrows are pointing to the direction of translation of the corresponding genes).
  • the ColE1 origin (Ori)
  • the Pr promoter ( ⁇ Pr)
  • the intergenic region between the cyaC and truncated ‘cyaA genes is detailed in the lower part.
  • 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, CACTE5Cys-Ova, harboring a unique cysteine inserted within the genetically inactivated catalytic domain was constructed by inserting an appropriate double strand oligonucleotide between the BsiwI and KpnI sites of pCACT-Ova-E5 [Guermonprez et al, 2000].
  • 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′-pyridyidithio) propionamide (Biotin-HPDP, PIERCE) according to the manufacturer's 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 ⁇ 1 ⁇ cm ⁇ 1 (binding studies) or using the Biorad protein assay system (CAMP accumulation and cell death studies).
  • 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. Sera were precipitated from immune serum by ammonium sulfate (33%).
  • 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 5 ⁇ 10 ⁇ 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].
  • All binding assays were performed in DMEM 4.5 mg/ml glucose (Life Technologies) without serum in 96 well culture plates (Costar). 2 ⁇ 10 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 pi at 4° C. Then, plates were centrifuged at 1500 rpm for 5 min and supernatants were removed. Cells were incubated at 4° C.
  • biotinylated anti-CyaA rabbit polyclonal antibodies ⁇ fraction (1/400) ⁇ in DMEM, 50 ⁇ l/well
  • a control (non-immune or pre-immune) rabbit serum as a saturating agent (1/50).
  • 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. 5 ⁇ 10 5 cells were preincubated in 96 well plates in 100 pi/well with or without 10 ⁇ g/ml of specific mAbs at 4° C.
  • Microtiterplates were coated with CAMP-BSA conjugates diluted at ⁇ fraction (1/4.000) ⁇ in Na 2 CO 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 ⁇ fraction (1/2.500) ⁇ in PBS-BSA 2% and incubated at 37° C. for 3 h.
  • CyaA cellular specificity toward a population of leukocytes we choose three representative murine cell lines expressing various combination of ⁇ 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.
  • A.2.2 CyaA Saturable Binding is Specifically Blocked by Anti-CD11b mAbs
  • a second anti-CD11b mAb (clone 5c6) also inhibited binding of CyaA (FIGS. 2 c,d ). Similar results were obtained with FSDC, an immature dendritic cell line expressing CD11b (FIGS. 2 a, c ), and the J774A.1 macrophage cell line (FIGS. 2 b, d ).
  • 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 (FIG. 3). Preincubation of neutrophils with the 44 or M1/70 anti-CD11b mAbs led to, a respectively complete or partial inhibition of the binding of CyaA (FIG.
  • A.2.3 CyaA-Mediated cAMP Increase and Toxicity are Specifically Blocked by an Anti-CD11b mAb
  • FIG. 4 b shows that anti-CD11b mAb J774A.1 dramatically reduced the cell death induced by CyaA (88% inhibition). The cell death induced by CyaA was unaffected when J774A.1 were preincubated with mAbs that did not block toxin binding to cells (anti-CD11a, CD11c or CD18 or a control mAb).
  • CyaA has been considered for a long time, as independent of any receptor binding. This is based on the observations that i) 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.
  • 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.
  • CyaA 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.
  • actinomycetemcomitans and P. haemolytica leukotoxin 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].
  • CyaA recognizes another ⁇ 2 integrin (CD11b/CD18) whose cellular distribution is different.
  • 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.
  • the pOVA synthetique peptide originated from NEOSYSTEM and was diluted in PBS at 1 mg/ml.
  • splenocytes were restimulated in 10 ml CM (see below) with irradiated spleen cells (2.5 ⁇ 10 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).
  • 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 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.
  • APC were washed twice and put in coculture with B3Z.
  • Purified mAbs used were against CD11b (M1/70 ratlgG2b, ⁇ ) or isotype-matched control and originated from Pharmingen (San Diego, USA).
  • mAbs used were against CD11b (M1/70 ratlgG2b, ⁇ ) or isotype-matched control and originated from Pharmingen (San Diego, USA).
  • TSC total spleen cells
  • LDF low density
  • HDF high density fractions
  • TSC total spleen cells
  • 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 appart and termed low density and high density fractions.
  • CD11c staining was performed at 4° C.
  • the chicken ovalbumin, H-2K 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 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 (FIG. 6 a ).
  • Contaminant LPS possibly acting as adjuvants, are not involved in the stimulation of CTL responses CyaAOVA since C57BL10ScSn and the LPS-hyporesponsive mice C57BL10ScCr displayed similar OVA-specific-CTL response after CyaAOVA injection (not shown).
  • results shown in FIG. 7 d show that DC (CD11c + ) fraction was the more efficient APC.
  • the macrophage/granulocyte (CD11b high+ CD11c ⁇ ) and the B cells (CD45R + ) sorted from the same fraction were very inefficient for the stimulation of B3Z.
  • CD11c + CD8 ⁇ + lymphoid subset presented equally well the pOVA synthetic peptide to B3Z.
  • CyaAOVA binds efficiently to CD11c + CD8 ⁇ ⁇ that expresses high levels of CD11b, less efficiently to CD11c + CD8 ⁇ + that expresses low levels of CD11b and very weakly to CD11c ⁇ CD8 ⁇ + T cells that do not express CD11b (FIG. 9 b ). It is noticeable that CyaOVA binds efficiently to a low percentage of CD11c ⁇ CD8 ⁇ ⁇ cells in correlation with the presence of CD11b high+ into this CD11c ⁇ population. Thus, CyaOVA biotine binding is mediated by CD11b (as for CyaA WT) and predicts the ability of a given cell type to present CyaOVA.
  • 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).
  • the maturation phase is now widely assumed as a prerequisite for T cell priming.
  • In vitro studies have highlighted the role of CD4 + T cells in signalling DC maturation, notably via CD40L-CD40 interaction (Bell et al., 1999).
  • CD8+ T cells priming after the crosspriming of cellular Ag
  • CD4 + 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 plausible to speculate that detoxified CyaA could be endowed of intrinsic adjuvanticity.
  • a recombinant CyaA toxin that contains a single cysteine residue genetically inserted within the catalytic domain of CyaA is produced.
  • ACTM235 The recombinant CyaA toxin, ACTM235, has been previously characterized (Heveker and Ladant, 1997).
  • 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.
  • a synthetic peptide corresponding to a CD8 + 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).
  • 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 ElIman'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.
  • 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 ⁇ 1 .cm ⁇ 1 .
  • 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.
  • PASAKAVYNFATCGT 15 amino acid long polypeptide sequence
  • the plasmid encoding this recombinant toxin is a derivative of pCACT-ova-E5 (Guermonprez et al.
  • 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 FIG. 12.
  • the plasmid encoding this recombinant toxin is a derivative of pCACT-ova-E5 modified by the insertion between the BsiWI and KpnI restriction sites of an appropriate synthetic double stranded oligonucleotide encoding the ASCGSIINFEKLGT sequence.
  • the CyaAE5-CysOVA protein was expressed and purified as described previously in A.1.1.
  • 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.
  • NPys CTL peptides coupled to CyaAE5-CysOVA Name of epitopes Amino-acid sequence of peptides CEA 571 Cys(NPys)-GGYLSGANLNL Gp100 Cys(NPys)-GGITDQVPFSV MelanA Cys(NPys)-GGEAAGIGILTV Tyrosinase Cys(NPys)-GGYMDGTMSQV
  • 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.
  • the cytotoxic activity of the effector cells was measured on 51 Cr-labeled EL4 target cells pulsed or not with 50 UM of the OVA peptide.
  • CyaA373-1706 protein was produced in E. coli by using a novel expression vector pTRAC-373-1706 (FIG. 16). It is a derivative of plasmid pDL1312 (Ladant 1995), constructed by replacing the neurocalcin gene of plasmid pDLi312 by the cyaC gene (that encodes CyaC which is involved in the conversion of proCyaA into the active toxin by post-translational palmitoylation of Lys 860 and 983 of CyaA) and the 3′ part of the cyaA gene that encodes the C-terminal domain—codons 373 to 1706 of CyaA (see FIG. 16).
  • Both cyaC and ‘cyaA genes are placed in the same transcriptional unit under the control of the A phage Pr promoter.
  • the 3′ end of the cyaC gene was modified to introduce before its stop codon, a ribosome binding site to enhance the translation initiation of the downstream ‘cyaA gene (FIG. 16).
  • the resulting modification of the CyaC polypeptide (the last 3 amino acid Gly-Thr-Ala at the C-terminus of CyaC were replaced by the Asn-Arg-Glu-Glu sequence) had no effect on its ability to acylate CyaA.
  • the 5′ end of the cyaA gene (coding for the catalytic domain of the toxin (upstream of the unique BstBI site of cyaA) was deleted and replaced by an appropriate synthetic double stranded oligonucleotide encoding the MGCGN sequence (FIG. 16).
  • the pTRAC-373-1706 also encodes the thermosensitive A repressor Cl 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.
  • 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.
  • 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 + 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).
  • CAMP synthesis toxic effect
  • 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.
  • CyaA373-1706 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 + cells in vivo.
  • 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.
  • Dendritic cells are the principal cells in mouse spleen bearing immunogenic fragments of foreign proteins. Journal of Experimental Medicine 172, no. 1:383-6.
  • Lymphocyte function-associated antigen 1 is a receptor for Pasteurella haemolytica leukotoxin in bovine leukocytes. Infection & Immunity 68, no. 1:72-9.
  • a conditioned dendritic cell can be a temporal bridge between a CD4+ T-helper and a T-killer cell. Nature 393, no. 6684:474-8.

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