US20160296610A1 - Therapeutic cancer vaccine based on stress proteins rendered immunogenic - Google Patents

Therapeutic cancer vaccine based on stress proteins rendered immunogenic Download PDF

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US20160296610A1
US20160296610A1 US15/029,997 US201415029997A US2016296610A1 US 20160296610 A1 US20160296610 A1 US 20160296610A1 US 201415029997 A US201415029997 A US 201415029997A US 2016296610 A1 US2016296610 A1 US 2016296610A1
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cells
stress
proteins
stress proteins
tumour
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BenoÏtè René Eugène PINTEUR
Gilles Guy DEVILLERS
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Brenus Pharma SA
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/13—Tumour cells, irrespective of tissue of origin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001176—Heat shock proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5152—Tumor cells

Definitions

  • the present invention relates to a novel therapeutic approach to the treatment of cancer based on the stress proteins, “chaperone proteins”, including such types as HSP and/or GRP or other proteins that are involved in the resistance mechanisms (LRPs, CTL4, PD-L1, etc.). It concerns in particular a method for preparing pharmaceutical compositions or therapeutic vaccines, the said pharmaceutical compositions or therapeutic vaccines themselves, and a treatment method for therapeutic treatment of cancer.
  • the immune system is based on two defense mechanisms, namely, innate immunity, which is rapid but nonspecific; and acquired immunity, which is slower, but specific and has a memory.
  • innate immunity which is rapid but nonspecific
  • acquired immunity which is slower, but specific and has a memory.
  • the three phases theory includes the first phase, referred to as elimination phase, during which the immune system fights against tumour proliferation entailing the involvement of tissue and environmental changes that are associated with the tumour (mobilisation of non-specific cells (macrophage, NK, s DC)), secretion of anti-proliferative, apoptotic, angiostatic molecules, production of cytokines, mobilization and activation of CD4 and CD8.
  • the “sensitive” tumour cells are eliminated and the immune selection of the most resistant cells is brought about.
  • the mechanisms of resistance that are set in motion are resistance to apoptosis, the secretion of inhibiting cytokines (TGF- ⁇ , IL-10, PGE2, IDO), alteration of the antigen presentation (partial or complete loss of expression of major histocompatibility complex (MHC) class I), the secretion of neutralising molecules, and MICA and MICB expression, a “counter-attack” of the cell mediated immune system by expression of Fas-L, PD-L1 leading to the death of T lymphocytes.
  • TGF- ⁇ , IL-10, PGE2, IDO inhibiting cytokines
  • MHC major histocompatibility complex
  • MICA and MICB expression a “counter-attack” of the cell mediated immune system by expression of Fas-L, PD-L1 leading to the death of T lymphocytes.
  • This phase corresponds to the remission phase observed during the treatments, it is also a selection phase for selecting the most virulent clones by their capacity to resist the immune defense system or the treatment.
  • escape phase the third phase, referred to as escape phase, the cells that are resistant to the various protective mechanisms of the immune system having developed one or more means of escape proliferate in the absence of any control. The cancer cells then develop a tumour mass that is the clinical manifestation of the physiological escape phenomenon. A related escape phenomenon is also observed in the advanced and metastatic stages of the cancers during the phenomena of resistance to the treatments.
  • the anti-cancer treatments include surgery, wherein the objective is primarily to remove or reduce the tumour, but this “mechanical” action does not have any real inhibitory effect on the process of carcinogenesis and is generally supplemented by various different treatment therapies aimed at “eliminating” the origin of the cancer.
  • Radiotherapy is intended to bring about an alteration in the DNA of rapidly proliferating cells, which is the case with the tumour cells.
  • the side effects of radiotherapy are twofold: even the healthy cells are irradiated, thereby causing the destruction thereof or genetic alterations which can lead to the “cancerization” thereof, and the tumour cells develop resistance to radiation-induced apoptosis by means of production of Chaperone proteins or chaperones (HSP, GRP, etc.) resulting in an escape phenomenon.
  • Chaperone proteins or chaperones HSP, GRP, etc.
  • Chemotherapy is intended to eliminate the tumour cells by acting either on the cells themselves, or by inhibiting specific metabolic pathways: direct interaction with DNA (electrophilic agents, intercalating agents, splitting agents), indirect interaction with DNA (inhibitors of DNA synthesis such as antimetabolites, topoisomerase inhibitors; inhibitors of spindle formation), neovascularisation inhibitors, proteasome inhibitors.
  • direct interaction with DNA electrophilic agents, intercalating agents, splitting agents
  • indirect interaction with DNA inhibitortors of DNA synthesis such as antimetabolites, topoisomerase inhibitors; inhibitors of spindle formation
  • neovascularisation inhibitors proteasome inhibitors.
  • Immunotherapy comprises passive immunotherapy which consists of providing a significant amount of effectors, and active immunotherapy whereof the objective is to induce a specific immune response.
  • Passive immunotherapy is based on the injection of antibodies so as to block a receptor, induce cell lysis, stimulate cytotoxicity, lift the inhibition of apoptosis (cetuximab-Erbitux®, Bevacizumab-Avastin®, Rituximab-Mabthera®, etc).
  • cytokines is another strategy to stimulate protective immune responses against the tumour (IL2, Inf ⁇ , etc).
  • the objective of active immunotherapy or immunisation by vaccination is to immunize patients against cancer in accordance with different strategies: activation of the immune system by activation of dendritic cells, TLR agonists, tumour cell lysate, non-proliferating tumour cells either modified or not.
  • These approaches constitute the new advanced-therapy medicinal products (ATMPs). They are promising and give good results, even more so if they are coupled with other treatments.
  • the first active immunotherapy treatment to have been approved by the Food and Drug Administration (FDA) is Sipuleucel-T Provenge® based on the activation of the T lymphocytes (TLs) of the patient by culture in the presence of specific tumour antigens.
  • FDA Food and Drug Administration
  • escape phenomena are observed that are similar to the natural escape phenomena as described in the 3Es theory involving the immune system.
  • the various different anti-tumour mechanisms are all supported by the underlying strategy of the direct or indirect attack of tumor cells, with the ultimate step being the lysis of the cell or apoptosis thereof.
  • the apoptosis resistance factor thus seems to be a key phenomenon because it induces a direct escape (inhibition of induced apoptosis) or indirect escape by protecting the tumour cells against intermediate mechanisms of destruction.
  • the mechanism of resistance to stress, and in particular to heat shock appears to be universal and present in all living organisms (bacteria, plants, animals).
  • the function of the HSPs is characterized by the protective and reparative activity of certain proteins and enzymes by forming molecular complexes that inhibit the denaturation and the formation of “improper bonds” following as a result of “metabolic attacks” (hypoxia, low carbohydrate concentration, etc), aggressive physical attacks (thermal, radiation), or chemical/medication attacks.
  • the objective of the present invention is to provide a comprehensive overall approach, that is to say, which provides the ability to act simultaneously on multiple resistance mechanisms, preferably the entire set of these mechanisms which are developed by the tumour cells.
  • the objective of the invention is also to provide for the possibility of this approach being adapted to the stress or stresses to which the tumour cells are subjected in vivo, in particular in the course of the treatment protocol applied to a patient.
  • the objective of the invention is also to propose an approach that provides for a standardization related to the use of cell lines.
  • the objective of the invention therefore also relates to ensuring that this approach takes into account the type and/or the grade of the tumour cells, in particular those of the patient being treated.
  • the objective of the invention is thus also to propose such an approach that may in and of itself constitute a therapeutic protocol, or which is an adjuvant protocol of another therapeutic protocol.
  • the further objective of the invention is also to propose such an approach that is patient-specific.
  • the objective of the present invention is to stimulate the immune system by means of stress proteins that have been rendered immunogenic, in particular through haptenisation, in order to bring about the elimination of cells expressing the said resistance factors (stress proteins).
  • This immunological treatment is aimed at eliminating or controlling the cells that can express these factors of resistance.
  • the therapeutic approach proposed is intended in particular to be used against cancer on the basis of the following points:
  • the invention thus concerns in particular a method for producing a composition that contains stress proteins induced by the application of a stress in vitro on a culture of tumour cells. More particularly, the said method produces such stress proteins in immunogenic form, that is to say capable of being recognized by the immune system of the patient and of leading the latter to develop an immune response against these proteins and the tumour cells that present such proteins.
  • the method advantageously includes a step of inactivation of the tumour cells in order to render them non-proliferative.
  • the invention also relates to compositions, in particular pharmaceutical compositions or therapeutic vaccines that contain such proteins.
  • the invention also relates to methods of treatment against cancer by means of administration of such compositions, by way of principle therapeutic strategy or combined (in particular adjuvant) with another treatment protocol.
  • the invention relates to, and the method provides the ability to prepare, a pharmaceutical composition which comprises a plurality or all or the majority of the stress proteins expressed by the tumour cells under the conditions of the stress applied to them.
  • This constitutes an advantage of the resulting product obtained which, by being representative of a plurality of stress proteins, allows the pharmaceutical composition to include a whole range of stress proteins rendered immunogenic that constitute further immunogenic elements against which the immune system of the patient will be able to react, and then target a whole panel of tumour cells and counteract the mechanisms of resistance that have variously been developed by these tumour cells under the pressure from the immune system and/or from the treatments applied.
  • these stress proteins will be found in the compositions according to the invention in one or more of the following forms: in the free state, in the state associated with the membrane of a tumour cell (including intra-membrane), presented at the surface of a tumour cell, present within the interior of a tumour cell, associated with a fragment of tumour cell or with a tumour peptide.
  • composition according to the invention therefore will preferably include one or more of the identified stress proteins documented to date and at a given point in time.
  • the following section presents several families thereof that are known to the person skilled in the art.
  • HSP70 The HSPs are classified into six families according to their molecular weight, but their conformations and their characteristics within the same family seem to be variable according to their origin.
  • HSP70s which include HSP72 and 73 among others
  • HSP70 the distinction can be made between those that respond to heat shock (HSP70) and those that are very weakly affected by the heat stress (HSP70).
  • the HSP25/HSP27 weakly expressed in normal cells, are overexpressed in tumour cells, and would play a role in the resistance to certain treatments and promote the growth and development of the proliferative tumour cells. Another role would be the inhibition of apoptosis by interaction with the pathway involving Caspase-3, regulation in the production of TN F- ⁇ , etc.
  • HSP47s The family of HSP47s is the one most recently identified, and has a role that is yet poorly defined. It would mainly participate in the development of connective tissues, in particular during the production of collagen. Being included among the “Heat Shock Proteins” its involvement in the resistance of tumour cells is likely, in particular, its participation during the neovascularization of tumours. HSP40 would also play a role of “co-chaperone” in association with HSP70.
  • the HSP60 (chaperonin) have been identified in bacteria (GroEL for Growth E. Coli large protein), and found in plants. These chaperone proteins participate in the tertiary conformation of the protein structure. This family of HSPs would play a role in immunity and in particular in autoimmune diseases.
  • HSP70s The family of HSP70s is one of the most studied families and it is along with HSP90, the ones that are the most widespread. There exist several subclasses that would be either expressed by the cells in the ‘normal’ state, or induced directly following a stress.
  • the characteristic features of these HSPs include their ability to interact with other proteins during their translation (folding), their participation in membrane transport, the inhibition of apoptosis as chaperone protein.
  • the chaperone activity of HSP70 is complex and entails the involvement of several factors such as HSP40, Hip (Hsc Interactive Protein), Hop (Hsc-HSP90 Organizing Protein) that enable the ATPase activity necessary for the formation of “chaperon-substrate” complexes.
  • HSP-70s The anti-apoptosis action of HSP-70s is, it seems multifaceted and involves numerous inhibition/activation mechanisms on the pro-Caspase 9,8,3 pathways (inhibition), interaction with the p53 pathway (stabilization of the anti-P53 protein), inhibition of pro-apoptotic factors (Bax), regularization of cytochrome C, etc.
  • HSP90s are the most expressed constitutively and are present in all eukaryotes. They also come to be overexpressed during the phenomenon of stress and are targeted at the stabilization and the renaturation of proteins. Several isoforms have been identified according to their locations, their origins and their functionalities.
  • the HSP90s after dimerization have the ability to interact with the co-chaperones and thus form protein complexes that allow for the mutation and protection of molecules involved in tumoral transformation of cells (stabilization of specific proteins): P90 participates in the regulation of apoptosis (inhibition of Bcl-2, and Apaf-1), participates in cell proliferation and in metastasis (hTERT, MMP2), inhibits the p53, participates in the mutation by alteration then stabilization of the proteins.
  • the HSP110s (or 105) are mainly induced as a result following stress. They would participate in the protection of ribosomes that are particularly sensitive during heat shocks. Their protective effect is mainly the dissociation of protein aggregates, their re-solubilization which allows for the reactivation of these proteins.
  • the HSP110s would seem to be a subgroup of the HSP70s with identical binding domains and therefore certain similar properties.
  • the GRPs constitute the other major group of stress proteins. Generally linked to the HSPs, their main role in the mechanisms of resistance is related to the protection of protein structures. Thus GRP78 (BiP) would have a chaperone role with malformed proteins to be destroyed normally which thereby explains the resistance of cancer cells. GRP75 known under the name “mortalin” would have an antiproliferative action in normal cells which, following its deregulation would participate in the cancerization of cells, while resulting in the opposite effect by inactivation of p53 participating in the regulation of apoptosis. GRP94 also participates in the protection of incompletely assembled proteins whose three-dimensional conformation was imperfect. Related to HSP70 it participates in the resistance to certain anti-cancer agents.
  • Multi Drug Resistance Other protein resistance factors, described as “Multi Drug Resistance”, such as GP170, MPRs (seven different ones), LRPs, and more recently VMP1 participate in the therapeutic escaping.
  • An object of the invention therefore relates to a method for preparing a composition comprising stress proteins of tumour cells, which include the following steps:
  • tumour cells ii) subjecting the tumour cells under i) to a stress and ensuring the result that these cells produce stress proteins in response to the stress;
  • the culture medium may be a medium that makes it possible to maintain the viability of the cells and/or the growth or multiplication thereof.
  • the media that are well-known to the person skilled in the art may be used. These media generally comprise a base culture medium “Minimum Essential Medium” that allow for cell survival wherein it is possible to add one or more mitogenic factors by means of serum or by using defined growth factors (“synthetic serum-free” medium).
  • the step ii) is preferably applied to a culture medium comprising tumour cells in growth phase or plateau phase.
  • the person skilled in the art can readily determine that the cells are in one of these phases. In the first place, it is possible to determine in advance the culture conditions allowing to attaining these phases after a determined time period. It is also possible to determine that these phases have been attained by means of the following methods: cell growth curve, viability, doubling time, metabolite assay, or nutrient consumption, etc.
  • the stress is applied in vitro. It may be of any kind, in terms of chemical or physical nature, and the time period for application of the stress may vary in proportions that depend on the type of stress applied, the nature and application time period for each being chosen in order to ensure that the tumour cells produce stress proteins to a level that is appropriate for the invention, for example so as to take into account the anticancer protocol being applied to the patient.
  • the following methods may be used:
  • the conditions for each type of stress can be easily determined, with the aim of stressing these living cells in order to obtain the production of stress proteins, without inducing apoptosis or the death of the tumour cells or of too many of them, as has been indicated here above.
  • the methods of Western blot and FACS cytometry for example make it possible to monitor the production of particular stress proteins, for example HSP, by using antibodies directed against these proteins. Such antibodies are available, as will be discussed further below in respect of some examples of HSP. It is also possible to check by means of routine tests in vitro (for example dose-effect tests) whether the conditions of a stress are too drastic, resulting in the death of the tumour cells.
  • a centrifugation is carried out after the first stress, then a wash, a centrifugation, then followed by addition of fresh medium, lag phase, then the subsequent stress, etc.
  • the person skilled in the art has at their disposable the means enabling it, such as analysis by flow cytometry, for example, FACS, ELISA, Western blot, etc. They can confirm the expression or production of the types of stress proteins, from their membership in a particular family, and even identify and quantify the particular type.
  • the step iii) may be followed by a step iii-i) in which the resulting outcome of the previous step is treated in accordance with one or more of the following modalities: concentration of solid and proteinaceous materials, including stress proteins and tumour cells and fragments thereof, separation or purification of stress proteins or tumour cells, separation or purification of tumour cells and stress proteins, extraction of stress proteins.
  • concentration of solid and proteinaceous materials including stress proteins and tumour cells and fragments thereof, separation or purification of stress proteins or tumour cells, separation or purification of tumour cells and stress proteins, extraction of stress proteins.
  • the stress proteins may be divided into intracellular, extracellular free, intramembrane (cell or cell fragment), at the surface of a cell or a cell fragment.
  • the stressed cancer cells include intracellular stress proteins and surface stress proteins.
  • step iv) a treatment process is carried out that makes it possible to render the stress proteins immunogenic or immunocompetent, that is to say capable of generating in vivo an immune response against them.
  • the time interval between the end of the step of applying the stress and the beginning of the subsequent step is sufficient for the tumour cells to have produced the stress proteins.
  • This interval can be determined by varying the said time interval and determining or measuring the change in the expression of stress proteins.
  • the methods of Western blot and flow cytometry e.g. FACS, using the specific antibodies for the proteins to be monitored (see below), may be employed.
  • This interval is preferably several hours, and it may in particular be comprised between about 5 hours and about 24 hours.
  • This interval may be less than 5 hours, it is possible to determine it by monitoring the production of stress proteins, by using the methods described herein, Western blot and FACS cytometry. It may also be longer than 24 hours, but such a long period may not be necessary, this can also readily be determined using the methods of the abovementioned measures.
  • a molecule that provides the means to confer immunogenicity to the stress proteins is introduced into the product resulting from iii) or iii-i).
  • This step iv) in vitro provides the ability for example to chemically bond the said molecule to the proteins present, whether the latter are free or present on the surface or within the interior of a cell membrane or a cell structure, for example a tumour cell or a tumour cell fragment.
  • the molecule for example of haptenisation, is a molecule that is not naturally present in the tumour cells or in its environment (“non-naturally occurring molecule”).
  • haptens immunogenic by combining an immunogenic molecule (a “carrier”) and a hapten (in the case in hand the stress proteins) bonded in a covalent manner to this molecule.
  • any carrier molecule or mixture of known carrier molecules may be used.
  • DNFB 2,4-Dinitrofluorobenzene
  • EDA N-iodoacetyl-N′-(5-sulfonic-naphthyl) ethylene diamine
  • aniline p-amino benzoic acid
  • the “carrier” molecules are capable of penetrating the membrane of the cell and reaching the cytosol.
  • the free stress proteins, the intracellular stress proteins and those bonded to the cell membranes are haptenised.
  • the haptenisation step includes the incubation in vitro of the stressed cells and the carrier molecule. Typically, the incubation may last from around 15 minutes to around 1 hour, in particular around 20 minutes to around 40 minutes.
  • This step is preferably carried out under mild agitation that provides the ability to maintain the cells in suspension or under a certain agitation condition.
  • the step that is used to render the proteins immunogenic (for example haptenisation) step iv) may be followed by a step iv-i) in which the resulting outcome of the previous step is treated in accordance with one or more of the following modalities: concentration of solid and proteinaceous materials, including stress proteins and tumour cells and fragments thereof, separation or purification of stress proteins or tumour cells, separation or purification of tumour cells and stress proteins, extraction of stress proteins.
  • concentration of solid and proteinaceous materials including stress proteins and tumour cells and fragments thereof, separation or purification of stress proteins or tumour cells, separation or purification of tumour cells and stress proteins, extraction of stress proteins.
  • the tumour cells may be more or less degraded or fragmented.
  • the main objective of the invention is not to degrade or fragment these cells, but to lead them to express their panoply of stress proteins. This signifies that a certain proportion of tumour cells may remain viable.
  • a step that serves the purposes of inactivating the viable cells may be provided for.
  • This step referred to as the inactivation step is carried out downstream of the application of the stress and after a time period that is sufficient for the cells to have expressed the stress proteins.
  • the product derived from the step that is used to render the proteins immunogenic step iv) or step iv-i) may be treated, in particular inactivated, (step v)), in order for the tumour cells possibly present to be rendered non-proliferative.
  • step v it is the product derived from the stress step which may be treated, in particular inactivated, (step v)) in order for the tumour cells possibly present to be rendered non-proliferative.
  • the inactivation is placed between steps ii) and iv).
  • any inactivation treatment known to the person skilled in the art may be employed, provided that it ensures the means that cause the tumour cells to lose their ability to proliferate in vivo when the composition is administered to the patient.
  • This inactivation can be achieved in particular by means of a chemical treatment process (ethanol fixation) or by means of a physical treatment process, for example high dose radiation (for example about 25 gray). It is possible to determine the right conditions for inactivation, by carrying out cell culture tests, in order to determine the conditions that lead to a total lack of viability. It is also possible to use propidium iodide which provides the ability to distinguish between living cells and dead cells, as is known to the person skilled in the art. It is also possible for example, to use the test with propidium iodide followed by culturing as described here below.
  • the product derived from the step iv) or from the step v) is formulated by mixing of the product rendered immunogenic (haptenized protein material and/or haptenised cells and/or haptenized cell fragments) with a pharmaceutically acceptable vehicle or excipient and possibly an adjuvant.
  • the step includes the mixing with a pharmaceutically acceptable vehicle or excipient and an adjuvant.
  • the method provides the ability to prepare a pharmaceutical composition which comprises a plurality, or all, or the majority of the stress proteins expressed by the tumour cells under the conditions of the stress applied to them.
  • tumour cells used in step i) may be patient cells, derived from biopsies, possibly cultured or maintained live, possibly pools derived from various different patients, in particular allogeneic cells (same cell type); cells from the patient (autologous cells); allogeneic cells from pre-established lines or produced from patient cells, or mixtures of such cells; mixtures of these different types of cells.
  • these whole cells are advantageously from cell lines, and in particular from lines of the same type as the tumour of the patient to be treated (allogeneic cells).
  • These cells are “stimulated” by one of the stress factors, thereby enabling the overexpression of resistance factors.
  • a line may be subjected to a heat shock, a chemical agent, an irradiation, a metabolic stress or to a plurality of these stresses during the culturing thereof.
  • the “stimulated” lines or the raw untreated or treated product derived from the stress protocol are then rendered immunogenic in order to enable the recognition of the stress proteins by the immune system.
  • several lines, in particular allogeneic may be used.
  • groups of cells are subjected to various different stresses. It is possible for example to subject a first portion of the cells to one type of stress protocol selected from those described here above, and then subject a second portion of the cells to one of the other stress protocols (nature of stress may be different or same nature of stress but with different conditions), etc. They may then be used in the form of a kit-of-parts for administration, which may be simultaneous, separate or staggered over time. They may also be mixed.
  • a pharmaceutical composition is made available which contains stress proteins that are potentially specific to each of the mechanisms of resistance depending on the type and/or the stress conditions applied (for example irradiation, thermal, chemical, metabolic, etc.).
  • the method is applied to a mixture of allogeneic tumour cells, that is to say of the same tumour type, or by way of a variant, the method is applied separately to at least two populations of allogeneic tumour cells, then the products obtained that have been rendered immunogenic are mixed in order to constitute the pharmaceutical composition.
  • the invention therefore makes it possible to provide such a solution which allows for a targeted approach based on the type of tumour cell, that is to say, the use of different cell types, but all deriving from the same type of cancer in order to potentiate the common immune reactions against the resistance factors expressed by all of these cells of one same given type of tumour (allogeneic cells), or expressed specifically by certain tumour cells.
  • the cells derived from this tumour are maintained in a medium thereby providing for their viability and/or growth, and then subjected to one (or more) of the stresses cited.
  • the autologous cells thus stimulated express the stress proteins, which are then rendered immunocompetent, for example, during the subsequent haptenisation.
  • the method is applied to a mixture of autologous tumour cells, that is, say, to the tumour cells originating from the patient to be treated, or by way of a variant, the method is applied separately to at least two populations of autologous tumour cells. They may then be used in the form of a kit-of-parts for administration, which may be simultaneous, separate or staggered over time. They may also be mixed.
  • the invention therefore makes it possible to provide a personalized therapeutic approach, specific to the individual who has developed the tumour in order to maintain the factors of resistance and the protein environment specific to the patient's tumour by using autologous cells.
  • the previous solutions are combined, with the kit-of-parts or mixtures of autologous cells and allogenic cells, the autologous and allogeneic cells preferably being allogeneic to each other, or with the mixture of products thereof rendered immunogenic according to the method of invention.
  • the cells When working from the patient tumour cells, it is preferable to begin with the cells isolated from a biopsy or resection.
  • the composition containing immunogenic stress proteins, and/or, preferably and, inactivated tumour cells is frozen or lyophilized.
  • the object of the present invention therefore also relates to a composition that may be obtained or produced by the method of the invention.
  • compositions of the invention are characterized by the fact that they include stress proteins that are rendered immunogenic in accordance with the invention, in particular haptenised.
  • This composition may also be characterized by the fact that it includes tumour cells which are those that have produced the stress proteins in response to the stress according to step ii), in particular tumour cells and/or debris or fragments of such cells that have been produced by the said method.
  • This composition may comprise immunogenic stress proteins, in particular haptenised, that are free and/or immunogenic stress proteins, in particular haptenised, that are presented on the surface or within the interior of the tumour cells or fragments thereof.
  • the compositions of the invention include inactivated tumour cells and immunogenic stress proteins, which are in particular haptenised.
  • compositions include a plurality, or all, or the majority of the stress proteins expressed by the tumour cells in vitro following as a result of a stress applied to them, and they are in an immunogenic form, in particular haptenised.
  • stress proteins when talking about “stress proteins”, reference is being made to at least two different stress proteins, preferably 2, 3, 4, 5, 6, 7, 8, 9, or 10, or even more stress proteins, this varying based on the cell type and on the stress or stresses applied in vitro.
  • the composition contains one or more heat shock proteins, in particular chosen from those described here above, for example HSP27, HSP70 or HSP90; HSP27, HSP70 and HSP90; HSP27 and HSP70; HSP90 and HSP27; HSP90 and HSP70.
  • this or these HSPs is/are rendered immunogenic, in particular haptenised.
  • the composition comprises one or more proteins with pleiotropic resistance to drugs, in particular proteins with resistance to chemotherapy (MRP, multidrug resistance-associated protein), for example GP170, the MPRs (seven different proteins), the LRPs and/or VMP1.
  • MRP proteins with resistance to chemotherapy
  • GP170 proteins with resistance to chemotherapy
  • MPRs even different proteins
  • LRPs Long RPs
  • VMP1 proteins with resistance to chemotherapy
  • the composition comprises proteins with radiation resistance or even proteins with resistance to metabolic stress.
  • the composition comprises GRPs, such as GRP78 (BiP), GRP75 and/or GRP94.
  • GRPs such as GRP78 (BiP), GRP75 and/or GRP94.
  • the composition may include stress proteins of several of these categories.
  • the invention relates more particularly to a pharmaceutical composition or a therapeutic vaccine or an immunogenic composition comprising stress proteins that are rendered immunogenic, preferably haptenised.
  • These stress proteins may be in the free state, in the state associated with the membrane of a tumour cell (including intra-membrane), presented on the surface of a tumour cell, present within the interior of a tumour cell, and/or associated with a fragment of tumour cell or with a tumour peptide.
  • the composition comprises stress proteins that are rendered immunogenic, preferably haptenised.
  • the composition comprises tumour cells which are those that have produced stress proteins in response to the stress according to the step ii), including inactivated tumour cells.
  • These cells may include and advantageously do include intracellular stress proteins, that are haptenised or otherwise rendered immunogenic in accordance with the invention. In vivo, these intracellular immunogenic stress proteins will in particular be presented to the immune system by the antigen presenting cells.
  • composition contains tumour cells and/or fragments of these cells, containing and/or bearing immunogenic stress proteins, that are preferably haptenised.
  • the composition contains both immunogenic stress proteins, that are free and preferably haptenised, and tumour cells and/or fragments of these cells, containing and/or bearing immunogenic stress proteins, that are preferably haptenised.
  • these cells are rendered incapable of proliferation, this being referred to as inactivation.
  • composition may also include a pharmaceutically acceptable vehicle or excipient.
  • the composition comprises a population of immunogenic stress proteins, that are preferably haptenised and/or cells containing such proteins, which have been subjected to a first protocol of stress, and at least one other population of immunogenic stress proteins, that are preferably haptenised and/or cells containing such proteins, which have been subjected to another protocol of stress.
  • the composition comprises a population of immunogenic stress proteins, that are preferably haptenised and/or cells containing such proteins, and at least one other population of immunogenic stress proteins, that are preferably haptenised and/or cells containing such proteins, the populations being derived from initial allogeneic tumour cells and these cells having been subjected to a protocol of stress as a mixture or separately.
  • the composition comprises immunogenic stress proteins, that are preferably haptenised and/or cells containing such proteins, derived from initial autologous tumour cells.
  • the composition comprises immunogenic stress proteins, that are preferably haptenised and/or cells containing such proteins, that are allogeneic and autologous.
  • the composition comprises immunogenic stress proteins, that are preferably haptenised and/or cells containing such proteins, that are allogenic and autologous, for simultaneous, separate or staggered over time administration.
  • the composition may be constituted of multiple doses of stress proteins that have been rendered immunogenic and/or cells containing allogeneic stress proteins that have been rendered immunogenic, in particular derived from different lines (1, 2, 3, . . . lines) and multiple doses of stress proteins that have been rendered immunogenic and/or cells containing autologous stress proteins that have been rendered immunogenic.
  • compositions according to the invention are useful as anti-cancer drugs.
  • they are useful as anti-cancer drugs for administration to a patient who has undergone an anti-cancer treatment. As explained here above, this treatment would have resulted in a stress and therefore the production of stress proteins. The patient thus bears tumour cells presenting stress proteins.
  • compositions are useful as anti-cancer drugs for administration to a patient who is undergoing or will undergo anti-cancer treatment.
  • this treatment is likely to result in a stress and therefore the production of stress proteins.
  • the patient whose treatment is ongoing either already has or is suspected to have tumour cells presenting the stress proteins.
  • the composition in addition comprises an adjuvant.
  • the adjuvant is a substance which acts by increasing the efficacy of a vaccine. This substance may act by accelerating, by prolonging and/or by augmenting the immune responses specific to an immunogen when this substance is used in combination with this specific immunogen.
  • the person skilled in the art may refer in particular to Recent Advances in Vaccine Adjuvants, Singh M & O'Hagan DT, Pharmaceutical Research 2002, Volume 19, issue 6, pp 715-728.
  • cytokines and other immunomodulation molecules that is, chemokines and co-stimulatory factors
  • adjuvant substances derived from microorganisms and plants or which are chemically synthesized.
  • the adjuvants can act not only as immunostimulatory adjuvants but also as a vaccine delivery system.
  • These delivery systems are generally particulates and mention may be made, in particular, of emulsions, microparticles (for example based on polylactide-glycolide PLG), ISCOMs, liposomes, which serve the purpose mainly of orienting and guiding the immunogens to the antigen presenting cells.
  • the immunostimulatory adjuvants they are mainly molecules of microbiological origin, for example lipopolysaccharide, monophosphoryl lipid A, CpG DNA, which activate the immune system.
  • Adjuvants may also be used for delivery by means of the mucosal pathway, for example, bacterial enterotoxins, in particular deriving from E. coli , heat-labile enterotoxins, detoxified mutants such as K63 or R72.
  • adjuvants By way of representative useable adjuvants mention may be made, among others, of: aluminum hydroxide, saponins (for example Quillaja saponin or Quil A; see Vaccine Design, The Subunit and Adjuvant Approach, 1995, edited by Michael F.
  • pharmaceutically acceptable is used to refer to a vehicle or excipient that can be used for administering an immunogen to a patient without risk of any allergic or other adverse reaction while also preserving the characteristic features of the product during storage thereof.
  • This includes, for example, alone or in mixture, water, saline solutions, phosphate buffers, protein compounds, dextrose, sucrose, glycerol, DMSO, ethanol and the like. Mention may be made for example, of a saline solution with 0.9% NaCl, a phosphate buffer, a mixture based on commercially available preservative solutions.
  • the composition according to the invention is intended to be frozen, in particular between ⁇ 20° C. and ⁇ 80° C. Then at least one cryoprotective agent is added to the composition, and/or the composition is prepared in the usual manner in order for it to tolerate the freezing without damage to its components, in particular the tumour cells.
  • composition according to the invention is intended to be lyophilized (freeze-dried). Then as may be necessary, at least one usual excipient for lyophilization is added.
  • composition of the vehicle or excipient and/or the choice of the adjuvant may vary depending on the route of administration, as is known to the person skilled in the art.
  • the product developed by the invention may have a variable composition, however insofar as it has characteristic features such as can be retained, it is that it includes stress proteins that have been induced in vitro by the subjecting of live tumour cells to one or more controlled stress(es), that these stress proteins are found at high concentration levels due to their overexpression by these tumour cells in response to the stress, and that generally these stress proteins are multiple in type, that these stress proteins in the final product that can be administered to the patient, are haptenised or otherwise treated so as to render them immunocompetent (immunogenic).
  • the principle underlying the invention is based on the capacity and ability of the immune system to develop a specific response against the resistance factors of the tumour cell (stress proteins) after having been stimulated by the presence of stress proteins that have been rendered immunocompetent (immunogens), for example being haptenised.
  • This immune reaction eliminating the tumour cells completes the actions initiated during the prior treatments (adjuvant action of the composition according to the invention) or enables the activation of the immune response as the principal treatment.
  • the object of the invention also relates to a method of therapeutic treatment.
  • This method comprises administration of an effective amount of a composition according to the invention in order to provoke the immune response.
  • the dose administered may for example comprise from 10 5 to 10 7 , in particular at least 10 6 ⁇ 0.5 cells per dose.
  • This quantity of cells can be expressed as total tumour cells, in tumour-originated carbon (TOC) or in terms of protein quantification.
  • TOC tumour-originated carbon
  • the dosage regimen may include one or more dose administrations separated over time.
  • the regimen may provide for 2, 3, 4, 5, 6, 7, 8, 9 or 10 administrations spaced out over an interval of 1 to 10 days between each administration.
  • any route of administration may be employed.
  • routes that may be cited include oral, nasal, rectal, and parenteral routes.
  • the parenteral route is preferred, in particular via intramuscular-, intravenous-, intraperitoneal-, subcutaneous-, and intradermal injection. Intradermal injection is particularly selected.
  • the treatment may be supplemented with the administration of immunostimulant(s) of any type, thus providing for the potentiation thereof (cytokine, growth factor, immunomodulator, adjuvant . . . ), whether or not the therapeutic composition has already been adjuvanted.
  • immunostimulant(s) of any type, thus providing for the potentiation thereof (cytokine, growth factor, immunomodulator, adjuvant . . . ), whether or not the therapeutic composition has already been adjuvanted.
  • the method for treatment may include the combination of a treatment according to the invention with a conventional anti-cancer treatment protocol: surgery, radiation therapy, chemotherapy and/or immunotherapy.
  • the compositions according to the invention are administered to the patient before, during or after one or more of these protocols.
  • composition according to the invention comprising radiation-induced stress proteins and/or cells that express radiation-induced stress proteins, prior to a radiation therapy treatment and/or administration of the composition according to the invention, comprising chemically induced stress proteins and/or cells that express the chemically induced stress proteins, by using preferably the anti-cancer therapeutic molecule as initiator of the stress, prior to a chemotherapy treatment with this molecule.
  • FIG. 1 is a Western Blot showing the induction of the expression of HSP70 by a thermal stress.
  • the main calibration spots and HSP70 spots have been encircled, with the latter being identified by the reference numeral 1.
  • FIG. 2 is a Western Blot showing the induction of HSP70 by heat stress and the haptenisation of HSP70, with in parallel a Western Blot performed on a ⁇ -actin control. In order to ensure ease of reading, the main spots have been encircled. Identifying reference numerals: HSP70 (2); HSP70 haptenised (3); ⁇ -actin (4); ⁇ -actin haptenised (5); Molecular Weight Marker (6); CT26WT Cells treated for 1 hour at 37° C. (7); CT26WT Cells treated for 1 hour at 42° C.
  • FIGS. 3 and 4 are graphs each representing the evolving change over time of the volume of tumours induced in mice between the control mice injected with an excipient (“Veh” for “vehicle”) and the mice treated with vaccines according to the invention (“The” for “Therapeutic”).
  • FIGS. 5 and 6 are graphs showing, for the same groups represented in the FIGS. 1 and 2 , the weight of the tumour in grams induced in the mice at the time of resection (“Veh” for “vehicle” or excipient; “The” for Therapeutic).
  • the cell lines are derived from commercially available pre-established lines (ATCC type) or resulting from the constitution of the line derived from the patient sample, characterized, and tested.
  • ATCC type commercially available pre-established lines
  • the lines that are used include Caov, OVCAR-3, ES-2, and OV-3, for an ovarian cancer therapeutic target. They are cultured separately in the recommended conditions.
  • the patient cells are isolated from the patient and cultured in a medium and under the appropriate conditions.
  • the cells from the cell lines and patient are subjected during the course of their growth phase or their plateau phase to a stress so as to enable the overexpression of factors of resistance (stress proteins).
  • the factors of resistance (stress proteins) expressed by the allogeneic cells are chemically labeled or tagged with Dinitrophelyl.
  • stress proteins genes expressed by the allogeneic cells are chemically labeled or tagged with Dinitrophelyl.
  • sulphanilic acid N-iodoacetyl-N′-(5-sulfonic-naphthyl) ethylene diamine (EDA), aniline, or p-amino benzoic acid).
  • the allogeneic cells comprising the haptenised stress proteins are then rendered non-proliferative by high dose irradiation (25 gray).
  • high dose irradiation 25 gray
  • use may be made of ethanol fixation between 10% and 50% v/v or any other method that provides for the inhibition of cell proliferation while maintaining the cell structure intact.
  • the haptenised cellular stress proteins of allogeneic origin are distributed in the form of cell suspension in a formulation medium that is suitable for therapeutic use and allows for the preservation thereof at low temperature ( ⁇ 20° C., ⁇ 80° C.), and then distributed in doses comprising from 1 to 5 10 6 inactivated and haptenised cells per dose corresponding to a therapeutic dose expressed in protein amount “HSP positive” and/or in organic matter amount.
  • An immunizing adjuvant may be present, for example BCG, GM-CSF, IL2.
  • the cells are derived from the tumour of the patient after resection.
  • the biological material is transported in a specific kit that makes possible its preservation while ensuring optimal viability of the cells.
  • the cells of the biopsy are dissociated by a suitable mechanical method, and then placed in suspension in a nutrient medium that provides for their growth or viability only.
  • tumour cells are used as they are or following a selection by means of cell sorting.
  • tumour cells in the expansion phase or in the stationary phase are subjected to a stress as defined in the production of allogeneic cells.
  • the factors of resistance (stress proteins) expressed by autologous cells are chemically tagged by a method similar to that described for the allogeneic cells in Example 1.
  • the autologous cells including the haptenised stress proteins are rendered non-proliferative by the same method as that used for the allogeneic cells in Example 1.
  • the haptenised stress proteins integrated in the autologous cells are distributed in the form of a cell suspension in a formulation medium that is suitable for therapeutic use and provides for their preservation and then distributed in therapeutic doses.
  • the therapeutic treatment regimen includes the administration of:
  • the administration of the product is by way of an intradermal injection. It is also possible to use another route of administration and in particular per os.
  • the product is administered alone or in synergy with any other therapy that allows for a potentiation of the treatment.
  • the CT26-WT line is a mouse colon carcinoma line, available under ATCC® CRL-2638TM.
  • the cells grow easily and rapidly (doubling time 22 hours).
  • the HL60 line is a human cell line (Caucasian promyelocytic leukemia) available from Sigma-Aldrich®. The cells are frozen after the expansion.
  • the original cells had been received in frozen form.
  • the cells were thawed, after which they were put to culture in flasks, in an appropriate culture medium.
  • the culture medium was changed on D+1.
  • a count of the live cells was performed, then the concentration was adjusted to about 2 ⁇ 10 6 living cells/ml of culture medium.
  • a pool of 200 ⁇ 10 6 cells was recovered, and then washed after confirming that the cells were at approximately 50% confluence.
  • the suspension was then divided into 50 ml tubes based on 25 ml per tube. The tubes were then immersed in a water bath heated to 42° C. The tubes were allowed to remain for about 1 hour in the water bath.
  • the cells were then transferred into the flasks at a rate of 2 ⁇ 10 6 cells. Thereafter the cells were incubated for 14 hours at 37° C. The cell concentration was then adjusted to 5 ⁇ 10 6 living cells/ml.
  • the cells were then haptenised with a solution of 2,4-dinitrofluorobenzene (DNFB) 0.07%.
  • DNFB 2,4-dinitrofluorobenzene
  • the haptenisation is revealed by labeling with FACS.
  • FACS flow cytometry was chosen as the analytical technique, it provides the ability to give a percentage of cells haptenised in relation to the total number of cells.
  • the cell suspension is adjusted to a concentration of 2 to 5 ⁇ 10 6 cells/ml with freezing medium and stored in a freezer at ⁇ 80° C. ⁇ 3° C. for a minimum of 24 hours before testing them.
  • the frozen cells were then subjected to irradiation by X-rays at 25 grays, in order to inactivate the tumour cells.
  • anti-HSP27 antibodies use was made of the commercially available anti-HSP27 antibodies, anti-HSP70 antibodies, anti-HSP 90 ⁇ / ⁇ antibodies.
  • commercially marketed antibodies that are usable are available from Santa Cruz Biotechnology Inc. and BD Biosciences.
  • the haptenised cells were thawed, they were subsequently washed and the concentration was adjusted from 1 to 2 ⁇ 10 6 cells/ml. The following were deposited in a V bottom 96-well plate:
  • control antibody anti-KLH FITC
  • HSPs An overexpression of HSPs and more specifically of the HSP27 and 70 in the stressed cells is noted.
  • tumour doses consisted in bringing about expansion of the CT26.WT cells, followed thereafter by a freezing in the culture medium supplemented with 5% DMSO and at a rate of 6.25 ⁇ 10 4 cells in 250 ⁇ l final.
  • the production of vaccine doses consisted in bringing about the expansion of the CT26.WT cells, then after 2 passages the cells were subjected to a heat stress for 1 hr at +42° C. followed by a recovery time of 14 h at 37° C. Then, the cells were haptenised (by DNFB, dinitrofluorobenzene), then frozen at ⁇ 80° C. based on 6.25 ⁇ 10 5 cells in 250 ⁇ l final and finally the vaccine doses were irradiated (25 Gray).
  • Tumour doses production of the tumour in animals
  • mice BALB/c male and female mice aged 6 to 8 weeks, obtained from Charles River, were used. 5 mice were placed per 16 ⁇ 19 ⁇ 35 cm cage under controlled temperature (22 ⁇ 2° C., in alternating lighting conditions (12 h cycles of day and 12 hours of dark) and supplied with water and food ad libitum. The mice were acclimated for at least 1 week before starting the experiments.
  • tumour model is the subcutaneous CT26 carcinoma (CT26WT, ATCC® CRL-2638TM), which is a syngeneic tumour model commonly used for the study of therapeutic applications against cancer in animals, in particular for testing immunotherapy protocols and study the immune response.
  • CT26WT subcutaneous CT26 carcinoma
  • ATCC® CRL-2638TM a syngeneic tumour model commonly used for the study of therapeutic applications against cancer in animals, in particular for testing immunotherapy protocols and study the immune response.
  • mice 50 female mice were divided into 5 groups of 10. Each mouse was injected subcutaneously (SC) on day 0 (DO) with 5 ⁇ 10 4 CT26WT tumour cells.
  • SC subcutaneously
  • DO subcutaneously
  • CT26WT tumour cells 5 ⁇ 10 4

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FR3011850B1 (fr) 2018-04-06
CA2927616C (fr) 2023-03-28
CA2927616A1 (fr) 2015-04-23
US20190038731A1 (en) 2019-02-07
EP3057981B1 (fr) 2021-11-10
CN105722853A (zh) 2016-06-29

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