WO2007127204A2 - Méthodes et compositions concernant une immunostimulation - Google Patents

Méthodes et compositions concernant une immunostimulation Download PDF

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Publication number
WO2007127204A2
WO2007127204A2 PCT/US2007/009947 US2007009947W WO2007127204A2 WO 2007127204 A2 WO2007127204 A2 WO 2007127204A2 US 2007009947 W US2007009947 W US 2007009947W WO 2007127204 A2 WO2007127204 A2 WO 2007127204A2
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WIPO (PCT)
Prior art keywords
inhibitor
dpp8
selective
cancer
dpp4
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Ceased
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PCT/US2007/009947
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WO2007127204A3 (fr
Inventor
Michael I. Jesson
Paul A. Mclean
Glenn T. Miller
Barry Jones
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Point Therapeutics Inc
Dara Biosciences Inc
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Point Therapeutics Inc
Dara Biosciences Inc
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Publication of WO2007127204A2 publication Critical patent/WO2007127204A2/fr
Anticipated expiration legal-status Critical
Publication of WO2007127204A3 publication Critical patent/WO2007127204A3/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/4035—Isoindoles, e.g. phthalimide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/426—1,3-Thiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4453—Non condensed piperidines, e.g. piperocaine only substituted in position 1, e.g. propipocaine, diperodon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • A61K31/4725—Non-condensed isoquinolines, e.g. papaverine containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4741—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having oxygen as a ring hetero atom, e.g. tubocuraran derivatives, noscapine, bicuculline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

  • the invention relates to methods for inducing immunostimulation via DPP8/9 inhibition.
  • Abnormal cell proliferation is usually characterized by an increased rate of division and in some cases uncontrolled growth.
  • a proliferative cell disorder is a tumor.
  • primary malignant tumors are particularly problematic given their tendency to invade surrounding tissues and metastasize to distant organs in the body.
  • the most frequently used methods for treating neoplasia, especially solid tumor forms of neoplasia include surgical procedures, radiation therapy, drug therapies, and combinations of the foregoing. These methods involve significant risk (e.g., of infection, death) to the patient. More importantly, the probability of eliminating all malignant cells is small particularly if the zone of malignant growth is not well defined or if the primary tumor has metastasized by the time of surgery. Achieving therapeutic doses effective for treating the cancer is often limited by the toxic side effects of the anti-cancer agent on normal, healthy tissue. An ideal anti-cancer agent or therapy has tissue specificity, thereby reducing side-effects on normal (dividing) cells.
  • the invention relates in part to the finding that inhibition of dipeptidyl peptidases (DPP) 8 and/or 9 results in cytokine induction including IL-I induction.
  • DPP dipeptidyl peptidases
  • the invention therefore provides methods and compositions for immune stimulation using DPP8/9 inhibitors, including selective and/or specific DPP8/9 inhibitors, as stand alone therapies or in combination with other agents.
  • the invention provides compositions and methods of use in the prevention and treatment of disorders that would benefit from enhanced immunostimulation.
  • the enhanced immunostimulation may activate macrophages and other antigen presenting cells, and is therefore useful in enhancing immune responses that involve such cells including antibody dependent cell-mediated cytotoxicity and antigen presentation.
  • DPP8/9 inhibitors to stimulate cytokine and chemokine production endogenously is beneficial since exogenous administration of some of these factors, such as for example, IL-I, has been associated with toxicity.
  • Production of IL-I endogenously can be used to induce cytokines in a controlled manner, and thereby overcome toxicity problems.
  • induction of these cytokines from cells in vivo also indicates that feedback loops normally operating in vivo may be operative and can control cytokine levels.
  • Immune therapies include but are not limited to passive immune therapies such as immunoglobulin administration, and active immune therapies such as vaccination with antigens alone or antigens in the context of dendritic cells.
  • the methods are intended to treat or prevent various indications that would benefit from an enhanced immune response.
  • the DPP8/9 inhibitors are administered with an antibody or antibody fragment, with an antigen and optionally with an adjuvant, or as stand alone compositions.
  • the immune response that is stimulated is a cell- mediated immune response involving T cells, NK cells, macrophages, and the like.
  • the immune response that is stimulated is a humoral response involving B cells and antibody production. Both types of responses can co-exist in yet other embodiments.
  • the immune response is an innate immune response, while in others it is an adaptive immune response.
  • the invention provides a method for stimulating an immune response comprising administering to a subject in need thereof a DPP8/9 inhibitor in an amount effective to stimulate an immune response.
  • the invention provides a method for stimulating an immune response in a subject comprising administering to a subject in need of immune stimulation a DPP8/9 inhibitor and an antibody or antibody fragment, in an amount effective to stimulate an immune response.
  • the subject may be one in need of immune stimulation is a subject having or at risk of developing cancer or it may be a subject having or at risk of developing an infection.
  • the invention provides a method for stimulating an immune response in a subject comprising administering to a subject in need of immune stimulation a DPP8/9 inhibitor and an antigen, in an amount effective to stimulate an antigen-specific immune response.
  • the method further comprises administering an adjuvant to the subject.
  • the adjuvant may be alum, cholera toxin, CpG immunostimulatory nucleic acids, MPL, MPD, or QS-21.
  • the antigen is a cancer antigen.
  • the immune response is antibody dependent cell-mediated cytoxicity.
  • the immune response is a cell-mediated immune response and/or a humoral (i.e., antibody-mediated) immune response.
  • the immune response may be an innate immune response or an adaptive immune response, in other embodiments.
  • the immune response is an antigen specific immune response.
  • the invention provides a method of treating a subject at risk of developing an infection comprising identifying a subject at risk of developing an infectious disease, and administering an amount effective of a DPP8/9 inhibitor to the subject.
  • the invention provides a method for treating a subject having or at risk of developing a condition characterized by abnormal cell proliferation comprising administering to a subject in need thereof a DPP8/9 inhibitor in an amount effective to inhibit the condition.
  • the invention provides a method for treating a subject having or at risk of developing a condition characterized by abnormal cell proliferation comprising administering to a subject in need thereof a DPP8/9 inhibitor and a DPP4 inhibitor in an amount effective to inhibit the condition.
  • the method further comprises administering a fibroblast activation protein (FAP) inhibitor to the subject.
  • FAP inhibitor may be a selective or a specific inhibitor but it is not so limited.
  • the invention provides a method for treating a subject having or at risk of developing a condition characterized by abnormal cell proliferation comprising administering to a subject in need thereof a DPP8/9 inhibitor and a fibroblast activation protein (FAP) inhibitor in an amount effective to inhibit the condition.
  • the method further comprises administering a DPP4 inhibitor to the subject.
  • the DPP4 inhibitor may be a selective or a specific inhibitor but it is not so limited.
  • the invention provides a method for improving the efficacy of anticancer treatment comprising administering to a subject in need thereof an anti-cancer agent and a DPP8/9 inhibitor in an amount effective to improve the effect of the anti-cancer agent.
  • the anti-cancer agent is a chemotherapeutic agent.
  • the chemotherapeutic agent is docetaxel (TAXOTERE), cisplatin, gemcitabine, pemetrexed (ALIMTA), erlotinib (TARCEVA), gefitinib (IRESSA), temozolomide (TEMODAR), carboplatin, cyclophosphamide or doxorubicin.
  • the anti-cancer agent is an immunotherapeutic agent.
  • the immunotherapeutic agent is an antibody.
  • the antibody is rituximab (RITUXAN), bevacizumab (AV ASTIN), cetuximab (ERBITUX), trastuzumab (HERCEPTIN), tositumomab (BEXXAR), or alemruzumab (CAMPATH).
  • the efficacy of antibodies or fragments thereof can be enhanced through their use in conjunction with a DPP8/9 inhibitor.
  • DPP8/9 inhibitor a DPP8/9 inhibitor.
  • the subject is refractory to a prior anti-cancer treatment.
  • the prior anti-cancer treatment is different from the anti-cancer agent.
  • the prior anti-cancer treatment comprises a chemotherapeutic agent.
  • the prior anti-cancer treatment comprises an immunotherapeutic agent.
  • the invention provides a method for treating a subject having or at risk of developing a condition characterized by abnormal cell proliferation comprising administering to a subject in need thereof a DPP8/9 inhibitor in an amount effective to inhibit the condition, wherein the inhibitor is not a specific DPP4 inhibitor.
  • the invention provides a method for treating a subject having or at risk of developing a condition characterized by abnormal cell proliferation comprising administering to a subject in need thereof a DPP8/9 inhibitor in an amount effective to inhibit the condition, wherein the inhibitor is not
  • a and Ai may be L- or D-amino acid residues such that each A in A m (i.e., where m>l) may be a different amino acid residue from every other A in A m ; the C bonded to B is in the L-configuration; and each Xi and X2 is, independently, a hydroxyl group or a group capable of being hydrolyzed to a hydroxyl group in aqueous solution at physiological pH.
  • the invention provides a method for treating a subject having or at risk of developing an interferon (IFN)-responsive condition.
  • the method comprises administering to a subject in need of such treatment a DPP8/9 inhibitor in an amount effective to induce a therapeutically or prophylactically effective amount of IL-I in the subject.
  • the method may further comprise identification of a subject having or at risk of developing an IFN-responsive condition.
  • the IFN may be IFN ⁇ , IFN ⁇ -2b, IFN ⁇ or IFN ⁇ , but is not so limited.
  • the condition may be an IFN ⁇ -responsive condition, and may be selected from the group consisting of viral infections and associated diseases, and cancer .
  • the subject is HIV positive.
  • the IFN-responsive condition is a chronic infection selected from the group consisting of a chronic hepatitis B infection, chronic hepatitis C infection, chronic Epstein Barr Virus infection, and tuberculosis.
  • Other disorders include hepatocellular carcinoma, Kaposi's Sarcoma (AIDS-related), thick primary melanomas, and regional lymph node metastases.
  • the disorder is refractive (i.e., resistant) to prior therapy (e.g., drug treatment).
  • the disorder is drug resistant.
  • the disorder is multiple sclerosis. IFN- responsive conditions are not intended to be so restricted however.
  • the method may further comprise administering to the subject a second active agent selected from the group consisting of IFN ⁇ , pegylated IFN, IFN ⁇ -2b, acyclovir, lobucavir, ganciclovir, L-deoxythymidine, clevudine, a therapeutic vaccine, phosphonoformate (PFA), ribavirin (RBV), thymosin alpha- 1, 2 3-dideoxy-3-fluoroguanosine (FLG), famciclovir, lamivudine, adefovir dipivoxil, entecavir, emtricitabine, and hepatitis B-specif ⁇ c immunoglobulin.
  • a second active agent selected from the group consisting of IFN ⁇ , pegylated IFN, IFN ⁇ -2b, acyclovir, lobucavir, ganciclovir, L-deoxythymidine, clevudine, a therapeutic vaccine, phospho
  • the invention provides a method for treating a subject having or at risk of developing cancer comprising administering to a subject in need of such treatment an enzyme inhibitor selected from the group consisting of a tyrosine kinase inhibitor, a CDK inhibitor, a MAP kinase inhibitor, and an EGFR inhibitor, and a DPP8/9 inhibitor in an amount effective to inhibit the cancer.
  • an enzyme inhibitor selected from the group consisting of a tyrosine kinase inhibitor, a CDK inhibitor, a MAP kinase inhibitor, and an EGFR inhibitor, and a DPP8/9 inhibitor in an amount effective to inhibit the cancer.
  • a method for treating a subject having or at risk of developing cardiovascular disease comprising administering to a subject in need of such treatment a DPP8/9 inhibitor in an amount effective to induce an effective amount of IL-I .
  • the method may further comprise identifying a subject in need of such treatment.
  • the invention provides a method for preventing drug resistance in a subject.
  • the method involves administering to a subject receiving an anti-microbial agent, a DPP8/9 inhibitor in an amount effective to reduce the risk of resistance to the anti-microbial agent.
  • the subject is one having or is at risk of developing an infection.
  • the bacterial infection is a Pseudomonas infection.
  • Other drug resistant microbes and the drugs to which they are resistant include Staphylococcus aureus (penicillin), Streptococcus pneumoniae (penicillin), gonorrhea (penicillin), and Enterococcus faecium (penicillin).
  • the anti-microbial agent is selected from the group consisting of an anti-bacterial agent, an anti- viral agent, an anti-fungal agent, and an antiparasitic agent.
  • the invention provides a method for shortening a vaccination course.
  • the method may involve, in one embodiment, administering to a subject in need of immunization a DPP8/9 inhibitor in an amount effective to induce an antigen-specific immune response to a vaccine administered in a vaccination course, wherein the vaccination course is shortened by at least one immunization.
  • the vaccination course is shortened by one immunization, two immunizations, three immunizations, or more.
  • the method may involve, in another embodiment, administering to a subject in need of immunization a DPP8/9 inhibitor in an amount effective to induce an antigen-specific immune response to a vaccine administered in a vaccination course, wherein the vaccination course is shortened by at least one day.
  • the vaccination course is shortened by one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, one month, two months or more.
  • Immunizations that can be modified in this way include but are not limited to newborn immunizations for HBV; immunizations at for example two months of age for Polio, DTaP, Hib, HBV, Pneumococcus; immunizations at for example four months of age for Polio, DTaP, Hib, Pneumococcus; immunizations at for example six months of age for Polio, DTaP, Hib, HBV, Pneumococcus; immunizations at for example 12-15 months of age for Hib, Pneumococcus, MMR, Varicella; immunizations at for example 15-18 months of age for DTaP; immunizations at for example 4-6 years of age for Polio, DPT, MMR; immunizations at for example 11-12 years of age for MMR; immunizations at for example 14-16 years of age for tetanus-diphtheria (i.e., Td) (
  • Vaccination courses that can be shortened by the method of the invention include but are not limited to: HBV: Hepatitis B vaccine (3 total doses currently recommended); Polio: Inactivated polio vaccine (4 total doses currently recommended); DTaP: Diphtheria/tetanus/acellular Pertussis (3-in-l vaccine; 5 total doses currently recommended); Hib: Haemophilus influenzae type b conjugate vaccine (4 total doses currently recommended); Pneumococcus (Prevnar): Protects against certain forms of Strep.
  • DPP8/9 inhibitors can be used together with oral polio vaccine.
  • the DPP8/9 inhibitor may be a selective or a specific DPP8/9 inhibitor.
  • the inhibitor is a selective DPP8/9 inhibitor that inhibits DPP8/9 to a greater extent than it inhibits at least DPP4.
  • the inhibitor is a selective DPP8/9 inhibitor that inhibits DPP8/9 to a greater extent than it inhibits at least DPP4 and FAP.
  • the DPP8/9 inhibitor is a selective inhibitor.
  • the selective DPP8/9 inhibitor may be
  • Lys[Z(NO 2 )]-pyrrolidide (8, X CH 2 ) Lankas et al., Diabetes 2005, 54, 2988.
  • Lys[Z(NO 2 )]-piperidide (10, X CH 2 CH 2 ) Lankas et al., Diabetes 2005, 54, 2988.
  • R 2 is nicotinonitrile
  • Arg-Pro-Ala-Tyr (SEQ ID NO: 1) (see WO02/31 134), Met-Phe-Ala-Tyr (SEQ ID NO: 2) (see WO02/31134), or Ile-Phe-Ala-Tyr (SEQ ID NO: 3) (WO02/31 134).
  • the subject has a condition characterized by abnormal cell proliferation, while in others the subject is at risk of developing a condition characterized by abnormal cell proliferation.
  • the condition may be a cancer.
  • the condition or the cancer may be a carcinoma such as but not limited to non-small cell lung cancer, pancreatic cancer, or colorectal cancer.
  • the condition or the cancer may be melanoma.
  • the condition or the cancer may be a sarcoma such as but not limited to osteosarcoma or fibrosarcoma.
  • the condition or the cancer may be a leukemia or a lymphoma.
  • the condition or the cancer may be chronic lymphocytic leukemia.
  • the condition or the cancer may be refractory to a prior treatment.
  • the condition or the cancer may be a primary tumor. In some embodiments, the cancer expresses normal or below- normal levels of DPP8 and/or 9 or does not express DPP8 and/or 9.
  • the method further comprises administering to the subject a second therapeutic agent that is an anti-cancer agent.
  • the anti-cancer agent may be a chemotherapeutic agent such as but not limited to docetaxel, cisplatin, gemcitabine, pemetrexed (ALIMTA), erlotinib (TARCEVA), gefitinib (IRESSA), temozolomide (TEMODAR), carboplatin, cyclophosphamide or doxorubicin.
  • the method further comprises administering to the subject a second therapeutic agent that is an immunotherapeutic agent.
  • the immunotherapeutic agent may be an antibody or an antibody fragment including but not limited to rituximab (RITUXAN), bevacizumab (AVASTIN), cetuximab (ERBITUX), trastuzumab (HERCEPTIN), tositumomab (BEXXAR), or alemtuzumab (CAMPATH).
  • the immunotherapeutic agent may an antigen such as a cancer antigen or a microbial antigen such as a bacterial antigen, a viral antigen, a fungal antigen, a parasitic antigen or a mycobacterial antigen.
  • the cancer antigen may be MART-1/Melan-A, gplOO, adenosine deaminase-binding protein (ADAbp), FAP, cyclophilin b, colorectal associated antigen (CRC)--C017-l A/GA733, carcinoembryonic antigen (CEA), CAP-I, CAP-2, etv6, AMLl, prostate specific antigen (PSA), PSA-I, PSA-2, PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor/CD3-zeta chain, and CD20.
  • a cancer antigen may be MART-1/Melan-A, gplOO, adenosine deaminase
  • the cancer antigen may also be selected from the group consisting of MAGE-A 1 , MAGE-A2, MAGE- A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-AlO, MAGE-Al 1, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-Cl, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5).
  • the cancer antigen is selected from the group consisting of GAGE-I, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9.
  • the cancer antigen is selected from the group consisting of BAGE, RAGE, LAGE-I, NAG, GnT- V, MUM-I, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCASl, ⁇ -fetoprotein, E-cadherin, ⁇ -catenin, ⁇ -catenin, ⁇ -catenin, pl20ctn, gplOO Pmel117 , PRAME, NY-ESO-I, cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, pi 5, gp75, GM2 ganglioside, GD2 gangli
  • APC adenomatous poly
  • the methods may further comprise treating the subject with a therapy selected from the group consisting of surgery, radiation and chemotherapy.
  • the DPP8/9 inhibitor and the antigen (or the antibody) are administered prior to treating the subject with a therapy selected from the group consisting of surgery, radiation and chemotherapy. In another embodiment, the DPP8/9 inhibitor and the antigen (or antibody) are administered after treating the subject with a therapy selected from the group consisting of surgery, radiation and chemotherapy. In yet another embodiment, the DPP8/9 inhibitor and the antigen (or antibody) are administered before and after treating the subject with a therapy selected from the group consisting of surgery, radiation and chemotherapy. In some embodiments, the method further comprises administering to the subject a second therapeutic agent that is a cytokine.
  • the cytokine may be G-CSF or GM-CSF, or it may be M-CSF.
  • the method further comprises administering to the subject a DPP4 inhibitor.
  • the DPP4 inhibitor may be a selective inhibitor or a specific inhibitor, although it is not so limited.
  • the DPP4 inhibitor may be a selective DPP4 inhibitor such as
  • the DPP4 inhibitor has an IC50 for DPP8/9 that is at least 3 fold higher than its IC50 for DPP4. In some embodiments, the DPP4 inhibitor has an IC50 for DPP8/9 that is at least 5 fold higher than its IC 50 for DPP4. In some embodiments, the selective DPP8/9 inhibitor is administered prior to the
  • the selective DPP8/9 inhibitor and the DPP4 inhibitor are administered to the subject substantially simultaneously. In some embodiments, the selective DPP8/9 inhibitor and the DPP4 inhibitor are administered to the subject in an alternating manner. In some embodiments, the method further comprises administering to the subject a boroProline compound having a structure of
  • T is a boronate group of the formula
  • each D 1 and D 2 independently, is a hydroxyl group or a group which is capable of being hydrolyzed to a hydroxyl group in aqueous solution at physiological pH;
  • X comprises an amino acid or a peptide which mimics the site of a substrate recognized by a post-prolyl cleaving enzyme;
  • Y is
  • each R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , and R 8 is a hydrogen.
  • the boroProline compound is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • the selective DPP8/9 inhibitor is administered prior to the boroProline compound. In some embodiments, the selective DPP8/9 inhibitor and the boroProline compound are administered to the subject substantially simultaneously. In some embodiments, the selective DPP8/9 inhibitor and the boroProline compound are administered to the subject in an alternating manner.
  • the subject has or is at risk of developing an infection such as a bacterial infection, a viral infection, a fungal infection, a parasitic infection or a mycobacterial infection.
  • the method further comprises administering to the subject an anti-microbial agent such as an anti-bacterial agent, an anti-viral agent, an anti-fungal agent, an anti-parasitic agent, or an anti-mycobacterial agent.
  • the bacterial infection may be an E. coli infection, a Staphylococcal infection, a Streptococcal infection, a Pseudomonas infection, Clostridium difficile infection, Legionella infection, Pneumococcus infection, Haemophilus infection, Klebsiella infection, Enterobacter infection, Citrobacter infection, Neisseria infection, Shigella infection, Salmonella infection, Listeria infection, Pasteurella infection, Streptobacillus infection, Spirillum infection, Treponema infection, Actinomyces infection, Borrelia infection, Corynebacterium infection, Nocardia infection, Gardnerella infection, Campylobacter infection, Spirochaeta infection, Proteus infection, Bacteriodes infection, H. pylori infection, or anthrax infection.
  • the mycobacterial infection may be tuberculosis or leprosy respectively caused by the M. tuberculosis and M. leprae species, but is not so limited.
  • the viral infection may be a Herpes simplex virus 1 infection, a Herpes simplex virus 2 infection, cytomegalovirus infection, hepatitis A virus infection, hepatitis B virus infection, hepatitis C virus infection, human papilloma virus infection, Epstein Barr virus infection, rotavirus infection, adenovirus infection, influenza A virus infection, respiratory syncytial virus infection, varicella-zoster virus infections, small pox infection, monkey pox infection, SARS infection or avian flu infection.
  • the viral infection is not an HIV infection.
  • the fungal infection may be candidiasis, ringworm, histoplasmosis, blastomycosis, paracoccidioidomycosis, crytococcosis, aspergillosis, chromomycosis, mycetoma infections, pseudallescheriasis, or tinea versicolor infection.
  • the parasite infection may be amebiasis, Trypanosoma cruzi infection, Fascioliasis, Leishmaniasis, Plasmodium infections, Onchocerciasis, Paragonimiasis, Trypanosoma brucei infection, Pneumocystis infection, Trichomonas vaginalis infection, Taenia infection, Hymenolepsis infection, Echinococcus infections, Schistosomiasis, neurocysticercosis, Necator americanus infection, or Trichuris trichuria infection.
  • the antigen is a microbial antigen.
  • a microbial antigen is an antigen derived from an infectious pathogen, and may include the entire pathogen.
  • the antigen may be peptide, lipid, or carbohydrate in nature, but it is not so limited.
  • the microbial antigen is selected from the group consisting of a bacterial antigen, a mycobacterial antigen, a viral antigen, a fungal antigen, and a parasitic antigen.
  • the bacterial, viral, fungal, parasitic and mycobacterial antigen may be derived from any of the bacterial, viral, fungal, parasitic and mycobacterial species recited herein, respectively.
  • the antibody or antibody fragment is conjugated (covalently or otherwise) to a toxin derived from plant, fungus, or bacteria.
  • the toxin may be selected from the group consisting of A chain toxin, deglycosylated A chain toxin, ribosome inactivating protein, ⁇ -sarcin, aspergillin, restrictocin, ribonuclease, diptheria toxin and Pseudomonas exotoxin, but is not so limited.
  • the antibody or antibody fragment is conjugated to a chemotherapeutic agent, a radioisotope or a cytotoxin.
  • the chemotherapeutic agent may be selected from the group consisting of an anti-metabolite, an anthracycline, a vinca alkaloid, an antibiotic, an alkylating agent, and an epipodophyllotoxin, but is not so limited.
  • the DPP8/9 inhibitors may be formulated together or separately from the other agent, depending on the administration regimen, route, and like.
  • compositions comprising one or more of the foregoing agents including combinations of DPP8/9 inhibitors, DPP4 inhibitors, FAP inhibitors, antibodies, antigens, adjuvants, cytokines, anti-bacterial agents, anti-viral agents, anti-fungal agents, anti-parasitic agents, anti-mycobacterial agents, and the like.
  • the invention provides in yet another aspect a composition comprising an effective amount of a DPP8/9 inhibitor and an antibody or antibody fragment.
  • the composition further comprises a pharmaceutically acceptable carrier.
  • the effective amount is an amount to stimulate antibody dependent cell-mediated cytoxicity.
  • the effective amount is an amount to treat or prevent cancer.
  • the effective amount is an amount to treat or prevent an infectious disease.
  • the antibody or antibody fragment is an antibody, and it can be selected from the group listed above.
  • the invention provides a composition comprising an effective amount of a DPP8/9 inhibitor and a cancer antigen.
  • the effective amount is an amount to treat or prevent cancer.
  • the cancer antigen may be a peptide antigen, or a lipid antigen, but it is not so limited.
  • the cancer antigen can be selected from the groups recited above.
  • kits comprising these agents, compositions and/or combinations.
  • the compositions may be provided in a housing such as a container, a box, or a bag.
  • the housing may also contain instructions for use of the composition either thereon or therein.
  • the instructions for use indicate how the contents of the housing are to be used, including timing and dose of administrations.
  • FIG. 1 demonstrates activation of caspase-1 and induction of IL-I ⁇ by PT-100 (talabostat, L-valinyl-L-boroproline) and the DPP8/9 selective inhibitors PTX-1200 (cyclohexyl glycine-isoindoline) and PTX-1210 (l-Bis(4-fluorophenyl)methyl piperazine/aspartate/isoindoline) in cultures of PMA-differentiated THP-I human monocytic cells.
  • PT-100 talabostat, L-valinyl-L-boroproline
  • PTX-1200 cyclohexyl glycine-isoindoline
  • PTX-1210 l-Bis(4-fluorophenyl)methyl piperazine/aspartate/isoindoline
  • THP-I cells were incubated (37°C) in ⁇ 3kDa factor in multiwell plates with PT-100, PTX- 1200 or PTX-1210, each at a concentration of 10 ⁇ M, or without addition of a compound (control).
  • Caspase-1 activity was measured by the Ac-WEHD-AFC fluorogenic assay (A) and secreted IL- I ⁇ by ELISA (B) at the time points indicated on the abscissae.
  • FIG. 2 demonstrates activation of caspase-1 by PT-100 (talabostat, L-valinyl-L- boroproline) and the DPP8/9 selective inhibitor PTX-1210 (l-Bis(4-fluorophenyl)methyl piperazine/aspartate/isoindoline) in cultures of undifferentiated THP-I human monocytic cells.
  • THP-I cells were incubated (37 0 C) in multiwell plates with PT-100, or PTX-1210, each at a concentration of 10 ⁇ M, or without addition of a compound (control).
  • Caspase-1 activity was measured by the Ac-WEHD-AFC fluorogenic assay at the time points indicated on the abscissae. It is to be understood that the Figures are not required for enablement of the invention.
  • the invention relates in part to the finding that inhibition of dipeptidyl peptidase 8 and/or 9 (DPP8/9) results in induction of IL-I and various other cytokines and chemokines.
  • DPP8/9 inhibition results in activation of caspase I which in turn results in at least IL-I beta secretion. This is followed by the upregulation of a number of cytokines and chemokines for example by autocrine and/or paracrine pathways.
  • cytokines and chemokines include but are not limited to IL-I alpha, IL-6, G-CSF, MCP-1/CCL2, MCP-2/CCL8, MCP- 3/MARC/CCL7, MCP-5/CCL12, MRP-1/CCL6, JBE, MIP-lalpha/CCL3, MIP-I beta/CCL4, MIP-2/CXCL2, MIG/CXCL9, JE, TARC/CCL17, Lymphotactin/XCLl, IL-8/KC/CXCL1, ENA78/CXCL5, LIX, eotaxin/CCLl 1, IP-10/CXCLlO, MDC/CCL22, IFNbeta, and thrombospondin.
  • IL-I beta may also upregulate expression of endothelial adhesion molecules which can in turn facilitate migration of neutrophils.
  • DPP8/9 inhibitors are able to cause the release of neutrophils from the bone marrow via the induction of IL-I beta.
  • the invention therefore provides methods for stimulating immune responses by inhibiting DPP8/9. These methods can form the basis of various therapies that are immune responsive including but not limited to cancer and infectious disease.
  • the invention contemplates the use of DPP8/9 inhibitors alone or in combination with other DPP inhibitors or other secondary agents.
  • the methods are intended to stimulate an immune response in a subject.
  • the immune response may be a cell-mediated immune response and/or a humoral (i.e., antibody-mediated) immune response.
  • the immune response may be an innate immune response or an adaptive immune response.
  • the immune response is an antigen specific immune response.
  • the DPP8/9 inhibitors can be used to stimulate or enhance antibody dependent cell-mediated cytoxicity (ADCC), particularly when administered with an antibody or antibody fragment.
  • the immunostimulatory DPP8/9 inhibitors of the invention can be used in a number of conditions as described in greater detail herein. These conditions are generally those that benefit from immunostimulation, rather than those that require immunosuppression (e.g., autoimmune diseases, allograft transplant rejection, etc.).
  • the methods of the invention use DPP8/9 inhibitors.
  • a DPP8/9 inhibitor is a compound that inhibits the enzymatic activity of DPP8 and/or DPP9.
  • Various methods provided herein employ selective or specific DPP8/9 inhibitors.
  • a selective DPP8/9 inhibitor is a compound that inhibits DPP8/9 (i.e., DPP8 and/or DPP9) to a greater extent than it does at least one (including two or three) other post-prolyl cleaving enzyme.
  • a selective DPP8/9 inhibitor inhibits DPP8/9 to a greater extent than it does DPP4, or fibroblast activation protein (FAP), or PEP, or DPP2.
  • FAP fibroblast activation protein
  • PEP fibroblast activation protein
  • DPP2 fibroblast activation protein
  • the selective DPP8/9 inhibitor may inhibit DPP8/9 to a greater extent than it does two or three other post-prolyl cleaving enzymes.
  • a selective DPP8/9 inhibitor may inhibit DPP8/9 to a greater extent than it does DPP4 and FAP, or DPP4 and PEP, or FAP and PEP, or DPP2 and DPP4, etc.
  • a selective DPP8/9 inhibitor may inhibit DPP8/9 to a greater extent than it does DPP4, FAP and PEP, or DPP2, DPP4 and FAP, or DPP2, FAP and PEP, etc.
  • a specific DPP8/9 inhibitor is a compound that inhibits DPP8/9 to a greater extent than it does at least non-DPP8/9 post-prolyl cleaving enzymes DPP2, DPP4, FAP and PEP.
  • Enzyme inhibition may be measured as the concentration of compound required to inhibit enzymatic activity in vitro by 50%. This measure is referred to herein as the IC 50 , and it is usually expressed as a submolar value (e.g., nM). Accordingly, a selective DPP8/9 inhibitor has a lower IC 50 for DPP8/9 than for at least one non-DPP8/9 post-prolyl cleaving enzyme, including DPP4 and fibroblast activation protein (FAP). A specific DPP8/9 inhibitor has a lower IC 50 for DPP8/9 than for non-DPP8/9 post-prolyl cleaving enzymes DPP2, DPP4, FAP and PEP.
  • IC 50 concentration of compound required to inhibit enzymatic activity in vitro by 50%. This measure is referred to herein as the IC 50 , and it is usually expressed as a submolar value (e.g., nM).
  • a selective DPP8/9 inhibitor has a lower
  • the selective DPP8/9 inhibitor has an IC 50 for DPP 8/9 that is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 7-fold, at least 10-fold, at least 50-fold, at least 100-fold, or at least 1000 fold less than the IC 50 for at least one other post-prolyl cleaving enzyme.
  • the specific DPP8/9 inhibitor has an IC 50 for DPP8/9 that is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 7-fold, at least 10-fold, at least 50-fold, at least 100-fold, or at least 1000 fold less than the IC 50 for post-prolyl cleaving enzymes DPP2, DPP4, FAP and PEP.
  • a DPP4 inhibitor is a compound that inhibits the enzymatic activity of DPP4.
  • a selective DPP4 inhibitor is a compound that has a lower IC 50 for DPP4 than for at least one other post-prolyl cleaving enzyme. The definition is similar to that for selective DPP8/9 inhibitors as taught herein.
  • a specific DPP4 inhibitor is a compound that has a lower IC 50 for DPP4 than for post-prolyl cleaving enzymes DPP2, DPP8, DPP9, FAP and PEP.
  • FAP inhibitors including selective and specific FAP inhibitors.
  • a FAP inhibitor is a compound that inhibits the enzymatic activity of FAP.
  • a selective FAP inhibitor is a compound that has a lower IC 50 for DPP4 than for at least one other post-prolyl cleaving enzyme. The definition is similar to that for selective DPP8/9 inhibitors as taught herein.
  • a specific FAP inhibitor is a compound that has a lower IC50 for FAP than for post-prolyl cleaving enzymes DPP2, DPP4, DPP8, DPP9 and PEP.
  • PEP inhibitors including selective and specific PEP inhibitors.
  • a PEP inhibitor is a compound that inhibits the enzymatic activity of PEP.
  • a selective PEP inhibitor is a compound that has a lower IC50 for PEP than for at least one other post-prolyl cleaving enzyme. The definition is similar to that for selective DPP8/9 inhibitors as taught herein.
  • a specific PEP inhibitor is a compound that has a lower IC 50 for PEP than for other post-prolyl cleaving enzymes.
  • Post-prolyl cleaving enzymes are enzymes that cleave peptides (including proteins) at the carboxy terminal of preferably a proline residue.
  • Post-prolyl cleaving enzymes can have endopeptidase and/or exopeptidase activity.
  • Post-prolyl cleaving enzymes with exopeptidase activity include dipeptidyl peptidases.
  • Dipeptidyl peptidases are enzymes that cleave dipeptides from the amino terminus of a peptide (e.g., a protein) provided that the penultimate residue is a proline (and sometimes an alanine). These enzymes cleave at the carboxy side of the penultimate residue.
  • Post-prolyl cleaving enzymes include but are not limited to DPP2, DPP4, DPP8, DPP9, fibroblast activation protein (FAP) and prolylendopeptidase (PEP).
  • DPP2, DPP4, DPP8 and DPP9 have exopeptidase activities.
  • PEP has endopeptidase activity.
  • FAP has both exo- and endopeptidase activity. All of these enzymes are known in the art.
  • DPP8/9 inhibitors including selective and specific DPP8/9 inhibitors are as follows:
  • Lys[Z(NO 2 )]-pyrrolidicle (8, X CH 2 ) Lankas et at., Diabetes 2005, 54, 2988.
  • Lys[Z(NO 2 )]-piperidide (10, X CH 2 CH 2 ) Lankas et al., Diabetes 2005, 54, 2988.
  • DPP4 inhibitors including selective and specific DPP4 inhibitors are as follows:
  • DPP4 inhibitors are Saxagliptin (BMS), Sitagliptin (Merck MK-0431), (2R)-4- oxo-4-[3-(trifluoromethyl)-5,6-dihydro[l,2,4]triazolo[4,3- ⁇ ]pyrazin-7(8H)-yl]-l-(2,5- difluorophenyl)butan-2-amine fumarate and hydrochloride salts (des-fluoro analog of sitagliptin; Lankas et al.
  • X-boroPro compounds such as those described herein are also DPP4 inhibitors (e.g., Val-boroPro).
  • FAP inhibitor is acetyl-Gly-boroPro (Edosada et al. J Biol Chem. 2006 Mar 17;281(11):7437-44). Requirements for FAP specificity are discussed in Edosada et al. FEBS Lett. 2006 Mar 6;580(6):1581-6.
  • X-boroPro compounds such as those described herein are also FAP inhibitors (e.g., Val-boroPro).
  • Table A lists a number of selective and specific inhibitors for various post-prolyl cleaving enzymes (i.e., comparison of IC50 values of a given compound for a number of post- prolyl cleaving enzymes will identify selective and specific inhibitors). Comparison between compounds is not intended as not all compounds are tested under identical conditions.
  • inhibitors include isoleucylthiazolidide and cyclohexylglycylpyrrolidide.
  • T is a reactive group such as those recited above (e.g., a boronate group of the formula
  • each D 1 and D 2 independently, is a hydroxyl group or a group which is capable of being hydrolyzed to a hydroxyl group in aqueous solution at physiological pH);
  • X comprises an amino acid or a peptide which mimics the site of a substrate recognized by a post-prolyl cleaving enzyme.
  • X further comprises an N-terminal blocking group.
  • Y may be
  • Each R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , and R 8 may be a group which does not interfere significantly with site-specific recognition of the compound by a post-prolyl cleaving enzyme.
  • T is a boronate group, each D 1 and D 2 , independently, is OH; each R 1'8 is H; and X comprises an amino acid and a blocking group (e.g., N-blocking group) such as an acetyl group.
  • a blocking group e.g., N-blocking group
  • blocking groups can vary widely. In one blocking reaction, a hydrogen atom of the amino terminal amino group is replaced, generally in a dehydration reaction. Blocking groups can be
  • blocking groups may be employed not only to protect amino-terminal groups (and thereby act as N-terminal blocking groups), but also may be used to protect side chains of amino acid residues (e.g., side chains of Lys and Arg). Similarly, amino acid residues having acidic or hydroxy side chains can be protected using t-butyl, benzyl, or other suitable esters or ethers as blocking groups.
  • a and Aj may be a naturally or non- naturally occurring amino acid residue, peptide or peptidomimetic such that when A is an amino acid residue each A in A m (i.e., where m >1) may be a different amino acid residue from every other A in A m and when A is a peptide or peptidomimetic m is 1 ; the C bonded to B is preferably in the R-configuration; and each Xi and X 2 is, independently, a hydroxyl group or a group capable of being hydrolyzed to a hydroxyl group in aqueous solution at physiological pH.
  • Ai may be valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan, serine, threonine, aspartate, glutamate, asparagine, glutamine, lysine, arginine, histidine, cysteine, alanine or proline.
  • Ai is an L- amino acid residue, and optionally every A in A n , is an L- amino acid residue.
  • m is 0.
  • Xi and X 2 are hydroxyl groups.
  • the proline residue may be replaced with another amino acid residue such as, for example, alanine.
  • derivatives of these compounds in which the organo boronate is replaced with an organo phosphonate, a fluoroalkylketone, an halomethyl ketone, a diazomethyl ketone, a dimethylsulphonium salt, an alphaketo carboxylic acid, an alphaketo ester, an alphaketo amide, an alpha-diketone, an acyloxymethyl ketone, an aldehyde, an epoxysuccinyl, an N-peptidyl-O-acylhydroxylamine, an azapeptide, a fluoroolefin, a peptidyl (alpha- aminoalkyl) phosphonate ester, or a nitrile are also contemplated by the invention.
  • the proline residue attached to the reactive group is referred to as a pyrrolidine ring.
  • the pyrrolidine may be replaced with an azetidine or a thiazolidine.
  • the pyrrolidine-T moiety is replaced with a 4-cyano thiazolidine.
  • the dipeptide moiety can be an isostere.
  • m is an integer between 0 and 10, inclusive;
  • are naturally or non-naturally occurring amino acids, peptides or peptidomimetics; wherein if A is an amino acid residue, it can be a different amino acid residue in each repeating bracketed unit; the C bonded to B is in the R-configuration; and each Xi and X 2 is, independently, a hydroxyl group or a group capable of being hydrolyzed to a hydroxyl group in aqueous solution at physiological pH.
  • a and Ai may be alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, aspartate, glutamate, asparagine, glutamine, lysine, arginine, histidine, cysteine, methionine, or proline.
  • A) is an L- amino acid residue, and optionally every A is an L- amino acid residue.
  • the amino acid residues may be naturally and non-naturally occurring amino acids.
  • naturally occurring amino acids are glycine (GIy), and the L-forms of alanine (Ala), valine (VaI), leucine (Leu), isoleucine (He), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), cysteine (Cys), methionine (Met), serine (Ser), threonine (Thr), lysine (Lys), arginine (Arg), histidine (His), aspartic acid (Asp), glutamic acid (GIu), asparagine (Asn), glutamine (GIn) and proline (Pro).
  • Non-naturally occurring amino acids include the D-forms of Ala, VaI, Leu, He, Phe, Tyr, Trp, Cys, Met, Ser, Thr, Lys, Arg, His, Asp, GIu, Asn, GIn, and Pro.
  • non-naturally occurring amino acids include 2-azetidinecarboxylic acid or pipecolic acid (which have 6-membered, and 4-membered ring structures respectively), 4-hydroxy-proline (Hyp), 5-hydroxy-lysine, norleucine (NIe), 5- hydroxynorleucine (Hyn), 6-hydroxynorleucine, ornithine, cyclohexylglycine (Chg), N- Methylglycine (N-MeGIy), N-Methylalanine (N-MeAIa), N-Methylvaline (N-MeVaI), N- Methylleucine (N-MeLeu), N-Methylisoleucine (N-MeIIe), N-Methylnorleucine (N-MeNIe), N-Methyl-2-aminobutyric acid (N-Me Abu) and N-Methyl-2-aminopentanoic acid (Hy
  • the boroPro inhibitors can be provided in linear or cyclic form or as mixtures thereof, as described in U.S. Patent No. 6,355,614, issued March 12, 2002.
  • the proportion of linear (versus cyclic) forms in these mixtures may vary (e.g., 0-100%) depending on the formulation. In some embodiment, at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the inhibitor is in the linear form.
  • the boroPro inhibitors and derivatives may be provided as prodrugs that are converted
  • a prodrug of for example "A-boroPro”, as used herein, is a compound that is metabolized in vivo to A-boroPro or that disintegrates (e.g., upon contact with stomach acid) to form A-boroPro.
  • Some prodrugs are converted into A-boroPro via hydrolysis or oxidation in vivo. These include alcohol precursors of A-boroPro that are oxidized in vivo (e.g., in the liver) and a boroxine derivative of A-boroPro, as well as esters of Glu-boroPro and related compounds.
  • Prodrugs of A-boroPro also include cyclized versions of the molecule.
  • prodrugs includes compounds that are converted to A-boroPro by enzymes. These enzymes may be post-prolyl cleaving enzymes (e.g., DPP4) or non-post- prolyl cleaving enzymes. Examples of this class of prodrug moieties are disclosed in U.S. Patent Nos. 5,462,928 issued October 31, 1995; and 6,100,234 issued August 8, 2000; and published PCT applications WO 91/16339 published October 31, 1991; WO 93/08259 published April 29, 1993; and WO 03/092605, published November 13, 2003, among others.
  • the length of such prodrug compounds may be 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 50, 100 or more residues in length (whereby the length includes A and proline residues). Multiples of 3 are also contemplated.
  • the boroPro inhibitors when provided as a plurality or in a mixture, may be provided in a substantially optically pure form. That is, at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% of these inhibitors in a mixture (or composition) possess boron-bearing carbon atoms that are in the L-configuration. Methods for synthesizing substantially optically pure isomers of boroPro inhibitors are disclosed in published PCT application WO 93/08259.
  • the mixture of R- and S-enantiomers of boron substituted pyrrolidine contains at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the R-enantiomer of boron substituted pyrrolidine.
  • the DPP8/9 inhibitors exclude the inhibitors described in U.S. Patent 6890904. The teachings of these inhibitors is incorporated by reference herein.
  • the DPP8/9 inhibitor including a selective or specific DPP8/9 inhibitor is used together with Glu-boroPro, Gln-boroPro, Arg-boroPro, Phe-boroPro, Lys- boroPro, and/or acetyl-Gly-boroPro.
  • the DPP8/9 inhibitors, including selective or specific DPP8/9 inhibitors and the X-boroPro compound may be used at their maximum tolerated dose. In these instances, the DPP8/9 inhibitor may be administered before, before and during, during and after, before and after, or before, during and after administration of the X-boroPro compound.
  • the DPP8/9 inhibitors including selective or specific DPP8/9 inhibitors are used together with Val-boroPro, Ile-boroPro, Met-boroPro, Leu-boroPro, cyclopentylglycine boroPro
  • boroPro compound in these instances may be used at its maximum tolerated dose or at a sub-therapeutic dose.
  • a sub-therapeutic dose is a dose that is less than the dose that produces the maximal, medically acceptable, therapeutic result in the subject when administered alone.
  • the sub-therapeutic dose may be equal to or less than 95%, 90%, 80%,
  • Therapeutic doses for human subjects can be derived from clinical trial data. Alternatively, therapeutic doses can be determined using murine model systems. In some instances, the sub-therapeutic dose of for example VaI -boroPro may be a dose that reduces tumor size but does not induce cytokine or chemokine production.
  • the invention further provides methods for identifying compounds as DPP8/9 inhibitors including selective or specific DPP8/9 inhibitors and using such compounds in the methods provided herein.
  • DPP8/9 inhibition assays include IC50 assays (as described herein) as well as cytokine and/or chemokine induction assays as described in the Examples as well as in U.S. Published Application 2005-0084490 Al, published on April 21, 2005, particularly in Examples 1, 5, 6, and 7, and such assay descriptions are incorporated by reference herein.
  • Compounds that may be tested for DPP8/9 inhibition include but are not limited to those described in US Pat. No. 4,318,904; US Pat. No. 4,499,082; US Pat. No. 4,652,552; US Pat. No. 4,963,655; US Pat. No. 5,093,477; US Pat. No. 5,187,157; US Pat. No. 5,242,904; US Pat. No. 5,250,720; US Pat. No. 5,288,707; US Pat. No. 5,296,604; US Pat. No. 5,384,410; US Pat. No. 5,444,049; US Pat. No. 5,527,923; US Pat. No. 5,543,396; US Pat. No. 5,624,894; US Pat. No.
  • the invention further provides methods for screening compounds for DPP8/9 inhibition followed by ability to stimulate immune responses as described herein.
  • the compounds may be screened for the ability to induce IL-I (e.g., IL-lbeta) levels as described in the Examples.
  • the compounds may be screened for the ability to induce levels of other cytokines and/or chemokines (e.g., IL-6).
  • IL-I e.g., IL-lbeta
  • cytokines and/or chemokines e.g., IL-6
  • DPP8/9 inhibitors and particularly selective or specific DPP8/9 inhibitors, act at least in part via neutrophil and macrophage activation and recruitment.
  • the invention provides methods of treating conditions amenable to immunotherapy (e.g., cancer or infectious disease) by inducing neutrophil and/or macrophage production and/or chemotaxis to affected sites in the body.
  • Neutrophil production can be induced using granulocyte stimulating factors.
  • Granulocyte stimulating factors are factors that increase the number of granulocytes, and preferably neutrophils, in the subject.
  • Increased granulocyte numbers generally result from increased differentiation from precursors including granulocyte restricted precursors such as CFC-G or oligo- or multi-lineage precursors such as CFC-GM or CFC-GEMM.
  • Granulocyte stimulating factors are known in the art and include but are not limited to granulocyte colony stimulating factor (G-CSF) and granulocyte/macrophage colony stimulating factor (GM-CSF).
  • G-CSF granulocyte colony stimulating factor
  • GM-CSF granulocyte/macrophage colony stimulating factor
  • macrophage production can be induced using macrophage stimulating factors.
  • Macrophage stimulating factors are factors that increase the number of macrophages in the subject.
  • Increased macrophage numbers generally result from increased differentiation from precursors including macrophage restricted precursors such as CFC-M or oligo- or multi-lineage precursors such as CFC-GM or CFC-GEMM.
  • An example of a macrophage stimulating factor is macrophage colony stimulating factor (M-CSF) which is known in the art.
  • M-CSF macrophage colony stimulating factor
  • the invention contemplates the administration of a granulocyte and/or a macrophage stimulating factor prior to the administration of a DPP8/9 inhibitor including a selective or specific DPP8/9 inhibitor.
  • chemoattractants are agents that induce movement of cells along a concentration gradient (e.g., from a region of low chemoattractant concentration to a region of higher chemoattractant concentration). Examples of chemoattractants are provided in Table B.
  • the chemoattractants are administered locally to an affected site (e.g., a melanoma lesion). The chemoattractants may also be administered locally in a sustained release formulation.
  • the invention intends to treat subjects having or at risk of developing a condition characterized by abnormal mammalian cell proliferation.
  • subject means a mammal including humans, nonhuman primates, dogs, cats, sheep, goats, horses, cows, pigs and rodents.
  • an abnormal mammalian cell proliferation disorder or condition refers to a cell population (e.g., a tumor) which exhibits an abnormal (e.g., increased) rate of division as compared to its normal cellular counterparts, or which grows in a factor independent manner.
  • Conditions characterized by an abnormal mammalian cell proliferation include but are not limited to solid and non-solid benign, pre-malignant or malignant conditions.
  • the condition may be a cancer.
  • the cancer may be carcinoma, sarcoma or melanoma.
  • a subject having a cancer is a subject that has detectable cancerous cells.
  • a subject at risk of developing a cancer is one who has a higher than normal probability of developing cancer. These subjects include, for instance, subjects having a genetic abnormality that has been demonstrated to be associated with a higher likelihood of developing a cancer, subjects having a familial disposition to cancer, subjects exposed to cancer causing agents (i.e., carcinogens) such as tobacco, asbestos, or other chemical toxins, and subjects previously treated for cancer and in apparent remission.
  • cancer causing agents i.e., carcinogens
  • Carcinomas include but are not limited to basal cell carcinoma, biliary tract cancer, bladder cancer, breast cancer, cervical cancer, choriocarcinoma, CNS cancer, colon and rectum cancer, kidney or renal cell cancer, larynx cancer, liver cancer, small cell lung cancer, non-small cell lung cancer (NSCLC, including adenocarcinoma, giant (or oat) cell carcinoma, and squamous cell carcinoma), oral cavity cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer (including basal cell cancer and squamous cell cancer), stomach cancer, testicular cancer, thyroid cancer, uterine cancer, rectal cancer, cancer of the respiratory system, and cancer of the urinary system.
  • NSCLC non-small cell lung cancer
  • Sarcomas are rare mesenchymal neoplasms that arise in bone (osteosarcomas) and soft tissues (fibrosarcomas).
  • Sarcomas include liposarcomas (including myxoid liposarcomas and pleomorphic liposarcomas), leiomyosarcomas, rhabdomyosarcomas, malignant peripheral nerve sheath tumors (also called malignant schwannomas, neurofibrosarcomas, or neurogenic sarcomas), Ewing's tumors (including Ewing's sarcoma of bone, extraskeletal (i.e., not bone) Ewing's sarcoma, and primitive neuroectodermal tumor), synovial sarcoma, angiosarcomas, hemangiosarcomas, Iymphangiosarcomas, Kaposi's sarcoma, hemangioendothelioma,
  • Melanomas are tumors arising from the melanocyte system of the skin and other organs.
  • Examples of melanoma include lentigo maligna melanoma, superficial spreading melanoma, nodular melanoma, and acral lentiginous melanoma.
  • the cancer may be a leukemia or a lymphoma.
  • leukemia examples include acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphoid (or lymphocytic) leukemia (CLL), T cell leukemia, B cell leukemia, Hodgkin's lymphoma, Non-Hodgkin's lymphoma, B cell lymphoma, and myeloma.
  • AML acute myeloid leukemia
  • ALL acute lymphoid leukemia
  • CML chronic myeloid leukemia
  • CLL chronic lymphoid leukemia
  • T cell leukemia B cell leukemia
  • Hodgkin's lymphoma Hodgkin's lymphoma
  • Non-Hodgkin's lymphoma Non-Hodgkin's lymphoma
  • B cell lymphoma and myeloma.
  • the cancer may be bone cancer, brain cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, cancer of the head and neck, gastric cancer, intra-epithelial neoplasm, neuroblastoma, retinoblastoma, rhabdomyosarcoma.
  • the cancer is breast cancer, colorectal cancer, chronic lymphocytic leukemia, non-small cell lung cancer, Non-Hodgkin's lymphoma, melanoma or prostate cancer.
  • the condition may be a primary tumor with undetectable metastatic lesions, or it may be a tumor that has metastasized.
  • the compositions and methods provided herein therefore are intended to treat primary tumors and/or metastases. In some instances, treatment may be of a primary tumor in the absence of detectable metastatic lesions in the subject.
  • the cancer may or may not express DPP8/9.
  • Some cancers to be treated according to the invention are DPP8/9 negative (as measured by mRNA and/or protein expression assays). Others may be DPP8/9 positive but with an expression level that is regarded as normal when compared to the expression level of DPP8/9 in normal tissue counterparts.
  • the invention intends to treat hormone-insensitive cancers.
  • Hormone insensitive cancers are cancers that do not require hormones for survival. Examples of these cancers include breast cancer, prostate cancer, testicular cancer, and some forms of ovarian cancer. Examples of hormones include estrogen, progesterone, and testosterone.
  • the anti-cancer effect of DPP8/9 inhibitors is a T cell independent effect (i.e., it occurs independently of T cell involvement). In some instances, such a T cell independent effect can be observed in osteosarcoma, non-small cell lung carcinoma, head and neck cancer, and pancreatic cancer. In these and other cancer types, the anti-cancer effect of DPP8/9 inhibitors appears may involve innate immune responses including those involving neutrophils.
  • the anti-cancer effect of DPP8/9 inhibitors involves T cells including CTLs. These effects can be observed in fibrosarcoma, T cell lymphoma and mastocytoma. In still other embodiments, both T cells and neutrophils are believed to be involved in the anti-cancer effects of DPP8/9 inhibitors. These effects can be observed in B cell lymphoma and melanoma.
  • the cancers to be treated may be refractory cancers.
  • a refractory cancer as used herein, is a cancer that is resistant to the ordinary standard of care prescribed. Refractory cancers may appear initially responsive to a treatment (and then recur), or they may be completely non-responsive to the treatment.
  • the ordinary standard of care will vary depending upon the cancer type, and the degree of progression in the subject. It may be a chemotherapy, or surgery, or radiation, or a combination thereof. Those of ordinary skill in the art are aware of such standards of care or are able to readily discern the ordinary standard of care based on FDA guidelines. Subjects being treated according to the invention for a refractory cancer therefore may have already been exposed to another treatment for their cancer.
  • refractory cancers include but are not limited to leukemia, melanoma, lung cancer including non-small cell lung cancer, pancreatic cancer and Non- Hodgkin's lymphoma.
  • the ordinary standard of care may comprise one or more chemotherapeutic or immunotherapeutic agents including but not limited to platinum- containing regimens such as cisplatin and carboplatin, docetaxel (TAXOTERE), pemetrexed (ALIMTA), 5-fluorouracil, gemcitabine, cyclophosphamide, doxorubicin, rituximab (RITUXAN), HERCEPTIN, and the like, either alone or in combination, concurrently or consecutively administered during treatment of the subject.
  • the methods provided herein may be used as first line therapy or as subsequent line therapy (e.g., second, third or fourth line therapy).
  • the invention can also be used to treat cancers that are immunogenic.
  • Cancers that are immunogenic are cancers that are known to (or likely to) express immunogens on their surface or upon cell death. These immunogens are in vivo endogenous sources of cancer antigens and their release can be exploited by the methods of the invention in order to treat the cancer. These cancers therefore are also known to respond to immunotherapy such as vaccine or antibody therapy.
  • cancers examples include melanoma, renal cell cancer, colorectal cancer, breast cancer, ovarian cancer, prostate cancer, leukemia such as T cell leukemia, B cell leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, common (pre-B) acute lymphocytic leukemia, chronic lymphocytic leukemia (CLL), and lymphoma such as Non-Hodgkin's lymphoma, T-cell lymphoma, and B-cell lymphoma.
  • leukemia such as T cell leukemia, B cell leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, common (pre-B) acute lymphocytic leukemia, chronic lymphocytic leukemia (CLL), and lymphoma such as Non-Hodgkin's lymphoma, T-cell lymphoma, and B-cell lymphoma.
  • One category of benign or precancerous conditions characterized by abnormal cell proliferation is proliferative dermatologic disorders. These include conditions such as keloids, actinic keratosis, bowenoid actinic keratosis seborrheic keratosis, hemangiomas, papilloma virus infection (e.g.
  • verruca vulbaris verruca plantaris, verruca plana, condylomata, etc.
  • eczema hypertrophic actinic keratosis, arsenical keratosis, hydrocarbon keratosis, thermal keratosis, radiation keratosis, viral keratosis, Bowen's disease, erythroplaquia of queyrat, oral erythroplaquia, leukoplakia, and intraepidermal epithelialoma.
  • An precancerous lesion is a lesion that has a propensity to develop into a cancerous condition.
  • the epithelial lesions may develop into an invasive form of squamous cell carcinoma and may pose a significant threat of metastasis.
  • compositions and methods of the invention are useful in some instances for improving the efficacy of or replacing existing therapies (e.g., cancer therapies) including but not limited to surgical procedures, radiation therapies, chemotherapies, immunotherapies and/or hormone therapies (e.g., the ordinary standard of care therapies).
  • existing therapies e.g., cancer therapies
  • the DPP8/9 inhibitors including selective or specific DPP8/9 inhibitors can be used in combination with surgery, radiation, chemotherapy, immunotherapy and/or hormone therapy to treat subjects according to the invention.
  • the second therapy i.e., the non-DPP8/9 inhibitor therapy
  • the second therapy may be administered before, concurrent with, or after treatment with DPP8/9 inhibitor.
  • the DPP8/9 inhibitors including the selective or specific DPP8/9 inhibitors may be administered, according to some embodiments, with a second therapeutic agent.
  • the second therapeutic agent may be an anti-cancer agent or an anti-microbial agent but it is not so limited.
  • the second therapeutic agent may be but is not limited to a chemotherapeutic agent or an immunotherapeutic agent.
  • the immunotherapeutic agent may be but it not limited to an antigen (e.g., in the form of a vaccine), an antibody or an antibody fragment, an adjuvant, a cytokine, or a chemokine.
  • An anti-cancer agent is an agent that at least partially inhibits the development or progression of a cancer, including inhibiting in whole or in part symptoms associated with the cancer even if only for the short term.
  • the anti-cancer agent may be a chemotherapeutic agent.
  • the compositions of the invention can further include chemotherapeutic agents such as but not limited to those currently in use with the antibodies recited herein.
  • chemotherapeutic agents can be categorized as DNA damaging agents and these include topoisomerase inhibitors (e.g., etoposide, ramptothecin, topotecan, teniposide, mitoxantrone), anti-microtubule agents (e.g., vincristine, vinblastine), anti-metabolic agents (e.g., cytarabine, methotrexate, hydroxyurea, 5-fluorouracil, floxuridine, 6-thioguanine, 6-mercaptopurine, fludarabine, pentostatin, chlorodeoxyadenosine), DNA alkylating agents (e.g., cisplatin, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chorambucil, busulfan, thiotepa, carmustine, lomustine, carboplatin, dacarbazine, procarbazine), DNA strand break in
  • DPP4 inhibitor and/or a FAP inhibitor and optionally with cisplatin in the treatment of melanoma e.g., metastatic melanoma
  • melanoma e.g., metastatic melanoma
  • the DPP8/9 inhibitor is used alone or in conjunction with a DPP4 inhibitor and/or a FAP inhibitor and optionally with docetaxel (TAXOTERE) and/or pemetrexed (ALIMTA) in the treatment of non-small cell lung carcinoma.
  • TAXOTERE docetaxel
  • ALIMTA pemetrexed
  • the DPP8/9 inhibitor is used alone or in conjunction with a DPP4 inhibitor and/or a FAP inhibitor and optionally with rituximab (RITUXAN) in the treatment of chronic lymphocytic leukemia.
  • the DPP8/9 inhibitor is used alone or in conjunction with a DPP4 inhibitor and/or a FAP inhibitor and optionally with gemcitabine in the treatment of pancreatic cancer.
  • Some important chemotherapies include but are not limited to docetaxel
  • TXOTERE e.g., in non-small cell lung carcinoma
  • ALARMTA pemetrexed
  • gemcitabine e.g., gemcitabine
  • cisplatin e.g., in lymphoma and melanoma
  • carboplatin cyclophosphamide
  • doxorubicin dacarbazine
  • 5-FU paclitaxel
  • TARCEVA erlotinib
  • IRESSA gefitinib
  • TEMODAR temozolomide
  • imatinib mesylate GLEEVAC
  • REVLIMTD lenalidomide
  • REVIMID REVIMID
  • TEMODAR temozolomide
  • TAALOMID thalidomide
  • VELCADE rituximab
  • RITUXAN rituximab
  • HERCEPTIN trastuzumab
  • AVASTIN bevacizumab
  • Adozelesin Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate;
  • Aminoglutethimide Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin;
  • Azacitidine Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Bortezomib
  • VELCADE Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone;
  • Caracemide Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin;
  • Cedefingol Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate;
  • Cyclophosphamide Cytarabine; dacarbazine; Dactinomycin; Daunorubicin; Decitabine; Dexormaplatin; Dezaguanine; Diaziquone; Docetaxel (TAXOTERE); Doxorubicin;
  • Droloxifene Dromostanolone; Duazomycin; Edatrexate; Eflornithine; Elsamitrucin;
  • Enloplatin Enpromate; Epipropidine; Epirubicin; Erbulozole; Erlotinib (TARCEVA),
  • Esorubicin Estramustine; Etanidazole; Etoposide; Etoprine; Fadrozole; Fazarabine;
  • Fenretinide Floxuridine; Fludarabine; Fluorouracil; Flurocitabine; Fosquidone; Fostriecin; Gefitinib (IRESSA), Gemcitabine; Hydroxyurea; Idarubicin; Ifosfamide; Ilmofosine; Imatinib mesylate (GLEEVAC); Interferon alpha-2a; Interferon alpha-2b; Interferon alpha-nl;
  • Lometrexol Lomustine; Losoxantrone; Masoprocol; Maytansine; Mechlorethamine; Megestrol; Melengestrol; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Metoprine;
  • Meturedepa Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin;
  • Mitosper Mitosper; Mitotane; Mitoxantrone; Mycophenolic Acid; Nocodazole; Nogalamycin;
  • Pentamustine Pentomone; Peplomycin; Perfosfamide; Pipobroman; Piposulfan; Piritrexim Isethionate; Piroxantrone; Plicamycin; Plomestane; Porfimer; Porfiromycin; Prednimustine;
  • Procarbazine Puromycin; Pyrazofurin; Riboprine; Rogletimide; Safingol; Semustine;
  • the chemotherapeutic agent and/or any of the other active agents may be administered in a sub-therapeutic dose, as defined herein.
  • the DPP8/9 inhibitor is used in combination with IL-
  • one or more of the following chemotherapies can be administered to the subject in order to achieve a potentiated effect: doxorubicin, cisplatin, indomethacin, ketoconazole, mitomycin-D, porf ⁇ romycin, cyclophosphamide, and carboplatin.
  • the anti-cancer agent may be an immunotherapeutic agent.
  • the immunotherapeutic agent may be an antibody or an antibody fragment.
  • antibodies such as but not limited to bevacizumab (AVASTIN), trastuzumab (HERCEPTIN), alemtuzumab (CAMPATH, indicated for B cell chronic lymphocytic leukemia,), gemtuzumab (MYLOTARG, hP67.6, anti-CD33, indicated for leukemia such as acute myeloid leukemia), rituximab (RITUXAN), tositumomab (BEXXAR, anti-CD20, indicated for B cell malignancy), MDX-210 (bispecific antibody that binds simultaneously to HER-2/neu oncogene protein product and type I Fc receptors for immunoglobulin G (IgG) (Fc gamma RI)), oregovomab (OVAREX, indicated for ovarian cancer), edrecolomab (PANOREX), daclizumab (ZENAPAX), palivizumab (SYNAGIS, indicated for respiratory conditions such as RSV
  • the invention embraces a number of classes of antibodies and fragments thereof including but not limited to antibodies directed to cancer antigens (as described above), cell surface molecule, stromal cell molecules, extracellular matrix molecules, and tumor vasculature associated molecules.
  • a cell surface molecule is a molecule that is expressed at the surface of a cell. In addition to an extracellular domain, it may further comprise a transmembrane domain and a cytoplasmic domain. Examples include HER 2, CD20, CD33, EGF receptor, HLA markers such as HLA-DR, CD52, CDl, CEA, CD22, GD2 ganglioside, FLK2/FLT3, VEGFR, and the like.
  • the antibody or antibody fragment may be specific for CD20.
  • examples of such antibodies include rituximab (RITUXAN), ibritumomab tiuxetan (ZEVALIN), tositumomab (BEXXAR).
  • the antibody or antibody fragment may be specific for CD22. Examples include epratuzumab (L YMPHOCIDE) and MDX-210.
  • the antibody or antibody fragment may be specific for EGF receptor. Examples include cetuximab (ERBITUX, IMC-C225), trastuzumab (HERCEPTIN), MDX-210, MDX-447, EMD-72000, and IOR EGF/R3.
  • the antibody or antibody fragment may be specific for CD33.
  • Examples include gemtuzumab (MYLOTARG, CMA 676) and SMART M 195 (ZAMYL).
  • the antibody or antibody fragment may be specific for CD52.
  • Examples include alemtuzumab (CAMPATH, LDP-03).
  • the antibody or antibody fragment may be specific for HLA-DR.
  • Examples include lym-1 (ONCOLYM) and SMART IDlO Ab (REMITOGEN).
  • the antibody or antibody fragment may be specific for ⁇ V ⁇ 3 integrin. Examples include VITAXIN.
  • the antibody or antibody fragment may be specific for TAG.
  • the antibody or antibody fragment may be specific for CDl . Examples include IOR-T6.
  • An extracellular matrix molecule is a molecule found in the extracellular matrix. Examples include but are not limited to collagen, glycosaminoglycans (GAGs), proteoglycans, elastin, fibronectin and laminin.
  • a tumor vasculature associated molecule is a molecule expressed by vasculature of a tumor (i.e., a solid cancer rather than a systemic cancer such as leukemia). As with a cancer antigen, a tumor vasculature associated molecule may be expressed by normal vasculature however its presence on vasculature of a tumor makes it a suitable target for anti-cancer therapy.
  • the tumor vasculature associated molecule is expressed at a higher level in tumor vasculature than it is in normal vasculature.
  • examples include but are not limited to endoglin (see U.S. Pat. No. 5,660,827), ELAM-I, VCAM-I, ICAM-I, ligand reactive with LAM-I, MHC class II antigens, aminophospholipids such as phosphatidylserine and phosphatidylethanolamine (as described in U.S. Pat. No. 6,312,694), VEGFRl (FIt-I) and VEGFR2 (KDR/Flk-1), and other tumor vasculature associated antigens such as those described in U.S. Pat. No.
  • Antibodies to endoglin are described in U.S. Pat. No. 5,660,827 and include TEC-4 and TEC-11, and antibodies that recognize identical epitopes to these antibodies.
  • Antibodies to aminophospholipids are described in U.S. Pat. No. 6,312,694.
  • Antibodies that inhibit VEGF are described in U.S. Pat. No. 6,342,219 and include 2C3 (ATCC PTA 1595).
  • Other antibodies that are specific for tumor vasculature include antibodies that react to a complex of a growth factor and its receptor such as a complex of FGF and the FGFR or a complex of TGF ⁇ and the TGF ⁇ R.
  • Antibodies of this latter class are described in U.S. Pat. No. 5,965,132, and include GV39 and GV97.
  • the antibody or antibody fragment may be specific for a stromal cell molecule such as but not limited to FAP and CD26.
  • the antibodies embraced by the invention include those recited explicitly herein and also those that bind to the same epitope as those recited herein.
  • the antibody or antibody fragment may be specific for CA 125. Examples include oregovomab (OVAREX, B43.13).
  • the antibody or antibody fragment may be specific for MUC-I. Examples include BREVAREX.
  • the antibody or antibody fragment may be specific for PSA. Examples include PROSTAREX.
  • the antibody or antibody fragment may be specific for CA 19.9. Examples include GIVAREX.
  • the antibody or antibody fragment may be specific for Ep-CAM (EPG40 antigen, 17-1 A). Examples include celogovab (ONCOSCINT, OVl 03) and 3622 W94.
  • the antibody or antibody fragment may be specific for VEGF.
  • Examples include bevacizumab (AVASTIN). Examples include MDX-220.
  • the antibody or antibody fragment may also be an anti-idiotypic antibody. Examples include anti- idiotypic mAb mimic of high molecular weight proteoglycan (I-Mel-1), MELIMMUNE-I, melanoma (IDEC); anti-idiotypic mAb mimic of high molecular weight proteoglycan (I-Mel- 2), MELIMMUNE-2, melanoma (IDEC); and anti-idiotypic mAb mimic of ganglioside GD3 epitope, BEC2, small cell lung carcinoma, melanoma (ImClone Systems).
  • the antibody or antibody fragment may be infliximab (inflammatory bowel disease and rheumatoid arthritis) or etanercept (rheumatoid arthritis).
  • the antibody or antibody fragment may be conjugated (covalently or otherwise) to a toxin derived from plant, fungus, or bacteria.
  • the toxin may be selected from the group consisting of A chain toxin, deglycosylated A chain toxin, ribosome inactivating protein, ⁇ - sarcin, aspergillin, restrictocin, ribonuclease, diptheria toxin and Pseudomonas exotoxin, but is not so limited.
  • the antibody or antibody fragment may also be conjugated to a chemotherapeutic agent, a radioisotope or a cytotoxin.
  • the chemotherapeutic agent may be selected from the group consisting of an anti-metabolite, an anthracycline, a vinca alkaloid, an antibiotic, an alkylating agent, and an epipodophyllotoxin, but is not so limited.
  • the antibody or antibody fragment may be administered on a first day of multi-day cycle, with the DPP8/9 inhibitor (e.g., the selective or specific DPP8/9 inhibitor) administered on the remaining days of the cycle.
  • the cycle may be a 2, 3, 4, 5, 6, 7, or more day cycle.
  • the DPP8/9 inhibitor may be administered once, twice, or more times per day.
  • the antibody or antibody fragment is administered on the first day of a seven day cycle, followed by a twice daily administration of a DPP8/9 inhibitor on each of the remaining days of the seven day cycle.
  • Other therapeutic agents may also be administered during the cycle.
  • the DPP4 and/or the FAP inhibitor may be administered on the same days as the DPP8/9 inhibitor, on alternating days (e.g., days 2, 4 and 6 of a seven day cycle), and the like.
  • the multi-day cycle may be repeated twice, thrice, four times, or more. It may also be repeated for various lengths of time, including but not limited to a week, a month, two months, or more.
  • the DPP8/9 inhibitors may be used to stimulate immune responses to a cancer antigen. This may be accomplished using the inhibitors together with the cancer antigen itself or an antibody (or antibody fragment) specific to that antigen.
  • Cancer antigens are antigens that are expressed in cancerous cells at levels that are higher than the expression level in normal counterpart cells.
  • the cancer antigen may be peptide, lipid or carbohydrate in nature or some combination thereof.
  • Cancer antigens can be prepared from cancer cells either by preparing crude extracts of cancer cells, for example, as described in Cohen, et al., 1994, Cancer Research, 54:1055, by partially purifying the antigens, by recombinant technology, or by de novo synthesis of known antigens.
  • Cancer antigens include but are not limited to antigens that are recombinantly expressed, an immunogenic portion of, or a whole tumor or cancer. Such antigens can be isolated or prepared recombinantly or by any other means known in the art.
  • cancer antigens can be classified in a variety of ways.
  • cancer antigens include antigens that are encoded by mutant cellular genes such as oncogenes (e.g., activated ras oncogene) or suppressor genes (e.g., mutant p53), antigens that are fusion proteins resulting from internal deletions or chromosomal translocations; and antigens encoded by viral genes such as those carried on RNA and DNA tumor viruses.
  • cancer antigens include antigens encoded by genes that have undergone chromosomal alteration. Many of these antigens are found in lymphoma and leukemia. Even within this classification, antigens can be characterized as those that involve activation of quiescent genes.
  • BCL-I and IgH Mantel cell lymphoma
  • BCL-2 and JgH Follicular lymphoma
  • BCL-6 Diffuse large B-cell lymphoma
  • TAL-I and TCRO or SIL T-cell acute lymphoblastic leukemia
  • c-MYC and IgH or IgL Burkitt lymphoma
  • MUN/IRF4 and IgH Myeloma
  • PAX-5 (BSAP) (Immunocytoma).
  • cancer antigens that involve chromosomal alteration and thereby create a novel fusion gene and/or protein include RARa, PML, PLZF, NPM or NuMA (Acute promyelocyte leukemia), BCR and ABL (Chronic myeloid/acute lymphoblastic leukemia), MLL (HRX)
  • HLF Bacillus fibroblastic leukemia
  • NPM Acute leukemia
  • ALK Anaplastic large cell leukemia
  • NPM MLF-I (Myelodysplastic syndrome/acute myeloid leukemia).
  • cancer antigens are specific to a tissue or cell lineage. These include cell surface proteins such as CD20, CD22 (Non-Hodgkin's lymphoma, B-cell lymphoma, Chronic lymphocytic leukemia (CLL)), CD52 (B-cell CLL), CD33 (Acute myelogenous leukemia (AML)), CDlO (gplOO) (Common (pre-B) acute lymphocytic leukemia and malignant melanoma), CD3/T-cell receptor (TCR) (T-cell lymphoma and leukemia), CD79/B-cell receptor (BCR) (B-cell lymphoma and leukemia), CD26 (Epithelial and lymphoid malignancies), Human leukocyte antigen (HLA)-DR, HLA-DP, and HLA-DQ (Lymphoid malignancies), RCAS 1 (Gynecological carcinomas, bilary adenocarcinomas and ductal aden
  • Tissue- or lineage- specific cancer antigens also include epidermal growth factor receptors (high expression) such as EGFR (HERl or erbBl) and EGFRvIII (Brain, lung, breast, prostate and stomach cancer), erbB2 (HER2 or HER2/neu) (Breast cancer and gastric cancer), erbB3 (HER3) (Adenocarcinoma), and erbB4 (HER4) (Breast cancer).
  • epidermal growth factor receptors high expression
  • EGFR HERl or erbBl
  • EGFRvIII Brain, lung, breast, prostate and stomach cancer
  • erbB2 HER2 or HER2/neu
  • HER3 HER3
  • HER4 erbB4
  • Tissue- or lineage- specific cancer antigens also include cell-associated proteins such as Tyrosinase, Melan-A/MART-1, tyrosinase related protein (TRP)- l/gp75 (Malignant melanoma), Polymorphic epithelial mucin (PEM) (Breast tumors), and Human epithelial mucin (MUCl) (Breast, ovarian, colon and lung cancers).
  • TRP tyrosinase related protein
  • PEM Polymorphic epithelial mucin
  • MUCl Human epithelial mucin
  • Tissue- or lineage-specific cancer antigens also include secreted proteins such as Monoclonal immunoglobulin (Multiple myeloma and plasmacytoma), Immunoglobulin light chains (Multiple Myeloma), ⁇ -fetoprotein (Liver carcinoma), Kallikreins 6 and 10 (Ovarian cancer), Gastrin-releasing peptide/bombesin (Lung carcinoma), and Prostate specific antigen (Prostate cancer).
  • Monoclonal immunoglobulin Multiple myeloma and plasmacytoma
  • Immunoglobulin light chains Multiple Myeloma
  • ⁇ -fetoprotein Liver carcinoma
  • Kallikreins 6 and 10 Ovarian cancer
  • Gastrin-releasing peptide/bombesin Lung carcinoma
  • Prostate specific antigen Prostate cancer
  • CT antigens that are expressed in some normal tissues such as testis and in some cases placenta. Their expression is common in tumors of diverse lineages and as a group the antigens form targets for immunotherapy.
  • tumor expression of CT antigens include MAGE-Al, -A3, -A6, -A12, BAGE, GAGE, HAGE, LAGE-I, NY-ESO-I, RAGE, SSX-I, -2, -3, -4, -5, -6, -7, -8, -9, HOM-TES- 14/SCP-l, HOM-TES-85 and PRAME.
  • CT antigens and the cancers in which they are expressed include SSX-2, and -4 (Neuroblastoma), SSX-2 (HOM-MEL-40), MAGE, GAGE, BAGE and PRAME (Malignant melanoma), HOM-TES- 14/SCP-l
  • SSX-4 Oledendrioglioma
  • HOM-TES- 14/SCP-l MAGE-3 and SSX-4 (Astrocytoma), SSX member (Head and neck cancer, ovarian cancer, lymphoid tumors, colorectal cancer and breast cancer), RAGE-I, -2, -4, GAGE-I, -2, -3, -4, -5, -6, -7 and -8 (Head and neck squamous cell carcinoma (HNSCC)), HOM-TES 14/SCP-l, PRAME, SSX-I and CT-7 (Non-Hodgkin's lymphoma), and PRAME (Acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML) and chronic lymphocytic leukemia (CLL)).
  • ALL acute lymphoblastic leukemia
  • AML acute myelogenous leukemia
  • CLL chronic lymphocytic leukemia
  • cancer antigens are not specific to a particular tissue or cell lineage. These include members of the carcinoembryonic antigen (CEA) family: CD66a, CD66b, CD66c, CD66d and CD66e. These antigens can be expressed in many different malignant tumors and can be targeted by immunotherapy.
  • CEA carcinoembryonic antigen
  • cancer antigens are viral proteins and these include Human papilloma virus protein (cervical cancer), and EBV-encoded nuclear antigen (EBNA)-I (lymphomas of the neck and oral cancer).
  • cervical cancer Human papilloma virus protein
  • EBNA EBV-encoded nuclear antigen-I (lymphomas of the neck and oral cancer).
  • cancer antigens are mutated or aberrantly expressed molecules such as but not limited to CDK4 and beta-catenin (melanoma).
  • antigens include HER 2 (pi 85), CD20, CD33, GD3 ganglioside, GD2 ganglioside, carcinoembryonic antigen (CEA), CD22, milk mucin core protein, TAG-72, Lewis A antigen, ovarian associated antigens such as OV-TL3 and MOvI 8, high Mr melanoma antigens recognized by antibody 9.2.27, HMFG-2, SM-3, B72.3, PR5C5, PR4D2, and the like.
  • CEA carcinoembryonic antigen
  • CD22 milk mucin core protein
  • TAG-72 Lewis A antigen
  • Lewis A antigen ovarian associated antigens
  • OV-TL3 and MOvI 8 high Mr melanoma antigens recognized by antibody 9.2.27, HMFG-2, SM-3, B72.3, PR5C5, PR4D2, and the like.
  • cancer antigens include MART-1/Melan-A, gplOO, adenosine deaminase- binding protein (AD Abp), FAP, cyclophilin b, colorectal associated antigen (CRC)-COl 7- 1 A/GA733, carcinoembryonic antigen (CEA), CAP-I, CAP-2, etv6, AMLl, prostate specific antigen (PSA), PSA-I 5 PSA-2, PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor/CD3-zeta chain, and CD20.
  • AD Abp adenosine deaminase- binding protein
  • FAP cyclophilin b
  • CRC colorectal associated antigen
  • CEA carcinoembryonic antigen
  • CAP-I CAP-2
  • etv6 etv6, AMLl
  • PSA prostate specific antigen
  • PSMA prostate-specific membrane antigen
  • the cancer antigen may also be selected from the group consisting of MAGE-Al, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-AlO, MAGE-Al 1, MAGE-A 12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C 1 , MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5).
  • the cancer antigen is selected from the group consisting of GAGE-I, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9.
  • the cancer antigen is selected from the group consisting of BAGE, RAGE, LAGE-I, NAG, GnT-V, MUM-I, CDK4, tyrosinase, p53, MUC family, HER2/neu, p2 Iras, RCAS 1 , ⁇ -fetoprotein, E-cadherin, ⁇ -catenin, ⁇ -catenin, ⁇ -catenin, pl20ctn, gplOO Pmel " 7 , PRAME, NY-ESO-I, cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, pi 5, gp75, GM2 ganglioside, GD2
  • the cancer antigen is VEGF, Anti-idiotypic mAb (GD3 ganglioside mimic), CD20, CD52, Anti-idiotypic mAb (CEA mimic), ERBB2, EGFR, CD22, ERBB2 X CD65 (fc ⁇ RI), EpCam, PEM and CD33.
  • Cancer or tumor antigens can also be classified according to the cancer or tumor they are associated with (i.e., expressed by).
  • Cancers or tumors associated with tumor antigens include acute lymphoblastic leukemia (etv ⁇ ; amll; cyclophilin b), B cell lymphoma (Ig- idiotype); Burkitt's (Non-Hodgkin's) lymphoma (CD20); glioma (E-cadherin; ⁇ -catenin; ⁇ - catenin; ⁇ -catenin; pl20ctn), bladder cancer (p21ras), biliary cancer (p21ras), breast cancer (MUC family; HER2/neu; c-erbB-2), cervical carcinoma (p53; p21ras), colon carcinoma
  • the antigens may be administered in a substantially purified form.
  • substantially purified refers to a compound which is substantially free of other compounds such as proteins, lipids, carbohydrates or other materials with which it is naturally associated.
  • One skilled in the art can purify viral or bacterial compounds such as polypeptides using standard techniques such as for example protein purification.
  • the substantially pure polypeptide will often yield a single major band on a non-reducing polyacrylamide gel. In the case of partially glycosylated polypeptides or those that have several start codons, there may be several bands on a non-reducing polyacrylamide gel, but these will form a distinctive pattern for that polypeptide.
  • the purity of the viral or bacterial polypeptide can also be determined by amino-terminal amino acid sequence analysis.
  • the vaccine methods and compositions described herein similarly envision the use of nucleic acid based vaccines in addition to peptide based vaccines.
  • the art is familiar with nucleic acid based vaccines.
  • the invention seeks to enhance other forms of immunotherapy including dendritic cell vaccines.
  • These vaccines generally include dendritic cells loaded ex vivo with antigens such as tumor-associated antigens.
  • the dendritic cells can be incubated with the antigen, thereby allowing for antigen processing and expression on the cell surface, or the cells may simply be combined with the antigen prior to injection in vivo.
  • the dendritic cells may be activated in vitro and then re-infused into a subject in the activated state.
  • DPP8/9 inhibitors can be combined with the dendritic cells in all of these embodiments.
  • dendritic cell based vaccines include autologous tumor antigen-pulsed dendritic cells (advanced gynaecological malignancies); blood-derived dendritic cells loaded ex vivo with prostate cancer antigen (Provenge; Dendreon Corporation); blood-derived dendritic cells loaded ex vivo with antigen for multiple myeloma and other B-cell malignancies (Mylovenge; Dendreon Corporation); and blood-derived dendritic cells loaded ex vivo with antigen for cancers expressing the HER-2/neu proto-oncogene (APC8024; Dendreon Corporation); xenoantigen (e.g., PAP) loaded dendritic cells, and the like.
  • APC8024 HER-2/neu proto-oncogene
  • the second therapeutic agent may also be a hormone therapy agent, particularly for breast and gynecological cancers.
  • DPP8/9 inhibitors can be used in combination with tamoxifen or aromatase inhibitor arimidex (i.e., anastrozole), or simply for disorders responsive to either (e.g., breast cancer).
  • the invention further provides a method for treating a subject having or at risk of developing cancer comprising administering to a subject in need of such treatment an enzyme inhibitor selected from the group consisting of a tyrosine kinase inhibitor, a CDK inhibitor, a MAP kinase inhibitor, and an EGFR inhibitor, and a DPP8/9 inhibitor including a selective or specific DPP8/9 inhibitor in an amount effective to inhibit the cancer.
  • an enzyme inhibitor selected from the group consisting of a tyrosine kinase inhibitor, a CDK inhibitor, a MAP kinase inhibitor, and an EGFR inhibitor
  • a DPP8/9 inhibitor including a selective or specific DPP8/9 inhibitor in an amount effective to inhibit the cancer.
  • the tyrosine kinase inhibitor may be Genistein (4',5,7-trihydroxyisoflavone), Tyrphostin 25 (3,4,5- trihydroxyphenyl), methylene]-propanedinitrile, Herbimycin A, Daidzein (4',7- dihydroxyisoflavone), AG- 126, trans- 1 -(3 '-carboxy-4'-hydroxyphenyl)-2-(2",5"-dihydroxy- phenyl)ethane, or HDBA (2-Hydroxy5-(2,5-Dihydroxybenzylamino)-2-hydroxybenzoic acid.
  • the CDK inhibitor may be p21, p27, p57, pl5, pl6, pl8, or pl9.
  • the MAP kinase inhibitor may be KY12420 (C 23 H 24 O 8 ), CNI-1493, PD98059, or 4-(4-Fluorophenyl)-2-(4- methylsulfinyl phenyl)-5-(4-pyridyl) lH-imidazole.
  • the EGFR inhibitor may be erlotinib (TARCEVA), gefitinib (IRESSA), WHI-P97 (quinazoline derivative), LFM-Al 2 (leflunomide metabolite analog), ABX-EGF, lapatinib, canertinib, ZD-6474 (ZACTIMA), AEE788, and AG1458.
  • DPP8/9 inhibitors can also be used in combination with VEGF inhibitors or antagonists.
  • VEGF inhibitors include bevacizumab (AVASTIN), ranibizumab (LUCENTIS), pegaptanib (MACUGEN), sorafenib, sunitinib (SUTENT), vatalanib, ZD-6474 (ZACTIMA), anecortave (RETAANE), squalamine lactate, and semaphorin.
  • Iymphokine activated killer cells LAKs
  • DPP8/9 inhibitors can be combined with such cells either as an addition to the activating Iymphokine or in place of it.
  • the invention also embraces the use of adjuvants.
  • adjuvants derived from microorganisms, such as bacillus Calmette-Guerin, heighten the immune response and enhance resistance to tumors in animals.
  • Adjuvants that may be combined with the DPP8/9 inhibitors include alum, immunostimulatory oligonucleotides such as CpG oligonucleotides, QS-21, and the like.
  • Other aspects of the invention are directed to treatment of subject having or at risk of developing an infectious disease.
  • a DPP8/9 inhibitor can be administered after viral, bacterial mycobacterial, fungal, or parasitic infection in order to stimulate innate immunity (i.e., immunity mediated by neutrophils, macrophages, NK cells and eosinophils) and/or adaptive immunity (i.e., immunity mediated by T cells and B cells).
  • innate immunity i.e., immunity mediated by neutrophils, macrophages, NK cells and eosinophils
  • adaptive immunity i.e., immunity mediated by T cells and B cells.
  • a DPP8/9 inhibitor can be used prophylactically to prevent infection including during periods of heightened risk, including for example flu season, epidemics, and travel to places where the risk of pathogen exposure is high.
  • Many of the cytokines and chemokines induced by DPP8/9 inhibition can prime a subject and prepare it for passive exposure to a pathogen. The rate at which a DPP8/9 inhibitor stimulates these cytokines and chemok
  • infectious disease and "microbial infection” are used interchangeably and intended to convey an infection by any microbe including but not limited to a bacterium, a mycobacterium, a virus, a fungus, a parasite, and the like.
  • the infectious disease may be a bacterial infection, a mycobacterial infection, a viral infection, a fungal infection or a parasitic infection.
  • Subjects having an infectious disease are those that exhibit symptoms of infectious disease (e.g., rapid onset, fever, chills, myalgia, photophobia, pharyngitis, acute lymphadenopathy, splenomegaly, gastrointestinal upset, leukocytosis or leukopenia) and in whom infectious pathogens or byproducts thereof can be detected.
  • Tests for diagnosing infectious diseases are known in the art and the ordinary medical practitioner will be familiar with these laboratory tests which include but are not limited to microscopic analyses, cultivation dependent tests (such as cultures), and nucleic acid detection tests.
  • a subject at risk of developing an infectious disease is one that is at risk of exposure to an infectious pathogen.
  • Such subjects include those that live in an area where such pathogens are known to exist and where such infections are common.
  • These subjects also include those that engage in high risk activities such as sharing of needles, engaging in unprotected sexual activity, routine contact with infected samples of subjects (e.g., medical practitioners), people who have undergone surgery, including but not limited to abdominal surgery, etc.
  • Examples of bacterial infections include E.
  • coli Streptococcal infections, Staphylococcal infections, Pseudomonas infections, Clostridium difficile, Legionella infections, Pneumococcus infection, Haemophilus infections (e.g., Haemophilus influenzae infections), Klebsiella infections, Enterobacter infections, Citrobacter infections, Neisseria infections (e.g., N. meningitidis infection, N. gonorrhoeae infection), Shigella infections, Salmonella infections, Listeria infections (e.g., L.
  • Pasteurella infection e.g., Pasteurella multocida infection
  • Streptobacillus infection Spirillum infection
  • Treponema inection e.g., Treponema pallidum infection
  • Actinomyces infection e.g., Actinomyces israelii infection
  • Borrelia infection Corynebacterium infection
  • Nocardia infection Gardnerella infections (e.g., Gardnerella vaginalis infection)
  • Campylobacter infections e.g., Campylobacter fetus infection
  • Spirochaeta infections Proteus infections
  • Bacteriodes infections H. pylori, and anthrax.
  • viral infections include HIV infection, Herpes simplex virus 1 and 2 infections (including encephalitis, neonatal and genital forms), human papilloma virus infection, cytomegalovirus infection, Epstein Barr virus infection, Hepatitis virus A, B and C infections, rotavirus infection, adenovirus infection, influenza A virus infection, respiratory syncytial virus infection, varicella-zoster virus infections, small pox infection, monkey pox infection, SARS infection, and avian flu virus infection.
  • HIV infection Herpes simplex virus 1 and 2 infections (including encephalitis, neonatal and genital forms), human papilloma virus infection, cytomegalovirus infection, Epstein Barr virus infection, Hepatitis virus A, B and C infections, rotavirus infection, adenovirus infection, influenza A virus infection, respiratory syncytial virus infection, varicella-zoster virus infections, small pox infection, monkey pox infection, SARS infection, and avian flu virus infection.
  • the subject being treated according to the invention is HIV negative.
  • fungal infections include candidiasis infection, ringworm, histoplasmosis infection, blastomycosis infections, paracoccidioidomycosis infections, crytococcosis infections, aspergillosis infections, chromomycosis infections, mycetoma infections, pseudallescheriasis infection, and tinea versicolor infection.
  • Examples of parasite infections include both protozoan infections and nematode infections. These include amebiasis, Trypanosoma cruzi infection (i.e., Chagas' disease), Fascioliasis (e.g., Facioloa hepatica infection), Leishmaniasis, Plasmodium infections (e.g., malaria causing Plasmodium species infections, e.g., P. falciparum, P. knowlesi, P.
  • Onchocerciasis i.e., Sleeping sickness
  • Pneumocystis infection e.g., Pneumocystis carinii infection
  • Trichomonas vaginalis infection Taenia infections
  • Hymenolepsis infections e.g., Hymenolepsis nana infection
  • Echinococcus infections e.g., Schistosomiasis (e.g., Schistosoma mansoni infection)
  • neurocysticercosis e.g., Schistosoma mansoni infection
  • Necator americanus infection i.e., Sleeping sickness
  • Pneumocystis infection e.g., Pneumocystis carinii infection
  • Trichomonas vaginalis infection Trichomonas vaginalis infection
  • Taenia infections e.g., Hymenolepsis nana infection
  • Echinococcus infections e.
  • infections that can be treated according to the methods of the invention include Chlamydia infection, Mycobacterial infection such as tuberculosis and leprosy, and Rickettsiae.
  • DPP8/9 can be used in the treatment of infections regardless of whether the infectious agent produces a deleterious post prolyl cleaving enzyme, as described in US Patent No. 4935493.
  • the infectious agent lacks a deleterious post-prolyl cleaving enzyme.
  • DPP8/9 inhibitors can be used in the treatment of human papillomavirus (HPV) infection.
  • HPV human papillomavirus
  • the current therapy for HPV is injection of IFN into a lesion and/or surgical ablation.
  • DPP8/9 inhibitors are similarly useful in combination with HPV vaccines currently in development such as HPV virus-like particle (VLP)-based vaccine (see, for example, Virology 2000 Jan 20;266(2):237-45).
  • a DPP8/9 inhibitor can be administered as a stand alone therapy or in combination with an anti-infective agent (i.e., an anti-microbial).
  • the anti-infective agent may be an antibacterial agent, an anti-mycobacterial agent, an anti-viral agent, an anti-fungal agent and an anti-parasitic agent.
  • the anti-infective agent may be an immunotherapeutic agent including antigens, antibodies, cytokines, chemokines, adjuvants and the like.
  • Antigens associated with infectious diseases that can be used in the methods of the invention include whole bacteria, whole virus, whole fungi, whole parasites, and fragments thereof. Examples include non-infectious human papillomavirus-like particles (VLP) (which can be used as a cancer antigen as well, particularly for cervical cancer), and the like.
  • VLP non-infectious human papillomavirus-like particles
  • a DPP8/9 inhibitor be used with normal and hyper-immune globulin therapy.
  • Normal immune globulin therapy utilizes an antibody product from the serum of normal blood donors. This pooled product contains low titers of antibody to a wide range of antigens such as those of infectious pathogens (e.g., bacteria, viruses such as hepatitis A, parvovirus, enterovirus, fungi and parasites).
  • Hyper-immune globulin therapy utilizes antibodies which are prepared from the serum of individuals who have high titers of an antibody to a particular antigen. The antibodies may be those that are currently used or in development for treating infectious diseases.
  • Examples include zoster immune globulin (useful for the prevention of varicella- zoster in immunocompromised children and neonates), human rabies immunoglobulin (useful in the post-exposure prophylaxis of a subject bitten by a rabid animal), hepatitis A or B immune globulin (useful in the prevention of hepatitis A or B virus, especially in a subject exposed to the virus), RSV immune globulin (useful in the treatment of respiratory syncitial virus infections), tetanus immunoglobulin; measles immunoglobulin (useful in the prevention of infection in immunocompromised or adult subjects); rubella immunoglobulin (useful in the prevention of infection in pregnant female subjects).
  • antibodies for infectious diseases include anti-shiga toxin antibodies, anti- staphylococcal antibodies (Virion Systems), and the like.
  • An example of an anti-viral that is an antibody is palivizumab (SYNAGIS) which is used prophylactically for RSV infections in infants and children.
  • the DPP8/9 inhibitor can be used together with an anti-infective that is a chemotherapeutic.
  • anti-bacterial s examples include ⁇ -lactam antibiotics, penicillins (such as natural penicillins, aminopenicillins, penicillinase-resistant penicillins, carboxy penicillins, ureido penicillins), cephalosporins (first generation, second generation, and third generation cephalosporins), and other ⁇ -lactams (such as imipenem, monobactams,), ⁇ -lactamase inhibitors, vancomycin, aminoglycosides and spectinomycin, tetracyclines, chloramphenicol, erythromycin, lincomycin, clindamycin, rifampin, metronidazole, polymyxins, sulfonamides and trimethoprim, and quinolines.
  • penicillins such as natural penicillins, aminopenicillins, penicillinase-resistant penicillins, carboxy penicillins, ureido penicillins
  • cephalosporins first generation, second generation,
  • Anti-bacterials include: Acedapsone; Acetosulfone Sodium; Alamecin; Alexidine; Amdinocillin; Amdinocillin Pivoxil; Amicycline; Amifloxacin; Amifloxacin Mesylate; Amikacin; Amikacin Sulfate; Aminosalicylic acid; Aminosalicylate sodium; Amoxicillin; Amphomycin; Ampicillin; Ampicillin Sodium; Apalcillin Sodium; Apramycin; Aspartocin; Astromicin Sulfate; Avilamycin; Avoparcin; Azithromycin; Azlocillin; Azlocillin Sodium; Bacampicillin Hydrochloride; Bacitracin; Bacitracin Methylene Disalicylate; Bacitracin Zinc; Bambermycins; Benzoylpas Calcium; Berythromycin; Betamicin Sulfate; Biapenem; Biniramycin; Biphenamine Hydrochloride; Bispyrithione Mag
  • Cefepime Cefepime Hydrochloride
  • Cefetecol Cefixime
  • Cefmenoxime Hydrochloride
  • Cefmetazole Cefmetazole Sodium; Cefonicid Monosodium; Cefonicid Sodium;
  • Cefpiramide Cefpiramide Sodium; Cefpirome Sulfate; Cefpodoxime Proxetil; Cefprozil;
  • Cefroxadine Cefsulodin Sodium; Ceftazidime; Ceftibuten; Ceftizoxime Sodium; Ceftriaxone
  • Chloramphenicol Sodium Succinate Chlorhexidine Phosphanilate; Chloroxylenol;
  • Chlortetracycline Bisulfate ChlortetracycHne Hydrochloride; Cinoxacin; Ciprofloxacin;
  • Ciprofloxacin Hydrochloride Cirolemycin; Clarithromycin; Clinafloxacin Hydrochloride; Clindamycin; Clindamycin Hydrochloride; Clindamycin Palmitate Hydrochloride;
  • Cyclacillin Cycloserine; Dalfopristin; Dapsone; Daptomycin; Demeclocycline;
  • Demeclocycline Hydrochloride Demecycline; Denofungin; Diaveridine; Dicloxacillin; Dicloxacillin Sodium; Dihydrostreptomycin Sulfate; Dipyrithione; Dirithromycin;
  • Erythromycin Lactobionate Erythromycin Propionate; Erythromycin Stearate; Ethambutol Hydrochloride; Ethionamide; Fleroxacin; Floxacillin; Fludalanine; Flumequine; Fosfomycin;
  • Fosfomycin Tromethamine Fumoxicillin; Furazolium Chloride; Furazolium Tartrate;
  • Hetacillin Hetacillin Potassium; Hexedine; Ibafloxacin; Imipenem; Isoconazole; Isepamicin;
  • Lomefloxacin Hydrochloride Lomefloxacin Mesylate; Loracarbef; Mafenide; Meclocycline;
  • Meclocycline Sulfosalicylate Megalomicin Potassium Phosphate; Mequidox; Meropenem;
  • Nifurthiazole Nitrocycline; Nitrofurantoin; Nitromide; Norfloxacin; Novobiocin Sodium;
  • Penicillin V Benzathine; Penicillin V Hydrabamine; Penicillin V Potassium; Pentizidone
  • Rosaramicin Propionate Rosaramicin Sodium Phosphate
  • Rosaramicin Stearate Rosoxacin
  • Roxarsone Rosithromycin
  • Sancycline Sanfetrinem Sodium
  • Sarmoxicillin Sarmoxicillin
  • Sulfacytine Sulfadiazine; Sulfadiazine Sodium; Sulfadoxine; Sulfalene; Sulfamerazine; Sulfameter; Sulfamethazine; Sulfamethizole; Sulfamethoxazole; Sulfamonomethoxine;
  • Sulfamoxole Sulfanilate Zinc; Sulfanitran; Sulfasalazine; Sulfasomizole; Sulfathiazole;
  • Sulfazamet Sulfisoxazole; Sulfisoxazole Acetyl; Sulfisoxazole Diolamine; Sulfomyxin;
  • Temafloxacin Hydrochloride Temocillin; Tetracycline; Tetracycline Hydrochloride; Tetracycline Phosphate Complex; Tetroxoprim; Thiamphenicol; Thiphencillin Potassium;
  • Tiodonium Chloride Tiodonium Chloride; Tobramycin; Tobramycin Sulfate; Tosufloxacin; Trimethoprim; Trimethoprim Sulfate; Trisulfapyrimidines; Troleandomycin; Trospectomycin Sulfate;
  • Anti-mycobacterials include Myambutol (Ethambutol Hydrochloride), Dapsone (4,4'- diaminodiphenylsulfone), Paser Granules (aminosalicylic acid granules), Priftin (rifapentine), Pyrazinamide, Isoniazid, Rifadin (Rifampin), Rifadin IV, Rifamate (Rifampin and Isoniazid),
  • Anti-virals include amantidine and rimantadine, ribivarin, acyclovir, vidarabine, trifluorothymidine, ganciclovir, zidovudine, retinovir, and interferons. Anti-virals further include Acemannan; Acyclovir; Acyclovir Sodium; Adefovir;
  • Famciclovir Famotine Hydrochloride; Fiacitabine; Fialuridine; Fosarilate; Foscarnet Sodium; Fosfonet Sodium; Ganciclovir; Ganciclovir Sodium; Idoxuridine; Kethoxal; Lamivudine;
  • Ribavirin Rimantadine Hydrochloride
  • Saquinavir Mesylate Somantadine Hydrochloride
  • Anti-fungals include imidazoles and triazoles, polyene macrolide antibiotics, griseofulvin, amphotericin B, and flucytosine.
  • Antiparasites include heavy metals, antimalarial quinolines, folate antagonists, nitroimidazoles, benzimidazoles, avermectins, praxiquantel, ornithine decarboxylase inhibitors, phenols (e.g., bithionol, niclosamide); synthetic alkaloid (e.g., dehydroemetine); piperazines (e.g., diethylcarbamazine); acetanilide
  • quinolines e.g., iodoquinol (diiodohydroxyquin)
  • nitrofurans e.g., nifurtimox
  • diamidines e.g., pentamidine
  • tetrahydropyrimidine e.g., pyrantel pamoate
  • sulfated naphthylamine e.g., suramin
  • anti-infectives include Difloxacin Hydrochloride; Lauryl Isoquinolinium Bromide; Moxalactam Disodium; Ornidazole; Pentisomicin; Sarafloxacin Hydrochloride;
  • Protease inhibitors of HIV and other retroviruses Integrase Inhibitors of HIV and other retroviruses; Cefaclor (Ceclor); Acyclovir (Zovirax); Norfloxacin (Noroxin); Cefoxitin
  • the invention also provides a method for preventing drug resistance in a subject.
  • the method involves administering to a subject receiving an anti-microbial agent, a DPP8/9 inhibitor (e.g., a selective or specific DPP8/9 inhibitor) in an amount effective to reduce the risk of resistance to the anti-microbial agent.
  • a DPP8/9 inhibitor e.g., a selective or specific DPP8/9 inhibitor
  • the subject may be one having or at risk of developing an infectious disease.
  • the subject may have already received an anti-microbial agent, and the infectious disease may have been resistant to such agent.
  • Examples of drug resistant microbes and in some instances the drugs to which they are resistant include Staphylococcus aureus (methicillin), Enterococcus faecium (vancomycin), Streptococcus pneumoniae (penicillin), gonorrhea (penicillin), Acinetobacter baumannii, Escherichia coli, Klebsiella species, Aspergillus, and Pseudomonas aeruginosa.
  • the invention therefore provides methods for using a DPP8/9 inhibitor (e.g., selective or specific DPP8/9 inhibitor) in combination with various vaccines either currently being used or in development, whether intended for human or non-human subjects.
  • vaccines for human subjects and directed to infectious diseases include the combined diphtheria and tetanus toxoids vaccine; pertussis whole cell vaccine; the inactivated influenza vaccine; the 23-valent pneumococcal vaccine; the live measles vaccine; the live mumps vaccine; live rubella vaccine; Bacille Calmette-Guerin (BCG) tuberculosis vaccine; hepatitis A vaccine; hepatitis B vaccine; hepatitis C vaccine; rabies vaccine (e.g., human diploid cell vaccine); inactivated polio vaccine; meningococcal polysaccharide vaccine; quadrivalent meningococcal vaccine; yellow fever live virus vaccine; typhoid killed whole cell vaccine; cholera vaccine; Japanese B encephalitis
  • the invention provides a method for shortening a vaccination course.
  • shortening a vaccination course refers to reducing either the number of vaccine administrations (e.g., by injection) or the time between vaccine administrations. This is accomplished by stimulating a more robust immune response in the subject.
  • the method may involve, in one embodiment, administering to a subject in need of immunization a DPP8/9 inhibitor (e.g., a selective or specific DPP8/9 inhibitor) in an amount effective to induce an antigen-specific immune response to a vaccine administered in a vaccination course, wherein the vaccination course is shortened by at least one immunization.
  • a DPP8/9 inhibitor e.g., a selective or specific DPP8/9 inhibitor
  • the vaccination course is shortened by one immunization, two immunizations, three immunizations, or more.
  • the method may involve, in another embodiment, administering to a subject in need of immunization a DPP8/9 inhibitor (e.g., a selective or specific DPP 8/9 inhibitor) in an amount effective to induce an antigen-specific immune response to a vaccine administered in a vaccination course, wherein the vaccination course is shortened by at least one day.
  • the vaccination course is shortened by one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, one month, two months or more.
  • a DPP8/9 inhibitor e.g., a selective or specific DPP8/9 inhibitor
  • Immunizations that can be modified in this way include but are not limited to newborn immunizations for HBV; immunizations at for example two months of age for Polio, DTaP, Hib, HBV, Pneumococcus; immunizations at for example four months of age for Polio, DTaP, Hib, Pneumococcus; immunizations at for example six months of age for Polio, DTaP, Hib, HBV, Pneumococcus; immunizations at for example 12-15 months of age for Hib, Pneumococcus, MMR, Varicella; immunizations at for example 15-18 months of age for DtaP; immunizations at for example 4-6 years of age for Polio, DPT, MMR; immunizations at for example 11-12 years of age for MMR; immunizations at for example 14-16 years of age for tetanus-diphtheria (i.e., Td) (
  • a recommended vaccination course for tetanus/diphtheria includes a primary immunization series given in adults if not received as a child, followed by routine booster doses of tetanus-diphtheria (Td) every 10 years.
  • the method of the invention will allow for a shortened series of vaccinations at the first time point, and may in some instances obviate the need for subsequent booster shoots.
  • hepatitis vaccination commonly requires three administrations spaced at least two weeks, and sometimes one month, apart in order to develop full immunity. Using the methods of the invention, it is possible either to reduce the number of injections from three to two or one, or to reduce the time in between injections from weeks or months to days or weeks.
  • Vaccination courses that can be shortened by the method of the invention include but are not limited to: HBV: Hepatitis B vaccine (3 total doses currently recommended); Polio: Inactivated polio vaccine (4 total doses currently recommended); DTaP: Diphtheria/tetanus/acellular Pertussis (3-in-l vaccine; 5 total doses currently recommended); Hib: Haemophilus influenzae type b conjugate vaccine (4 total doses currently recommended); Pneumococcus (Prevnar): Protects against certain forms of Strep.
  • a DPP8/9 inhibitor e.g., a selective or specific DPP8/9 inhibitor
  • MMR measles/mumps/rubella
  • Td Adult tetanus/diphtheria (2-in-l vaccine; for use in people over age 7).
  • a DPP8/9 inhibitor e.g., a selective or specific DPP8/9 inhibitor
  • oral polio vaccine e.g., a selective or specific DPP8/9 inhibitor
  • Prion diseases are neurodegenerative diseases that involve aggregates of prion proteins that have an abnormal conformation.
  • Normal prion protein is usually present in the cell membrane of many tissues, particularly neuronal tissue.
  • the abnormally conformed prion protein is believed to be directly involved in converting normally conformed prion protein into more of the abnormally conformed prion protein, which then self-assembles into aggregates that are damaging to neuronal tissue anatomy and function.
  • Some prion diseases are transmissible.
  • prion diseases include Creutzfeldt- Jakob disease (CJD), bovine spongiform encephalopathy (BSE), and scrapie.
  • the CJD may be iatrogenic CJD (iCJD), variant CJD (vCJD) or sporadic CJD (sCJD).
  • Subjects at risk of prion diseases include subjects that have ingested "infected" animal products (e.g., a subject that has eaten meat from an animal having "Mad-cow” disease). Further reference to prion diseases, subjects at risk thereof and diagnosis of subjects having prior disease can be found in published PCT Application WO 2004/007743, published January 22, 2004, and these specific descriptions are incorporated by reference herein.
  • the invention further provides a method for stimulating an immune response in a chronically immunocompromised or immunosuppressed subject comprising administering to a subject in need thereof a DPP8/9 inhibitor (e.g., a selective or specific DPP8/9 inhibitor).
  • a DPP8/9 inhibitor e.g., a selective or specific DPP8/9 inhibitor
  • Chronic immunosuppression can arise from pharmaceutical use such as the use of deliberate anti-inflammatories such as cox-1 or cox-2 inhibitors celecoxib (Celebrex), rofecoxib (Vioxx), naproxen (Naprosyn), non-steroidal anti-inflammatory drugs (NSAIDS) such as ibuprofen (Motrin, Advil), fenoprofen, indomethacin, and valdecoxib (Bextra), and aspirin; substance abuse such as the alcoholism, intravenous drug use, morphine use, and chronic infections or disease states such as gingivitis, osteomyelitis, diabetes types I and II, chronic granulomas, Pneumocystis carinii pneumonia (PCP) infection, recurrent fungal/yeast infections, non-Hodgkin's lymphoma, and Kaposi's Sarcoma.
  • CEP pallasarcoma
  • PCP chronic granulomas
  • PCP P
  • the invention further provides a method for stimulating an immune response in a genetically immunocompromised subject comprising administering to a subject in need thereof a DPP8/9 inhibitor including a selective or specific DPP8/9 inhibitor.
  • Genetically immunocompromised subjects harbor a genetic mutation that renders them immunocompromised even in the absence of an infectious or exogenous procedure. These subjects include those having a genetic deficiency such as SCID, agammaglobulinemia (e.g., Bruton's agammaglobulinemia and congenital hypogammaglobulinemia), common variable immunodeficiency (CDG), selective immunoglobulin A deficiency, congenital disorder of glycosylation (CDG), or common variable immunodeficiency (CVID).
  • SCID genetic deficiency
  • agammaglobulinemia e.g., Bruton's agammaglobulinemia and congenital hypogammaglobulinemia
  • CDG common variable immunodefic
  • the subjects to be treated according to the invention are immunocompetent.
  • Some methods of the invention involve screening of subjects to determine immunocompetency prior to treatment. This can be accomplished by harvesting immune cells (e.g., peripheral blood lymphocytes) from the subject and testing such cells in vitro for responsiveness to stimuli such as mitogens (e.g., PHA or ConA). This can also be accomplished by analyzing the cellular composition of blood, bone marrow, spleen, thymus, lymph node, or other hematopoietic and/or lymphoid tissue in the body of the subject.
  • immune cells e.g., peripheral blood lymphocytes
  • mitogens e.g., PHA or ConA
  • the invention further provides a method for treating a subject having or at risk of developing an interferon (IFN)-responsive condition.
  • the IFN may be IFN ⁇ , IFN ⁇ -2b, IFN ⁇ or IFN ⁇ , but is not so limited.
  • the method comprises administering to a subject in need of such treatment a DPP8/9 inhibitor including a selective or specific DPP8/9 inhibitor.
  • the inhibitor may be administered in an amount effective to induce IL-I.
  • the method may further comprise identification of a subject having or at risk of developing an IFN-responsive condition.
  • the condition is an IFN ⁇ -responsive condition, including but not limited to viral infections and associated diseases, and cancer.
  • the IFN-responsive condition is a chronic infection selected from the group consisting of multiple sclerosis, chronic hepatitis B infection, chronic hepatitis C infection, chronic Epstein Barr Virus infection, and tuberculosis
  • the viral infection may be an HIV infection.
  • Other conditions include hepatocellular carcinoma, Kaposi's Sarcoma (AIDS-related), thick primary melanomas, and regional lymph node metastases.
  • the condition is drug resistant.
  • These methods may further comprise administering to the subject a second therapeutic agent selected from the group consisting of IFN ⁇ , pegylated IFN, IFN ⁇ -2b, acyclovir, lobucavir, ganciclovir, L-deoxythymidine, clevudine, an antigen or antibody (or fragment thereof) (e.g., hepatitis B-specific immunoglobulin), phosphonoformate (PFA), ribavirin (RBV), thymosin alpha- 1, 2 3-dideoxy-3-fluoroguanosine (FLG), famciclovir, lamivudine, adefovir dipivoxil, entecavir, and emtricitabine.
  • a second therapeutic agent selected from the group consisting of IFN ⁇ , pegylated IFN, IFN ⁇ -2b, acyclovir, lobucavir, ganciclovir, L-deoxythymidine, clevudine
  • the invention further provides a method for treating a subject having or at risk of developing cardiovascular disease comprising administering to a subject in need of such treatment a DPP8/9 inhibitor (e.g., a selective or specific DPP8/9 inhibitor) in an amount effective to induce an effective amount of IL-I .
  • a DPP8/9 inhibitor e.g., a selective or specific DPP8/9 inhibitor
  • the invention intends to treat conditions that are not autoimmune diseases such as arthritis or SLE.
  • the invention does not intend to ameliorate allograft transplantation.
  • the agents of the invention are administered in effective amounts.
  • An effective amount is a dosage of the agent sufficient to provide a medically desirable result.
  • the effective amount will vary with the particular condition being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of the treatment, the nature of the concurrent or combination therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgment.
  • an effective amount to inhibit the condition would be an amount sufficient to reduce or halt altogether the abnormal mammalian cell proliferation so as to slow or halt the development of or the progression of a cell population such as, for example, a tumor, and/or to inhibit in whole or in part symptoms associated with the condition.
  • Inhibition can be assessed by the size of a cell mass (e.g., a tumor), or by the presence and/or frequency of cancer cells in the blood or other body fluid or tissue (e.g., a biopsy).
  • some inhibitors are administered in an amount effective to inhibit the condition while reducing in whole or in part side effects such as those associated with post-prolyl cleaving enzymes including Class I inhibitors.
  • the DPP8/9 inhibitor is administered in an effective amount to induce apoptosis via activation of caspases 3 and/or 7 in the absence of IL-I beta induction.
  • Some embodiments of the invention relate to the administration of inhibitors substantially simultaneously.
  • the term “substantially simultaneously” means that the inhibitors (or other agents) are administered at the same time or within minutes of each other (e.g., within 10 minutes of each other).
  • the term embraces joint administration as well as consecutive administration. If the administration is consecutive, it is separated in time for only a short period (e.g., the time it would take a medical practitioner to administer two compounds separately).
  • concurrent administration and substantially simultaneous administration are used interchangeably.
  • the invention contemplates a variety of inhibitor administration dosings, schedules and regimens.
  • the DPP8/9 inhibitor may be administered in a single or in multiple administrations per day. In some important embodiments, the inhibitor is administered in two administrations per day. Any administration per day may include additional inhibitors such as DPP4 and/or FAP inhibitors. One or all administrations per day may include one, two, three or more inhibitor classes. For example, the first administration of a day may include the DPP8/9 inhibitor and the second administration of the day may include the DPP4 and/or FAP inhibitor (optionally with the DPP8/9 inhibitor).
  • the invention further contemplates administration of a DPP8/9 inhibitor (e.g., a selective or specific DPP8/9 inhibitor) on alternating days.
  • a DPP8/9 inhibitor e.g., a selective or specific DPP8/9 inhibitor
  • the DPP8/9 inhibitor may be administered on day 2, day 4 and day 6 of a weekly cycle.
  • administration of the DPP8/9 inhibitor may be interrupted for a day or more depending on the presence and magnitude of side effects. The entire time course may therefore be shifted based on the length of the interruption.
  • a DPP8/9 inhibitor is administered in combination with a DPP4 inhibitor and/or a FAP inhibitor and are administered alone or in combination so as to maximize the therapeutic benefit and minimize the side effect(s) of each, both or all inhibitor classes. In some embodiments, this is accomplished by providing a molar excess of DPP4 inhibitor (including selective or specific DPP4 inhibitor) and/or a FAP inhibitor (including selective or specific FAP inhibitor) relative to a DPP8/9 inhibitor (including a selective or specific DPP8/9 inhibitor).
  • the molar ratio of DPP8/9 inhibitor to DPP4 and/or F AP inhibitor can be 1 :2, 1 :3, 1 :4, 1:5, 1 :6, 1:7, 1 :8, 1 :9, 1 :10, 1:20, 1:30, 1 :40, 1 :50, 1:60, 1 :70, 1 :80, 1 :90 or 1 : 100.
  • the molar ratio as used herein refers to the total moles of the DPP8/9 inhibito ⁇ total moles of DPP4 and/or FAP inhibitor.
  • the DPP4 and/or FAP inhibitor can also be administered in a greater than 100 molar excess (as compared to the DPP8/9 inhibitor) (e.g., the DPP8/9 inhibitor: DPP4 and/or FAP inhibitor molar ratio is 1 : 150, 1 :200, etc.).
  • the invention further embraces the administration of different inhibitors at different times.
  • a DPP8/9 inhibitor e.g., a selective or specific DPP8/9 inhibitor
  • a DPP4 inhibitor and/or a FAP inhibitor is administered before a DPP8/9 inhibitor (e.g., a selective or specific DPP8/9 inhibitor).
  • the inhibitors are administered in an alternating manner.
  • the DPP8/9 inhibitor e.g., the selective or specific DPP8/9 inhibitor
  • the DPP4 inhibitor and/or the FAP inhibitor is administered first, followed by the DPP4 inhibitor and/or the FAP inhibitor, followed by the DPP8/9 inhibitor, followed by the DPP4 inhibitor and/or the FAP inhibitor, etc.
  • the DPP8/9 inhibitor may be administered on days 1, 3, 5, 7, etc. and the DPP4 inhibitor and/or the FAP inhibitor may be administered on days 2, 4, 6, 8, etc.
  • the DPP8/9 inhibitor is administered on a number of days (e.g., 2, 3, 4, 5, 6, 7, or more days), followed by administration of the DPP4 inhibitor and/or the FAP inhibitor for a number of days (e.g., 2, 3, 4, 5, 6, 7, or more days).
  • the number of days in which the DPP8/9 inhibitor is administered may be equal to or different from the number of days in which the DPP4 inhibitor and/or the FAP inhibitor is administered.
  • the timing of administration may also depend upon whether a second therapy is used in combination with the DPP8/9 inhibitor (e.g., the selective or specific DPP8/9 inhibitor).
  • the inhibitor is used in combination with a chemotherapeutic or an immunotherapeutic agent that is administered on day 1 of a treatment cycle.
  • the inhibitor may be administered on days 2-7 of the same treatment cycle.
  • the cycle may be 7, 14, 21, 28, 35, 42, 49 days or longer.
  • the cycle may be performed once, twice, three times, four times, or more, optionally with rest periods (e.g., days or weeks in which no therapy is administered) in between.
  • the cycle may be a 21 day cycle in which the immunotherapeutic or chemotherapeutic agent is administered on days 1 and 8, the inhibitor is administered on days 2-7 and 9-14, and days 15- 21 are rest days.
  • the cycle may be a 21 day cycle in which the immunotherapeutic or chemotherapeutic agent is administered on days 1, 8 and 15, and the inhibitor is administered on days 2-7, 9-14 and 15-21.
  • the DPP8/9 inhibitor (e.g., the selective or specific DPP8/9 inhibitor) may be administered prior to the administration of the chemotherapeutic or immunotherapeutic agent, with administration of the DPP4 inhibitor (e.g., the selective or specific DPP4 inhibitor) and//or the FAP inhibitor (e.g., the selective or specific FAP inhibitor) during and/or after administration of the chemotherapeutic or immunotherapeutic agent.
  • the DPP4 inhibitor e.g., the selective or specific DPP4 inhibitor
  • the FAP inhibitor e.g., the selective or specific FAP inhibitor
  • routine schedule refers to a predetermined designated period of time.
  • the routine schedule may encompass periods of time which are identical or which differ in length, as long as the schedule is predetermined.
  • the routine schedule may involve administration on a daily basis, every two days, every three days, every four days, every five days, every six days, a weekly basis, a monthly basis or any set number of days or weeks there-between, every two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, etc.
  • the predetermined routine schedule may involve administration on a daily basis for the first week, followed by a monthly basis for several months, and then every three months after that. Any particular combination would be covered by the routine schedule as long as it is determined ahead of time that the appropriate schedule involves administration on a certain day.
  • the agents may be formulated separately or together with each other or with secondary therapeutic agents. Being formulated together means that the agents are present in the same composition prior to administration to the subject. Being formulated separately means the agents are present in separate and distinct compositions prior to administration to the subject. In this latter instance, however, they may be commingled immediately prior to administration to the subject.
  • the inhibitor can be administered to a subject by any route that delivers the inhibitor to the affected site, either directly or indirectly.
  • routes of administration include but are not limited to oral, topical (including intranasal, ocular, vaginal, rectal, transdermal, etc.), and parenteral (including intramuscular, intravenous, subcutaneous, etc.), by inhalation and intratracheal. Delivery may be local (e.g., mucosal) or systemic.
  • the administration route of the inhibitor(s) and other therapeutic agents may be the same or it may be different. In some embodiments, the inhibitors are administered orally, and the other therapeutic agent is administered by a non-oral route.
  • compositions are sterile compositions that comprise effective amounts of active agent, such as the inhibitors of the invention, preferably in a pharmaceutically-acceptable carrier.
  • pharmaceutically-acceptable carrier means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a human or other subject contemplated by the invention.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • the components of the pharmaceutical compositions are commingled in a manner that precludes interaction that would substantially impair their desired pharmaceutical efficiency.
  • the inhibitors may be administered rjer se (neat) or in the form of a salt.
  • the salts are preferably pharmaceutically acceptable. Salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic. Salts can also be alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.
  • Suitable buffering agents include acetic acid and a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v).
  • Suitable preservatives include benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).
  • the compounds, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • Pharmaceutical parenteral formulations include aqueous solutions of the active ingredients in water-soluble form.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of active ingredients may be prepared as oil-based suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • a suitable compound for sustained release delivery is GELFOAM, a commercially available product consisting of modified collagen fibers.
  • compositions also may comprise suitable solid or gel phase carriers or excipients.
  • suitable solid or gel phase carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
  • the active agents may be formulated in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • a suitable vehicle e.g., sterile pyrogen-free water
  • the agents can be formulated readily by combining with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion.
  • Pharmaceutical compositions for oral use can be obtained as solid excipients, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • the oral formulations may also be formulated in saline or buffers for neutralizing internal acid conditions or may be administered without any carriers.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added.
  • Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.
  • compositions may take the form of tablets, lozenges, gum, dissolvable sheets or films, pastes (such as tooth pastes), washes (such as mouth washes), formulated in conventional manner.
  • the compounds may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • compositions may also be formulated for topical delivery (e.g., to the skin) by commingling the active ingredients with bases such as creams, ointments or lotions.
  • the compounds may also be formulated as a depot preparation.
  • Such long acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • the preferred vehicle is a biocompatible microparticle or implant that is suitable for implantation into the mammalian recipient.
  • exemplary bioerodible implants that are useful in accordance with this method are described in PCT International Application No. PCT/US/03307 (Publication No. WO 95/24929, entitled “Polymeric Gene Delivery System", claiming priority to U.S. patent application serial no. 213,668, filed March 15, 1994).
  • PCT/US/0307 describes a biocompatible, preferably biodegradable polymeric matrix for containing a biological macromolecule. The polymeric matrix may be used to achieve sustained release of the agent in a subject.
  • the agent may be encapsulated or dispersed within the biocompatible, preferably biodegradable polymeric matrix disclosed in PCT/US/03307.
  • the polymeric matrix preferably is in the form of a microparticle such as a microsphere (wherein the agent is dispersed throughout a solid polymeric matrix) or a microcapsule (wherein the agent is stored in the core of a polymeric shell).
  • Other forms of the polymeric matrix for containing the agent include films, coatings, gels, implants, and stents.
  • the size and composition of the polymeric matrix device is selected to resulfin favorable release kinetics in the tissue into which the matrix device is implanted.
  • the size of the polymeric matrix devise may be further selected according to the method of delivery which is to be used, typically injection into a tissue or administration of a suspension by aerosol into the nasal and/or pulmonary areas.
  • the polymeric matrix composition can be selected to have both favorable degradation rates and also to be formed of a material which is bioadhesive, to further increase the effectiveness of transfer when the devise is administered to a vascular or pulmonary surface.
  • the matrix composition also can be selected not to degrade, but rather, to release by diffusion over an extended period of time.
  • Both non-biodegradable and biodegradable polymeric matrices can be used to deliver agents to the subject.
  • Biodegradable matrices are preferred.
  • Such polymers may be natural or synthetic polymers. Synthetic polymers are preferred.
  • the polymer is selected based on the period of time over which release is desired, generally in the order of a few hours to a year or longer. Typically, release over a period ranging from between a few hours and three to twelve months is most desirable.
  • the polymer optionally is in the form of a hydrogel that can absorb up to about 90% of its weight in water and further, optionally is cross-linked with multivalent ions or other polymers.
  • agents may be delivered using the bioerodible implant by way of diffusion, or more preferably, by degradation of the polymeric matrix.
  • exemplary synthetic polymers which can be used to form the biodegradable delivery system include: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, poly-vinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate
  • non-biodegradable polymers examples include ethylene vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.
  • biodegradable polymers include synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid), and poly(lactide-cocaprolactone), and natural polymers such as alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion.
  • Bioadhesive polymers of particular interest include bioerodible hydrogels described by H.S. Sawhney, CP. Pathak and J.A. Hubell in Macromolecules, 1993, 26, 581-587, the teachings of which are incorporated herein, polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).
  • the invention provides a composition of the inhibitors for use as a medicament, methods for
  • Other delivery systems can include timed release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of agent, increasing convenience to the subject and the physician.
  • Many types of release delivery systems are available and known to those of ordinary skill in the art. They include the above-described polymeric systems, as well as polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Patent 5,075,109.
  • Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides; hydrogel release systems; silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like.
  • lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides
  • hydrogel release systems silastic systems
  • peptide based systems such as wax, but are not limited to: (a) erosional systems in which the agent is contained in a form within a matrix such as those described in U.S. Patent Nos. 4,452,775, 4,675,189 and 5,736,152 and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Patent Nos.
  • a long-term sustained release implant may be particularly suitable for treatment Of chronic conditions, such as a the suspected presence of dormant metastases.
  • Long-term release are used herein, means that the implant is constructed and arranged to delivery therapeutic levels of the active ingredient for at least 30 days, at least 60 days and more preferably for several months.
  • Long-term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described above.
  • kits that minimally comprise a DPP8/9 inhibitor optionally formulated for oral administration in one container.
  • the kit contains another container having antibody or antibody fragment, preferably formulated for administration by injection.
  • the kit may comprise another container having an antigen, or a cocktail of antigens.
  • Example 1 Assays for inhibition of the dipeptidyl peptidase activity of DPP4, DPP8/9, FAP and DPP2 by amino boronic dipeptide, cyanopyrrolidine and isoindoline compounds
  • Production of soluble DPP4 enzyme Production of plasmid DNA encoding soluble DPP4 was carried out essentially as described in published PCT application WO 2005/071073 published August 4, 2005. Plasmids encoding soluble recombinant DPP4 were introduced into 293T epithelial cells (CRL-11268, ATCC) by transient transfection. These cells express little or no DPP4 endogenously. 293T cells were plated at 6 x 10 6 in 10 cm plates one day before transfection. Cells were transfected with 10 or 20 ⁇ g purified plasmid DNA using Lipofectamine 2000 (InVitrogen) according to manufacturer's instructions.
  • tissue culture medium was changed to Freestyle 293 Expression medium (serum free, InVitrogen) containing penicillin and streptomycin. Supernatant was harvested at 4-6h intervals (6 ml) or after overnight incubation (10 ml).
  • DPP8 cDNA was obtained by PCR of cDNA derived from a Caco2 cell line using KOD XL polymerase using 2 primer pairs: DPP8-Sfi (5' GTA GTC GGCC CAGCC GGCC ATG GCA GCA GCA ATG GAA ACA GA A-3 ' SEQ ID NQ: 4) and DPP8-R (5' GTAGTCA GCGGCCGC TCA TGA GGC TTG TAG AGC ATC CCA TAC AAT GT-3' SEQ ID NO: 5), and DPP8-Sfi and DPP8-R2 (5' GTAGTCA GCGGCCGC TTA TAT CAC TTT TAG AGC AGC AAT ACG TGA TCC AAG 3' SEQ ID NO: 6).
  • the PCR product was digested with Sfil and Notl and the Sfil-Notl fragment cloned in pSecTag-2B. This placed the DPP8 downstream of the resident secretion sequence in pSecTag-2B.
  • the complete coding sequence was obtained in stages by splicing together sections with no PCR errors to give an intermediate plasmid (#187).
  • the 5' section was then removed with Nhel (which cut upstream of the secretion sequence in pSecTag-2B, and inside DPP8) and replaced by a 780 nt 5' Nhel PCR fragment which deleted the secretion sequence to give plasmid #197 so that wild-type DPP8 could be expressed.
  • the primer pair DPP8-cyto 5'-CCAAGCTG GCTAGC TCA AAC AGA CACCATG GCAGCAGCAATGGAAACAGAA-S' SEQ ID NO: 7) and DPP8-R were used in this procedure.
  • a second plasmid (#237) expressing a DPP8-myc- 6xHis fusion protein was made by replacing a section at the 3' end of plasmid #197 with a PCR product in which the stop codon was deleted and an Apal site placed to give in frame fusion with the tag sequence in pSecTag- 2B.
  • two plasmids were made in pSecTag-2B vector (Invitrogen); the first contained wild-type DPP8 sequence (plasmid #197), the second was a myc 6xHis fusion at the C-terminus of DPP8 (plasmid #237).
  • DPP9 cDNA was obtained by PCR using oligo dT primed cDNA from RNA isolated from human bone marrow stromal cells. KOD XL polymerase was used with the PCR primer pair: DPP9-cyto (5'-CCA AGC TGTCTAGATC AAACAGACACCATG GCCACCACCGGGACCCCAACGGCCGA-3' SEQ ID NO: 8) and DPP9-R (5'-GTAGTCA GCGGCCGC TCA GAG GTA TTC CTG TAG AAA GTG CAG CAA CGT-3' SEQ ID NO: 9).
  • the 5' primer introduced a Xbal site which is compatible with the Nhel site in the pSecTag-2B vector.
  • Nhel site in DPP9 made Nhel unusable.
  • the resulting PCR product was isolated and digested with Xbal and Notl restriction enzymes.
  • the 2.6 kb fragment was isolated by gel electrophoresis under standard conditions (0.8% agarose, TAE) and ligated to Nhel and Notl digested pSecTag-2B vector.
  • DNA sequence of a plasmid (#392) prepared from one of the ampicillin-resistant transformants contained the appropriate size insert, and was determined to encode the correct DPP9 primary sequence. Three silent PCR-induced mutations were observed.
  • Recombinant PEP. PEP encoding cDNA was obtained by PCR of oligo dT primed cDNA from RNA isolated from human bone marrow stromal cells.
  • KOD XL polymerase was used with the primer pair: PEP-F (5'-CCA AGC TGG GCT AGC CAA ACA GAC ACC ATG CTG TCC CTT CAG TAC CCC GAC GTG-3' SEQ ID NO: 10) and PEP-tag (5'-GTA GTC AGC GGG CCC TGG AAT CCA GTC GAC GTT CAG GCA CCG-3' SEQ ID NO: 11 ).
  • the PCR program was 94 0 C, 40 sec followed by 30 cycles of 94 0 C for 15 sec, 54 0 C for 20 sec, and 62 0 C for 4 min.
  • a Nhel restriction site was placed upstream of the ATG start codon underlined in primer sequence above, and an Apal site was placed at the end of the coding sequence to give an in phase fusion with the myc and 6xHis tags in the pSecTag-2B vector (InVitrogen).
  • the PCR product was isolated and digested with Nhel and Apal restriction enzymes.
  • the 2.2 kb fragment was isolated by standard gel electrophoresis (0.8% agarose,TAE) and ligated to Nhel and Apal digested pSecTag-2B vector.
  • DNA sequence of a plasmid (#409) prepared from one of the ampicillin-resistant transformants contained the appropriate size insert and was determined to encode the correct PEP primary sequence with no PCR-induced mutations.
  • Production of soluble DPP8, DPP9 and PEP enzymes 293T cells were transfected with the appropriate recombinant plasmid using Lipofectamine 2000 (InVitrogen). 5-6 x 10 6 cells were cultured in a 10 cm dish one day prior to transfection. Culture medium was DMEM, 10% fetal calf serum, penicillin, streptomycin and glutamine (all at standard concentrations).
  • the extract was affinity purified on 2 nickel columns using the B-Per 6XHis Fusion Protein Purification Kit (Pierce). Material was eluted from the columns in a 4 mL fraction of immidazole buffer according to the manufacturer's instructions. Purified enzyme was diluted 120-fold in HEPES saline assay buffer (pH 8.1) containing 0.1% BSA.
  • a truncated soluble recombinant FAP was made from a cDNA clone of human FAP and fused to a secretion signal in the secretion vector pSecTag 2B. This construct allowed production of soluble FAP in 293T mammalian cells.
  • the strategy was based on the N-terminal sequence of serum DPP IV (see above) and was designed to produce a truncated FAP in which a signal/leader sequence is joined to the residue in FAP analogous to the amino-terminus of serum DPP IV.
  • the transmembrane sequence was deleted in order to render the recombinant FAP soluble.
  • the sequence of the amino terminus of human serum DPP IV and the analogous site for FAP are as follows. The transmembrane anchors of the naturally occurring integral membrane proteins are underlined.
  • hDPP IV MKTPWKVLLGLLGAAALVTIITVPVVLLNKGTDDATADSRKXYT'LTDY ⁇ KN—
  • hFAP MKTWVKIVFGVATSAVLALLVMCIVLRPSRVHNSEENTMPALfgKBl ⁇ lNG-
  • the leader sequence was provided by the secretion plasmid vector pSecTag2-B (InVitrogen) in which an immunoglobulin kappa light chain secretion signal is situated upstream of a polylinker allowing the insertion of human or mouse FAP cDNA in phase.
  • the Sfil restriction site in the vector was chosen in order to minimize the number of residues contributed by the vector in the N-terminal portion of recombinant FAP as shown below.
  • Mature FAP contained 6 residues derived from the vector.
  • the DNA sequence of the vector-FAP junction encoding the 6 vector-derived residues DAAQPA included in mature secreted FAP was as follows.
  • This recombinant FAP also contains a single mutation at threonine 229 (T229M mutation) and therefore lacks one of the six glycosylation sites due to destruction of the N-x-T glycosylation motif at N227.
  • Soluble recombinant FAP was produced by transient transfection of 293T cell (CRL-11268, ATCC) epithelial cells that do not express
  • 293T cells were plated at 6 x 10 6 in 10 cm diameter plates one day before transfection. Cells were transfected with 10 or 20 ⁇ g purified plasmid DNA using Lipofectamine 2000 (InVitrogen) according to manufacturer's instructions. After overnight incubation with Lipofectamine/ DNA, the tissue culture medium was changed to Freestyle 293 Expression medium (serum free; InVitrogen) containing penicillin and streptomycin. Culture supernatant was collected 66-72 hours after transfection and frozen prior to purification on MacroPrep High Q Support (BioRad) ion exchange resin.
  • Lipofectamine 2000 InVitrogen
  • a total of 1.5 litres of expression medium containing secreted rhFAP was diluted in 50 mM Tris, pH 8.1 to give a NaCl concentration of 75-100 mM.
  • the diluted material was run over a 5 mL High Q column, the column washed with 50 mM Tris, 75 mM NaCl, pH 8.1 , and eluted with 50 mM Tris, 400 mM NaCl, pH 8.1. Material that initially flowed through the column was reapplied, washed and eluted. Reapplication of eluted material was performed twice.
  • the pooled eluted material was diluted 3-fold in 50 mM Tris, pH 8.1 to give a final salt concentration of approximately 133 mM, and concentrated approximately 75-fold using a Centricon Plus-80 concentrator (Millipore). Purified enzyme was diluted 267-fold in HEPES saline assay buffer (pH 8.1) containing 0.1% BSA. Human DPP2. Soluble DPP2 purified from human placenta was purchased from
  • IC 50 values for inhibition of dipeptidyl peptidase activity of amino boronic dipeptide and cyanopyrrolidine compounds were preincubated for 5 min. at room temperature with the amino boronic dipeptides L-valinyl- L-boroproline (talabostat [PT-IOO]), L-glutamyl-L-boroproline, L-isoleucyl-L-boroproline, or L-norleucyl-L-boroproline, or the cyanopyrrolidine compounds l-[2-[5-cyanopyridin-2- yl)amino]ethylamino]acetyl-2-cyano-(5)-pyrrolidine (cyanopyrrolidine 1) or l-[[(3-hydroxy- l-adamantyl)amino]acetyl]-2-cyano-(S')-pyrrolidine (cyanopyrrolidine 2) or with no addition.
  • Each compound was added at a concentration of 10 "4 , 10 "5 , 10 “6 , 10 “7 , 10 “8 , 10 “9 or 10 '10 M.
  • the fluorogenic substrate Ala-Pro-7-amino-4-trifiuoro-methyl coumarin (Ala-Pro-AFC, Enzyme Systems Products was added at a final concentration of 0.4 mM, and the reaction mixture incubated for a further 15 min. at room temperature. Reactions were stopped by addition of 1 M sodium acetate and fluorescence was measured in a SpectroMax Gemini XS fluorometer (Molecular Devices, Sunnyvale, CA) using an excitation wavelength of 400 nm and an emission wavelength of 505 nm.
  • reaction mixtures were buffered to the optimal pH for each dipeptidyl peptidase as follows: FAP and DPP 8, pH 8.1; DPP 4, pH 7.6; DPP 2, pH 5.5.
  • IC 50 values were determined from enzyme- inhibition curves obtained by plotting fluorescence units against talabostat concentration. Determination of IC 50 values for inhibition of dipeptidyl peptidase activity or isoindoline compounds PTX- 1200 and PTX-1210.
  • Isoindoline inhibitors 2 with a (S)- cyclohexylglycine (PTX-1200) or a l-(4,4'-difluor-benzhydryl)-piperazine (PTX-1210) at the P2 site were dissolved, respectively, as a 0.07M stock in 50% DMSO or as 0.1 M in 100% DMSO. Secondary 1 mM stocks were made in ImM HCl for routine use. Inhibitors were stored at -20 0 C. Dilutions for inhibition assays were in water.
  • HEPES/NaCl (pH 8.1) assay buffer 50 mM HEPES, 140 mM NaCl, 0.1% bovine serum albumin
  • assay buffer 50 mM HEPES, 140 mM NaCl, 0.1% bovine serum albumin
  • 5 ⁇ l of inhibitor compound at a concentration 20-fold higher than the final concentration in complete reaction mixture was added, and the plate incubated for 5 min to allow inhibitor binding.
  • the enzyme reaction was then started by addition of 5 ⁇ l of 8 mM Ala-Pro- AFC to provide a 0.4 mM final concentration.
  • IC 50 S were approximately 10-fold higher for DPP 2, FAP and PEP with the exception of an IC 50 of 1 nM for inhibition of DPP 2 by NorLeu-boroPro.
  • Glu-boroPro was also a non- selective inhibitor of DPP 8, DPP 9, DPP-IV, DPP 2 and FAP; but, compared to the other amino boronic dipeptides, it was a relatively weak inhibitor of PEP.
  • the cyanopyrrolidine compounds 1 and 2 were shown to be selective inhibitors of DPP-IV, and the isoindolines PTX- 1200 and PTX 1210 to be extremely selective inhibitors of DPP 8 and DPP 9 (Table 2).
  • the THP-I human monocytic cell line (American Type Culture Collection, Manassas, VA) was propagated in RPMI 1640 (Life Technologies, Inc., Rockville, MD) supplemented with heat-inactivated fetal bovine serum (10%), HEPES (lO raM), glutamine (1 mM), penicillin (100 units/ml), streptomycin (100 ⁇ g/ml), and this medium was also used for the caspase-1 and IL-I ⁇ induction assay cultures.
  • THP-I cells were differentiated prior to caspase-1 activation and IL- l ⁇ induction assays by incubation (37 0 C) in 5 ⁇ g/ml phorbol myristate acetate (PMA, Sigma, St. Louis, MO) for 3 hours.
  • Conditioned medium was obtained from cultures of MM45T.Sp cells (American Type Culture Collection) in fully supplemented RPMI 1640 and fractionated by Amicon Centriplus YM-3 (Millipore, Billerica, MA) filtration to produce a filtrate containing material of ⁇ 3kDa in molecular size.
  • the filtrate was reconstituted with heat-inactivated fetal bovine serum (10%) and is referred to as ⁇ 3kDa factor.
  • PMA-differentiated THP-I cells 2.5 x 10 5 PMA-differentiated THP-I cells were added to wells of 24- well plates (Corning Inc., Life Sciences, Acton, MA) and after incubation for 18 hours the compounds PT-100, PTX- 1200 or PTX-1210 with ⁇ 3kDa factor were added to the culture wells.
  • Supernatant concentrations of IL-l ⁇ were determined in duplicate by Quantikine ELISA (R&D Systems, Minneapolis, MN) at 120, 180 and 240 minutes after addition of compounds.
  • the post-proline dipeptidyl aminopeptidases DPP8 2 and DPP9 3 ' 4 are members of the DPP-IV gene family that are expressed as soluble proteins localized in the cytoplasm. 5 Both DPP8 and DPP9 are expressed ubiquitously in human tissues and in leucocytes. 5 Talabostat inhibits all the enzymatically active members of the DPP4 gene family: DPP4 itself, DPP2, DPP8, DPP9 and FAP.
  • DPP8 and DPP9 in order to investigate the role of DPP8 and DPP9 in the induction of IL-l ⁇ by talabostat in monocytes, the activities of the DPP8/9-selective isoindoline compounds PTX-1200 and PTX-1210 were compared with the activity of talabostat in cultures of the THP-I human monocytic cell line.
  • DPP 8 and DPP 9 are closely related, and PTX- 1200 and PTX-1210 inhibit these dipeptidyl peptidases with IC 50 values in range of 25 to 105 nM, whereas IC 50 values for inhibition of DPP-IV, FAP, and DPP 2 are greater than 30,000 nM.
  • THP-I cells are similar to primary monocytes, and they constitutively express high levels of inactive pro-caspase-1 but not IL-I ⁇ in either its immature (pro-IL-l ⁇ ) or mature form.
  • 6 THP-I cells can be stimulated to differentiate into adherent cells that express cytoplasmic pro-IL-l ⁇ by incubation with phorbol myristate acetate (PMA).
  • PMA phorbol myristate acetate
  • Talabostat at a concentration of 10 ⁇ M was shown to stimulate IL-I ⁇ secretion by THP-I cells that had been differentiated by prior PMA treatment (FIG. IB).
  • IL-I ⁇ secreted IL- l ⁇ could be detected in the supernatant of cultured cells at 120 minutes after addition of talabostat, and the supernatant IL- l ⁇ concentration increased steadily over a 4- hour incubation period.
  • both PTX- 1200 and PTX-1210 at the 10 ⁇ M concentration stimulated IL- l ⁇ secretion with similar kinetics (FIG. IB).
  • the secretion of IL-I ⁇ by monocytes is critically dependent upon the activity of the cytoplasmic cysteine protease caspase-1, also known as IL-I ⁇ converting enzyme or ICE.
  • Caspase-1 cleaves the 31 kDa pro-form of IL-I ⁇ to produce biologically active, 17 kDa mature IL- l ⁇ which is secreted from the monocyte by a pathway that does not involve the classical endoplasmic reticulum to golgi pathway.
  • 9 Caspase-1 itself is expressed as an inactive 45-kDa zymogen that requires cleavage to form an enzymatically active complex of 10-kDa and 20-kDa chains that can process pro-IL-l ⁇ .
  • Activation of caspase-1 appears to occur within a multicomponent protein complex called the inflammasome; but little is known about the biochemical interactions that regulate inflammasome formation and caspase-1 activation within the monocyte.
  • DPP 8 or DPP 9 were investigated in THP-I cells using the selective inhibitors.
  • Talabostat, PTX-1200 and PT-1210 were all shown to activate caspase-1 by addition of the synthetic, fluorogenic caspase-1 substrate Ac-WEHD-AFC to the cultures 2 hours after addition of the compounds (FIG. IA).
  • the kinetics of caspase 1 activation were similar for all 3 compounds and also resembled the kinetics of IL-I ⁇ secretion (FIG. 1).
  • Dipeptidyl peptidase 9 has two forms, a broad tissue distribution, cytoplasmic location and DPIV-like peptidase activity. Biochim. Biophys. Acta. 2004; 1679: 18-28

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Abstract

L'invention concerne des méthodes d'immunostimulation qui impliquent une inhibition de DPP8 et/ou 9.
PCT/US2007/009947 2006-04-24 2007-04-24 Méthodes et compositions concernant une immunostimulation Ceased WO2007127204A2 (fr)

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EP2014651A1 (fr) * 2007-07-12 2009-01-14 Exonhit Therapeutics SA Composants et procédés de modulation de Rho GTPases
WO2009109927A3 (fr) * 2008-03-05 2010-02-25 Tel Hashomer Medical Research Infrastructure And Services Ltd. Agonistes du récepteur glp-1 et ingrédients pharmaceutiques actifs apparentés pour le traitement du cancer
EP1353684B1 (fr) * 2001-01-02 2011-05-04 KeyNeurotek Pharmaceuticals AG Utilisation d'inhibiteurs de la dipeptidylpeptidase iv (ec 3.4.14.5) ainsi que de l'aminopeptidase n (ec 3.4.11.2) pour le traitement de processus neurodegeneratifs d'origine ischemique
US20110135651A1 (en) * 2008-08-19 2011-06-09 Children's Hospital Medical Center Method and composition for enhancing hematopoietic stem cell mobilization
EP2366394A1 (fr) * 2010-03-17 2011-09-21 IMTM GmbH Caractérisation et validation d'inhibiteurs et de ligands de la dipeptidyl-aminopeptidase de type IV (DP IV)
WO2018187698A3 (fr) * 2017-04-07 2018-11-15 Trustees Of Tufts College Polythérapies utilisant des agents anticancéreux dépendant de la caspase-1 et des antagonistes de pge2
RU2711380C2 (ru) * 2015-07-16 2020-01-16 Байоксэл Терапьютикс, Инк. Новый подход к лечению рака с применением иммуномодуляции
US11096924B2 (en) 2016-09-07 2021-08-24 Trustees Of Tufts College Combination therapies using immuno-dash inhibitors and PGE2 antagonists
WO2022187241A1 (fr) * 2021-03-01 2022-09-09 Washington University Compositions et procédés de ciblage de card8
US11530242B2 (en) 2018-07-17 2022-12-20 Unm Rainforest Innovations EGFRvIII immunogen and methods for using same
US11559537B2 (en) 2017-04-07 2023-01-24 Trustees Of Tufts College Combination therapies using caspase-1 dependent anticancer agents and PGE2 antagonists
WO2024107832A3 (fr) * 2022-11-16 2024-07-11 Onkosxcel Therapeutics, Llc Régime de traitement pour le traitement de cancers
US20240252466A1 (en) * 2022-12-22 2024-08-01 Merck Sharp & Dohme Llc Cyclohexylgycine derivatives as selective cytotoxic agents

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US6100234A (en) * 1997-05-07 2000-08-08 Tufts University Treatment of HIV
US20030130199A1 (en) * 2001-06-27 2003-07-10 Von Hoersten Stephan Dipeptidyl peptidase IV inhibitors and their uses as anti-cancer agents
EP1745293A1 (fr) * 2004-04-28 2007-01-24 Bayer HealthCare AG Agents utilises dans le diagnostic et le traitement des maladies associees a la dipeptidyl-peptidase 9 (dpp9)

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EP1353684B1 (fr) * 2001-01-02 2011-05-04 KeyNeurotek Pharmaceuticals AG Utilisation d'inhibiteurs de la dipeptidylpeptidase iv (ec 3.4.14.5) ainsi que de l'aminopeptidase n (ec 3.4.11.2) pour le traitement de processus neurodegeneratifs d'origine ischemique
EP2014651A1 (fr) * 2007-07-12 2009-01-14 Exonhit Therapeutics SA Composants et procédés de modulation de Rho GTPases
WO2009109927A3 (fr) * 2008-03-05 2010-02-25 Tel Hashomer Medical Research Infrastructure And Services Ltd. Agonistes du récepteur glp-1 et ingrédients pharmaceutiques actifs apparentés pour le traitement du cancer
US20110135651A1 (en) * 2008-08-19 2011-06-09 Children's Hospital Medical Center Method and composition for enhancing hematopoietic stem cell mobilization
EP2366394A1 (fr) * 2010-03-17 2011-09-21 IMTM GmbH Caractérisation et validation d'inhibiteurs et de ligands de la dipeptidyl-aminopeptidase de type IV (DP IV)
WO2011113895A3 (fr) * 2010-03-17 2011-12-08 Imtm Gmbh Caractérisation et validation d'inhibiteurs et de ligands de la dipeptidyl aminopeptidase iv (dp iv)
US11564986B2 (en) 2015-07-16 2023-01-31 Onkosxcel Therapeutics, Llc Approach for treatment of cancer via immunomodulation by using talabostat
RU2711380C2 (ru) * 2015-07-16 2020-01-16 Байоксэл Терапьютикс, Инк. Новый подход к лечению рака с применением иммуномодуляции
US11096924B2 (en) 2016-09-07 2021-08-24 Trustees Of Tufts College Combination therapies using immuno-dash inhibitors and PGE2 antagonists
US11583516B2 (en) 2016-09-07 2023-02-21 Trustees Of Tufts College Dash inhibitors, and uses related thereto
US11957657B2 (en) 2016-09-07 2024-04-16 Trustees Of Tufts College Combination therapies using immuno-dash inhibitors and PGE2 antagonists
US12478609B2 (en) 2016-09-07 2025-11-25 Trustees Of Tufts College Combination therapies using immuno-DASH inhibitors and PGE2 antagonists
US11559537B2 (en) 2017-04-07 2023-01-24 Trustees Of Tufts College Combination therapies using caspase-1 dependent anticancer agents and PGE2 antagonists
WO2018187698A3 (fr) * 2017-04-07 2018-11-15 Trustees Of Tufts College Polythérapies utilisant des agents anticancéreux dépendant de la caspase-1 et des antagonistes de pge2
US11530242B2 (en) 2018-07-17 2022-12-20 Unm Rainforest Innovations EGFRvIII immunogen and methods for using same
WO2022187241A1 (fr) * 2021-03-01 2022-09-09 Washington University Compositions et procédés de ciblage de card8
WO2024107832A3 (fr) * 2022-11-16 2024-07-11 Onkosxcel Therapeutics, Llc Régime de traitement pour le traitement de cancers
US20240252466A1 (en) * 2022-12-22 2024-08-01 Merck Sharp & Dohme Llc Cyclohexylgycine derivatives as selective cytotoxic agents
US12310945B2 (en) * 2022-12-22 2025-05-27 Merck Sharp & Dohme Llc Cyclohexylglycine derivatives as selective cytotoxic agents

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