Patent Grant
9902947
The invention is directed to means, based on CyaA-carried polypeptide(s), for use in the immunotherapeutic treatment of first determined pathological condition(s) diagnosed in a mammalian host by eliciting a T cell immune response against a first group of epitopes contained in said polypeptide(s) and in the prophylaxis against second determined pathological condition(s) in the same mammalian host by eliciting a T cell memory immune response against a second group of epitopes contained in said polypeptide(s), said immune responses being obtained after administration of said vector-carried polypeptide(s) into said host, wherein said prophylaxis against second determined pathological condition(s) is not observed when said second group of epitopes is not contained in said administered vector-carried polypeptide(s). In a particular embodiment, the invention is directed to means, based on CyaA-carried polypeptide(s), for use (i) in the immunotherapeutic treatment of first determined pathological condition(s) diagnosed in a mammalian host, by eliciting a T cell immune response against a first group of epitopes contained in said polypeptide(s), (ii) in the prophylaxis against second determined pathological condition(s) in the same mammalian host, by eliciting a T cell memory immune response against a second group of epitopes contained in said polypeptide(s) and (iii) in the prevention against the re-occurrence of said first determined pathological condition(s), by eliciting a T cell memory immune response against said first group of epitopes contained in said polypeptide(s), said immune responses being obtained after administration of said vector-carried polypeptide(s) into said host, wherein said prophylaxis against second determined pathological condition(s) is not observed when said second group of epitopes is not contained in said administered vector-carried polypeptide(s).
One of the key toxins produced by Bordetella pertussis, the pathogen responsible for the whooping cough, is its Adenylate Cyclase (CyaA). It was shown using a murine model that CyaA is required by the bacteria during the early phase of lung colonization (Goodwin and Weiss 1990). CyaA exhibits a unique mechanism of eukaryotic cell invasion by delivering in a dedicated fashion its catalytic domain into cell cytosol (Simsova, Sebo et al. 2004). Detoxified and recombinant CyaA used as a vaccine vector displays the exquisite ability to target CD11b/CD18-expressing antigen presenting cells (APC), as for example dendritic cells or Langerhans cells (Guermonprez et al. 2001). These residents APC are key innate cells in the initiation of antigen-specific T-cell responses following intradermal immunization approaches (Merad, Ginhoux et al. 2008). After specific binding to CD11b+ APC, CyaA carrying either a viral or tumour antigen can deliver its antigenic cargo in a dedicated manner (Preville, Ladant et al. 2005). Based on this unique technology, the Applicant has developed a clinical stage bivalent vaccine to cure HPV infected patients: ProCervix. ProCervix is a bivalent therapeutic vaccine prepared with a mixture of two different CyaA vaccines: one embedding the HPV16 E7 protein and the other one carrying the HPV18 E7 protein.
Indeed Human papillomavirus (HPV) infections are generally long lasting and a compromised host immune response can lead to the development of cervical cancer, especially with high-risk HPV such as HPV18 and HPV16. E7 oncoproteins of HPV are expressed all along the replicative cycle of the virus, thus making them chosen targets for T-cell mediated immunotherapy (Iwasaki 2010). B-cell mediated immunity to the viral capsid proteins has been shown to be sufficient for a prophylactic protection against HPV infection (Stanley 2010). However, innate and T-cell mediated immunities are critical to cure patients that are already infected by the virus (Frazer 2009). Besides, available HPV prophylactic vaccines are not efficient to treat already HPV-infected patients and to cure the disease, thus highlighting the importance to develop therapeutic vaccines that would elicit antigen-specific T cell responses against HPV antigens (Trimble and Frazer 2009).
Prophylactic vaccines are based on the development of a B-cell mediated immunity. By contrast, therapeutic vaccines aim to develop strong and robust pro-inflammatory CD4+ and CD8+ T cell responses for an efficient treatment of chronic infection (virus, bacteria, etc.) or of cancer (Bachmann and Jennings 2010). Many studies have described both in mouse model and in human patients that induction of an antigen-specific T cell immunity can be correlated with a protection against diseased cells, either infected cells or tumour cells (Pulendran, Li et al. 2010; Sallusto, Lanzavecchia et al. 2010). By measuring qualitative and quantitative aspects of the CD8+ T cell response induced post-therapeutic vaccination in tumour bearing mice, it is possible to predict the therapeutic outcome, i.e., the progression or the regression of the tumour in individual animals (Rosato, Zoso et al. 2006). After a successful therapeutic vaccination that leads to a complete elimination of diseased cells, a key aspect of this immunotherapy would be its potential to generate long-term memory T lymphocytes in order to protect the patient against secondary pathogen infection. Memory T lymphocytes can be classified in two main cellular subsets: TEM (Effector Memory) and TCM (Central Memory). TEM are the first memory T cells to be generated following the clonal contraction phase of the immune response which fit with the elimination of the pathogen. TEM are CD62L− CCR7− and preferentially reside in peripheral tissues, such as the skin, gut and lungs, where they provide a first line of defence for the host (Woodland and Kohlmeier 2009). Over time, TEM progressively differentiate towards a TCM phenotype: CD62L+ CCR7−. These T lymphocytes are preferentially localized in secondary lymphoid organs (Kaech, Hemby et al. 2002; Ahmed, Bevan et al. 2009). Nevertheless both TEM and TCM are found in the circulation. A critical aspect of CD8 memory response efficacy is the speed at which CD8+ memory T lymphocyte acquire lytic potential and thus eliminate infected cells upon a novel infection by the same pathogen in order to prevent the spread of the infection and in turn the associated disease development. It has been shown that mice, that have been able to clear Lymphocytic choriomeningitis virus (LCMV) acute infection via the development of antigen-specific CD8+ T-cell responses, also developed memory CD8+ T-cells able to rapidly eliminate infected cells (Barber et al., 2003).
Based on this knowledge, the inventors have extended the approach of therapeutic vaccination involving T cell mediated immune response to devise a new concept of therapeutic and prophylactic treatment of pathological condition(s) by way of combining the administration of active ingredients in a designed multivalent immunogenic composition involving vectorized epitopes.
In particular, the inventors designed multivalent therapeutic vaccines suitable to induce, in a single patient, an immunotherapeutic treatment against a diagnosed pathology while mounting a robust prophylactic response against antigens or epitopes that are not related to said treated pathology, and optionally mounting a protective and preventive response against the re-occurrence of said treated pathology.
Indeed, the use of CyaA-based multivalent therapeutic approaches highlights the potential of CyaA-carried polypeptide(s) to treat and possibly eradicate a determined infection or a cancer while providing in the same patient a strong immune response, preferably protective T-cell memory response, against targeted epitopes contained in said polypeptide(s) against which a prophylactic protection is sought, and optionally a protective and preventive response against the re-occurrence of said determined infection or cancer.
The invention is directed to means for use (i) in the immunotherapeutic treatment of first determined pathological condition(s) diagnosed in a mammalian host and (ii) in the prophylaxis against second determined pathological condition(s) in the same mammalian host, and (iii) optionally in the prevention of the re-occurrence of said first determined pathological condition(s). The immune response is obtained by (i) eliciting a T cell immune response against a first group of epitopes contained in polypeptide(s) designed as active ingredient(s) to treat said diagnosed pathological condition(s) and (ii) by eliciting a T cell memory immune response against a second group of epitopes contained in polypeptide(s) designed as active ingredient(s) to prevent the onset or the development of said second determined condition(s), and (iii) optionally by eliciting a T cell memory immune response against said first group of epitopes to prevent the re-occurrence of said first determined pathological condition(s), said immune responses being obtained after administration of said vector-carried polypeptide(s) into said host, wherein said prophylaxis against second determined pathological condition(s) is not observed when said second group of epitopes is not contained in said administered vector-carried polypeptide(s). These means include:
(1) as active ingredients, vector-carried polypeptide(s), wherein said vector carrying the polypeptide(s) consists in a CyaA protein or a fragment thereof suitable to present said polypeptide(s) to the immune system in a mammalian host;
(2) a composition comprising or containing such active ingredients including a composition comprising the vector-carried polypeptide(s) as defined in (1) in combination with a pharmaceutically acceptable vehicle or formulation; and
(3) a composition comprising a first vector-carried polypeptide(s) containing a first group of epitopes and a second separate vector-carried polypeptide(s) containing the second group of epitopes.
Thus, in a first embodiment the invention concerns vector-carried polypeptide(s), wherein said vector carrying the polypeptide(s) consists in a CyaA protein or a fragment thereof suitable to present said polypeptide(s) to the immune system in a mammalian host, for use (i) in the immunotherapeutic treatment of first determined pathological condition(s) diagnosed in said mammalian host by eliciting a T cell immune response against a first group of epitopes contained in said polypeptide(s) and (ii) in the prophylaxis against second determined pathological condition(s) in the same mammalian host by eliciting a T cell memory immune response against a second group of epitopes contained in said polypeptide(s), and (iii) optionally in the prevention against the re-occurrence of said first determined pathological condition(s) by eliciting a T cell memory immune response against said first group of epitopes contained in said polypeptide(s), said immune responses being obtained after administration of said vector-carried polypeptide(s) into said host, wherein said prophylaxis against second determined pathological condition(s) is not observed when said second group of epitopes is not contained in said administered vector-carried polypeptide(s).
The invention also concerns a composition comprising, in combination with a pharmaceutically acceptable vehicle, a vector-carried polypeptide(s), wherein said vector carrying the polypeptide(s) consists in a CyaA protein or a fragment thereof suitable to present said polypeptide(s) to the immune system in a mammalian host, for use (i) in the immunotherapeutic treatment of first determined pathological condition(s) diagnosed in said mammalian host by eliciting a T cell immune response against a first group of epitopes contained in said polypeptide(s) and (ii) in the prophylaxis against second determined pathological condition(s) in the same mammalian host by eliciting a T cell memory immune response against a second group of epitopes contained in said polypeptide(s), and (iii) optionally in the prevention against the re-occurrence of said first determined pathological condition(s) by eliciting a T cell memory immune response against said first group of epitopes contained in said polypeptide(s) said immune responses being obtained after administration of said composition into said host, wherein said prophylaxis against second determined pathological condition(s) is not observed when said second group of epitopes is not contained in the vector-carried polypeptide(s) of said administered composition.
The invention also relates to a composition comprising, optionally in combination with a pharmaceutically acceptable vehicle:
for use in (i) combined immunotherapeutic treatment against pathology associated with said first group of epitopes and (ii) immunoprophylaxis treatment associated with said second group of epitopes and (iii) optionally prevention against the re-occurrence of said first determined pathological condition(s), wherein said prophylaxis against second determined pathological condition(s) is not observed when said second group of epitopes is not contained in the vector-carried polypeptide(s) of said administered composition.
In a specific embodiment, said composition with (a), (b) and (c) is for use (i) in the immunotherapeutic treatment of first determined pathological condition(s) diagnosed in said mammalian host by eliciting a T cell immune response against the first group of epitopes and (ii) in the prophylaxis against second determined pathological condition(s) in the same mammalian host by eliciting a T cell memory immune response against the second group of epitopes contained in said polypeptide(s), and (iii) optionally in the prevention against the re-occurrence of said first determined pathological condition(s) by eliciting a T cell memory immune response against said first group of epitopes contained in said polypeptide(s), said immune responses being obtained after administration of said composition into said host.
The term “CyaA” means an Adenylate Cyclase (or adenylcyclase) of a Bordetella species, in particular a CyaA from Bordetella pertussis, Bordetella parapertussis or Bordetella bronchiseptica. The adenylate cyclase protein of Bordetella pertussis is a toxin of 1706 residues (SEQ ID NO:1), comprising a N-terminal catalytic domain of 400 amino acid residues and a C-terminal part of 1306 residues. The C-terminal part is responsible for the binding of the toxin to target cell membrane and subsequent delivery of the catalytic moiety into the target cell cytosol. The sequence of Bordetella pertussis CyaA is provided in
The expression “CyaA fragment” means a part of the CyaA protein, optionally encompassing all or part of the C-terminal part of the full-length CyaA protein, provided said fragment is able to present said polypeptide(s) to the immune system in a mammalian host, i.e., is able to lead to the induction of specific immune response(s) against epitopes contained in the vector-carried polypeptide(s) or to favour said response. In particular, said CyaA fragment is able to specifically bind to CD11b-expressing cells and optionally to deliver said polypeptide(s) into the cell cytosol. Said fragment encompasses a CyaA protein which has been truncated (deletion from the N-terminal and/or C-terminal extremities) or a full-length protein with internal deletion of amino acid residue(s). Thus, a particular fragment of CyaA protein according to the invention consists of amino acid residues 372 to 1706 of B. pertussis CyaA protein (truncation of the first 371 residues). Another particular fragment is the B. pertussis CyaA protein wherein amino acid residues 225 to 234 have been deleted, thus providing a CyaA fragment consisting of residues 1 to 224 and 235 to 1706 (internal deletion). The term “specifically” means within the context of the present invention that the adenylate cyclase or its fragment, when used as a vector molecule, is preferentially directed to CD11b-expressing cells, thereby offering means to target the polypeptide(s) at the surface of said cells or within said cells in a selective way with respect to other cells (not expressing CD11b).
The term “CyaA” also encompasses a CyaA protein or its fragments which is modified, preferably by one or more amino acid substitution(s), internal insertion of amino acid(s) or internal deletion of amino acid(s), in order to give rise to a detoxified or a non-toxic product or a product devoid of enzymatic (invasive and cytotoxic) activity. Thus, such a CyaA protein (or its fragments) has no catalytic activity, but the capacity to present said polypeptide(s) to the immune system in a mammalian host, and optionally the specific binding to CD11b/CD18 receptor and/or the process of translocation of the catalytic domain of the original CyaA protein, is(are) not affected. An example of a well-known non-toxic CyaA protein is a Bordetella pertussis CyaA in which the dipeptide Leu-Gln has been inserted in-frame between residues Asp188 and 11e189 (essential part of the catalytic site). The absence of toxicity or enzymatic activity of this CyaA protein (or its fragments) may be assayed as disclosed in Ladant et al. (1992). The capacity of the CyaA protein (or its fragments) to target CD11b/CD18 cells can be assayed especially according to the methods disclosed in EP03291486 or in WO02/22169. Furthermore, the capacity of the CyaA protein (or its fragments) to translocate the antigenic polypeptide into the cytosol of target cell can be assayed by applying the method described in WO02/22169.
In a further particular embodiment, the term “CyaA” also encompasses, the CyaA protein (or its fragments) which has been modified by post-translational modifications, for example by post translational palmitoyation of at least one of its residues, in particular the two internal lysine residues (lysines 860 and 983). This (these) post translational modification(s) may be obtained by co-expression of the cyaA and cyaC genes. Thus, the CyaA protein or a fragment thereof, within the vector-carried polypeptide(s), may be a CyaA protein or a fragment thereof which is the result of the co-expression in a cell, especially in a recombinant cell, of both cyaA and cyaC genes.
The term “vector” or “vector molecule” used in the present application encompasses the full-length CyaA protein, or fragments thereof, modified or not, as detailed herein.
By “CD11b-expressing cells”, it is meant cells that express the CD11b/CD18 receptor on their surface (CD11b+). In particular, these cells are granulocytes/neutrophils, macrophages, NK cells, subsets of T CD8+, subsets of B cells, Langerhans cells, dendritic cells and myeloid dendritic cells.
The expression “vector-carried polypeptide(s)” means that the CyaA protein (or its fragments) carries at least one polypeptide which is heterologous with respect to CyaA, in particular which is not a CyaA fragment as defined herein, and especially does not immunologically cross-react with CyaA. The expression “at least one” means one polypeptide or more, in particular 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 polypeptide(s), preferably 1, 2 or 3 polypeptide(s).
The term “carries” encompasses various structures associating the CyaA protein or a fragment thereof in accordance with the present invention and the polypeptide(s). Such structures may be obtained as a result of:
When a CyaA protein (or its fragments) carries more than one polypeptide, these polypeptides may be either all carried by chemical coupling, all carried by genetic link (preferably all genetically inserted), or one(s) of them is(are) carried by chemical coupling whereas the other(s) is(are) carried by genetic link. In a particular embodiment, when all the polypeptides are genetically inserted into CyaA, preferably into permissive sites of CyaA, the polypeptides are inserted into different sites, preferably different permissive sites. In another embodiment, when all the polypeptides are genetically inserted into CyaA, preferably into permissive sites of CyaA, the polypeptides are inserted into the same site, preferably into the same permissive site.
The term “polypeptide” refers to a concatenation of amino acids, and is at least 9 amino acids residues and is in particular from 9 to 500 residues, 9 to 200, from 9 to 100, from 9 to 50 residues, or from 30 or 50 to 500 or to 200 residues, or from 100 to 500 or from 100 to 200 residues in length. Within the invention, the term “polypeptide” means a polypeptide which is able, once carried by the vector molecule, to induce an immune response, in particular a T cell immune response, against epitope(s) contained in this polypeptide. The polypeptide(s) contained in the vector molecule(s) may be derived from a tumor antigen, i.e., a peptide expressed by tumor or by cancerous cells, whether the tumor is self or induced by a pathogen; the tumor antigen may be self (in particular of human origin) or from the pathogen inducing the tumor.
The term “tumor antigen” encompasses the following groups of tumor antigens, and the polypeptide(s) contained in the vector molecule(s) of the invention may be chosen in at least one of the following groups: (a) oncofetal tumor antigens, (b) oncoviral tumor antigens, (c) overexpressed/accumulated tumor antigens, expressed in a wide variety of normal tissues and overexpressed in tumors, (d) shared tumor-specific antigens or cancer-Testis antigens, expressed in many tumors but not in normal tissues (including BAGE family, GAGE family, MAGE family, SAGE family and XAGE family), (e) lineage-restricted tumor antigens, (f) mutated tumor antigens, resulting from point mutations in genes that are ubiquitously expressed; and (g) differentiation tumor antigens, expressed in the normal tissue of origin of the tumors but which are not tumor-specific.
In a particular embodiment, when more than one polypeptide is used in a single vector molecule for elicitation of immunotherapeutic response or a single vector molecule for elicitation of an immunoprophylactic response, all the polypeptides are derived from tumor antigens.
In another embodiment, the polypeptide(s) contained in the vector molecule(s) may also or alternatively be derived from an antigen of pathogen, i.e., an antigen which is produced by the pathogen in the infected mammalian host, and possibly processed in the cells of said infected mammalian host or a component of said pathogen. Examples of pathogen antigens are a bacterial antigen, a viral antigen, a fungus antigen or a parasite antigen. In these examples, as a particular embodiment, one can distinguish pathogens involved in tumorigenesis (oncopathogens) and pathogens which are not involved in tumorigenesis. Examples of pathogens are intracellular pathogens, in particular pathogens inducing T-cell immune response(s) in their host. Thus, the polypeptide(s) may be derived from, but not limited to, Chlamydia, Plasmodium, Leishmania, Mycobacterium tuberculosis, HIV, HPV, HBV, HCV, adenoviruses, EBV, herpes virus, HTLV.1 virus and CMV. In a particular embodiment, the polypeptide(s) contained in the vector molecule(s) may be derived from a surface protein of the pathogen (such as the envelope protein of HIV) or derived from a polypeptide interacting with the machinery of the infected cell (such as E6 or E7 of HPV).
In a particular embodiment, when more than one polypeptide is used, in a single vector molecule or in a combination of vectors, all the polypeptides are derived from antigens of pathogens, possibly of distinct pathogens, genus or species.
In a particular embodiment, the polypeptides contained in the vector molecule(s) are all derived from bacterial antigens, are all derived from viral antigens, are all derived from fungus antigens or are all derived from parasite antigens. In another embodiment, the distinct polypeptides contained in the vector molecule(s) described herein are derived independently from a bacterial antigen, a viral antigen, a fungus antigen or a parasite antigen. In another embodiment, the polypeptides, in a single vector molecule or in a combination of vectors, are derived from tumor and derived from a pathogen.
The expression “derived from”, with respect to a polypeptide carried by a vector molecule, means either the full-length antigenic protein, or a fragment of this antigenic protein, or a synthetic, non-natural polypeptide carries epitope(s) consisting of several parts of the antigenic protein fused together or a synthetic, non-natural polypeptide consisting of one or several part(s) of several proteins fused together, provided that the fragment or the synthetic, non-natural polypeptide is able to induce, once carried by the vector molecule(s), an immune response, in particular a T cell immune response, against an antigenic determinant contained in this fragment or polypeptide. According to this definition, the polypeptide(s) carried by the vector molecule(s) is or comprises a unique epitope, or is or comprises a group of epitopes. The expression “group of epitopes” encompasses at least one epitope, i.e., one epitope or more, in particular between 10 and 500 epitopes, between 50 and 200 epitopes and between 80 and 150 epitopes. In a particular embodiment, the expression “group of epitopes” means 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 epitopes. In a particular embodiment, the polypeptide(s) carried by the vector molecule(s) is or comprises a polyepitope, i.e., a polypeptide with at least two epitopes, in particular at least two T-cell epitopes. Epitopes within the present invention are, either linear or conformational, preferably linear, and are any amino acid sequence involved in the induction of a cell-mediated immune response, especially a T cell immune response. Accordingly, epitopes in the vector-carried polypeptide(s) described herein include those which are processed by APC (Antigen Presenting Cells) in a host, especially T epitopes recognized in association with class I MHC (Major Histocompatibility Complex) molecules, such as epitopes which target cells are CD8+ T lymphocytes or T epitopes recognized in association with class II MHC molecules, such as those which target cells are CD4+ T lymphocytes. In a particular embodiment, the polypeptide(s) also contains B epitope(s) involved in humoral response. In a particular embodiment, the polypeptide(s) carried by the vector molecule(s) consists or comprises several different or several identical epitopes.
According to the invention, the vector-carried polypeptide(s) contain(s) at least two different groups of epitopes, i.e., one group of epitopes is able to elicit a T cell immune response to enable an immunotherapeutic treatment of first determined pathological condition(s) diagnosed in a mammalian host, whereas the second group of epitopes is able to elicit a T cell memory immune response to enable the prophylaxis against second determined pathological condition(s) in the same mammalian host. In a particular embodiment, the first group of epitopes is optionally able to elicit, in addition to a T cell response providing immunotherapeutic treatment of first determined pathological condition(s) diagnosed in a mammalian host, a T cell memory immune response against said first group of epitopes contained in said polypeptide(s) to enable the prevention against the re-occurrence of said first determined pathological condition(s).
According to the invention, in the absence of said second group of epitopes in said vector-carried polypeptide(s), the prophylaxis against second determined pathological condition(s) is not observed. This means that the second group of epitopes as defined herein is necessary to obtain the prophylaxis against second determined pathological condition(s).
In a particular embodiment, the second group of epitopes [within the vector-carried polypeptide(s)] is necessary and sufficient to obtain the prophylaxis against second determined pathological condition(s); in this embodiment, the contribution of the sole second group of epitopes, in the elicited immune reponse, is sufficient to obtain the prophylaxis against second determined pathological condition(s). In other words, there is causal relationship between the administration of said second group of epitopes and said prophylactic response.
In another embodiment, the second group of epitopes [within the vector-carried polypeptide(s)] is necessary to obtain the prophylaxis against second determined pathological condition(s), but is not sufficient or benefits from the contribution of the first group, meaning that the prophylaxis against second determined pathological condition(s) is obtained following a synergy between the immune response elicited against the second group of epitopes and the immune response elicited against the first group of epitopes. In this later case, the contribution of both groups of epitopes is necessary to obtain the prophylaxis against second determined pathological condition(s).
The necessary contribution of the second group of epitopes in the prophylaxis against second determined pathological condition(s) may be put in evidence by comparing the effect of the administration of the vector-carried polypeptide(s) of the invention on said second determined pathological condition(s) [the term prophylaxis is defined below] in the two following groups of mammals, in particular in two following groups of mice: (1) mammals administered with vector-carried polypeptide(s) of the invention that do(es) not bear a second group of epitopes as defined in the application and (2) mammals administered with vector-carried polypeptide(s) of the invention that bear(s) a second group of epitopes as defined in the application.
As understood by the expression “wherein said prophylaxis against second determined pathological condition(s) is not observed when said second group of epitopes is not contained in said administered vector-carried polypeptide(s)”, it is excluded that the first group of epitopes as defined herein be sufficient (i.e., alone) to obtain the prophylaxis against second determined pathological condition(s).
Thus, in a particular embodiment, the amino acid sequence of the first group of epitopes (or polypeptide(s) consisting of this first group of epitopes) is different from the amino acid sequence of the second group of epitopes (or polypeptide(s) consisting of this second group of epitopes). The term “different” means that both sequences differ by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, calculated over the entire length of the sequence of the polypeptides (global alignment calculated for example by the Needleman and Wunsch algorithm). In an alternative definition or in a particular embodiment of said “different sequences”, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the epitopes of the first group have a sequence which is different from the sequence of the epitopes of the second group. In a further particular embodiment, the first and second groups of epitopes (or the polypeptide(s) consisting of the first group of epitopes and the polypeptide(s) consisting of the second group of epitopes) have no epitope sequence in common. In a particular embodiment, the T-cell immune response obtained against the first group of epitopes is effective against the pathological condition(s) associated with said first group of epitopes and is not effective or not specifically effective against a pathological condition(s) associated with the second group of epitopes. Hence, in a particular embodiment, the T-cell immune response obtained against a group of epitopes is specific for this group, i.e., the T cells involved in the immune response against this group of epitopes does not recognize the other group of epitopes.
In a particular embodiment, the polypeptide(s) contain at least one epitope from HPV strains, especially HPV strains selected among the genus Alpha-papillomavirus, Beta-papillomavirus, Gamma-papillomavirus, Delta-papillomavirus, Epsilon-papillomavirus, Zeta-papillomavirus, Eta-papillomavirus, Theta-papillomavirus, Iota-papillomavirus, Kappa-papillomavirus, Lambda-papillomavirus, Mu-papillomavirus, Nu-papillomavirus, Xi-papillomavirus, Omikron-papillomavirus and Pi-papillomavirus. In a particular embodiment, papillomaviruses having a human tropism, such as strains from genus Alpha-papillomavirus, Beta-papillomavirus, Gamma-papillomavirus, Mu-papillomavirus or Nu-papillomavirus, are preferred. In a more particular embodiment, the polypeptide(s) contain at least one epitope from HPV strains from the Alpha-papillomavirus genus, especially strain from HPV species 7 and 9 of the Alpha-papillomavirus genus (de Villiers et al. Virology 2004). Thus, the polypeptide(s) contain at least one epitope from HPV highly oncogenic type species such as HPV16, HPV18, HPV31, HPV33, HPV35, HPV45, HPV52 or HPV58. Among these type species, HPV18 and HPV16 are of particular interest. In an embodiment, the polypeptides carried by the vector molecule(s) described herein are from different HPV strains or different HPV type species chosen among the ones disclosed above. In another particular embodiment, and whatever the HPV strains or HPV type species, the polypeptides are derived from the L1, L2, E1, E2, E4, E5, E6 or E7 proteins, with a particular interest for polypeptides bearing epitopes from E6 or E7 proteins of HPV strains or HPV type species. In a particular embodiment, the polypeptides carried by the vector molecule(s) described herein are from the same HPV protein but from different HPV strains or different HPV type species chosen among the ones disclosed above. In a particular embodiment of the invention, E6 or E7 proteins of HPV16 and E6 or E7 proteins of HPV18 are used for the design of polypeptides, more preferably the E7 protein of HPV16 and HPV18 HPV type species. According to a particular embodiment of the invention, a vector molecule carries several polypeptides, preferably by genetic insertion, each of them containing or consisting of one or several epitopes of one or several HPV proteins of at least two distinct HPV strains or two distinct HPV type species. Thus, a particular embodiment is vector-carried polypeptides consisting of a CyaA protein or fragment thereof carrying a polypeptide or several polypeptides encompassing epitopes derived from the E6 or the E7 protein from both HPV16 and HPV18 HPV type species. In another embodiment, the invention concerns a composition comprising a first vector molecule carrying a polypeptide or several polypeptides encompassing epitopes derived from the E6 or the E7 protein from HPV16 (the first vector-carried polypeptides with a first group of epitopes) and a separate vector molecule carrying a polypeptide or several polypeptides encompassing epitopes derived from the E6 or the E7 protein from HPV18 (the separate vector-carried polypeptides with a second group of epitopes). When several polypeptides are carried by a single vector molecule, these polypeptides may consist of different fragments of the same protein, for example of an E7 or E6 protein, which are inserted in different sites, in particular permissive sites, of the vector molecule.
Thus, a composition used within the present invention comprises a vector-carried polypeptide(s) whose polypeptide(s) is/are derived from the E7 protein of HPV16 and a vector-carried polypeptide(s) whose polypeptide(s) is/are derived from the E7 protein of HPV18. An example of such composition comprises:
(a) a first vector molecule carrying a first polypeptide which is a fragment comprising residues 1 to 29 or a fragment consisting of residues 1 to 29 of E7 of HPV16 and carrying a second polypeptide which is a fragment comprising residues 43 to 98 or a fragment consisting of residues 43 to 98 of E7 protein of HPV16. In a preferred embodiment, the first polypeptide is the first 29 amino acid residues of HPV16-E7 and is inserted between codons 319 and 320 of CyaA, and the second polypeptide consists of residues 43 to 98 of HPV16-E7 and is inserted between codons 224 and 235 of CyaA (exemplified in the pKTRACE5-HPV16E7Δ30-42 vector); and
(b) a separate vector molecule carrying a first polypeptide which is a fragment comprising residues 1 to 31 of E7 of HPV18 or a fragment consisting of residues 1 to 31 of E7 of HPV18, and a second polypeptide which is a fragment comprising residues 43 to 105 of E7 of HPV18 or a fragment consisting of residues 43 to 105 of E7 of HPV18. In a preferred embodiment, the first polypeptide is the first 31 amino acid residues of HPV18-E7 and is inserted between codons 319 and 320 of CyaA, and the second polypeptide consists of residues 43 to 105 of HPV18-E7 and is inserted between codons 224 and 235 of CyaA (exemplified in the pKTRACE5-HPV18E7Δ32-42 vector).
In another embodiment, the polypeptide(s) contain at least one epitope derived from the MAGE A3 tumor antigen, such as a polypeptide consisting of residues 97 to 178 of MAGE A3 (SEQ ID NO:8), or such as a polypeptide consisting of residues 190 to 221 fused to residues 242 to 295 of MAGE A3 (SEQ ID NO:9). In a particular embodiment, a vector-carried polypeptide(s) of the invention (or a composition contained it) consists of a CyaA protein, preferably B. pertussis CyaA, in which two polypeptides derived from MAGE A3 have been inserted in two different sites. Such a vector-carried polypeptide(s) consists of the B. pertussis CyaA, wherein (1) a first polypeptide consisting of residues 97 to 178 of MAGE A3 has been inserted between codons 319 and 320 of CyaA and (2) a second polypeptide consisting of residues 190 to 221 fused to residues 242 to 295 of MAGE A3 has been inserted between codons 234 and 235 of CyaA. A particular example of a CyaA-MAGE A3 vector is provided in
The vector-carried polypeptide(s) as defined herein, as such or in a composition, is/are used to obtain in a same mammalian host, especially in a human host, (i) an immunotherapeutic treatment of first determined pathological condition(s) diagnosed in said mammalian host by eliciting a T cell immune response against a first group of epitopes contained in said polypeptide(s) and (ii) the prophylaxis against second determined pathological condition(s) by eliciting a T cell memory immune response against a second group of epitopes contained in said polypeptide(s), and (iii) optionally the prevention against the re-occurrence of said first determined pathological condition(s) by eliciting a T cell memory immune response against said first group of epitopes contained in said polypeptide(s), said immune responses being obtained after administration of said vector-carried polypeptide(s) into said host, wherein said prophylaxis against second determined pathological condition(s) is not observed when said second group of epitopes is not contained in said administered vector-carried polypeptide(s). According to an embodiment of the invention, the polypeptide(s), as defined herein, carried by the vector molecule(s), are chosen according to the groups of epitopes which is sought and may be classified according to two groups:
The invention lies from the observations that:
(i) an immunotherapeutic treatment of first determined pathological condition(s) diagnosed in a mammalian host can be obtained by eliciting a T cell immune response against a first group of epitopes contained in polypeptide(s) carried by a vector molecule;
(ii) the prophylaxis against second determined pathological condition(s) in the same mammalian host can be obtained by eliciting a T cell memory immune response against a second group of epitopes contained in polypeptide(s); and
(iii) optionally, the prevention against the re-occurrence of said first determined pathological condition(s) can be obtained by eliciting a T cell memory immune response against said first group of epitopes contained in said polypeptide(s) carried by a vector molecule;
said second group of epitopes being either carried by the same vector molecule, or carried by a separate vector molecule, and administered in the same composition as and simultaneously with the first vector molecule.
Therefore, the vector molecule as such as defined herein or in a composition, or the vector molecules of a composition as defined herein carries at least one, preferably one, polypeptide comprising epitope(s) of the first group and at least one polypeptide comprising epitope(s) of the second group. In other embodiments, the vector molecule as such as defined herein or in a composition or the vector molecules of a composition as defined herein carries/carry (a) one polypeptide comprising epitope(s) of the first group and at least one polypeptide comprising epitope(s) of the second group, (b) one polypeptide comprising epitope(s) of the first group and at least one polypeptide comprising epitope(s) of the second group selected among 1, 2, 3, 4, 5 or 6 polypeptides, (c) at least one polypeptide comprising epitope(s) of the first group selected among 2 or 3 polypeptides and at least one polypeptide comprising epitope(s) of the second group and (d) at least one polypeptide comprising epitope(s) of the first group selected among 2 or 3 polypeptides and at least one polypeptide comprising epitope(s) of the second group selected among 1, 2, 3, 4, 5 or 6 polypeptides.
When one vector-carried polypeptide(s) is used as such or in a composition, the vector may carries one polypeptide comprising epitope(s) of the first group and epitope(s) of the second group, i.e., that said second group of epitopes is contained in the same polypeptide as the first group of epitopes.
In another embodiment, said first and second group of epitopes are contained in different polypeptides. The polypeptide comprising epitope(s) of the first group and the polypeptide comprising epitope(s) of the second group may be carried by the same vector molecule. In another embodiment, the polypeptide comprising epitope(s) of the first group and the polypeptide comprising epitope(s) of the second group may be carried by different vector molecules and included in a same composition. When a composition, containing a first and a second, separate, vector-carried polypeptide(s), is used, the first vector molecule-carries at least one polypeptide comprising epitope(s) of the first group, meaning that one polypeptide comprising epitope(s) of the first group at the minimum is carried by said first vector molecule. In the same composition, the second vector molecule carries at least one polypeptide comprising epitope(s) of the second group, meaning that one polypeptide comprising epitope(s) of the second group at the minimum is carried by said second separate vector molecule. Finally, optional one or more (e.g. 1, 2, 3, 4, 5 or 6) vector-carried polypeptide(s) may also be included in said composition, whatever the group of epitopes contained in this/these vector-carried polypeptide(s).
By “immune response”, it is meant a cell-mediated immune response, especially a T-cell mediated immune response. In a particular embodiment, said T-cell mediated immune response is a cell-mediated cytotoxic immune response CTL, especially a CD8+ immune response. In the case of polypeptide(s) derived from tumor antigen, the immune response is preferably a tumor-specific cytotoxic immune response, involving tumor-specific cytotoxic lymphocytes. In a particular embodiment, said T-cell mediated immune response is a CD4+ immune response, especially a T-helper immune response. In a particular embodiment, the immune response, in particular the immune response(s) induced against the epitopes of the second group (as defined herein), following the administration of the vector-carried polypeptide(s) or composition defined herein, is a memory T cell immune response. The expression “immune response” may also encompass, in addition to a cell-mediated immune response as defined above, a humoral immune response (production of antibodies). The immune responses discussed within the present application are obtained after administration, into the host, of the vector-carried polypeptide(s) of the invention, as such or within a composition.
The expression “immunotherapeutic treatment” refers to the treatment of a (first) determined pathological condition(s) diagnosed in a mammalian host, by eliciting especially a T-cell immune response against a first group of epitopes contained in the polypeptide(s) carried by the administered vector molecule(s) as defined herein. The use of the vector-carried polypeptide(s) or composition as defined herein aims at improving the clinical condition(s) of a mammalian host, in need thereof, who has been diagnosed as infected by a pathogen or as suffering from a pathological state, such as tumor, or aims at the elimination of the causative agent or organism of the disease, or at lowering said agent or organism. In a situation of pathogen infection, the immunotherapeutic treatment results in a significant decrease in the pathogen load at the site of infection or at the site of replication of this pathogen, in particular in the plasma or in the mucosa of the host, and possibly results in a pathogen load, such as plasma load, which is less than what can be detected when measured or, at lowering the size or the development of the tumor if any.
The expression “immunoprophylaxis” or “prophylaxis” refers to a response that prevents or protects against the exposure, infection, onset or the development of a second determined pathological condition(s) or disease or clinical consequences thereof in the same mammalian host by eliciting especially a T cell memory immune response against a second group of epitopes contained in the polypeptide(s) carried by the administered vector molecule(s) as defined herein. The use of the vector-carried polypeptide(s) or composition as defined herein results in a prophylactic immune response against future infection, future malignant events or diseases and accordingly prevents the occurrence of a pathological state in said mammalian host.
The efficacy of the response in conferring prophylaxis can be assayed by detecting marker of the pathological state. In a particular embodiment, the efficacy criteria are selected to reach statistical relevancy.
The expression “prevention against the re-occurrence” refers to the elicitation of an immune response to prevent a novel future exposure, novel future infection, novel future onset or novel future development of the first determined pathological condition(s), conditions which have been previously diagnosed and have been treated following the administration of the vector-carried polypeptide(s) or composition as defined herein.
The expression “mammalian host” encompasses all mammals, in particular primates and humans (e.g., patient).
It is understood that, following the administration(s) of the vector-carried polypeptide(s) or composition as defined herein, both the T cell immune response against a first group of epitopes and the T cell memory immune response against a second group of epitopes and optionally response against said first group of epitopes, are induced in the same mammalian host, within a particular window of time. Thus, the at least one administration of the vector-carried polypeptide(s) or composition as defined herein leads to the induction of a T cell immune response against a first group of epitopes and a T cell memory immune response against a second group of epitopes and optionally a T cell memory immune response against said first group of epitopes, which can be put in evidence and/or measured from 1 month to 12 months or more post administration (in particular at 2, 3, 6, 9 or 12 months), though T-cell involved in one of these or in these both immune responses may still be put in evidence several years post administration. The expression “at least one administration” or “administering once” means that the vector-carried polypeptide(s) or composition as defined herein is administered into the mammalian host, especially the patient, once or more, preferably once or twice. Each administration consists in at least one vector-carried polypeptide(s), provided that a first group of epitopes and a second group of epitopes contained in at least one polypeptide(s) carried by at least one vector molecule(s), with at least one epitope of said second group of epitopes, are administered to the mammalian host at the same time. If appropriate, the second and possible subsequently administrations (prime-boost) are carried out with the same vector-carried polypeptide(s) or with the same composition, regarding the polypeptide(s), as the first administration. The experiments reported hereinafter show that an immunotherapeutic treatment and prophylaxis, and optionally prevention against the re-occurrence, may be obtained following a single administration of the vector-carried polypeptide(s) or composition as defined herein.
In a particular embodiment, the invention relates to the vector-carried polypeptide(s) or composition as defined herein for use (i) in the immunotherapeutic treatment of first pathological condition(s) consecutive to a pathogen infection, in particular consecutive to a bacterial or viral infection, diagnosed in a mammalian host and (ii) in the prophylaxis against second pathological condition(s) consecutive to an infection by a different pathogen, in particular consecutive to an infection by a different bacteria or different virus (or different strain thereof) and (iii) optionally in the prevention against the re-occurrence of said first pathological condition(s), wherein said prophylaxis against said second determined pathological condition(s) is not observed when the second group of epitopes linked to said different pathogen (e.g., different bacteria or different virus) is not contained in said administered vector-carried polypeptide(s). In the particular case of pathological condition(s) consecutive to different virus strains, especially different HPV strains or different HPV type species, the invention relates to the vector-carried polypeptide(s) or composition as defined herein for use (i) in the immunotherapeutic treatment of first pathological condition(s) consecutive to the infection by a first virus, first HPV strain or first HPV type strains, diagnosed in a mammalian host and (ii) in the prophylaxis against second pathological condition(s) consecutive to the infection by a second different virus, second different HPV strain or second different HPV type strain, and (iii) optionally in the prevention against the re-occurrence of said first pathological condition(s), wherein said prophylaxis against second determined pathological condition(s) is not observed when the second group of epitopes linked to said second different virus, second different HPV strain or second different HPV type strain is not contained in said administered vector-carried polypeptide(s). In a preferred embodiment, the invention relates to vector-carried polypeptide(s) or composition as defined herein, comprising a polypeptide which is derived from the E7 protein of HPV16 and a polypeptide which is derived from the E7 protein of HPV18, for use either: (1) (i) in the immunotherapeutic treatment of first pathological condition(s) consecutive to the infection by HPV16, diagnosed in a mammalian host and (ii) in the prophylaxis against second pathological condition(s) consecutive to the infection by HPV18 and (iii) optionally in the prevention against the re-occurrence of said first pathological condition(s), wherein said prophylaxis against second determined pathological condition(s) is not observed when the polypeptide derived from the E7 protein of HPV18 is not contained in said administered vector-carried polypeptide(s); or (2) (i) in the immunotherapeutic treatment of first pathological condition(s) consecutive to the infection by HPV18, diagnosed in a mammalian host and (ii) in the prophylaxis against second pathological condition(s) consecutive to the infection by HPV16 and (iii) optionally in the prevention against the re-occurrence of said first pathological condition(s), wherein said prophylaxis against second determined pathological condition(s) is not observed when the polypeptide derived from the E7 protein of HPV16 is not contained in said administered vector-carried polypeptide(s).
In a particular embodiment, the invention relates to the vector-carried polypeptide(s) or composition as defined herein for use (i) in the immunotherapeutic treatment of first pathological condition(s) consecutive to first tumor cells, diagnosed in a mammalian host and (ii) in the prophylaxis against second pathological condition(s) consecutive to second tumor cells, whose origin and/or histology is different from the first tumor cells, and (iii) optionally in the prevention against the re-occurrence of said first pathological condition(s), wherein said prophylaxis against second determined pathological condition(s) is not observed when the second group of epitopes linked to said second tumor cells is not contained in said administered vector-carried polypeptide(s).
In a particular embodiment, the invention relates to the vector-carried polypeptide(s) or composition as defined herein for use (i) in the immunotherapeutic treatment of first pathological condition(s) consecutive to a pathogen infection, in particular consecutive to a bacterial or viral infection, diagnosed in a mammalian host and (ii) in the prophylaxis against second pathological condition(s) consecutive to the development of tumor cells and (iii) optionally in the prevention against the re-occurrence of said first pathological condition(s), wherein said prophylaxis against second determined pathological condition(s) is not observed when the second group of epitopes linked to said tumor cells is not contained in said administered vector-carried polypeptide(s).
In a particular embodiment, the invention relates to the vector-carried polypeptide(s) or composition as defined herein for use (i) in the immunotherapeutic treatment of first pathological condition(s) consecutive to the development of tumor cells, diagnosed in a mammalian host and (ii) in the prophylaxis against second pathological condition(s) consecutive to a pathogen infection, in particular consecutive to a bacterial or viral infection, and (iii) optionally in the prevention against the re-occurrence of said first pathological condition(s), wherein said prophylaxis against second determined pathological condition(s) is not observed when the second group of epitopes linked to said different pathogen (e.g., different bacteria or different virus) is not contained in said administered vector-carried polypeptide(s).
The invention also relates to a method to obtain in a same mammalian host, both (i) an immunotherapeutic treatment of first determined pathological condition(s) diagnosed in a mammalian host, especially by eliciting a T cell immune response against a first group of epitopes, and (ii) the prophylaxis against second determined pathological condition(s), especially by eliciting a T cell memory immune response against a second group of epitopes, wherein said prophylaxis against second determined pathological condition(s) is not observed when said second group of epitopes is not contained in said administered vector-carried polypeptide(s), said method comprising administering, at least once, to said mammalian host either:
(1) a vector-carried polypeptide(s), wherein said vector carrying the polypeptide(s) consists in a CyaA protein or a fragment thereof suitable to present said polypeptide(s) to the immune system in a mammalian host, said first group of epitopes and second group of epitopes being contained in at least one polypeptide(s) carried by said vector, and at least one epitope of said second group of epitopes being different from the epitopes of the first group of epitopes;
(2) a composition comprising the vector-carried polypeptide(s) as defined in (1) in combination with a pharmaceutically acceptable vehicle; or
(3) a composition comprising (a) a first vector-carried polypeptide(s) wherein said first vector carrying the polypeptide(s) consists in a CyaA protein or a fragment thereof suitable to present said polypeptide(s) to the immune system in a mammalian host, said first group of epitopes being contained in at least one polypeptide(s) carried by said first vector and (b) a second, separate, vector-carried polypeptide(s) wherein said second vector molecule carrying the polypeptide(s) consists in a CyaA protein or a fragment thereof suitable to present said polypeptide(s) to the immune system in a mammalian host, said second group of epitopes being contained in at least one polypeptide(s) carried by said second vector.
In an embodiment, the invention also relates to a method to obtain in a same mammalian host, (i) an immunotherapeutic treatment of first determined pathological condition(s) diagnosed in a mammalian host, especially by eliciting a T cell immune response against a first group of epitopes, (ii) the prophylaxis against second determined pathological condition(s), especially by eliciting a T cell memory immune response against a second group of epitopes and (iii) optionally the prevention against the re-occurrence of said first determined pathological condition(s), especially by eliciting a T cell memory immune response against said first group of epitopes, wherein said prophylaxis against second determined pathological condition(s) is not observed when said second group of epitopes is not contained in said administered vector-carried polypeptide(s), said method comprising administering at least once to said patient either:
(1) a vector-carried polypeptide(s), wherein said vector carrying the polypeptide(s) consists in a CyaA protein or a fragment thereof suitable to present said polypeptide(s) to the immune system in a mammalian host, said first group of epitopes and second group of epitopes being contained in at least one polypeptide(s) carried by said vector, and at least one epitope of said second group of epitopes being different from the epitopes of the first group of epitopes;
(2) a composition comprising the vector-carried polypeptide(s) as defined in (1) in combination with a pharmaceutically acceptable vehicle; or
(3) a composition comprising (a) a first vector-carried polypeptide(s) wherein said first vector carrying the polypeptide(s) consists in a CyaA protein or a fragment thereof suitable to present said polypeptide(s) to the immune system in a mammalian host, said first group of epitopes being contained in at least one polypeptide(s) carried by said first vector and (b) a second, separate, vector-carried polypeptide(s) wherein said second vector molecule carrying the polypeptide(s) consists in a CyaA protein or a fragment thereof suitable to present said polypeptide(s) to the immune system in a mammalian host, said second group of epitopes being contained in at least one polypeptide(s) carried by said second vector.
The compositions as defined herein may, in a particular embodiment, comprise a pharmaceutically acceptable vehicle or formulation, or physiologically acceptable diluent, which is chosen among buffering agents, saline, phosphate buffered saline, dextrose, glycerol, water, ethanol and the like and combinations thereof.
Moreover, the vector-carried polypeptide(s) or the composition as defined herein as means to obtain an immunotherapeutic treatment and the prophylaxis may be combined or mixed with, or the composition as defined herein as means to obtain an immunotherapeutic treatment and the prophylaxis may further comprise, at least one immunopotentiator, such as at least one adjuvant, preferably one adjuvant, and/or a surfactant and/or immunomodulatory substances (such as cytokines or chemokines). Various adjuvants are known in the art and include Complete Freund's Adjuvant (CFA), Incomplete Freund's Adjuvant (IFA), montanide ISA (incompletre seppic adjuvant), muramyl peptides such as muramyl dipeptide (MDP) MDP-Lys (L18) (Nα-acetylemuramyl-L-alanyl-D-isoglutaminyl-Nesteoroyl-L-lysine), zinc sulphate, colloidal iron hydroxide, calcium phosphate or calcium chloride, CpG oligodeoxynucleotides (CPG ODN) such as CPG ODN 1826 and CPG ODN 2007, MF59 which is a detergent stabilized oil-in water emulsion containing 5% squalene (w/v), 0.5% Tween® 80 (w/v) and 0.5% Span (w/v) in water, TLR4 ligands (such as MPL, GLA) TLR3 ligands (such as Hiltonol®), polysaccharides (such as Inulin) and liposomes (such as cationic liposomes, ISCOMs).
In a particular embodiment, the at least one adjuvant is chosen among molecules which have the capacity to activate T-cell immune response, in particular T-cell memory response. Preferred adjuvants are the ones that bind or are agonist to TLR (Toll like receptor) 3, 4, 7, 8 and/or 9 into immune cells (such as APC). In a particular embodiment, the adjuvant is a TLR ligand, in particular a TLR ligand selected from the group consisting of TLR ligands of class 3, such as poly-ICLC, TLR ligands of class 4, TLR ligands of class 9, such as CpG, and TLR ligands of class 7/8, such as Imiquimod. Examples of adjuvants are Imiquimod sold as a cream containing 5% Imiquimod (Aldara™) and Poly-ICLC sold by Oncovir (Inc, WA, US) as Hiltonal®.
By “combined”, it is meant that the vector-carried polypeptide(s) or the composition as defined herein and the immunopotentiator are both put in contact with the host, at different times and/or by different modes of administration, preferably at the same site of contact. In a particular embodiment, the vector-carried polypeptide(s) or the composition as defined herein is injected into the host and the immunopotentiator (such as an adjuvant) is applied topically, e.g., cutaneously (onto the skin), to the host. For example, the vector-carried polypeptide(s) or the composition as defined herein is injected into the host and the immunopotentiator (such as an adjuvant) is applied onto the skin of the host following the injection, at the site of injection. In contrast, “mixed” means that the vector-carried polypeptide(s) or the composition as defined herein and the immunopotentiator are in the same formulation when administered.
It is noteworthy that in the present application, when an immunopotentiator (such as an adjuvant) is mixed or combined with the vector-carried polypeptide(s) or the composition as defined herein, the immunopotentiator is used, at least, each time the vector-carried polypeptide(s) or the composition of the invention is administered into the host. In a particular embodiment, the vector-carried polypeptide(s) or the composition of the invention is administered twice, and the immunopotentiator is applied (either mixed or combined, preferably cutaneously), at the site of administration of the vector-carried polypeptide(s) or the composition of the invention, the day of each administration. In another particular embodiment, the vector-carried polypeptide(s) or the composition of the invention is administered twice, and the immunopotentiator is applied (either mixed or combined, preferably cutaneously), at the site of administration of the vector-carried polypeptide(s) or the composition of the invention, the day of each administration and the day following the day of each administration. In a particular embodiment, the vector-carried polypeptide(s) or the composition of the invention is administered twice, and the adjuvant (preferably Imiquimod, such as Aldara™) is applied cutaneously at the site of administration of the vector-carried polypeptide(s) or the composition of the invention, the day of each administration and the day following the day of each administration.
The vector-carried polypeptide(s) as defined herein or the composition as defined herein as means to obtain an immunotherapeutic treatment and the prophylaxis and optionally the prevention against the re-occurrence, may additionally be combined or mixed, in administration regimens, with other active compounds suitable to treat pathogen infection, tumor cells or pathological condition(s) associated with this infection or tumor, such as antitumoral or antiviral active compounds.
The vector-carried polypeptide(s) or compositions defined herein can be injected in a patient via different routes: subcutaneous (s.c.), intradermal (i.d.), intramuscular (i.m.) or intravenous (i.v.) injection, oral administration and mucosal administration, especially intranasal administration or inhalation. In a particular embodiment, the vector-carried polypeptide(s) or compositions defined herein is/are administered intradermally.
In a particular embodiment, the vector-carried polypeptide(s) or compositions defined herein, either mixed or combined with at least one immunopotentiator or not, whatever the administration routes, is administered at a site, independent upon the (first) determined pathological condition(s) diagnosed in the mammalian host, i.e., for a tumor, at a site other than the one of the tumor development (for example other than mucosa), and for a pathogen, at a site other than the pathogen replication site.
The vector-carried polypeptide(s) or compositions defined herein may be in a solid form (cachet, powder, gelule, pill, suppository, quick release tablet, gastro-resistant tablet, delayed release tablet), a powder form, preferably after lyophilization (lyophilized form or lyophilized powder form) which needs to be reconstituted for example with diluents(s) before injection, or in a liquid form, such as an injectable solution or injectable suspension.
The quantity of vector-carried polypeptide(s) to be administered (dosage) depends upon the subject to be treated, including considering the condition of the patient, the state of the individual's immune system, the route of administration and the size of the host. The conventional dosages range from 1 to 1200 μg, 100 to 1000 μg, 200 to 1000 μg, 500 to 1000 μg. A particular dosage is chosen from the group consisting of 100, 200, 300, 400, 500, 600, 700, 800, 900 and 1000 μg. In another embodiment, conventional dosages range from 1 to 100 μg, 1 to 50 μg and 1 to 10 μg of vector-carried polypeptide(s). These examples can be modified by one skilled in the art, depending on circumstances.
Each specific embodiment may be combined with any particular embodiment.
Mice
Six weeks-old female C57BL/6 mice (H-2b) are purchased from Charles River Laboratories. Mice are housed under pathogen-free conditions with water and food ad libitum. Procedures involving animals and their care are conformed to Genticel guidelines that comply with national and international laws and policies and that are reviewed by the local ethical committee.
Tumour Cell Lines
TC-1 (tissue culture number one) tumour cells (Lin, Guarnieri et al. 1996) were prepared by transformation of C57BL/6 primary mouse lung cells with HPV16 E6 and E7 oncogenes and activated human c-Ha-Ras oncogene. The cells used in this study have been obtained from the ATCC. TC1 cells are thawed before each experiment and are then cultured and expanded in vitro during at least 10 days before injection.
EG7, the OVA-transfected EL4 lymphoma murine cell line (C57BL/6 genetic background), are used to induce solid tumour expressing the Ovalbumin protein. This model is widely described and used as a murine tumour model of cancer (Schreiber, Deyev et al. 2009). EG7 cells are thawed before each experiment and are then cultured and expanded in vitro during at least 10 days before injection.
Tumour Cells Inoculation
On day 0, C57BL/6 mice are injected with TC-1 cells (0.5×106 cells per mouse for HPV_TUR008, 1×106 cells per mouse for the others studies) diluted in 100 μL of PBS 1× via the subcutaneous route in the flank. In some experiments mice are injected at day 60 with EG7 cells diluted in 100 μL of PBS 1× via the subcutaneous route in the flank.
Vector Preparation
(1) the MAGE A397-178 epitope inserted between residues 319 and 320 of B. pertussis CyaA: LGDNQIMPKAGLLIIVLAIIAREGDCAPEEKIPKKLLTQHFVQENY LEYRQVPGSDPACYEFLWGPRALVETSYVKVLHHMVKISG (SEQ ID NO: 8); and
(2) the MAGE-A3190-295* epitope corresponding to residues 190-221 fused to residues 242-295 of the MAGE A3 sequence, inserted between residues 224 and 235 of CyaA: TFPDLESEFQAALSRKVAELVHFLLLKYRAREPVTKAEMLGSVVGN WQYFFPVIFSKASSSLQLVFGIELMEVDPIGHLYIF (SEQ ID NO:9).
Vaccine Administration
On day 11, after tumour measurement, mice with detectable solid tumours are vaccinated by intradermal (id) injection into ears dermis (both ears are injected). In some experiments, mice are receiving a two-shot vaccination at day 11 and day 39 post-TC-1 tumour cells inoculation. Regarding ProCervix, mice were administered with 5 μg of CyaA-HPV16E7Δ30-42 and 5 μg of CyaA-HPV18E7Δ32-42.
Adjuvant Molecules
Aldara™ is a pharmaceutical formulation of a molecule activating innate immunity via a preferential binding on TLR7 into immune cells as for example APC. This active molecule is Imiquimod, a small synthetic compound. Aldara™ is marketed as a cream containing 5% Imiquimod in 250 mg single-dose use packets (12.5 mg of active Imiquimod). The dose inducing an adjuvant effect for ProCervix mouse vaccination is 25 mg of Aldara™ per immunisation site so respectively 1.25 mg of active Imiquimod per site of injection (50 mg for one mouse). A topical (cutaneous) application of Aldara™ is done the day of the immunisation and 24 h after the immunisation. Individual tubes are prepared, each containing 25 mg of Aldara™ for application on ear skin (2 tubes for one mouse). In order to finely evaluate the real quantity of Aldara™ cream applied on each ProCervix injection site (corresponding to each ear for an individual mouse); each eppendorf tube is weighted before and after deposit of the cream inside. After Aldara™ cream application on ear skin, the weight of eppendorf tubes is re-evaluated to calculate approximately the quantity of cream rubbed into the skin. For each tube, all the cream content is rubbed for at least 15 seconds and until complete skin penetration.
Poly-ICLC (TLR3 agonist) was provided by Oncovir (Inc, WA, US) in vials containing 1 mL of 2 mg/mL opalescent sterile solution. The Poly-ICLC is left in the original recipient and stored at +4° C. Poly-ICLC for injection contains 2 mg/mL of poly-IC stabilized with 1.5 mg/mL poly-L-Lysine and 5 mg/mL sodium carboxymethylcellulose in 0.9% sodium chloride solution and adjusted to pH 7.6-7.8 with sodium hydroxide.
Tumour Measurement
Different parameters are taken into account to evaluate tumour development in mice:
The method for measuring cytotoxicity of CD8+ T cells in vivo has been extensively described (Barchet, Oehen et al. 2000; Ingulli 2007). Briefly, syngeneic splenocytes from naive mice are labeled with different concentrations of CFSE (Carboxyfluorescein succinimidyl ester, Molecular Probes Invitrogen) and either pulsed in vitro with relevant H-2b restricted peptides or let unpulsed. Both peptide-pulsed and unpulsed target cells populations are adoptively transferred intravenously into syngeneic vaccinated hosts and the loss of peptide-pulsed targets is measured by flow cytometry (BD FACSCalibur) into the spleen. The percentage of killing is estimated from the reduction in the ratio of the percentage of pulsed target cells to unpulsed cells, corrected by the initial ratio (see below). Cellular preparations are analyzed by flow cytometry before injection to check-CFSE loading of the different target cells and get reference values (real percentage of each cellular population) for the calculation of the in vivo killing. The three target cells populations are then injected intravenously at a 1:1:1 ratio to each vaccinated mice. The percentage of in vivo killing is calculated as described elsewhere with the following formula (Barber, Wherry et al. 2003)
PERCENT KILLING=100−([(% peptide pulsed in vaccinated/% unpulsed in vaccinated)/(% peptide pulsed before injection/% unpulsed before injection))×100)
IFN-γ ELISpot (Enzyme-Linked-Immunospot) Assay
Frequencies of HPV16 E749-57 and HPV18 E7AS43-49 IFN-γ producing specific CD8+ T cells are evaluated on ex-vivo restimulated splenocytes by an IFN-γ ELISpot assay:
IFN-γ secretion is monitored by a sandwich based ELISpot revealed by BCIP/NBT using streptavidin-AKP. Data were analyzed on a Bioreader 5000-Pro S (Biosys).
Therapeutic/Prophylactic Vaccinations
The therapeutic scheme, detailed below, is summarized in
At day 0, two groups of mice (groups 1 and 2) were inoculated on the right flank with TC-1 cells (1×106 cells per mouse). Then, mice received two vaccinations, the first one at day 11 and the second one at day 39, with Placebo (PBS 1×+urea) (group 1) or with Placebo+Aldara™ (group 2). Groups 1 and 2 are negative control.
At day 0, four groups of mice (groups 3 to 6) were inoculated on the right flank, with TC-1 cells (1×106 cells per mouse) (groups 3 to 6); then, mice received two vaccinations, the first one at day 11 and the second one at day 39, with CyaA-MAGEA397-178/193-295/CyaA-HPV16 E7 (5 μg of each CyaA-based vector) in presence of Aldara™ (group 3), with CyaA-cysOVA/CyaA-HPV16 E7 (5 μg of each CyaA-based vector) in presence of Aldara™ (group 4) or with Aldara™-adjuvanted ProCervix (groups 5 and 6, as positive control for TC-1 tumour elimination). The right flank of these mice was monitored until day 100 (
When it is used, Aldara™ is applied topically (cutaneously), at the site of vaccination, the day of vaccination and the day following the vaccination (i.e., at days 12 and 40).
Mice Bearing HPV16 E7-Expressing Solid Tumours Vaccinated by ProCervix Display High Tumour Regression Rate and Improved Survival
Mice vaccinated at day 11 with PBS and which received Aldara™ application did not display inhibition of tumour growth with the exception of 2 mice out of 10 that fully eliminated the tumor before day 50 (
Mice vaccinated at day 11 with unadjuvanted ProCervix also displayed important tumour outgrowth without tumour-free mice at day 50 (
ProCervix Therapeutic Vaccination Promotes the Development of Both HPV16 E7 and HVP18 E7 Specific Functional Memory CTL
It was described by Rafi Ahmed and colleagues in a murine model of acute viral infection with LCMV that memory CD8+ T cells are able to exhibit rapid lytic potential in vivo (Barber, Wherry et al. 2003). Based on these observations, we decided to investigate if mice that have fully cleared the TC-1-induced solid tumours, following administration of ProCervix at day 11, display functional antigen-specific memory CD8+ T cells after eradication of the tumour.
To this end, at day 60 post-TC-1 cells inoculation, remaining tumour free mice in each group were adoptively transferred with CFSE-loaded syngeneic splenocytes in order to measure in parallel the in vivo cytotoxicity (as described above in the Material and Methods section) of memory CD8+ T cells against the two following H-2b restricted epitopes: HPV16 E749-57 and HPV18 E7AS43-49. In the groups of mice vaccinated by ProCervix either adjuvanted by Aldara™ or adjuvanted by Poly-ICLC, 5 tumour-free mice were taken (randomly selected in the group) to perform the in vivo killing assay. In all tested mice, which have all fully eradicated the tumour, we detected strong cytotoxicity against HPV16 E749-57-pulsed target cells (
Taken together, our data demonstrate the exquisite efficiency of ProCervix at inducing, with only one injection, functional CD8-dependent memory response against both HPV16 E7 and HPV18 E7 antigens. A one shot ProCervix vaccination is able to induce the differentiation of a pool of antigen-specific memory CD8+ T lymphocytes that confer to vaccinated mice, both a long term protection against a secondary challenge with syngeneic grafted cells expressing HPV16 E7 (antigen expressed by tumours) and in parallel a protection against a new challenge with HPV18 E7 expressing syngeneic grafted cells (antigen which is not expressed by tumours and which is only delivered by the CyaA-HPV18E7Δ32-42 vector).
Bivalent CyaA-HPV16E7Δ30-42/CyaA-MAGE A3 Therapeutic Vaccination Provides Eradication of HPV16 E7-Expressing Solid Tumours and Protection Against the Development of MAGE-A3 Induced-Tumours
Mice vaccinated with CyaA-HPV16E7Δ30-42/CyaA-MAGE A3+Aldara as transcutaneous adjuvant (group 3) and mice vaccinated with Aldara-adjuvanted ProCervix (groups 5 and 6) cleared TC1-induced solid tumours within 40 days: 9 out of 10 mice in group 3 (
At day 60, survival mice from groups 3 and 5 were inoculated, in their left flank, with B16-tumour cells expressing the MAGE A3 protein. None of the mice, that were vaccinated with CyaA-HPV16E7Δ30-42/CyaA-MAGE A3 in the presence of Aldara™ and that have cleared TC1-induced tumours, showed B16-MAGEA3 tumour development after challenge (0 out of 9 mice developed B16-MAGEA3 tumours;
In contrast, mice vaccinated with Aldara™-adjuvanted ProCervix and that have cleared TC1-induced tumours develop B16-MAGEA3 tumours (6 out of 8 mice developed B16-MAGEA3 tumours;
In conclusion, these experiments demonstrate that bivalent CyaA may be used, following one administration, both to eradicate TC-1 cells (HPV18E7-specific immune response), and to prevent the development of B16-MAGEA3 tumor cells (MAGE A3-specific T cell mediated memory response). Moreover, results obtained after day 60 showed that the prophylactic effect obtained against the B16-MAGEA3 tumor cells did not impair the therapeutic effect obtained against TC-1 cells (eradiction of TC1 cells until day 100;
Bivalent CyaA-HPV16E7Δ30-42/CyaA-cysOVA Therapeutic Vaccination Provides Eradication of HPV16 E7-Expressing Solid Tumours and Protection Against the Development of EG7-OVA Induced-Tumours
Mice vaccinated with CyaA-HPV16E7Δ30-42/CyaA-CysOVA in presence of Aldara™ as transcutaneous adjuvant (group 4) and mice vaccinated with Aldara-adjuvanted ProCervix (group 5 and 6) cleared TC1-induced solid tumours within 40 days: 9 out of 10 mice in group 4 (
At day 60, survival mice from groups 4 and 6 were inoculated, in their left flank, with EG7-OVA tumour cells (malignant syngeneic cells expressing the ovalbumin protein). In group 6, mouse 6004 developed a tumor after day 60
(
In conclusion, these experiments demonstrate that bivalent CyaA may be used, following one administration, both to eradicate TC-1 cells (HPV18E7-specific immune response), and to prevent the development of EG7-OVA-induced solid tumours. Moreover, results obtained after day 60 showed that the prophylactic effect obtained against the EG7-OVA tumor cells did not impair the therapeutic effect obtained against TC-1 cells (eradiction of TC1 cells until day 100;
The novel concept brought in the present application, with the bivalent ProCervix therapeutic vaccination is, in one hand, to treat for example HPV16 infected patients, eradicate the infection and, in the other hand, to provide T-cell mediated memory responses for both HPV16 E7 and HPV18 E7 antigens, thus establishing for the ProCervix-vaccinated patients a long term protection against possible re-infection with HPV16 and also against later infection with HPV18. This was confirmed with two other bivalent vaccinations (CyaA-HPV16E7αΔ-42/CyaA-MAGE A3 and CyaA-HPV16E7Δ30-42/CyaA-CysOVA) that have been shown, in the one hand, to treat HPV16 infected mice (eradiction of TC1 cells), and in the other hand, to provide protection against the development of B16-MAGE A3 tumors or EG7-OVA tumors respectively.
Using a preclinical murine model of cervical carcinoma [TC-1 tumour cells (Lin, Guarnieri et al. 1996; Zwaveling, Ferreira Mota et al. 2002)], it was shown that therapeutic vaccination of mice bearing solid HPV16 E7-expressing tumours with ProCervix combined to an adjuvant molecule [TLR agonists as Aldara™ (Johnston and Bystryn 2006; Heib, Becker et al. 2007; Schon and Schon 2008) or Poly-ICLC (Longhi, Trumpfheller et al. 2009)] leads to an efficient tumour regression.
Moreover, it was shown that vaccine-induced tumour clearance can be correlated with the presence of long lasting HPV16 E7-specific CTL memory responses in mice that have fully eradicated the tumour. Unexpectedly, it was also put in evidence that in these tumour-free mice, ProCervix therapeutic vaccination generates, in parallel, functional HPV18 E7-specific CTL memory responses. Both HPV16 E7 and HPV18 E7 memory CTL displayed a lytic potential in vivo.
The observations that CyaA-carried polypeptide(s) are able to generate a preventive T-cell memory response(s) against a second group of epitopes in a mammalian host (prophylactic immune response) while enabling to generate a immunotherapeutic treatment of first determined pathological condition(s) diagnosed in said mammalian host by eliciting a T cell immune response against a first group of epitopes, is surprising. Indeed, it is well known that competition exists between different epitopes, either regarding access to APC, processing and presentation by APC and availability of cytokines. This phenomenon leads to a hierarchy of dominant and subdominant epitopes, and enables T-cell immune response to be activated and other T-cell immune response(s) to be suppressed. This phenomenon was expected in the present situation where T cells recognizing the first group of epitopes already exist in the patient before administration of the vector-carried polypeptides (since one or some epitopes of the first group have already been presented to the host immune system), whereas native T cells have to be activated regarding the second group of epitopes. Interestingly, the present invention has shown that, in contrast to what it is expected, the preventive immune response against the second group of epitopes contained in a CyaA-carried polypeptides seems not to be disfavoured with respect to the therapeutic immune response against the first group of epitopes. These results mean that no competition is observed between the immune response induced against the first group of epitopes and the immune response induced against the second group of epitopes. Consequently, the inventors have shown that CyaA-carried polypeptide(s) is/are sufficiently efficient to elicit a T cell immune response within an immunotherapeutic treatment of first determined pathological condition(s) diagnosed in a mammalian host and to elicit a T cell memory immune response within the prophylaxis against second determined pathological condition(s) in the same mammalian host.
| Number | Date | Country | Kind |
|---|---|---|---|
| 11305069 | Jan 2011 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2012/051027 | 1/24/2012 | WO | 00 | 10/8/2013 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2012/101112 | 8/2/2012 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20070072266 | Preville et al. | Mar 2007 | A1 |
| Number | Date | Country |
|---|---|---|
| 1576967 | Sep 2007 | EP |
| 1 894 941 | Mar 2008 | EP |
| WO 2005053738 | Jun 2005 | WO |
| WO 2008025848 | Mar 2008 | WO |
| WO 2008026071 | Mar 2008 | WO |
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| Number | Date | Country | |
|---|---|---|---|
| 20140037670 A1 | Feb 2014 | US |
