Patent Application
20090304688
The present invention relates, generally, to methods and compositions for increasing the efficiency of therapeutic antibodies.
Various therapeutic strategies in human beings are based on the use of therapeutic antibodies. These include, for instance, the use of therapeutic antibodies developed to deplete target cells, particularly diseased cells such as virally-infected cells, tumor cells or other pathogenic cells. Such antibodies are typically monoclonal antibodies, of IgG species, typically with human IgG1 or IgG3 Fc portions. These antibodies can be native or recombinant antibodies, and are often “humanized” mouse antibodies (i.e. comprising functional domains from various species, typically an Fc portion of human or non human primate origin, and with a variable region or complementary determining region (CDR) of mouse origin). Alternatively, the monoclonal antibody can be fully human through immunization in transgenic mice having the human Ig locus, or obtained through cDNA libraries derived from human cells.
A particular example of such therapeutic antibodies is rituximab (Mabthera®, Rituxan®), which is a chimeric anti-CD20 monoclonal antibody made with human γ1 and κ constant regions (therefore with human IgG1 Fc portion) linked to murine variable domains conferring CD20 specificity. In the past few years, rituximab has considerably modified the therapeutical strategy against B lymphoproliferative malignancies, particularly non-Hodgkin's lymphomas (NHL). Other examples of humanized IgG1 antibodies include alemtuzumab (Campath-1H®), which is used in the treatment of B cell malignancies, and trastuzumab (Herceptin®), which is used in the treatment of breast cancer. Additional examples of therapeutic antibodies under development are disclosed in the art.
The mechanism of action of therapeutic antibodies is still a matter of debate. Injection of antibodies leads to depletion of cells bearing the antigen specifically recognized by the antibody. This depletion can be mediated through at least three mechanisms: antibody mediated cellular cytotoxicity (ADCC), complement dependent lysis, and direct antitumor inhibition of tumor growth through signals given via the antigen targeted by the antibody.
While these antibodies represent a novel and efficient approach to human therapy, particularly for the treatment of tumors, they do not always exhibit a strong efficacy. For instance, while rituximab, alone or in combination with chemotherapy, was shown to be effective in the treatment of both low-intermediate and high-grade NHL, 30% to 50% of patients with low grade NHL have no clinical response to rituximab. It has been suggested that the level of CD20 expression on lymphoma cells, the presence of high tumor burden at the time of treatment, or low serum rituximab concentrations may explain the lack of efficacy of rituximab in some patients. Nevertheless, the actual causes of treatment failure remain largely unknown.
Further, the use of therapeutic antibodies can be limited by side effects caused by their administration. For example, side effects such as fever, headaches, nausea, hypotension, wheezing, rashes, infections, and numerous others can appear in patients, potentially limiting the possible amount or frequency with which the antibodies can be administered.
Thus, it would be very advantageous to increase the efficacy of therapeutic antibodies, or to be able to achieve therapeutic efficacy using reduced doses of the antibodies that are less likely to produce side effects. The present invention addresses these and other needs.
The present invention discloses novel approaches to enhance the efficacy of therapeutic antibodies. Indeed, the present invention provides novel compositions and methods that overcome the current difficulty related to the efficacy of therapeutic antibodies, particularly those intended to deplete a target cell (e.g. tumor cell, infected cell, inflammation-mediating cell, etc.). It is shown in the present invention that Vγ9δ2 T cells from an individual can effect the efficiency of therapeutic mAb (monoclonal antibody) when γδ T cells are activated and/or expanded, pointing to a synergy of therapeutic antibodies and γδ T cell activator therapies. Such conjoint use of a γδ T cell activator and therapeutic antibody leads to greater target cell lysis than observed using the therapeutic antibody along. It has also been observed that activation and/or expansion of γδ T cells can re-establish target cell lysis in cases where very little or no lysis was observed prior to activation and/or expansion of γδ T cells.
Therefore, the invention discloses methods of treatments of a subject in which a γδ T cell activator compound is co-administered with the therapeutic antibody to the subject. The inventors demonstrate here that the efficiency of a therapeutic antibody can be greatly enhanced by the co-administration, e.g., co-injection, of such a γδ T cell activator, preferably a phosphoantigen, that activates cell responses such as lysis, cytokine release and/or induces the proliferation of these cells. Optionally the phosphoantigen is further administered in conjunction with a cytokine. Preferably, the cytokine will be any cytokine capable of giving rise to the proliferation of γδ T cells when administered in conjunction with a γδ T cell activator, for example IL-2.
The present invention concerns a pharmaceutical composition comprising a γδ T cell activator, a therapeutic antibody and a pharmaceutically acceptable carrier. The present invention also concerns a kit or a product containing a γδ T cell activator and a therapeutic antibody as a combined preparation for simultaneous, separate or sequential use in the treatment of a disease.
The present invention also concerns the use of a γδ T cell activator and a therapeutic antibody for the preparation of a medicament for treating a disease.
The invention also concerns the use of a γδ T cell activator for increasing the efficiency of a treatment with a therapeutic antibody. In particular, the present invention concerns the use of a γδ T cell activator for the preparation of a drug for increasing the efficiency of a treatment involving the administration of a therapeutic antibody in a subject, wherein the administration of said therapeutic antibody is administered to said subject prior to, simultaneously with, or following, a therapeutically-effective amount of a γδ T cell activator.
In a particular aspect, the present invention provides a method of treatment of a disease or of eliminating a cell in a human subject in need thereof, comprising: a) administering to said subject a γδ T cell activator; and, b) administering to said subject a therapeutic antibody.
In another aspect, the present invention provides a method of eliminating a cell, comprising: a) bringing said cell into contact with a therapeutic antibody; and, b) bringing said cell into contact with an activated γδ T cell. In one aspect, the method is carried out in vitro and the cell to be eliminated is in a biological sample. Optionally, step (a) comprises adding an activated γδ T cell to the sample, or comprises adding a γδ T cell and a γδ T cell activator to the sample. In another aspect, the method is carried out in vivo, and said cells to be eliminated are in a mammalian subject. Optionally, step (a) comprises administering to said subject an activated γδ T cell, or administering to said subject a γδ T cell activator. Optionally, in said in vivo or in vitro methods, said activated γδ T cell has been brought into contact with a γδ T cell activator prior to addition to the sample or administration to the subject.
Preferably, the therapeutic antibody targets diseased cells such as virally-infected cells, tumor cells, cells underlying an autoimmune disorder or other pathogenic cells. It can be a monoclonal, human, humanized or chimeric antibody or an antigen binding fragment thereof. In a preferred embodiment, the therapeutic antibody is an anti-CD20 antibody (e.g. rituximab), and anti-HER2/Neu antibody (e.g. herceptin) or and anti-CD52 antibody (e.g. campath).
Preferably, the γδ T cell activator is a compound of formula (I):
wherein Cat+ represents one (or several, identical or different) organic or mineral cation(s) (including proton);
m is an integer from 1 to 3;
B is O, NH, or any group capable to be hydrolyzed;
Y═O−Cat+, a C1-C3 alkyl group, a group -A-R, or a radical selected from the group consisting of a nucleoside, an oligonucleotide, a nucleic acid, an amino acid, a peptide, a protein, a monosaccharide, an oligosaccharide, a polysaccharide, a fatty acid, a simple lipid, a complex lipid, a folic acid, a tetrahydrofolic acid, a phosphoric acid, an inositol, a vitamin, a co-enzyme, a flavonoid, an aldehyde, an epoxyde and a halohydrin;
A is O, NH, CHF, CF2 or CH2; and,
R is a linear, branched, or cyclic, aromatic or not, saturated or unsaturated, C1-C50 hydrocarbon group, optionally interrupted by at least one heteroatom, wherein said hydrocarbon group comprises an alkyl, an alkylenyl, or an alkynyl, preferably an alkyl or an alkylene, which can be substituted by one or several substituents selected from the group consisting of: an alkyl, an alkylenyl, an alkynyl, an epoxyalkyl, an aryl, an heterocycle, an alkoxy, an acyl, an alcohol, a carboxylic group (—COOH), an ester, an amine, an amino group (—NH2), an amide (—CONH2), an imine, a nitrile, an hydroxyl (—OH), a aldehyde group (—CHO), an halogen, an halogenoalkyl, a thiol (—SH), a thioalkyl, a sulfone, a sulfoxide, and a combination thereof.
In a more preferred embodiment, the γδ T cell activator is a compound of formula (II):
in which X is an halogen (preferably selected from I, Br and Cl), B is O or NH, m is an integer from 1 to 3, R1 is a methyl or ethyl group, Cat+ represents one (or several, identical or different) organic or mineral cation(s) (including the proton), and n is an integer from 2 to 20, A is O, NH, CHF, CF2 or CH2, and Y is O−Cat+.
In particular, the γδ T cell activator can be BrHPP, C—BrHPP or N—BrHPP. In an other more preferred embodiment, the γδ T cell activator is a compound of formula (XII):
in which R3, R4, and R5, identical or different, are a hydrogen or (C1-C3)alkyl group, W is —CH— or —N—, R6 is an (C2-C3)acyl, an aldehyde, an (C1-C3)alcohol, or an (C2-C3)ester, Cat+ represents one (or several, identical or different) organic or mineral cation(s) (including the proton), B is O or NH, m is an integer from 1 to 3, A is O, NH, CHF, CF2 or CH2, and Y is O−Cat+.
In particular, the γδ T cell activator can be HDMAPP, C—HDMAPP or N—HDMAPP.
Preferably, the γδ T cell activator can also be an aminophosphonate of formula XVII:
with R′ being a linear, branched, or cyclic, aromatic or not, saturated or unsaturated, C1-C50 hydrocarbon group, wherein said hydrocarbon group comprises an alkyl, an alkylenyl, or an alkynyl, preferably an alkyl or an alkylene, which is substituted by one or several substituents selected from the group consisting of: an amine, an amino group (—NH2), an amide (—CONH2), an imine, and a combination thereof.
In a preferred embodiment, R′ of formula XVII is a linear, branched, or cyclic, aromatic or not, saturated or unsaturated, C1-C10 hydrocarbon group, which is substituted by an amine, an amino group, a pyridine group, a pyrimidine group, a pyrrole group, an imidazole group, a pyrazole group, a triazole group.
In a still more preferred embodiment, R′ of formula XVII is selected from the group consisting of:
In particular, the γδ T cell activator can be selected from the group consisting of pamidronate, alendronate, ibandronate, risedronate and zoledronate.
In another aspect of any of the embodiments, any one of the γδ T cell activator listed herein can be excluded, including but not limited to a aminophosphonates, for example zoledronate can be excluded.
In a particular embodiment, the disease requires the depletion of the targeted cells, preferably the diseased cells such as virally-infected cells, tumor cells, cells underlying an autoimmune disorder or other pathogenic cells. The disease can be selected from the group consisting of a cancer, an auto-immune disease, an inflammatory disease, and an infectious (e.g. bacterial or viral) disease. The disease also concerns a graft rejection, more particularly allograft rejection, and graft versus host disease (GVHD).
In particular, an object of the present invention is to provide an efficient combination treatment with an anti-CD20 antibody, preferably rituximab and a γδ T cell activator which is more efficient for the depletion of B-lymphomas than rituximab alone.
In particular, another object of the present invention is to provide an efficient combination treatment with an anti-CD20 antibody, preferably rituximab and a γδ T cell activator which delays the reconstitution of B cell population, thereby improving the effectiveness of B-cell depletion therapy in vivo. The present invention provides a combination treatment of a γδ T cell activator and a therapeutic antibody for improving the depletion of B-lymphomas. In another aspect, the present invention also provides a combination treatment of a γδ T cell activator and a therapeutic antibody for delaying the reconstitution of B cell population. Preferably, the therapeutic antibody is an anti-CD20 antibody, such as rituximab.
Therapeutic antibodies such as anti-CD20 antibodies (e.g. rituximab), anti-HER2/Neu antibody (e.g. herceptin) or and anti-CD52 antibody (e.g. campath) are now commonly used in the treatment of cancer, and include a wide range of biological mechanisms. Therapeutic antibodies agents are in most cases the first line of treatment. Nevertheless, therapeutic antibodies are not effective for all patients and depending on the situation, a large percentage of patients is unresponsive or refractory. Moreover, once patients are treated with therapeutic antibodies their tumors may “escape” and become yet more resistant to other therapies. There has therefore been an active search for drug combinations in order to improve treatment.
The present invention relates to compositions and methods useful for treating a cancer in mammals, including humans. The methods and compositions typically comprise use of a therapeutic antibody and a γδ T cell activator, such that the composition is effective for treating a cancer. Preferably the composition enhances the effect of the therapeutic antibody prevents or delays the escape of a tumor from classic antibody therapy.
The present invention also provides a scheme of administration of a combination of a γδ T cell activator and a therapeutic antibody for the treatment of a disease, comprising administering the therapeutic antibody prior to the γδ T cell activator. Preferably, the therapeutic antibody is administered once weekly, for a treatment cycle of about a few weeks, typically around 3 weeks and the γδ T cell activator is administered conjointly with the therapeutic antibody. Most preferably the γδ T cell activator is administered substantially at the same time than the second administration of the therapeutic antibody. Optionally, a cytokine is administered for a period comprised between 3 and 10 days, the first cytokine administration taking place on the same day as the γδ T cell activator administration. Preferably, the cytokine is IL-2 and IL-2 is administered for 5 consecutive days with each γδ T cell activator administration.
However, these therapeutic antibody therapies do not completely eradicate the tumor, and while they manage to control the growth of a tumor for a period of time the tumor eventually escapes control and is then resistant to the therapeutic antibody therapy and/or other therapies. A means to prevent the escape of the tumor would be advantageous.
In another aspect, the invention encompasses a method for killing or inhibiting a proliferating (e.g. tumor) cell, for enhancing the anti-tumor effect of an antibody therapy, for enhancing the anti-tumor effect of a γδ T cell activating therapy, for preventing the escape of a tumor from control by an antibody therapy, and/or for preventing resistance of a tumor to antibody therapy, in a mammal, the method comprising: conjointly administering to the mammal a γδ T cell activating compound and a therapeutic antibody. Also provided is the use of a γδ T cell activator for the manufacture of a pharmaceutical composition or medicament, wherein said pharmaceutical composition or medicament is used or administered in combination with a therapeutic antibody. Also encompassed are related pharmaceutical compositions and kits comprising such compositions.
The present invention provides improved means of preventing the escape of a tumor, particularly a solid tumor. The method of the invention therefore also provides methods of prolonging or enhanced survival in a human patient with a tumor. The method also provides a means for preventing the progression of a tumor treated with a therapeutic antibody. In another embodiment the invention provides a method of preventing a tumor or a tumor cell from becoming resistant to treatment with a therapeutic antibody.
Where “comprising” is used, this can preferably be replaced by “consisting essentially of”, more preferably by “consisting of”.
As used in the specification, “a” or “an” may mean one or more. As used in the claim(s), when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one. As used herein “another” may mean at least a second or more.
Where hereinbefore and hereinafter numerical terms are used, they are meant to include the numbers representing the upper and lower limits. For example, “between 1 and 3” stands for a range “from and including 1 up to and including 3”, and “in the range from 1 to 3” would stand for “from and including 1 up to and including 3”. The same is true where instead of numbers (e.g. 3) words denoting numbers are used (e.g. “three”).
“Weekly” stands for “about once a week” (meaning that more than one treatment is made with an interval of about one week between treatments), the about here preferably meaning ±1 day (that is, translating into “every 6 to 8 days”); most preferably, “weekly” stands for “once every 7 days”.
“3-weekly” or “three-weekly” stands for “about once every three weeks” (meaning that more than one treatment is made with an interval of about three weeks between treatments), the about here preferably meaning ±3 days (that is, translating into every 18 to 24 days); most preferably, “weekly” stands for “once every 21 days” (=every third week).
The term “about” or “approximately” usually means within 20%, more preferably within 10%, and most preferably still within 5% of a given value or range. Alternatively, especially in biological systems (e.g., when measuring an immune response), the term “about” means within about a log (i.e., an order of magnitude) preferably within a factor of two of a given value.
As used herein, the terms “conjoint”, “in combination” or “combination therapy”, used interchangeably, refer to the situation where two or more therapeutic agents affect the treatment or prevention of the same disease. The use of the terms “conjoint”, “in combination” or “combination therapy” do not restrict the order in which therapies (e.g., prophylactic or therapeutic agents) are administered to a subject with the disease. A first therapy can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapy to a subject with a disease.
As used herein, the terms “substantially at the same time” usually means, at the same time, or within a short period of time such as 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours.
Combination of Therapeutic Antibodies and γδ T Cell Activators
The present invention concerns a pharmaceutical composition comprising a γδ T cell activator and a therapeutic antibody. The pharmaceutical composition can further comprising a pharmaceutically carrier. It can also comprise other active agent. In particular, the composition can further comprise a cytokine, preferably an interleukin, more preferably IL-2 (aldesleukin, Proleukin®). Indeed, IL-2 can provide improved in vivo expansion of γδ T cells. Furthermore, the compositions of this invention may further comprise or may be used in combination with other active agents or therapeutic programs such as chemotherapy or other immunotherapies, either simultaneously or sequentially. The present invention also concerns kits comprising a therapeutic antibody and a γδ T cell activator. In addition, the present invention concerns a product containing a γδ T cell activator and a therapeutic antibody as a combined preparation for simultaneous, separate or sequential use in the treatment of a disease. More particularly, the treatment of the disease requires the depletion of the targeted cells, preferably the diseased cells such as virally-infected cells, tumor cells, cells underlying an autoimmune disorder, or other pathogenic cells. Preferably, the disease is a cancer, infectious or immune disease. More preferably, the disease is selected from the group consisting of a cancer, an auto-immune disease, an inflammatory disease, and an infectious (e.g. bacterial or viral) disease. The disease also concerns a graft rejection, more particularly allograft rejection, and graft versus host disease (GVHD).
In addition, the present invention concerns a pharmaceutical composition comprising a therapeutic antibody and γδ T cells activated by a γδ T cell activator. γδ T cells activated by a γδ T cell activator can be prepared as described in the patent application US 2005-0196385, the disclosure of which is incorporated herein by reference. The present invention also concerns kits comprising a therapeutic antibody and γδ T cells activated by a γδ T cell activator. The present invention concerns a product containing a therapeutic antibody and γδ T cells activated by a γδ T cell activator as a combined preparation for simultaneous, separate or sequential use in the treatment of a disease.
Compositions of this invention may comprise any pharmaceutically acceptable carrier or excipient, typically buffer, isotonic solutions, aqueous suspension, optionally supplemented with stabilizing agents, preservatives, etc. Typical formulations include a saline solution and, optionally, a protecting or stabilizing molecule, such as a high molecular weight protein (e.g., human serum albumin).
The invention also concerns the use of a γδ T cell activator and a therapeutic antibody for the preparation of a medicament for treating a disease. The present invention further concerns a method for treating a disease in a subject comprising administering a γδ T cell activator and a therapeutic antibody to the subject. The administration of the γδ T cell activator and the therapeutic antibody can be simultaneous, separate or sequential. The method can further comprise the administration of a cytokine, in particular of IL-2. More particularly, the treatment of the disease requires the depletion of the targeted cells, preferably the diseased cells such as virally-infected cells, tumor cells or other pathogenic cells.
The invention also concerns the use of a therapeutic antibody and γδ T cells activated by a γδ T cell activator for the preparation of a medicament for treating a disease. The present invention further concerns a method for treating a disease in a subject comprising administering a therapeutic antibody and γδ T cells activated by a γδ T cell activator to the subject. The administration of γδ T cells activated by a γδ T cell activator and the therapeutic antibody can be simultaneous, separate or sequential. The method can further comprise the administration of a cytokine, in particular of IL-2.
The contemplated diseases include neoplastic proliferation of hematopoietic cells. Optionally, said diseases are selected from the group consisting of lymphoblastic leukemia, acute or chronic myelogenous leukemia, Hodgkin's lymphoma, Non-Hodgkin's lymphoma, myelodysplastic syndrome, multiple myeloma, and chronic lymphocytic leukemia. Said diseases also include ENT cancers, colorectal cancers, breast cancer, epithelial cancer. Said diseases include viral infection, such as CMV infection, and hepatitis B. Said diseases include inflammatory diseases, such as Crohn disease, rheumatoid arthritis, asthma, psoriasis, multiple sclerosis or diabetes. In particular, any disease listed in the table 1 can be treated.
According to the methods and compositions of the present invention, compounds, preferably γδ T cell activators and therapeutic antibodies are administered in an “efficient” or “therapeutically effective” amount. Preferably, the therapeutically effective amount will be an amount of a therapy (e.g., a therapeutic agent) which is sufficient to ameliorate a disease or condition, or one or more symptoms thereof, or prevent the advancement of the disease or condition, or improve the therapeutic effect(s) of another therapy (e.g., a therapeutic agent or other physical treatment). Effective doses will also vary, as recognized by those skilled in the art, depending on the diseases treated, route of administration, excipient usage, and the possibility of co-usage with other therapeutic treatments such as use of other agents.
Preferably, treating an individual or subject comprises the reduction or amelioration of the progression, severity, and/or duration of a disease or condition, or one or more symptoms thereof that results from the administration of one or more therapies (e.g., one or more prophylactic and/or therapeutic agents).
Preferably, preventing a disease or condition in an individual or subject comprises the prevention of the recurrence, onset, or development of a disease or condition, or one or more symptoms. thereof in a subject, said prevention resulting from a therapy (e.g., the administration of a prophylactic or therapeutic agent), or a combination therapy (e.g., the administration of a combination of prophylactic or therapeutic agents).
In preferred embodiments, treating a cancer comprises preventing the development of a cancer, reducing the symptoms of cancer, and/or inhibiting the growth, reducing the size and/or inducing the destruction of an established cancer. In other aspects, a medicament is administered to a subject at risk of developing a cancer for the purpose of reducing the risk of developing the cancer.
In one embodiment, the method of treatment according to the present invention further comprises an additional step in which the activity or number of γδ T cells in the subject is assessed prior or subsequent to the administration of the γδ T cell activator. In another embodiment, the additional step involves i) obtaining γδ T cells from the subject prior to the administration; ii) incubating the γδ T cells in the presence of one or more target cells that are recognized by the therapeutic antibody, in the presence or absence of the γδ T cell activator; and iii) assessing the effect of the γδ T cell activator on the ability of the γδ T cells to deplete the target cells; wherein a detection that the γδ T cell activator enhances the ability of the γδ T cells to deplete the target cells indicates that the compound is suitable for use in the method, and that the method is suitable for use with the subject.
The present invention also concerns a method of killing target cells in a subject comprising administering a γδ T cell activator and a therapeutic antibody targeting the target cells to the subject. In particular, the target cells can be cancer cells, infected cells, antibody-coated cells. The present invention further concerns a method for increasing the killing of target cells in a subject comprising administering a γδ T cell activator to the subject, wherein a therapeutic antibody targeting the target cells is administered to the subject. The therapeutic antibody targeting the target cells can be administered before, simultaneously or after the γδ T cell activator. The present invention concerns in addition a method of killing target cells in a subject comprising administering a therapeutic antibody targeting the target cells and γδ T cells activated by a γδ T cell activator to the subject.
The invention also concerns the use of a γδ T cell activator (or activated γδ T cells) for increasing the efficiency of a treatment with a therapeutic antibody. In particular, the present invention concerns the use of a γδ T cell activator (or activated γδ T cells) for the preparation of a drug for increasing the efficiency of a treatment involving the administration of a therapeutic antibody in a subject, wherein the administration of said therapeutic antibody is administered to said subject prior to, simultaneously with, or following, a therapeutically-effective amount of a γδ T cell activator (or activated γδ T cells). The present invention also concerns a method for increasing in a subject the efficiency of a treatment with a therapeutic antibody, wherein a γδ T cell activator (or activated γδ T cells) is administered to the subject prior to, simultaneously with, or following the administration of a therapeutic antibody.
In one embodiment, the candidate compound enhances the ability of the therapeutic antibody to destroy the target cells by 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more.
The present invention also comprises a method for reducing the dosage of a therapeutic antibody, e.g. an antibody that depletes a target cell, in particular by administering a γδ T cell activator (or activated γδ T cells). For example, co-administration of a therapeutic antibody and a γδ T cell activator (or activated γδ T cells) allows a lower dose of the therapeutic antibody to be used. Such antibodies can be used at a 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% lower dose than the recommended dose in the absence of the compound.
In an important embodiment of the invention, the use of the γδ T cell activator (or activated γδ T cells) can allow therapeutic efficacy to be achieved with reduced doses of therapeutic antibodies. The use (e.g., dosage, administration regimen) of therapeutic antibodies can be limited by side effects, e.g., in the case of rituximab, fever, headaches, wheezing, drop in blood pressure, and others. Accordingly, while in many patients a standard dose of the therapeutic antibodies will be administered in combination with γδ T cell activators (or activated γδ T cells), thereby enhancing the efficacy of the standard dose in patients needing ever greater therapeutic efficacy, in other patients, e.g., those severely affected by side effects, the administration of the γδ T cell activators (or activated γδ T cells) will allow therapeutic efficacy to be achieved at a reduced dose of therapeutic antibodies, thereby avoiding side effects. In practice, a skilled medical practitioner will be capable of determining the ideal dose and administrative regimen of the therapeutic antibody and the γδ T cell activator (or activated γδ T cells) for a given patient, e.g. the therapeutic strategy that will be most appropriate in view of the particular needs and overall condition of the patient. Numerous references are available to guide in the determination of proper dosages, for both the therapeutic antibodies and the γδ T cell activators (or activated γδ T cells), e.g., Remington: The Science and Practice of Pharmacy, by Gennaro (2003), ISBN: 0781750253; Goodman and Gilmans The Pharmacological Basis of Therapeutics, by Hardman, Limbird & Gilman (2001), ISBN: 0071354697; Rawlins E. A., editor, “Bentley's Textbook of Pharmaceutics”, London: Bailliere, Tindall and Cox, (1977); and others.
In one embodiment, a medical practitioner can gradually lower the amount of the therapeutic antibody given in conjunction with the administration of any of γδ T cell activator (or activated γδ T cells); either in terms of dosage or frequency of administration, and monitor the efficacy of the therapeutic antibody; e.g. monitor γδ T cell activity; monitor the presence of target cells in the patient, monitor various clinical indications, or by any other means, and, in view of the results of the monitoring, adjust the relative concentrations or modes of administration of the therapeutic antibodies and/or γδ T cell activator to optimize therapeutic efficacy and limitation of side effects.
Suitable doses of the γδ T cell activators and/or therapeutic antibodies can also generally be determined in vitro or in animal models, e.g. in vitro by incubating various concentrations of a therapeutic antibody in the presence of target cells, γδ T cells, and varying concentrations of one or more γδ T cell activators, and assessing the extent or rate of target cell depletion under the various conditions, using standard assays (e.g. as described in the examples section). Alternatively, varying dosages of the therapeutic antibodies can be given to animal models for diseases treatable with the antibodies (e.g. an animal model for NHL in the case of rituximab), along with varying dosages of the γδ T cell activators, and the efficacy of the antibodies (e.g. as determined by any suitable clinical, cellular, or molecular assay or criterion) in treating the animals can be assessed.
The composition or product according to the present invention may be injected directly to a subject, typically by intra-venous, intra-peritoneal, intra-arterial, intramuscular or transdermic route. Several monoclonal antibodies have been shown to be efficient in clinical situations, such as Rituxan (Rituximab) or Xolair (Omalizumab), and similar administration regimens (i.e., formulations and/or doses and/or administration protocols) may be used with the composition of this invention. The γδ T cell activators (or activated γδ T cells) and therapeutic antibodies can be administered by the same route or by different routes.
Therefore, the invention provides a method for determining a therapeutically-effective, reduced dose of a therapeutic antibody, e.g., an antibody intended to cause the depletion of a target cell, the method comprising i) co-incubating a first concentration of the therapeutic antibody with target cells and γδ T cells in the absence of a γδ T cell activator; ii) co-incubating a second, lower concentration of the therapeutic antibody with target cells and with γδ T cells in the presence of a γδ T cell activator; iii) determining if the depletion of target cells observed in step ii) is as great as the depletion observed in step i). If it is observed that step ii) is as efficacious as step i), then the relative concentrations of the γδ T activator and the therapeutic antibody can be varied, and depletion observed, in order to identify different conditions that would be suitable for use in a given patient, e.g., maximizing target cell depletion, lowered dose of therapeutic antibody, or lowered dose of the compound, depending on the particular needs of the patient.
In another aspect, the present invention provides a method of selecting a γδ T cell activator for administration in combination with a therapeutic antibody, said method comprising: i) providing a candidate γδ T cell activator; ii) incubating the therapeutic antibody with target cells specifically recognized by the therapeutic antibody in the presence of γδ T cells and in the presence or absence of the candidate compound; and iii) assessing the effect of the candidate compound on the ability of the γδ T cells to deplete the target cells; wherein a detection that the candidate compound enhances the ability of the γδ T cells to deplete the target cells indicates that the candidate compound is suitable for use in the method.
Within the context of the present invention, a subject or patient includes any mammalian subject or patient, more preferably a human subject or patient.
In particular, an object of the present invention is to provide an efficient combination treatment with rituximab and a γδ T cell activator which is more efficient for the depletion of B-lymphomas than rituximab alone. B-lymphomas affect B cell showing a CD20+ phenotype, anti-CD20 antibodies, such as rituximab, are now currently used in therapy to treat patients having such cancer. Anti-CD20 antibodies and in particular rituximab are known to have a cytotoxic effect named ADCC (antibody dependant cell-mediated cytotoxicity), this effect is directed towards B cells expressing CD20 marker. Previous studies have shown that patients can tolerate an important B cell depletion without any serious side effects, thus an advantageous way to treat a B cell cancer would be to selectively eradicate cancerous B cells and let the B cell regenerate from the patient's bone marrow. A mean to obtain an enhanced and prolonged B cell depletion would thus be of interest to obtain better therapeutic results. The inventors have surprisingly found out, as explained in example 2 that the combination of an anti-CD20 antibody, such as rituximab and a γδ T cell activator leads to an enhanced and prolonged B cell depletion, compared to an anti-CD20 antibody, such as rituximab, alone.
In particular, another object of the present invention is to provide an efficient combination treatment with rituximab and a γδ T cell activator which delays the reconstitution of B cell population, thereby improving the effectiveness of B-cell depletion therapy in vivo.
Administration of the Combination Treatment
In one embodiment, the therapeutic antibody and the γδ T cell activator (or the activated γδ T cells) are administered into the subject simultaneously. In another embodiment, the γδ T cell activator (or the activated γδ T cells) is administered to the subject within several week (e.g. 2, 3, 4, 5, or 6 weeks), preferably within one week of the administration of the therapeutic antibody. In a first embodiment, the γδ T cell activator (or the activated γδ T cells) is administered to the subject before the therapeutic antibody. In a second embodiment, the therapeutic antibody is administered to the subject before the γδ T cell activator (or the activated γδ T cells). The γδ T cell activator (or the activated γδ T cells) and the therapeutic antibody are administered so that the synergic effect can be obtained.
The γδ T cell activator can be administered only once to the individual. In another aspect, the γδ T cell activator is administered in multiple doses, the administration of successive doses of the γδ T cell activator is separated by at least 2, 3 or 4 or more weeks, each new administration of the γδ T cell activator defining a cycle of treatment. The number of cycles of treatment will be determined by the skilled artisan, depending on the specific responsiveness and health of each patient. Generally, the γδ T cell rate (number of γδ T cells), is allowed to return to substantially basal rate prior to a second administration of the compound. At least about one week, but more preferably at least about two weeks, or up to eight weeks are required for a patient's γδ T cell rate to return to substantially basal rate. For example, the γδ T cell activator can be administered only once to the individual, or preferably only once within (e.g. during or after) a particular course of therapeutic antibody, which is practice will usually mean that the γδ T cell activator is administered no more than once per month or once every 2, 3 or 6 months. The γδ T cell activator is administered during the therapeutic antibody treatment. The γδ T cell activator can be administered for several cycles during the therapeutic antibody. More preferably, the γδ T cell activator is administered for at least two cycles, or more preferably for at least three cycles.
The therapeutic antibody is usually administered about once a week for a treatment cycle of about a few weeks, typically around 3 weeks, as shown in example 2. In one embodiment, the therapeutic antibody is administered for a period of time of 3 to 5 weeks, typically round 21 days. Preferably, the therapeutic antibody is administered prior to the γδ T cell activator.
The invention provides here a specific scheme of administration that has been proven efficient in relevant in vivo models such as the cynomolgus macaque, as set forth in example 2.
The γδ T cell activator is administered once, conjointly with the therapeutic antibody. Preferably the γδ T cell activator is administered substantially at the same time than one antibody administration. Most preferably, the γδ T cell activator is administered substantially at the same time than the second administration of the therapeutic antibody, typically within 48 hours of the second antibody administration, (e.g. around day 7 or 8 of the treatment cycle). Most preferably, the γδ T cell activator is administered once, within 48 hours of the second antibody administration. In one example, the γδ T cell activator can be used conjointly with a therapeutic antibody treatment such that the γδ T cell activator is be administered on day 7 or 8, day 0 being the first day of administration of the therapeutic antibody. It will be appreciated, however, that the γδ T cell activator need not be administered between each two successive doses of therapeutic antibody, and that successive administrations of the γδ T cell activator will be separated by at least 2, 3 or 4 or more weeks. In another embodiment, a cytokine is further administered for a period comprised between 3 and 10 days, the first cytokine administration taking place on the same day as the γδ T cell activator administration. Preferably, the cytokine is IL-2 and IL-2 is administered for 5 consecutive days with each γδ T cell activator administration.
Co-Treatment with Cytokine
In embodiments where the γδ T cell activator is used conjointly with a therapeutic antibody, the methods of the invention optionally comprise further administering a cytokine. While the compounds of the invention may be used with or without further administration, in a preferred aspect a cytokine can be administered, wherein said cytokine is capable of increasing the expansion of a γδ T cell population treated with a γδ T cell activator compound, preferably wherein the cytokine is capable of inducing an expansion of a γδ T cell population which is greater than the expansion resulting from administration of the γδ T cell activator compound in the absence of said cytokine. A preferred cytokine is an interleukin-2 polypeptide.
A cytokine having γδ T cell proliferation inducing activity, most preferably the interleukin-2 polypeptide, is administered at low doses, typically over a period of time comprised between 1 and 10 days. The γδ T cell activator is preferably administered in a single dose, and typically at the beginning of the γδ T cell activator treatment.
In preferred aspects, a cytokine, most preferably IL-2, is administered daily for up to about 10 days, preferably for a period of between about 3 and 10 days, or most preferably for about 5 days. Preferably, the administration of the cytokine begins on the same day (e.g. within 24 hours of) as administration of the γδ T cell activator. It will be appreciated that the cytokine can be administered in any suitable scheme within said regimen of between about 3 and 10 days. For example, in one aspect the cytokine is administered each day, while in other aspects the cytokine need not be administered on each day. In a preferred embodiment, the cytokine IL-2 can be administered for 5 days beginning on day 8.
Preferably, IL-2 is administered at a low dose, e.g. a dose that is lower than therapeutic standard of 18 million internaitonal unit per square meter (MIU/m2) daily. Preferably, the IL-2 dose will be less than 5 MIU/m2 daily, corresponding to less than 10 MIU total daily in human. Preferably, the dose will be comprised between 1 MIU total daily in human (0.5 MIU/m2) and 8 MIU total daily in human (4 MIU/m2).
Therapeutic Antibodies
The present invention deals with the use of a γδ T cell activating compound in combination with therapeutic antibodies. Any of a large variety of therapeutic antibodies can be used in the present invention.
The term “antibody,” as used herein, refers to polyclonal and monoclonal antibodies. Depending on the type of constant domain in the heavy chains, antibodies are assigned to one of five major classes: IgA, IgD, IgE, IgG, and IgM. Several of these are further divided into subclasses or isotypes, such as IgG1, IgG2, IgG3, IgG4, and the like. An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are termed “alpha,” “delta,” “epsilon,” “gamma” and “mu,” respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. IgG and/or IgM are the preferred classes of antibodies employed in this invention, with IgG being particularly preferred, because they are the most common antibodies in the physiological situation, because they are most easily made in a laboratory setting, and because IgGs are specifically recognized by Fc gamma receptors. Preferably the antibody of this invention is a monoclonal antibody. Particularly preferred are humanized, chimeric, human, or otherwise-human-suitable antibodies.
Within the context of this invention, the term “therapeutic antibody or antibodies” designates more specifically any antibody that functions to target cells in a patient and optionally to deplete targeted cells in a patient. In particular, therapeutic antibodies specifically bind to antigens present on the surface of the target cells, e.g. tumor specific antigens present predominantly or exclusively on tumor cells. Preferably, therapeutic antibodies include human Fc portions, or are capable of interacting with human Fc receptors. Therapeutic antibodies can target cells by any means, e.g. ADCC or otherwise, and can be “naked,” i.e. with no conjugated moieties, or they can be conjugated with compounds such as radioactive labels or toxins.
For the purposes of the present invention, a “humanized” antibody refers to an antibody in which the constant and variable framework region of one or more human immunoglobulins is fused with the binding region, e.g. the CDR, of an animal immunoglobulin. Such humanized antibodies are designed to maintain the binding specificity of the non-human antibody from which the binding regions are derived, but to avoid an immune reaction against the non-human antibody.
A “chimeric antibody” is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity. In preferred embodiments of the present invention, the chimeric antibody nevertheless maintains the Fe region of the immunoglobulin, preferably a human Fc region, thereby allowing interactions with human Fc receptors on the surface of target cells.
The present compounds can be used to enhance the ability of therapeutic antibodies to deplete target cells that express an antigen that is specifically recognized by the therapeutic antibodies. Accordingly, any disease or condition that is caused or exacerbated at least in part by cells that can be targeted by a therapeutic antibody can be treated using the herein-described methods. Specific examples of target cells include tumor cells, virus-infected cells, allogenic cells, pathological immunocompetent cells (e.g., B lymphocytes, T lymphocytes, antigen-presenting cells, etc.) involved in allergies, autoimmune diseases, allogenic reactions, etc., or even healthy cells (e.g., endothelial cells in an anti-angiogenic therapeutic strategy). Most preferred target cells within the context of this invention are tumor cells and virus-infected cells. The therapeutic antibodies may, for instance, mediate a cytotoxic effect or cell lysis, particularly by antibody-dependent cell-mediated cytotoxicity (ADCC).
The therapeutic antibody has a human immunoglobulin constant domain (Fc) of the G1, G2, G3 or G4 subtype (e.g. an IgG1, IgG2, IgG3, IgG4), and/or bind to CD16. Each of these subtypes have been demonstrated in at least some case to be depleting. In preferred embodiments, the therapeutic antibody comprises a human Fc region of the G1 or G3 subtype, or a human Fc region of the G2 or G4 subtype in case where the Fc region or the antibody have been modified or so produced as to increase the ability of the therapeutic antibody to deplete a target cell. In another embodiment, the therapeutic antibody is an antibody or a fragment thereof. The therapeutic antibodies may be polyclonal or monoclonal. Preferably, the therapeutic antibody is a monoclonal antibody or fragment thereof. In one embodiment, the therapeutic antibodies can be antibody fragments or derivatives such as, inter alia, a Fab fragment, a Fab′2 fragment, a CDR and a ScFv. Preferably a fragment is an antigen-binding fragment. In one embodiment, the therapeutic antibody is a human, humanized or chimeric antibody or a fragment thereof. Essentially, any therapeutic antibody, whether “naked” or conjugated with a radiolabel, toxin, or other moiety, or whether full length or a fragment; or whether a true antibody or a modified derivative of an antibody, can be used. In another embodiment, the therapeutic antibody is not conjugated with a radioactive or toxic moiety.
The therapeutic antibodies may be produced by hybridomas or by recombinant cells engineered to express the desired variable and constant domains. The antibodies may be single chain antibodies or other antibody derivatives retaining the antigen specificity and the lower hinge region or a variant thereof. These may be polyfunctional antibodies, recombinant antibodies, humanized antibodies, fragments or variants thereof. Therapeutic antibodies which comprise an antibody fragment may also include but are not limited to bispecific antibodies; one example a suitable bispecific antibody comprises an antigen binding region specific for CD16 and an antigen binding region specific for a tumor antigen. Other antibody formats comprising fragments include recombinant bispecific antibody derivatives combining the binding regions of two different antibodies on a single polypeptide chain, also referred to as BiTE™ (Kufer P, et al TRENDS in Biotechnology 2004; 22 (5): 238-244; and Baeuerle et al, Current Opinion in Molecular Therapeutics 2003; 5(4): 413-419, the disclosures of which are incorporated herein by reference.
Therapeutic antibodies are generally specific for surface antigens, e.g., membrane antigens. Most preferred therapeutic antibodies are specific for tumor antigens (e.g., molecules specifically expressed by tumor cells), such as CD20, CD52, ErbB2 (or HER2/Neu), CD33, CD22, CD25, MUC-1, CEA, KDR, αVβ3, etc., particularly lymphoma antigens (e.g., CD20). The therapeutic antibodies have preferably human or non human primate IgG1 or IgG3 Fc portion, more preferably human IgG1.
Typical examples of therapeutic antibodies of this invention are rituximab, alemtuzumab and trastuzumab. Such antibodies may be used according to clinical protocols that have been authorized for use in human subjects. Additional specific examples of therapeutic antibodies include, for instance, epratuzumab, basiliximab, daclizumab, cetuximab, labetuzumab, sevirumab, tuvurimab, palivizumab, infliximab, omalizumab, efalizumab, natalizumab, clenoliximab, etc. Other examples of preferred therapeutic antibodies for use in accordance with the invention include anti-ferritin antibodies (US Patent Publication no. 2002/0106324), anti-p140 and anti-sc5 antibodies (WO 02/50122), the disclosures of each of the above reference being incorporated herein by reference. Other examples of therapeutic antibodies are listed in the following table, any of which (and others) can be used in the present methods. It will be appreciated that, regardless of whether or not they are listed in the following table or described elsewhere in the present specification, any antibody that can target cells, and optionally deplete the targeted cell, e.g. by ADCC, can benefit from the present methods, and that the following Table 1 is non exhaustive, neither with respect to the antibodies listed therein, nor with respect to the targets or indications of the antibodies that are listed.
Therefore, the therapeutic antibody can be an antibody selected from the antibodies in Table 1 or an antibody binding the same antigen. The efficient amount of therapeutic antibodies administered to the subject can be between about 0.1 mg/kg and about 20 mg/kg. The efficient amount of antibody depends however of the form of the antibody (whole Ig, or fragments), affinity of the mAb and pharmacokinetics parameter that must be determined for each particular antibodies.
In a preferred embodiment, the antibody is the Rituximab. In a more particular embodiment, the antibody is rituximab, and said antibody is administered at a dosage of less than 375 mg/m2 per week. In another embodiment, the antibody is Campath. In a more particular embodiment, the antibody is Campath, and the antibody is administered at a dosage of less than 90 mg per week.
In another aspect of any of the embodiments, any one of the antibodies listed herein can be excluded, including but not limited to anti-EGFR antibodies or anti-VEGF antibodies, for example Cetuximab can be excluded.
γδ T Cell Activators
The term “γδ T cell activator” designates a molecule, preferably artificially produced, which can activate γδ T lymphocytes. It is more preferably a ligand of the T receptor of γδ T lymphocytes. The activator may by of various natures, such as a peptide, lipid, small molecule, etc. It may be a purified or otherwise artificially produced (e.g., by chemical synthesis, or by microbiological process) endogenous ligand, or a fragment or derivative thereof, or an antibody having substantially the same antigenic specificity.
A phosphoantigen that is a γδ T cell activator preferably increases the biological activity or causes the proliferation of γδ T cells, preferably increasing the activation of γδ T cells, particularly increasing cytokine secretion from γδ T cells or increasing the cytolytic activity of γδ T cells, with or without also stimulating the proliferation or expansion of γδ T cells. Accordingly, the γδ T cell activator is administered in an amount and under conditions sufficient to increase the activity γδ T cells in a subject, preferably in an amount and under conditions sufficient to increase cytokine secretion by γδ T cells and/or to increase the cytolytic activity of γδ T cells. Cytokine secretion and cytolytic activity can be assessed using any appropriate in vitro assay.
In any exemplary assay, cytokine secretion can be determined according to the methods described in Espinosa et al. (J. Biol. Chem., 2001, Vol. 276, Issue 21, 18337-18344), describing measurement of TNF-α release in a bioassay using TNF-α-sensitive cells. Briefly, 104 γδ T cells/well were incubated with stimulus plus 25 units of IL2/well in 100 μl of culture medium during 24 h at 37° C. Then, 50 μl of supernatant were added to 50 μl of WEHI cells plated at 3×104 cells/well in culture medium plus actinomycin D (2 μg/ml) and LiCl (40 mM) and incubated for 20 h at 37° C. Viability of the TNF-α-sensitive cells and measured with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. 50 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma; 2.5 mg/ml in phosphate-buffered saline) per well were added, and after 4 h of incubation at 37° C., 50 μl of solubilization buffer (20% SDS, 66% dimethyl formamide, pH 4.7) were added, and absorbance (570 nm) was measured. Levels of TNF-α release were then calculated from a standard curve obtained using purified human rTNF-α (PeproTech, Inc., Rocky Hill, N.J.). Interferon-γ released by activated T cells was measured by a sandwich enzyme-linked immunosorbent assay. 5×104 γδ T cells/well were incubated with stimulus plus 25 units of IL2/well in 100 μl of culture medium during 24 h at 37° C. Then, 50 μl of supernatant were harvested for enzyme-linked immunosorbent assay using mouse monoclonal antibodies (BIOSOURCE, Camarillo, Calif.).
A preferred assay for cytolytic activity is a 51Cr release assay. In exemplary assays, the cytolytic activity of γδ T cells is measured against autologous normal and tumor target cell lines, or control sensitive target cell lines such as Daudi and control resistant target cell line such as Raji in 4h 51Cr release assay. In a specific example, target cells were used in amounts of 2×103 cells/well and labeled with 100 Ci 51Cr for 60 minutes. Effector/Target (E/T) ratio ranged from 30:1 to 3.75:1. Specific lysis (expressed as percentage) is calculated using the standard formula
[(experimental-spontaneous release/total-spontaneous release)×100].
As discussed, the methods of the invention can generally be carried out with any γδ T cell activator that is capable of stimulating γδ T cell activity. This stimulation can be by direct effect on γδ T cells as discussed below using compounds that can stimulate γδ T cells in a pure γδ T cell culture, or the stimulation can be by an indirect mechanism, such as treatment with pharmacological agents such as bisphosphonates which lead to IPP accumulation. Preferably, a γδ T cell activator is a compound capable of regulating the activity of a γδ T cell in a population of γδ T cell clones in culture. The γδ T cell activator is capable of regulating the activity of a γδ T cell population of γδ T cell clones at millimolar concentration, preferably when the γδ T cell activator is present in culture at a concentration of less than 100 mM. Optionally a γδ T cell activator is capable of regulating the activity of a γδ T cell in a population of γδ T cell clones at millimolar concentration, preferably when the γδ T cell activator is present in culture at a concentration of less than 10 mM, or more preferably less than 1 mM. Regulating the activity of a γδ T cell can be assessed by any suitable means, preferably by assessing cytokine secretion, most preferably TNF-α secretion as described herein. Methods for obtaining a population of pure γδ T cell clones is described in Davodeau et al, (1993) and Moreau et al, (1986), the disclosures of which are incorporated herein by reference. Preferably the activator is capable of causing at least a 20%, 50% or greater increase in the number of γδ T cells in culture, or more preferably at least a 2-fold increase in the number of γδ T cells in culture.
In one embodiment, the activator may be a synthetic chemical compound capable of selectively activating Vγ9Vδ2 T lymphocytes. Selective activation of Vγ9Vδ2 T lymphocytes indicates that the compound has a selective action towards specific cell populations, preferably increasing activation of Vγ9Vδ2 T cells at a greater rate or to a greater degree than other T cell types such as Vδ1 T cells, or not substantially not activation other T cell types. Such selectivity can be assessed in vitro T cell activation assays. Such selectivity, as disclosed in the present application, suggests that preferred compounds can cause a selective or targeted activation of the proliferation or biological activity of Vγ9Vδ2 T lymphocytes.
Preferred Phosphoantigens
The γδ T cell activator is preferably a non-peptide antigen. In yet other embodiments, the compound is any other activator of γδ T cells, including compounds that act directly or indirectly (e.g. by activating directly an immune cell other than γδ T cells).
γδ T cell activators useful in the present invention comprise the compounds of Formula (I):
wherein Cat+ represents one (or several, identical or different) organic or mineral cation(s) (including proton);
m is an integer from 1 to 3;
B is O, NH, or any group capable to be hydrolyzed;
Y═O−Cat+, a C1-C3 alkyl group, a group -A-R, or a radical selected from the group consisting of a nucleoside, an oligonucleotide, a nucleic acid, an amino acid, a peptide, a protein, a monosaccharide, an oligosaccharide, a polysaccharide, a fatty acid, a simple lipid, a complex lipid, a folic acid, a tetrahydrofolic acid, a phosphoric acid, an inositol, a vitamin, a co-enzyme, a flavonoid, an aldehyde, an epoxyde and a halohydrin;
A is O, S, NH, CHF, CF2 or CH2; and,
R is a linear, branched, or cyclic, aromatic or not, saturated or unsaturated, C1-C50 hydrocarbon group, optionally interrupted by at least one heteroatom, wherein said hydrocarbon group comprises an alkyl, an alkylenyl, or an alkynyl, preferably an alkyl or an alkylene, which can be substituted by one or several substituents selected from the group consisting of: an alkyl, an alkylenyl, an alkynyl, an epoxyalkyl, an aryl, an heterocycle, an alkoxy, an acyl, an alcohol, a carboxylic group (—COOH), an ester, an amine, an amino group (—NH2), an amide (—CONH2), an imine, a nitrile, an hydroxyl (—OH), a aldehyde group (—CHO), an halogen, an halogenoalkyl, a thiol (—SH), a thioalkyl, a sulfone, a sulfoxide, and a combination thereof.
In a particular embodiment, the substituents as defined above are substituted by at least one of the substituents as specified above.
Preferably, the substituents are selected from the group consisting of: an (C1-C6)alkyl, an (C2-C6)alkylenyl, an (C2-C6)alkynyl, an (C2-C6)epoxyalkyl, an aryl, an heterocycle, an (C1-C6)alkoxy, an (C2-C6)acyl, an (C1-C6)alcohol, a carboxylic group (—COOH), an (C2-C6)ester, an (C1-C6)amine, an amino group (—NH2), an amide (—CONH2), an (C1-C6)imine, a nitrile, an hydroxyl (—OH), a aldehyde group (—CHO), an halogen, an (C1-C6)halogenoalkyl, a thiol (—SH), a (C1-C6)thioalkyl, a (C1-C6)sulfone, a (C1-C6)sulfoxide, and a combination thereof.
More preferably, the substituents are selected from the group consisting of: an (C1-C6)alkyl, an (C2-C6)epoxyalkyl, an (C2-C6)alkylenyl, an (C1-C6)alkoxy, an (C2-C6)acyl, an (C1-C6)alcohol, an (C2-C6)ester, an (C1-C6)amine, an (C1-C6)imine, an hydroxyl, a aldehyde group, an halogen, an (C1-C6)halogenoalkyl, and a combination thereof.
Still more preferably, the substituents are selected from the group consisting of : an (C3-C6)epoxyalkyl, an (C1-C3)alkoxy, an (C2-C3)acyl, an (C1-C3)alcohol, an (C2-C3)ester, an (C1-C3)amine, an (C1-C3)imine, an hydroxyl, an halogen, an (C1-C3)halogenoalkyl, and a combination thereof. and a combination thereof. Preferably, R is a (C3-C25)hydrocarbon group, more preferably a (C5-C10)hydrocarbon group.
In the context of the present invention, the term “alkyl” more specifically means a group such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, heneicosyl, docosyl and the other isomeric forms thereof. (C1-C6)alkyl more specifically means methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl and the other isomeric forms thereof. (C1-C3)alkyl more specifically means methyl, ethyl, propyl, or isopropyl.
The term “alkenyl” refers to an alkyl group defined hereinabove having at least one unsaturated ethylene bond and the term “alkynyl” refers to an alkyl group defined hereinabove having at least one unsaturated acetylene bond. (C2-C6)alkylene includes a ethenyl, a propenyl (1-propenyl or 2-propenyl), a 1- or 2-methylpropenyl, a butenyl (1-butenyl, 2-butenyl, or 3-butenyl), a methylbutenyl, a 2-ethylpropenyl, a pentenyl (1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl), an hexenyl (1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl), and the other isomeric forms thereof. (C2-C6)alkynyl includes ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, or 5-hexynyl and the other isomeric forms thereof.
The term “epoxyalkyl” refers to an alkyl group defined hereinabove having an epoxide group. More particularly, (C2-C6)epoxyalkyl includes epoxyethyl, epoxypropyl, epoxybutyl, epoxypentyl, epoxyhexyl and the other isomeric forms thereof. (C2-C3)epoxyalkyl includes epoxyethyl and epoxypropyl.
The “aryl” groups are mono-, bi- or tri-cyclic aromatic hydrocarbons having from 6 to 18 carbon atoms. Examples include a phenyl, α-naphthyl, β-naphthyl or anthracenyl group, in particular.
“Heterocycle” groups are groups containing 5 to 18 rings comprising one or more heteroatoms, preferably 1 to 5 endocyclic heteroatoms. They may be mono-, bi- or tri-cyclic. They may be aromatic or not. Preferably, and more specifically for R5, they are aromatic heterocycles. Examples of aromatic heterocycles include pyridine, pyridazine, pyrimidine, pyrazine, furan, thiophene, pyrrole, oxazole, thiazole, isothiazole, imidazole, pyrazole, oxadiazole, triazole, thiadiazole and triazine groups. Examples of bicycles include in particular quinoline, isoquinoline and quinazoline groups (for two 6-membered rings) and indole, benzimidazole, benzoxazole, benzothiazole and indazole (for a 6-membered ring and a 5-membered ring). Non aromatic heterocycles comprise in particular piperazine, piperidine, etc.
“Alkoxy” groups correspond to the alkyl groups defined hereinabove bonded to the molecule by an —O— (ether) bond. (C1-C6)alkoxy includes methoxy, ethoxy, propyloxy, butyloxy, pentyloxy, hexyloxy and the other isomeric forms thereof. (C1-C3)alkoxy includes methoxy, ethoxy, propyloxy, and isopropyloxy.
“Alcyl” groups correspond to the alkyl groups defined hereinabove bonded to the molecule by an —CO— (carbonyl) group. (C2-C6)acyl includes acetyl, propylacyl, butylacyl, pentylacyl, hexylacyl and the other isomeric forms thereof. (C2-C3)acyl includes acetyl, propylacyl and isopropylacyl.
“Alcohol” groups correspond to the alkyl groups defined hereinabove containing at least one hydroxyl group. Alcohol can be primary, secondary or tertiary. (C1-C6)alcohol includes methanol, ethanol, propanol, butanol, pentanol, hexanol and the other isomeric forms thereof. (C1-C3)alcohol includes methanol, ethanol, propanol and isopropanol.
“Ester” groups correspond to the alkyl groups defined hereinabove bonded to the molecule by an —COO— (ester) bond. (C2-C6)ester includes methylester, ethylester, propylester, butylester, pentylester and the other isomeric forms thereof. (C2-C3)ester includes methylester and ethylester.
“Amine” groups correspond to the alkyl groups defined hereinabove bonded to the molecule by an —N— (amine) bond. (C1-C6)amine includes methylamine, ethylamine, propylamine, butylamine, pentylamine, hexylamine and the other isomeric forms thereof. (C1-C3)amine includes methylamine, ethylamine, and propylamine.
“Imine” groups correspond to the alkyl groups defined hereinabove having a (—C═N—) bond. (C1-C6)imine includes methylimine, ethylimine, propylimine, butylimine, pentylimine, hexylimine and the other isomeric forms thereof. (C1-C3)imine includes methylimine, ethylimine, and propylimine.
The halogen can be Cl, Br, I, or F, more preferably Br or F.
“Halogenoalkyl” groups correspond to the alkyl groups defined hereinabove having at least one halogen. The groups can be monohalogenated or polyhalogenated containing the same or different halogen atoms. For example, the group can be an trifluoroalkyl (CF3—R). (C1-C6)halogenoalkyl includes halogenomethyl, halogenoethyl, halogenopropyl, halogenobutyl, halogenopentyl, halogenohexyl and the other isomeric forms thereof. (C1-C3)halogenoalkyl includes halogenomethyl, halogenoethyl, and halogenopropyl.
“Thioalkyl” groups correspond to the alkyl groups defined hereinabove bonded to the molecule by an —S— (thioether) bond. (C1-C6)thioalkyl includes thiomethyl, thioethyl, thiopropyl, thiobutyl, thiopentyl, thiohexyl and the other isomeric forms thereof. (C1-C3)thioalkyl includes thiomethyl, thioethyl, and thiopropyl.
“Sulfone” groups correspond to the alkyl groups defined hereinabove bonded to the molecule by an —SOO— (sulfone) bond. (C1-C6)sulfone includes methylsulfone, ethylsulfone, propylsulfone, butylsulfone, pentylsulfone, hexylsulfone and the other isomeric forms thereof. (C1-C3)sulfone includes methylsulfone, ethylsulfone and propylsulfone.
“Sulfoxyde” groups correspond to the alkyl groups defined hereinabove bonded to the molecule by an —SO— (sulfoxide) group. (C1-C6)sulfoxide includes methylsulfoxide, ethylsulfoxide, propylsulfoxide, butylsulfoxide, pentylsulfoxide, hexylsulfoxide and the other isomeric forms thereof. (C1-C3)sulfoxide includes methylsulfoxide, ethylsulfoxide, propylsulfoxide and isopropylsulfoxide.
“Heteroatom” denotes N, S, or O.
“Nucleoside” includes adenosine, thymine, uridine, cytidine and guanosine.
In a particular embodiment, the hydrocarbon group is a cycloalkylenyl such as a cyclopentadiene or a phenyl, or an heterocycle such as a furan, a pyrrole, a thiophene, a thiazole, an imidazole, a triazole, a pyridine, a pyrimidine, a pyrane, or a pyrazine. Preferably, the cycloalkylenyl or the heterocycle is selected from the group consisting of a cyclopentadiene, a pyrrole or an imidazole. In a preferred embodiment, the cycloalkylenyl or the heterocycle is substituted by an alcohol. Preferably, said alcohol is a (C1-C3)alcohol.
In an other embodiment, the hydrocarbon group is an alkylenyl with one or several double bonds. Preferably, the alkylenyl group has one double bond. Preferably, the alkylenyl group is a (C3-C10)alkylenyl group, more preferably a (C4-C7)alkylenyl group. Preferably, said alkylenyl group is substituted by at least one functional group. More preferably, the functional group is selected from the group consisting of an hydroxy, an (C1-C3)alkoxy, an aldehyde, an (C2-C3)acyl, or an (C2-C3)ester. In a more preferred embodiment, the hydrocarbon group is butenyl substituted by a group —CH2OH. Optionally, said alkenyl group can be the isoform trans (E) or cis (Z), more preferably a trans isoform (E). In a most preferred embodiment, the alkylenyl group is the (E)-4-hydroxy-3-methyl-2-butenyl. In an other preferred embodiment, the alkylenyl group group is an isopentenyl, an dimethylallyl or an hydroxydimethylallyl.
In an additional embodiment, the hydrocarbon group is an alkyl group substituted by an acyl. More preferably, the hydrocarbon group is an (C4-C7)alkyl group substituted by an (C1-C3)acyl.
In a further preferred embodiment, R is selected from the group consisting of:
wherein n is an integer from 2 to 20, R1 is a (C1-C3)alkyl group, and R2 is an halogenated (C1-C3)alkyl, a (C1-C3)alkoxy-(C1-C3)alkyl, an halogenated (C2-C3)acyl or a (C1-C3)alkoxy-(C2-C3)acyl. Preferably, R1 is a methyl or ethyl group, and R2 is an halogenated methyl (—CH2—X, X being an halogen), an halogenated (C2-C3)acetyl, or (C1-C3)alkoxy-acetyl. The halogenated methyl or acetyl can be mono-, di-, or tri-halogenated. Preferably, n is an integer from 2 to 10, or from 2 to 5. In a more preferred embodiment, n is 2. In a most preferred embodiment, n is 2, R1 is a methyl and R2 is an halogenated methyl, more preferably a monohalogenated methyl, still more preferably a bromide methyl. In a particularly preferred embodiment, n is 2, R1 is a methyl, R2 is a methyl bromide. In a most preferred embodiment, R is 3-(bromomethyl)-3-butanol-1-yl.
wherein n is an integer from 2 to 20, and R1 is a methyl or ethyl group. Preferably, n is an integer from 2 to 10, or from 2 to 5. In a more preferred embodiment, n is 2 and R1 is a methyl.
wherein R3, R4, and R5, identical or different, are a hydrogen or (C1-C3)alkyl group, W is —CH— or —N—, and R6 is an (C2-C3)acyl, an aldehyde, an (C1-C3)alcohol, or an (C2-C3)ester. More preferably, R3 and R5 are a methyl and R4 is a hydrogen. More preferably, R6 is —CH2—OH, —CHO, —CO—CH3 or —CO—OCH3. Optionally, the double-bond between W and C is in conformation trans (E) or cis (Z). More preferably, the double-bond between W and C is in conformation trans (E).
The group Y can allow to design a prodrug. Therefore, Y is enzymolabile group which can be cleaved in particular regions of the subject. The group Y can also be targeting group. In a preferred embodiment, Y is O−Cat+, a group -A-R, or a radical selected from the group consisting of a nucleoside, a monosaccharide, an epoxyde and a halohydrin. Preferably, Y is an enzymolabile group. Preferably, Y is O—Cat+, a group -A-R, or a nucleoside. In a first preferred embodiment, Y is O—Cat+. In a second preferred embodiment, Y is a nucleoside.
In a preferred embodiment, Cat+ is H+, Na+, NH4+, K+, Li+, (CH3CH2)3NH+.
In a preferred embodiment, A is O, CHF, CF2 or CH2. More preferably, A is O or CH2.
In a preferred embodiment, B is O or NH. More preferably, B is O.
In a preferred embodiment, m is 1 or 2. More preferably, m is 1.
In one particular embodiment, synthetic γδ T cell activators comprise the compounds of Formula (II):
in which X is an halogen (preferably selected from I, Br and Cl), B is O or NH, m is an integer from 1 to 3, R1 is a methyl or ethyl group, Cat+ represents one (or several, identical or different) organic or mineral cation(s) (including the proton), and n is an integer from 2 to 20, A is O, S, NH, CHF, CF2 or CH2, and Y is O−Cat+, a nucleoside, or a radical -A-R, wherein R is selected from the group of 1), 2) or 3). Preferably, Y is O−Cat+, or a nucleoside. More preferably, Y is O−Cat+. Preferably, R1 is a methyl. Preferably, A is O or CH2. More preferably, A is O. Preferably, n is 2. Preferably, X is a bromide. Preferably, B is O. Preferably, m is 1 or 2. More preferably, m is 1.
For example, synthetic γδ T cell activators comprise the compounds of Formula (III) or (IV):
wherein X, R1, n, m and Y have the aforementioned meaning.
In one preferred embodiment, synthetic γδ T cell activators comprise the compounds of Formula (V):
in which X is an halogen (preferably selected from I, Br and Cl), R1 is a methyl or ethyl group, Cat+ represents one (or several, identical or different) organic or mineral cation(s) (including the proton), and n is an integer from 2 to 20. Preferably, R1 is a methyl. Preferably, n is 2. Preferably, X is a bromide.
In a most preferred embodiment, synthetic γδ T cell activators comprise the compound of Formula (VI) (also named Phosphostim):
In an other most preferred embodiment, synthetic γδ T cell activators comprise the compound of Formula (VII):
In an other most preferred embodiment, synthetic γδ T cell activators comprise the compound of Formula:
In one particular embodiment, synthetic γδ T cell activators comprise the compounds of Formula (VIII):
in which R1 is a methyl or ethyl group, Cat+ represents one (or several, identical or different) organic or mineral cation(s) (including the proton), B is O or NH, m is an integer from 1 to 3, and n is an integer from 2 to 20, A is O, S, NH, CHF, CF2 or CH2, and Y is O−Cat+, a nucleoside, or a radical -A-R, wherein R is selected from the group of 1), 2) or 3). Preferably, Y is O−Cat+, or a nucleoside. More preferably, Y is O−Cat+. Preferably, R1 is a methyl. Preferably, A is O or CH2. More preferably, A is O. Preferably, n is 2. Preferably, B is O. Preferably, m is 1 or 2. More preferably, m is 1.
For example, synthetic γδ T cell activators comprise the compounds of Formula (IX) or (X):
wherein R1, n, m and Y have the above mentioned meaning.
In one preferred embodiment, synthetic γδ T cell activators comprise the compounds of Formula (XI):
in which R1 is a methyl or ethyl group, Cat+ represents one (or several, identical or different) organic or mineral cation(s) (including the proton), and n is an integer from 2 to 20. Preferably, R1 is a methyl. Preferably, n is 2.
In a most preferred embodiment, synthetic γδ T cell activators comprise the compound of Formula (XI):
Preferably x Cat+ is 1 or 2 Na+.
In one particular embodiment, synthetic γδ T cell activators comprise the compounds of Formula (XII):
in which R3, R4, and R5, identical or different, are a hydrogen or (C1-C3)alkyl group, W is —CH— or —N—, R6 is an (C2-C3)acyl, an aldehyde, an (C1-C3)alcohol, or an (C2-C3)ester, Cat+ represents one (or several, identical or different) organic or mineral cation(s) (including the proton), B is O or NH, m is an integer from 1 to 3, A is O, S, NH, CHF, CF2 or CH2, and Y is O−Cat+, a nucleoside, or a radical -A-R, wherein R is selected from the group of 1), 2) or 3). Preferably, Y is O−Cat+, or a nucleoside. More preferably, Y is O−Cat+. Preferably, A is O or CH2. More preferably, A is O. More preferably, R3 and R5 are a methyl and R4 is hydrogen. More preferably, R6 is —CH2—OH, —CHO, —CO—CH3 or —CO—OCH3. Preferably, B is O. Preferably, m is 1 or 2. More preferably, m is 1. Optionally, the double-bond between W and C is in conformation trans (E) or cis (Z). More preferably, the double-bond between W and C is in conformation trans (E).
For example, synthetic γδ T cell activators comprise the compounds of Formula (XIII) or (XIV):
wherein R3, R4, R5, R6, W, m, and Y have the above mentioned meaning. Preferably, W is —CH—. Preferably, R3 and R4 are hydrogen. Preferably, R5 is a methyl. Preferably, R6 is —CH2—OH.
In a most preferred embodiment, synthetic γδ T cell activators comprise the compound of Formula (XV):
In an other most preferred embodiment, synthetic γδ T cell activators comprise the compound of Formula (XVI):
In an other most preferred embodiment, synthetic γδ T cell activators comprise the compound of Formula:
In another example, phosphoantigen comprises a compound of Formula:
wherein R3, R4, R5, R6 and A have the above mentioned meaning, and R7 represents a hydrogen atom or a (C1-C3)alkyl group,
Preferably, R3, R4 and R6 are hydrogen. Preferably, R7 is a methyl. Preferably, R5 is —CH2—OH.
Preferably, A is CH2, NH or O.
In a preferred embodiment, a phosphoantigen comprises a compound of Formula:
These compounds may be produced according to various techniques known per se in the art, some of which being disclosed in PCT Publications nos. WO 00/12516, WO 00/12519, WO 03/050128, and WO 03/009855, the disclosures of which are incorporated herein by reference. In a most preferred embodiment, the synthetic γδ T cell activator is selected from the group consisting of HDMAPP, CHDMAPP, Epox-PP, BrHPP and CBrHPP, more preferably HDMAPP, CHDMAPP, BrHPP and CBrHPP, still more preferably HDMAPP.
Alternatively, although potentially less efficient, other activators for use in the present invention are phosphoantigens disclosed in WO 95/20673, alkenyl pyrophosphates such as isopentenyl pyrophosphate (IPP) (U.S. Pat. No. 5,639,653) and 3-methylbut-3-enyl pyrophosphonate (C—IPP). The disclosures of both references are incorporated herein by reference. Other compounds of interest include 2-methyl-3-butenyl-1-pyrophosphoric acid salts and other compounds in EP 1 153 928.
In particular, the γδ T cell activator can be HDMAPP, C—HDMAPP or N—HDMAPP.
Specific examples of compounds also include: (E)1-pyrophosphonobuta-1,3-diene; (E)1-pyrophosphonopenta-1,3-diene; (E)1-pyrophosphono-4-methylpenta-1,3-diene; (E,E)1-pyrophosphono-4,8-dimethylnona-1,3,7-triene; (E,E,E)1-pyrophosphono-4,8,12-trimethyltrideca-1,3,7,11-tetraene; (E,E)1-triphosphono-4,8-dimethylnona-1,3,7-triene; 4-triphosphono-2-methylbutene; α,β, -di-[3-methylpent-3-enyl]-pyrophosphonate; 1-pyrophosphono-3-methylbut-2-ene; α,γ-di-[3-methylbut-2-enyl]-triphosphonate; α,β-di-[3-methylbut-2-enyl]-pyrophosphonate; allyl-pyrophosphonate; allyl-triphosphonate; α,γ-di-allyl-pyrophosphonate; α,β-di-allyl-triphosphonate; (E,E)4-[(5′-pyrophosphono-6′-methyl-penta-2′,4′-dienyloxymethyl)-phenyl]-phenyl-methanone; (E,E)4-[(5′-triphosphono-6′-methyl-penta-2′,4′-dienyloxymethyl)-phenyl]-phenyl-methanone; (E,E,E)[4-(9′-pyrophosphono-2′,6′-dimethyl-nona-2′,6′,8′-trienyloxymethyl)-phenyl]-phenyl-methanone; (E,E,E)[4-(9′-pyrophosphono-2′,6′,8′-trimethyl-nona-2′,6′,8′-trienyloxymethyl)-phenyl]-phenyl-methanone; 5-pyrophosphono-2-methypentene; 5-triphosphono-2-methypentene; α,γ-di-[4-methylpent-4-enyl]-triphosphonate; 5-pyrophosphono-2-methypent-2-ene; 5-triphosphono-2-methypent-2-ene; 9-pyrophosphono-2,6-dimethynona-2,6-diene; 9-triphosphono-2,6-dimethynona-2,6-diene; α,γ-di-[4,8-dimethylnona-2,6-dienyl]-triphosphonate; 4-pyrophosphono-2-methybutene; 4-methyl-2-oxa-pent-4-enyloxymethylpyrophosphate; 4-methyl-2-oxa-pent-4-enyloxymethyltriphosphate; α,β-di-[4-methyl-2-oxa-pent-4-enyloxymethyl]-pyrophosphate; and α,γ-di-[4-methyl-2-oxa-pent-4-enyloxymethyl]-triphosphate.
In other particular embodiments, the phosphoantigen can be selected from the group consisting of: 3-(halomethyl)-3-butanol-1-yl-diphosphate; 3-(halomethyl)-3-pentanol-1-yl-diphsophate; 4-(halomethyl)-4-pentanol-1-yl-diphosphate; 4-(halomethyl)-4-hexanol-1-yl-diphosphate; 5-(halomethyl)-5-hexanol-1-yl-diphosphate; 5-(halomethyl)-5-heptanol-1-yl-diphosphate; 6-(halomethyl)-6-heptanol-1-yl-diphosphate; 6-(halomethyl)-6-octanol-1-yl-diphosphate; 7-(halomethyl)-7-octanol-1-yl-diphosphate; 7-(halomethyl)-7-nonanol-1-yl-diphosphate; 8-(halomethyl)-8-nonanol-1-yl-diphosphate; 8-(halomethyl)-8-decanol-1-yl-diphosphate; 9-(halomethyl)-9-decanol-1-yl-diphosphate; 9-(halomethyl)-9-undecanol-1-yl-diphosphate; 10-(halomethyl)-10-undecanol-1-yl-diphosphate; 10-(halomethyl)-10-dodecanol-1-yl-diphosphate; 11-(halomethyl)-11-dodecanol-1-yl-diphosphate; 11-(halomethyl)-11-tridecanol-1-yl-diphosphate; 12-(halomethyl)-12-tridecanol-1-yl-diphosphate; 12-(halomethyl)-12-tetradecanol-1-yl-diphosphate; 13-(halomethyl)-13-tetradecanol-1-yl-diphosphate; 13-(halomethyl)-13-pentadecanol-1-yl-diphosphate; 14-(halomethyl)-14-pentadecanol-1-yl-diphosphate; 14-(halomethyl)-14-hexadecanol-1-yl-diphosphate; 15-(halomethyl)-15-hexadecanol-1-yl-diphosphate; 15-(halomethyl)-15-heptadecanol-1-yl-diphosphate; 16-(halomethyl)-16-heptadecanol-1-yl-diphosphate; 16-(halomethyl)-16-octadecanol-1-yl-diphosphate; 17-(halomethyl)-17-octadecanol-1-yl-diphosphate; 17-(halomethyl)-17-nonadecanol-1-yl-diphosphate; 18-(halomethyl)-18-nonadecanol-1-yl-diphosphate; 18-(halomethyl)-18-eicosanol-1-yl-diphosphate; 19-(halomethyl)-19-eicosanol-1-yl-diphosphate; 19-(halomethyl)-19-heneicosanol-1-yl-diphosphate; 20-(halomethyl)-20-heneicosanol-1-yl-diphosphate; 20-(halomethyl)-20-docosanol-1-yl-diphosphate; 21-(halomethyl)-21-docosanol-1-yl-diphosphate; and 21-(halomethyl)-21-tricosanol-1-yl-diphosphate.
More particularly, the phosphoantigen can be selected from the group consisting of: 3-(bromomethyl)-3-butanol-1-yl-diphosphate (BrHPP); 5-bromo-4-hydroxy-4-methylpentyl pyrophosphonate (CBrHPP); 3-(iodomethyl)-3-butanol-1-yl-diphosphate (IHPP); 3-(chloromethyl)-3-butanol-1-yl-diphosphate (ClHPP); 3-(bromomethyl)-3-butanol-1-yl-triphosphate (BrHPPP); 3-(iodomethyl)-3-butanol-1-yl-triphosphate (IHPPP); α,γ-di-[3-(bromomethyl)-3-butanol-1-yl]-triphosphate (diBrHTP); and α,γ-di-[3-(iodomethyl)-3-butanol-1-yl]-triphosphate (diHITP).
In another particular embodiment, the phosphoantigen can be selected from the group consisting of: 3,4-epoxy-3-methyl-1-butyl-diphosphate (Epox-PP); 3,4,-epoxy-3-methyl-1-butyl-triphosphate (Epox-PPP); α,γ-di-3,4,-epoxy-3-methyl-1-butyl-triphosphate (di-Epox-TP); 3,4-epoxy-3-ethyl-1-butyl-diphosphate; 4,5-epoxy-4-methyl-1-pentyl-diphosphate; 4,5-epoxy-4-ethyl-1-pentyl-diphosphate; 5,6-epoxy-5-methyl-1-hexyl-diphosphate; 5,6-epoxy-5-ethyl-1-hexyl-diphosphate; 6,7-epoxy-6-methyl-1-heptyl-diphosphate; 6,7-epoxy-6-ethyl-1-heptyl-diphosphate; 7,8-epoxy-7-methyl-1-octyl-diphosphate; 7,8-epoxy-7-ethyl-1-octyl-diphosphate; 8,9-epoxy-8-methyl-1-nonyl-diphosphate; 8,9-epoxy-8-ethyl-1-nonyl-diphosphate; 9,10-epoxy-9-methyl-1-decyl-diphosphate; 9,10-epoxy-9-ethyl-1-decyl-diphosphate; 10,11-epoxy-10-methyl-1-undecyl-diphosphate; 10,11-epoxy-10-ethyl-1-undecyl-diphosphate; 11,12-epoxy-11-methyl-1-dodecyl-diphosphate; 11,12-epoxy-11-ethyl-1-dodecyl-diphosphate; 12,13-epoxy-12-methyl-1-tridecyl-diphosphate; 12,13-epoxy-12-ethyl-1-tridecyl-diphosphate; 13,14-epoxy-13-methyl-1-tetradecyl-diphosphate; 13,14-epoxy-13-ethyl-1-tetradecyl-diphosphate; 14,15-epoxy-14-methyl-1-pentadecyl-diphosphate; 14,15-epoxy-14-ethyl-1-pentadecyl-diphosphate; 15,16-epoxy-15-methyl-1-hexadecyl-diphosphate; 15,16-epoxy-15-ethyl-1-hexadecyl-diphosphate; 16,17-epoxy-16-methyl-1-heptadecyl-diphosphate; 16,17-epoxy-16-ethyl-1-heptadecyl-diphoshate; 17,18-epoxy-17-methyl-1-octadecyl-diphosphate; 17,18-epoxy-17-ethyl-1-octadecyl-diphosphate; 18,19-epoxy-18-methyl-1-nonadecyl-diphosphate; 18,19-epoxy-18-ethyl-1-nonadecyl-diphosphate; 19,20-epoxy-19-methyl-1-eicosyl-diphosphate; 19,20-epoxy-19-ethyl-1-eicosyl-diphosphate; 20,21-epoxy-20-methyl-1-heneicosyl-diphosphate; 20,21-epoxy-20-ethyl-1-heneicosyl-diphosphate; 21,22-epoxy-21-methyl-1-docosyl-diphosphate; and 21,22-epoxy-21-ethyl-1-docosyl-diphosphate.
In a further particular embodiment, the phosphoantigen can be selected from the group consisting of: 3,4-epoxy-3-methyl-1-butyl-diphosphate (Epox-PP); 3,4,-epoxy-3-methyl-1-butyl-triphosphate (Epox-PPP); α,γ-di-3,4,-epoxy-3-methyl-1-butyl-triphosphate (di-Epox-TP); and uridine 5′-triphosphate-(3,4-epoxy methyl butyl) (Epox-UTP).
In another preferred embodiment, the phosphoantigen can be selected from the group consisting of: (E)-4-hydroxy-3-methyl-2-butenyl pyrophosphate (HDMAPP) and (E)-5-hydroxy-4-methylpent-3-enyl pyrophosphonate (CHDMAPP).
These compounds may be produced according to various techniques known per se in the art, some of which being disclosed in PCT Publications nos. WO 00/12516, WO 00/12519, WO 03/050128, WO 02/083720 and WO 03/009855, the disclosures of which are incorporated herein by reference.
In one preferred embodiment, the phosphoantigen is a γδ T cell activator and is a compound described in any one of PCT publication nos. WO 00/12516, WO 00/12519, WO 03/050128, WO 02/083720, WO 03/009855 and WO 05/054258, the disclosures of which Formulas and specific structures as well as synthesis methods are incorporated herein by reference. In another preferred embodiment, the phosphoantigen is a γδ T cell activator and is a compound selected from the group consisting of HDMAPP, CHDMAPP, NHDMAPP, H-angelylPP, Epox-PP, BrHPP and CBrHPP.
In further embodiments it will be appreciated that the present invention and particularly method for making crystalline phases is suitable for use with structurally related compounds to the ones mentioned specifically herein. In preferred embodiments, the invention also encompasses nucleotides and nucleotide analogs or derivatives or nucleotide-like compounds as well as a bisphosphonate compounds.
The γδ T cell activator can also be an aminophosphonate, preferably an aminophosphonate of Formula XVII:
with R′ being a linear, branched, or cyclic, aromatic or not, saturated or unsaturated, C1-C50 hydrocarbon group, wherein said hydrocarbon group comprises an alkyl, an alkylenyl, or an alkynyl, preferably an alkyl or an alkylene, which is substituted by one or several substituents selected from the group consisting of: an amine, an amino group (—NH2), an amide (—CONH2), an imine, and a combination thereof.
In a preferred embodiment, R′ of Formula XVII is a linear, branched, or cyclic, aromatic or not, saturated or unsaturated, C1-C10 hydrocarbon group, which is substituted by an amine, an amino group, a pyridine group, a pyrimidine group, a pyrrole group, an imidazole group, a pyrazole group, a triazole group.
In a still more preferred embodiment, R′ of Formula XVII is selected from the group consisting of:
Preferably, a compound of the bisphophonate type is selected from the group consisting of the following compounds or a pharmaceutically acceptable salt thereof, or any hydrate thereof: 3-amino-1-hydroxypropane-1,1-diphosphonic acid(pamidronic acid), e.g. pamidronate (APD); 3-(N,N-dimethylamino)-1-hydroxypropane-1,1-diphosphonic acid, e.g. dimethyl-APD; 4-amino-1-hydroxybutane-1,1-diphosphonic acid(alendronic acid), e.g. alendronate; 1-hydroxy-ethidene-bisphosphonic acid, e.g. etidronate; 1-hydroxy-3-(methylpentylamino)-propylidene-bisphosphonic acid, ibandronic acid, e.g. ibandronate; 6-amino-1-hydroxyhexane-1,1-diphosphonic acid, e.g. amino-hexyl-BP; 3-(N-methyl-N-pentylamino)-1-hydroxypropane-1,1-diphosphonic acid, e.g. methyl-pentyl-APD (=BM 21.0955); 1-hydroxy-2-(imidazol-1yl)ethane-1,1-diphosphonic acid; 1-hydroxy-2-(3-pyridinyl)ethane-1,1-diphosphonic acid(risedronic acid), e.g. risedronate, including N-methyl pyridinium salts thereof, for example N-methyl pyridinium iodides such as NE-10244 or NE-10446, 3-[N-(2-phenylthioethyl)-N-methylaminol-1-hydroxypropane-1,1-diphosphonic acid; 1-hydroxy-3-(pyrrolidin-1-yl)propane-1,1-diphosphonic acid, e.g. EB 1053 (Leo); 1-(N-phenylaminothiocarbonyl)methane-1,1-diphosphonic acid, e.g. FR 78844 (Fujisawa); 5-benzoyl-3,4-dihydro-2H-pyrazole-3,3-diphosphonic acid tetraethyl ester, e.g. U-81581 (Upjohn); 1-hydroxy-2-(imidazo[1,2-a]pyridin-3-yl)ethane-1,1-diphosphonic acid, e.g. YM 529; and 1,1-dichloromethane-1,1-diphosphonic acid(clodronic acid), e.g. clodronate. Preferably the bisphosphonate are compounds which lead to activation of γδ T cells.
In particular, the γδ T cell activator can be selected from the group consisting of pamidronate, alendronate, ibandronate, risedronate and zoledronate.
Further aspects and advantages of this invention are disclosed in the following experimental section, which should be regarded as illustrative and not limiting the scope of this application.
Preferably, dosage (single administration) of a γδ T cell activator compound of formula I to XVII for treatment is between about 1 μg/kg and about 1.2 g/kg. It will be appreciated that the above dosages related to a group of compounds, and that each particular compound may vary in optimal doses, as further described herein for exemplary compounds. Nevertheless, compounds are preferably administered in a dose sufficient to significantly increase the biological activity of γδ T cells or to significantly increase the γδ T cell population in a subject. Said dose is preferably administered to the human by intravenous (i.v.) administration during 2 to 180 min, preferably 2 to 120 min, more preferably during about 5 to about 60 min, or most preferably during about 30 min or during about 60 min. In preferred exemplary compounds, a compound of formula I to XVII is administered in a dosage (single administration) between about 0.1 mg/kg and about 1.2 g/kg, preferably between about 10 mg/kg and about 1.2 g/kg, more preferably between about 5 mg/kg and about 100 mg/kg, even more preferably between about 5 μg/kg and 60 mg/kg.
1. In Vitro Efficacy Results
Assay for Cytolytic Activity
A first experiment consists in the assessment of the tumoral cell death. Peripheral Vγ9δ2 T cells from healthy donors have been tested for lytic capacity towards several tumoral cell lines measured in standard cytotoxicity assay (4 h 51Cr release). Tumoral cell lines were isotopically labelled with 51Cr. Release of 51Cr has been determined after 4 hours of co-culture. Specific lysis (expressed as percentage) is calculated using the standard formula [(experimental-spontaneous release/total-spontaneous release)×100].
Three experimental conditions have been used in order to compare tumoral cell death:
The experiments have been performed at least in triplicate. (*) and (**) mean highly significant, < 1/100 and < 1/1000, respectively.
Tumoral cell lines tested are the following: NCBE, GRANTA, RL, Karpas-422, RAJI, DAUDI, and Es-Moult. The tested cell lines are indicated in the
The results are shown in
It has been observed for two different therapeutic antibodies that the tumoral cell death is higher by using a combination of a therapeutic antibody and a γδ T cell activator. Therefore, the therapeutic antibody and the γδ T cell activator have a synergic effect on the death of tumoral cells.
Assay for Cytolytic Cells Determination
A second experiment consists in the assessment of the cytotoxic capacity of Vγ9δ2 T cells. Tumoral cell lines were co-cultured as previously described with γδ T cells (therapeutic antibody alone 10 μg/ml, activated Vγ9δ2 T cells by phosphantigen alone, or both). The experiments determined the number of Vγ9δ2 T cells having cytotoxic activity. Vγ9δ2 T cells having cytotoxic capacity are known to express CD107a on their surface after coming into contact with a target cell susceptible to lysis. CD107a+ cells have been measured by flow cytometry.
Tumoral cell lines tested are the following: NCBE, RL, Karpas-422, RAJI, DAUDI, GRANTA, FKBR3, and Es-Moult. The tested cell lines are indicated in
The results are shown in
It has been observed for two different therapeutic antibodies that the number of cytotoxic Vγ9δ2 T cells is increased following a combination treatment with a therapeutic antibody and a γδ T cell activator.
2. Pre-Clinical Data in Primates
8 purpose bred Cynomolgus monkeys (Macaca fascicularis), were treated with rituximab and BrHPP, in a GLP study. Six animals received the combination of rituximab and BrHPP. The control group consisted of two animals treated with rituximab alone (no BrHPP).
Cynomolgus monkeys (3/gender) received 4 weekly intravenous injections (5 mL/kg, 30-min or 1-hour infusion) of rituximab at 5 mg/kg, 3 intravenous injections (15 mL/kg, 30-min infusion) of BrHPP at 90 mg/kg separated by 3-week intervals, the first administration of BrHPP being on the same day as the second rituximab injection and administered together with subcutaneous IL-2 (1 million IU, equivalent to 4 million IU per m2, 450 μL dose volume) for 5 consecutive days starting on the day of BrHPP administration.
Control animals (1/gender) received 4 weekly intravenous infusions of rituximab at 5 mg/kg together with vehicle injections in place of BrHPP and IL-2.
No persistent signs of intolerance or acute toxicity were observed throughout the study, and the combination treatment is considered safe.
Based on early cytokine release dosages (within 4 hours after each administration of rituximab or BrHPP), it seems that there is no apparent excessive induction of pro-inflammatory cytokines. The cytokine profile is identical and the levels produced are consistent with those expected after administration of each compound on its own.
Immuno-monitoring of blood lymphocyte populations confirmed that γδ T lymphocytes can be efficiently and repeatedly amplified in vivo by BrHPP in rituximab-treated animals, with a level and kinetics of response at least as good as what is generally observed with BrHPP alone (
Moreover, in BrHPP+rituximab treated animals, B cell depletion is rapid, efficient and considerably higher than in the animals treated with rituximab alone. Furthermore, reconstitution of B cell in blood is slower in BrHPP+rituximab treated animals than in the rituximab alone treated animals (
All these observations confirm that the combination of rituximab and BrHPP is more efficient for the depletion of B-lymphomas than rituximab alone, and delays the reconstitution of B cell population, thereby improving the effectiveness of B-cell depletion therapy in vivo.
Conclusion. The interaction between BHPP and rituximab appears pharmacologically beneficial (B-cell depletion) with an improved effect compared to each compound in monotherapy. Moreover, no major signs of toxicity, especially indicative of dramatic increase in cytokine release due to the combination treatment, occurred applying the clinical dosing regimen to monkeys.
| Number | Date | Country | Kind |
|---|---|---|---|
| 06291146.6 | Jul 2006 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2007/057217 | 7/12/2007 | WO | 00 | 2/20/2009 |
| Number | Date | Country | |
|---|---|---|---|
| 60924208 | May 2007 | US |
